microsoft word hostin nb 9-2.doc nova biotechnologica 9-2 (2009) 205 chlorine production for water disinfection by the means of photovoltaic panels stanislav hostin 1, peter benedikovič2, anna michalíková2 1department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (stanislav.hostin@ucm.sk) 2slovak university of technology in bratislava, faculty of materials science and technology in trnava, institute of safety and environmental engineering, department of environmental engineering, botanická 49, trnava, sk-917 08, slovak republic (anna.michalikova@stuba.sk) abstract: in this contribution a possibility of electrochemical production of chlorine for water disinfection, by using photovoltaic panels from solar energy, is described. a simple way of chloride production by means of a photovoltaic panel, comparable with classical electrical power source was performed on an experimental device. by using photovoltaic panel with nominal output 50 w and solar irrigation 380 – 550 w/m2 chlorine production was 0.3 mg/min, which represents amount of chlorine sufficient for disinfections of approximately 4000 l water per day. key words: chlorine production, disinfection of water, solar energy, photovoltaic panel 1. introduction water disinfection is important to provide for a hygienic quality of water. it is not only a concern of drinking, service, heating and cooling waters in industry but also waters for public and private pools. in all these cases a potential infection with pathogenic micro-organism legionella pneumophilla must be prevented. water disinfection must be acceptable from point of view of environmental impacts, efficiency and costs (verčimáková et al., 2008). a broad scale of disinfection agents and techniques are available. each of them have advantages and disadvantages. for disinfection of drinking water chlorine, chlordioxide, ozone, cationic silver particles, uv light and ultrafiltration are the most frequently used. to prepare service water, for example for pools, chlorine, calcium hypochlorite (pellets, solution), sodium hypochlorite (solution produced by electrolysis of sodium chloride), bromine, peroxide, iodine, silver, trichlorisocyuronic acid can be used (bartram et al., 2005). a frequent way of chlorine production is electrolysis of sodium/potassium chloride solutions. during this process sodium/potassium hydroxide are also produced. the main technologies are mercury, diaphragmatic and membrane electrolysis. in these procedures the base material is mainly sodium chloride or less frequently potassium chloride. each of the mentioned technologies represents a different way of chlorine 206 hostin, s. et al. accumulation that is produced in the space near anode and hydrogen and hydroxide accumulation produced nearby cathode (reed, 2004). for the diaphragmatic method, which was used in this work, a summary chemical reaction of electrolysis of sodium chloride can be written as follows: 222 222 hnaohclohnacl ++→+ photovoltaic panels (pv panels) represent nowadays modern and available devices for a direct production of electric energy from the solar source. principles, materials, properties, types of pv panels are described in many foreign and native publications where various applications in industry and household are presented (appleyard, 2009; ďuricová et al., 2008). in this work we describe an application of pv panels for electrochemical production of chlorine which is subsequently used for disinfection, preferably service water. 2. material and methods 2.1 chlorine production a hermetic cell (electrolyzer) containing a carbon (6 cm2 area) and an iron (15 cm2 area) with a final volume 750 ml was constructed (fig. 1). an asbestine diaphragm was localized inside of the cell, 500 ml of a brine containing 7% nacl was used as an electrolyte. the hermetic cell contained two outlets provided with plastic tubes. one outlet served for releasing of hydrogen into atmosphere, chlorine was collected into a beaker containing 500 ml water using a second outlet. electrolyser was connected to electric circuit according to a scheme presented in fig. 2. in the first series of experiments a variable laboratory source of unidirectional current type uhs 401-1 (ac 220 v, 2 a / dc 1....15 v, 4 a) was used. a photovoltaic (pv) panel, model str 36×50, produced by solatec, ltd., czech republic was used as a provider of solar energy. pv panel was localized on a south-west oriented flat roof under 35° angle. the panel of 1005 × 453 × 34 mm size, 5.9 kg mass was constructed from a monocrystalline silicium, contained 36 primary cells, working temperature -35 -85 °c. electric parameters for solar irradiation: 1000w/m2, spectrum am 1.56, temperature 25 °c, nominal output 50 w, optimal output 50 w, nominal voltage 12 v, optimal voltage 17.4 v, off-load voltage 21.4 v, short circuit current 3.27 a, optimal current 2.97 a. electric characteristics of electrolysis (voltage, current) were monitored using wattmeter lutron dw 6090 connected via a serial boundary to pc. in case of pv panel, a rheostat with cylindrical coil, total resistance 100 ω for maximum voltage 500v and current 3a, was used. 2.2 determination of chlorine concentration in water concentration of chlorine soluble in water during chlorination was determined spectrophotometrically according to (jonson and overby, 1969). a yellownova biotechnologica 9-2 (2009) 207 orange color of a reaction product of chlorine and o-tholuidine was measured at 440 nm on a digital spectrophotometer genesystm 8 connected to pc. a mixture of k2cr2o7 and k2cro4 was used as a calibration curve. fig. 1. a hermetic electrolytic cell for chlorine production. fig. 2. a scheme of connection of a hermetic cell for chlorine production. 2.3 determination of solar irradiation this was done by an indicator of intensity of solar irradiation, type fl a613-gs, localized nearby the pv panel connected to the data logger almeno 5690-1m (ahlborn). 208 hostin, s. et al. 3. results and discussion 3.1 study of chlorine production using a laboratory electric power source in the first series of experiments a hermetic electrolytic cell was connected to a regulated laboratory electric power source and an electro-chemical production of chlorine was tested. optimal electric characteristics of electrolysis were determined as a constant voltage 6 v and current 0.6 a. fig. 3 presents results of water chlorination. it was found that maximal concentration of chlorine in water was 4.2 mg/dm3 and this was achieved after 25 minutes of electrochemical chlorine production. 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 5 10 15 20 25 30 electrolysis time [ min ] c hl or in e co nc en tr at io n [ m g/ l ] fig. 3. electrolytic production of chlorine using a laboratory electric power source. 3.2 study of chlorine production using a photovoltaic panel in a next series of experiments a hermetic electrolytic cell was connected to a photovoltaic panel which served as a source of electric energy. irregularities of this energy source (dependence on intensity of solar irradiation) were compensated by rheostat. the constant voltage 6 v and 0.6 a current were used similarly as in the case of laboratory electric power source. results of water chlorination are presented in fig. 4. it was found that maximal concentration of chlorine in water was 4.2 mg/dm3 which was achieved after 25 minutes of electrochemical chlorine production. both energy sources provide the maximal concentration of chlorine in water 4.2 mg/dm3 after 25 minutes of electrolysis. however, in case of pv panel the initial chlorine concentration is about 2.5 times higher. measurement of intensity of solar irradiation represents figure 5. from the kinetics of chlorine production it was calculated that the tested hermetic cell connected to the photovoltaic panel ( nominal output 50 w and solar irradiation nova biotechnologica 9-2 (2009) 209 380 -550 w/m2 ) can provide 0.3 mg of chlorine per minute what represents amount of chlorine sufficient for disinfection of approximately 4000 l water per day. 2 2.5 3 3.5 4 4.5 0 5 10 15 20 25 30 electrolysis time [ min ] c hl or in e co nc en tr at io n [ m g/ l ] fig. 4. electrolytic production of chlorine using a photovoltaic panel. 200 250 300 350 400 450 500 550 600 14:45 14:52 15:00 15:07 15:14 15:21 15:28 15:36 15:43 15:50 15:57 time [ h ] s ol ar ir ri ga tio n [ w /m 2 ] fig. 5. solar irradiation during chlorination of water using a photovoltaic panel. 4. conclusion it was found that pv panel can be used as an alternative of a laboratory electric power source to provide electric energy for an electrolytic chlorine production in a hermetic cell. pv panel can be recommended, after some improvements and economic calculations, for practical application. 210 hostin, s. et al. acknowledgement: this work was supported by projects under the contract no. vega 1/0798/08, development and utilization of small hydroenergetic power source combined with solar systems in machine technologies. references appleyard, s.: assessing the use of simple dye-sensitized solar cells for drinking water chlorination by communities with limited resources. renew. energ., 34, 2009, 1651-1654 bartram, j., mia, k. l., lenton, r., wright, a.: focusing on improved water and sanitation for health. lancet, 365, 2005, 810-812 ďuricová, i., hostin, s., ondruška, m., michalíková, a., gerulová, k.: the catalogue of projects, solar laboratory, mtf stu trnava, 2008, 58 pp., isbn 978-80-969443-8-5 jonson, j. d., overby, r.: stabilized neutral orthotolidine, snort, colorimetric method for chlorine. anal. chem., 41, 1969, 1744-1750 reed, r.h.: the inactivation of microbes by sunlight, solar disinfection as a water treatment process. adv. appl. microbiol., 54, 2004, 333-365 verčimáková, k., rybár, r., trojan, p.: utilization of uv radiation for water disinfection in solar reservoirs. acta mont. slov., 13, 2008, 343-349 microsoft word vranovicova nbc 12-2.doc nova biotechnologica et chimica 12-2 (2013) 70 doi 10.2478/nbec-2013-0008 © university of ss. cyril and methodius in trnava magnetostructural j-correlations in fe(iii) complexes – a revision beata vranovičová1, roman boča1,2 1department of chemistry, faculty of natural sciences university of ss. cyril and methodius, sk-917 01 trnava, slovak republic (roman.boca@stuba.sk) 2institute of inorganic chemistry, faculty of chemical and food technology, slovak university of technology, bratislava, sk-812 37, slovak republic abstract: the magnetostructural correlations in fe(iii) complexes, originally outlined by gorun and lippard has been revised. other correlation variants have been tested and the dataset enlarged for more recent entries possessing the fe-o-fe bridge including dinuclear, tetranuclear and hexanuclear fe(iii) complexes. the resulting relationships confirm that instead of the original suggestions, the correlation could stay as a linear relationship which covers the possibilities of positive values of exchange coupling constants. key words: exchange coupling, x-ray structure, magnetostructural correlation, fe(iii) complexes 1. introduction relationships between structural and magnetic parameters attract attention of scientists for a long time. invented by hatfield (crawford et al., 1976; hatfield et al., 1985), the exchange coupling constant occurring in the heisenberg form of the spin hamiltonian ˆ ˆˆ ( )ab ab a bh j= − ⋅s s (1) depends upon certain structural parameters such as bond lengths and bond angles. in the series of dinuclear cu(ii) complexes of the [cu(oh)2cu] type the values of j correlate with the bonding angle α = cu-o-cu along a straight line with the negative slope as shown in fig. 1. (notice, the original definition of the j-constant utilizes a numerical prefactor −2, so that all the data below are rescaled to the definition that matches eq. (1)). the critical angle when the positive coupling constant alters to the negative value is close to 98 deg. thus the ground state s = 1 (j > 0) and/or s = 0 (j < 0) can be controlled by the bonding angle α. the correlations of the above type have been extended also to some other types of dinuclear cu(ii) complexes, such as alkoxidoor phenoxido-bridged complexes (thompson et al., 1996). a further progress has been achieved by proposing the magnetostructural correlations in dinuclear cr(iii) complexes of the [cr(oh)2cr] type by hodgson et al. (glerup et al., 1983; hodgson et al., 1985). in dinuclear mn(iv) complexes of the [mn(o)2mn] type a magnetostructural correlation has been outlined by law et al. (2000). contrary to the previous cases, gorun and lippard (1991) proposed a magnetostructural correlation in fe(iii) oxido-bridged complexes in a different form: the value of –j has been found to correlate with the shortest fe-o contact (abbr. p) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:00 utc nova biotechnologica et chimica 12-2(2013) 71 along a decreasing exponential function: 1[cm ] exp( )j a bp−− = ⋅ with the constants a = 8.763 × 1011 and b = −12.663. cu-o-cu /o 94 96 98 100 102 104 106 j/ cm -1 -600 -400 -200 0 200 400 fig. 1. magnetostructural correlation in [cuii(oh)2cuii] complexes by hatfield et al. (crawford et al., 1976) regression line: 1 o[cm ] 78.33 [ ] 7653j α− = − ⋅ + , r2 = 0.98. 2. results and discussion the dataset compiled by gorun and lippard (1991) has been subjected to a non-linear regression using contemporary software. to this end the correlation displayed in fig. 2 has been reconstructed; the regression line is 1[cm ] exp( )j a bp−− = ⋅ − with parameters a = 2.360 × 1010 and b = 10.661 and it is drawn by bold-solid line. the outer bands refer to the confidence and prediction intervals (95 %), respectively. p(fe-o)/10−10 m 1.75 1.80 1.85 1.90 1.95 2.00 2.05 2.10 − j /c m -1 0 20 40 60 80 100 120 140 fig. 2. magnetostructural correlation for [feiii(o)feiii] complexes by gorun and lippard (1991). regression line: 1 10 10[cm ] (2.360 × 10 ) exp( 10.661 [10 m])j p− −− = ⋅ − ⋅ , r2 = 0.95. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:00 utc 72 vranovičová, b. and boča, r. in can be seen that the data is aggregated to two clusters: one for short p ~ 1.8 å referring to a strongly negative j; the second cluster is around “normal” p ~ 1.95 – 2.00 å and it is associated with weakly negative j. the correlation of the above type excludes the occurrence of positive j (no such a data has been reported so far). there is a clear difference between the j-α correlations proposed for cu(ii) complexes and j-p correlations proposed for fe(iii) ones. therefore we tested some other variants of the magnetostructural correlations for fe(iii) complexes. p(fe-o)/10−10 m 1.75 1.80 1.85 1.90 1.95 2.00 2.05 2.10 j/ cm -1 -140 -120 -100 -80 -60 -40 -20 0 fig. 3. redrawn magnetostructural j-p correlation for fe(iii) complexes in a linear mode. first, the original (−j)-p correlation has been redrawn to the usual (+j)-p picture and subjected to the linear regression (fig. 3). the resulting regression line is 1 10[cm ] 525.7 [10 m] 1055j p− −= ⋅ − , r2 = 0.92, and it can be concluded that the correlation coefficient adopts the acceptable value. moreover, the linear relationship allows passing to the region of positive j. there are three datapoints in the intermediate region (p = 1.85 – 1.95) that do not fall the straight line (highlighted by a dot). however, a detailed inspection to the dataset reveals that these data refer to triangulo-[μ3-o-fe3] complexes where the bond angles are fixed to 120 deg so that the bond lengths are more constrained. there is another datapoint that does not match the linear correlation referring to the [fe(salen)cl]2 complex (marked by a cross). omission of these four points improves the regression to r2 = 0.958 that is a better value relative to the original exponential regression. second, the correlation j-α has also been tested for fe(iii) oxido-bridged complexes and the results are displayed in fig. 4. the problematic datapoint is marked by a cross and excluded from the correlation: 1 o[cm ] 3.98 [ ] 396j α− = − ⋅ + , r2 = 0.70. having some more datapoints at the disposal from recent structural and magnetic investigations (nesterov et al., 2012; chygorin et al., 2012; nesterova et al., 2013), the original dataset has been enlarged and subjected the linear regression (fig. 5). finally, the regression line is 1 10[cm ] 520.9 [10 m] 1047j p− −= ⋅ − , r2 = 0.93. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:00 utc nova biotechnologica et chimica 12-2(2013) 73 α(fe-o-fe) /o 90 100 110 120 130 140 150 160 170 j/ cm -1 -140 -120 -100 -80 -60 -40 -20 0 fig. 4. probing of magnetostructural correlation j-α in fe(iii) complexes using original dataset. a point excluded from the correlation is marked by a cross. p(fe-o)/10−10 m 1.75 1.80 1.85 1.90 1.95 2.00 2.05 2.10 j/ cm -1 -200 -150 -100 -50 0 fig. 5. magnetostructural correlation in fe(iii) complexes using enlarged dataset. two points excluded from the correlation are marked by cross. 3. conclusions it has been demonstrated that the original (exponential) magnetostructural jcorrelation for fe(iii) complexes with μ-oxido bridge suffers of a number of weaknesses. it can be substituted by a linear dependence that is approximately of the same quality. the original dataset can be enlarged by points obtained from recent magnetic and structural studies. the data need be filtered for the quality of the x-ray structure determination, different structural motif, and also the quality of experimental magnetic data-taking and theoretical analysis. acknowledgements: slovak grant agencies (vega 1/0233/12, apvv-0014-11) are acknowledged for the financial support. this article was created with the support of the ministry of education, science, research and sport of the slovak republic within the research and development operational programme for the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:00 utc 74 vranovičová, b. and boča, r. project "university science park of stu bratislava", itms 26240220084, co-funded by the european regional development fund. references crawford, v. h., richardson, h. w. , wasson, j. r., hodgson, d. j., hatfield, w. e.: relation between the singlet-triplet splitting and the copperoxygen-copper bridge angle in hydroxo-bridged copper dimers. inorg. chem., 15 (9), 1976, 2107-2110. glerup, j., hodgson, d. j., pedersen, e.: a novel correlation between magnetism and structural parameters in superexchange coupled chromium(iii) dimers. acta. chem. scand., 37, 1983, 161-164. gorun, m. s., lippard, j. s.: magnetostructural correlations in magnetically coupled (μ-oxo)diiron (ii) complexes. inorg. chem.,, 30, 1991, 1625-1630. hatfield, w. e. in: willett, r. d., gatteschi, d., kahn, o. (eds.), magneto-structural correlations in exchange coupled systems, nato asi series. reidel, dordrecht, 1985, p. 555. hodgson, d. j. in: willet, r. d., gatteschi, d., kahn, o. (eds.), magnetostructural correlations in exchange coupled systems, nato asi series. reidel, dordrecht, 1985, p. 501. chygorin, e. n., nesterova, o. v., rusanova j. a., kokozay, v. n., bon, v. v., boča, r., ozarowski, a.: novel heterometallic schiff base complexes featuring unusual tetranuclear {coiii2fe iii 2(μ-o)6} and octanuclear {coiii4fe iii 4(μ-o)14} cores: direct synthesis, crystal structures, and magnetic properties. inorg. chem., 51, 2012, 386-396. law, n. a., kampf, j. w., pecoraro, v. l.: a magneto-structural correlation between the heisenberg constant, j adn the mn-o-mn angle in [mniv(μ-o)]2 dimers. inorg. chim. acta, 297, 2000, 252-264. nesterov, d. s., chygorin, e. n., kokozay, v. n., bon, v. v., boča, r., kozlov, y. n., shuľpina, l. s., jezierska, j., ozarowski, a., pombeiro, a. j. l., shuľpin, g. b.: heterometallic coiii4fe iii 2 schiff base complex: structure, electron paramagnetic resonance, and alkane oxidation catalytic activity. inorg. chem., 51 (16), 2012, 9110-9122. nesterova, o. v., chygorin, e. n., kokozay, v. n., bon, v. v., omelchenko, i. v., shishkin, o. v., titiš, j., boča, r., pombeiro, a. j. l., ozarowski, a.: magnetic, high-field epr studies and catalytic activity of schiff base tetranuclear cuii2fe iii 2 complexes obtained by direct synthesis. dalton trans., 42, 2013, 16909. thompson, l. k., mandal, s. k., tandon, s. s., bridson, j. n., park, m. k.: magnetostructural correlations in bis(μ2-phenoxide)-bridged macrocyclic dinuclear copper(ii) complexes. influence of electron-withdrawing substituents on exchange coupling. inorg. chem., 35, 1996, 3117-3125. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:00 utc microsoft word mikulasova nb 9-2.doc nova biotechnologica 9-2 (2009) 161 antimicrobial effects of essential oils from tanacetum vulgare l. and salvia officinalis l., growing in slovakia mária mikulášová1, štefánia vaverková2 1institute of cell biology, faculty of natural sciences, comenius university, mlynská dolina, bratislava, sk-842 15, slovak republic (mikulasovam@fns.uniba.sk) 2department of pharmacognosy and botany, faculty of pharmacy, comenius university, odbojárov 10, bratislava, sk83232, slovak republic (vaverkova@fpharm.uniba.sk) abstract: possible antimicrobial properties of essential oils isolated from tanacetum vulgare l., and salvia officinalis l., harvested from five different locations in slovakia, were examined using the disc agar diffusion method and by the microdilution method. gc/ms analysis of the essential oil from tanacetum vulgare l. resulted in the identification of 16 compounds constituting 82.1% of the total oil. gram-positive bacteria, mainly bacillus subtilis, were more susceptible to essential oils from both plants than were gramnegative species. tested essential oils posses also anti-yeast activity. the shares of the constituents in the essential oils as well as their antimicrobial activity differed in dependence on the locality. key words: essential oils, tanacetum, salvia, antimicrobial 1. introduction essential oils are volatile compounds of plant secondary metabolism, and may act as phytoprotective or insecticide agents. these compounds also have antibacterial and antifungal activity, which is important both for food preservation and control of human and plant diseases of microbial origin. the quality and yield of essential oils is influenced by many factors, such as fertilizer and ph of soil (alvarezcastelianos and pascual-villalobos, 2003), chemotype or subspecies (goren et al., 2001), choice of plant part (keskitalo et al., 2001), harvesting season (cornu et al., 2001), the choice and stage of drying conditions (tateo and riva, 1991) and extraction method (scalia et al., 1999). the content of some components of essential oils varies according to the geographic location. thus, 1,8cineole was the most abundant in the oil of sage from the usa (18.0%), camphor in the oil of romania (26.5%), caryophallene in the oil from the usa (10.0%), alphathujone in the oil from italy (45.8%), beta-thujone in the oil from romania (23.1%) (boelens and boelens, 1997). the composition of the essential oils from various chemotypes of tanacetum vulgare was studied in detail on populations of wild growing species in finland (keskitalo et al., 2001), norway (dragland et al., 2005), hungary, poland, italy, and others. in our previous work (vaverková et al., 2006) we examined qualitative properties of the essential oil obtained from tanacetum vulgare l. and we confirmed that the shares of the constituents in the essential oils differ in dependence on the location. the purpose of this work was to estimated and compared antimicrobial activity of extracts from tanacetum vulgare and salvia sp. from some geographic locations in slovakia. 162 mikulášová, m. and vaverková, s. 2. material and methods 2.1 plant material and gc/ms analyses of essential oils tanacetum vulgare l. and salvia officinalis l. were used as a testing material. collection of the plant has been carried in the phase of full flowering flower heads. twenty grams of dry flower heads were subjected to hydrodistilation for 3.5 hours in accordance to the european pharmacopoea. isolated oil was diluted in n-hexane and dried over anhydrous sodium sulphate. oil samples were analyzed using a hewlett packard hp 5971. a mass selective detector directly coupled to a gas chromtaograph hp 5890 series ii fid was used. a capillary column db-wax/26m x 0.20 mm, 0.2 mm film thickness (hewlett packard, usa) was used. detailed agro-ecological characteristics of individual locations are archived at the workplace of authors. 2.2 microbial strains bacterial strains escherichia coli ccm 3988, proteus mirabilis ccm 1941, staphylococcus aureus ccm 3953, micrococcus luteus ccm 732, bacillus subtilis ccm 1718 and the yeasts saccharomyces cerevisiae, candida albicans, candida parapsiloides and rhodotorula glutinis were obtained from the czech collection of microorganisms, brno, czech republic. 2.3 antimicrobial activity disc diffusion method nutrient agar plates were swabbed with the respective broth culture of the organisms (diluted to 0.5 mcfarland standard with saline). filter paper discs (6 mm in diameter) were impregnated with 5 μl of the extract and placed on the inoculated plates. these plates, after staying at 4°c for 2 h, were incubated at 37°c for 24 h for bacteria and at 28°c for 48 h for the yeasts. the diameters of the inhibition zones were measured in millimeters. determination of minimum inhibitory concentration was performed using broth microdilution method. the overnight growing culture of bacteria was filtered and 2% suspension of bacteria was prepared. 180 µl of this suspension and 20 µl of double diluted solutions of eo were placed into the wells of 96 well microtitre plates and cultivated for 24 h on reciprocal shaker at 37°c. the time course of absorbance (a630) was determined at 120 min intervals in triplicates. 3. results and discussion 3.1 chemical composition of the essential oil the following compounds were identified using gc/ms analysis in essential oil from tanacetum vulgare l., which constituted 82.1 % of total oil: α-pinene, camphene, sabinene, β-pinene, myrcene, 1,8-cineole, artemisia ketone, β-tujone, nova biotechnologica 9-2 (2009) 163 camphor, borneol, umbellulone, d-carvone, chrysantenyl-acetate, bornyl-acetate, thymol, germacrene and carvacrol. comparison of these results with those mentioned in literature indicates that these compounds are in the most cases the main components of tanacetum essential oils. the shares of the constituents in the essential oils differed in dependence on the location. 3.2 antimicrobial activity as seen in fig. 1, tested oils from tanacetum vulgare l., and salvia officinalis l. showed better activity against gram-positive bacteria than against gram-negative ones. a 0 5 10 15 20 25 t1 t2 t3 t4 t5 zo ne o f i nh ib it io n (m m ) b 0 5 10 15 20 25 s1 s2 s3 zo ne o f i nh ib it io n (m m ) e.coli p.mirabilis s.aureus m.luteus b.subtilis fig. 1. antibacterial activity of essential oils from tanacetum vulgare l. (a) and salvia officinalis l. (b) from different locations. 164 mikulášová, m. and vaverková, s. among the bacteria tested bacillus subtilis was the most sensitive to all eos. other gram-posititve bacteria show lower susceptibility to the essential oils. from gramnegative bacteria only escherichia coli was more susceptible to eo from salvia officinalis l. oil from one chemotype of tanacetum vulgare l. (t3) had very strong effect on both gram-negative bacteria, but oils from another chemotypes had only very slightly effect. according to kalodera et al. (1997), oils from tanacetum are effective on gram-negative bacteria. however, most studies investigating the antibacterial effects of essential oils confirmed, that they are more active against grampositive than gram-negative bacteria (randrianarivelo et al., 2009). our results indicate that this effect differs due to different chemotype of plant, as is state later. the comparative evaluation of essential oils from two distinct species of plants (tanacetum vulgare l., and salvia officinalis l.) showed variations in the level of activity against bacteria. these variations were evident from zones of inhibition as well from mic values. mic of essential oils from tanacetum ranged in all bacteria from <0.6 % to >>2.5 %. three essential oils from salvia had strong effect on micrococcus luteus and bacillus subtilis (mic <0.6 %). in gbacteria escherichia coli and proteus mirabilis mics of salvia essential oils ranged from <0.6 % to 2.5 %. samples of essential oils t1-t5 originated from the same plant species, were isolated using the same procedures and differ only by the geographic location of plants. as is evident from the comparison of inhibition zones (fig. 1) and mic values their antibacterial properties are different. t4 and t5 possess the lowest antibacterial effect and sample t3 posses the best one. mic of this eo in e. coli, proteus mirabilis and bacillus subtilis is <0.6%, only in micrococcus luteus is 2.5 %. essential oils were tested also for their effects on the yeasts. as is evident from the values of inhibition zones in fig. 2, the more susceptible to eo is candida albicans. also on this yeast, the most effective essential oils are t3 from tanacetum and s1 from salvia. a 0 2 4 6 8 10 12 14 16 t1 t2 t3 t4 t5 zo ne o f i nh ib it io n (m m ) nova biotechnologica 9-2 (2009) 165 b 0 2 4 6 8 10 12 14 16 18 s1 s2 s3 zo ne o f i nh ib it io n (m m ) s.cerevisiae c.albicans c.parapsilosis r.glutinis fig. 2. the effect of essential oils from tanacetum vulgare l. (a) and salvia officinalis l. (b) from different locations on the growth of yeasts. 4. conclusions from this study it can be concluded, that essential oils from tanacetum vulgare l. and salvia officinalis l. posse antibacterial and anti-yeast activity. it is known, that the chemical composition of essential oils from plants species can vary according to the geographical origin and harvesting period. individual components of essential oils exhibit different degrees of antimicrobial activity; therefore variation in composition between batches of essential oils from plants from different locations can be sufficient to cause variability in their antimicrobial effects. references alvarez-castelianos, p.p., pascual-villalobos, m.j.: effect of fertilizer on yield and composition of flowerhead essential oil of chrysanthemum coronarium (asteraceae) cultivated in spain. ind. crop prod., 17, 2003, 77-81. boelens, m. h., boelens, h.: chemical and sensory evaluation of three sage oils. perfum. flavor., 22, 1997, 9-39. cornu, a., camat, a.p, martin, b., coulon, j.p., lamaison, j.l., berdagué, j.l.: solid-phase micro-extraction of volatile components from natural grassland plants. j. agric. food chem., 49, 2001, 203-209. dragland, s., rohloff, j., mordal, r., iversen, t.h.: harvest optimization and essential oil production in five tansy (tanacetum vulgare l.) genotypes under a northern climate. j. agric. food chem., 53, 2005, 4946-53. goren, n., demirci, b., baser, k.h.c.: composition of the essential oils of tanacetum spp. from turkey. flavour frag. j., 16, 2001, 191-194. 166 mikulášová, m. and vaverková, s. kalodera, z., pepeljnak, s., blaževič, n., petrak, t.: chemical composition and antimicrobial activity of tanacetum parthenium essential oil. pharmazie, 52, 1997, 885-886. keskitalo, m., pehu, e., simon, j.e.: variation in volatile compounds from tansy (tanacetum vulgare l.) related to genetic and morphological differences of genotypes. biochem. system. ecol., 29, 2001, 267-285. randrianarivelo, r., sarter, s., odoux, e., brat, p., lebrun, m., romestand, b., menut, c., andrianoelisoa, h.s., raherimandimby, m., danthu, p.: composition and antimicrobial activity of essential oils from cinnamosma fragrans. food chem., 114, 2009, 680-684. scalia, s., giuffireda, l., pallado, p.: analytical and preparative supercritical fluid extraction of chamonile flowers and its comparison with conventional methods. j. pharm. biomed. anal., 21, 1999, 549-558. tateo, f., riva, g.: influence of the drying process on the quality of essential oils in artemisia absinthum. mitt.geb. leven. hyg., 82, 1991, 607-614. vaverková, š., mikulášová, m., habán, m., sloboda, p.: a study of qualitative properties of the essential oils of tanacetum vulgare l. čes. slov. farm., 55, 2006, 181-185. microsoft word kavsek nb 9-3.doc nova biotechnologica 9-3 (2009) 265 multivariate statistical methods for characterization of waste water quality darja kavšek1, darinka brodnjak vončina2 1regional technological centre zasavje, chemical-technological laboratory, nasipi 48, 1420 trbovlje, slovenia (darjakavsek@gmail.com) 2faculty of chemistry and chemical engineering, university of maribor, smetanova 17, 2000 maribor, slovenia (darinka.brodnjak@uni-mb.si) abstract: the aim of this work is focused on water quality classification of the waste waters and evaluation of pollution by the monitoring measurements during period 2006-2008. environmental monitoring was performed in the region of trbovlje, slovenia, with two sampling sites and 15 chemical and physicochemical water quality parameters (ph, temperature, suspended solids, settling matter, chemical oxygen demand, biochemical oxygen demand, aox (adsorbable organic halogens), total phosphorus, ammonium, nitrite, sulphate, chloride, fluoride, sulphide and mineral oil content) monitored in monthly periods (total of 60 objects x 15 variables). for handling the results different chemometric methods were employed, such as basic statistical methods for the determination of mean and median values, standard deviations, minimal and maximal values of measured parameters and their mutual correlation coefficients, the principal component analysis (pca), cluster analysis (ca), and linear discriminant analysis (lda). monitoring of general pollution of waste waters and following measuring parameters which are above permitted concentration level can be used for searching of pollution source and for planning prevention measures from pollution, as well. the study allows drawing new information from the data sets such as patterns of similarity between sampling locations, sources of pollution in the environment, seasonal behavior of chemical contents and time trends. key words: waste waters, water quality, chemometrics, principal component analysis, classification. 1. introduction the experimental data set, carried out throughout the years 2006-2008, was composed of analytical parameters from 2 waste water sources, first is the cave water from savski rov and the second is the industrial wastewater. through this period the quality of the water was followed and the classification has been made according to sampling sites. from 28 variables 15 variables, carrying the most useful information, were selected and investigated. the aim of this work is to find the correlation between sampling sites and the variables obtained by chemical measurements, which can be used to construct a fast decision model for separating different waste water quality samples. chemometric methods have been often used for the classification and comparison of different water samples (simeonova and simeonov, 2007; massart et al., 1997). some examples are, for instance, the differentiation of water sources in a vast southwest area of paris by principal component analysis (pca) (de luca et al., 2008), application of chemometric techniques to the analysis of suquia river water quality (alberto et al., 2001), identification of sources of bottom waters in the weddel sea by pca and target estimation (lindegren and josefson, 1998), 266 kavšek, d. and brodnjak-vončina, d. determination of correlation of chemical and sensory data in drinking waters by factor analysis (meng et al., 1997), to name just a few. chemometric methods have been used also for evaluating environmental data of lagoon water (carrer and leardi, 2006), san francisco bay and estuary (jarman et al., 1997), and muggia bay in northern adriatic sea (barbieri et al., 1998). the quality of the waste waters was studied through the years 2006-2008. altogether 15 preselected characteristic features were measured for 60 samples collected and analysed during this period. several chemometric methods were applied in order to visualize multivariate data and to enable a quick classification of samples, regarding the source location within the studied time period. 2. materials and methods a standard method was used for sampling (iso 5667-01:1996 (e)). water was collected in polyethylene bottles 0.5 m below the surface. all glass and plastic ware used for sampling and analyses were rinsed with milli-q water. standard analytical methods (water quality, 1980, 1996, 1998, 2000, 2002, 2004, 2005) were used for the determination of 15 physico-chemical variables. all reagents were analytical grade. the milli-q system was used for purifying the water. the 60 samples are characterized by 15 physico-chemical variables: (1) ph, (2) water temperature, (3) suspended solids, (4) settling matter, (5) chemical oxygen demand (6) biochemical oxygen demand, (7) adsorbable organic halogens (aox), (8) total phosphorus, (9) ammonium, (10) nitrite, (11) sulphate, (12) chloride, (13) fluoride, (14) sulphide, and (15) mineral oil content. the results of all measurements have been investigated by different chemometric methods (massart et al., 1997): the basic statistical methods for the determination of mean and median values, standard deviations, minimal and maximal values of measured variables and their mutual correlation coefficients. the pca was applied for grouping of water samples due to measured variables. all the calculations and plots in the following (pca) section were done with the teach/me software (teach/me datalab 2.002, 1999) using teach/me data analysis option. 3. results and discussion 3.1 statistical screening of data first the mean and median values and standard deviation were determined. after excluding 2 samples, which were discovered as outliers, namely, first sample with very high content of sulphate (more ten times higher than in all other samples, but still under the legislation permitted level) and the second one with high content of suspended solids. the mutual correlation was sought for all measured variables. the maximal correlation coefficient of the data was found between measurements of sulphate content and ph (r = -0.82) and between suspended solids and settling matter (r = 0.77). the correlation between suspended solids and settling matter is expected to be high. the negative correlation between sulphate content and ph shows that samples from two sampling sites are mainly differentiated according to these two parameters. nova biotechnologica 9-3 (2009) 267 fig. 1. dendrogram of 58 samples at 2 different sampling sites (classes). cluster analysis resulted in a dendrogram shown in fig. 1, where all 58 samples are divided into a number of clusters, depending on the level of similarity. clustering is based on ward distance. first group of samples, namely sampling site “savski rov” (the right-most cluster) is well distinguished from second sampling site. clusters of samples from sampling sites 1 and 2 are well defined. according to the content of pollution parameters from the previous mentioned sampling sites it can be concluded that sample 13 from sampling site 1 is different from all others and is clustered in cluster 2. 3.2 principal component analysis (pca) pca was performed in order to get an overall impression about the correlation of 58 water samples, described with physical and chemical variables, with the quality of water in different sampling sites. pca was applied on the matrix composed of 58x15 elements. 58 rows represent waste water samples composed of 15 variables. data were additionally pre-processed in two different ways. first the "column centering" of the data was used and second, the autoscaling of individual variables was performed, called "column standardization". the pca with column standardized data were further analysed for formed clusters. it was found from the score plots of the first and the second pcs that samples are well separated according to sampling sites. clusters of samples from sampling sites 1 and 2 are well defined. the scores and loadings plots of pca of the waste water samples represented with 15 variables are shown in figure 2. it is evident from figure 2 that samples from two sampling sites are well separated. the inspection of particular parameters shows that the content of sulphate is much higher for all samples from sampling site 1, while ph for all samples from sampling site 1 is always lower than for the sampling site 2 (fig. 4). 268 kavšek, d. and brodnjak-vončina, d. fig. 2. pca for all waste water samples from 2 different sampling sites denoted by class numbers 1 and 2. fig. 3. pca for 58 waste water samples from 2 different sampling sites; loadings in 15 pc axes are shown. fig. 4. ph value for all samples from two different sampling sites. nova biotechnologica 9-3 (2009) 269 separation of the two classes is obtained by pc1 (labels 1, 11 and 9, see fig. 3) which is accounted for the ph value and content of ammonium and sulphate, while the second principal component pc2 is accounted for suspended solids, settling matter and mineral oil content (labels 3, 4 and 15, see fig. 3). 3.3 linear discriminant analysis (lda) linear discriminant analysis confirms the predetermined classes. it was found from lda that samples are well separated according to sampling sites (fig. 5). plot of discriminant functions -6 -3 0 3 6 9 function 1 -2,3 -1,3 -0,3 0,7 1,7 2,7 f un ct io n 2 variety 0 1 2 centroids fig. 5. discriminant function for waste waters. 4. conclusions the study gives the opportunity to follow quality of waste waters at different sampling sites. monitoring of general pollution of waste waters and following measuring parameters which are above permitted concentration level can be used for searching of pollution source, for planning of prevention action and for the protection from pollution. references alberto, w.d., del pilar, d.m., valeria, a.m., fabiana, p.s., cecilia, h.a., de los angeles, b.m.: pattern recognition techniques for the evaluation of spatial and temporal variations in water quality, a case study: suquia river basin (cordoba-argentina). water res., 35, 2001, 2881-2894. barbieri, p., adami, g., favretto, a., reisenhofer, e., a chemometric survey of three sites in muggia bay (northern adriatic sea): meteorological effects on heavy metal patterns in surface coastal waters. fresenius j. anal. chem., 361, 1998, 349-352. carrer, s., leardi, r.: characterizing the pollution produced by an industrial area: chemometric methods applied to the lagoon of venice. sci. total environ., 370, 2006, 99-116. de luca, m., oliverio, f., ioele, d., husson, g. p., ragno, g.: monitoring of water quality in south paris district by clustering and simca classification. int. j. environ. anal. chem., 88, 2008, 1087-1105. 270 kavšek, d. and brodnjak-vončina, d. deutsche einheisverfahren zur wasser-, abwasserund schlammuntersuchung bestimmung des volumenanteils der absetzbaren stoffe im wasser und abwasser (h9), din 38409-h9-2:1980. deutsche einheitsverfahren zur wasser-, abwasserund schlammuntersuchung physikalische unf physikalisch-chemische bestimmung der temperatur (c4), din 38404:c4:2000. jarman, w.m., johnson, g.w., bacon, c.e., davis, j.a., risebrough, r.w., ramer, r.: levels and patterns of polychlorinated biphenyls in water collected from the san francisco bay and estuary, 1993-1995. fresenius j. anal. chem., 359, 1997, 254-260. massart, d.l., vandeginste, b.g.m., buydens, l.m.c., de jong, s. lewi, p.j., verbeke, j.s.: handbook of chemometrics and qualimetrics: part a, elsevier, amsterdam, 1997. meng, a.k., suffet, i.h.: a procedure for correlation of chemical and sensory data in drinking water samples by principal component factor analysis. environ, sci. technol., 31, 1997, 337-345. simeonova, p., simeonov, v.: chemometrics to evaluate the quality of water sources for human consumption. microchim. acta, 156, 2007, 315-320. teach/me, sdl software development lohninger; teach/me datalab 2.002, 1999, springer, berlin, developed by h. lohninger and the teach/me people. soil qualitydetermination of oil contentmethod by infrared spectrometry and gas chromatographic method, iso tr 11046 (e). water quality-samplingpart 10: guidance on sampling of waste water iso 5667-01: 1996 (e). water qualitydetermination of ph, iso 10523:1996 (e). water qualitydetermination of suspended solids by filtration through glass fibre filters, iso 11923:1998 (e). water qualitydetermination of chemical oxygen demand, iso 6060:1996 (e). water qualitydetermination of biochemical oxygen demand after n days (bodn)part 2: method for undiluted samples, iso 5815-2:2002 (e). water qualitydetermination of adsorbable organically bound halogens (aox), iso 9562:2005 (e). water qualitydetermination of phosphorusammonium molybdate spectrometric method, iso 6878:2004 (e). water qualitydetermination of ammoniumdistillation and titration method, iso 5664:1996 (e). water qualitydetermination of dissolved anions by liquid chromatography of ionspart 2: determination of bromide, chloride, nitrate, nitrite, orthophosphate and sulfate in waste water, iso 10304-2:1998(e). microsoft word nadaska nb 9-3.doc nova biotechnologica 9-3 (2009) 295 manganese fractionation analysis in specific soil and sediment samples gabriela nádaská, kristína polčová, juraj lesný department of biotechnology, university of ss cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (pisarcig@ucm.sk) abstract: manganese has been determined in soiland sediment samples taken from selected regions with high manganese concentrations anthropogenic and/or geogenic. the total content of manganese in chosen sedimentand soil samples has been determined applying faas after microwave digestion and the manganese fractions after sequential extraction procedures using galvanostatic stripping chronopotentiometry. the highest content of manganese has been determined in sediment from hôrka (6243.6 ± 56.2 mg kg-1), while the lowest value has been obtained in the sediment from kráľová (278.6 ± 3.9 mg kg-1). using a modified tessier’s procedure it was found, that manganese in sediments from kráľová is associated mainly with the carbonate fraction (>50%), while in sediments from lozorno and hôrka it is associated primarily with the mn and fe oxide fraction (80% and 42% respectively). key words: manganese, galvanostatic stripping chronopotentiometry, fractionation analysis 1. introduction manganese is an essential micronutrient for all organisms, playing an important role in tissue and bone formation, in reproductive functions and in carbohydrate and lipid metabolisms (anjos et al., 2007), but at higher levels it can be toxic. in man, chronic manganese poisoning affects the central nervous system, with symptoms resembling those of parkinson’s disease. manganese toxicity is a serious constraint to crop cultivation since manganese is taken-up by plants and can easily be passed into the food chain (khoo et al., 1996; banks et al., 2005; pearson and greenway, 2005). therefore, the determination of manganese in several matrices samples is very important for some areas, such as environmental chemistry and food control (lemos et al., 2008). measurement of total metal concentrations is useful to evaluate the heavy metal burden but their mobility depends strongly on their specific chemical forms or ways of binding. however, the determination of specific chemical species or binding forms is as a rule very complex and often hardly possible. for this reason, sequential extraction procedures are commonly applied because they provide information about the fractionation of metals in the different lattices of the solid sample which is a good compromise to give information on environmental contamination risk (marguí et al., 2004). for manganese determination highly sensitive methods are required. various techniques have been used for the determination of trace manganese in biological and environmental samples, such as spectrophotometry and atomic absorption spectrometry. however the electronalytical techniques such as stripping analysis, which incorporate a preconcentration step, e.g. stripping voltammetry and stripping 296 nádaská, g. et al. chronopotentiometry are the most used in trace metal determination in complex samples. these techniques have been shown to be suitable, versatile and rapid for multicomponent analysis, having good selectivity and adequate sensitivity (town and leeuwen, 2001; filipe et al., 2003). 2. materials and methods 2.1 samples soils and sediments analyzed in this study were collected from regions with high concentrations of anthropogenic and/or geogenic manganese at five sampling sites: kráľová (n 48°12'35''; e 17°48'02''), lozorno (n 48°32'83''; e 17°05'85''), kišovce (n 49°01'18''; e 20°37'95''), primovce (n 49°01'18''; e 20°37'95'') and hôrka (n 49°01'18''; e 20°39'06''). soil samples were taken from top layer (horizon a; 0-15 cm) and subsurface substrate (35-45 cm). the samples were air-dried, homogenized, screened through a 0.3 mm sieve, and stored for subsequent analysis. 2.2 mineralization soil and sediment samples (0.25 g) have been transferred into ptfe digestion vessels and digested with 2 ml of conc. hcl and 5 ml of conc. hno3 in a microwave oven multiwave mw 3000 (anton paar gmbh, austria) according to the following working conditions: 210°c; 30 min; 40 bars. the acid solution was diluted to 25 ml and the metal content was determined by faas using the atomic absorption spectrometer varian spectraa-200 (varian inc., australia). 2.3 sequential extraction procedure the sequential extractions have been performed using the modified tessier’s procedure on 1 g of soil and sediment samples. the relevant procedure is described in table 1. table 1. sequential extraction procedure. extracted fraction procedure f1exchangeable 1.0 g sample; 8 ml 1 m mgcl2 (ph=7), 1 h agitation; room temperature, centrifugation 4000 rpm, 5 min; rinsing of solid residue with 3 ml deionized h2o; centrifugation 4000 rpm, 5 min f2bound to carbonates solid residue from step 1; 8 ml 1 m naoac (ph=5), 5 h agitation; room temperature, centrifugation 4000 rpm, 5 min; rinsing of solid residue with 3 ml deionized h2o; centrifugation 4000 rpm, 5 min f3bound to fe-mn oxides/reducible solid residue from step 2; 8 ml 0.1 m nh2oh.hcl (ph=2), 16 h agitation; room temperature, centrifugation 4000 rpm, 5 min; rinsing of solid residue with 3 ml deionized h2o; centrifugation 4000 rpm, 5 min f4oxidisable solid residue from step 2; 8 ml 30% h2o2, 5 h agitation; room temperature, centrifugation 4000 rpm, 5 min; rinsing of solid residue with 3 ml deionized h2o; centrifugation 4000 rpm, 5 min nova biotechnologica 9-3 (2009) 297 2.4 galvanostatic stripping chronopotentiometry (scp) the amount of mn in obtained fractions after evaporation and dissolving in electrolyte has been determined by galvanostatic stripping chronopotentiometry using the flow-through system ecaflow model glp 150 (istran ltd., slovakia). for the determination, stripping chronopotentiometry on a macroporous electrode has been applied. at suitable positive potential mn(ii) is deposited from a slightly acidic solution on the electrode surface as a hydrate oxide: −+ − + ++→ ehohohmnohmn xx 3)()()( 332 2 2 in the next step the deposit is stripped applying a constant negative current. during this step the signal (chronopotentiogram) is recorded and the concentration of mn in the sample is evaluated. the compact flow-through electrochemical cell ecacell 104, ag/agcl reference electrode and carbon porous working electrode e-104 has been used. basic (carrier) electrolyt solution of 0.1 mol dm-3 na2so4 + 0.01 mol dm -3 nah2po4 and reagent solution of 0.1 mol dm-3 hcl has been used. standard solutions 50 μg dm-3, 100 μg dm-3 and 200 μg dm-3 mn(ii) were prepared from certified reference material (merck). deionised water (conductivity of 0.054 μs cm-1) has been used for all solutions. in all experiments analytical grade reagents were used. table 2 shows the applied experimental parameters for mn determination using scp. table 2. experimental conditions for the determination of mn (www.istran.sk). d e p o s i t i o n p o t e n t i a l 1 1 0 0 m v d e p o s i t i o n t i m e 6 0 s i n i t i a l p o t e n t i a l 1 1 0 0 m v f i n a l p o t e n t i a l 5 0 0 m v d i s s o l u t i o n c u r r e n t 2 5 μa t i m e o f s t a b i l i z a t i o n 1 0 s m a x . t i m e o f s t r i p p i n g 1 2 0 s r e g e n e r a t i o n p o t e n t i a l 4 0 0 m v t i m e o f r e g e n e r a t i o n 1 0 s v o l u m e o f s a m p l e 1 m l „ s t a n d b y “ p o t e n t i a l 6 5 0 m v 3. results and discussion the total mn concentrations and the manganese fractions obtained by the chosen sequential extraction procedures are shown in tables 3-5. samples from kráľová contain anthropogenic manganese markedly derived from waste dump of a former nickel smelter in sereď. manganese in sediments has been found only in carbonate fraction. this is caused evidently by higher ph values of the water reaching 8.2. manganese in soil samples from the same site has been bound to fe-mn oxides in higher proportion, while significant differences of its abundance in different soil horizons have not been observed. 298 nádaská, g. et al. the total mn concentrations of investigated soil samples show similar values as well. our results show for manganese concentrations in soils in comparison with the ones in sediments higher values. we assume that for soils floods may act as a not negligible source of mn. in the same time by lower ph values caused by acid rains the soluble mn species reach the deeper soil horizons. table 3. concentrations of mn in samples of stream sediments after modified tessier’s extraction procedure. concentration of mn ± sd in fractions [mg kg-1] sample f1 f2 f3 f4 total concentration of mn ± sd [mg kg-1] kráľová 141.4 ± 2.9 278.6 ± 3.9 lozorno 48.2 ± 3.6 645.8 ± 22.1 779.9 ± 10.9 hôrka 594.9 ± 2.1 2586.8 ± 56.1 219.5 ± 0.2 6243.6 ± 56.2 kišovce 209.9 ± 12.8 50.3 ± 6.7 2.4 ±0 .8 617.3 ± 13.6 primovce 969.1 ± 5.2 516.6 ± 11.3 45.2 ± 0.4 3219.8 ± 61.2 0 10 20 30 40 50 60 70 80 90 primovce (3219.8) kišovce (617.3) hôrka (6243.6) lozorno (779.9) kráľová (278.6) f1-exchangeable fraction f2-fraction bound to carbonates f3-fraction bound to fe-mn oxides f4-oxidisable fraction m n fr ac tio n [% ] fig. 1. percentual abundance of mn in particular fractions using modified tessier’s extraction procedure in sediment samples (data under the graph represent total manganese concentrations [mg kg-1] determined by faas). table 4. concentrations of mn in soil samples taken from horizon a after modified tessier’s extraction procedure. concentration of mn ± sd in fractions [mg kg-1] sample f1 f2 f3 f4 total concentration of mn ± sd [mg kg-1] kráľová 101.2 ± 4.0 145.9 ± 1.8 14.7 ± 0.6 733.2 ± 10.9 lozorno 27.4 ± 0.9 471.6 ± 3.3 590.1 ± 7.08 kišovce 145.4 ± 9.2 309.6 ± 5.4 85.7 ± 27.9 1339.7 ± 26.8 primovce 498.9 ± 6.4 620.9 ± 23.9 167.6 ± 4.1 2426.6 ± 16.9 nova biotechnologica 9-3 (2009) 299 0 10 20 30 40 50 60 70 80 m n fr ac tio n [% ] f1-exchangeable fraction f2-fraction bound to carbonates f3-fraction bound to fe-mn oxides f4-oxidisable fraction primovce (2426.6) kišovce (1339.7) lozorno (590.1) kráľová (733.2) fig. 2. percentual abundance of mn in particular fractions of the modified tessier’s extraction procedure in soil samples taken from horizon a (data under the graph represent total manganese concentrations [mg kg-1] determined by faas). table 5. concentrations of mn in soil samples taken from subsurface substrate after modified tessier’s extraction procedure. concentration of mn ± sd in fractions [mg kg-1] sample f1 f2 f3 f4 total concentration of mn±sd [mg kg-1] kráľová 79.9 ± 5.4 198.3 ± 6.1 32.3 ± 0.6 757.7±12.1 lozorno 4.5 ± 0.4 343.2 ± 5.3 446.6±15.2 kišovce 81.8 ± 6.6 65.5 ± 1.2 8.9 ± 0.5 488.1±8.3 primovce 298.9 ± 32.3 952.8 ± 126.6 248.3 ± 15.2 2620.8±28.8 0 10 20 30 40 50 60 70 80 m n fr ac tio n [% ] f1-exchangeable fraction f2-fraction bound to carbonates f3-fraction bound to fe-mn oxides f4-oxidisable fraction primovce (2620.8) kišovce (488.1) lozorno (446.6) kráľová (757.7) fig. 3. percentual abundance of mn in particular fractions of the modified tessier’s extraction procedure in soil samples taken from subsurface substrate (data under the graph represent total manganese concentrations [mg kg-1] determined by faas). 300 nádaská, g. et al. acidic soils are typical for zahorie region. this is evidently caused by chemically inactive soil constituents. both, iron and manganese as well, are at oxidizing conditions in higher oxidation states and so they are present in precipitated chemical forms. in such conditions major proportions of iron and manganese in sediments as well as in investigated soil horizons are bound to fe and mn oxides. the highest contents of total manganese have been measured in samples taken from hôrka, kišovce and primovce. this region is known as one with high geogenic manganese concentrations. at lower ph values the carbonate system consists of hydrogen carbonates predominantly and manganese is in such conditions soluble. according to our observation sediments near hôrka contain extremely high manganese concentrations. this fact is avowedly caused by former mining activity. we found that manganese is in this region predominantly bound to mn-fe oxides caused apparently by oxidation of manganese originated from mining activity. fig. 4. typical chronopotentiograms obtained by manganese determination using scp. 4. conclusions results of fractionation experiments prove that the type of manganese coupling to the sedimentand soil matrix is strongly affected by factors connected to geological bedrock as well as to relevant ph values. in regions with low ph values the carbonate system consists mostly from hydrogen carbonates and at such conditions manganese is soluble. for this reason manganese in sediments from lozorno and hôrka is present in fraction bound to fe-mn oxides, namely 80% and 42% respectively. in regions with higher ph values the solubility of carbonates is low. manganese in sediment from kráľová is bound to carbonates, namely >50%. standard 100 μg dm-3 standard 200 μg dm-3 standard 50 μg dm-3 sample nova biotechnologica 9-3 (2009) 301 references anjos, a.p, ponce, l.c., cadore, s., baccan, n.: determination of manganese by flame atomic absorption spectrometry after its adsorption onto naphthalene modified with 1-(2-pyridylazo)-2-naphtol (pan). talanta, 71, 2007, 1252-1256. banks, c.g., kruusma, j., moore, r.r., tomčík, p., peters, j., davis, j., lovrić, š.k., compton, r.g.: manganese detection in marine sediments: anodic vs. cathodic stripping voltammetry. talanta, 65, 2005, 423-429. beinrohr, e.: determination of mn in clean as well as turbid water samples, application list no.74. istran ltd., slovakia. http://www.istran.sk/en/ download/start-download/english/application-lists/33-application-listno.74.html filipe, o.m.s, brett, c.m.a.: cathodic stripping voltammetry of trace mn(ii) at carbon film electrodes. talanta, 61, 2003, 643-650. khoo, s.b., soh, m.k., cai, q., khan, m.r., guo, s.x.: differential pulse cathodic stripping voltammetric determination of manganese(ii) and manganese(vii) at the 1-(2-pyridylazo)-2-naphtol-modified carbon paste electrode. electroanalysis, 9, 1997, 45-51. lemos, v.a., baliza, p.x., carvalho, a.l., oliveira, r.v., teixeira, l.s.g., bezerra, m.a.: development of a new sequential injection in-line cloud point extraction system for flame atomic absorption spectrometric determination of manganese in food samples. talanta, 77, 2008, 388-393. marguí, e., salvadó, v., queralt, i., hidalgo, m.: comparison of threestage sequential extraction and toxicity characteristic leaching tests to evaluate metal mobility in mining wastes. anal. chim. acta, 524, 2004, 151-159. pearson, g.p, greenway, g.m.: recent developments in manganese speciation. trends anal. chem., 24, 2005, 803-809. town, r.m., leeuwen, h.p.: fundamental features of metal ion determination by stripping chronopotentiometry. j. electroanal. chem., 509, 2001, 58-65. microsoft word fargasova nb 9-2.doc nova biotechnologica 9-2 (2009) 107 cr and ni simultaneous phytotoxicity and mutagenicity assay agáta fargašová, jana lištiaková department of ecosozology and physiotactics, faculty of natural sciences, comenius university in bratislava, mlynská dolina, sk-842 15 bratislava, slovak republic (fargasova@fns.uniba.sk) abstract: for genotoxicity study simultaneous phytotoxicity and mutagenicity assay with vicia sativa l. var. klára was used. for phytotoxicity the following rank orders of growth inhibition can be arranged: for roots: ni(ii) > cr(vi) > cr(iii); for shoots: ni(ii) > cr(vi) ≥ cr (iii). for mutagenicity assay root tips of v. sativa were used and chromosome aberrations were determined at least in 500-anatelophases. all tested metals exerted in v. sativa a significant increase of chromosomal aberration rate in applied concentrations. maximum of aberrations invoked cr(vi) and the rank order of aberrations fall was: cr(vi) > ni(ii) > cr(iii). genotoxic effects of metals were determined by analysis of micronuclei frequency in the pollen tetrads of tradescantia plants. none of tested metal significantly stimulated micronuclei frequency and genotoxic effect was decreased in order: cr(vi) ≥ ni(ii) > cr(iii). key words: chromium, nickel, vicia sativa, phytotoxicity, chromosomal aberation assay, tradescantia micronucleus assay 1. introduction vascular plants have been found to be highly effective for recognizing and predicting metal stress in the environment (growth inhibition, reduction of biomass production, changes in water absorption and translocation) (shanker et al., 2005; szárazová et al., 2008). for genotoxicity studies are plants highly responsible and sensitive. their beneficial interest is that seeds and pollen grains can be easy storage away and offer cheap, relative easy and accurate toxicological assessment (kristen, 1997). by their ability to accumulate toxic substances, they indicate metal presence in the environment even in very low concentration (chandra, 2004). contamination of soil and water by cr and ni are of particular recent concern. the impact of cr contamination on the physiology of plants depends on the metallic species responsible for its mobilization, uptake and toxicity in the plant system (bennicelli et al., 2004). while cr is not considered an essential element for plant nutrition (sharma et al., 1995), ni is classified an essential trace element (brown et al., 1987); and although it is found everywhere in the environment, it usually occurs only in trace amounts. 2. materials and methods simultaneous phytotoxicity and mutagenicity assay was carried out on plant species vicia sativa l. var. klára according to miadoková et al. (2005). after 24 h of soaking at 25 °c in distilled water or solution with metal concentration equal to ic50 value the seeds of v. sativa were allowed to germinate in petri dishes (diameter = 18.5 108 fargašová, a. and lištiaková, j. cm) with filter paper soaked with the same concentration of tested metal as that used for soaking. phytotoxicity was assayed after 72 h of the dark cultivation in the thermostat at 25 °c by the same way as described previously svetková and fargašová (2007) for s. alba. the roots and shoots of v. sativa seedlings were measured and the growth inhibition percentage was assessed. the seedling roots used for chromosome and genome mutability evaluation were fixed and permanent slides were prepared by the feulgen method. chromosome aberrations were determined at least in 500-anatelophases. for statistic analysis the student’s t-test was used. the procedures for maintaining the tradescantia plants and for analyzing micronuclei frequency in the tetrads have been described by mišík et al. (2006; 2007). tradescancia paludosa clone 03 was standardly cultivated at the department of botany, faculty of natural sciences, comenius university in bratislava. inflorescence were harvested at the 8-10-buds stage and immersed into 500 ml of tested metal solutions (100 mg/l cro3 and nicl2, 1000 mg/l cr(no3)3) for 12 h. as control tap water was used. the 24 h reconvalescence, during which inflorescence peduncles were dipped in 500 ml of tap water, succeeded to 12 h exposure. then the buds were fixed for 24 h in ethanol : acetic acid (3 : 1). the fixed material was stored in 70% ethanol. slides were prepared from the fixed material using the aceto-carmine squash technique. micronuclei were scored in the early tetrad stages of pollen mother cells. in the present study, 15 to 20 inflorescences comprised a sample. three hundred tetrads were scored from each of five slides prepared from a treatment sample for a total of 1,500 tetrads per plot. data were recorded as the number of micronuclei (mcn) per 100 tetrads. a change of frequency of mcn/100 tetrads was considered statistically significant (at p < 0.05) if the difference between the mean of the control population and the mean of the treated population was at least twice as large as the standard error of the difference between the two means (ma et al., 1994; mišík et al., 2007). for samples of tested metals nicl2.6h2o, cr(no3)3.9h2o and crco3 of analytical grade p.a. were obtained from lachema, brno, czech republic. all experiments for growth inhibition were set up in a completely randomized design with three replicates. chronic toxicity was assessed as inhibition of roots and shoots growth and results were evaluated by gryck-haustein method and ic25, ic50 and ic75 concentrations were determined. the results were statistically evaluated by using toxicity program. 3. results and discussion toxic effects of heavy metals, mainly during chronic exposure, are not visible immediately hence ecotoxicological studies request also assessment of genotoxicity. genotoxicological effect is developed then the concentration is low order than that for fytotoxicity effect (mičieta and murín, 1998). for phytotoxicity and clastogenicity study v. sativa seedlings were used. phytotoxicity was determined through ic25, ic50 and ic75 values and for roots and shoots the strongest inhibitory effect had ni(ii) (fig. 1). no significant differences were confirmed between cr(iii) and cr(vi) adverse effects on v. sativa shoot growth. on the basis of these values, and their statistical evaluation, metals can be arranged in the following rank orders of inhibition: for roots: ni(ii) > cr(vi) > cr(iii); for shoots: ni(ii) > cr(vi) ≥ cr (iii). nova biotechnologica 9-2 (2009) 109 fig. 1. ic25, ic50 and ic75 values and their 95% confidence intervals (ci) (ml/l) for vicia sativa l. after 72 h application; the mean of three determinations with a standard deviation 6% or less for mutagenicity assay root tips of v. sativa were used and chromosome aberrations were determined at least in 500-anatelophases. all tested metals exerted in v. sativa a significant increase of chromosomal aberration rate in applied concentrations (table 1). from all tested metals cr(vi) invoked maximum of aberrations in anatelophase cells. the rank order of aberrations fall was: cr(vi) > ni(ii) > cr(iii). table 1. potential clastogenicity evaluation of cr and ni in vicia sativa l. (n = 500) metal concentration (mg/l) metal ni cr number of aberrations ± sd percentage of aberrations ± sd control <0.07 <0.01 7 ± 0.69 2.33 ± 0.23 ni(ii) 16.46 ± 2.16 10 ± 0.75 ** 3.33 ± 0.25 ** cr(iii 114.41 ± 13.36 8 ± 0.75 * 2.67 ± 0.25 * cr(vi) 69.69 ± 8.66 13 ± 0.69 ** 4.33 ± 0.23 ** sd – standard deviation; ** significant differences in comparison with control at p <0.01; * significant differences in comparison with control at p <0.05; control – sterile distilled water genetic variation in susceptibility to environmental agents and metals can be considered as differences in metabolism of these agents in various organisms (omenn, 1991). in addition, dna target size and dna content are also important in determining genotoxic hazard of metals. as described kovalchuk et al. (1998) and chauhan et al. (1998) genotoxicity can be obtained as a result of multipolar anaphase and c-mitose or damage of protein synthesis in the presence of dna 110 fargašová, a. and lištiaková, j. toxicant. simultaneous toxicity and clastogenity of wastes with cr and ni content was also confirmed for v. sativa by miadoková et al. (1999) and for v. faba and allium cepa by chandra et al. (2004; 2005). chromozomal fragments and bridges created in cr(vi) presence indicated as introduced quian (2004) that cro3 affecting dna structure and conformation. table 2. micronuclei frequency in the tradescantia pollen tetrads after treatment with cr and ni solutions (n = 1,500) metal concentration (mg/l) metal ni cr number of micronuclues ± sd percentage of micronucleus ± sd control <0.07 <0.01 43 ± 13.74 2.89 ± 0.92 ni(ii) 24.71 ± 0.25 59 ± 17.48 3.93 ± 1.17 cr(iii 130.00 ± 1.30 47 ± 16.61 3.13 ± 1.11 cr(vi) 52.00 ± 0.52 60 ± 15.98 4.00 ± 1.07 sd – standard deviation for determination of cr and ni genotoxic effects was also used analysis of micronuclei frequency in the pollen tetrads of tradescantia plants. as it is evident from table 2 none of tested metal significantly stimulated, in comparison with the control, micronuclei frequency and genotoxic effect was decreased in order: cr(vi) ≥ ni(ii) > cr(iii). tradescantia micronucleus test (trad-mcn) belongs together with allium cepa l. and vicia faba l. tests with root tips to most frequently used genotoxicity tests on plants (majer et al., 2005) and it is very popular now for in situ biomonitoring of air pollution (mišík et al., 2006; 2007). results obtained during our genotoxicity tests are in good agreement with those introduced by knasmüller et al. (1998) when cro3, crcl3 and nicl2 up to concentration 10 mm did not evoke genotoxic effects. the same conclusion also introduced majer et al. (2005) for cr(iii). higher genotoxicity of cr(vi) than cr(iii) determined during our experiments also described němeček et al. (2002). rossman (1995) introduced that molecular mechanism of dna damage by cr(vi) involve induction of dna-dna and dnaprotein cross-links and genotoxic effect can be also increased by reactive oxygen produced during intracellular reduction. for ni(ii) no genotoxic effects confirmed for bacteria rossman (1995) and patierno and costa (1987) introduced that mutations after ni applications are also the result of dna damage and dna-protein cross-links formation. acknowledgements this study was supported by grants vega 1/4361/07, vega 1/0238/08 and apvv – 0231-07. references bennicelli, r., stepniewska, z., banach, a., szajnocha, k., ostrowski, j.: the ability of azolla caroliniana to remove heavy metals (hg(ii), cr(iii), cr(vi)) from municipal waste water. chemosphere, 55, 2004, 141146. nova biotechnologica 9-2 (2009) 111 brown, p.h., welch, r.m., cary, e.e.: nickel: a micronutrient essential for higher plants. plant physiol., 85, 1987, 801−803 chandra, s., chauhan, l.k.s., pande, p.n., gupta, s.k.: cytogenetic effects of leachates from tannery solid waste on the somatic cells of vicia faba. environ. toxicol., 19, 2004, 129-133. chandra, s., chauhan, l.k.s., murthy, r.c., saxena, p.n., pande, p.n., gupta, s.k.: comparative biomonitoring of leachates from hazardous solid waste of two industries using allium test. sci. total environ., 347, 2005, 46-52. chauhan, l.k.s., saxena, p.n., sundararaman, v., gupta, s.k.: diuron-induced cytological and ultrastructural alterations in the root meristem cells of allium cepa. pestic. biochem. physiol., 62, 1998, 152-163. knasmüller, s., gottmann, e., steinkellner, h., fomin, a., pickl, c., paschke, a., god, r., kundi, m.: detection of genotoxic effects of heavy metal contaminated soils with plant bioassays. mutat. res., 420, 1998, 37-48. kovalchuk, o., kovalchuk, i., arkhipov, a., telyuk, p., hohn, b., kovalchuk, l.: the allium cepa chromosome aberration test reliably measures genotoxicity of soils of inhabited areas in the ukraine contaminated by the chernobyl accident. mutat. res., 415, 1998, 47-57. kristen, u.: use of higher plants as screens for toxicity assessment. toxicol. vitro, 11, 1997, 181-191. ma, t.h., cabrera, g.l., chen, r., gill, b.s., sandhu, s.s., vanderberg, a.l., salamone, m.f.: tradescantia micronucleus bioassay. environ. health perspect., 95, 1994, 157-189. majer, b.j., grummt, t., uhl, m., knasmüller, s.: use of plant bioassays for the detection of genotoxins in the aquatic environment. acta hydrochim. hydrobiol., 33, 2005, 45-55. miadoková, e., dúhová, v., vlčková, v., sládková, l., suchá, v., vlček, d.: genetic risk assessment of acid waste water containing heavy metals. gen. physiol. biophys., 18, 1999, 92-98. miadoková, e., svidová, s., vlčková, v., dúhová, v., pražmáriová, e., tothová, k., naďová, s., kogan, g., rauko, p.: the role of natural biopolymers in genotoxicity of mutagens/carcinogens elimination. biomed. pap. med. fac. univ. palacky olomouc czech rep., 149, 2005, 493-496. mičieta, k., murín, g.: tree species of genus pinus suitable as bioindicators of polluted environment. water air soil pollut., 104, 1998, 413-422. mišík, m., solenská, m., mičieta, k., mišíková, k., knasmüller, s.: in situ monitoring of clastogenicity of ambient air in bratislava, slovakia using the tradescantia micronucleus assay and pollen abortion assays. mutat. res./genet. toxicol. environ. mutagen., 605, 2006, 1-6. mišík, m., mičieta, k., solenská, m., mišíková, k., pisarčíková, h., knasmüller, s.: in situ biomonitoring of the genotoxic effects of mixed industrial emissions using the tradescantia micronucleus and pollen abortion tests with wild life plants: demonstration of the efficacy of emission controls in an eastern european city. environ. pollut., 145, 2007, 459-466. 112 fargašová, a. and lištiaková, j. němeček, j., podlešáková, e., vácha, r.: transfer of trace elements with low soil mobility into plants. rostl. výr., 48, 2002, 45-50. omenn, g.s.: future research direction in cancer ecogenetics. mutat. res., 247, 1991, 283-291. patierno, s.r., costa, m.: effect of nickel ii on nuclear protein binding in intact mammalian cells. cancer biochem. biophys., 9, 1987, 113-126. quian, x.: mutagenic effects of chromium trioxide on root tip cells of vicia faba. j. zhejiang univ. sci., 5, 2004, 1570-1576. rossman, t.g.: metal mutagenesis. in: r.a. goyer, g.c. cherian (eds.) toxicology of metals. springer, new york, 1995, 373-430. shanker, a.k., cervantes, c., loza-tavera, h., avudainayagam, s.: chromium toxicity in plants. environ. int., 31, 2005, 739-753. sharma, d.c., chatterjee, c., sharma, c.p.: chromium accumulation and its effects on wheat (triticum aestivum l. c.v. dh 2204) metabolism. plant sci., 111, 1995, 145−151 svetková, k., fargašová, a.: phytotoxicity of washing waste waters from cutlery production line. bull. environ. contam. toxicol., 79, 2007, 109-113. szárazová, k., fargašová, a., hiller, e., velická, z., pastierová, j.: phytotoxic effects and translocation of cr and ni in washing wastewaters from cutlery production line to mustard (sinapis alba l.) seedlings. fresenius environ. bull., 17, 2008, 58-65. microsoft word uhrovcik et al nbc12-2.doc nova biotechnologica et chimica 12-2 (2013) 93 doi 10.2478/nbec-2013-0011 © university of ss. cyril and methodius in trnava possibility of the spectrophotometric determination of europium by means of arsenazo iii jozef uhrovčík, monika gyeváthová, juraj lesný department of ecochemistry and radioecology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (jozef.uhrovcik@ucm.sk) abstract: the concentration of eu(iii) cations in model aqueous solutions can be quantified by means of arsenazo iii reagent. absorbance of the solution was measured at the wavelength λmax = 655 nm. molar absorptivity reached the value ε655 = 5.5±0.2 · 104 cm-1 mol-1 · dm3. beer's law was obeyed in the range from 0 to 2 mg · dm-3 eu(iii). the value of limit of detection was established by application of 3σ approach and reached the value of 20.9 μg · dm-3. repeatability of analysis expressed by relative standard deviation does not exceed the value of ± 8% and apparent recovery lay in acceptable range from 91 to 106 %. stoichiometry between eu(iii) and arsenazo iii in media of relevant solution was 1:1. the absorbance of the solutions within the linear range of the proposed method maintained a constant value for 60 minutes. described procedure can be utilized to determination of eu(iii) concentration in real samples, but it is necessary eliminate interfering ions. cations like la(iii), sm(iii), th(iv), u(vi) and complexing agent edta cause significant error at the determination of eu(iii) in model solution. presented spectrophotometric method could be applied for the determination of europium in the minerals and water samples, however after a suitable separation and preconcentration of target analyte. key words: europium, arsenazo iii, spectrophotometry, limit of detection. 1. introduction europium is a soft silvery white metal which belongs to the lanthanide family. it was discovered in 1896 and isolated in 1901 by eugène-anatole demarçay (heiserman, 1992); its atomic number is 63 and atomic weight is 151.96. europium can exist in two oxidation states, namely eu(ii) and eu(iii) and the latter represents its most stable oxidation form. majority of europium is obtained from monazite sand, which is a mixture of phosphates of calcium, thorium, cerium and most of the other rare earth metals. europium can be separated from the rare earth metals first of all by precipitation (blazejak-ditges, 1970). the result of this process is formation of europium oxide, eu2o3. europium in metal state can be prepared by mixing with powdered lanthanum metal in a tantalum crucible. various methods have been developed for the determination of rare earth metals including europium. they include spectrofluorimetry (takatatsu and sato, 1979; yamada et al., 1982), polarography (fu et al., 1993), voltammetry (mlakar and branica, 1991), atomic emission spectroscopy with inductively coupled plasma (daskalova et al., 1996) and mass spectrometry with inductively coupled plasma (zang et al., 1995). however, all these procedures are troublesome or the instruments are not readily available. therefore, there is the need for suitable laboratories equipped with inexpensive instrumentation, which allows the determination of europium to be carried bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:08 utc 94 uhrovčík, j. et al. out in a fast and cheap way without sacrificing precision. spectrophotometric method is an alternative that satisfies these requirements and can be afforded by most laboratories. in the present time exists some spectrophotometric methods suggested for the determination of traces of europium in different types of samples, for example (fang et al., 1999; poluektov et al., 1971; stoyanov et al., 2011). arsenazo iii is reported to be suitable primarily for the photometric determination of lanthanides and actinides (savvin, 1961; savvin 1964). it is commercially available and soluble in water and diluted acids. the reagent has ability to form stable chelates and can work in strongly acidic medium eliminating the chances of partial hydrolysis of metal ions to be determined. its sensitivity due to color reaction is quite high (0.05–0.01 μg cm-3 of the element). the high sensitivity and stability is coupled with a well-defined contrast in color transition from pinkish (reagent) to blue-green or raspberry-red (complex with arsenazo iii) (basargin et al., 2000). in this paper is described the spectrophotometric method for determination of europium using arsenazo iii as chromogenic reagent in buffer medium. 2. material and methods 2.1 chemicals and reagents all used reagents were of analytical grade and were prepared in deionized water. stock solution of eu(iii) with mass concentration m = 10.0±0.1 mg · dm-3 was prepared by appropriate dilution of standard solution (fluka analaytical, austria). 100 cm3 of given solution was stabilized by addition of 0.5 cm3 concentrated nitric acid (merck, germany). stock solution of arsenazo iii (fluka analytical, austria) with concentration 0.05% (w./v) was prepared by weighting and dissolution of appropriate amount of complexing agent in deionized water. buffer solutions in the ph range from 2.20 to 4.00 were prepared by suitable mixing of diluted hydrochloric acid with molar concentration c = 1 mol · dm-3 (mikrochem, slovakia) and solution of potassium hydrogen phthalate with molar concentration c = 0.1 mol · dm-3 (merck millipore, germany). stock solutions of interfering cations and anions with given mass concentrations were prepared by appropriate dilution of their standard solutions (merck, germany). 2.2 apparatus a double wavelength/double beam spectrophotometer cary winuv 50 with 10 mm quartz rectangular cuvette (varian inc., australia) was employed for the absorbance measurements. the ph of studied solutions and buffer solutions was measured and adjusted by ph-meter inolab 730 (wtw gmbh, germany). for the production of deionized water with specific conductivity < 0.054 μs · cm-1 was used ultrapure water system simplicity 185 (millipore, germany). 2.3 recommended procedure place the appropriate amount of pretreated sample containing < 20 μg eu(iii) into 10 cm3 volumetric flask, add 2 cm3 of buffer solution with ph = 2.60 and 0.8 cm3 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:08 utc nova biotechnologica et chimica 12-2 (2013) 95 of arsenazo iii solution; then fill with deionized water to mark and mix thoroughly. measure the absorbance of this solution against the reagent blank at the wavelength 655 nm. obtain the europium quantity from a calibration curve, which has been prepared with known amounts (0-20 μg) of europium. it is necessary to use technique of standard additions or technique of external calibration with matrix-matched standards in the case of existence of matrix effect. 3. results and discussion the absorption maximum of the complex between eu(iii) and arsenazo iii was observed at the wavelength 610 and 655 nm, respectively. the latter wavelength was used for the determination due to the lower absorbance of background and higher value of analytical signal of formed complex. the position of measured peak (λ = 655 nm) was not affected by the value of ph in the range from 2.20 to 4.00 as shown in fig. 1. 600 625 650 675 700 725 750 775 800 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 a bs or ba nc e λ [nm] ph = 2.20 ph = 2.60 ph = 3.00 ph = 3.40 ph = 3.80 ph = 4.00 fig. 1. absorption spectra of eu(iii)-arsenazo iii complex at the ph value of solutions; blank corrected. c eu(iii) = 1 mg · dm-3; buffer solution v = 2 cm3, arsenazo iii solution v = 1 cm3. the effect of buffer solution ph on the absorbance of relevant complex in the range from 2.20 to 4.00 has been investigated. it has been found out that the maximum absorbance for eu(iii)-arsenazo iii complex was occurred at ph = 2.60. on the basis of this finding, ph of measured solutions was adjusted to this value and used for subsequent measurements. the optimum volume of arsenazo iii, necessary for maximum absorbance, is represented by 0.8 cm3. the raspberry-red colour of formed complex develops instantaneously. the validity of beer’s law was verified in the concentration range from 0 to 2 mg dm-3 eu(iii) (fig. 2). however, beyond that, a deviation from the linearity was observed. the molar extinction coefficient and sandell's sensitivity of the complex were calculated from the regression equation and reached the value ε655 = 5.5±0.2 10 4 cm-1 ·mol-1 ·dm3 and 0.02 μg cm-2, respectively. coefficient of determination r2 was equal to 0.9952. upper boundary of beer’s law validity was bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:08 utc 96 uhrovčík, j. et al. established by application of qc parameter (van loco et al., 2002). results for determination of repeatability of measurement and trueness of measurement are given in table 1. trueness of measurement was verified by spike-recovery procedure. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.2 0.4 0.6 0.8 a bs or ba nc e at λ = 6 55 n m m eu(iii) [mg . dm-3] fig. 2. absorbance of eu(iii)-arsenazo iii complex as a function of europium mass concentration; blank corrected. table 1. determination of repeatability and trueness for studied analytical procedure (n = 10). m eu(iii) [mg · dm-3] apparent recovery [%] relative standard deviation [%] 0.5 91.1 ±7.8 1.0 100.7 ±3.0 1.5 106.1 ±2.3 0 20 40 60 80 100 120 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 a bs or ba nc e at λ = 6 55 n m t [min.] fig. 2. time dependence of absorbance for eu(iii)-arsenazo iii complex; blank corrected. (●) – 0.5 mg dm3; (●) – 1.0 mg dm-3; (●) – 1.5 mg dm-3. the intercept of calibration line has proven to be statistically negligible against the hypothetical value 0 (significance level α = 0.05). the student t-test was used for this bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:08 utc nova biotechnologica et chimica 12-2 (2013) 97 purpose. limit of detection calculated according to 3σ criterion reached the value 20.9 μg · dm-3. the optical density was found constant within the linear range for 60 minutes under normal laboratory conditions (fig. 2). a gradual decrease in the absorbance was then observed. composition of eu(iii)-arsenazo iii complex under presented experimental design, equal to 1:1, was established by mole ratio method (fig. 3). same results gained rohwer and hosten (1997). 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 a bs or bn ac e at λ = 6 55 n m n arsenazo iii / n eu(iii) fig. 3. determination of eu(iii)-arsenazo iii composition by mole ratio method; blank corrected. c eu(iii) = 6.85 μmol · dm-3. the effect of selected ions on europium determination has been checked. the results are listed in table 2. the mass concentration of eu(iii) was maintained at the level m = 1 mg ·dm-3. the mass ratio between eu(iii) and studied cations and anions is given in relevant table. a ±5 % variation in the absorbance is taken as tolerable limit within experimental error. table 2. impact of selected cations and anions on the eu(iii) determination in model solutions. anion mass ratio deviation [%] cation mass ratio deviation [%] cation mass ratio deviation [%] edtac 1:15 −21.9 ca(ii)a 10:1 +0.1 sr(iii)b 10:1 +0.2 fluoridec 1:10 +0.8 cu(ii)b 10:1  +0.8 th(iv)b 2:1  +26.0 phosphatec 1:10  +2.0 fe(iii)a 10:1  +2.5 u(vi)b 2:1  +24.1 sulphatec 1:10  +2.2 la(iii)b 10:1  +13.2   oxalatee 1:10  −2.7 mg(ii)a 2:1 +1.1 tartrated 1:10  +1.4 sm(iii)a 10:1 +11.0 a as chloride, b as nitrate, c as sodium salt, d as acid, e as ammonium salt. results indicate that la(iii), sm(iii), th(iv), u(vi) cations and edta anion interfere seriously. all interfering ions, except edta, have increased the absorbance. cation-exchange resin, for example ag50w-x8 (strelow, 1980) should be applied for removal of above mentioned interfering ions from matrix of real sample containing analyte of interest. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:08 utc 98 uhrovčík, j. et al. 4. conclusions simple, sensitive and fast spectrophotometric procedure has been developed for determining eu(iii) using arsenazo iii as chromogenic reagent. a calibration curve was linear over the range up to 2.0 mg · dm-3 eu(iii) and the limit of detection reached the value 20.9 μg · dm-3. elements from the lanthanide and actinide family interfere seriously and should be removed. the method is applicable for europium determination in ores and water with reasonable accuracy, but it is necessary to use proper type of cation-exchange resins or another type of separation technique. references basargin, n. n., ivanov, v. m., kuznetsov, v. v., mikhailova, a. v.: 40 years since the discovery of the arsenazo iii reagent. j. anal. chem., 55, 2000, 204-210. blazejak-ditges, d.: über die photometrische bestimmung des cergehaltes legierter stähle. z. anal. chem., 251, 1970, 11-15. daskalova, n., velichkov, s., slavova, p.: spectral interferences in the determination of traces of scandium, yttrium and rare earth elements in “pure” rare earth matrices by inductively coupled plasma atomic emission spectrometry part iii. europium. spectrochim. acta, 51b, 1996, 733-768. fang, g. z., pan, j. m., zhou, w. l., xu, b. l.: study on the spectrophotometric determination of rare earths with a new chromogenic reagent dibromo-pmethyl-chlorosulfonazo (dbmcsa). chinese chem. lett., 10, 1999, 851-854. fu, x. t., wang, c. m., zhang, y. s.: polarographic study of the eu(iii)triethylenetetraaminehexaacetic acid complex and determination of europium by oscillopolarography. anal. chim. acta, 272, 1993, 221-225. heiserman, d. l.: exploring chemical elements and their compounds, mcgrawhill, new york, 1992, 371 pp. mlakar, m., branica, m.: voltammetric study of europium(iii) in the presence of 2-thenoyltrifluoroacetone. anal. chim. acta, 247, 1991, 89-95. poluektov, n. s., kirilov, a. i., makarenko, o. p., vlasov, n. a.: determination of total rare earth content in natural waters by extraction – spectrophotometric method (english translation). zavod. lab., 37, 1971, 536-537. rohwer, h., hosten, e.: ph dependence of the reactions of arsenazo iii with the lanthanides. anal. chim. acta. 339, 1997, 271-277. savvin, s. b.: analytical use of arsenazo iii. determination of thorium, zirconium, uranium and rare earth elements. talanta, 8, 1961, 673-685. savvin, s. b.: analytical applications of arsenazo iii–ii* determination of thorium, uranium, protactinium, neptunium, hafnium and scandium. talanta, 11, 1964, 1-6. stoyanov, a., chivireva, n. a., stoyanova, i. v., timukhin, e. v., antonovich, v. p.: indirect photometric determination of europium(ii) in some inorganic materials containing aliovalent europium species. j. anal. chem., 66, 2011, 470-475. strelow, f. w. e.: quantitative separation of lanthanides and scandium from barium, strontium and other elements by cation-exchange chromatography in nitric acid. anal. chim. acta. 120, 1980, 249-254. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:08 utc nova biotechnologica et chimica 12-2 (2013) 99 takatatsu, t., sato, a.: spectrofluorimetric determination of europium and samarium with mixtures of 2-thenoyltrifluoroacetone and tri-n-octylphosphine oxide in aqueous solutions containing triton x-100. anal. chim. acta, 108, 1979, 429-432. van loco, j., elskens, m., croux, ch., beernaert, h.: linearity of calibration curves: use and misuse of the correlation coefficient. accredit. qual. assur., 7, 2002, 281-285. yamada, s., kano, k., ogawa, t.: time discrimination in the laser fluorimetry and ultratrace determination of europium(iii) and samarium(iii) with 4,4,4trifluoro-1-(2-thienyl)-1,3-butanedione. anal. chim. acta, 134, 1982, 21-29. zang, s., murachi, s., imasak, t., wataabe, m.: determination of rare earth impurities in ultrapure europium oxide by inductively-coupled plasma mass spectrometry. anal. chim. acta, 314, 1995, 193-201. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:08 utc microsoft word lehotay nb 9-3.doc nova biotechnologica 9-3 (2009) 341 imprinted polymers in analytical chemistry jozef lehotay1, miroslava lachová1, ján mocák2 1institute of analytical chemistry, faculty of chemical and food technology, slovak university of technology, radlinského 9, bratislava, sk-812 37, slovak republic (jozef.lehotay@stuba.sk) 2department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic abstract: molecularly imprinted polymers were prepared and tested in different way. 1-methyl-2piperidinoethylester of 4-decyloxyphenylcarbamic acid was used as template for imprints formation. acrylamide, 4-vinylpyridine and methacrylic acid as monomers and methanol and acetonitrile as a porogene were used. non-imprinted polymers (nip) were prepared for each imprinted polymer by the same procedure. polymers were employed as sorbents for solid-phase extraction (spe). in this work the influence of polymerization mixture composition on polymer properties, such as capacity and selectivity, was investigated. the influence of alkoxy-chain length and position on benzene ring on the selectivity of polymers was also investigated. key words: hplc, molecularly imprinted polymer, spe. 1. introduction molecular imprinting is a technique for preparing chemically selective binding sites, which recognize a particular molecule, in a macroporous polymer matrix. the producing of molecularly imprinted polymers (mips) involves polymerization in solution of a target molecule (template) with functional and crosslinking monomers. removal of the template molecule from a resultant polymer leaves behind specific recognition sites that are complementary to the template and enable the polymer to selectively rebind the imprint molecule from a mixture of closely related compounds. the mips can be produced in a covalent or non-covalent manner. in the case of covalent approach of molecular imprinting covalent bonds between the template and polymerizable monomers are formed. in order to remove the template from the polymer to liberate the binding sites, these covalent bonds have to be chemically cleaved. non-covalent approaches are based on the formation of a prepolymerization complex between monomers and the template trough noncovalent bonds such as ionic interactions or hydrogen bonding. this enables the removal of the template simply by solvent extraction. because mips have outstanding advantages such as predetermined selectivity, simple and convenient preparation, robustness in organic solvents and acidic or basic reagents, and durability to high temperature, molecular imprinting has drawn extensive attention in recent years (zhang et al., 2001). the advantages, applications and developments in molecular imprinting were mentioned in several reviews (kandimalla and ju, 2004 chapuis, 2006; ramström et al., 2001, andersson, 2000; ensing and de boer, 1999; haupt and mosbach, 1998). 342 lehotay, j. et al. a technology, using selective solid phases based on mips in mispe (molecularly imprinted spe), is still investigated by research groups. analysts can use two approaches to obtain the required selectivity: non-selective and selective adsorption of mips for spe. if the analyte can be trapped on the mip by ionic or hydrophobic interactions, users can load an aqueous sample directly and retain it by using nonselective interactions. if the trapping is performed in water, the non-selectively bound components will be removed by washing with organic solvent, and the analyte of interest retained on the mip will switch from non-selective to selective binding (ensing and majors, 2002). mips have been already applied in biological, pharmaceutical (bereczki et al., 2001; theodoridis and manesiotis, 2002; xie et al., 2003; feng et al., 2004; caro et al., 2004) and environmental analysis (baggiani et al., 2001; mena et al., 2002; chapuis et al., 2003; zhon and lai, 2004). there are also some works where mispe was used for sample preparation of herb or tea (xie et al., 2001; blahova et al., 2004). in this study, the influence of functional monomer and porogen, respectively, on the sorptive properties was investigated. the structurally related compounds (analytes with alkoxy-chain with in different position or with different length) were utilized to study the selectivity of mips. all mips were prepared by non-covalent approach by bulk polymerization and used as the sorbent for solid-phase extraction. 2. material and methods 2.1 chemicals 1-methyl-2-piperidinoethylesters of alkoxyphenylcarbamic acid (fig. 1) were synthesized on pharmaceutical faculty in bratislava. acetonitrile, methanol, toluene, methacrylic acid and diethylamine were purchased from merck, 4-vinylpyridine was obtained from aldrich, acrylamide and azobisizobutylonitrile were obtained from fluka, and acetic acid was purchased from lachema. (a) nh coo or ch ch3 ch2 n h cl + (b) nh coo or ch ch3 ch2 n fig. 1. structure of compounds used in research, free base (a) and hydrochloride (b). template molecule: r= c10h21 in 4position (4-dpca). other analytes used in research: r= c10h21 in 2position (2-dpca). 2.2 hplc analysis an hp 1100 system (hewlett-packard, germany), consisting of a pump with a degasser, a diode-array detector (dad) a 50 μl injector and a hp chemstation were used. analyses were carried out on the analytical column separon sgx c18 (125 x 4 mm, 7 μm) (watrex, usa) at laboratory temperature. mobile phase consisted of methanol, acetonitrile, acetic acid and diethylamine (80:20:0.1:0.1) at a flow rate of nova biotechnologica 9-3 (2009) 343 0.5 ml/min. izokratic elution was used. diode-array detector worked in the range of 190 – 400 nm and the chromatograms were acquired at wavelength of 240 nm. 2.3 polymer preparation the molecularly imprinted polymer was prepared according to zhang et al. (2001) method. for synthesis of mips template (1-methyl-2-piperidinoethylester of 4decyloxyphenylcarbamic acid – 4-dpca) was applied as hydrochloride and base, respectively. the base was used mainly for polymers prepared in acetonitrile and toluene because of better solubility of base template in these solvents. the composition of polymerization mixtures is shown in table 1. table 1. composition of polymerization mixtures. polymer form of template molecule porogene monomer mip1 base methanol acrylamide mip2 hydrochloride methanol acrylamide nip1,2 methanol acrylamide mip3 base acetonitrile acrylamide nip3 acetonitrile acrylamide mip4 base toluene acrylamide nip4 toluene acrylamide mip5 hydrochloride methanol 4-vinylpyridine nip5 methanol 4-vinylpyridine mip6 base acetonitrile 4-vinylpyridine nip6 acetonitrile 4-vinylpyridine mip7 base toluene 4-vinylpyridine nip7 toluene 4-vinylpyridine mip8 hydrochloride methanol methacrylic acid mip9 base methanol methacrylic acid nip8,9 methanol methacrylic acid mip10 base acetonitrile methacrylic acid nip10 acetonitrile methacrylic acid mip11 base toluene methacrylic acid nip11 toluene methacrylic acid 2.4 evaluation of mip the cartridge capacity of each mip and nip was tested in methanol, acetonitrile, water and toluene. prior to applying the solution of derivative of 4-dpca, the polymer was pre-equilibrated with 5 ml of methanol and then with 5 ml of solvent in which the capacity was studied. 4-dpca solution (0.5 μg/ml) was applied onto the cartridge (effluent was collected in 1 ml fractions) until a release was detected. each fraction was measured by hplc. in the case of toluene, 5 ml of 4-dpca solution dissolved in toluene (0.5 μg/ml in the case of mip4, mip7, nip4 and nip7;) was applied. in the case of mip11 and nip11, 20 ml of 4-dpca (5 μg/ml) was applied onto the 344 lehotay, j. et al. cartridges. then the cartridges were dried and the adsorbed analyte was desorbed by methanol acetic acid mixture (95:5). effluent was dried, resolved in methanol and measured by hplc. for polymers´capacity, the template was used in the same form (free base or hydrochloride) as it was used during polymerization. the selelectivity of mip3, mip 9, mip10 and mip11 for 1-methyl-2piperidinoethylester of 4-methoxyphenylcarbamic acid (4-mpca) and 2decyloxyphenylcarbamic acid (2-dpca) was tested. the selectivity of mip3 and mip11 in acetonitrile and mip9 and mip10 in methanol was determined. the procedure and the solution concentration were the same as described for 4-dpca. 3. results and discussion 3.1 capacity of polymers as it was described above, the capacities of mips were evaluated in different solvents. the same procedure was performed on mips and nips, respectively, and the resultant values of polymer capacities for template molecules are shown in table 2. table 2. binding capacities of prepared polymers. rsd = 4.5 – 13.5 %, n = 3. capacity (µg of analyte/100 mg of polymer) polymer acetonitrile methanol toluene mip1 (aa-meoh) 0.1 0 mip2 0 0 nip1,2 0.1 0.1 mip3 (aa-acn) 2.5 0.5 nip3 0.4 0.4 mip4 (aa-tol) 0.7 0.1 1.2 nip4 0.5 0 0.9 mip5 (4vp-meoh) 0.2 0.1 nip5 0.3 0 mip6 (4vp-acn) 0.5 0.2 nip6 0.6 0 mip7 (4vp-tol) 0.7 0.1 0.4 nip7 0.8 0.1 0.4 mip8 (maa-meoh) 3.8 2.0 mip9 28.0 8.0 nip8,9 4.0 1.9 mip10 (maa-acn) 20.4 20.2 nip10 10.7 5.9 mip11 (maa-tol) 76.0 32.0 61.1 nip11 9.7 6.2 6.6 rsd = 4.5 – 9.5 %, n = 3. polymers prepared with hydrochloride form as a template were tested for both forms (hydrochloride and base, respectively) and the values of capacities of each polymer (mip1, 5, 8) were similar for both forms of template. nova biotechnologica 9-3 (2009) 345 as it was mentioned in the introduction, the type of functional monomer and porogen plays important role during the formation of pre-polymerization complex. it is obvious from table 2, that the specific capacity of polymers prepared with 4vinylpyridine as a functional monomer is very low. therefore 4-vinylpyridine is not suitable monomer for this type of template. methacrylic acid seems to be the most convenient monomer for our template. all mips, with the exception of mip8, prove the ability to bind the template molecule specifically. different capacity of mip8 in comparison to mip9 demonstrates the influence of the template form on the ability to form the pre-polymerization complex. the mip9 was prepared using by the base form and the mip8 was prepared with hydrochloride form. when we compare the mips prepared with acrylamide, only mip3 is able to bind the template specifically. it indicates the influence of porogene on the formation of pre-polymerization complex. acetonitrile is less polar solvent in comparison to methanol. in this case less polar solvents optimize the interactions between monomer and template. more polar solvents such as methanol and water cancel the hydrogen bonding between monomer and template. the difference between acetonitrile and methanol is also in the way of creation of the hydrogen bonds – donor and acceptor. the solvent used in the process of capacity determination is also very important. the nature of solvent employed for this step influences the relative swelling of the polymer. swelling of the polymer causes changes in the binding site cavities. solvent is also responsible to solvatation process. it means that the size and shape of template molecule depends on the solvent. combination of these factors affects the change of capacity of mip. the highest values of binding capacities were obtained using water (not presented in table) for sample loading for all mips and also for nips. the whole amount of loaded template (100 µg) was sorbed onto the sorbents. in aquaeous environments, hydrogen-bonding and electrostatic interactions could be disrupted, and hydrophobic interactions, which are non-specific, could govern analyte retention (haginaka 2005) this allows to use water samples for extraction, of course, after sample loading, the cartridge should be dried and washed with selective organic solvent able to disrupt the non-specific interaction of analyte with polymer. 3.2 capacity of mips for structurally related compounds the procedure of determination of capacity of mip3, mip9, mip10 and mip11 for structurally related compounds was the same as for template. the influence of length of alkoxychain on benzene ring was tested using by 4-mpca. the influence of position of alkoxy-chain was investigated by measuring of mips´ capacity for 2dpca. the polymer capacities of mips and relevant nips are shown in table 3. as it is obvious from table 3, the capacity of mip3 is lower for analyte with shorter alkoxy-chain (4-mpca) than for template. therefore the length of alkoxychain impacts the capacity of analyte and mip3 can recognize template molecule from structurally related compounds. the influence of alkoxy chain position on benzene ring was also tested. the difference between capacity values of the template (4-dpca) and the analyte with 346 lehotay, j. et al. table 3. binding capacities of prepared polymers. capacity (µg of analyte/100 mg of polymer) ester of pdecyloxyphenylcarb. acid (template) – 4dpca ester of pmethoxyphenylcarb. acid – 4mpca ester of odecyloxyphenylcarb. acid – 2dpca mip3 (aa-acn) 2.5 1.3 1.9 nip3 0.4 0.3 0.4 mip9 (maa-meoh) 8.0 9.2 4.8 nip8,9 1.9 1.9 1.8 mip10 (maa-acn) 20.2 18.1 10.2 nip10 5.9 3.2 6.2 mip11 (maa-tol) 76.0 75.1 76.2 nip11 9.7 10.2 8,0 rsd = 4.5 – 9.1 %, n = 3. alkoxy chain in ortoposition (2-dpca) is not very leap. but in the case of mip9 and mip10, the capacity of mips is two times higher for template than for the analyte with alkoxy-chain in orto-position. it demonstrates that mip9 and mip10 have the different selectivity for compounds with decyloxy-group in other position on benzene ring. references andersson, l.i.: molecular imprinting for drug bioanalysis: a review on the application of imprinted polymers to solid-phase extraction and binding assay. j. chromatogr. b 2000, 739, 163-173. baggiani, c., giovannoli, c., anfossi, l., tozzi, c.: molecularly imprinted solid-phase extraction sorbent for the clean-up of chlorinated phenoxyacids from aqueous samples. j. chromatogr. a, 938, 2001, 35-44. bereczki, a., tolokán, a., horvai, g., horváth, v., lanza, f., hall, a.j., sellergren, b.: determination of phenytoin in plasma by molecularly imprinted solid-phase extraction. j. chromatogr. a, 930, 2001, 31-38. blahová, e., lehotay, j., skačáni, i.: the use of molecularly imprinted polymer for selective extraction of (+)-catechin. j. liq. chromatogr. rel. tech., 27, 2004, 2715-2731. caro, e., marcé, r.m., cormack, p.a.g., sherrington, d.c., borrul, f.: a new molecularly imprinted polymer for the selective extraction of naproxen from urine samples by solid-phase extraction. j. chromatogr. b, 813, 2004, 137143. chapuis, f., dichon, v., lanza, f., sellerfgre, s., hennon, m.c.: optimization of the class-selective extraction of triazines from aqueous samples using a molecularly imprinted polymer by a comprehensive approach of the retention mechanism. j. chromatogr. a, 999, 2003, 23-33. ensing, k., de boer, t.: tailor-made materials for tailor-made applications: application of molecular imprints in chemical analysis. trends anal. chem., 18, 1999, 138-145. nova biotechnologica 9-3 (2009) 347 ensing, k., majors, r.e.: selective sorbents for solid-phase extraction based on molecularly imprinted polymers. lc-gc europe, 15, 2002, 16-25. feng, s.y., lai, e.p.c., dabak-zlotorzynska, e., sadeghi, s.: molecularly imprinted solid-phase extraction for the screening of antihyperglycemic biguanides. j. chromatogr. a, 1027, 2004, 155-160. haginaka, j., selectivity of affinity media in solid-phase extraction of analytes. trends anal. chem. 2005, 24, 407-415. haupt, k., mosbach, k.: plastic antibodies: developments and applications. trends biotech., 16, 1998, 468-475. kandimalla, v.b., ju, h.: molecular imprinting: a dynamic technique for diverse applications in analytical chemistry. anal. bioanal. chem., 380, 2004, 587-605. mena, m.l., martínez-ruiz, p., reviejo, a.j., pizarrón, j.m.: molecularly imprinted polymers for on-line preconcentration by solid phase extraction of pirimicarb in water samples. anal. chim. acta, 451, 2002, 297-304. ramström, o., skudar, k., haines, j., patel, p., brüggemann, o.: food analyses using molecularly imprinted polymers. j. agric. food chem., 49, 2001, 2105-2114. theodoridis, g., manesiotis, p.: selective solid-phase extraction sorbent for caffeine made by molecular imprinting. j. chromatogr. a, 948, 2002, 163-169. xie, j., chen, l., li, ch., xu, x.: selective extraction of functional components derived from herb in plasma by using a molecularly imprinted polymer based on 2,2-bis(hydroxymethyl)butanol trimethacrylate. j. chromatogr. b, 788, 2003, 233242. xie, j., zlu, l., luo, h., zhon, l., li, ch., xu, x.: direct extraction of specific pharmacophoric flavonoids from gingko leaves using a molecularly imprinted polymer for quercetin. j. chromatogr. a, 934, 2001, 1-11. zhang, t., liu, f., chen, w. wang, j., li, k.: influence of intramolecular hydrogen bond of templates on molecular recognition of molecularly imprinted polymers. anal. chim. acta, 450, 2001, 53-61. zhon, s.n., lai, e.p.c., miller, j.d.: analysis of wheat extracts for ochratoxin a by molecularly imprinted solid-phase extraction and pulsed elution. anal. bioanal. chem., 378, 2004, 1903-1906. microsoft word ondas nb 9-2.doc nova biotechnologica 9-2 (2009) 183 conversion of corn fiber into fuel ethanol vladimír ondáš, hana novanská, viera horváthová department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (vladimir.ondas@gmail.com) abstract: corn fiber due to its chemical composition (up to 20% starch, 50 60% non-starch polysaccharides) and availability has potential to serve as a substrate for manufacture of various products, including fuel ethanol. this paper deals with assessment of fiber-to-ethanol conversion. the water/dry fiber ratio in suspensions was 10/1. enzyme liquefaction and saccharification of residual starch in corn fiber was carried out in two steps with thermostable α-amylase (20 min, 120°c) and mixture of pullulanase and glucomalyse (24 hours, 60°c). procedures resulted in release of 57.7±1.6 mg of glucose per gram of dry fiber basis. it responds to the dextrose equivalent expression to 96.7±2.2%. by fermentation of the starch hydrolysates by yeasts saccharomyces cerevisiae ccy-11-3 (5% v/v inoculum, 28°c, 72 hours) 0.48 g of ethanol per gram of glucose in hydrolysates was obtained. the solids after starch hydrolysis were separated by filtration and processed by acid pretreatment (0.1 g of conc. hcl/g of biomass/5 ml of water, 120°c, 20 min) with subsequent enzyme hydrolysis (24 hours, 60°c) by the multienzyme preparations containing cellulases and hemicellulases. overall yield of reducing sugars after these two steps was 740.7±3.9 mg/gram of dry corn fiber basis. fermentation of lignocellulosic hydrolysates by yeasts pichia stipitis ccy-39-50-1 and candida shehatea ccy-29-68-4 (in both cases 5% v/v inoculum, 28°c, 72 hours) resulted in 0.38 and 0.12 g of ethanol per gram of reducing sugars. the results indicate that applied pretreatment methods and used microorganisms are able to produce ethanol from corn fiber. key words: corn fiber, enzyme hydrolysis, acid pretreatment, pentoseand hexose-fermenting yeasts 1. introduction corn fiber is a potential raw material for the production of various products, including fuel ethanol, because it is available in countries in which corn grains are processed (mosier et al., 2005; atkin, et al., 2008; noureddini et al., 2009). it is obtained in the process of wet milling of corn. corn fiber, similar to other lignocellulosic materials is the complex of polysaccharides (35% hemicelluloses, 20% cellulose, up to 20% starch) and lignin. the main component of corn fiber is the outer corn grain layer – pericarp and residual part of starchy endosperm (gáspár et al., 2007). the first step of bioethanol production is enzymatic saccharification, in which polysaccharides are converted to monomeric sugars. in contrast to starch or cellulose, containing only hexose – glucose, hemicelluloses contain also pentoses, like xylose and arabinose. in the second step yeasts are used for the fermentation of these sugars (réczey et al., 1996, ballesteros et al., 2004; palmarola-adrados et al., 2005). the most used pentose-fermenting microorganisms are various yeasts like pichia stipitis, candida shehatea, pachysolen tannophilus, but also other strains of this species are able to ferment xylose (zhao et al., 2005; maris et al., 2006). beside naturally occurring pentose-ferment yeasts, also genetically improved e. coli strains or zymomonas mobilis strains are able to convert pentose to ethanol (jarboe et al., 2007). 184 ondáš, v. et al. conversion of the starch along with the lignocellulosic components in the corn fiber would increase ethanol yields from corn wet mill by 13% (grohmann and bothast, 1997) and is promising if the value of the corn fiber as an animal feed product is not severely affected (schell et al., 2004). the aim of this study was to investigate the possibility of corn fiber to ethanol conversion by traditional methods of lignocellulose substrate processing. 2. materials and methods 2.1 materials microbial strains saccharomyces cerevisiae ccy-11-3, pichia stipitis ccy 39-50-1 and candida shehatea ccy 29-68-4 were obtained from the culture collection of yeasts at institute of chemistry (slovak academy of sciences, slovakia), subcultured on slant agar media and stored in a refrigerator at a temperature about 4 °c. corn fiber was received from amylum slovakia s.r.o (boleráz, slovakia) and stored at – 20°c. the following commercial enzymes were used: thermostable αamylase (termamyl 120l), glucoamylase (amg 300l), thermostable pullulanase (promozyme d2), cellulose (celluclast 1,5l) and β-glucosidase (viscozyme l). all enzymes were obtained from novozymes (denmark). 2.2 corn fiber destarching a 10% (w/w) non-ground corn fiber dry matter aqueous solution was prepared. thermostable α-amylase for starch liquefaction (0.3 µl/g starch) was added in two steps and the mixture was boiled at up to 120°c for 20 min. after cooling to about 60°c was glucoamylase (0.8 µl/g starch) and thermostable pullulanase (7.2 µl/ g starch) added and incubated at 60°c for 24 hours. the solids were recovered by filtration. schematic view of this process in the context of other processes is depicted in fig. 1. 2.3 pretreating of destarched corn fiber with hydrochloric acid solids after destarching were mixed with 5 ml of water and 0.1 g of concentrated hydrochloric acid. mixture was heated in a pressure device at 120°c for 20 minutes. after cooling to room temperature, the solids were recovered by filtration. schematic view of this process in the context of other processes is illustrated in fig. 1. 2.4 enzymatic hydrolysis of cellulose in pretreated corn fiber the mixture of cellulolytic enzymes (119 µl/g dry matter of lignocellulose) and βglucosidase (595 µl/g dry matter of lignocellulose) was inoculated to 5% acetate buffer solution of destarched and pretreated solids of corn fiber. the reaction was allowed to proceed for 24 hours at 60°c and stirring at speed rate 120 rpm. schematic view of this process in the context of other processes is shown in fig. 1. nova biotechnologica 9-2 (2009) 185 fig. 1. schematic view of processes involved in the processing of corn fiber to ethanol. 2.5 fermentation liquid hydrolysate from corn fiber destarching was inoculated with 5% inoculum of yeasts saccharomyces cerevisiae, fermentation medium components were added and ph was adjusted to 5.0. liquid hydrolysate from enzyme hydrolysis of destarched and pretreated corn fiber was inoculated with 5% inoculum of yeasts pichia stipitis or candida shehatea, fermentation medium components were added and ph was adjusted to 5.0. fermentation was carried out at 28°c and lasts 72 hours. schematic view of this process in the context of other processes is depicted in fig. 1. 2.6 analytical procedures the dry matter content of corn fiber, destarched corn fiber and pretreated corn fiber was determined by drying of the sample at 105°c. glucose content was determined 186 ondáš, v. et al. using biola test glukosa god 1500 (lachema, czech republic) and reducing sugars were determined using dinitrosalicylic acid (dns). analyses of glucose, maltose, maltotriose and oligosaccharides were performed by high performance liquid chromatography (hplc) with an hpx-87c column (bio-rad laboratories, hercules, usa) at 80°c with water as the mobile phase at a flow rate of 0.5 ml/min. 3. results and discussion destarching of corn fiber with enzymes was carried out in two steps using thermostable α-amylase for liquefaction and mixture of pullulanase and glucoamylase for saccharification. kinetics of saccharification is shown in table 1. results are averages of three determinations table 1. saccharification kinetics of liquefied starch in suspension with 10% content of corn fiber dry matter. reducing sugars glucose hour mg/ml hydrolysate mg/g dry matter mg/ml hydrolysate mg/g dry matter de (%) 0 3.0±0.2 24.4±1.6 0.6±0.05 5.2±0.4 8.7±0.3 2 5.1±0.1 41.8±0.8 4.4±0.1 35.8±0.8 59.9±1.5 4 5.8±0.2 47.2±1.6 4.6±0.1 37.8±0.8 63.3±1.6 6 6.1±0.1 50.0±0.8 5.5±0.2 44.5±1.6 74.5±1.6 8 6.6±0.1 53.8±0.8 6.4±0.1 52.3±0.8 90.0±1.9 24 9.6±0.1 78.5±0.8 7.1±0.2 57.7±1.6 96.7±2.2 de (%) = dextrose equivalent in the first eight hours the dextrose equivalent achieved 90%, and in the next sixteen hours it rose to almost 97%. similar time course of saccharification is described in the work of leathers (2003). glucose represents about 64.6% of all sugars in hydrolysate, maltotriose 17.2% and last 18% other oligosaccharides. we supposed that lower proportion of glucose was caused by reduced availability of starch as a result of the particle size of substrate. destarching of corn fiber leads to better access of pretreatment or hydrolysing agents to non-starch polysaccharides or other substrates. because corn fiber contains about 35% of hemicelluloses, 18% of cellulose and 8% of lignin, it is necessary to pretreat it before enzymatic hydrolysis of polysaccharides to achieve the maximum release of fermentable sugars. acid hydrolysis is one of the possible pretreatment ways (shibanuma et al., 1999; saha, 2003; noureddini et al., 2009). in our experiments 1 g of destarched corn fiber was supplemented with 0.1 g of concentrate hydrochloric acid and 5 ml of water. the mixture was heated in a pressure device at up to 120°c for 20 minutes. after heating the device was nova biotechnologica 9-2 (2009) 187 allowed to cool to room temperature and solids were separated from liquid hydrolysate by filtration. obtained results are listed in table 2. results are averages of three determinations. table 2. concentration of reducing sugars and glucose after pretreatment of destarched corn fiber with hydrochloric acid. reducing sugars glucose mg/ml hydrolysates mg/ml hydrolysates mg/ml hydrolysates mg/g dry matter hydrochloric acid 35.2 ± 1.3 587 ± 3 7.0 ± 0.4 117 ± 1 because most of the pretreatment strategies do not lead to complete hydrolysis of polysaccharides, an important step in processing any lignocellulosic material is the enzymatic hydrolysis of cellulose or hemicelluloses or both polysaccharides. in addition, enzymatic processes are carried out under mild conditions and thus lower consumption of energy. the risk of fermentation inhibitors creation is also minimal. processing of destarched corn fiber with cellulytic and hemicellulytic enzymes in our experiments lasts 24 hours and temperature was maintained at 60°c. the results are summarized in table 3 and are averages of three determinations. table 3. concentration of reducing sugars and glucose after enzymatic hydrolysis of destarched corn fiber and destarched and pretreated corn fiber. reducing sugars glucose substrate mg/ml hydrolysates mg/ml hydrolysates mg/ml hydrolysates mg/g dry matter destarched corn fiber 3.7 ± 0.1 61.7 ± 2.2 1.5 ± 0.1 25.8 ± 0.9 destarched and pretreated corn fiber 8.2 ± 0.3 154 ± 1 2.81 ± 0.1 52.9 ± 0.3 by hplc (results provided by amylum slovakia s.r.o., boleráz) was determined that oligosaccharides represent more than 60% of all sugars in enzyme hydrolysates of destarched and pretreated corn fiber. other saccharides were maltose and maltotriose. in next part of the research fermentation of starch and lignocellulosic hydrolysates prepared from corn fiber was carried out. for the fermentation of starch hydrolysates yeasts saccharomyces cerevisiae were used and for lignocellulosic hydrolysates yeasts pichia stipitis and candida shehatea were used. fermentation conditions were the same for all three yeast strains – ph 5.0, 5% inoculum, 28°c, 72 hours. results are shown in table 4 (starch hydrolysates), table 5 and table 6 (lignocellulosic hydrolysates). 188 ondáš, v. et al. table 4. yield of ethanol after fermentation by saccharomyces cerevisiae of starch hydrolysates with 10% content of corn fiber dry matter. amount of waterless ethanol substrate g/ 100 ml fermentation medium g/g glucose g/g dry matter conversion [%]* corn fiber 0.56 0.48 0.03 94.2 * the degree of conversion is value relative to the maximum theoretical yield of ethanol (0.51 g ethanol from 1 g of glucose) (ballesteros et al., 2004). bura et al. (2002), for the fermentation of starch from corn fiber also used yeasts saccharomyces cerevisiae, but with substrate pretreated by so2 steam explosion. in those conditions yeasts saccharomyces cerevisiae converted saccharides in hydrolysates with efficiency from 90 to 96%. also our conversion efficiency falls within this range. table 5. yield of ethanol after fermentation by pichia stipitis and candida shehatae of lignocellulosic hydrolysate of destarched corn fiber pretreated with hydrochloric acid. amount of waterless ethanol conversion [%]* microorganism g/ 100 ml fermentation medium g/g reducing sugars g/g dry matter pichia stipitis 0.16 0.44 0.02 84.2 candida shehatae 0.05 0.14 0.01 28.4 * the degree of conversion is value relative to the maximum theoretical yield of ethanol (0.51 g ethanol from 1 g of glucose) (ballesteros et al., 2004). table 6. yield of ethanol after fermentation by pichia stipitis and candida shehatea of hydrolysate of corn fiber pretreated by acid prehydrolysis and enzymatic hydrolysis. amount of waterless ethanol conversion [%]* microorganism g/ 100 ml fermentation medium g/g reducing sugars g/g dry matter pichia stipitis 0.85 0.38 0.09 73.9 candida shehatea 0.27 0.12 0.03 22.9 * the degree of conversion is value relative to the maximum theoretical yield of ethanol (0.51 g ethanol from 1 g of glucose) (ballesteros et al., 2004). nova biotechnologica 9-2 (2009) 189 we have to state that fermentation of destarched corn fiber hydrolysates by candida shehatea was efficient not enough. one of the problems causing such low values may also be poor adaptation of yeast strain candida shehatea ccy 29-68-4 to the fermentation conditions such as temperature, ph, presence of inhibitors, the composition of the fermentation medium or the need for aeration. to define optimal conditions for the strain of candida shehatea ccy 29-68-4 undertaking of further experiments is needed. 4. conclusions although agricultural residues are plentiful and readily accessible, improved methods are needed for their conversion to fermentable sugars and subsequently to ethanol or other value-added products. this is also a case of pretreatment method based on acid hydrolysis at lower temperature (under 150°c). also using of pentosefermenting yeasts, like pichia stipitis and candida shehatea needs very careful determination of fermentation conditions. acknowledgments: this research was supported by the slovak research and development agency within the project apvv lpp-0251-07. references atkin, d.e., rigsby, l.l.: corn fiber: structure, composition, and response to enzymes for fermentable sugars and coproducts. appl. biochem. biotechnol., 144, 2008, 59-68. ballesteros, m., olive, j.m., negro, m.j., manzanares, p., ballesteros, i.: ethanol from lignocellulosic materials by a simultaneous saccharification and fermentation process (sfs) with kluyveromyvces marxianus cect 10875. process biochem., 39, 2004, 1843-1848. bura, r., mansfield, s.d., saddler, j.n., bothast, r.j.: so2-catalyzed steam explosion of corn fiber to ethanol production. appl. biochem. biotechnol., 98, 2002, 59-66. gáspár, m., kálmán, g., réczey, k.: corn fiber as a raw material for hemicellulose and ethanol production. process biochem., 42, 2007, 1135-1139. grohmann, k., bothast, r.: saccharification of corn fiber by combined treatment with dilute sulphuric acid and enzymes. process biochem., 32, 1997, 405-415. jarboe, l.r., grabar, t.b., yomano, l.p., shanmugan, k.t., ingram, l.o.: development of ethanologenic bacteria. adv. biochem. engin/biotechnol., 108, 2007, 237-261. leathers, t.d.: bioconversion of maize residues to value-added coproducts using yeast – like fungi. fems yeast res., 3, 2003, 133-140. mosier, s.n., hendrickson, r., brewer, m., ho, n., sedlak, m., dreshel, r., welch, g., dien, b.s., aden, a., ladisch, r.m.: industrial 190 ondáš, v. et al. scale-up of ph-controlled liquid hot water pretreatment of corn fiber for fuel ethanol production. appl. biochem. biotechnol., 125, 2005, 77-85. noureddini, h., byun, j., yu, t.: stagewise dilute-acid pretreatment and enzyme hydrolysis of distillers' grains and corn fiber. appl. biochem. biotechnol., 2009 (in press). palmarola-adrados, b., chotěborská, p., galbe, m., zacchi, g.: ethanol production from non-starch carbohydrates of wheat bran. bioresour. technol., 96, 2005, 843-850. réczey, k., szengyel, z., eklund, r., zacchi, g.: cellulase production by trichoderma reesei. bioresour. technol., 57, 1996, 20-25. saha, b.c.: hemicellulose bioconversion. ind. microbiol. biotechnol., 30, 2003, 279-291. schell, d.j., riley, c.j., dowe, n., farmer, j., ibsen, k.n., ruth, m.f., toon, s.t., lumpkin, r.e.: a bioethanol process development unit: initial operating experiences and results with corn fiber feedstock. bioresour. technol., 91, 2004, 179-188. shibanuma, k., takamine, k., maseda, s., osaki, s., abe, j., hizukuri, s.: partial acid hydrolysis of corn fiber for the ethanol production of l-arabinose. j. appl. glycosci., 46, 1999, 249-256. van maris, a.j.a., abbott, d.a., bellissimi, e., brink, j., kuyper, m., luttik, m.a.h., wisselink, h.w., scheffers, w.a., dijken, j.p., pronk, j.t.: alcoholic fermentation of carbon sources in biomass hydrolysates by saccharomyces cerevisiae: current status. antonie van leeuwenhoek, 90, 2006, 391-418. zhao, j., wang, m., yang, z.: measurement of inhibitory effect of furfural and furfural alcohol using coupled redox mediators. enzym. microb. tech., 37, 2005, 246-253. microsoft word mocak nb 9-3.doc nova biotechnologica 9-3 (2009) 319 current calculation of irreversible electrode reaction mechanisms in linear sweep voltammetry ján mocák, estera rábarová department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (jan.mocak@ucm.sk, estera.rabarova@ucm.sk) abstract: application of exponential infinite series gives highly accurate analytical solution contributing to the theory of linear sweep voltammetry for single scan experiments. we have calculated theoretical dimensionless current function (usually denoted as π1/2χ(bt)) at relevant potentials for irreversible charge transfer without a coupled chemical reaction. for this purpose several transformation techniques were used, which convert the derived infinite series into summable sequences. since infinite series of further electrochemical mechanisms with irreversible electrode reaction have similar features (particularly those comprising preceding and catalytic chemical reaction), the same approach can be successfully applied also for further electrochemical mechanisms. the respective infinite series are divergent in the most important potential region at and after voltammetric peak therefore their transformation by epsilon and levin transform techniques was used. necessary arbitrary precision arithmetic (apa) was implemented by ubasic. the results were compared to the customary solution of nicholson and shain, who computed the current-potential curves by means of numerical solution of the integral equations but with a much lower precision. our results were obtained in a broad potential range including the potential regions where the series are divergent. obtained current functions are precise to 12 valid decimal numbers, which is utilizable for evaluation of the results achieved by various faster but less precise digital simulation techniques. key words: linear sweep voltammetry, irreversible charge transfer, dimensionless current function, infinite series, arbitrary precision mathematics. 1. introduction mathematical description of current – potential curves, which create an essential analytical signal in linear sweep voltammetry, is complex end still not sufficiently treated in electrochemical literature, in spite of its value. the relevant calculations originate from a system of parabolic differential equations describing the concentrations of the reduced r and oxidized o forms of the studied electrochemical system as well as concentrations of further species eventually taking part in the overall electrochemical mechanism. different mechanisms differ in the detailed composition of initial and boundary conditions. an effective way of calculation submitted nicholson and shain (nicholson and shain, 1964) who solved eight electrochemical mechanisms by converting the boundary value problem into integral equations, which may be further solved either by the derived infinite series or directly using a numerical solution. of them only the series solution is considered analytical from the mathematical point of view since it provides an arbitrary precise result in the region where the series converge. the main problem with the series solution is that they are generally not convergent in the most desired potential region close to the maximum current (peak), which is basic for quantitative analysis. therefore very 320 mocák, j. and rábarová, e. many numerical ways of calculation have been described in electrochemical literature, mostly indicated as and belonging to digital simulation (britz, 2005; britz, 2009; rudolph et al., 1994; mocak and feldberg, 1994). this work is focussed to the uncomplicated irreversible electrode reaction (mechanism ii denoted by nicholson and shain (1964). severe problems with the series convergence can be overcome (at least in a sufficient extent) by transformation of divergent series into summable sequences. 2. theory, problem formulation and tools 2.1 convergence acceleration and resummation in applied mathematics, natural and technical sciences, various methods are used for the convergence acceleration of slowly convergent sequences or series and for the summation of divergent series. their basic idea is to extract hidden information contained in partial sums of a specific slowly convergent or divergent series, and to use that information in order to make a qualified estimate about new (usually higherorder) partial sums which eventually converge to some limit. in many cases, this “qualified estimate” leads to spectacular numerical results which represent a drastic improvement over a term-by-term summation of the original series, even if the series is formally convergent. for further discussion it is useful to consider a sequence {{sn}} = {{s0, s1, . . .}} with elements sn or the terms an = sn − sn−1 of an infinite series. sequence transformations are important tools for the convergence acceleration of slowly convergent sequences or series and also for the summation of divergent series (brezinski, 2000). the basic idea is to construct from a given sequence {{sn}} a new sequence {{s′n}} = t({{sn}}) where each s′n depends on a finite number of elements sn1, . . . , snm. often, the sn are the partial sums of an infinite series. the aim is to find a transformation t such that {{s′n}} converges faster than sn or, after all, it is capable to sum {{sn}}. a common approach is to rewrite sn as sn = s + rn (1) where s is the limit (or antilimit in the case of divergence) and rn is the remainder. the aim then is to find a new sequence {{s′n}} such that s′n = s + r′n , r′n /rn → 0 for n → ∞ (2) thus, the sequence {{s′n}} converges faster to the limit s (or diverges less violently) than {{sn}}. 2.2 employed ways of series transformation a large number of mainly nonlinear sequence transformations for the acceleration of convergence and the summation of divergent series are discussed in review (brezinski, 2000). some of the sequence transformations are well established nova biotechnologica 9-3 (2009) 321 in mathematical literature; among them wynn’s epsilon algorithm (wynn, 1956; wynn, 1966) and levin’s sequence transformations (levin, 1973; smith and ford, 1979; 1982), which were used in this work. wynn's epsilon algorithm or epsilon (ε-) transformation is the nonlinear recursive scheme utilising the partial sums of the original series (0)0ε = s0, (1) 0ε = s1, …, ( ) 0 nε = sn, and defined by the following equations (wynn, 1956): ( ) 1 nε − = 0, ( ) 0 nε = sn , n ≥ 0 (3) ( )n kε = ( 1) 2 n kε + − + 1 / ( ) 1 n kε −δ , k > 0 (4) where the forward difference operator δ is applied to superscripts: ( ) 1 n kε −δ = ( 1) 1 n kε + − − ( )1 n kε − (5) wynn found out that only the elements of the ε table with even subscripts (2n) are applicable for the results calculation (they give so-called shanks transformation) whereas the elements of the ε table with odd subscripts (2n+1) are only auxiliary quantities. each calculation element has a rhombus structure containing four terms and a recursive calculation using a single 1-dimensional array is possible (wynn, 1965). epsilon algorithm allows a simple and efficient series transformation and stimulated an enormous amount of research in this field. transformations t({{sn}}, {{rn}}) that depend not only on the sequence elements or partial sums sn but also on an auxiliary sequence containing the estimates of the remainder rn are of levin-type if they are linear in the sn, and nonlinear in the rn. remainder estimates are usually denoted ωn and provide an easy-to-use possibility to utilize asymptotic information on the problem sequence for the construction of highly efficient sequence transformations. levin transformations are based on the ratio of two series. the first series (in numerator) is a function of sn values of the original sentence divided by ωn, the second series (in denominator) depends on 1/ωn (figure 1). several variants of levin transformation differ in the way how ωn is estimated. in this work two variants were found most useful, particularly levin u and levin d transformations. they all can be solved recursively by using only a 3-term recurrence formulas, which are separately expressed for numerator as well as denominator and the final result is achieved as the ratio of the lastly calculated numerator and denominator terms. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 1 1 0 1 1 0 1 1 1 1 1 1 1 kk j n j k j n jn k n n kk j k j n j sk n j j n k l s , k n j j n k ω ω ω − + − = + − − = + + +⎛ ⎞ − ⎜ ⎟ + +⎝ ⎠ = + +⎛ ⎞ − ⎜ ⎟ + +⎝ ⎠ ∑ ∑ l fig. 1. general calculation scheme of levin transformations; remainder estimates ωn = (n+1)an and ωn = an+1 for the u and d transformations, respectively. 322 mocák, j. and rábarová, e. 2.3 solved problems and corresponding series investigated irreversible electrochemical reaction is identical to the mechanisms ii defined by nicholson and shain (nicholson and shain, 1964) where all employed symbols, common in voltammetry, are defined. the corresponding reaction scheme and the derived series are: r – z e− k ⇒ o (6, 7) ( ) ( ) ( ) 1 00 ii =1 * ( ) 1 exp ln 1 ( ) ( ) j j a j a s d bj n f rt i bt e e rt n f kj π πβ π χ β ∞ + ⎡ ⎤ = = − − +⎢ ⎥ − ⎢ ⎥⎣ ⎦ ∑ ! the summation of the series was performed by epsilon and levin u transformations (more suitable at larger potential values). computer program for recursive calculation of this mechanism was written in ubasic programming language, which allows implementation of arbitrary precision arithmetic (apa) and uses all numbers with very many decimal places (koyama, 2000). the potential scale utilized by nicholson and shain and their successors is defined as: e*(n-s) = (e – e0)αza + (rt/f) ln [(πdo b)1/2/ ks ] (8) however, oldham and coworkers in several important papers concerning irreversible electrode reaction used a simpler potential scale (e.g. in dalrymplealford, 1977) where π1/2 was omitted from the argument of logarithm: e*(oldh) = (e – e0)αza + (rt/f) ln [(do b)1/2/ ks ] (9) so that this scale is shifted by (rt/f) ln(π)1/2 = 14.705546966777 mv or by 0.572364943 when the dimensionless potential scale is used as another alternative, frequently utilized in electrochemistry. 3. results and discussion 3.1 current – potential dependence for irreversible electrode reaction using real unit potential scale for the sake of space the obtained results are demonstrated in a tabulated form. table 1 brings the calculated values of the current function π1/2χ(bt) designated here also as the dimensionless current i*, which was used in our previous papers (mocak, 2002; mocak and bond, 2004) and in the pioneer works of reinmuth. potential scales of nicholson-shain (n-s) as well as oldham (oldh) are implemented using real millivolt units. our precise values located in the third and seventh column (using n-s scale) can be compared to the values shown in the columns two and six, which were nova biotechnologica 9-3 (2009) 323 calculated numerically 45 years ago by nicholson and shain (1964). one should highly appreciate them (especially considering the computer level that time) since the deviation on the third rounded decimal place is observed only in four cases (emphasized in italics). it is surprising that the results of sivakumar and basha (2005), who investigated the same problem and used four decimal figures for the tabulated current function, were less precise behind the peak potential than the results of nicholson and shain and their potential range was even smaller than that of nicholson and shain. table 1. calculated dimensionless current for irreversible electrode oxidation using nicholson-shain and oldham potential scales expressed in real millivolt units. e*, mv π1/2χ(bt) (n-s) i* (n-s) i* (oldh) e*, mv π1/2χ(bt) (n-s) i* (n-s) i* (oldh) -300 0.00001505 0.00000849 -10 0.462 0.46201793 0.37464546 -250 0.00010536 0.00005944 -5 0.480 0.48053005 0.40852572 -200 0.00073716 0.00041603 0 0.492 0.49178853 0.43859990 -160 0.003 0.00348738 0.00197054 5 0.496 0.49574697 0.46329925 -150 0.00513823 0.00290546 10 0.493 0.49301358 0.48139777 -140 0.008 0.00756454 0.00428200 15 0.485 0.48470520 0.49222092 -120 0.016 0.01632951 0.00927948 20 0.472 0.47221381 0.49576253 -110 0.024 0.02391140 0.01363423 25 0.457 0.45696130 0.49266598 -100 0.035 0.03488516 0.01999080 30 0.441 0.44020946 0.48407130 -90 0.050 0.05062198 0.02922079 35 0.423 0.42295691 0.47137873 -80 0.073 0.07288591 0.04252075 40 0.406 0.40591713 0.45600522 -70 0.104 0.10376342 0.06146949 45 0.38955001 0.43919904 -60 0.145 0.14535833 0.08802158 50 0.374 0.37411700 0.42194196 -50 0.199 0.19905868 0.12433319 60 0.34644141 0.38861355 -45 0.23037469 0.14676318 70 0.323 0.32294382 0.35892542 -40 0.264 0.26422510 0.17225545 80 0.30310390 0.33350738 -35 0.300 0.29992874 0.20082435 100 0.27192236 0.29386329 -30 0.337 0.33648220 0.23230306 120 0.24872422 0.26514046 -25 0.372 0.37256762 0.26628454 150 0.22320466 0.23462457 -20 0.406 0.40661969 0.30206924 200 0.19446262 0.20168254 -15 0.437 0.43696362 0.33863453 250 0.17485343 0.17997456 3.2 current – potential dependence for irreversible electrode reaction using dimensionless potential scale in principle the same calculation approach was used for obtaining the dimensionless current i* related to the dimensionless potential e* defined again in two potential scales, those introduced by nicholson-shain (n-s) and oldham (oldh), respectively. tabulated results are exhibited in table 2. assuming t = 298.15 k, one dimensionless potential unit corresponds to the value rt / f = 25.692606 mv (mocak, 2002). it is worth mentioning that assuming temperature t = 298.15 k, one dimensionless potential unit corresponds to the value rt / f = 25.692606 mv (mocak, 2002). 324 mocák, j. and rábarová, e. table 2. calculated dimensionless current for irreversible electrode oxidation using dimensionless nicholson-shain and oldham potential scales. e* i* (oldh) i* (n-s) e* i* (oldh) i* (n-s) -10.0 0.00004540 0.00008046 0.5 0.48846716 0.48888145 -9.0 0.00012339 0.00021869 1.0 0.49177335 0.45470458 -8.0 0.00033535 0.00059424 1.5 0.46069675 0.37002530 -7.0 0.00091105 0.00161366 2.0 0.41718128 0.30856070 -6.0 0.00247262 0.00437423 3.0 0.34050384 0.26830872 -5.0 0.00669276 0.01180127 4.0 0.28933513 0.24060300 -4.0 0.01798448 0.03143348 5.0 0.25529883 0.22028455 -3.0 0.04739313 0.08092026 6.0 0.23121011 0.20457946 -2.0 0.11864417 0.19088973 7.0 0.21311328 0.19194529 -1.0 0.26145051 0.36765286 8.0 0.19886406 0.18147266 -0.5 0.35430163 0.44848739 9.0 0.18724114 0.17259233 0.0 0.43859990 0.49178853 10.0 0.17750549 0.17750549 4. conclusions the infinite series solution represents at present time the only alternative of analytical solution (from mathematical standpoint) of the corresponding set of differential equations pertinent to irreversible charge transfer (6) in stationary electrode voltammetry and facilitates very precise calculation of voltammetric current even in the potential region far after the peak. even though the corresponding infinite series (7) behaves very badly at large positive potentials, levin transformations and the use of arbitrary precision arithmetic prevents catastrophic loss of precision in calculation and enables very accurate calculation even at 250 mv (using n-s scale). in the indicated potential region all calculated dimensionless current values were precise at minimum to 12 decimal places even though the tables exhibited above contain only the numbers with 8 decimal places to be sufficiently lucid. for practical comparison with the calculations obtained in other mathematical ways even a smaller number of decimal places is needed. in any case, the resulting current – potential data obtained in this work may serve as the standard values utilizable for precision assessment of other, perhaps faster computational tools like digital simulation. acknowledgement: the support of this work by slovak grant agencies by means of grants vega 1/1005/09 and 1/0066/09, and vvce-0004-07 is highly acknowledged. references brezinski, c.: convergence acceleration during 20th century. j. comput. appl. math., 122, 2000, 1-21. britz, d.: digital simulation in electrochemistry. lect. notes phys., 666, 3rd edition, springer-verlag, berlin, 2005, 338 pp. nova biotechnologica 9-3 (2009) 325 britz, d.: digital simulation in electrochemistry, 3rd ed. electronic version: http://www.scribd.com/doc/17687737/digital-simulation-in-electrochemistry-3rded-dieter-britz, 2009. koyama, y.: ubasic. kanazawa univ. japan, ftp://rkmath/rikkyolac.jp/pub/ubibm; ubasic ver. 8.8f, download: http://www.rkmath.rikkyo.ac.jp/~kida/ubasic.htm, 2000. levin, d.: development of non-linear transformations for improving convergence of sequences. int. j. comput. math., b 3, 1973, 371-388. mocak, j., feldberg, s.w.: the richtmyer modification of the fully implicit finite-difference algorithm for simulations of electrochemical problems. j. electroanal. chem., 378, 1994, 31-37. mocak, j.: voltammetric current-potential calculations using infinite series solution. electrochem. comm., 4, 2002, 803-807. mocak, j., bond, a.m.: use of mathematica software for theoretical analysis of linear sweep voltammograms. j. electroanal. chem., 561, 2004, 191-202. nicholson, r.s., shain, i.: single scan and cyclic methods applied to reversible, irreversible, and kinetic systems. anal. chem., 36, 1964, 706-723. dalrymple-alford, p., goto, m., oldham, k.b.: shapes of derivative neopolarogram. j. electroanal. chem., 55, 1977, 1-15. rudolph, m., reddy, d.p., feldberg, s.w.: a simulator for cyclic voltammetric responses. anal. chem., 66, 1994, a589-a600. sivakumar, s., basha, c.a.: evaluation of the current function in linear sweep voltammetry by pade approximation and epsilon convergence. russian j. electrochem., 41, 2005, 421-438. smith, d.a., ford, w.f.: acceleration of linear and logarithmic convergence. siam j. numer. anal., 16, 1979, 223-240. smith, d.a., ford, w.f.: numerical comparisons of nonlinear convergence accelerators. math. comput., 38, 1982, 481-499. wynn, p.: on a device for computing the em(sn) transformation. math. tables aids comput., 10, 1956, 91-96. wynn, p.: a note on programming repeated applications of the ε-algorithm. rev. franc. trait. inf. – chiffres, 8, 1965, 23-62. wynn, p.: on the convergence and the stability of the epsilon algorithm. siam j. numer. anal., 3, 1966, 91-122. microsoft word hegedusova nb 9-2.doc nova biotechnologica 9-2 (2009) 125 use of phytoremediation techniques for elimination of lead from polluted soils alžbeta hegedűsová1, silvia jakabová1, andrea vargová1, ondrej hegedűs2, tímea judit pernyeszi3 1department of chemistry, constantine the philosopher university in nitra, tr. a. hlinku 1, nitra, sk949 01, slovak republic (ahegedusova@ukf.sk, sjakabova@ukf.sk, avargova@ukf.sk) 2regional authority of public health in nitra, štefánikova 58, nitra, sk949 63, slovak republic (nr.hegedus@uvzsr.sk) 3department of analytical and environmental chemistry university of pécs, ifjúság u. 6, pécs, hu-7624, hungary (ptimea@ttk.pte.hu) abstract: the effect of chelating agent – edta (ethylene-diamine-tetra-acetic acid) was used for induced phytoextraction to increase intensity of lead transfer from roots to aboveground parts of garden pea. pot experiments with contaminated soil substrata (50 mg pb.kg-1 and 100 mg pb.kg-1) were established for experimental purposes in growth chamber. the results showed that application of 5 and 10 mmol edta.kg-1 to experimental variants with 100 mg pb.kg-1 doubled the increase of lead uptake by pea roots in comparison with variants without edta addition, which was statistically confirmed. intensive lead transfer was observed from roots to aboveground parts of pea after application of 5 and 10 mmol edta.kg-1 in variant with 50 mg pb.kg-1 (40-fold increase), as well as in variant with 100 mg pb.kg-1 (17-fold increase). the results showed that induced phytoextraction can improve the mobility of lead from soil to plant roots. application of 5 mmol edta.kg-1 resulted to 40-fold increase of lead transfer to green plant parts, despite the fact, that garden pea does not belong to conventional metal hyperaccumulating plant species. following the results, pea could be used for decontamination of arable soil. the optimal edta concentration seems to be 5 mmol.kg-1. therefore, application of 10 mmol edta.kg-1 decreased root mass about 55%, which resulted to decrease the intensity of lead uptake. key words: induced phytoextraction, edta, lead, garden pea 1. introduction the territory of slovakia belongs to long term polluting areas with transfer of contaminants from large industrial and power plant complexes with regional effects. these negative effects affect an extraordinary heterogenous soil cover and increase the content of risk elements to amounts exceeding their limits. accumulation of risk elements in soils affects its ability to produce hygienically unobjectionable foodstuffs, which applies especially for the content of heavy metals with a high degree of biotoxicity from various sources (hegedűsová et al., 2000). decontamination of areas, polluted with toxic metals, presents one of very important research topics and cleaning of the areas by conventionally used physico-chemical methods is financially very expensive and often non-ecologic. modern decontamination technologies, generally called phytoremediation offer possibilities of removing heavy metals from soils (huang and cunningham, 1996; lasat, 2002; simon et al., 2003). the most often used phytoremediation techniques are especially phytoextraction and phytostabilitation. 126 hegedűsová a. et al. during the process of phytoextraction, plants absorb contaminants from soil through the root system and store them mainly in the green biomass and partially in root biomass. application of chelating agents increases the bioavailability of the elements, which is known as an induced phytoextraction process. the most effective is using the plant species with larger biomass production (mcgrath et al., 2002). obtained biomass is processed by several different techniques including microbiological (composting), thermic (ashing or combustion), or chemical (extraction) ones. some of plants species, called hyperaccumulators, bind the metals in high amounts, and therefore, they are used in phytoremediation. tolerance of plants to heavy metals can be explained by binding of metal to cell wall, membrane tolerance to metals, active transport of metals in plant cells, presence of metal-tolerant enzymes, accumulation of metals in vacuole and chelatation of metals with organic or inorganic ligands. lead does not belong to essential elements; moreover, it is highly toxic for animals and humans. similarly to other heavy metals, ions of lead are also potential cancerogenic agents. lead disturbs intermediate metabolism, inhibits the exchange of saccharides in nerve tissue, affects the changes in metabolism of porphyrins and influences the inhibition of homeostasis. it mainly interferes with enzymes on their sulfhydryl groups. the highest risk of intake and retention of this xenobiotic element is from foodstuff intake, therefore approximately 60 % of lead in human organism is ingested and 30 % comes from inhalation (kafka and punčochářová, 2002). common source of lead is soil. the content of lead in main soil types in slovakia varies in wide interval between 10 and 60 mgpb.kg-1. natural values of pb in soil rise after accumulation of dust around roads and from wet deposition after rainfalls. in soil, degradation by microorganisms does not occur, because contact with oxygen leads to oxidation (fodor, 2003; fodor et al., 1996; grčman et al., 2003). the aim of this work was to follow the effect of selected chelating agent – edta (ethylene-diamine-tetra-acetic acid) within decontamination of soil to increase transfer of lead from model contamined soil to aboveground parts of garden pea (pisum sativum l.) using the technique of induced phytoextraction. 2. materials and methods 2.1 plant material pot experiment was established with garden pea (pisum sativum l., variety oskar, very early, marrowfat). from nutritional aspect, peas are well known for being a rich source of highly bioavailable protein. composition of the proteins is similar to proteins of animal origin. ions of heavy metals usually attack tiol-groups, and also binding on carboxyl or amino groups of amino acids occurs in pea proteins. this could explain why pea has higher uptake of metals than cereals. n-compounds exceed 90 % in pea; moreover, main fraction present globulins (45 – 60 %) and other fractions (albumins, insoluble proteins and non-protein fraction) present approximately 15 – 20 % of all nitrogen. content of proteins varies in dependence on variety properties, soil, nutrition, climatic conditions and level of maturity. pea seeds are important source of vitamins nova biotechnologica 9-2 (2009) 127 c, e and b group, derivatives of folic acid, called folates, and macro elements like phosphorus, potassium, magnesium, and calcium. pea is also interesting from the aspect of water soluble or non soluble fibre. 2.2 pot experiments with induced phytoextraction model pot experiments were realized in nine experimental variants in growth chamber with controlled condition at the department of botany and genetics constantine the philosopher university in nitra. growing medium was clean soil substrata in an amount 1000g in each pot. lead solution was applied in two concentrations (50 and 100 mg pb.kg-1) by spraying of soil 4 weeks before sowing. garden pea was sowed directly to pots in amount 20 seeds per pot. growing conditions were following: air temperature 20 ºc, air humidity 60 – 70 %, soil humidity 75 %. light was set to12 h of light and 12 h of darkness, intensity of radiation was max. 50 000 lx). in the sixth and seventh week of growth, chelating agent edta was applied as the solutions with the total addition 5 and 10 mmol.kg-1. after eight weeks of growth all experimental plants were harvested and submitted to analysis. 2.3 sample preparation and determination of lead by et-aas technique dry and homogenized plant samples in amount 1 g were wet digested in vessels za-1 with digesting mixture 1 cm3 deionized water, 2 cm3 h2o2 and 5 cm 3 hno3. soil extracts in were prepared according to standard stn iso 11466:2001. solutions of roots, aboveground plant parts and soil extracts were submitted to quantitative determination of lead using the method et-aas. determination was done on atomic absorption spectrometer spectraa240fs (varian, mulgrave virginia, australia). the technique requires lead hollow cathode lamp to operate at a wavelength of 217.0 nm with a slit width set to 1.0 nm and an electrodeless discharge lamp set at 5 ma current. injected sample volume was 10 μl and palladium matrix modifier pb(no3)2 with concentration 0.1 mol.dm -3 digested in 0.1% hno3 and ascorbic acid (1%) were used as modifiers. 2.4 evaluation of results evaluation of the results was done with the method of calibration curve. the results were statistically evaluated using the single-factor analysis of variance and tukey b test in spss program. 3. results and discussion effect of chelating agent edta to enhance lead uptake from soil to plant and metal transfer in the plant was followed. model concentrations of lead were proposed according to results of soil monitoring on southern slovakia (hegedűsová et al., 2000). in variants with pb addition 50 mg pb.kg-1 and 100 mg pb.kg-1 without edta treatment pea roots accumulated lead in amount 0.22 mg pb.kg-1 and 0.27 mg pb.kg-1 128 hegedűsová a. et al. respectively. transfer of lead did not continue from roots to aboveground parts of plant in variant without application of edta. on the other hand, addition of chelating agent (edta) enhanced the content of the metal in all plant tissues in comparison to variant without edta treatment. pb content in pea roots from pots with 50 mg pb.kg-1 increased about 30% (1.3-fold) after application 5 mmol edta.kg-1. non-significant enhancement of pb was found after application of 10 mmol edta.kg-1 in comparison with edta-nontreated variant (table 1, fig.1). additions of edta resulted to statistically significant increase of lead transfer from soil to pea roots within the soil treatment with 100 mg pb.kg-1 and 5 mmol edta.kg-1. the increase presented 150 % in comparison with edta non treated variant. 100% increase of pb content was found in variant with application 100 mg pb.kg-1 and 10 mmol edta.kg-1 compared with variant without chelate (table 1, fig. 2). statistically significant increase of lead in aboveground plant parts of pea was also found in variants with 50 mg. pb kg-1 in combination with edta treatment. application of 5 and 10 mmol edta.kg-1 resulted to 44-fold and 36-fold increase of pb respectively. lead content in aboveground parts rised approximately 18-times in variant with 100 mg pb.kg-1 in combination with two applied concentrations of chelate (table 1, fig.1, fig.2). there is an assumption that root exudes in a mixture of selected plant species played an important role in the process of metal dissolving in soil and make easier the uptake of metals by pea roots. luo et al. (2008) experimentally proved enhancement of metal uptake (in pea, placed in mixed culture with barley. concentrations of pb, cu, zn, cd and fe in the shoots were higher than in the case of growing pea in isolation. these results presented the evidence that natural chelates, produced by plants themselves, enhance the bioavailability of heavy metals in soil solution and support their uptake by plants. table 1. content of lead in roots and aboveground parts of garden pea in dependence on soil treatment by lead and edta (pot experiments in growth chamber, nitra, 2008). content of pb [mg.kg-1] roots aboveground parts variant pb/edta xmin xm xmax xmin xm xmax c 0.042 0.05a 0.05 0.02 0.03a 0.04 50/0 0.15 0.22ab 0.33 0.02 0.03a 0.03 50/5 0.16 0.29b 0.43 0.8 1.33c 0.18 50/10 0.31 0.33b 0.39 0.96 1.08bc 1.19 100/0 0.21 0.27b 0.40 0.28 0.04a 0.58 100/5 0.59 0.67c 0.78 0.55 0.73b 1.02 100/10 0.32 0.54c 0.79 0.51 0.71b 0.97 legend: c – control variant without application of pb and edta, xmin – minimum value, xmax – maximum value, xm– mean value, a, b, c, d, e – single-factor analysis of variance, tukey b test – statistically significant difference is between the mean values which have different index in the same group, significance level p<0.05, n (number of duplicates) = 9. nova biotechnologica 9-2 (2009) 129 fig. 1. transfer of lead to roots and aboveground parts of garden pea in soil with addition 50 mg pb.kg-1 in dependence on applied amounts of edta (pot experiments in growth chamber, nitra, 2008) fig. 2. transfer of lead to roots and aboveground parts of garden pea in soil with addition 100 mg pb.kg-1 in dependence on applied amounts of edta (pot experiments in growth chamber, nitra, 2008) many studies dealt with increase of heavy metal accumulation in plants within the process of induced phytoextraction. result of experiment of huang and co-workers (1997) confirmed that application of 1g edta.kg-1 to soil contamined with 2500 mg pb.kg-1 enhanced lead transfer to roots and aboveground plant parts about 120-times. five synthetic chelating agents (edta, hedta, dtpa, egta, eddha) were examined and just edta showed the most intensive desorption properties. 3.1 biomass production the biomass production and content of dry mass was followed in all experimental variants. the results showed the statistically significant difference only in variants 130 hegedűsová a. et al. treated with 10 mmol edta.kg-1 (table 2). this amount of chelating agent affected 24 to 33 % decrease of root biomass production (calculations were done with dry mass values). therefore we also supposed decrease of lead uptake intensity by root system. table 2. dry mass of roots and aboveground plant parts of pea in all variants (pot experiments in growth chamber, nitra, 2008). dry mass (g/pot) variant pb/edta roots aboveground parts c 0.73a 3.33a 50/0 0.70a 3.33a 50/5 0.68a 2.99a 50/10 0.53b 3.09a 100/0 0.67a 3.33a 100/5 0.57a 3.33a 100/10 0.45b 2.95a note: single-factor analysis of variance, tukey b test (p<0.05). 3.2 transport indexti transport index (table 3) was calculated as a ratio between pb content in aboveground part and its content in root. table 3. transport index. variant ti 50/0 14.00 50/5 4.59 50/10 3.27 100/0 0.15 100/5 1.09 100/10 1.31 note: ti – transport index (aboveground parts /roots) the results showed that concentration of 5 mmol edta.kg-1 enhanced pb transport from roots to aboveground parts 33-times in variant with 50 mg pb.kg-1. lower transport indexes (1.09 and 1.31) were calculated in variants with addition 100 mg pb.kg-1, which were probably connected with decrease of weight of root system. 3.3 lead balance in plant lead balance in pea plant showed 56 to 62% accumulation of the metal in roots in variants without edta treatment. induced phytoextraction was activated after additions 5 and 10 mmol edta.kg-1, which resulted in enhanced of the metal mobility nova biotechnologica 9-2 (2009) 131 from roots. moreover, 95% and 88 % of metal content was translocated to aboveground parts in variants with application 50 and 100 mg pb.kg-1 respectively (table 4). table 4. lead balance in pea plant (pot experiments in growth chamber, nitra, 2008). withdrawn amount of pb /pot/dry mass roots aboveground parts variant [μg] [%] [μg] [%] c 0.03 23.07 0.10 76.93 50/0 0.15 62.05 0.09 37.50 50/5 0.18 5.02 3.41 94.98 50/10 0.18 4.78 3.59 95.22 100/0 0.19 55.88 0.15 44.12 100/5 0.39 13.00 2.61 87.00 100/10 0.27 11.74 2.03 88.26 the lowest transfer of lead from roots to aboveground parts of pea in variant with addition 100 mg pb.kg-1 could be caused by lower production of root biomass after application of chelate. 4. conclusions despite the fact that in the recent years decreased the amount of emissions in slovakia as a result of certain system precautions, the problem of heavy metal pollution of the environment still exist. hygienic safety of agricultural soils was the basic assumption for growth of hygienic safety foodstuffs. the results from the study of induced phytoextraction process on garden pea plants (pisum sativum l.) using the chelating agent edta allowed us to conclude the following: 1. content of lead in plants in experimental variants with pb addition increased after application of chelating agent. uptake of lead to aboveground parts of pea increased using edta concentrations 5 and 10 mmol edta. kg-1 in the range from 36 to 44fold (in variant with 50 mg pb.kg-1) and to 18-fold (in variant with 100 mg pb.kg-1). 2. statistical significant enhancement of lead transfer from soil to roots was observed only in variant with 100 mg.kg-1 pb addition and present 2 to 2.4 fold increase. 3. efficiency of pb translocation from roots to aboveground parts of pea was evaluated from the calculations of transport indexes. the highest values of transport indexes were observed in pea plants, grown on soil with addition 50 mg pb.kg-1 in both variants treated with edta. 4. the balance of lead content showed enhancement of metal mobility from roots after treatment with edta. moreover, 95% of absorbed metal was translocated to aboveground parts. 132 hegedűsová a. et al. 5. decrease of dry mass production was observed only on root biomass after treatment with 10 mmol edta.kg-1, which resulted to decrease of lead uptake from soil. we can conclude that the most effective continual phytoextraction of lead using garden pea was in variant with 50 mg pb.kg-1 after application 5 mmol edta.kg-1. theoretical calculations showed that garden pea is able to absorb 45-times more lead after chelate treatment than in the case without treatment. pea biomass could be used as an fuel and lead could be obtained back in metal form. another manner how to manage with the biomass is composting, which can reduce its volume. ackowledgment: the work was supported by the project vega 1/4370/07. references fodor, f.: ólom és kadmiumstressz növényekben. bot. közlem., 90, 2003, 107-120. fodor, f., sárvári, é., láng, f., sziget, z., cseh, e.: effects of pb and cd on cucumber depending on the fe-complex in the culture solution. j. plant. physiol., 148, 1996, 434-439. grčman, h., vodník, d., velikonja-bolta, š., leštan, d.: heavy metals in the environment. j. environ. qual., 32, 2003, 500-506. hegedűsová, a., hegedűs, o., vollmannová, a.: kontaminácia poľnohospodárskych pôd a zelenín ťažkými kovmi na južnom slovensku. hort. sci., 27, 2000, 57-64. huang, j.w., cunningham, s.d.: lead phytoextraction: species variation in lead uptake and translocation. new phytol., 134, 1996, 75-84. huang, j.w., chen, j., berti, w. r., cunningham, s.d.: phytoremediation of lead-contaminated soils: role of synthetic chelates in lead phytoextraction. environ. sci. technol., 31, 1997, 800-805. kafka, z., punčochářová, j.: těžké kovy v přírodě a jejich toxicita. chem. listy, 96, 2002, 611 617. lasat, m.: phytoextraction of toxic metals. j. environ. qual., 31, 2002, 109-120. luo, ch., shen, z., li, x.: root exudates increase metal accumulation in mixed cultures: implications for naturally enhanced phytoextraction. water air soil pollut., 193, 2008, 147 – 154. mcgrath, s.p. zhao, f.j., lombi, e.: phytoremediation of metals, metalloids, and radionuclides. adv. agron., 75, 2002, 1-56. simon, l., szegvári, i., csillag j.: impact of picolinic acid on the chromium accumulation in fodder radish and komatsuna. plant soil, 254, 2003, 337-348 microsoft word gasparova revision.doc nova biotechnologica 8-1 (2008) 79 synthesis and plant growth activity of 2-(4-oxochromen-3-yl)benzothiazolium and benzoxazolium bromides mária henselová1, renata gašparová2, margita lácová3 1department of plant physiology, faculty of natural sciences, comenius university, mlynská dolina b-2, sk-842 15 bratislava, slovak republic 2department of chemistry, faculty of natural sciences, university of ss. cyril and methodius, námestie jozefa herdu 2, sk-917 01 trnava, slovak republic 3department of organic chemistry, faculty of natural sciences, comenius university, mlynská dolina ch-2, sk-842 15 bratislava, slovak republic abstract: a facile synthesis of novel 2-(4-oxochromen-3-yl) benzothiazolium bromides 4 and benzoxazolium bromide 5 using the one-pot condensation of substituted 4-oxochromene-3-carboxaldehydes 1 with 2-methylbenzothiazole (2a) or 2-methylbenzoxazole (2b) and benzyl bromide is described. the effects of benzothiazolium bromides 4 on the growth stimulation of the cucumber and retardant activity on corn seedlings were investigated. they inhibited growth of cucumber (root and hypocotyl) and shoots of corn at the range of 10 – 100 ppm and stimulated at 0.1 – 1 ppm concentrations. keywords: plant growth activity, benzothiazolium, benzoxazolium salts, 4-oxochromene-3-carboxaldehydes 1. introduction benzothiazole derivatives represent a large group of heterocyclic substances, which were tested for a different biological activity (sutoris et al., 1988; kráľová et al., 1994; el-shaaer et al., 1998). 2-r substituted benzotiazole derivatives manifested antibacterial and antifungal activities (afsah and nayer 1996; magdolen et al., 2000; buffa et al., 2001) and are reported also to be active as antineoplastics agent (krieg and billitz 1996). according šimonová et al., (2005) ones were characterized as biologically active substances with dominant auxin-like growth promoting activity. 3-r substituted benzothiazole derivatives influenced the prolongation of growth, stimulated the plant regeneration, activity of peroxidase, and may induce the dedifferentiation and morphogenesis of plants in in vitro conditions (sutoris et al., 1993; havel et al., 1994). benzothiazole derivatives were also observed as protective agents against uv-induced mutagenesis in euglena gracilis foltínová et al., (2003) and also against of cadmium stress henselová et al., (2007); klecová šimonová et al. (2008) in phytollaca dioica, vigna radiata and brassica juncea plants. a large number of 4-oxochromene derivatives occur in nature and exhibit variety of biological activities, e.g. antialgal (kráľová et al., 1998), antifungal, antimycobacterial and antiparasitic (caujolle et al., 1993; lácová et al., 1995; gašparová et al., 1997; nawrot-modranka et al., 2006), however, none of 80 henselová, m. et al. them have been reported to have plant growth regulation activity. this paper described the auxin and retardant-like growth activity of newly synthesized oxochromene benzothiazole compounds. 2. materials and methods 2.1 chemistry scheme 1 shows the synthetic pathway to benzothiazolium and benzoxazolium salts. more synthetic details as well as the characteristic data of synthesized compounds are described (kleštinec et al., 2008). general procedure for synthesis 4a-5 a stirred mixture of 4-oxochromene-3-carboxaldehyde 1a (1 mmol), 2methylbenzothiazole 2a (1 mmol) (or 2-methyloxazole 2b) and benzylbromide (1 mmol) in anhydrous nitromethane (2 dm3) was irradiated in microwave oven at 270 w for 10 min. after cooling, the solid product was filtered off, washed with warm acetone and crystallized from acetonitrile to give 45 – 81% yields of products. o o cho r1 x n ch3 c6h5 o or 1 n x br c6h5br + 1 3a x = s 3b x = o 4a-4f x = s 5 x = o x n ch3 scheme 1 c6h5ch2br ch3no2 ch3no2 2a x = s 2b x = o r1 = h, 6-ch3, 6-cl, 6-br, 6-no2, 6-oh, 7-oh 2.2 study of the plant growth activity the solutions of salts 4 5 were prepared in dimethylsulfoxide (dmso). the final concentration of dmso in experiments, including the control, was held constant at 1% by volume. this concentration of dmso had no detectable effect on the growth of plants under experimental conditions. for determination of stimulated growth activity like indole-3-acetic acid (iaa) we used the bioassay as described by merrill (1989) in this modification: twenty seeds of cucumber (cucumis sativus l. cv. regina) were placed in 12 cm petri-dishes on two layers of filter paper whatman № 2, wetted with 15 ml of distilled water (control), or of the indicated benzothiazole nova biotechnologica 8-1 (2008) 81 solution at 0.1, 1 and 10 ppm concentrations. the petri-dishes were placed to a dark box and incubated at 28 °c. the length of the root and hypocotyl seedlings was measured to the nearest milimeter after 7 days. the stimulated effect of tested compounds was evaluated and compared with the control and standard iaa in the range from 0.001 to 10 ppm concentrations. for corn bioassay in nutrient culture the seeds of corn (zea mays l. cv. greta) were pregerminated by soaking them in water for 24 hours. twenty-five seeds were placed on wet filter paper in the petri-dishes of 20 cm diameter for 48 hours at 25 °c to the dark box. seeds selected for treatment had a coleoptile emergence of 10 to15 mm. for determination of retardant activity-like abscisic acid (aba) we used the seedlings bioassay as described by dathe et al., (1978): twenty seedlings were chosen per every variant and placed after two seedlings in glass flasks containing 4 ml of half nutrient solution prepared according brown and dalton (1970) with application of testing compounds in 1, 10 and 100 ppm concentrations. the treated and untreated corn plants were cultivated under glass cover in climatic box (temperature 25/15 °c during 16 hours photoperiod and light intensity of 60 μmol·m-2·s-1 with a relative humidity of 80%). the level of the solutions in the flasks was maintained daily by adding nutrient solution. the length of the corn shoots was measured after 7 days and compared with the control and standard aba. the standards iaa and aba come from sigma, st. louis, mo, usa. all dates are presented as arithmetic averages of the individual experiments. bioassay results were statistically evaluated using the student’s t-test. 3. results and discussion 3.1 study of inhibition of germination and early growth of cucumber and corn seedlings it is obvious from our experimental data that the biological growth effects depend on concentrations of tested compounds. the cucumber growth test and corn bioassay have been used as a specific and sensitive tests for verification of auxin and retardant activities of tested benzothiazole compounds. the differences in auxin (iaa) and retardant (aba) like growth activities between tested derivatives are evident from tables 1 and 2. in general, the inhibition activity was observed at higher (10-100 ppm), and the stimulation activity at lower (0.1-1 ppm) concentrations. all tested compounds showed concentration-dependent stimulation activity in growth elongation of cucumber hypocotyl and root (table 1). growth elongation of hypocotyls was increased from 6% (compound no.5) to 42% (no. 4a). in the case of root growth, maximum stimulation was achieved following the application of 0.1ppm concentration from 14.3% (no. 4e) to 45% (no. 4c). their effects were similar not only to those of synthetic auxin iaa, but also 2,4-dichlorophenoxyacetic acid (york et al., 1984; sutoris et al., 1993) which stimulated elongation of pea stem and wheat coleoptile segments. the highest significant growth stimulation was achieved following application-tested substances in a concentration range of 0.1 to 1 ppm for both hypocotyl and root. compounds no. 4b and 5 have no stimulation effect on root growth (table 1). however, all the compounds were weaker, than iaa, which 82 henselová, m. et al. exhibited maximum effect (6165%) at a concentration one hundred times lower (0.001 ppm). similar effects achieved sutoris et al., (1993) at testing of 3– phenoxycarbonyl-methyland 3-aryloxycarbonyl-methyl-2-benzothiazolinones and (šimonová et al., (2005) at 2-r substituted benzothiazole derivatives. in the range of 10-3 10-7 m concentrations the compounds showed different auxin-like effects on elongation growth of wheat coleoptile segments and the formation and number of adventitious roots and the length of hypocotyl in mung bean. tab. 1. effect of 4a-5 benzothiazole compounds and indole-3-acetic acid (iaa) on root and hypocotyl length in cucumber cv. regina. compound r1 concentration [ppm] mean length [mm] % of control root hypocotyl root hypocotyl control –– 63.5 36.6 100.0 100.0 4a h 0.1 1.0 10.0 83.0 77.0 63.0 52.0 48.5 44.7 130.7a 121.2a 99.2 142.1a 132.5a 122.1 4b 6-ch3 0.1 1.0 10.0 59.0 52.2 40.0 44.7 38.2 26.2 92.9 82.2a 63.0a 122.1a 104.4 71.6a 4c 6-cl 0.1 1.0 10.0 92.0 71.8 45.8 46.2 39.8 26.8 144.9a 113.1b 72.1a 126.2a 108.7 73.2a 4d 6-br 0.1 1.0 10.0 80.0 63.0 61.0 50.5 47.0 10.7 126.0a 99.2 96.1 137.9a 128.4a 111.2 b 4e 6-no2 0.1 1.0 10.0 72.6 51.8 48.6 47.0 44.2 35.2 114.3b 81.6a 76.5a 128.4a 120.8a 96.2 4f 6-oh 0.1 1.0 10.0 89.7 79.7 64.0 49.2 47.5 43.2 141.2a 125.5a 100.8 134.4a 129.8a 118.0a 4g 7-oh 0.1 1.0 10.0 74.6 70.4 48.8 41.2 35.8 33.7 117.5b 110.9 b 76.8 b 112.6a 97.8 92.1 5 6-ch3 0.1 1.0 10.0 61.2 39.0 31.2 38.8 25.4 19.2 96.4 61.4a 49.1a 106.0 69.4a 52.4a iaa standard 0.001 0.01 0.1 1.0 10.0 104.8 94.8 86.4 79.8 32.2 58.9 51.2 44.6 43.1 27.9 165.0a 149.3a 136.1a 125.7a 50.7a 160.9a 139.9a 121.8a 117.7a 76.2a a values are significantly different at the 1%; b values are significantly different at the 5% level against the control as determined by student’s t-test. present studies thus show that the compounds retarded shoot growth of corn, although not necessarily at the same rate (table 2). the majority of tested compounds showed retardation activity in elongation shoot growth of corn seedlings in the range nova biotechnologica 8-1 (2008) 83 of 10 -100 ppm concentrations. this effect, in dependence on the compound structure, amounted up to 82% at no.5 (table 2). at the highest concentration (100 ppm) the compounds exhibited a morfogenic effect evident in the dwarfing of corn coleoptile and cucumber hypocotyle. it is known that the synthetic auxins at higher concentrations causes inhibition of growth, stem twisting, and other formative effects in susceptible plants (chang and foy 1983; fargašová 1994). on the other hand however, stimulates both root and shoot growth of many plants at rates lower, what confirmed also our results. the effect of tested substances was comparable with that of aba (table 2) with exception of compounds no. 4b and 5 that exhibited higher retardant activity (63-82%) in compare to the standard (42.8%) at the same concentration of 100 ppm. three compounds (4a, 4e and 4f) had no retardant effect on corn shoot growth in the range of tested concentrations (table. 2). tab. 2. effect of 4a – 5 benzothiazole compounds and abscisic acid (aba) on shoot length of corn cv. greta. compound concentration (ppm) mean length of the shoot (mm) % of control control –— 121.4 100.0 4a 1 10 100 155.5 143.0 142.0 128.1a 117.8a 116.9a 4b 1 10 100 113.0 65.0 45.0 93.1 53.5a 37.1a 4c 1 10 100 185.0 141.0 98.5 152.4a 116.1a 81.1a 4d 1 10 100 125.0 142.0 93.0 102.9 116.9a 76.6a 4e 1 10 100 135.0 145.0 121.5 111.2 b 119.4a 100.1 4f 1 10 100 132.0 146.0 143.5 108.7 120.3a 118.2a 5 1 10 100 126.0 101.0 22.0 103.8 83.2a 18.1a aba standard 1 10 100 95.4 77.4 45.2 78.6a 63.7a 57.2a a values are significantly different at the 1%; b values are significantly different at the 0.5% level against the control as determined by student’s t-test. the enhancement of growth of cucumber and corn seedlings at the lower concentrations and, on the other hand, their inhibition effect at higher concentrations 84 henselová, m. et al. observed in tested of benzothiazole derivatives are according to merrill’s characteristic growth pattern for auxins. auxins generally showed the best induction of adventitious roots and stimulated the elongation growth on various plants (müller 2000; wang et al., 2003; kollárová et al., 2005). mode(s) of action of growth stimulators, especially auxin-like substances as were tested compounds, have often been hypothesized as being involved in the regulation of synthesis or degradation of endogenous auxin indole-3-acetic acid (iaa) in plants (davies 1995). 4. conclusions the effects of 3-benzyl-2-[(6-r-4-oxochromen-3-yl)ethenyl]benzothiazolium bromides 4a – 4g and 3-benzyl-2-[(6-r-4-oxochromen-3-yl)ethenyl]benzoxazolium bromide and 5 were tested on the germination and early growth of cucumber and corn seedlings. the tested oxochromene benzothiazole derivatives may be characterized as biologically active substances with auxin and retardant growth promoting activity. they inhibited growth of cucumber (root and hypocotyl) and shoots of corn at the range of 10 – 100 ppm and stimulated at 0.1 – 1 ppm concentrations. the most effective compounds with stimulating activity on cucumber seedlings was 4a (r = h), 4f (r = oh) and the best retardant activity on corn seedlings showed 5 (r = ch3) and 4b (r = ch3). acknowledgement: the authors are grateful for financial support to the slovak research and development agency by way of project no. apvt-20-005204 and to the vega grant agency of slovak ministry of education by way of project no. 1/3584/06. references afsah, s.a., nayer, s.a.: synthesis of some styryl chain substituted benzothiazolium asymmetric cyanines: spectra and antimicrobial studies. asian j. chem., 8, 1996, 419-424. brown, b.t., dalton, l.k.: morphactin–like activity of benzilate esters in arabidopsis thaliana.“ in: d.j. carr (ed.), plant growth substances. springerverlag, berlin-heidelberg-new york. 1970, 318-323. buffa, r., zahradník, p., foltínová, p.: computer aided benzothiazole derivativess. synthesis, structure and biological study of new push-pull conjugated benzothiazolium salts. heterocyclic commun., 7, 2001, 331-336. caujolle, r., baziard-mouysset, g., favrot, j. d., payard, m., loiseau, p. r., amarouch, h., linas, m. d., seguela, j. p., loiseau, p. m.: synthesis, antiparasitic and antifungal activities of arylalkyl and arylvinylthiazolines. eur. j. med. chem., 28, 1993, 29 35. dathe, w., schneider, g., sembdner, g.: endogenous gibberellins and inhibitors in caryopses of rye. phytochemistry, 17, 1978, 963-966. davies, p.j.: plant hormones, physiology, biochemistry and molecular biology. kluwer academic publishers, dordrecht-boston-london 1995, 833 p. el-shaaer, h.m., foltínová, p., lácová, m., chovancová, j., stankovičová, h.: synthesis, antimicrobial activity and bleaching effect of nova biotechnologica 8-1 (2008) 85 some reaction products of 4-oxo-4h-benzopyran-3-carboxaldehydes with aminobenzothiazoles and hydrazides. il farmaco, 53, 1998, 224-232. fargašová, a.: toxicity determination of selected plant growth hormones on germination and root growth of sinapis alba seeds. biologia (bratislava), 49, 1994, 109-112. foltínová, p., magdolen, p. zahradník, p.: inhibition of uv-induced mutagenesis in euglena gracilis by benzothiazole derivatives. pharmazie, 58, 2003, 4. gašparová, r., lácová, m., el-shaaer, h.m., olderová, ž.: synthesis and antimycobacterial activity of some new 3-heterocyclic substituted chromones. farmaco, 52, 1997, 251-253. havel, l., konečná, h., snášelová, v., procházka, s., blažková, j.: regeneration and peroxidases activity in cabbage tissue culture under the influence of benzolinon and auxins. rost. výr., 40, 1994, 783 791. henselová, m., machlicová, a., bujdoš, m.: benzolinone – induced tolerance of phytolacca dioica l. plants to cadmium stress. proceedings of 27th international symposium „industrial toxicology ´07“ stu bratislava, 2007, 174179. chang, i.k., foy, c.l.: rapid growth responses of dwarf corn coleoptile sections to picloram. pestic. biochem. physiol.,19, 1983, 203-209. klecová-šimonová, e., henselová, m., masarovičová, e., zahradník, p.: resistance increasing of vigna radiata and brassica juncea plants to cd-stress after application of benzothiazole derivative. sborník z konference „vliv abiotických a biotických stresorů na vlastnosti rostlin 2008“. vúrv praha, 2008, 234-238. (in slovak). kleštinec, m., gašparová, r., lácová, m.: in preparing kollárová, k., henselová, m., lišková, d.: effect of auxins and plant oligosaccharides on the root formation and elongation growth of mung bean hypocotyls. plant growth reg., 46, 2005, 1-9. kráľová, k., mitterhauszerová, l., halgaš, j.: effect of some benzothiazolium salts on chlorophyl production in chlorella vulgaris. biol. plant., 36, 1994, 477 – 479. kráľová, k., šeršeň, f., gašparová, r., lácová, m.: effect of 3-r2ch22-[2-(6-r1-chromon-3-yl)ethenyl]benzothiazolium halides on photosynthetic processes. chem. pap., 52, 1998, 776 779. krieg, m., billitz, j.: structurally modified trimethyne thiacarbocyanine dyes. effect of n-alkyl substituents on antineoplastic behavior. mol. biochem. pharmacol., 51, 1996, 1461-1467. lácová, m., stankovičová, h., odlerová, ž.: chromonyl-aminosalicylic acid derivatives as possible antimycobacterial agents. farmaco, 50, 1995, 885-888. magdolen, p., zahradník, p., foltínova, p.: synthesis, antimicrobial testing and qsar study of new 2-phenylethenylbenzothiazolium salts substituted by cyclic amines. pharmazie, 55, 2000, 803-810. merrill, g.b.: eupatoriochromene and encecalin, plant-growth regulators from yellow starthistle (centaurea solstitialis l). j. chem. ecol., 15, 1989, 2073-2087. 86 henselová, m. et al. müller, j.l.: indole-3-butyric acid in plant growth and development. plant growth reg., 32, 2000, 219-230. nawrot-modranka, j., nawrot, e., graczyk, j.: in vivo antitumor, in vivo antibacterial activity and alkylating properties of phosphorohydrazine derivatives of coumarin and chromone. eur. j. med. chem., 41, 2006, 1301-1309. šimonová, e., henselová, m., zahradník, p.: benzothiazole derivatives substituted in position 2 as biologically active substances with plant growth regulation activity. plant soil environ., 51, 2005, 496-505. sutoris, v., bajči, p., sekerka, v., halgaš, j.: benzothiazolium salts which increase sugar and chlorophyl contents in plants. chem. pap., 42, 1988, 249 261. sutoris, v., gáplovský, a., sekerka, v.: benzothiazole compounds. xlvi. 3,4-disubstitued 2-benzothiazolinones with plant growth regulation activity. chem. pap., 47, 1993, 260-263. wang, s., taketa, s., ichii, m., xu, l., xia, k., zhou, x.: lateral root formation in rice (oryza sativa l.): differential effects of indole-3-acetic acid and indole 3-butyric acid. plant growth reg., 41, 2003, 41-47. york, w.s., darvill, a.g., albersheim, p.: inhibition of 2,4dichlorophenoxyacetic acid-stimulated elongation of pea stem segments by a xyloglucan oligosaccharide. plant physiol., 75, 1984, 295-297. microsoft word chmelova nbc 12-2.doc nova biotechnologica et chimica 12-2 (2013) 120 doi 10.2478/nbec-2013-0014 © university of ss. cyril and methodius in trnava repeated-batch production of laccase by ceriporiopsis subvermispora daniela chmelová1, miroslav ondrejovič2 1department of biochemistry and biotechnology, faculty of biotechnology and food sciences, slovak university of agriculture in nitra, tr. a. hlinku 2, 949 76 nitra, slovak republic (daniela.chmelova@gmail.com) 2department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, 917 01 trnava, slovak republic abstract: the aim of this study was to set parameters of repeated-batch cultivation of ceriporiopsis subvermispora for laccase production and evaluate the efficiency of this type of cultivation for production of selected enzyme. the suitable conditions for repeated-batch cultivation were designed on the base of study of batch cultivation of white-rot fungus c. subvermispora. c. subvermispora was cultivated in media with different concentration of casein hydrolysate as nitrogen source and glucose as carbon source. a suitable concentration of casein hydrolysate to stimulate the laccase production was 1.5 and 2.5 g/l. laccase production was started at certain critical concentration of glucose (5 g/l). in order to improve laccase production by repeated-batch cultivation of c. subvermispora, glucose was tested in concentration 10 g/l and casein hydrolysate in concentration 1.5 g/l. during a repeated-batch cultivation was measured increase laccase activities from 177.8 to 266 u/l. it was also observed, the cultivation time needed to reach maximum laccase production was shortened to 10 days. keywords: ceriporiopsis subvermispora, laccases, repeated-batch cultivation. 1. introduction laccase is key enzyme participating in the breakdown of lignin during degradation lignocellulose in the nature. in comparison with other ligninolytic enzymes, laccase has wide substrate specificity for oxidation of various phenolic compounds, are more stable and not require the presence of cofactors (hammel and cullen, 2008). therefore, laccase founds more applications in various sectors. laccase activity is essential for biodegradation of organic pollutants with phenyl ring such as polyamines, aminophenols, aryldiamines, polycyclic aromatic hydrocarbons (pozdnyakova et al., 2004), organophosphorus pesticides and synthetic dyes (torres et al., 2003). laccase can be produced by various organisms, but significant producers of this enzyme are white-rot fungi such as ceriporiopsis subvermispora (rűttimann et al., 1992). laccase production by c. subvermispora is affected by various factors such as temperature, ph, medium composition and presence of elicitors in cultivation medium (chmelová et al., 2011). because laccase production starts in negative environmental conditions, parameters of fermentation process designed for laccase production are essential for achievement of sufficient yields. significant factor influenced the production of laccase is an availability of carbon and nitrogen sources. although easily utilizable carbon or nitrogen source allow an increase in laccase production, its production occurs only after exhaustion of the source (galhaup et bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:16 utc nova biotechnologica et chimica 12-2 (2013) 121 al., 2002; thiruchelvam and ramsay, 2007). in the literature, laccase was produced mostly by batch fermentation. this process does not allow sufficient using of sources for laccase production because most sources are used for biomass production. repeated-batch cultivation was reported as a promising method for high laccase production (birhanli and yesilada, 2010). fungal biomass can be reused for a long time in this cultivation (birhanli et al., 2013). the present study was carried out to investigate the laccase production by white-rot fungus c. subvermispora in batch cultivation media with different concentration of nitrogen or carbon sources, then the parameter selection of repeated-batch cultivation of c. subvermispora for laccase production and the evaluation of its effectiveness compared to batch cultivation. 2. materials and methods 2.1 chemicals 3,5-dinitrosalicylic acid 98 % (acros organics, usa), malt agar (biomark, india), k2hpo4. 12 h2o p.a., kh2po4 p.a., nacl p.a., cacl2. 2 h2o p.a., zncl2 p.a., glucose p.a., feso4. 7 h2o p.a., mgso4. 7 h2o p.a., cuso4. 5 h2o p.a., casein hydrolysate, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) 98 % (sigma aldrich, g), mnso4. 4 h2o p.a. (slavus, sk). 2.2 microorganism culture of ceriporiopsis subvermispora attc 90467 was provided from the centraalburea voor schimmelcultures (netherlands). the culture was maintained on malt agar and stored at 4 °c. in all cases, the suspension of fungal mycelium was prepared by scraping of plaque (1 cm2) of the growth culture from agar plate using microbiological loop in sterile deionized water (10 ml). 2.3 medium composition for batch cultivation, 50 ml of basic mineral medium (table 1) containing glucose as carbon source (5, 10, 25, 50, 75 and 100 g/l) and casein hydrolysate as nitrogen source (0.5, 1.5, 2.5 and 5.0 g/l) was inoculated with 5 ml of fungal mycelium suspension. inoculated cultivation medium was shaken (min. 200 rpm) at 30 °c. table 1. composition of basic mineral medium (aguiar et al., 2006). components of medium concentration mgso4. 7 h2o 0.5 g/l nacl 0.1 g/l cacl2. 2 h2o 0.1 g/l cuso4. 5 h2o 0.1 mg/l feso4. 7 h2o 0.2 mg/l mnso4. 4 h2o 0.02 mg/l zncl2 0.15 mg/l bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:16 utc 122 chmelová, d. and ondrejovič, m. for repeated-batch cultivation, 200 ml of basic mineral medium (table 1) containing glucose (10 g/l) and casein hydrolysate (1.5 g/l) was inoculated with 20 ml of fungal mycelium suspension. inoculated cultivation medium was maintained shaken (min. 200 rpm) at 30 °c. after achieving of maximal laccase activity, cultivation medium was replaced by fresh cultivation medium and biomass in fresh cultivation medium was cultivated at 30 °c with shaking (min. 200 rpm). replacement of cultivation medium was repeated two times. 2.4 concentration of glucose residual glucose was determined in cultivation media using dns (3,5dinitrosalicylic acid) method (miller, 1959). 0.8 ml of dns reagent was added to 0.1 ml of supernatant. reaction mixture was incubated in boiling water batch for 5 minutes. the mixture was cooled down to room temperature and 8 ml of distilled water was added. the absorbance of the reaction mixture (200 µl) was measured at 540 nm using a microplate reader (biotek el 800, fisher, ge). 2.5 activity of laccase activity of laccase was determined with abts (2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic acid) as the substrate. the assay mixture contained 150 µl of 50 mmol/l phosphate buffer (ph 5.0) with 1 mm abts and 50 µl of enzyme extracts. oxidation of abts was monitored by measuring of absorbance at 405 nm (shin et al., 1987). activity of laccase was expressed in unit (u) as the amount of enzymes able to convert 1 mg of abts per minute. 3. results and discussion composition and availability of nutrients play a significant role in laccase production (galhaup et al., 2002). production of laccase is a part of secondary metabolism of white-rot fungi (howard et al., 2003). secondary metabolism starts in response to negative environmental conditions as a lack of nutrients (carbon or nitrogen sources), the presence of specific organic compounds (catechol, syringaldazine, 2,5-xylidine, veratrylalcohol) (chmelova et al., 2011) or growth conditions (ph, temperature, pressure, ionic strength) (howard et al., 2003). in our study, we tested laccase production ability of white-rot fungus c. subvermispora under various concentrations of carbon and nitrogen sources. c. subvermispora was cultivated in medium intended for the production of biomass (aguiar et al., 2006). cultivation medium was composed from basic mineral medium (table 1), carbon source and nitrogen source. glucose was selected as a readily consumed carbon source, because it is a cheap and easily available substrate (galhaup et al., 2002). for efficient laccase production by fungi, the selection of suitable nitrogen source is essential (galhaup et al., 2002; thiruchelvam and ramsay, 2007). the organic nitrogen substrates such as peptone, casein hydrolysate or malt-extract supported better fungal biomass production and enzyme activities as bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:16 utc nova biotechnologica et chimica 12-2 (2013) 123 compared to the inorganic nitrogen substrates (levin et al., 2008; chmelová et al., 2011). therefore, casein hydrolysate was used as nitrogen source. for determination of effect of carbon and nitrogen sources on laccase production, c. subvermispora was cultivated in medium with glucose (10 g/l) and variable concentrations of casein hydrolysate (0.5, 1.5, 2.5 and 5.0 g/l). during cultivation, glucose concentration as indicator of metabolic activity and laccase activity were determined (fig. 1). fig. 1. concentration of glucose and activity of laccase during growth of white-rot fungus c. subvermispora in cultivation medium with glucose as carbon source (10 g/l) and casein hydrolysate as nitrogen source in a concentration a – 0.5 g/l, b – 1.5 g/l, c – 2.5 g/l and d – 5.0 g/l; ♦ concentration of glucose and ■ – activity of laccase. from fig. 1, laccase activity in cultivation media with different concentrations of casein hydrolysate varied considerably (from 21.1 to 169 u/l). the highest laccase activity (169 u/l) was determined in medium with 2.5 g/l of casein hydrolysate (fig. 1-c). in medium with higher concentration of casein hydrolysate (5.0 g/l), laccase bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:16 utc 124 chmelová, d. and ondrejovič, m. production was lower (fig. 1-d). time for maximum laccase production increased with increasing of casein hydrolysate concentration. laccase production achieved maximum enzyme activity in 13th (159 u/l), 17th (169 u/l) and 19th (62 u/l) day of cultivation at casein hydrolysate concentration 1.5, 2.5 and 5 g/l, respectively. production of laccase was observed when a glucose concentration in the medium declined to critical level (approximately 5 g/l) with the exception of the medium with the lowest concentration of casein hydrolysate (0.5 g/l). in this medium, the laccase activity was achieved the maximum after 16th day of cultivation, while the glucose concentration did not decrease to critical level (5 g/l) and maximum laccase activity was only 21 u/l. low casein concentration led to deficient laccase production. in this case, laccase production was started probably due to lack of nitrogen not carbon source. similar results were described in other studies (galhaup et al., 2002; levin et al., 2010), when laccase activity was mainly detectable in the phase when glucose in culture medium was almost exhausted. these results suggest that suitable a carbon:nitrogen ratio for an effective laccase production is 6.7:1. carbon:nitrogen ratio depend on fungal species, for example phanerochaete flavido-alba requires low carbon:nitrogen ratio (pérez et al., 1996) and pycnoporus cinnabarinus high c/n ratio (eggert et al., 1996). the effect of carbon source concentration on laccase production was studied in media with variable glucose concentrations (5; 10; 25; 50; 75 and 100 g/l) and with maintained carbon:nitrogen ratio (6.7:1). during cultivation of c. subvermispora, glucose concentration and laccase activity were determined. results are shown in fig. 2. activity of laccase was increased with increasing glucose concentration until a glucose concentration 50 g/l. activity of laccase reached a maximum at 15th (50.6 u/l), 12th (221 u/l), 26th (239.1 u/l) and 35th (352 u/l) days in cultivation media with glucose concentration 5, 10, 25 and 50 g/l, respectively (fig. 2a-d). laccase activity was not measured throughout the cultivation in media with 75 and 100 g/l of glucose (fig. 2e,f). this could be caused by an excessive concentration of glucose which prevents the start of secondary metabolism of c. subvermispora (lee et al., 2004). in these media, it was not observed decreasing of glucose concentration under critical level neither after 50 days. this could be due to the exhaustion of nitrogen source and stopping the growth of white-rot fungus c. subvermispora. we found that, although laccase production increased with increasing glucose also cultivation time required to maximum laccase production was extended. therefore, in repeated-batch process, cultivation media were composed of glucose in concentration 10 g/l and casein hydrolysate in concentration 1.5 g/l. repeated-batch cultivation represents a potential alternative mode of cultivation, in which medium or some part of the medium is drawn and fresh medium is refilled periodically without changing fungal biomass (wesenberg et al., 2003). this process allows the use of fungal biomass repeatedly and achieves better results, compared with batch cultivation (birhanli and yesilada, 2006). the best selected conditions were used for the following repeated-batch cultivation of fugal biomass. in order to improve laccase production by c. subvermispora, glucose was tested as a carbon source (10 g/l) and casein hydrolysate as nitrogen source (1.5 g/l). this medium was added to biomass of c. subvermispora. during cultivation was bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:16 utc nova biotechnologica et chimica 12-2 (2013) 125 determined glucose concentration as indicator of metabolic activity and laccase activity. after achieving of maximal laccase activity, the cultivation medium was replace by fresh medium with starting concentration of glucose and casein hydrolysate. the results of repeated-batch cultivation of c. subvermispora are shown in fig. 3. fig. 2. concentration of glucose and activity of laccase during growth of white-rot fungus c. subvermispora in cultivation medium with casein hydrolysate as nitrogen source and glucose as carbon source in carbon:nitrogen ratio (6.7:1; w/w) with glucose concentration a – 5 g/l, b – 10 g/l, c – 25 g/l, d – 50 g/l; e – 75 g/l and f – 100 g/l; ♦ concentration of glucose; ■ – activity of laccase. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:16 utc 126 chmelová, d. and ondrejovič, m. fig. 3. repeated-batch cultivation of c. subvermispora in cultivation medium with 10 g/l of glucose and 1.5 g/l of casein hydrolysate at 30 °c and ph 5.0; ♦ concentration of glucose and ■ – activity of laccase. from fig. 3, the laccase production can be periodically increased during long-term cultivation by periodical replacing the cultivation medium for medium with started concentration of glucose and casein hydrolysate. maximum laccase activity obtained by repeated-batch cultures of c. subvermispora was 177.8 u/l during first cultivation phase after 15 days, 233.5 u/l during second cultivation phase after 13 days and 266 u/l during third cultivation phase after 10 days. it was also observed, the cultivation time needed to reach maximum laccase production during repeated-batch cultivation was shortened and maximum laccase activity was increased. the advantage of this process is the reuse of fungal biomass for laccase production. similar results were observed in other studies (birhanli and yesilada, 2006; birhanli and yesilada, 2010). 4. conclusions the stimulatory effect of some components of cultivation medium on laccase production during batch cultivation was described. high amount of laccase could be obtained with repeated-batch cultivation by selecting the most appropriate culture conditions for laccase production (glucose concentration 10 g/l, casein hydrolysate concentration 1.5 g/l). our results show that repeated-batch cultivation is an easy and suitable process for high amount of laccase without a need for a new fungal biomass. acknowledgement: this work was supported by the grant no. fppv-08-2012. references aquiar, a., souza-cruz, p. b., ferraz, a.: oxalic acid, fe3+ reduction activity and oxidative enzymes detected in culture extracts recovered from pinus bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:16 utc nova biotechnologica et chimica 12-2 (2013) 127 taeda wood chips biotreated by ceriporiopsis subvermispora. enzyme microb. tech., 38, 2006, 873-878. birhanli, e., yesilada, o.: increased production of laccase by pellets of funalia trogii atcc 2008000 and trametes versicolor atcc 200801 in repeatedbatch mode. enzyme microb. tech., 39, 2006, 1286-1293. birhanli, e., yesilada, o.: enhanced production of laccase in repeated-batch cultures of funalia trogii and trametes versicolor. biochem. eng. j., 52, 2010, 3337. chmelová, d., ondrejovič, m., ondáš, v., šturdík, e.: influence of cultivation conditions on production of lignocellulolytic enzymes by ceriporiopsis subvermispora. biologia, 66, 2011, 748-754. eggert, c., temp, u., eriksson, k. e.: the ligninolytic system of the white rot fungus pycnoporus cinnabarinus: purification and characterization of the laccase. appl. environ. microbiol., 62, 1996, 1151-1158. ferraz, a., córdova, a. m., machuca, a.: wood biodegradation and enzyme production by ceriporiopsis subvermispora during solid-state fermentation of eucalyptus grandis. enzyme microb. tech., 32, 2003, 59-65. galhaup, c., wagner, h., hinterstoisser, b., haltrich, d.: increased production of laccase by the wood-degrading basidiomycete trametes pubescens. enzyme microb. tech., 30, 2002, 529-536. hammel, k. e., cullen, d.: role of fungal peroxidases in biological ligninolysis. curr. opin. plant biol., 11, 2008, 349-355. howard, r. l., abotsi, e., jansen van rensburg, e. l., howard. s.: lignocellulose biotechnology: issues of bioconversion and enzyme production. afr. j. biotechnol., 2, 2003, 602-619. kaal, e. e. j., field, j. a., joyce, t. w.: increasing ligninolytic enzyme activities in several white-rot basidiomycetes by nitrogen-sufficient media. bioresour. technol., 53, 1995, 133-139. lee, k. h., wi, s. g., singh, a. p., kim, y. s.: micromorphological characteristics of decayed wood and laccase produced by the brown-rot fungus coniophora puteana. j. wood sci., 50, 2004, 281-284. levin, l., herrmann, c., papinutti, v. l.: optimization of lignocellulolytic enzyme production by the white-rot fungus trametes trogii in solid-state fermentation using response surface methodology. biochem. eng. j., 39, 2008, 207-214. levin, l., melignani, e., ramos, a. m.: effect of nitrogen sources and vitamins on lignolytic enzyme production by some white-rot fungi. dye decolorization by selected culture filtrates. bioresour. technol., 101, 2010, 45544563. miller, g. l.: use of dinitrosalicylic reagent for the determination of reducing sugar. anal. chem., 31, 1959, 426-428. pérez, j., martínez, j., la rubia, t.: purification and partial characterization of a laccase from white rot fungus phanerochaete flavido-alba. appl. environ. microbiol., 62, 1996, 4263-4267. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:16 utc 128 chmelová, d. and ondrejovič, m. pozdnyakova, n. n., rodakiewicz-nowak, j., turkovskaya, o. v.: catalytic properties of yellow laccase from pleurotus ostreatus d1. j. mol. catal. b. enzym., 30, 2004, 19-24. rüttimann, c., schwember, e., salas, l., cullen, d., vicuna, r.: ligninolytic enzymes of the white rot basidiomycetes phlebia brevispora and ceriporiopsis subvermispora. biotechnol. appl. biochem., 16,1992, 64-76. shin, t., murao, s., matsumura, e.: a chromogenic oxidative coupling reaction of laccase: application for laccase and angiotensin i converting enzyme assay. anal. biochem., 166, 1987, 380-388. thiruchelvam, a. t., ramsay, j. a.: growth and laccase production kinetics of trametes versicolor in a stirred tank reactor. appl. environ. microbiol., 74, 2007, 547-554. torres, e., bustos-jaimes, i., le borgne, s.: potential use of oxidative enzymes for the detoxification of organic pollutants. appl. catal., b., 46, 2003, 115. wesenberg, d., kyriakides, i., agathos, n.: white-rot fungi and their enzymes for the treatment of industrial dye effluents. biotechnol. adv., 22, 2003, 161-187. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:16 utc microsoft word remenarova nb 9-3.doc nova biotechnologica 9-3 (2009) 239 biosorption of cationic dyes by1, by2 and bg4 by moss rhytidiadelphus squarrosus from binary solutions lucia remenárová1, martin pipíška1,2, miroslav horník1,2, jozef augustín1,2 1department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (pipiskam@ucm.sk) 2consortium for environmental biotechnology and environmental chemistry, hlavná 418, špačince, sk-919 51, slovak republic abstract: a biosorbent prepared from moss rhytidiadelphus squarrosus biomass was used for biosorption of cationic dyes – malachite green (bg4), auramine o (by2) and thioflavine t (by1) from binary aqueous solutions. sorption data obtained at non-equilibrium conditions were analyzed by sheindorfrebuhn-sheintuch (srs) equation (competitive model for binary systems derived from freundlich isotherm) and extended model of freundlich isotherm. following the comparison of coefficient of determination values (r2) as well as values of root mean squared error (rmse), the extended model of freundlich isotherm was more suitable for description of investigated binary systems bg4-by1 (r2bg4 = 0.983, r2by1 = 0.993) and bg4-by2 (r2bg4 = 0.976, r2by2 = 0.995). the competition coefficients aij, obtained from srs model can be considered as a way to quantify mutual competitive interactions. the competition coefficients indicated that the presence of by1 in binary system decreased the sorption of bg4 (aby1,bg4 = 0.835) while presence of bg4 (abg4,by1 = 0.208) has less pronounced competitive effect on the sorption of by1 onto biosorbent. competition coefficients obtained for binary system by1-bg4 indicate that bg4 (abg4,by2 = 0.186) was more significantly affected by the presence of by2 (aby2,bg4 = 1.167). finally, equations used in this work were represented by the three-dimensional biosorption isotherm surfaces. key words: cationic dyes, r. squarrosus, binary biosorption, equilibrium, isotherms 1. introduction pollution of water sources by many organic contaminants over the past decades has become an issue of growing importance. biosorption has become a well-established separation technique to remove diluted pollutants, offering the potential for regeneration, recovery and recycling of the adsorbed materials (mckay et al., 2004). most of the research is engaged in biosorption of cationic dyes in single systems, but as wastewater from the dye manufacturing and textile finishing contains usually mixture of dye components, bigger accent on multicomponent adsorption systems should be put. when more than one species is present in the sorption system, the evaluation, interpretation and representation of sorption data become much more complicated (see e.g. lee et al., 2004). but to provide additional information on the nature of biosortpion process, such as the fraction of adsorption sites being shared with each species, their relative affinities toward these sites, and the mutual interaction between the adsorbed species, is necessary to investigate the simultaneous sorption of two or more species (aksu et al., 2009). 240 remenárová, l. et al. our previous study concerned with the sorption of cationic dyes thioflavine t (by1), auramine o (by2) and malachite green (bg4) from single aqueous solutions onto moss biosorbent and freundlich and langmuir isotherm models were used to describe the equilibrium characteristics of monocomponent adsorption. the freundlich isotherm was found to represent the measured sorption data of bg4, by1 and by2 well (remenárová et al., 2009). within this context, the aim of our work was to study the sorption of cationic dyes from binary aqueous solutions by moss rhytidiadelphus squarrosus. on the basis of the results obtained from single systems, sheindorf-rebuhn-sheintuch (srs) equation and extended model of freundlich isotherm were chosen for analyzing equilibrium data and describing competitive effect of bg4, by1 and by2 in binary systems. 2. materials and methods 2.1 biosorbent preparation a biomass of moss r. squarrosus used in this work was collected in high tatras mountains, slovak republic. to remove the impurities, the biomass was washed twice in deionized water, then oven-dried for 72 h at a maximum of 45°c to avoid the degradation of binding sites. after drying, the moss biomass was milled and sieved for particle size <300 µm. 2.2 dyes cationic dyes malachite green (bg4, basic green 4 – malachite green oxalate, mr 927, c.i. 42 000, merck, d); auramine o (by2, basic yellow 2, mr 304, c.i. 41 000, aldrich, d) and thioflavine t (by1, basic yellow 1, mr 319, c.i. 49 005, fluka, d) were used in sorption experiments (fig. 1). the stock solutions of dyes were prepared in deionized water. working solutions of desired concentrations were obtained by successive dilutions. fig. 1. structure of the basic dyes: (a) malachite green, (b) auramine o and (c) thioflavine t. c a b nova biotechnologica 9-3 (2009) 241 2.3 batch equilibrium sorption experiments in binary systems batch biosorption experiments in binary systems bg4-by1 and bg4-by2 were carried out in series of solutions containing both dyes at initial concentrations within the range from 25 to 300 mg/l in the concentration ratio 2:1, 1:1 or 1:2. the ph was adjusted to 4.0 with 0.1 m hcl. biomass (1 g/l, dw) was added, and the mixture in flasks was agitated on a reciprocal shaker (120 rpm) for 4 h at 25°c. the dye concentrations (ceqbg4, ceqby1, ceqby2) in the solutions were determined spectrophotometrically (uv-vis spectrophotometer varian cary 50). all experiments were performed in duplicate. 2.4 equilibrium modeling for description of sorption equilibrium in binary systems sheindorf-rebuhnsheintuch (srs) equation (competitive model derived from freundlich isotherm) and extended model of freundlich isotherm were used. to calculate the corresponding parameters of adsorption isotherms, non-linear regression analysis was performed by the nlsf (origin´s nonlinear least square fitter) using program microcal origin 8.0 professional (originlab corporation, northampton, usa) with automatic initialization of parameters and graphpad prism 5.0 (graphpad software inc., usa). the 3d sorption surfaces for each binary system were obtained by plotting the experimental dye equilibrium concentrations ceq on the x and y axes, against the dye uptake qeq on the z axis. the tablecurve 3d 4.0 (systat software, inc., chicago, usa) software was used for this purpose. the coefficient of determination (r2) and root mean squared error (rmse) were used to assess the goodness of the fit. 2.5 scanning electron microscopy (sem) the surface structure of moss biosorbent was analyzing by scanning electron microscopy (sem) using vega tescan microscope and software quantax qx2 (version 1.6). 3. results and discussion moss biomass, washed with water, dried at 45°c and milled, represents easily fractionable, well sedimenting material with a large specific surface area, suitable for biosorption of dissolved solutes. the surface of the particles 300 – 600 μm fraction is shown in fig. 2a and 2b. biosorption in multicomponent systems is rather complicated due to the possible interactions among the solutes. the behavior of each species in a multicomponent system depends strongly on the number and properties of other species present. also the solution ph, the physical and chemical properties of both the sorbent and sorbate significantly influenced the sorption process (aksu and akpinar, 2000). 242 remenárová, l. et al. fig. 2. sem micrographs of biosorbent prepared from moss r. squarrosus. a) magnification 500×, b) magnification 2500×. sorption of dyes by r. squarrosus from binary system bg4-by1 and bg4-by2 at ph 4.0 is depicted in fig. 3 and fig. 4. 0 50 100 150 200 250 0 50 100 150 200 250 300 350 400 q eq b g 4 (μ m ol /g ) c eq bg4 (μmol/l) molar ratio by1 : bg4 0 : 1 2 : 1 3 : 1 5 : 1 0 100 200 300 400 500 600 700 0 50 100 150 200 250 300 350 molar ratio by1:bg4 1 : 0 1 : 2 1 : 3 1 : 5 q eq b y 1 (μ m ol /g ) c eq by1 (μmol/l) fig. 3. isotherms of the bg4 (a) and by1 (b) biosorption by r. squarrosus (1.0 g/l, d.w.) at 25°c and ph 4.0 from binary system at different initial molar [bg4]:[by1] ratios. it is evident that sorption of dyes increased with the increasing of dyes concentration in solution. sorption of bg4 from binary systems bg4-by1 and bg4by2 decreased with increasing concentration of by1 (fig. 3a) and by2 (fig. 4b) in solution. on the contrary, the presence of bg4 caused less pronounced decrease of both by1 (fig. 3b) and by2 (fig. 4a) sorption. this can be mainly attributed to the interactions between the basic dyes molecules on the biosorbent surface as reported turabik (2008). based on the literature review it is known that the most appropriate form to describe sorption equilibrium in binary systems is to adjust the experimental data to a b b a nova biotechnologica 9-3 (2009) 243 a mathematical model from which number of parameters can be obtained for quantitative interpretation of sorption equilibrium uptake (see e.g. srivastava et al., 2009; romera et al., 2008, turabik, 2008; teng et al., 2009). in our previous study we determined that the sorption of dyes bg4, by1 and by2 from single systems by r. squarrosus was well described by freundlich isotherm. therefore we used the extended freundlich model (fritz and schluender, 1974) and sheindorf-rebuhn-sheintuch (srs) equation (sheindorf et al., 1981) (competitive model for binary systems derived from freundlich isotherm) for description of equilibrium in binary systems. parameters of freundlich isotherm ki and ni are known from single system (remenárová et al., 2009). 0 200 400 600 800 1000 1200 0 50 100 150 200 250 300 350 400 q eq b y 2 (μ m ol /g ) c eq by2 (μmol/l) molar ratio by2:bg4 1 : 0 2 : 1 3 : 1 6 : 1 0 50 100 150 200 250 0 50 100 150 200 250 300 350 400 q eq b g 4 (μ m ol /g ) c eq bg4 (μmol/l) molar ratio by2 : bg4 0 : 1 2 : 1 3 : 1 6 : 1 fig. 4. isotherms of the by2 (a) and bg4 (b) biosorption by r. squarrosus (1.0 g/l, d.w.) at 25°c and ph 4.0 from binary system at different initial molar [bg4]:[by2] ratios. in 1974 fritz and schluender have discussed the extended form of the monocomponent freundlich isotherm for defining sorption from the binary mixtures which can be written as follows: 11 11 2,11, )/1( 1,1 1, z eq x eq xn eq eq cyc ck q + = + (1) 22 22 1,22, )/1( ,2 2, z eq x eq xn ieq eq cyc ck q + = + (2) where k1, k2, n1 and n2 are derived with the help of the mono-component isotherm. the rest of the parameters are determined using the non-linear regression analysis. the parameters q1 and q2 represent the amount of sorbed species 1 and 2 at equilibrium. also sheindorf et al. (1981) extended the freundlich isotherm to multi-solute systems to derive a freundlich-type multicomponent adsorption isotherm named sheindorf-rebuhn-sheintuch (srs) equation. the assumptions incorporated in the derivation are (1) each component individually follows the freundlich isotherm and (2) for each component in the multi-component adsorption, an exponential distribution of adsorption energies exists, the distribution which is equivalent to the distribution in the single-component system. b a 244 remenárová, l. et al. the srs equations for component 1 and 2 in binary system are written: 1 2121111 1)( −+= ncacckq (3) 12121222 2)( −+= nccackq (4) where α12 is the competition coefficient for the adsorption of component 1 in the presence of component 2. the values for competition coefficient α12 range from zero (complete lack of competition) to values greater than zero for a high degree of competition. wu et al. (2002) and wei and nakato (2006) laboured and successfully used this model to describe sorption of various contaminants from multisolute systems. both freundlich-type isotherms were used to fit the experimental data. 3-d sorption isotherm surfaces of bg4-by1 binary system predicted by the extended freundlich model and srs equation are shown in fig. 5 and 6. similar 3-d sorption isotherm surfaces of bg4-by2 binary system predicted by the extended freundlich model and srs equation were obtained (not shown). 0 50 100 150 200 ceq bg 4 (µ mo l/l) 010 020 030 040 050 060 0 ceq by1 (µmol/l) 0 0 100 100 200 200 300 300 400 400 q eq b g 4 (µ m ol /g ) q eq b g 4 (µ m ol /g ) 0 100 200 300 400 500 600 ceq by 1 (µ mo l/l) 0 50 100 150 200 ceq bg4 (µmol/l) 0 0 100 100 200 200 300 300 400 400 q eq b y 1 (µ m ol /g ) q eq b y 1 (µ m ol /g ) fig. 5. 3-d sorption isotherm surfaces of bg4-by1 binary system: (a) bg4 sorption (μmol/g), (b) by1 sorption (μmol/g). the surfaces are predicted by the extended freundlich model (eq 1 and 2), the symbols represent experimental data obtained at ph 4.0 and 25°c. the vertical bars represent 95% confidence interval. 0 50 100 150 200 ceq bg 4 (µ mo l/l) 010 020 030 040 050 060 0 ceq by1 (µmol/l) 0 0 100 100 200 200 300 300 400 400 q eq b g 4 (µ m ol /g ) q eq b g 4 (µ m ol /g ) 0 100 200 300 400 500 600 ceq by 1 (µ mo l/l) 0 50 100 150 200 ceq bg4 (µmol/l) 0 0 100 100 200 200 300 300 400 400 q eq b y 1 (µ m ol /g ) q eq b y 1 (µ m ol /g ) fig. 6. 3-d sorption isotherm surfaces of bg4-by1 binary system: (a) bg4 sorption (μmol/g), (b) by1 sorption (μmol/g). the surfaces are predicted by the srs equation (eq 3 and 4), the symbols represent experimental data obtained at ph 4.0 and 25°c. the vertical bars represent 95% confidence interval. a b a b nova biotechnologica 9-3 (2009) 245 parameters specified by the application of freundlich-type models are presented in table 1 and 2. by using of srs model the competition coefficients αij were obtained. higher competition coefficients values were estimated for sorption of by1 (aby1bg4 = 0.835 ± 0.111, table 1) and by2 (aby2bg4 = 1.167 ± 0.139, table 2) in the presence of bg4, indicating that both by1 and by2 affect adsorption of bg4 markedly. on the other hand, values of competition coefficients obtained for sorption of bg4 in the presence of by1 (abg4by1 = 0.208 ± 0.015, table 1) and in the presence of by2 (abg4by2 = 0.186 ± 0.015, table 2) were smaller and indicate that sorption of by1 and by2 would be least affected by competitive interactions of bg4. values of r2 and rmse are regarded as a measure of the goodness of fit of experimental data on the isotherms models. as can be seen from table 1 a 2 high coefficients of determination r2 and small values of rmse were obtained for both used models. this indicates that both models well describe the experimental biosorption data. to determine the model better describing experimental data we compared obtained experimental data (qeq exp) with data calculated from models (qeq cal). the analysis of the obtained scatter diagrams shows that experimental values were in a good agreement with values calculated from the extended freundlich model (diagrams not shown). agreement in case of the srs equation was lower. table 1. equilibrium parameters for bg4 and by1 biosorption from the binary mixtures bg4-by1 by moss r. squarrosus calculated from the extended freundlich model and srs model by non-linear regression analysis. extended freundlich model dye xi yi zi r2 rmse* (μmol/g) by1 0.858 ± 0.179 4.214 ± 1.960 0.304 ± 0.141 0.993 9.76 bg4 1.012 ± 0.137 0.025 ± 0.013 1.292 ± 0.169 0.983 13.22 srs model aby1bg4 abg4by1 by1 0.835 ± 0.111 1 0.972 18.75 bg4 1 0.208 ± 0.015 0.977 14.98 *root mean squared error table 2. equilibrium parameters for bg4 and by2 biosorption from the binary mixtures bg4-by2 by moss r. squarrosus calculated from the extended freundlich model and srs model by non-linear regression analysis. extended freundlich model dye xi yi zi r2 rmse* (μmol/g) by2 0.727 ± 0.095 4.126 ± 1.227 0.221 ± 0.069 0.995 8.94 bg4 1.140 ± 0.150 0.027 ± 0.016 1.340 ± 0.179 0.976 15.76 srs model aby2bg4 abg4by2 by2 1.167 ± 0.139 1 0.971 21.32 bg4 1 0.186 ± 0.015 0.973 16.57 *root mean squared error 246 remenárová, l. et al. 4. conclusions in this work the biosorption of cationic dyes from binary solutions onto moss rhytidiadelphus squarrosus was studied. sorption data obtained from binary systems by1-bg4 and by2-bg4 were analyzed by sheindorf-rebuhn-sheintuch (srs) equation and extended model of freundlich isotherm and presented in the form of 3d sorption isotherm surfaces. based on the r2 and rmse values both isotherms well described sorption of cationic dyes from binary systems. higher competition coefficients values were estimated for sorption of by1 and by2 in the presence of bg4, indicating that both by1 and by2 affect adsorption of bg4 markedly. the influence of bg4 on sorption of by1 and by2 from binary systems was less pronounced. it can be concluded that srs equation and extended freundlich model provide a suitable description of the experimental binary data. prepared moss biosorbent may be used for individual and simultaneous removal of basic dyes from effluents. references aksu, z., ertugrul, s., dönmez, g.: single and binary chromium (vi) and remazol black biosorption properties of phormidium sp. j. hazard. mat., 1168, 2009, 310-318. aksu, z. akpinar, d.: modelling of simultaneous biosorption of phenol and nickel(ii) onto dried aerobic activated sludge. sep. purif. technol., 21, 2000, 8799. fritz, w., schluender, e.u.: simultaneous adsorption equilibria of organic solutes in dilute aqueous solutions on activated carbon. chem. eng. sci., 29, 1974, 1279-1282. lee, h.s., suh, j.h., kim, i.b., yoon, t.: effect of aluminum in two-metal biosorption by an algal biosorbent. minerals eng., 17, 2004, 487-493. mckay, g., allen, s.j., porter, j.f.: adsorption isotherm models for basic dye adsorption by peat in single and binary component systems. j. colloid interface sci., 280, 2004, 322-33. remenárová, l., pipíška, m., horník, m., augustín, j.: sorption of cationic dyes from aqueous solutions by moss rhytidiadeplhus squarrosus: kinetics and equilibrium studies. nova biotechnol., 9, 2009, 53-61. romera, e., gonzález, f., ballester, a., blázquez, m.l., muñoz, j.a.: biosorption of heavy metals by fucus spiralis. bioresour. technol., 99, 2008, 684-4693. sheindorf, ch., rebhun, m., sheintuch, m.: a freundlich-type multicomponent isotherm. j. colloid interface sci., 79, 1981, s.136-142. srivastava, v.ch., mall, i.d., mishra. i.m.: competitive adsorption of cadmium(ii) and nickel(ii) metal ions from aqueous solution onto rice husk ash. chem. eng. process., 48, 2009, 370-379. teng, s.-x., wang, s.-g., liu, x.-w., gong, w.-x., sun, x.-f., cui, j.-j., gao, b.-y.: interaction between congo red and copper in a binary adsorption nova biotechnologica 9-3 (2009) 247 system: spectroscopic and kinetic studies. colloid. surface. a: physicochem. eng. aspects, 340, 2009, 86-92. turabik, m: adsorption of basic dyes from single and binary component systems onto bentonite: simultaneous analysis of basic red 46 and basic yellow 28 by first order derivative spectrophotometric analysis method. j. hazard. mat., 158, 2008, 52-64. wei, q., nakato, t.: competitive adsorption of phenols on organically modified layered hexaniobate k4nb6o17. miscropor. mesopor. mat., 96, 2006, 84-92. wu, ch.-h., kuo, ch.-y., lin, ch.-f., lo, s.-l.: modeling competitive adsorption of molybdate, sulfate, selenate, and selenite using a freundlich-type multicomponent isotherm. chemosphere, 47, 2002, 283-292. microsoft word maresova et al 2010-1.doc nova biotechnologica 10-1 (2010) 53 sorption of co2+, zn2+, cd2+ and cs+ ions by activated sludge of sewage treatment plant jana marešová, miroslav horník, martin pipíška, jozef augustín department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (jana.maresova@ucm.sk) abstract: sludges are byproducts of sewage treatment process. land application of sewage sludge is one of the final steps of waste water treatment, but solubilization of toxic metals restricts this method of sludge disposal. in our paper cobalt, zinc, cadmium and cesium sorption by suspension of non-treated activated sewage sludge (14 g/dm3, dry wt.) from waste water spiked with 60cocl2, 65zncl2, 109cdcl2 or 137cscl were determined in laboratory experiments at 20°c. activated sludge supplied by the municipal sewage treatment plant in zeleneč (trnava region, slovakia) showed high efficiency to sorb co2+, zn2+, cd2+ and cs+ ions from waste water ph 6-7. the process can be characterized by the concentration equilibrium (csolid/cliquid) typical for sorption processes. efficiency of the sorption increased in the order cs < co < zn < cd. metal sorption process was not inhibited by pretreatment of the sludge with 0.2% formaldehyde or thermal inactivation at 60°c, what confirms that the process was not dependent on metabolic activity of the sludge. cobalt, zinc, cadmium and cesium were easily removable from the sludge by washing with diluted hcl, edta or water solutions of the corresponding metal ions, but with low efficiency by deionized water. key words: activated sludge, sorption, cobalt, zinc, cadmium, cesium, radiotracer analysis 1. introduction several physico-chemical methods were used for removal of heavy metals from industrial liquid wastes, such as ion-exchange, chemical precipitation, chemical reduction and adsorption. there are still some problems associated with these methods since these are cost-expensive and they themselves can produce other wastes, which will limit their industrial applications. among the available treatment processes, the application of biological sorbents is the most promising due to the following reasons: requirement of chemicals for the treatment process is reduced, low operation costs, eco-friendly and cost-effective alternative of conventional techniques and high efficiency at low levels of contaminations. sludge of municipal waste water treatment plants are produced in huge amounts and generally represent one of the main problems of european countries (fytili and zabaniotou, 2008). many experimental studies are applied worldwide to determine the extractable trace metals in sludge to assess the bio-available metal fraction and the potential mobility of trace metals from polluted sludge (alvarez et al., 2002; fuentez et al., 2004; iaquinta et al., 2006). however, the activated sludge can be considered also as a biosorbent capable to bind toxic metals from liquid wastes of industrial origin. 54 marešová, j. et al. in our previous papers we described sorption characteristics of co, zn and cd binded by dry biomass of lichen e. prunastri (pipíška et al., 2008) and moss r. squarrosus (pipíška et al., 2010) biomass. the objective of this study was to obtain quantitative data of cobalt, zinc, cadmium and cesium sorption by nontreated activated sludge of the municipal wastewater treatment plant. 2. materials and methods 2.1 sorption and desorption experiments activated sludge supplied by a municipal sewage treatment plant in zeleneč (trnava region, slovakia) was kept in refrigerator at 4°c for 30 d and used for experiments. suspension of activated sludge ph 6.9 pre-concentrated by centrifugation contained 14.0 g/dm3 biomass (dry wt.). in batch experiments, the test tubes with 1 ml of activated sludge suspensions in waste water were spiked with 60cocl2, 65zncl2, 109cdcl2 or 137cscl (0.5-50 mmol/dm3) and shaken at 20°c. in time intervals, the biomass was separated by centrifugation (20 min at 12 000 × g), supernatant was removed and sediment was used for radiometric determination of sorbed metals. in desorption experiments, the biomass after metal sorption was separated (20 min at 12 000 × g) and resuspended by wortexing for 30 min in 1.0 ml deionized water, 0.1 m edta, 0.1 m hcl, 0.5 mm cocl2, 0.5 mm zncl2, 0.5 mm cdcl2 or 0.5 mm cscl. concentration of metals in biomass was estimated by similar way as in the sorption experiments. biomass dry weight was estimated by drying for 24 h at 60 °c. the prediction of co, zn, cd and cs speciation in the solution as a function of ph was performed using the visual minteq (version 2.53) program. 2.2 radiometric analysis the gamma spectrometric assembly using the well type scintillation detector 54bp54/2-x, nai(tl) (scionix, the netherlands) and the data processing software scintivision 32 (ortec, usa) were used for 60co, 65zn, 109cd and 137cs determination in sediment of sludge and supernatant fluids at the energy of γphotons [kev]: 60co1173.24, 65zn – 1115.52, 109cd – 88.04, 137cs – 661.62. counting time 600 s allowed obtaining data with measurement error <2 %, which do not reflect other sources of errors. standardized solutions of 65zncl2 (50 mg/dm 3 zncl2 in 3 g/dm 3 hcl), 60cocl2 (20 mg/dm 3 cocl2 in 3 g/dm 3 hcl), 109cdcl2 (50 mg/dm 3 cdcl2 in 3 g/dm3 hcl), 137cscl (20 mg/dm3 cscl in 3 g/dm3 hcl) were obtained from the czech institute of metrology (prague, czech republic). 3. results and discussion 3.1 metal sorption sorption experiments showed that non-treated activated sludge is able to bind high amounts of cobalt, zinc, cadmium and cesium (fig. 1). at c0 = 50 mmol/dm 3, nova biotechnologica 10-1 (2010) 55 sorptions q (μmol/g; dry wt.) 611, 995, 1076 and 374 for cobalt, zinc, cadmium and cesium respectively, were observed. however, at the initial concentration c0 = 50 mmol/dm3, the system was not saturated, and higher qmax values can be expected. 10 100 1000 10000 100000 0,01 0,1 1 10 100 1000 co zn cd cs q [μ m ol /g ] c0 [μmol/dm 3] fig. 1. cobalt (-), zinc (-o-o-o-), cadmium (-) and cesium (-) uptake q (μmol/g; dry wt.) by non-treated activated sludge in dependence on the initial cocl2, zncl2, cdcl2 and cscl concentration. specific radioactivity in solutions [bq/ml]: 60co – 195; 65zn – 156; 109cd – 192 and 137cs – 172. data after 1h reaction at 20°c. biomass concentration 14.0 g/dm3 (dry wt.). data are the arithmetic mean of three replicates. 3.2 the role of metabolic activity broad consortium of vital aerobic microbial population in activated sludge can participate in metal uptake driven by metabolic processes. on the other hand, dead microbial biomass and polymers such as proteins and polysaccharides can also bind metals on the basis of sorption processes or complexing. experiments showed that treatment of activated sludge by formaldehyde or thermal inactivation at 60°c had minimal effect on cobalt, zinc, cadmium and cesium uptake (fig. 2). it means, that metal uptake is not dependent on metabolic activity and physico-chemical processes play decisive role in cobalt, zinc and cadmium binding by non-treated activated sludge. the sorption of metals is attributed to the bacterial cell wall (fein 2006; vullo et al., 2008) and to the exopolymeric substances (pal and paul, 2008), which contain a large number of negatively charged functional groups such as carboxyl, phosphate and sulphate (wingender et al., 1999). exopolymers can be expected 56 marešová, j. et al. to buffer metal ion concentrations over wide ranges of concentrations and ph (bhaskar and bhosle, 2006). comprehensive data dealing with metal binding by activated sludge can be found in papers (milne et al., 2003; guibaud et al., 2009). 0 10 20 30 co zn cd cs q [μ m ol /g ] fig. 2. uptake of cobalt, zinc, cadmium and cesium q (μmol/g; dry wt.) by non-treated activated sludge ---, formaldehyde-treated sludge (0.5%, 30 min at 20°c, under wortexing) ---, and thermally treated sludge (30 min at 60°c) ---. specific radioactivity in solutions [bq/ml]: 60co – 198; 65zn – 150; 109cd – 167 and 137cs – 162. sorption 1 h under agitation at 20°c. reaction conditions see fig. 1. 3.3 the role of ph acidic ph region is not typical for municipal waste waters as well as activated sludges. it is generally known, that metal cation biosorption is diminished below ph 4 (pipíška et al., 2010). in acidic ph region species of cobalt and zinc exist as me2+ cations, and cesium as me+ cation (fig. 3). in the case of cadmium the main ionic form within ph 4 -8 beside cd2+ is cdcl+ with the ratio approx 60 to 40 (fig. 4). above ph 7 more complex ionic equilibrium takes place, in dependence of the concentration of anion such as chloride, carbonate and phosphate. the concentration of cd(oh)3 – anion is increasing above ph 9. during the sorption of metals by sludges from alkaline liquid wastes besides sorption of me2+ cations, also the existence of other than ionic metal forms with different solubility in water have to be taken into consideration. this fact is in agreement with our observations that sorption efficiency of activated sludge increases above ph 8 (not shown). strong sigmoidal ph dependence of cd2+ sorption by polysaccharides of activated sludge with inflection point at ph 8.6 was described by guibaud et al. (2009). nova biotechnologica 10-1 (2010) 57 3 4 5 6 7 8 9 10 11 12 0 20 40 60 80 100 (m en + / m e t ot al ) x 10 0 ph co2+ zn2+ cd2+ cs+ fig. 3. molar fraction of co2+, zn2+, cd2+ and cs+ in dependence on ph of synthetic waste water (iaquinta et al., 2006) at 25°c and c0 1.0 mmol/dm3. calculated by speciation program visual minteq ver. 2.53. 3 4 5 6 7 8 9 10 11 12 0 10 20 30 60 70 80 cdcl+ cdcl 2 cdso 4 cd(so4) 2 2 cdoh+ cd(oh) 2 cd 2 oh3+ cd(oh) 3 (m ex / m e t ot al ) x 10 0 ph fig. 4. speciation forms of cadmium in dependence on ph of synthetic waste water (iaquinta et al., 2006) at 25°c and c0 1.0 mmol//dm3. calculated by speciation program visual minteq ver. 2.53. 3.4 desorption high percentage of co, zn, cd and cs can be desorbed from the sludge by one step washing with cocl2, zncl2, cdcl2 and cscl solutions respectively, as well as with edta and hcl solutions (tab. 1; fig. 5b). at the same time only 6, 2 and 7 per cent of sorbed cobalt, zinc and cadmium, respectively, can be solubilized by washing the activated sludge with deionized water. it means that for releasing of the above mentioned metals certain, ionic force is necessary for replacing metal ions from binding sites. cesium was easily removable from the sludge even with deionized water. generally, cs+ ions show the highest mobility in biological systems resembling behavior of k+ ions. cadmium showed the highest affinity to the sludge and the lowest extractability with mineral acids and salt solutions (fig. 5a, b). the explanation of this 58 marešová, j. et al. phenomenon will require a more detailed study oriented toward speciation of cadmium in individual components of the sludge flocks. table 1. removable portion of metals from activated sludge by single step washing of sludge with 0.1 m hcl, 0.1 m edta or 0.5 mm cocl2, zncl2, cdcl2 or cscl. specific radioactivity in solutions [bq/ml]: 60co – 205; 65zn – 158; 109cd – 160 and 137cs – 146. calculations based on volume radioactivity of supernatant washing liquid. parameter co zn cd cs sorbed q (μmol/g; dry wt.) * 26±0.7 30±0.9 31±1.1 11±0.2 removed with water [%] 6 2 7 36 removed with 0.5 mm salt [%] 46 50 15 72 removed with 0.1 m hcl [%] 81 91 30 83 removed with 0.1 m edta [%] 90 92 73 81 * biosorption from c0 = 0.5 mmol/dm3 solutions at biomass concentration 14.0 g/ dm3 (dry wt.). 0,01 0,1 1 10 100 1000 10000 q [μ m ol /g ] c 0 [mmol/l] 5,0 50,00,5 0,1 1 10 100 1000 10000 q [μ m ol /g ] c 0 [mmol/l] 5,0 50,00,5 fig. 5a. cobalt (-), zinc (-), cadmium (-) and cesium (-) uptake q (μmol/g; dry wt.) by nontreated activated sludge in dependence on the initial cocl2, zncl2, cdcl2 and cscl concentration. specific radioactivity in solutions [bq/ml]: 60co – 195; 65zn – 156; 109cd – 192 and 137cs – 172. data after 1h reaction at 20 °c. biomass concentration 14.0 g/dm3 (dry wt.). fig. 5b. remaining metal uptake after 30 min washing of the sludge with cocl2, zn cl2, cd cl2 or cscl solution. data are the arithmetic mean of three replicates. according to hsiau and lo (1998), sequential extraction revealed that the percentages of the heavy metals of organically bound form and exchangeable form in chemically fixed sludge samples were in the order of cu > pb > cr > zn. however, mobility of cesium can be diminished by supplementing of activated sludge with clay materials or bentonites. in that case, cesium released from activated sludge will be bound irreversibly on clay minerals and will not be bio-available or leachable under natural conditions and even under more drastic conditions such as with alkali or acid solutions. clays (dyer et al., 2000) and bentonites (see e.g. a b nova biotechnologica 10-1 (2010) 59 galamboš et al., 2010; závodská and lesný, 2006) are well known sorbents for irreversible cesium binding, as well as for binding many heavy metals and metalloids. activated sludge disposes much higher capacity for metal sorption calculated for unit biomass, than the content of metals contained in municipal waste waters. it means that the sludge biomass is not saturated and can be used as sorbent for reversibly sorption of the metals from other liquid wastes. table 2. sorption capacities (mg/g, dry wt.) of naturally occurring sorbents. according to bailey et al. (1999), modified. sorbent cs co zn cd pb hg bark 32 182 400 modified cotton 1000 chitin 100 chitosan 558 796 1123 clay 16.5 58 microbial biomass 28 116 lignin 95 1865 150 modified wool 87 135 632 moss 46.7 peat 5.1 230 16.2 seaweed 215 344 33.3 18 1.2 xanthate zeolite 84.3 155.4 150.4 lichen biomass (1) 5.7 7.3 moss biomass (2) 7.2 12.2 19.4 green algae (3) 14.5 activated sludge(4) 24.5 activated sludge (5) 50 36 65 121 (1) pipíška et al. 2008; (2) pipíška et al., 2010; (3) horník et al., 2008; (4) choi and yun, 2006; (5) this paper. in tab. 2, the sorption capacity (q) values of activated sludge obtained in this paper are compared with q values of other sorbents published. as we can see, activated sludge showed the sorption capacity comparable with other low-cost sorbents and therefore the sludge could be used for lowering concentration of toxic metals originating from industrial effluents, subsequently dewatered or incinerated. 4. conclusions the non-treated activated sludge from aerobic phase of municipal waste water treatment plant contains the whole spectrum of metals. however, the sorption capacity of the sludge is much higher than actual concentration of heavy metals in treated waste waters. sludge can be used as an efficient sorbent for the removal of heavy metals from industrial liquid wastes. sorption capacity of activated sludge studied for co, cd, 60 marešová, j. et al. zn and cs from 0.5 mmol/dm3 solution was 36, 65, 121 and 50 mg/g (dry wt.), respectively. the ability of non-treated activated sludge to sorb co2+, zn2+, cd2+ and cs+ metal ions is based mainly on physical processes not dependent on the metabolic activity of the sludge microflora. recovery of the metals by washing with diluted hcl, edta and salt solutions decreases in the order: cs > co = zn > cd. the obtained data stress the potential of surplus production of activated sludge as a sorbent for binding of toxic and radiotoxic metals and metaloides from liquid industrial wastes. the next treatment of the sludge will depend on the concentration of sorbed metals and their toxicity. incineration will be one of the ways for final treatment. acknowledgement: we would like to thank juraj miština, m.a. for proofreading. department of english language, faculty of natural sciences, university of ss. cyril and methodius in trnava, slovak republic. references alvarez, e.a., mochon, m.c., sanchez, j.j., rodriguez, m.t.: heavy metal extractable forms in sludge from wastewater treatment plants. chemosphere, 47, 2002, 765-775. bailey, s.e., olin, t.j., bricka, r.m., adrian, d.d.: a review of potentially low-cost sorbents for heavy metals. wat. res., 33, 1999, 2469-2479. bhaskar, p.v., bhosle, n.b.: bacterial extracellular polymeric substance (eps): a carrier of heavy metals in the marine food-chain. environ. int., 32, 2006, 191-198. costley, s.c., wallis, f.m.: bioremediation of heavy metals in a synthetic wastewater using a rotating biological contactor. wat. res., 35, 2001, 3715-3723. choi, s.b., yun, y.s.: biosorption of cadmium by various types of dried sludge: an equilibrium study and investigation of mechanisms. j. hazard. mater. b, 138, 2006, 378–383. dyer, a., chow, j.k.k., umar, i.m.: the uptake of cesium and strontium radioisotopes onto clays. j. mater. chem., 10 (12), 2000, 2734-2740. fein, j.b.: thermodynamic modeling of metal adsorption onto bacterial cell walls: current challenges. adv. agron. 90, 2006, 179-202. fuentes, a., llorens, m., saez, j., aguilar, m., ortuno, f., meseguer, v.f.: phytotoxicity and heavy metals speciation of stabilised sewage sludges. j. hazard. mater., 108, 2004, 161-169. fytili, d., zabaniotou, a.: utilization of sewage sludge in eu. application of old and new methods a review. renew. sust. energ. rev., 12, 2008, 116-140. galamboš, m., kurčáková, j., rosskopfová, o., rajec, p.: adsorption of cesium and strontium on natrified bentonites. j. radioanal. nucl. chem., 2010, (in press). guibaud, g., van hullebusch, e., bordas, f., d´abzac, p., joussein, e.: sorption of cd (ii) and pb (ii) by exopolymeric substances (eps) extracted from activated sludges and pure bacterial strains: modelling of the metal/ligand ratio effect and role of the mineral fraction. bioresource technol., 100, 2009, 2959-2968. nova biotechnologica 10-1 (2010) 61 horník, m., pipíška, m., kočiová, m., augustín, j., lesný, j.: influence of chelating agents on 60co uptake by fresh water algae. cereal res. commun., 36, 2008, 419-422. hsiau, p., lo, s.: extractabilities of heavy metals in chemically-fixed sewage sludges. j. hazard. mater., 58, 1998, 73-82. iaquinta, m., stoller, m., merli, c.: development of synthetic wastewater from the tomato industry for membrane processing purposes. desalination, 200, 2006, 739-741. milne, c.j., kinniburgh, d.g., van riemsdijk, w.h., tipping, e.: generic nicadonnan model arameters for metal-ion binding by humic substances. environ. sci. technol., 37, 2003, 958-971. pal, a., paul, a.k.: microbial extracellular polymeric substances: central elements in heavy metal bioremediation. indian j. microbiol. 48, 2008, 49-64. pipíška, m., horník, m., remenárová, l., augustín, j., lesný, j.: biosorption of cadmium, cobalt and zinc by moss rhytidiadelphus squarrosus in the single and binary component systems. acta chim. slov., 2010 (in press). pipíška, m., horník, m., vrtoch, ľ., augustín, j., lesný, j.: biosorption of zn and co ions by evernia prunastri from single and binary metal solutions. chem. ecol., 24, 2008, 181-190. vullo, d.l., ceretti, h.m., daniel, m.a., ramirez, s.a.m., zalts, a.: cadmium, zinc and copper biosorption mediated by pseudomonas veronii 2e. bioresour. technol., 99, 2008, 5574-5581. wingender, j., neu, t.r., flemming, h.c.: what are bacterial extracellular polymeric substances? springer-verlag, heidelberg, germany, 1999, 171-200. závodská, l., lesný, j.: comparison of zn2+, cd2+ and sr2+ sorption characteristics for clinoptilolite and mordenite. in: szilágyi, m., szentmihályi, k. (eds.), trace elements in the food chain: proceedings: international symposium on trace elements in the food chain: budapest, 2006, 135-139. microsoft word maresova et al. nb 2010-2.doc nova biotechnologica 10-2 (2010) 157 cadmium sorption by dried plant biomass – reversibility studies kristína muráňová, jana marešová, miroslav horník, jozef augustín department of ecochemistry and radioecology, university of ss. cyril and methodius in trnava, j. herdu 2, trnava, sk-917 01, slovak republic (jana.maresova@ucm.sk) abstract: heavy metals such as cadmium are hazardous to biosystems and present possible human health risk. thus, the removal of cadmium from liquid wastes is of great importance from an environmental and industrial point of view. there is a tendency to use agricultural wastes for the sorption of toxic metals as an alternative to the existing conventional technologies. the aim of this work was to describe cd sorption and desorption equilibria by dried leaf biomass of tobacco (nicotiana tabacum l.), hop (humulus lupulus l.), pumpkin (cucurbita pepo l.), sunflower (helianthus annuus l.), cucumber (cucumis sativus l.), pepper (capsidum annuum l.), tomato (solanum lycopersicum l.) and vine-grape (vitis vinifera l.) using radiotracer technique with 109cdcl2. cadmium sorption q values (mg/g, d.w.) of all of plant biomass studied increased proportionally with the initial cdcl2 concentration within the range 0.01 – 10 mmol/l cdcl2. mean sorption capacity of dried leaf biomasses of eight plants for cd from the solution with the initial concentration c0 = 10 mmol/l cdcl2 in deionized water was q = 213 µmol/g (d.w.). by single step desorption of cd from leaf biomass for 30 minat 20 °c with deionized water, 0.1 mmol/l edta or 0.1 mmol/l hcl, up to 30 %, 85 % and 98 % of sorbed cd, respectively, was removed. obtained data can serve as a model for the prediction of sorption-desorption equilibria of biomass used for removing of cd from polluted waters and cd releasing back into waters containing other inorganic solutes. formation of cd2+, cdcl+, cdcl20, cdso40, cdoh+, cd2oh3+, cd(oh)20 and cd(oh)3species in dependence on ph values and the presence of clanions is also discussed. key words: cadmium, 109cdcl2, sorption, desorption, plant biomass 1. introduction cadmium is one of the most toxic metals found in effluents discharged from industries involved in metal plating, metallurgical alloying, mining, ceramics and other industrial activities. also, there is growing environmental concern about cd as being one of the most ecotoxic metals that exhibit highly adverse effects on soil biological activity, plant metabolism and the health of humans. usepa has also classified cadmium as group b1 carcinogen (usepa, 1999). the behaviour of cd in the environment and related health aspects has been reviewed by kabata-pendias and pendias (2001). broad spectrum of sorbents has received increased attention for heavy metal removal. data were published in numerous original papers and patents. for reviews see e.g. singh and tripathi (2007), sud et al. (2008). several physical and chemical methods have been used for removal of heavy metals from industrial liquid wastes, such as ion-exchange, chemical precipitation, chemical reduction and 158 muráňová, k. et al. adsorption. there are still problems associated with these methods mainly these are cost-expensive and can themselves produce other wastes, which has limited their industrial applications. among the available treatment processes, the application of biological sorbents is the most promising due to the following reasons: requirement of chemicals for the treatment process is reduced, lower operating costs, eco-friendly, higher efficiency at low levels of contaminations and cost-effective alternative of conventional techniques. in our previous papers we described sorption characteristics of co, zn and cd binding by dry biomass of lichen e. prunastri (pipíška et al., 2008) and moss r. squarrosus (pipíška et al., 2010) and by biomass of activated sludge of waste water treatment plant (marešová et al., 2010). the objective of this study was to obtain quantitative data of cadmium sorption by dried leaf biomass of common agricultural plants and reversibility of the sorption-desorption processes. 2. materials and methods 2.1 plant biomass leaves of sunflower (helianthus annuus l.), pumpkin (cucurbita pepo l.), cucumber (cucumis sativus l.), pepper (capsidum annuum l.), tomato (solanum lycopersicum l.) collected from plants growing in garden (trnava region) and leaves of vine-grape (vitis vinifera l.) from vineyard (malé karpaty region) in the middle of summer period. tobacco (nicotiana tabacum l.) and hop (humulus lupulus l.) leaves were obtained from plants cultivated in laboratory at 22 °c at light/dark period 12/12 h (2 000 lx). seeds of tobacco were germinated and grown in pots filled with granulated perlite watered with diluted hoagland medium ph 7.0 (hoagland, 1920). hop plants (variety osvald clone 72, genotype k-72/6/13) were obtained from the research institute of plant production in piešťany (sk). detached leaves were dried for 3 days at 60 °c, pulverized by rotatory knife blender and sieved by normalized sieves. fractions < 0.45 mm were used for experiments. 2.2 sorption experiments a batch equilibrium method was used to determine sorption of cd by dried leaf biomass of 8 plants. in plastic test tubes (10 ml) 0.05 g of dried biomass was mixed with 2 ml cdcl2 solution in deionized water (0.01 – 10 mmol/l), spiked with 109cdcl2 and placed on reciprocal shaker (200 min -1) at 20 °c. preliminary experiments showed that 10 min reaction time is sufficient for reaching of sorption equilibrium. for sorption experiments the initial ph value of solutions was adjusted to ph 5.0 with 1.0 m naoh. biomass was separated by centrifugation for 20 min at 2 800 × g and both sediments and liquid phase were analysed by gamma spectrometry. the experiments were conducted in triplicate. the amount of sorbed cd (μmol cd/g d.w.) was calculated according to the following equation: nova biotechnologica 10-2 (2010) 159 m ccv q eq )(* 0 −= (1) where q is the specific cd uptake (μmol cd/g biomass d.w.); v is the volume of cd solution (l); c0 is the initial concentration of cd in the solution (mmol/l); ceq is the concentration of cd in the solution in equilibrium (mmol/l) and m is mass of the dried biosorbent (g). 2.3 desorption experiments biomass (0.05 g, d.w.) after sorption experiments was separated by centrifugation for 20 min at 2 800 × g and resuspended by wortexing for 30 min in 5.0 ml deionized water, 1.0 ml 0.1 m edta or 1.0 ml 0.1 m hcl. cd concentrations in biomass and in extracts were estimated by similar way as in the sorption experiments. 2.4 radiometric and speciation analysis a gamma spectrometric assembly using the well type scintillation detector 54bp54/2-x, nai(tl) (scionix, nl) and data processing software scintivision-32 (ortec, usa) were used for cd determination in biomass and supernatants. a library of radionuclides was built by selecting characteristic γ-ray peaks (88.04 kev for 109cd, 661.66 kev for 137cs, 834.81 kev for 54mn and 1115.52 kev for 65zn) for energy and efficiency calibration. counting time 600 s allowed obtaining data with measurement error < 2 %, which do not reflect other source of errors. standardized solution of 109cdcl2 (3.94 mbq/ml, 50 mg/l cdcl2 in 3 g/l hcl) was obtained from the czech institute of metrology (prague, cr). cd speciation in the solution as a function of ph and concentration of clanions was performed using the visual minteq (ver. 3.0) modelling software. 3. results and discussions 3.1 cadmium sorption sorption experiments showed that dried leaf biomass is able to bind remarkable amounts of cadmium. q values (μmol/g, d.w.) at biomass concentration 25 g/l (d.w.) are proportional to the initial cdcl2 concentration within the range studied 0.01 – 10 mmol/l cdcl2. however as can be seen from the data in fig. 1 even at initial concentration c0 = 10 mmol/l cdcl2 plant sorbents were not saturated under given conditions and higher qmax values at saturation of sorbent can be expected. sorption capacity q of the dried leaf biomass of studied plants differs within the same order, ranging from 145 to 289 μmol/g (d.w.), despite of different taxonomic classification (tab. 1). in table 2, the value sorption capacities q (mg/g cd, d.w.) for naturally occurring sorbents are presented. we can conclude that studied plant biomasses showed similar results in comparison with others inorganic, organic and biological sorbents origin. 160 muráňová, k. et al. 1e-3 0,01 0,1 1 10 100 1e-3 0,01 0,1 1 10 100 0,1 1 10 100 1000 0,1 1 10 100 1000 tobacco hop pumpkin sunflower cucumber* cucumber** pepper tomato vine-grape q [μ m ol /g ] c 0 [mmol/l] fig. 1. cadmium sorption q (μmol/g; d.w.) by dried leaf biomass of different plants (25 g/l, d.w.) in dependence on initial cdcl2 concentration. data after 10 min reaction at 20 °c, ph 5.0 are the mean of three replicates. *c. sativus l. convar. longus,**c. sativus l. convar. viridis table 1. cadmium sorption q (μmol/g; d.w.) by dried leaf biomass (25 g/l, d.w.) of different plants after 10 min reaction in 10 mmol/l cdcl2 in deionized water at 20 °c, ph 5.0. data are the mean of three replicates. plant family q [μmol/g] humulus lupulus l. cannabaceae 288.8 ± 3.8 cucurbita pepo l. cucurbitaceae 283.1 ± 8.7 helianthus annuus l. asteraceae 268.4 ± 5.6 cucumis sativus l. convar. longus cucurbitaceae 228.4 ± 7.6 cucumis sativus l. convar. viridis cucurbitaceae 209.7 ± 6.1 capsicum annuum l. solanaceae 196.9 ± 4.3 nicotiana tabacum l. solanaceae 148.8 ± 4.0 solanum lycopersicum l. solanaceae 147.2 ± 4.0 vitis vinifera l. vitaceae 145.0 ± 2.0 table 2. sorption capacities q (mg/g cd; d.w.) of naturally occuring sorbents. (1) pipíška et al., 2010; (2) marešová et al., 2010; (3 ) bailey et al., 1999; (4) this paper. *c. sativus l. convar. longus,**c. sativus l. convar. viridis sorbent q [mg/g] ref. sorbent q [mg/g] ref. bark 32 3 activated sludge 121 2 chitosan 558 3 hop leaves 32.5 4 clay 16.5 3 pumpkin leaves 31.8 4 microbial biomass 28 3 sunflower leaves 30.2 4 modified wool 87 3 cucumber leaves* 25.7 4 peat 5.1 3 cucumber leaves** 23.6 4 seaweed 215 3 pepper leaves 22.1 4 xanthate 33.3 3 tobacco leaves 16.7 4 zeolite 84.3 3 tomato leaves 16.5 4 moss biomass 19.4 1 vine leaves 16.3 4 nova biotechnologica 10-2 (2010) 161 3.2 the influence of ph it is generally accepted that the ph optimum of biosorption for bivalent metal cations is within ph 4 – 6. in strong acid media the efficiency of cation sorption is minimal due to competitive effect of h+ ions. in alkaline region the situation is more complicated due to the existence of multiple cd species. data obtained by speciation program visual minteq shows that eight cd species can exist in alkaline solutions. molar fractions of cationic, anionic and nonionic forms of cd expressed in molar fraction x in dependence on ph value are shown in fig. 2. 2 3 4 5 6 7 8 9 10 11 12 0,0 0,2 0,4 0,6 0,8 cd2+ cdcl+ cdcl 2 0 cdso 4 0 cdoh+ cd 2 oh3+ cd(oh) 2 0 cd(oh) 3 m ol ar fr ac tio n x = [c dn + ] / [c dς n ] ph fig. 2. molar fraction x of cadmium species in dependence on solution ph at concentration 10 mmol/l cdcl20 and 20 °c. solution of inorganic salts corresponding to synthetic waste water according to iaquinta et al. (2006) (ppm): cl– 455; so42– 87; na+ 418; k+ 78; mg2+ 45; ca2+ 138. calculated by visual minteq ver. 3.0. as can be seen from the data in fig. 2, prevailing part of cd is present in cd2+ form (42 %) and cdcl+ form (51 %) and this proportion stay the same within ph range from ph 3.0 to ph 8.5. concentration of cd(oh)2 0 increases above ph 9. during the sorption of metals by sorbents from alkaline liquid wastes besides sorption of cd2+ cations also the existence of other than ionic metal forms with different solubility in water have to be taken into consideration. similar data was obtained also by guibaud et al. (2009), where sigmoidal ph dependence of cd2+ sorption by polysaccharides of activated sludge with inflection point at ph 8.6 was observed. 3.3 the influence of cl ions as can be seen from the data in fig. 3, molar fraction x of cd2+ significantly decreases in the presence of clions up to 10-1 mol/l nacl, as a result of the increase of cdcl+ molar fraction. at 1.0 mol/l nacl (5.8 %, w/v) concentration of cd2+ is minimal and prevailing cd species are non dissociated cdcl2 0 or cdcl+. in interactions of biosorbents with cd(ii) in the presence of clanions, what is typical for many kinds of liquid wastes, the existence of complex mixture of different cd species can be expected. mechanism of cd biosorption will then depend both on the ph values of liquid phase, as well as on the concentration of other solutes, mainly 162 muráňová, k. et al. clsalts, but also on the presence of other solutes able to form complexes with high values of stability constants log k. 1e-3 0,01 0,1 1 0,0 0,2 0,4 0,6 0,8 1,0 m ol ar fr ac tio n x = [c dn + ] / [c dς n ] cl[mol/l] cd2+ cdcl+ cdcl 2 0 fig. 3. molar fraction x of cd2+, cdcl+ and cdcl20 species in dependence on nacl concentration in 1.0 mol/l cdcl2 in deionized water and 20 °c. calculated by visual minteq ver. 3.0. 2 3 4 5 6 7 8 9 10 11 0,0 0,2 0,4 0,6 m ol ar fr ac tio n x = [c dn + ] / [c dς n ] 0.1 mmol/l ph cd2+ cdcl+ 2 3 4 5 6 7 8 9 10 11 0,0 0,2 0,4 0,6 ph cd2+ cdcl+ 1.0 mmol/l 2 3 4 5 6 7 8 9 10 11 0,0 0,2 0,4 0,6 m ol ar fr ac tio n x = [c dn + ] / [c dς n ] ph cd2+ cdcl+ 10.0 mmol/l 2 3 4 5 6 7 8 9 10 11 0,0 0,2 0,4 0,6 ph cd2+ cdcl+ 100.0 mmol/l fig. 4. ph dependence of molar fraction x of cd2+ and cdcl+ ions at different cdcl2 concentration and 20 °c in solution of inorganic salts simulating waste water according to iaquinta et al. (2006) (ppm): cl– 455; so42– 87; na+ 418; k+ 78; mg2+ 45; ca2+ 138. calculated by visual minteq ver. 3.0. nova biotechnologica 10-2 (2010) 163 3.4 desorption in dependence on the [biomass]: [extractant] ratio, different percentage of cadmium can be removed. by extracting with deionized water at the [biomass]: [extractant] = 1: 100 (w/v) ratio and in dependence on plant species up to 30 % of cd was removed by single step extraction at 20 °c. by extraction with 0.1 mol/l edta at [biomass]: [extractant] = 1: 20 (w/v) ratio, up to 85 % and with 0.1 m hcl up to 98 % was removed under identical conditions. by desorption with water, new concentration equilibrium expressed as p = [cd]sorbent: [cd]solution is formed. cadmium is released with higher efficiency from the binding sites of lower stability constants log k. stability constant of the metal complex can be described by the equation (2): log k = [ml] / [m]* [l] (2) where k is the stability constant, [m] is the concentration of cd2+ ions and [l] is the concentration of a ligand such as carboxylic or amino acids. 0,01 0,1 1 10 1 10 100 1000 10000 desorption with edta desorption with water p = [c d] bi om as s/ [c d] so lu tio n c0 [mmol/l] sorption fig. 5. sorbent to solution concentration ratio (mmol/kg)/(mmol/l) for cadmium in dependence on initial concentration of cdcl2. sorption process (–■–■–); desorption with deionized water (–o–o–) and desorption with 0.1 mol/l edta (–δ–δ–). experimental points are the mean values obtained from sorption experiments of 8 plants shown in fig. 1 (–■–■–); and of data obtained by desorption of biomass with deionized water (–o–o–) and 0.1 mol/l edta as described in materials and methods. metal ions can form more or less stable complexes with many biomolecules differing in stability constants log k. cd complexes with organic compounds can be divided into four groups. a: log k ~ 1 2 (carboxylic acids); b: log k ~ 5 6 (amino acids and citric acid); c: log k ~ 9 – 10 (cysteine, glutathione and other –sh compounds); d: log k ~ 16 18 (complexing ligands such as edta). classification is based on published data of cd and other bivalent metal complexes (martell and smith, 1964; srivastava et al., 1993; gunther and kastenholz, 2005). we can suppose that by extracting with deionized water only cd atoms bind into complexes of low log k values are released. characterization of the mode of cadmium 164 muráňová, k. et al. binding not extractable with the strongest ligands such as edta will require more detailed study. however differences in the efficiency of cd desorption with water and edta reflects the fact that broad spectrum of reactive groups differing in stability constants is taking place. 4. conclusions dried leaf biomass of eight vascular plants shows the sorption capacity for cadmium binding from cdcl2 water solution depending on the initial concentration of the solute, and is comparable with the sorption capacity of other sorbents of plant origin. sorbed cadmium is partially leachable with deionized water and with high efficiency by edta solution and diluted mineral acids. this supports the idea that in sorption processes cd complexes with different stability constants log k are formed. calculation by speciation program visual minteq showed, that both alkaline ph values and concentration of chloride anions determines the existence at least seven cd species with cationic, anionic and neutral charge. obtained data can serve for the prediction of cadmium transport in biosphere and at penetration into food chain from contaminated vegetable crops. references bailey, s.e., olin, t.j., bricka, r.m., adrian, d.d.: a review of potentially low-cost sorbents for heavy metals. water res., 33, 1999, 2469-2479. guibaud, g., van hullebusch, e., bordas, f., d´abzac, p., joussein, e.: sorption of cd (ii) and pb (ii) by exopolymeric substances (eps) extracted from activated sludges and pure bacterial strains: modelling of the metal/ligand ratio effect and role of the mineral fraction. bioresour. technol., 100, 2009, 29592968. gunther, k., kastenholz, b.: speciation of cadmium in the environment and food. in: cornelis, r., crews, h., caruso, j., heuman, k.g. (eds.), handbook of elemental speciation ii: species in the environment food, medicine and occupational health. john wiley and sons, ltd. 2005, 745 pp. hoagland, d.r.: optimum nutrient solutions for plants. science, 52, 1920, 562-564. iaquinta, m., stoller, m., merli, c.: development of synthetic wastewater from the tomato industry for membrane processing purposes. desalination, 200, 2006, 739-741. kabata-pendias, a., pendias, h.: trace elements in soils and plants. 3rd edition, crc press, boca raton, florida, usa, 2001, 403 pp. marešová, j., horník, m., pipíška, m., augustín, j.: sorption of co2+, zn2+, cd2+ and cs+ ions by activated sludge of sewage treatment plant. nova biotechnol., 10, 2010, 53-61. martell, a.e., smith, r.m.: stability constants of metal ion complexes. (special supplement no. 17). chemical society, london, 1964, 754 pp. nova biotechnologica 10-2 (2010) 165 pipíška, m., horník, m., remenárová, l., augustín, j., lesný, j.: biosorption of cadmium, cobalt and zinc by moss rhytidiadelphus squarrosus in the single and binary component systems. acta chim. slov., 57, 2010, 163-172. pipíška, m., horník, m., vrtoch, ľ., augustín, j., lesný, j.: biosorption of zn and co ions by evernia prunastri from single and binary metal solutions. chem. ecol., 24, 2008, 181-190. singh, s., tripathi, r.d. (eds.): environmental bioremediation technologies. springer-verlag, berlin 2007, 518 pp. srivastava, s.k., gupta, v.k., tiwari, b.b., ali, i.: electrophoretic determination of stability constants of zn(ii)and cd(ii)nitrilotriacetatepenicillamine mixed complexes. j. chromatogr., 635, 1993, 171175. sud, d., mahajan, g., kaur, m.p.: agricultural waste material as potential adsorbent for sequestering heavy metal ions from aqueous solutions – a review. bioresour. technol., 99, 2008, 6017-6027. usepa: us environmental protection agency, integrated risk information system (iris) on cadmium, national centre for environmental assessment, office of research and development, washington, dc, 1999. microsoft word melcakova 2010-1.doc nova biotechnologica 10-1 (2010) 33 biosorption of zinc from aqueous solution using algae and plant biomass iva melčáková, tomáš růžovič všb tu-ostrava, institute of environmental engineering, faculty of mining and geology, ostrava, czech republic (iva.melcakova@ vsb.cz, tomas.ruzovic.st@vsb.cz) abstract: in the present study, the sorption capacity of plant biomass has been studied; particularly the ability of biomass algae chlorella vulgaris, filamentous green algae spirogyra sp. and roots, stems and leaves of an invasive plant reynoutria japonica to bind up zn2+ ions. the results of this biosorption study revealed that the rate and extent of uptake were affected by ph level, contact time and initial metal concentration. the maximum uptake of metal ions was obtained at ph 6.0. the equilibrium sorption data for metal system at ph 6 were described by the langmuir isotherms model. for zn2+, sorption capacity qmax of 17 mg/g was achieved using biomass from leaves. removal of zn2+ with 1g of biosorbent from leaves was almost 77% when present in low concentrations, whereas it is lower at higher concentrations. keywords: chlorella vulgaris, spirogyra sp., reynoutria japonica, biosorption, zinc, isotherm 1. introduction metal pollution is one of the most important environmental problem today. three kinds of metals are of concern, including toxic metals (such as hg, cr, pb, zn, cu, ni, cd, as, co, sn etc.), precious metals (such as pd, pt, ag, au, ru etc.) and radionuclides (such as u, th, ra, am, etc.) (wang and chen, 2009). metals can be distinguished from other toxic pollutants, since are non-biodegradable and can accumulate in the living tissues, thus becoming concentrated throughout the food chain (williams et al., 1998). by far the greatest demand for metal sequestration comes from the need for immobilizing the metals “mobilized” by and partially lost through growing and everintensifying human technological activities. pollution of the environment by toxic metals arises as a result of many human activities like mining, metallurgy, electroplating, leather tanning, metal finishing, textile industry, and paper industry. effects of these metals on ecosystems are of large economic and public-health significance (volesky, 2001). biosorption and bioaccumulation belong to the group of biological methods suitable for heavy metal removal from wastewater (kaduková and virčíková, 2005). the biosorption is relatively new technology and could be considered for its economic edge as a possible alternative technique for metal recovery. in recent years, biosorption has been widely studied for the removal of metal ions, especially at the concentrations ranging from 1 to 100 mg/l, due to its lower costs and higher effectiveness than the conventional methods such as chemical precipitation and ion exchange (han et al., 2008). different biomass have been used to adsorb metal ions from the environment. bacteria, fungi, yeast, algae, and plants have proved to be 34 melčáková, i . and růžovič, t. potential metal sorbents (gupta et al., 2000; romera et al., 2007). while choosing biomaterial for metal sorption, its origin is a major factor to be taken into account; it can come from a) microorganisms as a by-product of fermentation industry, b) organisms naturally available in large quantities in nature and c) organisms cultivated or propagated for biosorption purposes using inexpensive media (ahluwalia and goyal, 2007). understanding the sorption of metal ions from aqueous solution is important for apllication in industrial water treatment. in this study we wanted to compare biosorption of zinc by laboratorly cultivated species chlorella vulgaris with ubiquitous and in large quantities in nature found species spirogyra sp. and reynoutria japonica. chlorella vulgaris is fast–growing edible algae, cultivated on large scale, and used as a food or feed supplement (chojnacka, 2007). several studies (bishnoi et al., 2007; gupta et al., 2006; gupta and sharma, 2008) has been reported about spirogyra sp., harvested from natural populations, which have exhibited its excellent ability to remove chromium, copper and lead from aqueous solutions. the cell wall of spirogyra sp. is very similar to that of terrestrial plants because its main components are cellulose and pectin. last species reynoutria japonica is an invasive ubiquitous plant investigated currently as a possible energetic plant. it is also known that the species of this genus are able to accumulate heavy metals from soil. zinc is the fourth among metals of the world in annual consumption. it is extensively used in the automobile industry, for the production of protective coatings for iron and steel, in cosmetics, powders, ointments, antiseptics, paints, varnishes, rubber and linoleum. zinc is also needed for manufacturing of parchment papers, glass, automobile tires, television screens, dry cell batteries and electrical equipment. the main sources of zn in the environment are zinc fertilizers, sewage sludges and mining and smelting (bradl et al., 2005). the average human body contains about 2 grams of zinc, which is essential for the normal activity of dna polymerization and for protein synthesis. soluble and astringent acid salts, such as znso4 in large doses (about 10 g), have caused internal organ damage and death (gupta and sharma, 2003). 2. materials and methods the culture of unicellular algae species, chlorella vulgaris (fig.1), the culture of filamentous green algae spirogyra sp. (fig. 2 and 3) and plant reynoutria japonica (fig.4) were used for laboratory experiments. 2.1 chlorella vulgaris cultivation chlorella vulgaris in log phase used in the experiments was inoculated in medium milieu bristol, modification 3n, at ph 7. the medium was sterilized by autoclaving at 121°c for 15 minute. medium was stored at 4°c until inoculated. cultures were grown in liquid media in 2 l glass erlenmeyer flasks, aerated with filtered air, and incubated at 25°c on a light table. samples were taken every 24 h using sterile glass pipettes. in the samples the cell counts were obtained using the thoma cellula, and the content of nova biotechnologica 10-1 (2010) 35 pigment chlorophyll a was determined by du®-64 spectrophotometer (beckman, memory pac +m). 2.2 preparation of biosorbents when chlorella vulgaris cultures reached the stationary phase, they were harvested and used for metal ion biosorption experiments. chlorella vulgaris was first cooled down in order to ease following centrifugation (10 minutes at 3000 rpm), and obtained biomass was extensively washed two times with water (1 l of de-ionized water per 1 g of biomass) to get rid of medium remnants. then it was dried in the oven (at 60°c) overnight and pulverized in a grinding mortar. acid pre-treatment was then done by washing the sorbent with 0.2 m solution of h2so4 (1 l of acid per 1 g of biomass for 90 minutes), washing twice again by de-ionized water, centrifuged once more and dried 24 hours at constant temperature of 60°c. filamentous green algae spirogyra sp. was collected from a fresh water pond. the sorbent was then prepared using the same procedure as described above. fig. 1. chlorella vulgaris fig. 2. spirogyra sp. fig. 3. spirogyra sp. fig. 4. reynoutria japonica 36 melčáková, i . and růžovič, t. all samples of reynoutria japonica used were collected from the same non-urban area in foothills of the lysá hora mountain, in the area of the moravskoslezské beskydy. this sampling area does not have any prior history of contamination by heavy metals. roots, stems and leaves of this plant were air-dried at room temperature. dried samples were ground and screened using a sieve shaker; uniform particle size fraction 1-2 mm was obtained. ion exchange resins manufactured for the same purpose generally feature particle sizes between 0.7 and 1.5 mm. biosorbent granule size usually ranges between 1 to 2 mm. particles of roots, stems and leaves were twice washed with 0.01 m of hcl (1 l of acid per 10 g of biomass), then with extensive volume of de-ionized water in order to remove soil or debris, and finally washed with distilled water. the biomass samples were then oven-dried at 90°c for one day. 2.3 chemicals zn2+ ions (znso4.7 h2o) were used in this study. test solutions containing this single ion were prepared by diluting proper amount of 1g/l stock solution of zinc ion in order to obtain desired concentrations. chemicals used were of analytical reagent grade and therefore used without further purification. 2.4 procedure of experiments 2.4.1 time course time course of zn2+ uptake by chlorella vulgaris, spirogyra sp. and reynoutria japonica was investigated. the sorbent with the final concentration 0.5 g/l of dried biomass of algae and 1 g/l of reynoutria japonica (leaves, stems, roots) was suspended in 200 ml or 500 ml of zinc ion solution. the flasks were placed on a shaker at 120 rpm at a room temperature (around 25°c) for 24 hours for algae and 6 hours for reynoutria japonica. the ph value of the solutions tended to drop during the equilibration and during the sorption experiments it was adjusted with 0.1 m solution of naoh. the temperature and ph were measured by a microcomputer meter. samples were taken from the solution at desired intervals and were filtered through membrane filter millipore 0.45 μm or, in the case of r. japonica, through filter paper ak-01 blue, dry. all samples were duplicated with the average presented in the results. the heavy metal concentrations in the resulting supernatant were measured by the atomic absorption spectrophotometry (aas) unicam 969, wavelength 213.9 nm. 2.4.2 effect of ph the experiment was conducted at concentration 100 mg/l of zinc ions for chlorella vulgaris and 10 mg/l for spirogyra sp. and reynoutria japonica, 1 g/l biosorbent dose in 50 ml and 500 ml zinc solution for six hours with ph varying from 4.0-6.0. ph of the solution was adjusted using 0.1 m naoh. nova biotechnologica 10-1 (2010) 37 2.4.3 adsorption isotherm in the present experiment we have determinate adsorption isotherms for zinc. amounts of 2 g/l dry acid-pre-treated biomass of algae and 1 g/l dry acid-pre-treated plant biomass (all three types: roots, stems and leaves) were suspended in samples of various concentrations (10-100 mg/l) of zn2+ solutions. the ph of solutions before and during equilibration was adjusted with 0.1 m and 0.01 m solutions of naoh. after 60 minutes of incubation, zinc samples were filtered in order to remove the biomass and metal concentration in supernatant was measured with aas. sorption capacity of the substrate (q) expressed in terms of metal amount sorbed on the unitary biosorbent mass (mg/g). this parameter has been calculated as indicated below (cimino et al., 2000). ( ) sccvq fi /−= (1) where: v – the volume of metal-bearing solution contacted with the sorbent some [l], ci – initial and residual concentrations of metal in the solution [mg/l], cf – residual concentrations of metal in the solution [mg/l], s – the amount of the added biosorbent [g]. 3. results and discussion 3.1 time course the kinetics experiments of zinc ions removal from solutions showed that biosorption is the equilibrium process, in which the equilibrium is reached after about 10 minutes. the ions are bound with the biomass steadily, and the final concentration of metal ions remained unchanged for 100 hours (chojnacka et al., 2005). to determine time course of biosorption of chlorella sp. biomass we have chosen zinc concentration of 100mg/l, based on our experience with previous experiments with chromium (chovancová, 2001). as zinc displayed different behaviour and the efficiency of its removal in high concentration was not sufficient, for following experiments with spirogyra sp. and reynoutria japonica we decreased concentrations to 10 mg/l. nevertheless, for both concentrations (100 mg/l and 10 mg/l) it can be concluded that at optimal ph value 6 (fig. 5 and 6) zinc binding onto algal and plant biomass is rapid (in the first 10 minutes). during the process, concentration of a metal ion gradually decreases and the equilibrium is reached; the concentration remains almost constant after approximately 100 minutes. adsorption slowed down in later stages because initially a large number of vacant surface sites may be available for adsorption and after some time, the remaining vacant surface sites may be difficult to occupy due to forces between the solute molecules of the solid and bulk phase (bishnoi et al., 2007). it is, however, necessary to say that in the case of higher concentration (100 mg/l) the equilibrium status has not been reached and after about 300 minutes zinc started to be separated out back into the solution (fig. 5). 38 melčáková, i . and růžovič, t. 0 2 4 6 8 10 12 14 0 200 400 600 800 1000 1200 1400 1600 time (minute) q (m g/ g) chlorella vulgaris fig. 5. time course of metal sorption of divalent zinc by chlorella vulgaris, ph 6.0, initial metal concentration 100 mg/l, room temperature 25°c. 0 1 2 3 4 5 6 7 8 9 0 50 100 150 200 250 300 350 400 time (minute ) q ( m g /g ) roots stems leaves spirogyra fig. 6. time course of metal sorption of divalent zinc by roots, stems and leaves of reynoutria japonica and spirogyra sp., ph 6.0, initial metal concentration 10 mg/l, room temperature 25°c. 3.2 effect of ph ph is one of the most important environmental factors influencing not only site dissociation, but also the solution chemistry of the heavy metals: hydrolysis, complexation by organic and/or inorganic ligands, redox reactions, and precipitation are strongly influenced by ph and, on the other side, strongly influence the speciation and the biosorption availability of the heavy metals (esposito et al., 2002). the effect of ph on the zinc biosorption capacity of biomass has been shown in fig. 7. the increase of sorption of zinc ions corresponds to ph values growing from 4.0 to 6.0, the latter being the optimum ph for biosorption of zinc ions by all types of biomass. acid-washed biosorbents can be viewed as natural ion-exchange materials that primary contain weak acid and basic groups (kratochvil and volesky, 1998). number of negatively charged active sites (functional groups such as carboxyl, amine, hydroxyl and phosphate groups) at higher ph increases, facilitating a higher electrical attraction to positively charged metal ions (klimmek et al., 2001; romera et al., 2007). chojnacka et al. (2005) identified in cyanobacteria spirulina sp. different functional groups at different ph values. in ph value range 5.0-9.0, which covers ph nova biotechnologica 10-1 (2010) 39 value used in our experiments, she identified with use of potentiometric titration carboxyl and phosphate group to be the main functional groups. our results of optimum ph value are in agreement with studies of romera et al. (2001), who studied untreated algae biomass of spirogyra insignis, and salehizadeh et al. (2003) and norton et al. (2004) studying bacteria. 0 2 4 6 8 10 12 14 16 0 1 2 3 4 5 6 7 ph q (m g/ g) roots stems leaves spirogyra sp. fig. 7. effect of ph on the sorption of zn2+ by roots, stems and leaves of r. japonica and spirogyra sp., ph 4.0-6.0, initial metal concentration 10 mg/l, room temperature 25°c. 3.3 determination of adsorption isotherms the fig. 8 displays metal uptake isotherms for zn2+ ions plotted against final metal concentration cf in aqueous solutions. when the initial zn 2+ concentration increased from 10 to 50 mg/l, uptake by chlorella vulgaris increased, and reached 15.9 mg/g. 0 5 10 15 20 25 0 20 40 60 80 100 120 final metal concentration in solution (mg/l) m et al u pt ak e (m g/ g) chlorella vulgaris spirogyra sp. reynoutria japonica roots reynoutria japonica stems reynoutria japonica leaves fig. 8 sorption isotherms for the sorption of zn2+ onto chlorella vulgaris, spirogyra sp. and roots, stems and leaves of reynoutria japonica biomass, ph 6.0, room temperature 25.0 o c. similar results were obtained with leaves of reynoutria japonica (uptake 19.5 mg/g), and spirogyra sp. biomass (uptake 14.5 mg/g), both at initial zinc concentration 75 mg/l. it is possible to conclude that in both low and high concentrations, leaves showed the best results. at initial zn2+ concentration 100 mg/l, decrease of sorption in 40 melčáková, i . and růžovič, t. all biomass types was recorded, with the exception of roots and stems of reynoutria japonica; however these two types of biomass displayed generally the lowest zinc uptake ability among all biomass investigated. the process of zinc sorption on the biosorbent was described by the langmuir adsorption model, which is widely used to analyze data for water and wastewater treatment applications. the langmuir model represents one of the first theoretical descriptions of nonlinear sorption and suggests that uptake occurs on a homogeneous surface by monolayer sorption without interaction between adsorbed molecules. in addition, the model assumes uniform energies of adsorption onto the surface and no transmigration of the adsorbate (sahmoune et al., 2008). the langmuir equation is given by eq. (2) ff bcbcqq += 1/max (2) where: qmax – maximal metal uptake [mg/g], b – a constant related to the affinity of the binding sites [l/mg], q – experimental metal uptake [mg/g], cf – residual concentrations of metal in the solution [mg/l]. q and b can be determined from the linear plot of cf:/q vs. cf (donmez et al., 1999). the main advantage of this model is the possibility of evaluation of qmax maximum possible quantity of a metal ion adsorbed per gram of adsorbent, and b – parameter related to the affinity of binding sites for a metal ion (michalak et al., 2007). in general, for good biosorbents, high qmax and high b are desirable (davis et al., 2003). the langmuir constants r2 for the zinc biosorption onto biomass of chlorella vulgaris, spirogyra sp. and reynoutria japonica are showed in fig. 9 and 10. the results indicate the highest applicability of the langmuir model for two biosorbents: spirogyra sp. with the best regression coefficient r2 = 0.9834, and leaves of reynoutria japonica r2 = 0.9807. based on qmax, it can be concluded that spirogyra sp. (qmax = 17.8 mg/g) has slightly higher adsorption capacity for zinc than leaves of reynoutria japonica (qmax =17.0 mg/g). as for parameter b, however, spirogyra sp. has significantly lower (b = 0.06) than leaves of reynoutria japonica (b = 0.26). hashim and chu (2004) suggest that a biosorbent with low qmax and high b could outperform a biosorbent with high qmax and low b, especially in cases where the metal ion to be removed is present at trace amounts. therefore, higher b value of leaves of reynoutria japonica indicates its higher affinity for zinc. ranking of sorptions of all sorbents studied, based on qmax, can be set up as follows: spirogyra sp. > leaves > stems > chlorella vulgaris > roots of reynoutria japonica. very similar results for spirogyra sp. were obtained by romea et al. (2007). in his experiments with untreated spirogyra sp. biomass maximum capacity for zinc was found to be 21.6 mg/g, and parameter b = 0.04. adsorption capacity for zinc by waste tea leaves biomass (similar type of biomass as leaves of reynoutria japonica) was found to be 11.8 mg/g (tee and khan, 1988). nova biotechnologica 10-1 (2010) 41 r2 = 0,9834 r2 = 0,9223 -2 0 2 4 6 8 10 12 0 10 20 30 40 50 60 70 80 90 cf c f/q chlorella spirogyra sp. fig. 9. sorption isotherms for the sorption of zn2+ onto chlorella vulgaris and spirogyra sp., ph 6.0, room temperature 25°c. r2 = 0,9128 r2 = 0,9807 r2 = 0,9779 0 2 4 6 8 10 12 14 16 0 20 40 60 80 100 120 cf c f/q roots stems leaves fig. 10. sorption isotherms for the sorption of zn2+ onto roots, stems and leaves of reynoutria japonica, ph 6.0, room temperature 25°c. 4. conclusions this work has demonstrated the possibility of utilization of biomass of algae chlorella vulgaris and spirogyra sp., and of a plant reynoutria japonica for biosorption of zinc ions from aqueous solutions. the binding capacity of zn2+ ions to biosorbent has been shown to depend upon ph, with the highest binding at ph 6.0. the biosorbents have good capacity for zinc adsorption, especially in low metal concentrations. there were no major differences in biosorption of zinc between cultured chlorella vulgaris and natural spirogyra sp. and reynoutria japonica. references ahluwalia, s.s., goyal, d.: microbial and plant derived biomass for removal of heavy metals from wastewater. bioresour. technol., 98, 2007, 2243-2257. 42 melčáková, i . and růžovič, t. bishnoi, n.r., kumar, r., kumar, s., rani, s.: biosorption of cr(iii) from aqueous solution using algal biomass spirogyra spp. j. hazard. mater., 135, 2007, 142–147. bradl, h.: heavy metals in the environment.1sted. london: elsevier, 2005, 282 pp. davis, t.a., volesky, b., mucci, a.: a review of the biochemistry of heavy metal biosorption by brown algae. water res. 37, 2003, 4311–4330. dönmez, g.c., aksu, z., ozturk, a., kutsal, t.: a comparative study on heavy metal biosorption characteristics of some algae. process biochem., 34, 1999, 885–892. cimino, g., passerini a., toscano, g.: removal of toxic cations and cr(vi) form aqueous solution by hazelnut shell. wat. res., 34, 2000, 2955-2962. esposito, a., pagnanelli, f., veglio, f.: ph-related equilibrium models for biosorption in single metal systems. chem. eng. sci., 57, 2002, 307-313. gupta, v.k., rastogi, a.: biosorption of lead from aqueous solution by green algae spirogyra sp.: kinetics and equilibrium studies. j. hazard. mater., 152, 2008, 407-414. gupta, v.k., rastogi, a., saini, v.k., jain, n.: biosorption of copper(ii) from aqueous solutions by spirogyra species. j. colloid interface sci., 296, 2006, 59-63. gupta, v.k., sharma, s.: removal of zinc from aqueous solutions using bagasse fly ash a low cost adsorbent. ind. eng. chem. res., 42, 2003, 6619-6624. haase, e.: pflanzen reiningen schwermetall-böden. umwelt (dusseldorf), 7-8, 1988, 342-344. hashim, m.a., chu, k.h.: biosorption of cadmium by brown, green and red seaweeds, chem. eng. j. 97, 2004, 249–255. chojnacka, k., chojnacki, a., górecka. h.: biosorption of cr3+, cd2+ and cu2+ ions by blue–green algae spirulina sp.: kinetics, equilibrium and the mechanism of the process. chemosphere, 59, 2005, 75-84. chojnacka, k.: using biosorption to enrich the biomass of chlorella vulgaris with microelements to be used as mineral feed supplement. world j. microbiol. biotechnol. 23, 2007, 1139-1147. chovancová,i.: uptake of trivalent chromium by biosorbent of algae of chlorella vulgaris, new trends in mineral processing iv, ostrava 28-30.6. 2001, 493-501. kaduková, j., virčíková, e.: comparison of differences between copper bioaccumulation and biosorption. environ. int., 31, 2005, 227– 232. klimmek, s., stan, h.-j., wilke, a., bunke, g. buchholz. r.: comparative analysis of the biosorption of cadmium, lead, nickel, and zinc by algae. environ. sci. technol., 35, 2001, 4283-4288. michalak, i., zielinska, a., chojnacka, k., matula, j.: biosorption cr (iii) by microalgae and macroalgae: equilibrium of the process. am. j. agri. biol. sci., 2, 2007, 284-290. romera, e., gonzalez, f., ballester, a., blazquez, m.l., munoz, j.a.: biosorption with algae: statistical review. crit. rev. biotechnol., 26, 2006, 223-35. salehizadeh, h., shojaosadati, s.a.: removal of metal ions from aqueous solution by polysaccharide produced from bacillus firmus. water res., 17, 2003, 4231–4235. nova biotechnologica 10-1 (2010) 43 tee, t.w., khan, a.r.m.: removal of lead, cadmium and zinc by waste tea leaves. environ. technol. lett., 9, 1988, 1223–32. váňa j., roth, j.: zabezpečení fytopaliva v oblasti elektrárny tušimice. dílčí zpráva. envicho s.r.o., chomutov, 1994. volesky, b.: detoxification of metal-bearing effluents: biosorption for the next century. hydrometallurgy, 59, 2001, 203-216. wang, j.,chen, c: biosorbents for heavy metals removal and their future. biotechnol. adv., 27, 2009, 195-226. williams, c.j., aderhol, d., edyvean, r.g.j.: comparison between biosorbents for the removal of metal ions from aqueous solutions, water res., 32, 1998, 216-224. nova biotechnol chim (2022) 21(2): e1322 doi: 10.36547/nbc.1322 1 nova biotechnologica et chimica chemical profiles and antibacterial activities of acetone extracts of globba macrocarpa hong thien van1,, tan viet pham1, ngoc an nguyen1, hanh thi dieu nguyen1, quoc hung nguyen2, thanh tho le2, van son le3 1institute of biotechnology and food technology, industrial university of ho chi minh city, no. 12 nguyen van bao street, go vap district, ho chi minh city, vietnam 2center of analytical services and experimentation hcmc, vietnam, no. 2 nguyen van thu, dakao ward, district 1, ho chi minh city, vietnam 3binh chau-phuoc buu nature reserve, bung rieng ward, xuyen moc district, ba ria-vung tau province, vietnam  corresponding author: vanhongthien@iuh.edu.vn article info article history: received: 13th december 2021 accepted: 7th april 2022 keywords: acetone extract antibacterial activities gc/ms globba macrocarpa abstract globba macrocarpa gagnep. is a rare species of globba genus (zingiberaceae family). the present study reported the chemical compositions and antibacterial effects of acetone extracts obtained from the g. macrocarpa rhizomes and aerial parts. by using gas chromatography mass spectrometry (gc/ms) assay, fifty and thirty-two chemical compounds were identified from rhizomes and aerial parts of the species, respectively. of those, germacrene d (15.25 %), 1h-indole, 4-(3-methyl-2butenyl)(14.33 %), (e)-β-farnesene (11.28 %), and 2-biphenylamine, 3-methyl (10.27 %) were the major constituents in the rhizome extract while the aerial part extract was characterized by the predominance of linolenic acid (19.89 %), palmitic acid (13.05 %), phytol (7.52 %), and neophytadiene (4.76 %). in addition, the rhizome extract had antibacterial activities against five 5 out of 6 oral bacteria, including pseudomonas aeruginosa, salmonella enteritidis, salmonella typhimurium bacillus cereus, and staphylococcus aureus. meanwhile, the aerial part extract was active against 4 out of 6 test bacteria, except for escherichia coli and s. typhimurium. introduction globba l. is a third largest genus belonging to zingiberaceae with over 100 species. this genus is distributed widely throughout tropical and subtropical of asia such as india, southern china, new guinea and southeast asia. globba plants are small perennial herbs, reaching to a height of 1 m, except for g. racemosa (about 3 m) (williams et al. 2004). members of globba consist of a large number of useful plants. for instance, the g. bulbifera rhizomes have been used to cure cough, asthma, and snakebite. g. multiflora was used to treat headache and rheumatic inflammation (jain 1995). the anti-inflammatory, antioxidant, and antipyretic activities were recorded as the biological activities of g. malaccensis (ngamrojanavanich et al. 2005). in addition, previous studies showed the phytochemical composition and bioactivities of several globba species (andila and tirt 2019; raj et al. 2020). globba macrocarpa gagnep. is a rare species of globba genus. this species was described for the first time by gagnepain (1901) and mainly mailto:vanhongthien@iuh.edu.vn.sk nova biotechnol chim (2022) 21(2): e1322 2 distributed in cambodia, thailand and vietnam (pham 2000; nguyen 2017). in 2020, we conducted some field trips to the binh chau-phuoc buu nature reserve, bung rieng ward, xuyen moc district, ba ria-vung tau province, and encountered a flowering population of g. macrocarpa. to date, the chemical compositions and bioactivities of g. macrocarpa are still unknown. the present study, thus, firstly reported the chemical constituents and antibacterial effects of acetone extracts from the g. macrocarpa rhizomes and aerial parts. experimental plant materials the samples of g. macrocarpa were collected from binh chau-phuoc buu nature reserve, bung rieng ward, xuyen moc district, ba ria-vung tau province, vietnam, location of about 10o32’19.4”e 107o26’57.4”n, 18 m in elevation (fig. 1). fig.1. globba macrocarpa. a – species in habitat, b – flowers, c – rhizome. bacterial strains in this study, the antibacterial properties of acetone extracts of the g. macrocarpa rhizome and aerial parts were investigated by using six bacterial strains such as salmonella typhimurium-atcc 13311, salmonella enteritidis-atcc 13976, pseudomonas aeruginosa-atcc 27853, escherichia coli-atcc 25922, staphylococcus aureus-atcc 25923, and bacillus cereus-atcc 11774. extraction procedures the rhizome and aerial parts of g. macrocarpa were dried at 50 oc until constant weight. the dried samples were pulverized. 250 ml of acetone 99 % solution was used to macerate 50 g of the dried powders at ambient temperature for three days. the studied samples were then filtered through the whatman paper. the process was repeated twice and filtrates were concentrated under the reduced pressure at 60 oc to obtain the brown extracts. the final extracts were subjected to sublimation drying to completely remove the remaining acetone (bobinaitė et al. 2013). gas chromatography/mass spectrometry (gc/ms) assay the trace 1310 gas chromatograph (thermo fisher scientific, waltham, usa) and isq 7000 single quadrupole mass spectrometer was used to identify the chemical compositions in the acetone nova biotechnol chim (2022) 21(2): e1322 3 extract. gc column db-5ms 30 m, 0.25 mm, 0.25 µm (agilent technologies, santa clara, usa) was used and carrier gas was helium with the column flow rate of 1.2 ml.min-1. the specimen was added into the system with the split ratio of 30 : 1, the splitless mode of 1 min, and the split flow of 36 ml.min-1, the inlet temperature of 250 oc. the initial temperature was set for 5 min at 80 oc. the temperature was then raised to 280 oc at the rate of 20 oc.min-1 and hold for 10 min. the electron ionization mode and the ion source temperature were 70 ev and 250 oc, respectively. the mass scan range was 29 – 650 m/z. the nist 2017 library version 2.3 (nist 2017) was used to identify the chemical constituents of studied samples. antibacterial assay the antibacterial properties of the acetone extract of the g. macrocarpa were conducted by disc diffusion assay following the clsi guideline (clsi 2010). luria-bertani broth was used to activate the bacterial strains until their turbidity was equivalent to 0.5 mcfarland. mueller hinton agar plate was inoculated with 0.1 ml of the bacterial culture by spread – plate technique before sterile paper discs containing 10 µl of the extract solution were placed on its surface. gentamycin antibiotic discs (10 µg.ml-1) (nam khoa biotek, ho chi minh city, vietnam) were used as the positive control. the plates were incubated at 37 oc for 18 – 24 h. the antibacterial effects of the studied samples were identified by recording the size of the zone of inhibition. three biological replicates were used for the experiment, and results were expressed as mean ± standard deviation (sd). differences between means groups were calculated by lsd procedure using the statgraphics centurion xv software (statgraphics technologies, inc., the plains, usa) with p < 0.05. results and discussion constituents of g. macrocarpa extracts the acetone extracts isolated from rhizomes and aerial parts of g. macrocarpa in this study contained a total of 50 and 32 components, respectively (table 1 and 2). accordingly, the rhizome extract was characterized by the predominance of germacrene d (15.25 %), 1h-indole, 4-(3-methyl-2-butenyl)(14.33 %), (e)-β-farnesene (11.28 %) and 2-biphenylamine, 3-methyl(10.27 %) (fig. 2a). meanwhile, the aerial part extract was found to be rich of linolenic acid (19.89 %), palmitic acid (13.05 %), phytol (7.52 %) and neophytadiene (4.76 %). previous studies have been reported the biological properties of some compounds obtained from the acetone extracts of g. macrocarpa rhizomes and aerial parts in this study. for instance, germacrene d, a major component from both rhizome and aerial part extracts, possessed the strong insecticidal activities against acyrthosiphon pisum, sitobion avenae, and myzus persicae (bruce et al. 2005). similarly, germacrene d had also potent larvicidal effects against three mosquitos such as anopheles gambiae, culex quinquefasciatus, and aedes aegypti with ld50 values of 1.8, 2.1 and 2.8 mg.cm-3 (ravi ands sita 2007). recently, schepetkin et al. (2020) demonstrated that germacrene d possessed the immunomodulatory activities. accordingly, germacrene d could inhibit neutrophil ca2+ mobilization, chemotaxis, and reactive oxygen species production with ic50 values of 0.51, 5.4 and 9.9 µg.ml-1 (schepetkin et al. 2020). in addition, (e)-β-farnesene, another component from both rhizome and aerial part acetone extracts of g. macrocarpa, showed the effects on natural enemies to cabbage aphid (brevicoryne brassicae) control in chinese cabbage fields (cui et al. 2012). also, this compound has been reported to possessed other biological activities, including repellent and aphicidal activities against myzus persicae (qin et al. 2016). furthermore, α-pinene and β-pinene, two bioactive compounds found in the rhizome and aerial part extracts, has been suggested as the larvicidal and insecticidal agents against aedes aegypti, lasioderma serricorne, and rhodnius nasutus (aparna et al. 2012; wu et al. 2015; carta et al. 2017). palmitic acid, a compound from both rhizome and aerial part acetone extracts of g. macrocarpa, possessed the anti-inflammatory activities (aparna et al. 2012), physiological role, nova biotechnol chim (2022) 21(2): e1322 2 metabolism and nutritional implications in human (carta et al. 2017). fig. 2. gc/ms chromatograms of studied extracts from g. macrocarpa rhizomes (a) and aerial parts (b) with major components. table 1. constituents of extract of g. macrocarpa rhizome. no. rt components [%] no. rt components [%] 1 2.15 tyranton 0.23 26 10.83 γ-amorphene 0.45 2 3.25 α-pinene 0.14 27 10.87 guaia-10(14),11-diene 0.40 3 3.54 camphene 0.69 28 10.91 α-bulnesene 0.22 4 4.06 sabinen 0.38 29 10.95 epicubebol 0.48 5 5.28 isosylvestrene 0.42 30 11.17 germacrene b 0.53 6 5.38 orthodene 0.28 31 11.21 tricyclo[5.1.0.0(2,4)]oct-5-ene-5propanoic acid, 3,3,8,8tetramethyl 0.34 7 5.63 β-ocimene 0.21 32 11.27 isoaromadendrene epoxide 0.59 8 6.36 3-(pyrrolidin-1-yl)cyclopent-2en-1-one 0.56 33 11.40 1h-indole, 4-(3-methyl-2butenyl) 1.65 9 6.93 triacetoneamin 0.14 34 11.51 1-epi-cubenol 0.24 10 7.10 4(1h)-quinolinone, octahydro-1methyl 0.39 35 11.56 aromadendrene oxide-(1) 0.12 11 7.42 alcanfor 0.51 36 11.60 benzeneacetoneitrile, αcyclopentyl 0.21 12 7.97 methyl salicylate 0.11 37 12.01 1h-indole, 4-(3-methyl-2butenyl) 14.33 13 8.12 dihydroanethole 0.77 38 12.38 3-(2-methylbut-3-enyl)-1h-indole 8.01 14 9.89 β-elemen 1.33 39 12.53 quinoline, 2-ter_butyl2.57 15 10.06 α-gurjunene 1.60 40 12.69 2-biphenylamine, 3-methyl10.27 16 10.17 caryophyllene 0.49 41 13.11 3-methyldiphenylamine 0.73 17 10.20 γ-elemene 0.43 42 13.32 palmitic acid 2.52 18 10.32 (e)-β-farnesene 11.28 43 13.41 benzenamine, 4-methyl-nphenyl 0.61 19 10.38 aristolene 0.13 44 14.18 9(e),11(e)-conuated linoleic acid 2.0 20 10.44 humulene 0.43 45 14.21 linolenic acid 1.44 21 10.55 isogermacrene d 0.40 46 15.95 palmitin, 2-mono0.63 22 10.58 (z)-α-farnesene 1.93 47 17.04 9,12-octadecadienoic acid (,),2,3dihydroxypropyl ester 0.93 23 10.62 germacrene d 15.25 48 17.23 stearin, 2-mono0.44 24 10.72 bicylogermacrene 1.35 49 24.13 stigmasterol 3.08 25 10.75 β-bisabolene 0.36 50 25.42 γ-sitosterol 0.94 4 nova biotechnol chim (2022) 21(2): e1322 3 table 2. constituents of extract of g. macrocarpa aerial part. no. rt components [%] no. rt components [%] 1 4.07 β-pinene 0.70 17 12.69 neophytadiene 4.76 2 4.24 cyclohexane, 1,2,4-trimethyl 0.38 18 12.72 3,7,11,15tetramethylhexadec-2-ene 1.05 3 6.37 3-(pyrrolidin-1-yl)cyclopent-2-en-1one 0.77 19 12.82 phytol 0.57 4 6.93 triacetonamin 0.21 20 12.69 neophytadiene 1.13 5 7.01 isophorone 1.62 21 13.32 palmitic acid 13.05 6 7.10 (5r,8ar)-5propyloctahydroindolizine 0.22 22 13.41 2-biphenylamine, 3-methyl3.17 7 8.07 pyrazine, 3-butyl-2,5-dimethyl 0.24 23 14.08 phytol 7.52 8 9.02 5h-1-pyrindine 0.12 24 14.18 linoleic acid 4.36 9 9.17 2-methoxy-4-vinylphenol 0.20 25 14.21 linolenic acid 19.89 10 10.31 (e)-β-farnesene 0.41 26 14.30 octadecanoic acid 1.48 11 10.57 trans-α-bergamotene 0.24 27 15.94 palmitin, 2-mono1.94 12 10.61 germacrene d 1.06 28 17.08 2-monolinolenin 3.93 13 10.68 α-farnesene 2.17 29 23.58 campesterol 0.55 14 11.26 (-)-spathulenol 0.30 30 24.10 stigmasterol 1.04 15 11.99 1h-indole, 4-(3-methyl-2-butenyl)2.84 31 25.39 β-sitosterol 5.80 16 12.52 3-(2-methylbut-3-enyl)-1h-indole 2.91 32 26.37 cholesteryl formate 0.70 several chemical compounds obtained from g. macrocarpa aerial parts and rhizomes in this study have been reported to possess the antimicrobial properties by previous studies. accordingly, simionatto et al. demonstrated that (e)-β-farnesene had antibacterial effects against bacillus subtilis, staphylococcus aureus, salmonella setubal, and pseudomonas aeruginosa (simionatto et al. 2007). in addition, linolenic acid showed strong bacterial activities against staphylococcus aureus, and streptococcus pyogenes (zheng et al. 2005). also, α-pinene presented the antimicrobial against a large amount of bacterial and fungal strains, including pseudomonas aeruginosa, staphylococcus aureus, escherichia coli, streptococcus faecalis, candida albicans, sclerotinia sclerotiorum, mycobacterium smegmatis, cylindrocarpon mali, aspergillus niger, and stereum purpureum (prudent et al. 1993). in another report, α-pinene has been also reported to possess the antibacterial activities against staphylococcus aureus, s. epidermidis, streptococcus pyogenes, and s. pneumonia. moreover, β-pinene had the antibacterial activity against gram-positive bacteria causing potential infectious endocarditis such as staphylococcus aureus, s. epidermidis, streptococcus pyogenes, and s. pneumonia (medeiros et al. 2007). as mentioned above, g. macrocarpa is a rare species. the phytochemicals and biological properties of this plant are limited. however, there are some publications for chemical compositions and antimicrobial effects of other member of the globba species using gs/ms assay. for instance, the major components of ethanol extracts isolated from g. candida rhizomes grown in indonesia included levoglucosan (19.07 %), allylhydrazone acetaldehyde (5.52 %), trans-2,3-epoxybutane (6.30 %), butan-3-enoic acid methyl ester (4.36 %) while the leaf extract has been reported to contain pinostrobin chalcone (75.63 %), 1-(1-(hydroxyphenyl-methyl)-cyclopropyl)-2-phenyl-ethano (2.4 %), benzenepentanal (2.08 %) as the major compounds (andila and tirta 2019). in addition, the chemical profiles of the essential oil extracted from the rhizomes of g. pendula from vietnam were found to be rich in -selinen (36.45 %) and ishwarane (10.76 %) (ngo et al. 2020). furthermore, the chemical compositions of rhizome essential oils of three globba species collected from india, including g. cernua, g. marantina, and g. ophioglossa have been reported (menon and dan 2009). as a result, caryophyllene was the most 5 nova biotechnol chim (2022) 21(2): e1322 2 components in the oils (19.3 – 24.2 %), followed by α-humulene, (z)-nerolidol, and (z,z)-farnesol (menon and dan 2009). antibacterial activities of g. macrocarpa extracts the extracts of g. macrocarpa rhizomes were found to be effective against five oral bacteria, except for e. coli (fig. 3 and table 3). as a result, the rhizome extract showed potent antibacterial effects against p. aeruginosa and s. aureus with the zone of inhibition of 19.0 ± 1.7 and 14.7 ± 2.5 mm, respectively which higher than that of positive control (table 3). also, the rhizome extract possessed moderate effects against b. cereus (14.2 ± 0.8 mm), s. enteritidis (11.2 ± 1.2 mm), and s. typhimurium (10.8 ± 1.1 mm). the data in table 3 and fig. 3 presented the antimicrobial effects of aerial part extracts of g. macrocarpa. among 6 tested bacteria, the extract was found to be effective against four studied bacteria, including p. aeruginosa, b. cereus, s. enteritidis, and s. aureus. generally, the aerial part extract possessed antibacterial effects weaker than that of rhizome extract. this extract only possessed the moderate effects against s. enteritidis with the zone of inhibition of 13.5 ± 1.3 mm while this sample presented the weak antibacterial activities against b. cereus (9.3 ± 0.3 mm), s. aureus (9.3 ± 0.3 mm), and p. aeruginosa (7.2 ± 0.3 mm). table 3. zone of inhibition of acetone extracts from g. macrocarpa against six oral bacteria. microorganism size of zone of inhibition [mm] rhizome aerial part gentamycin b. cereus 14.2 ± 0.8b 9.3 ± 0.3a 22.3 ± 0.6c e. coli 21.3 ± 1.5 p. aeruginosa 19.0 ± 1.7c 7.2 ± 0.3a 13.5 ± 1.8c s. enteritidis 11.2 ± 1.2a 13.5 ± 1.3a 20.3 ± 1.5b s. typhimurium 10.8 ± 1.1a 14.8 ± 1.1b s. aureus 14.7 ± 2.5b 9.3 ± 0.3a 12.3 ± 2.1ab a,b,cthe notable variation (p < 0.05) was represented by distinct superscript lower-case letters in the same row. fig. 3. antibacterial effects of the rhizome (r) and aerial part (a) extracts of g. macrocarpa. a – b. cereus; b – p. aeruginosa; c – s. aureus; d – s. enteritidis; e – s. typhimurium. (-) – negative control, (+) – positive control. 6 nova biotechnol chim (2022) 21(2): e1322 3 the chemical components of hexane and dichloromethane extracts obtained from different parts of g. schomburgkii collected from thailand have been reported. accordingly, hexane extract of rhizomes was characterized by the predominance of β-patchoulene (9.8 %), 3-acetoxy-5-pregnene (7.8 %), and γ-bicyclohomofarnesal (6.4 %) while phytol was the most abundant components in the hexane extract of stalks (13.8 %) and leaves (34.6 %). the major constituents in the hexane extract of flowers were β-caryophyllene (16.5 %) and γ-bicyclohomofarnesal (14.6 %). meanwhile, the most major constituent the dichloromethane extracts of rhizomes, stalks, leaves, and flowers were α-gurjunene (16.3 %), β-caryophyllene (11 %), phytol (19 %), and caryophyllene oxide (15.8 %), respective (doungchawee et al. 2019). in addition, the antimicrobial activities of the hexane, dichloromethane, and methanol extracts from four plant parts of g. schomburgkii have been investigated. all twelve extracts had an inhibitory effect on streptococcus sobrinus. the hexane and dichloromethane extracts displayed activity against streptococcus mutans, salmonella typhimurium, and staphylococcus aureus whereas aspergillus flavus was inhibited by the dichloromethane extracts (leaves, stalks, and flowers) and the methanol extracts of leaves (doungchawee et al. 2019). similarly, the dichloromethane extract and its fractions and sub-fractions obtained from g. schomburgkii rhizomes were mainly composed of γ-bicyclohomofarnesal (4.1 – 20.8 %), (e)-15,16dinorlabda-8(17),12-dien-14-one (7.6 – 58.2 %). moreover, crude dichloromethane extract and its fractions were found to be effective against four pathogenic bacteria, including s. aureus, m. luteus, e. coli, and p. aeruginosa (suekaew et al. 2020). conclusion this study identified 50 and 32 chemical compositions in the acetone extracts of g. macrocarpa rhizomes and aerial parts, in which some components have been reported to possess many biological properties. the rhizome extract was demonstrated to be active against 5 bacterial strains with the diameter of inhibition zones of p. aeruginosa (19.0±1.7 mm), s. aureus (14.7 ± 2.5 mm), b. cereus (14.2 ± 0.8 mm), s. enteritidis (11.2 ± 1.2 mm) and s. typhimurium (10.8 ± 1.1 mm) while the aerial part extract showed antibacterial effects s. enteritidis (13.5 ± 1.3 mm), s. aureus (9.3 ± 0.3 mm), b. cereus (9.3 ± 0.3 mm) and p. aeruginosa (7.2 ± 0.3 mm). the results from the present study can be used as reference for pharmaceutical products and relative fields from g. macrocarpa which could increase the economic valuation of this species. acknowledgement the authors would like to thank ms. thi ngọc anh nguyen, mr. tan loc tran, mr. tri phong nguyen, ms. hong bao nghi nguyen, ms. thuy tien le and ms. thi bao tran nguyen for their cooperation. conflict of interest the authors declare that they have no conflict of interest. references andila p, tirta i (2019) distribution and phytocomponent in the ethanol extract of globba candida gagnep. 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20: 21939-21945. zheng cj, yoo js, lee tg, cho hy, kim yh, kim wg (2005) fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids. febs letters 579: 5157-5162. 8 microsoft word mocak 9-1 2009.doc nova biotechnologica 9-1 (2009) 91 evaluation of iupac limit of detection and iso minimum detectable value electrochemical determination of lead ján mocák1, ivan janiga2, estera rábarová1 1department of chemistry, university of ss. cyril and methodius, nám. j. herdu 2, trnava, sk-917 01, slovak republic (jan.mocak@ucm.sk) 2department of theoretical bases of mechanical engineering, faculty of mechanical engineering, slovak university of technology, nám. slobody 17, sk-81231 bratislava, slovak republic abstract: the way of calculating the limit of detection recommended by iupac is compared to the minimum detectable value used by iso as one of the most important performance characteristics of a measurement process. in this work, theoretical analysis of both characteristics is given together with directions for their practical use. calculations are exemplified using electrochemical trace analysis of lead in surface water. key words: limit of detection, critical value, minimum detectable value, limit of quantification, determination of lead, voltammetric water analysis. 1. introduction capability of detection is an important performance characteristic of a measurement process. in chemistry, a representative characteristic of any analytical method is the smallest concentration or the mass of the analyte (the analyzed sample component) that can be detected with a specified degree of certainty. the related quantity is the limit of detection, lod, defined by international union for pure and applied chemistry (iupac, 1978) first of all as yd = μb + kd σb (1) where yd denotes the lod in the signal domain, μb is the expected mean blank value (the analyte-free sample), σb is the standard deviation of the blank and kd is a proportionality factor. the statistical quantities μb and σb are related to a very large set of observations and are unknown therefore they were in chemical practice commonly approximated by the sample quantities the arithmetic mean by of nb blank measurements, and the blank standard deviation sb. together with the lod further limits have also been invented and used in chemistry. among them, the limit of quantification (or determination), loq, refers to the smallest analyte concentration or mass, which can be quantitatively analysed with a reasonable reliability by a given procedure. its oldest, traditional definition (acs 92 mocák, j. et al. committee, 1980) is similar to that given for lod in the signal domain, but the numerical factor kq is used: yq = μb + kq σb (2) in practice however, a more important task is to know the concentration equivalents of the signal lod and loq values. for this purpose the slope q1 of the linear calibration line, y = q0 + q1x, expressing the dependence of the signal y on concentration x, is commonly used: lod = (yd −⎯ by ) / q1 = kd sb / q1 (3) loq = (yq −⎯ by ) / q1 = kq sb / q1 (4) it is noteworthy that the calibration model should be written in statistics using the variables denoted by capital letters, i.e. y = q0 + q1x, but it is frequently ignored in chemical literature for the sake of simplicity. the proportionality factors kd = 3 and kq = 10 have been traditionally accepted in this approach therefore this way of the lod and loq calculation will be designed in this paper as “traditional approach”. it is imperfect with regard to contemporary statistical theory mainly due to the following drawbacks (mocak et al., 1997): (1) the normal distribution is used for typically small sets of observations, supposing equality μb = by and σb = sb, (2) an exact intercept value is considered in calibration, supposing q0 = by , unaffected by random errors. despite these mistakes, mainly due to its simplicity and a long term usage, the traditional approach has been still used even though newer, more correct ways have been described in the literature, especially in the documents recommended by iupac (currie, 1994a, b; 1995; mocak et al., 1997). situation in this field is rather complicated also due to some terminological inconsistency introduced in the past. some authors (demanding equality of the α and β errors) used the proportionality factor 6 for the limit of detection and the factor of 3 assigned to the limit of decision; others suggested to keep the way of the lod calculation as explained above (with kd = 3) and utilized the proportionality factor 6 for the limit of identification, loi (alternatively named also as the limit of guarantee of purity). a more detailed discussion on this topic can be found e.g. in the iupac document (mocak et al., 1997). the aim of this work is to explain in a statistically correct way the calculation of the lod and further limits characterizing the measurement method in chemistry in the region of low concentrations (trace analysis) and, in addition, to compare these limits to analogical characteristics defined by international standards organization (iso, 2002), which is nowadays universally accepted as a leading metrological institution in many branches of science and technology. moreover, it will be recommended in the conclusion part of this work what performance characteristics should be implemented when evaluating measurements in chemical trace analysis and a guideline will be proposed regarding the use of appropriate terminology. nova biotechnologica 9-1 (2009) 93 2. theory 2.1. limit of detection obtained by upper limit approach the upper limit approach, ula, is fundamental newer way of the lod and loq calculations. it is based on the following details (mocak et al., 1997; mocak and bobrowski, 2000): (a) it makes use of the one-sided upper confidence limit for a future individual observation (green, 1978; massart et al., 1988), which defines the one-sided prediction band in calibration for predicting the maximum possible signal value, induced by random errors in measurement. the signal value corresponding to the zero analyte concentration (the blank) is here used at the 100(1−α) % probability level. (b) as the most suitable, the significance level (1−α) = 0.99 was recommended, which guarantees the best agreement of this calculation procedure with the previous traditional approach. (c) the signal variance is assumed constant (homoscedastic) therefore the sample standard deviation is considered constant at zero as well as non-zero concentrations (but not too far from zero). consequently, the residual standard deviation syx, expressing the regression error, is used instead of the blank standard deviation sb. (d) the values of the multiplication factors kd and kq are not fixed but depend on the number of experiments performed, which is reflected by the critical value of tdistribution utilized in calculation. (e) the concentration lod and loq values are estimated from the corresponding signals rigorously by regression using the inverse calibration model, x = f−1(y). it means that the lod and loq calculations are calibration model dependent. the concentration lod and loq values for the general straight line calibration model, y = q0 + q1x, are given as: lod = [t(n−2,1−α) syx / q1 ] [1 + 1/n + 2x / 1 ( = n i ∑ xi − x ) 2 ]1/2 (5) loq = 3 [t(n−2,1−α) syx / q1 ] [1 + 1/n + 2x / 1 ( = n i ∑ xi − x ) 2 ]1/2 (6) where n concerns the number of calibration points involved in the regression procedure, t(n−2,1−α) is the critical tvalue for the number of degrees of freedom (equal to n−2) and the significance level 0.99, xi is the i-th point of the concentration coordinate, x is the mean concentration of all points used in calibration. residual standard deviation syx represents the error in regression and is given by the squared deviation of ˆiy , calculated in regression, from yi, measured in calibration experiment: 2 2 0 1 1 1 1 1ˆ( ) ( ) 2 2 n n yx i i i i i i s y y y q q x n n= = = − = − − − −∑ ∑ (7) using this approach, the multiplication factor kd is defined as: kd(n,α) = t(n−2, 1−α) [1 + 1/n + 2x / 1 n i= ∑( xi − x ) 2 ] 1/2 (8) 94 mocák, j. et al. with regard to equations (5) and (6) there exists a simple relation between two multiplication factors: kq(n,α) = 3 kd(n,α). if the calibration experiment is performed in the equidistant manner, it is possible to utilize the corresponding entries of table 1 for a quick calculation of the lod and loq. moreover, the lod and loq values can be then calculated in a way similar to the traditional approach using simple multiplication formulae: lod = kd(n,α) syx / q1 ; loq = kq(n,α) syx / q1 = 3 lod (9a,b) the c(n) and b(n) terms introduced in table 1 depend exclusively on the concentration values and are independent of the signal values therefore they can be pre-calculated. the only condition to assure the validity of this simplified calculation procedure is to keep strictly the equidistant allocation of all calibration points, including the blank value (x = 0). 2.2. critical value and minimum detectable value a performance characteristic of any measurement process, characterizing the capability of detection, has been defined by (iso, 2000 a,b; 2007) as the minimum detectable value, mdv. another characteristic defined by iso is the critical value, cv. according to iso 11843-2, the concentration of analyte in the laboratory sample is named the state variable, z, since it represents the state of the material being analyzed. an analyte-free material is considered to be in the basic state. the difference between the state variable, z, and its value in the basic state is called the net state variable, denoted by x. the state variable or the net state variable cannot be observed directly, but they are related to an observable response variable, y, via calibration function f, defined by the mathematical relationship y = f(z) or y = f(x), representing the mathematical model of the measurement. in chemical calibration measurements, the response variable y is usually an instrument signal. the investigated analyte concentration is finally calculated by means of the evaluation function f−1, which represents the inverse of the calibration function, x = f−1(y). the iso critical value, xc, and the minimum detectable value, xd, of the net state variable are defined (janiga et al., 2004, 2006) by the equations cv = 2 0 1 1 1 ( 1 ) 1cc xx ˆ ˆy q x x t v, ˆ ˆq q i j s σ α − = = − + + (10) mdv = 2 1 1 ( , , ) 1d xx ˆ x x q̂ i j s σ δ ν α β= + + (11) where t(ν, 1−α) denotes a (1−α) % quantile of the tdistribution with ν = (i j – 2) degrees of freedom, i number of states (calibration standards), j number of parallel measurements, δ − non-centrality parameter of the non-central t-distribution, σ̂ − estimated residual standard deviation of the calibration line (identical to syx in part 2.1 for n = i j). nova biotechnologica 9-1 (2009) 95 table 1. the k d factors and auxiliary quantities used in the lod and loq calculation for α = 0.01 (onesided) and the number of experiments n assuming equidistant calibration* ) n ν (1+1/n)1/2 c(n) b(n) t(ν, 0.99) kd(ν, 0.99) 3 1 1.15470 0.50000 1.35401 31.821 43.086 4 2 1.11803 0.45000 1.30384 6.965 9.081 5 3 1.09545 0.40000 1.26491 4.541 5.744 6 4 1.08012 0.35714 1.23443 3.747 4.625 7 5 1.06905 0.32143 1.21008 3.365 4.072 8 6 1.06066 0.29167 1.19024 3.143 3.741 9 7 1.05409 0.26667 1.17379 2.998 3.519 10 8 1.04881 0.24546 1.15994 2.897 3.360 11 9 1.04447 0.22727 1.14812 2.821 3.239 12 10 1.04083 0.21154 1.13792 2.764 3.145 13 11 1.03775 0.19780 1.12904 2.718 3.069 14 12 1.03510 0.18571 1.12122 2.681 3.006 15 13 1.03280 0.17500 1.11430 2.650 2.953 16 14 1.03078 0.16544 1.10813 2.624 2.908 17 15 1.02899 0.15686 1.10258 2.602 2.869 18 16 1.02740 0.14912 1.09758 2.583 2.836 19 17 1.02598 0.14211 1.09304 2.567 2.806 20 18 1.02470 0.13571 1.08891 2.552 2.779 21 19 1.02353 0.12987 1.08512 2.539 2.756 22 20 1.02247 0.12451 1.08165 2.528 2.734 23 21 1.02151 0.11957 1.07844 2.518 2.715 24 22 1.02062 0.11500 1.07548 2.508 2.698 25 23 1.01980 0.11077 1.07274 2.500 2.682 26 24 1.01905 0.10684 1.07019 2.492 2.667 27 25 1.01835 0.10317 1.06781 2.485 2.654 28 26 1.01770 0.09975 1.06558 2.479 2.641 29 27 1.01709 0.09655 1.06350 2.473 2.630 30 28 1.01653 0.09355 1.06155 2.467 2.619 32 30 1.01550 0.08807 1.05800 2.457 2.600 34 32 1.01460 0.08319 1.05480 2.449 2.583 36 34 1.01379 0.07883 1.05200 2.441 2.568 38 36 1.01307 0.07490 1.04940 2.435 2.555 40 38 1.01242 0.07134 1.04710 2.429 2.543 ∞ ∞ 1.00000 0.00000 1.00000 2.326 2.326 *) t(ν, 0.99) denotes the critical tvalue ; ν = n − 2 ; b(n) = [1 + 1/n + c(n)] 1/2 ; ( ) ( ) ( ) .x/x/xx/xnc n i n i ii∑ ∑ = = −=−= 1 1 222 11 symbol sxx conventionally represents the sum 2 1 ( ) i i i xxs j x x = = −∑ (12) 96 mocák, j. et al. a considerable similarity of eqs. (5) and (10) is evident. moreover, it should be noted that for the sake of simplicity (appreciated by a practical chemist) some symbols in the lod calculation recommended by iupac (mocak et al., 1997) were simplified using e.g. q0, q1 and syx instead of 0q̂ , 1q̂ and σ̂ (or yxσ̂ ); another change valid for this work is using x for concentration instead of c. 3. material and methods all chemicals used were of analytical reagent grade; distilled and de-ionised water was used in all measurements. lead(ii) ions in surface water were determined by differential pulse anodic stripping voltammetry (dpasv) using an autolab/pgstat 20 electrochemical instrument, the netherlands. a standard three-electrode voltammetric cell with the static mercury drop electrode smde-1 (laboratorní přístroje, prague), a reference 3 mol/l silver-silver chloride electrode, and a platinum wire auxiliary electrode were used in all electrochemical measurements. the following parameters were set for the pb(ii) determination: deposition potential −1 v, equilibration time 10 s, modulation time 0.04 s, interval time 0.1 s, initial potential −0.8 v, end potential −0.2 v, step potential −0.002 v, modulation amplitude −0.05 v, and temperature 25 ± 0.5 °c. experimental data sampled by the electrochemical instrument were written to the computer hard disk and finally processed by microsoft excel and origin (microcal software, inc., northampton, ma) software. 4. results and discussion 4.1. comparison of iupac and iso measurement characteristics the calibration experiment was designed in the following way: (a) blank solution and seven standard solutions with a non-zero concentration were used in the concentration range 0–1.40 ppb pb(ii) (i = 8). (b) four replicate measurements (j = 4) of the signal (dpasv current) were performed four each solution (including blank) so that altogether n = i j = 8×4 = 32 measurements were performed. (c) two additional signal measurements were made for the calculations of the lod and loq values by “traditional approach” so that altogether six blank measurements were made and nb = 6 was used in eqs. (3) and (4). (d) the obtained values of signal and concentration were processed by linear regression and the corresponding quantities were calculated using the equations shown above. since the regression calculation in “traditional approach” is usually made without the blank measurements (which create here a distinct data set), the blank signals were not included so that n = i j = 7×4 = 28 in this case. the most important results and values characterizing the measurement performance, mainly lod, loq, cv and mdv, obtained for determination of lead in surface water, are summarised in table 2 together with the needed auxiliary quantities. nova biotechnologica 9-1 (2009) 97 several important inferences are available when exploring the results shown in table 2 (where in its entries more decimal figures are left than necessary only due to a better comparison): (a) exactly equal lod and cv values are not surprising since by a thorough inspection of eqs. (5) and (9) it can be found that these equations are identical. (b) in the given case, the lod calculated by traditional approach is not very different from the correct lod (and cv) value but it may be by chance more different, higher or lower, since it depends on the differences between sb and syx as well as 3 and t(ν, 0.99). (c) the found minimal detectable concentration is a bit smaller than the doubled lod value but the difference is small (0.73 %); it is explained by the fact that the non-centrality parameters δ of the non-central t-distribution are not too different from the doubled critical tvalues assuming equal α = β and the same (not too small) number of degrees of freedom. table 2. determination of lead by dpasv comparison of the lod and loq values with the critical value, cv, and the minimum detectable value, mdv * ) traditional approach ula (iupac recommended) iso nb 6 n 32 n 32 ⎯yb 19.2917 ν 30 ν 30 sb 0.47726 syx 0.58427 σ̂ 0.58427 3sb 1.4318 i 8 10sb 4.7726 j 4 q0 19.5186 q0 19.4067 q0 19.4067 q1 7.2437 q1 7.3557 q1 7.3557 t(ν, 0.99) 2.457 t(ν, 0.99) 2.457 t(ν, 0.95) 1.697 kd 3.741 δ(ν, 0.01,0.01) 4.879 kq 11.223 δ(ν, 0.05,0.05) 3.367 x 0.7000 x 0.7000 sxx 6.7200 sxx 6.7200 lod, ppb 0.1977 lod, ppb 0.2051 cv (xc), ppb 0.2051 loi, ppb 0.3953 2 lod, ppb 0.4102 mdv (xd), ppb 0.4072 loq, ppb 0.6589 loq, ppb 0.6153 *) the measured quantity with the corresponding unit of the blank signal mean by and the blank standard deviation sb is the maximum dpasv peak current in na. the ratio of the signal unit to the concentration unit (ppb) makes the slope unit. the loi and loq in traditional approach are defined as 6sb / q1 and 10sb / q1, resp. the residual standard deviation is defined as: 2 0 1 1 1 1ˆ ˆ ˆ( ) 2 i j ij i i j y q q x i j σ = = = − − ⋅ − ∑ ∑ . the most needed values δ can be found in table 3 otherwise 2 t(ν,1−α) may be approximately used. (d) if α = β = 0.05 were used in the iso approach then the 98 mocák, j. et al. resulting mdv would be smaller by 31 % however, it was justified in the iupac recommendation (mocak et al., 1997) that a better choice for chemical measurements should be α = 0.01 otherwise the lod and further limits used in chemical trace analysis would be too low, i.e. too optimistic. the best design of the calibration experiment is equidistant with at least one standard, except the blank, located in the region around the loq. the equidistant design allows using of very simple eqs. (9a,b) with the kd factor found in the last column of table 1. the usage of the calibration points at the concentration levels much higher than the limits of detection and quantification is sometimes made in practice but is not permissible (massart et al., 1988) because of errors caused by a distant extrapolation. therefore very often a special calibration experiment is necessary for determining the lod and loq, which is different from that used in a routine laboratory work. 4.2. recommendation what limits to use as mentioned in the first two parts of this work, there are nowadays known and defined several performance characteristics of a measurement process in chemical trace analysis due to the existence of iupac as well as iso standards, recommendations and guidelines. therefore it is reasonable to select those of them which are most important with regard to current trends of their utilization. consequently, we would like to propose three main performance characteristics for a general use: (1) the limit of detection, lod, as an equivalent of cv but much more widespread in chemical literature, (2) the minimum detectable value, mdv, the iso quantity widely widespread in science and technology, (3) the limit of quantification, loq, as an unique chemical characteristic relevant to the smallest measure at which quantitative analysis is possible. 5. conclusions newer way of calculation of the limit of detection, lod, and limit of quantification, loq, recommended by iupac (mocak et al., 1997), overcomes the problems caused by statistical incorrectness of by now still used traditional way of calculation and can be applied easily as shown in this paper. correct lod and loq values can be obtained by equations (5) and (6) or, which is the simplest way, using eqs. (8) and (9a,b) under condition that an equidistant calibration design is realized. in such a case the proportionality factor kd can be found from table 1 and the kq factor is computed as its triple. the iso defined critical value, cv, is identical to the lod defined by eq. (5). the minimum detectable value, mdv, is calculated by means of eq. (11) using the relevant non-centrality parameter δ of the non-central t-distribution, accessible in table 3 for common cases. otherwise the non-centrality parameter δ can be approximated by the doubled critical tvalues assuming equality α = β and the same number of degrees of freedom ν. under these conditions 2 lod represents a good mdv approximation. the calculation possibilities are demonstrated in table 2. nova biotechnologica 9-1 (2009) 99 table 3. values of the non-centrality parameter δ (v, α, β) of the non-central t-distribution for v degrees of freedom and significance levels α and β (here α = β ). v δ (v, 0.05, 0.05) δ (v, 0.01, 0.01) v δ (v, 0.05, 0.05) δ (v, 0.01, 0.01) 2 5.516 15.217 39 3.349 4.824 3 4.456 9.338 40 3.347 4.819 4 4.067 7.520 41 3.346 4.815 5 3.870 6.683 42 3.344 4.811 6 3.752 6.213 43 3.343 4.807 7 3.673 5.915 44 3.342 4.803 8 3.617 5.710 45 3.341 4.800 9 3.575 5.562 46 3.339 4.797 10 3.543 5.449 47 3.338 4.793 11 3.517 5.361 48 2.337 4.790 12 3.496 5.290 49 3.336 4.787 13 3.479 5.232 50 3.335 4.785 14 3.464 5.184 51 3.334 4.782 15 3.451 5.143 52 3.334 4.779 16 3.440 5.108 53 3.333 4.777 17 3.431 5.077 54 3.332 4.774 18 3.423 5.051 55 3.331 4.772 19 3.415 5.027 56 3.330 4.770 20 3.408 5.006 57 3.330 4.768 21 3.402 4.987 58 3.329 4.766 22 3.397 4.971 59 3.328 4.764 23 3.392 4.955 60 3.328 4.762 24 3.388 4.942 70 3.322 4.746 25 3.383 4.929 80 3.318 4.734 26 3.380 4.917 90 3.315 4.724 27 3.376 4.907 100 3.312 4.717 28 3.373 4.897 120 3.309 4.706 29 3.370 4.888 150 3.305 4.695 30 3.367 4.879 200 3.301 4.685 31 3.365 4.871 300 3.297 4.674 32 3.362 4.864 400 3.295 4.669 33 3.360 4.857 500 3.294 4.665 34 3.358 4.851 600 3.293 4.663 35 3.356 4.845 700 3.293 4.662 36 3.354 4.839 800 3.293 4.661 37 3.352 4.834 1000 3.292 4.659 38 3.350 4.829 ∞ 3.290 4.653 acknowledgement: financial support of this work by the grants vega 1/1005/09 and vvce-0004-07 is highly acknowledged. references acs committee of environmental improvement: guidelines for data acquisition and data quality evaluation in environmental chemistry. anal. chem., 52, 1980, 2242-2249. 100 mocák, j. et al. currie, l.a., horwitz, w.: iupac recommendations for defining and measuring detection and quantification limits. analysis, 22, 1994, m24-m26. currie, l.a., svehla, g.: nomenclature for the presentation of results of chemical analysis. pure appl. chem., 66, 1994, 595-608. currie l. a.: nomenclature in evaluation of analytical methods including detection and quantification capabilities. iupac recommendations 1995. pure appl. chem., 67, 1995, 1699-1723. green, j.r., margerison, d.: (1978). statistical treatment of experimental data. amsterdam. elsevier. chapter 14.3. iso 11843-2:2000: capability of detection − part 2: methodology in the linear calibration case. international organization for standardization (01-may-2000). 24 pp. corrigendum: iso 11843-2/cor1:2007. iupac, analytical chemistry division: nomenclature, symbol, units and their usage in spectrochemical analysis. ii. data interpretation. spectrochim. acta, 33 b, 1978, 241-246. iupac: comission v5 web site (1999): http://users.unimi.it/ape/v5. janiga, i., garaj, i., cisko, p.: estimation of minimum detectable value by using linear calibration with constant standard deviation. forum metricum slov., 8, 2004, 68-73. janiga, i., mocak, j., cisko, p., garaj, i., szarková, d.: detection of the mercury content in food and medicines by means of linear calibration funktion. forum statisticum slov., 2, 2006, 67-72. massart d.l., vandeginste, b.g.m., deming, s.n., micotte, y., kaufman, l.: chemometrics: a textbook. elsevier, amsterdam, 1988, p. 86-91; p. 113-114. mocak, j., varga, s., polak, p., gergely, s., izak, j.: calibration some newer chemometrical aspects in instrumental analysis. wissenschaftliche zeitsch., 32, 1990, 43-49. mocak, j., bond, a. m., mitchell, s., schollary, g.: a statistical overview of standard (iupac and acs) and new procedures for determining the limits of detection and quantification: application to voltammetric and stripping techniques. pure appl. chem., 69, 1997, 297-328. mocak, j., bobrowski, a. determination of cadmium and lead in water – new recommended way of evaluating the limits of detection and quantification. water sci. technol., 1, 2001, 19-26. stn iso 11843-2: detekčná schopnosť. časť 2: metodika lineárnej kalibrácie (in slovak). stn (slovak technical standard), bratislava, 2002. 46 nova biotechnologica et chimica 12-1 (2013) doi 10.2478/nbec-2013-0005 © university of ss. cyril and methodius in trnava application of isotopic dilution and single-step extractions for labile soil zinc determination vladimír frišták, martin pipíška, tatiana gablovičová, juraj lesný department of ecochemistry and radioecology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (vladimir.fristak@ucm.sk) abstract: concentration of available zinc from soils is the primary concern in assessment of its toxicity or essentiality for plants. this study evaluates the changes in chemical extractable zn from three slovak typical soils with simultaneous extractions as tools of zinc bioavailability. we found out that extractability of binding zinc decreased in order na2edta, mehlich 3, mehlich 2, nh4no3 and cacl2 for all soil samples. using flow-through stripping chronopotentiometry (scp) and atomic absorption spectrometry (gfaas) we found out that maximum of soil zinc was removed by organic ligands. lability of zn determined by isotopic dilution method using 65zn and γ-spectrometry showed the significant decrease of isotopic exchangeable zinc fraction (e-value) with decrease of soil reaction. obtained e-values of uppermost soil horizons showed the zinc lability ranged from 20 to 39%. our research confirmed the effect of soil reaction, composition and physico-chemical characteristics to zn lability. for further assessment of zinc bioavailability is needed to find the correlation and effects of structural changes and aging in studied soils. key words: soil, zn, bioavailability, extraction, e-value 1. introduction zinc is an essential trace element for the biological systems and is getting into the soil weathering of minerals and rocks forming the subsoil of the environment. major form is divalent ion zn2+ that is involved into sorption interactions of individual soil components. the increased concentrations of zinc can be potential risk of phytotoxicity. many elements involved as trace elements at higher than optimal concentrations can be toxic (frišták et al., 2013). soil contamination is not directly limited but can be reasonably associated with the negative anthropogenic effects as one of the major environmental problems. generally, the mobility of trace metals in soils is a function of its chemical forms (vijver et al., 2003). bioavailability may be defined as a fraction of metal that is available or can be made available for uptake of organisms (van gestel, 2008). mobility and bioavailability of heavy metals in soil depend on their speciation or fractionation. luoma and rainbow (2005) showed factors of metals bioavailability such as soil characteristics, routes exposure and physiological attributes. bioavailability can be also affected by the application of fertilizers and soil conditioners, which can significantly change the soil reaction (ph), thus uptake of element by plants acceptors. generally, the higher value of ph of the soil can cause more mobile and accessible elements forms for plant uptake (frišták et al., 2012). as a tool for estimation and determination of bioavailable metals fraction and metal fractionation in soils are commonly used simultaneous and sequential extraction bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:49 utc nova biotechnologica et chimica 12-1 (2013) 47 protocols as parts of fraction analysis. non selectivity of extracting agents and redistribution of elements among phases during extraction are disadvantages of these methods (tessier et al., 1979). despite a weak selectivity, chemical extraction methods are still often used to provide an understanding of mobility and bioavailability of metals (hullebusch et al., 2005). direct methods have generally insufficient sensitivity for metal speciation analysis and give the quantity of metal ions in solution together with those in the solid phase which are in equilibrium with the dissolved metal ions. therefore, determination of exchangeable metal fraction (evalue) represents quantification of ion pool from which ambient living organisms may take up the metal (sterckeman et al., 2009). the main aim of our paper was to describe distribution, leaching and bioavailability of zinc in soil samples from three typical slovak areas by series of simultaneous extraction protocols. for obtaining of relevant data and comparing the method the isotopic dilution with 65zn was used. 2. materials and method 2.1 experimental available fractions of zinc in samples of soil extract were measured using flowthrough stripping chronopotentiometry. experimental measurements were carried out on electrochemical analyser ecaflow model glp 150 (istran, ltd., bratislava, slovakia) equipped with electrochemical cell of type 104 with auxiliary, ag/agcl reference and e-104l graphite porous working electrodes. used parameters are given in table 1. obtained results were compared with reference values of gfaas measurements by atomic absorption spectrophotometer shimadzu aa-7000 (usa). for correction of background d2 lamp was used, after microwave digestion of the samples by the multiwave system mw 3000 (anton paar gmbh, aus). to calculate bioavailable zinc and obtain parameters of paired t-test, origin 8.0 professional (originlab corporation, northampton, usa) was used. table 1. operation parameters of electrochemical determination of zinc by galvanostatic stripping chronopotentiometry analysis (scp). parameter unit value deposition potential mv -1800 quiescence potential mv -1800 quiescence time 1 s 5 quiescence potential 2 mv -1400 quiescence time 2 s 30 terminal potential mv 100 stripping current µa 200 stand by potential mv 0 2.2 reagents and solutions all the chemicals used were of analytical reagent grade. deionized water (<0,05 μs/cm) prepared by simplicity 185 (millipore, france) was used for the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:49 utc 48 frišták et al. preparation of all solutions. for electrochemical analysis, carrier electrolyte r-008 (0.01 mol/l ch3cooh + 0.01 mol/l ch3coona + 0.2 mol/l nacl) and background electrolyte r-013 (0.1 mol/l hcl) were used. the bulk standard solution and calibration solutions of zinc were prepared in the background electrolyte from certified reference material (999 mg/l znno3, sigma-aldrich, germany). 2.3 sampling and sample preparation three typical slovak soil types were obtained from localities jaslovské bohunice (jb, calcic phaenozem), sihla (s, haplic cambisol) and borský mikuláš (bm, haplic arenosol). the related sampling sites are shown in figure 1. the samples were collected from horizon in depth of 0-20 cm. before the subsequent analytical procedures the soil samples were dried (at 22 °c), homogenised and sieved through a 2 mm sieve. samples were stored at dark and dry place. fig. 1 sampling sites of investigated soils. 2.4 physico-chemical characterisation of soil samples for actual soil ph, 5 g of air-dried soil samples was allowed to equilibrate in 25 ml of deionised water. flask was agitated on reciprocal shaker (150 rpm) for 1 h. to determine potential soil ph, 25 ml of 1 mol/l kcl was added to 5 g of air-dried soil samples and allowed to equilibrate for 1 h. after 1 h consolidation, ph values of supernatants were measured using a ph glass electrode (inolab ph/ion/cond 750, wtw, france). total carbonate content in soil samples was obtained by lime-analyser of own construction (gablovičová et al., 2012). the cation exchange capacity (cec) of samples was determined according to the stn iso 11260 modified by frišták et al. (2013). soil samples were suspended (83.3 g/l) in 0.1 mol/l bacl2 and mixed on a laboratory shaker for 1 h at 22°c and 150 rpm. phases were separated after centrifugation (5 min at 5000 rpm). this procedure was repeated twice. the next bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:49 utc nova biotechnologica et chimica 12-1 (2013) 49 step of cec determination consisted of suspending of soil sediments in 3 ml of 0.025 mol/l bacl2 and agitation for 19 h at 22°c. after phases separation, 3 ml of 0.02 mol/l mgso4 was added to sediments. after agitation (19 h at 22°c), centrifugation and phases separation, cec by standardized 0.02 mol/l na2edta was determined. the cec values were determined by chelatometric determination of mg2+ ions in liquid phase and calculated according equation: ( ) 3 00 10− − = εvmvvmcec (1) where cec is cation exchange capacity (meq/100g), m0 is molar concentration of magnesium added to the sample (mol/l), v0 is volume of solution of magnesium added to the sample (l), m is molar concentration of the magnesium in the leachate (mol l1), vv is volume of obtained extract (l) and ε is the conversion factor that has for the bivalent ions and amount 0.25 g of soil value 800 meq (100 g/mol). 2.5 total (aqua regia extractable) zn fraction pseudo total soil content of zinc was estimated by aqua regia digestion (3:1, v/v, hcl:hno3). this digestion procedure is considered adequate for analysing totalrecoverable heavy metals in soils (meers et al., 2007). residual elements that are not released by aqua regia digestion are mostly bound to silicate minerals and are considered unimportant for estimating the mobility and behaviour of metals (niskkavaara et al., 1997). 2.6 simultaneous extraction protocols mobile zn-fractions were obtained after simultaneous extractions with 0.01 mol/l cacl2, 1 mol/l nh4no3, 0.05 mol/l na2edta, mehlich 2 (0.2 mol/l ch3cooh + 0.2 mol/l nh4cl + 0.015 mol/l nh4f + 0.012 mol/l hcl) and mehlich 3 (0.2 mol/l ch3cooh + 0.25 mol/l nh4no3 + 0.013 mol/l hno3 + 0,015 mol/l nh4f + 0.001 mol/l edta). extraction conditions of all cationic exchange and complexation procedures are given in table 2. table 2. extraction conditions of single-step extraction protocols used in experiments extraction solution e:s * ratio equilibration time [h] ph reference 0.01 mol/l cacl2 10:1 2 6 donner et al. (2010) 1 mol/l nh4no3 10:1 2 6 meers et al. (2007) mehlich 3 10:1 2 2.5 mehlich (1984) mehlich 2 10:1 2 3 mehlich (1984) 0.05 mol/l na2edta 10:1 2 3 tica et al. (2011) *e:s is a ratio between extractant phase and solid phase simultaneous extractable fractions were obtained by mixing of soil samples (1 g) with 10 ml of extractant on laboratory shaker (150 rpm) for 2 h at 22 °c. after bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:49 utc 50 frišták et al. centrifugation (10 min, 8000 rpm), 5 ml of supernatant was added to bank with 20-30 ml of reagent solution r-013 and 0.5 ml 0.01 mol/l kmno4. the solution was heated to 80-95 °c for 5-10 min. the oxalic acid was added to reduce the excess of permanganate. the volume of samples was adjusted to 50 ml with the r-013 reagent solution. solid particles were removed by filtering, centrifugation or sedimentation. zinc analysis in purified extracts was subsequently performed using stripping chronopotentiometry by electrochemical analyser ecaflow model glp 150 (istran, ltd., bratislava, slovakia). obtained data were compared with data obtained by gfaas analyse. all the analyses were carried out in triplicates. 2.7 isotopic dilution with 65zn (e-values) isotopic dilution with 65zn was used to monitor changes in the labile zn fraction over time. the procedure was based on the method of goldberg and smith (1984) using 0.05 mol/l cacl2 for displacement of soil zinc and top horizons of studied soils. to 2.5 g of soil samples, 25 ml of 0.05 mol/l cacl2 was added. to minimize the microbial activity, 0.1 ml of chcl3 was added. flasks were agitated 5 days at 22 ± 2 °c and 150 rpm. calcium chloride increased the zn concentration in solution, making its determination more precise. process of chemical equilibrium was characterized by determination of exchangeable zinc in 5 ml aliquots by galvanostatic stripping chronopotentiometry (scp). these values were assigned to be the neutral salt extractable zn concentration. the suspensions were subsequently spiked with 0.1 ml of 65zn (as zncl2) solution of 25 kbq/ml (czech institute of metrology, praque, czech republic). spiked suspensions were shaken for 5 days. after centrifugation (5 min, 4000 rpm) radioactivity in 3 ml of solid-free solution was measured by gamma spectrometric assembly using a well-type scintillation detector 54bp54/2-x, nai(tl) (scionix, the netherlands) and the data processing software scintivision 32 (ortec, usa). all measurements were carried out in triplicates. the quantities of the isotopically exchangeable zn in soil samples (zne) were calculated according to equation: s s s e znr r zn ].[⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ = (2) where rs is radioactivity introduced in the suspension (bq/ kg d.w.) and rs is the radioactivity measured in reaction solution at the end of experiment (bq/ kg d.w.) and [zn]s is the mean concentration of extractable zn in the soil extract as determined by scp (mg/kg d.w.). 3. results and discussion the soil samples used in experiments varied widely in soil properties and characteristics (table 3). determination of soil ph noted the slightly acidic character of the soil sample s, and low alkaline character of soil sample jb. soil reaction of soil sample bm varied between 5.9 and 6.8, depending on the method of determination. the sub-alkaline ph of jb sample, likely played a crucial role in reducing the zn bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:49 utc nova biotechnologica et chimica 12-1 (2013) 51 levels in soil solution. a decrease of zn solubility with increasing ph has been reported by many authors (frišták et al., 2012; pardo and guadalix, 1994; zambella and adamo, 2010). carbonate content was in the range from 0.5 to 0.7 in all soil samples. kiekens (1990) studied the reversibility of the exchange reaction between ca and zn and found that a key fraction of zn was irreversibly bound by the soil components and following adsorption of zn2+ and desorption of ca2+ during zn2+/ca2+ exchange process. associated with the wide range in soil texture, cec value varied from 5.9 to 18.1 mmol/100g. specific adsorption reactions, adsorption sites dependent on ph and reversibility of zinc binding may contribute to selective or major zn adsorption (maes, 1973). the determination of pseudo total zinc concentrations in studied soil samples by gfaas analyses confirmed the increasing trend in order bm < s < jb. the uppermost parts of the rich soil on organic matter and humus contain increased concentration of heavy metals (gablovičová et al., 2012). table 3. physico-chemical properties of studied soil samples. soil sample pseudo total zn [mg/kg] phh2o phkcl cec [mmol/100g ] caco3 [%] jb 75 8.3 7.5 18.1 0.7 bm 12 6.8 5.9 5.9 0.5 s 61 4.6 3.8 6.0 0.5 application of aqua regia extraction confirmed the lowest concentration of zinc (12 mg/kg) in bm soil samples obtained from haplic arenosol area with high content of calcium clays, dust, sand, conglomerate, coal beds and seams of lignite. chemical extraction methods are widely used to assess the release of contaminants from soils, sludges, and sediments. as extracting agents in extraction procedures, strong acid and their mixtures (hno3, h2so4, hcl, aqua regia), neutral solution of salts (cacl2, mgcl2, nacl), pending puffer and complex-forming agents (na2edta) can be used (meers, 2007). however, no single method is recognized universally (tica et al., 2011). available zinc concentrations, expressed as a pore water concentration or concentrations extractable with agent of ion exchange were lower in comparison with other tested extracting agent (figure 2a). the other extraction protocols can be used for the assessment of potentially toxic metals released from soils to the water recipients and their potential availability to all biological systems (mclaughlin et al., 2000). for determination of soil zinc mobile fractions 5 extracting agents were used. we tried to quantify the zinc content in extracts using stripping chronopotentiometry. zinc was deposited on porous electrode from acidic solution with high efficiency dependent on deposition potential. obtained data were compared to values from gfaas as reference method (figure 2b). coefficient of determination (r2) of linear correlation was 0.998 and it showed the comparability of used analytical methods. paired t-test also confirmed nonsignificance of differences between used methods and thus statistical significance of obtained data of extractable zinc (α=0.05). the extractability of zn obtained with the nh4no3 protocols was in general higher than that obtained with the cacl2 protocols for all soil samples. low alkaline sample bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:49 utc 52 frišták et al. jb released zn easily in reaction system of nh4no3 in comparison to studied acidic agents. effect of this extraction protocol can be attributed to the possible complexation of zn by nh3 (lebourg et al., 1998) and higher ionic strength of extracting agent. extraction efficiency of acidic agents increased in order mehlich 2, mehlich 3 and na2edta for all soil samples. chelating effect of used extraction agent caused significant increasing of extractable zinc from exchange sites, carbohydrate and organic matter [9]. we confirmed the extraction efficiency of organic ligands on wide range of soil components. jb bm s 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 [z n] so lu te / [z n] to ta l . 10 0 [% ] soil samles a 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 c r ef er c exp b fig. 2. efficiency of simultaneous extraction protocols of zinc (mg/kg) from the three soil samples (jb, bm, s). extraction conditions: 2 h, 22°c, 150 rpm, 100g/l, extracting agents: cacl2 ( ), nh4no3 ( ), mehlich 2 ( ), mehlich 3 ( ), na2edta ( ). all experiments were carried out in triplicates. error bars represent standard deviation of the mean (±sd) (a). comparison obtained concentrations of extractable zinc determined by electrochemical analyses (cexp) with reference values determined by gfaas analyses (cref) (b). naidu and harter (1998) showed that metal extracted by a mixture of organic acids is well-correlated with the mobile metal fraction in the soil solution. metal fraction extracted by organic acids is more available for biological system compared to other complexing agents. low molecular weight organic acids such as ethylene diamine tetraacetic acid (edta) and their salts (na2edta) were reported to remove metals organically bound, occluded in oxides, and associated with secondary clay minerals (paya-perez et al., 1993). effect of soil reaction was less significant. we found out that simultaneous extraction protocols had maximal efficiency for low acidic soil sample of haplic arenosol (bm). thus we confirmed the effect of soil composition as a parameter of zinc availability from this typical slovak soil. for next investigation of labile or potentially available zn fraction in the studied soil samples, the isotopic dilution (e-value) was used. the studied samples of uppermost horizons contain the highest pseudo total concentration of zinc. the evalues reported in table 4 show how much the zn in soil samples was isotopically exchangeable within experimental equilibrium period. zinc availability in three soil samples ranged from 20 to 39%. these values were comparable with concentrations of na2edta extractable zinc. degryse and smolders (2006) reported e-values of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:49 utc nova biotechnologica et chimica 12-1 (2013) 53 zinc equivalent to 32-49 % lability in upper horizons of acid soils (ph 3.3-4.5). on the other hand, donner et al. (2010) showed the exchangeable fraction of zinc in the range from 12 to 15% for agricultural soils with ph from 5 to 6.6. our study confirmed decreasing concentration of labile soil zinc with increased soil reaction of samples in order haplic cambisol, haplic arenosol and calcic phaenozem. measurements by radioisotope dilution with 65zn demonstrate a significant of soils ph and variation of zn bioavailability depending on soil type and physic-chemical characteristics. further work is needed to assess the importance of soil amendments and long-term ageing in terms of zinc bioavailability. table 4. e-values of soil samples jb , bm and s depending on ph and total zn concentration [mg/kg]. all experiments were carried out in triplicates, mean ± sd. soil sample jb bm s ph 8.3 6.8 4.6 total zn [mg/kg] 75.0 ± 6.0 12.0 ± 0.9 61.0 ± 4.7 zn e-value [mg/kg] 15.1 ± 1.9 2.51 ± 0.2 23.8 ± 1.3 labile zn [%] 20.1 20.9 39.01 4. conclusions as tools of available zinc fraction determination, single-step extraction methods can be used. our paper confirmed the effect of physico-chemical properties (ph) of soil samples and applied extraction protocol to extractable zinc concentration. we found out that extractability of binding zinc decreased in order na2edta, mehlich 3, mehlich 2, nh4no3 and cacl2 for studied soil samples. flow-through stripping chronopotentiometry (scp) and atomic absorption spectrometry (gfaas) confirmed the maximum of removable soil zinc by organic ligands. lability of soil zn can be determined by isotopic dilution method using radioactive isotope 65zn and γspectrometry. determination showed the significant decrease of isotopic exchangeable zinc fraction (e-value) with decrease of soil reaction. e-values of studied soil samples showed the zinc ability in range from 20 to 39%. our study confirmed the effect of soil reaction, composition and physico-chemical characteristics to zn lability. acknowledgment: this work was financially supported by university of ss. cyril and methodius in trnava (project fppv-01-2013). references frišták, v., pipíška, m., horník, m., augustín, j., lesný, j.: sludge of wastewater treatment plants as co2+ ions sorbent. chem. pap., 67, 2013, 265-273. vijver, m.g., vink, j.p.m., miermans, c.j.h., van gestel, c.a.m.: oral sealing using glue: a new method to distinguish between intestinal and dermal uptake of metals in earthworms. soil biol. biochem., 35, 2003, 125-132. van gestel, c.a.m: physico-chemical and biological parameters determine metal bioavailability in soils. sci. total environ., 406, 2008, 385-395. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:49 utc 54 frišták et al. luoma, s.n., rainbow, p.s.: why is metal bioaccumulation so variable? biodynamic as a unifying concept. environ. sci. technol., 39, 2005, 1921-1931. frišták, v., valovčiaková, m., pipíška, m., augustín, j.: simultaneous and sequential extraction protocols as tools for determination of zinc bioavailability in 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niskavaara, h., reimann, c., chekushin, v., kashulina, g.: seasonal variability of total and easily leachable element contents in topsoils (0-5 cm) from eight catchments in the european arctic (finland, norway and russia). environ. pollut., 96, 1997, 261-274. donner, e., broos, k., heemsbergen, d., warne, m.s.j., mclaughlin, m.j., hodson, m.e., nortcliff, s.: biological and chemical assessments of zinc ageing in field soils. environ. pollut., 158, 2010, 339-345. mehlich, a.: mehlich-3 soil test extractant: a modification of mehlich-2 extractant, comm. soil sci. plant anal., 15, 1984, 1409-1416. tica, d., udovic, m., lestan, d.: immobilization of potentially toxic metals using different soil amendments. chemosphere, 85, 2011, 577-583. goldberg, s.p., smith, k.a.: soil manganese: e value, distribution of manganese 54 among soil fractions, and effects of drying. soil sci. soc. am. j., 48, 1984, 559-564. pardo, m.t., guadalix, m.e.: chemical factors affecting selenite sorption by allophanic soils. geoderma, 63, 1994, 43-52. zampella, m., adamo, p.: chemical composition and zn bioavailability of the soil solution extracted from zn amended variable charge soils. j. environ. sci., 22, 2010, 1398-1406. kiekens, l.: heavy metals in soils, blackie usa and canada halsted press, new york, 1990. maes, a.: ion exchange of some transition metal ions in montmorillonites and synthetic faujasites, doctoral thesis, louvain, 1973. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:49 utc nova biotechnologica et chimica 12-1 (2013) 55 mclaughlin, m.j., hamon, r.e., mclaren, r.g., speir, t.w., rogers, s.l.: review: a bioavailability-based rationale for controlling metal and metalloid contamination of agricultural land in australia and new zealand. aust. j. soil res., 38, 2000, 1037-1086. lebourg, a., sterckeman, t., ciesielski, h., proix, n.: trace metal speciation in three unbuffered salt solutions used to assess their bioavailability in soil. j. environ. qual., 27, 1998, 584-590. naidu, r., harter, r.d.: effects of different organic ligands on cadmium sorption and extractability from soils. soil sci. soc. am. j., 62, 1998, 644-650. paya-perez, a., sala, j., mousty, f.: comparison of icp-aes and icp-ms for the analysis of trace elements in soil extracts. int. j. environ. anal. chem., 51, 1993, 223-230. degryse, f., smolders, e.: mobility of cd and zn in polluted and unpolluted spodosols. eur. j. soil sci., 57, 2006, 122-133. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:49 utc microsoft word hornik et al nb 2008.doc nova biotechnologica 8-1 (2008) 55 bioaccumulation of 137cs and 60co in freshwater plants miroslav horník, martin pipíška, jozef augustín department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (hornikm@ucm.sk) abstract: contamination of the aquatic environment by the heavy metals and radionuclides has become a serious concern in the world. in our study, gamma-spectrometry of freshwater plants bacopa monnieri and egeria densa growing in cultivation media spiked with 137cscl and 60cocl2 was used for quantitative determination of bioaccumulation kinetic and distribution cs+ and co2+ ions in plant tissues. we found, that bioaccumulation of cs and co by fully immersed b. monnieri in hoagland media (hm) was dependent on ion concentration in medium. approx. 5-times lower cs uptake 2.9 nmol/g (d.w.) was obtained in plants cultivated in 20% hm than from deionized water. the maximal co uptake was 4-times higher than cesium uptake at the same conditions. both cs and co were localized mainly in roots. the highest immobilization from roots to shoots was found in the case of co uptake from deionized water with concentration ratio [co]leaves : [co]stem : [co]root = 1.00 : 5.33 : 56.8. cesium uptake by submerged plant e. densa was also strongly dependent on nutrients concentration in medium. however, in the case of cobalt uptake this dependence was less pronounced. nutrients concentration also had a significant influence on distribution of cs between stems and leaves of e. densa. cesium was localized in leaves, however with increasing of nutrients concentration in cultivation media cs was localized for account of stem. on the other hand, cobalt was immobilized mainly in leaves in whole range of nutrients concentration. obtained data can serve as a models for understanding of phytoaccumulation of radionuclides from open water ponds and water channels in the vicinity of nuclear power plants and monovalent and bivalent metals from industrial sources of contamination. keywords: cesium, cobalt, 137cs, 60co, bioaccumulation, freshwater plants 1. introduction heavy metals are toxic pollutants released into the surface and ground water as a result of different activities such as industries, mining, and agriculture. at present, a number of technologies can be used to remove of heavy metals from contaminated water such as filtration, reverse osmosis, solvent extraction, adsorption, chemical precipitation and ion-exchange (cheremisinoff, 2002). however, these methods are not efficient in removing low heavy metals concentrations, can be relatively expensive and may fail to achieve legal limits. contrary to this, phytoremediation, i.e. removal of pollutants by the use of plants offers a promising technology for heavy metal removal from waste water (sternberg, 2007; fiol et al., 2005). aquatic macrophytes have a great potential to accumulate heavy metals inside their plant body (prasad, 2007; otte and jacob, 2006). most studies on pollutant bioaccumulation in macrophytes are aimed at assessing removal efficiency or toxic effects without taking into account the metal bioaccumulation process by macrophytes, key knowledge not only to understand the behavior of macrophytes but also to optimize effluent depuration by means of artificial wetlands. wetlands have significant merits of low capital 56 horník, m. et al. and operating costs compare with conventional system as activated sludge, aerated lagoon system etc.. this technology has been used mainly in the case of mine waters (see e.g. lesley et al., 2008; lesley and younger, 2007; batty et al., 2005). the nuclear revolution (weapons testing or accidents at power production) has resulted in the large-scale release of cesium into the environment, in particular l34cs (τ = 2.07 y) and the long-lived radionuclide 137cs (τ = 30.2 y). radiocesium 137cs persists in aquatic systems, with environmental half-lives in monomitic lakes, meromitic lakes and flowing rivers 1.2, 6.7 and 1.4 years, respectively (avery, 1996). its almost unlimited solubility in aquatic systems and chemical similarity to potassium means that it can be easily assimilated by terrestrial and aquatic organisms (mould, 2000; kerpen, 1986). large variations in the activity concentrations of 137cs from chernobyl nuclear accident in fish and lake water were still being observed in the 1990s and even in the 2000s (saxén and ilus, 2008; saxén, 2007). cobalt usually occurs in the environment in association with other metals such as copper, nickel, manganese and arsenic. cobalt released by human activities comes mainly from: nickel, copper, silver, lead and iron mines and refineries; metal production facilities; industrial boilers that burn coal and oil; vehicles that burn gasoline; and incinerators that burn refuse and sewage sludge (perez-espinoza et al., 2005). cobalt, as microelement for methanogenic bacteria, not for higher plants, can be also trapped in anaerobic bottom sediments. cobalt 60co (τ = 5.27 y) is also present in low-level radioactive wastes (caron and mankarios, 2004). our previous papers were oriented to bioaccumulation of radionuclides by terrestrial vascular plants from defined water solutions (horník et al., 2007; barátová et al., 2006). the objectives of this study are the investigation of 137cs and 60co uptake and their distribution in roots, stems and leaves of freshwater plants bacopa monnieri and egeria densa, in order to evaluate the role of nutrient concentration in bioaccumulation processes. b. monnieri is an emergent, wetland macrophyte. it grows very fast with its creeping stem in wetlands as a weed. it is well known from ancient times for its medicinal properties having active ingredients like bacosides and bacopasides. e. densa is an introduced invasive species, mainly via the aquarium trade. also, this invasive submerged species is distributed in lakes and rivers of europe. 2. materials and methods 2.1 plant material freshwater plants bacopa monnieri and egeria densa were obtained from common aquarium shops. one week before experimentation, plants were pre-cultivated in 15 dm3 aquaria filled with 50% hoagland medium (hm), (hoagland, 1920) at artificial illumination with 12h/12h light/dark cycle (illumination with 2 tubes brilliant daylight 6000 k, 1 300 lm and tropic sun – 4 700 k, 1 000 lm; sera, d) and at 22±2°c. for experiments, healthy green plants of comparable weight (0.4-0.5 g nova biotechnologica 8-1 (2008) 57 fresh weight; f.w.) were used and three-times washed in deionized water before experiments. the following molar concentrations of salts were presented in full-strength hm (mm): mgso4.7h2o – 1.5; kno3 – 4.0; cacl2 – 4.0; nah2po4.2h2o – 1.87; na2hpo4.12h2o – 0.13; feso4.7h2o – 0.06; nano3 – 4.0; nh4cl – 3.17; nh4no3 – 2.0; h3bo3 – 0.14; na2moo4.2h2o – 0.0025; mnso4.5h2o – 0.21; znso4.7h2o – 0.023; cuso4.5h2o – 0.033; ph 6.5. 2.2 bioaccumulation experiments for bioaccumulation experiments, freshwater plants b. monnieri or e. densa (0.40.5 g; f.w.) were placed on the bottom in 100 cm3 erlenmayer flasks containing 50 cm3 deionized water or hm diluted with deionized water in the ratio 1:1, 1:3, 1:4, 1:7, 1:11 or 1:25, and spiked with both 137cscl and 60cocl2. cultivation was carried out at artificial illumination with 12h/12h light/dark cycle (2 000 lx) and 22±2°c under occasional shaking. in time intervals, aliquots were taken and the remaining radioactivity in the cultivation media was estimated. at the end of the experiments, plants were washed in deionized water, dried at 60°c during 48 h and incorporated radioactivity of 137cs or 60co in leaves, stems or roots was measured by gammaspectrometry. all the experiments were performed in triplicate series. 2.3 radiometric analysis for radiometric determination 137cs and 60co in plants and cultivation media gamma-spectrometric scintillation detector 54bp54/2-x with well type crystal nai(tl) (scionix, nl) with data processing software scintivision32 (ortec, usa) were used. counting time 600 s allowed obtaining data with measurement error <2 %, which do not reflect other source of errors. standardized solutions of 137cscl (5.723 mbq/cm3; 20 mg/dm3 cscl + 3 g/dm3 hcl) and 60cocl2 (5.571 mbq/cm 3; 20 mg/dm3 cocl2 + 3 g/dm 3 hcl) were obtained from czech metrology institute (cz). 2.4 speciation modeling prediction of cs and co speciation in the nutrient solutions as a function of the total salt concentrations, solution ph and temperature was performed using the software visual minteq ver. 2.53. this speciation model allows the calculation of the composition of solution, in regard to formation of metal complexes. 3. results and discussion freshwater plants may take up nutrients from the sediments by roots or may absorb nutrients from the water column by foliar uptake. determining the principal mode of toxic metals and radionuclides uptake by macrophytes is important because it affects the interpretation of the role of macrophytes in aquatic systems. if root 58 horník, m. et al. uptake is the principal mode, then the metals and radionuclides content of the macrophytes represents remobilization from the sediments. if foliar uptake is the principal mode, then the metals and radionuclides content of the macrophytes represents a reduction in the water column concentration with the macrophytes serving as at least a temporary sink for these pollutants (kelly and pinder iii, 1996). however, in these processes the speciation of metals and radionuclides in both water and sediment can play an important role. for example, madruga and carreiro (1992) found in tejo river water, that 60co was almost 100% in cationic forms, however, in the presence of sediment there was a decrease in proportion of cationic forms (to 50%), with some anionic forms appearing. 0 1 2 3 4 5 6 7 8 0 4 8 12 16 20 b io ac cu m ul at io n of c s+ [n m ol /g ]; d. w . time [d] 20% hm deionized water 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 b io ac cu m ul at io n of c o2 + [n m ol /g ]; d. w . time [d] 20% hm deionized water fig. 1. bioaccumulation kinetics of cs+ ions (a) and co2+ (b) by freshwater plant b. monnieri (25 g/dm3; f.w.). fully immersed plants were cultivated in deionized water or 20% hoagland medium (hm), ph 6.5 containing 0.12 µmol/dm3 cscl (17 kbq/dm3 137cscl) and 0.11 µmol/dm3 cocl2 (13 kbq/dm3 60cocl2) at light/dark cycle 12h/12h (2 000 lx) and at 22±2°c. total uptake of cs: deionized water – 8.3%; 20% hm – 0.9%. total uptake of co: deionized water – 35%; 20% hm – 6.3%. data represent average of three independent experiments. bioaccumulation kinetics of cs+ and co2+ ions by emergent freshwater plant bacopa monnieri are presented in fig. 1a, b. under given experimental condition, i.e. the initial concentration c0 = 0.12 µmol/dm 3 cscl and biomass concentration 25 g/dm3 (f.w.) at 22°c the maximum cesium uptake was observed after 24 h and was stable within the next 7 days. as can be seen from fig. 1a, bioaccumulation of cs is dependent on ion concentration in medium. cesium uptake 14.1 nmol/g (dry weight; d.w.) was obtained in deionized water after 8 days cultivation. approximately 5-times lower 2.9 nmol/g (d.w.) was found at plants cultivation in 20% hm, where concentration of competing k+ and nh4 + ions was 0.8 and 1.2 mmol/dm3, respectively. nevertheless, as can be calculated by visual minteq speciation program, cesium in 20% hm at ph 6.5 occurs practically as free cation (> 99% cs+). monovalent k+ and nh4 + ions act competitively on cesium uptake by plants. vascular plants can accumulate cs+ ions through both their leaves and through their roots. substrate affinity i.e. km values for cs + uptake in shoots and roots of winter wheat is 25.5 and 16.7 μmol/dm3 respectively. according to shaw et al. (1992) the measured transfer factor of roots is one order of magnitude greater than that of shoots. smith et al. (2003) in a whole-lake experiment at study to reduce the bioaccumulation of radiocesium 137cs in fish in lakes contaminated a b nova biotechnologica 8-1 (2008) 59 by the chernobyl accident found, that after the addition of 15 t of potassium chloride to lake svyatoe, kostiukovichy resulted in a decrease in activity concentration of 137cs to approximately 40% of pre-countermeasure values in a number of different fish species. however, the addition of 4 kg 133cscl into an 11.4-ha, 157 000 m3 reservoir previously contaminated with 137cs from past reactor operations at the us department of energy’s savannah river site near aiken, south carolina (usa) increased of 6.1 mbq of 137cs (1.9 mg 137cs) in the water column (pinder iii et al., 2006; 2005). these authors supposed, that possible sources for the increased 137cs included release from the sediments (the principal source), release from the approx. 26 000 kg of aquatic macrophytes that occupied 80% of the reservoir, and wash-in from the pond’s watershed. 0 2000 4000 6000 8000 10000 deionized water s pe ci fic r ad io ac tiv ity 1 37 c s [b q/ g] ; d .w . 20% hoagland medium root stem leaves 0 20000 40000 60000 80000 100000 120000 140000 deionized water s pe ci fic r ad io ac tiv ity 6 0 c o [b q/ g] ; d .w . 20% hoagland medium root stem leaves fig. 2. specific radioactivities of 137cs (a) and 60co (b) in root, stem and leaves of b. monnieri after 8 days cultivation of fully immersed plants in deionized water or 20% hm, ph 6.5 containing 0.12 µmol/dm3 cscl (17 kbq/dm3 137cscl) and 0.11 µmol/dm3 cocl2 (13 kbq/dm3 60cocl2) at light/dark cycle 12h/12h (2 000 lx) and at 22±2°c. data represent average of three independent experiments. bioaccumulation kinetic of co2+ ions by b. monnieri was identical with the process of cs bioaccumulation at the same conditions, but only in the case of plants cultivation in 20% hm (fig. 1b). cobalt in 20% hm at ph 6.5 occurs practically as free cation (> 93% co2+). when plants were cultivated in deionized water containing 0.11 µmol/dm3 cocl2, the bioaccumulation of cobalt gradually increased to the maximum 62 nmol/g (d.w.) in 5 day of cultivation, and then slowly decreased to the value 54 nmol/g (d.w.) in 8 day of plants cultivation. the bioaccumulation of co is also dependent on ion concentration in medium. it can be concluded, that cobalt was accumulated in more extent than cesium practically at the same conditions. adam and garnier-laplace (2003) found similar results at accumulation of 137cs and 60co by freshwater alga cyclotella meneghiana. from results of cesium distribution in plant organs of b. monnieri we found (fig. 2a), that cesium is mainly localized in roots than in stems and leaves with concentration ratio [cs]leaves : [cs]stem : [cs]root = 1.00 : 0.69 : 1.76 at plants cultivation in 20% hm and [cs]leaves : [cs]stem : [cs]root = 1.00 : 1.22 : 2.14 at plants cultivation in deionized water. similarly, cobalt was mainly immobilized in roots with concentration ratio [co]leaves : [co]stem : [co]root = 1.00 : 0.39 : 1.49 at plants cultivation in 20% hm (fig. 2b). in the case of plants cultivation in deionized water a b 60 horník, m. et al. cobalt was much more immobilized in roots with concentration ratio [co]leaves : [co]stem : [co]root = 1.00 : 5.33 : 56.8. its generally known, that at emergent species of water plants root uptake is presumed to be the dominant mechanism of nutrients absorption. in this regard, mineral nutrients, mainly macroelements (ca and mg), from 20% hm are probably preferentially accumulated by roots than cobalt. 0 1 2 3 4 5 6 7 8 0 10 20 30 40 b io ac cu m ul at io n of c s+ [n m ol .g -1 ]; d. w . time [d] deionized water 8,3% hm 12,5% hm 25% hm 50% hm 0 1 2 3 4 5 6 7 8 0 20 40 60 80 100 120 b io ac cu m ul at io n of c o2 + [n m ol .g -1 ]; d. w . time [d] deionized water 8,3% hm 12,5% hm 25% hm 50% hm fig. 3. bioaccumulation kinetics of cs+ (a) and co2+ (b) ions by e. densa (20 g/dm3; f.w.). fully immersed plants were cultivated in deionized water or diluted hm (8.3%, 12.5, 25% or 50%), ph 6.5 containing 0.12 µmol/dm3 cscl (17 kbq/dm3 137cscl) and 0.11 µmol/dm3 cocl2 (13 kbq/dm3 60cocl2) at light/dark cycle 12h/12h (2 000 lx) and 22±2°c. total cs uptake (%): deionized water – 46.4; 8.3% hm – 5.7; 12.5% hm – 6.4; 25% hm – 2.1; 50% hm – 3.3. total co uptake (%): deionized water – 70.3; 8.3% hm – 66.8; 12.5% hm – 72.0; 25% hm – 81.8; 50% hm – 70.6. data represent average of three independent experiments. 0 10 20 30 40 50 0 20 40 60 80 100 b io ac cu m ul at io n of c s+ [n m ol /g ]; d. w . hm concentration [%] 0 20 40 60 80 100 b io ac cu m ul at io n of c o2 + [n m ol /g ]; d. w . 0 10 20 30 40 50 0 20 40 60 80 100 0 20 40 60 80 100 ra tio [c o] fo rm /[c o] to ta l hm concentration [%] cs+ co2+ coso 4 cohpo 4 ra tio [c s] fo rm /[c s] to ta l fig. 4. a. dependence of bioaccumulation of cs+ (-■-) and co2+ (-○-) ions by e. densa (20 g/dm3; f.w.) on dilution of hoagland medium at ph 6.5 after 8 d cultivation of fully immersed plants at light/dark cycle 12h/12h (2 000 lx) and 22±2°c. for details see fig. 3. b. the percentage (ratio of concentration metal form to the total concentration of metal) of cesium in the form of cs+ and cobalt in the forms co2+ ions, coso4 or cohpo4 in dependence on dilution of hoagland medium at ph 6.5 and 22°c calculated by the speciation program visual minteq. as can be seen from fig. 3a,b and fig. 4a, cs+ uptake by freshwater submerged plant egeria densa is strongly dependent on ion concentration in medium. however in the case of cobalt uptake this dependence was less pronounced. cesium uptake 46% (32.8 nmol/g; d.w.) in deionized water decreased to 6% (6.0 nmol/g; d.w.) in 8.3% a b a b nova biotechnologica 8-1 (2008) 61 hm, where concentration of competing k+ and nh4 + ions was 0.33 and 0.5 mmol/dm3, respectively. on the contrary, uptake of co2+ ions increased with concentration of monovalent and bivalent cations in hm, however at highest concentration, i.e. 8 mmol/dm3 me+ ions and 3 mmol/dm3 me2+ ions in 50% hm cobalt uptake slightly decreased. the speciation of cesium and cobalt in individual cultivation media at ph 6.5 and 22°c can be seen in fig. 4b. 0 10 20 30 40 50 0 1 2 60co [6 0 c o] st em /[ 60 c o] le av es accum ulation in leaves hm concentration [%] accum ulation in stem 137cs 0 1 2 [1 37 c s] st em /[ 13 7 c s] le av es fig. 5. influence of dilution of hoagland medium on 137cs (-■-) and 60co (-○-) distribution in stems and leaves of e. densa (20 g/dm3; f.w.) after 8 d cultivation of fully immersed plants at light/dark cycle 12h/12h (2 000 lx) and 22±2°c. data expressed as the ratio of concentration in stems to the concentration in leaves (bq/g / bq/g; d.w.). for details see fig. 3 concentration of mineral nutrients had also a significant influence on distribution of 137cs between stems and leaves of submerged plant e. densa (fig. 5). in the case of plants cultivation in deionized water containing 0.12 µmol/dm3 cscl cesium was localized manly in leaves, however with increasing of nutrients concentration in cultivation media cesium was localized for account of stem. on the other hand, cobalt was immobilized mainly in leaves of e. densa in whole range of nutrients concentration. 4. conclusions cesium and cobalt in contaminated water systems undergo concentration equilibrium in contact with water plants, occurring in nearly all water bodies. obtained data from short-time laboratory experiments showed, that presence of monovalent and bivalent ions in defined cultivation media significantly influenced the bioaccumulation and distribution of 137cs+ and 60co2+ ions in freshwater plants bacopa monnieri and egeria densa. these results can serve for understanding of phytoaccumulation of radionuclides from open water ponds and water channels in the vicinity of nuclear power plants and monovalent and bivalent metals from industrial sources of contamination. 62 horník, m. et al. references adam, ch., garnier-laplace, j.: bioaccumulation of silver-110m, cobalt-60, cesium-137, and manganese-54 by the freshwater algae scenedesmus obliquus and cyclotella meneghiana and by suspended matter collected during a summer bloom event. limnol. oceanogr., 48, 2003, 2303-2313. avery, s.: fate of caesium in the environment: distribution between the abiotic and biotic components of aquatic and terrestrial ecosystems. j. environ. radioact., 30, 1996, 139-171. barátová, z., sekáčová, j., horník, m., pipíška, m., augustín, j.: bioaccumulation of 137cs and 60co by mustard sinapis alba l. and celery apium graveolens l.. nova biotechnol., vi-i, 2006, 7-18. batty, l.c., atkin, l., manning, d.a.c.: assessment of the ecological potential of mine-water treatment wetlands using a baseline survey of macroinvertebrate communities. environ. pollut., 138, 2005, 413-420. caron, f., mankarios, g.: pre-assessment of the speciation of 60co, 125sb, 137cs and 241am in a contaminated aquifer. j. environ. radioact., 77, 2004, 29-46. cheremisinoff, n.p.: handbook of water and wastewater treatment technologies. butterworth-heinemann, woburn, ma, usa, 2002, 631 p. fiol, n., serarols, j., poch, j., martínez, m., miralles, n., villaescusa, i.: low cost materials for metal uptake from aqueous solutions. in: lichtfouse, e., schwarzbauer, j., robert, d. 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(ed.) phytoremediation: methods and reviews, humana press, totowa, new jersey, usa, 2007, 185-203. microsoft word vidova 9-1 2009.doc nova biotechnologica 9-1 (2009) 63 multiplex pcr method for detection of variability in genes encoding the alpha proteins of streptococcus agalactiae barbora vidová1, 2, michal chotár2, jozef timko2 andrej godány2, 3 1institute of biochemistry, nutrition and health, slovak university of technology, sk-812 37 bratislava, slovak republic (barbora.vidova@savba.sk) 2institute of molecular biology, slovak academy of sciences sk-845 51, bratislava, slovak republic 3department of biotechnology, university of ss. cyril and methodius, sk-917 01 trnava, slovak republic abstract: the majority of streptococcus agalactiae strains express one or more surface-anchored proteins that vary by strain. these proteins, which are characteristic for s. agalactiae, and are able to induce protective antibodies, include the alpha c, rib proteins, alpha-like protein 2, and alpha-like protein 3. in this study was developed multiplex pcr method for detection of genes encoding these proteins, and its occurrence within a various s. agalactiae isolates of bovine origin. also are reported two new genes from bovine isolates of s. agalactiae amplified by pcr, encoding other putative members of the family, alphalike protein 6, and alpha-like protein 7. they contain an overall genetic organization highly similar to that of the alpha c and rib proteins. key words: streptococcus agalactiae, alpha-like family, variability, multiplex pcr 1. introduction the importance of streptococcus agalactiae as a major perinatal pathogen for invasive disease has been well documented (baker and edwards, 2000; zaleznik et al., 2000; ferrieri, 1997). the s. agalactiae first received attention as a cause of bovine mastitis, a disease defined as ‘inflammation of the mammary gland’, that cause the losses in a diary industry associated with clinical and subclinical mastitis, which arise from the costs of treatment, culling, death and decreased milk production (bradley, 2002). epidemiological studies of s. agalactiae infections are mainly based on capsule serotyping. the capsule is an antigenic determinant and a major virulence factor as it interferes with complement mediated killing (edwards et al., 1982). the s. agalactiae strains are classified into nine different serotypes based on differences in the capsular polysaccharide antigens ia, ib, and ii through viii (paoletti et al., 2000). serotype distribution varies with geographical region and ethnic origin, and the virulence of clinical isolates with similar capsular composition can also vary widely, suggesting that other bacterial virulence factors, except a capsule, are involved in the pathogenesis of s. agalactiae (spelleberg, 2000). in addition, several surface–anchored proteins that vary by strain have been described for s. agalactiae (moyo et al., 2002; kvam et al., 1995). 64 vidová, b. et al. the proteins include the immunoglobulin a-binding beta c protein (cβ) encoded by bac, the alpha c protein (cα) (bevanger and maeland, 1979) encoded by bca (michel et al., 1992), and the proteins r1, r3, and r4 (flores and ferrieri, 1996, 1989; lancefield and perlmann, 1952). the cα and the r proteins belong to a family of so-called “ladder-forming” proteins, a designation based on the banding patterns generated on western blotting (wästfelt et al., 1996). some of the best characterized protein antigens used as candidate vaccines belong to the alpha protein family, reviewed by lindahl et al. (2005). these major antigens are encoded by allelic genes. five different alleles, named alp2, alp3, rib, r28 and epsilon (alpha-like proteins) have been described (lachenauer et al., 2000; lachenauer and madoff, 1996; wästfelt et al., 1996; stålhammarcarlemalm et al., 1993). all these proteins are encoded by stable mosaic genes, generated by a recombination of modules at the same chromosomal locus (lachenauer et al., 2000). the proteins exhibit size variation between strains depending on the number of repeats in the corresponding gene. moreover, it has been demonstrated that, in the course of infection, the number of repeats inside the alpha c proteins can undergo internal deletions as a means for evading the host immune response (madoff et al., 1996). diversity of s. agalactiae strains has been analyzed using a broad range of methods. aside from serotyping and multilocus enzyme electrophoresis (mlee) (musser et al., 1989), a method based on primary structures of proteins, other typing methods used are dna based. these include ribotyping (blumberg et al., 1992), random amplified polymorphism (limansky et al., 1998), pulsed field gel electrophoresis (rolland et al., 1999; gordillo et al., 1993) and more recently, multilocus sequence typing (mlst) (jones et al., 2003). the study of surface proteins and their genes encoding sequences is important from the aspect of epidemiological analysis of the infections caused by streptococcus agalactiae, and simultaneously, surface proteins hold capability to be used in vaccines against this pathogen (maione et al., 2005; larsson et al., 2004; paoletti and madoff, 2002; brodeur et al., 2000). the aim of the present study was to analyse the gene content, which could indicate distribution of surface proteins, in the set of bovine s. agalactiae isolates. this was performed by multiplex pcr method for detection of genes bca, rib, alp2/3, alp4, and alp5 directly according the pcr products sizes. 2. materials and methods 2.1 bacterial strains and media the streptococcal isolates were obtained from different farms in slovakia (147 strains) and from czech republic (5 strains), and could be identified as streptococcus agalactiae belonging to lancefield’s serological group b. the identification was performed by pcr identification (chotár et al., 2006). s. agalactiae cultures were grown overnight on todd-hewitt (biomark) broth at 37°c on rotary shaker. nova biotechnologica 9-1 (2009) 65 2.2 preparation of bacterial cell suspension for pcr one milliliter of overnight culture was transferred in sterile tube and centrifuged at 10.000 rpm for 3 min, pellet was washed twice with sterile distilled water, once with 0.1 m phosphate buffered saline, ph 7.2 (pbs), and then resuspended in 1 ml of deionized sterile water to allow the burst of bacterial cells. samples of 2 μl of these preparations were used directly for pcr. 2.3 pcr conditions reactions were carried out in final volume of 20 µl. the reaction mixture contained 0.5 u dynazyme™ dna polymerase (finnzymes), 0.4 µl 10 mm dntp mix (finnzymes), 4 µl optimized dynazyme™ 10x reaction buffer (finnzymes). primers listed in table 1 were used in concentrations: 0.5 μm of universal forward primer saganf, and reverse primers were added as follows 0.16 μm of saga4, 0.4 μm of sagarib, 0.7 μm of sagaalpc, 0.9 μm of saga2/3, and 1.3 μm of saganr. all dna oligonucleotides were synthesized by sigma. table 1. oligonucleotide primers used in this study. primers sequences 5´-> 3´ size (bp) reverse: sagaalpc tat atg tgg tag tcg atc ttc acc 428 sagarib cac act gaa ctt tta aac caa gtg a 325 saga2/3 cat tca gat tat tat aat ata tag cac 630 saga4 tta atat gca ctg gat taa ctc cac 140 saganr cgc gga tcc atc ctc ttt ttt ctt aga aac 852 forward: saganf gga att cca taa tgt tta gaa ggt cta aaa a two microliters of bacterial suspension was used as a template. the amplification program consisted of denaturation at 96°c for 5 min, 30 cycles of denaturation at 96°c for one minute, annealing at 55°c for one minute and extension at 72°c for two minutes, followed by a final extension at 72°c for 8 minutes. ten microliters of pcramplified product was analyzed by electrophoresis in 0.9 % agarose gel stained with ethidium bromide. this was carried out in 1x bbe (0.65 m boric acid, 29 mm sodium tetra borate, 250 mm edta, ph 7.8). pcr amplicons were analyzed by uv transluminization. 2.4 dna sequence analysis pcr products were sequenced and compared with genebank sequences using blast (basic local alignment search tool) (altschul et al., 1990). the new sequences generated during this study have appeared in genebank with the following accession numbers: dq629924 (alp6) and dq629925 (alp7). previously published gene sequences used in this study and their genebank accession numbers are as follows: m97256 (bca), u58333 (rib), ay345596 (alp1), aj488912 (alp4) and ay461799 (alp5). 66 vidová, b. et al. 3. results and discussion alpha proteins of streptococcus agalactiae have typical repetition region consisted of about 80 amino acids, which are highly homologous (lindhal et al., 2005). while these repeats present extensive homology between alpha-protein-like proteins, the n-terminal portion is distinctive. the analysis of a clustalw amino acid multisequence alignment of the n-terminal portions of the alpc, rib, alp2, alp3, alp4 and alp5 identified distinctive strings for each protein, except for the alp2 and alp3 proteins, which are identical over the first half of their length. primer nucleotide sequences corresponding to the distinctive strings were used in a multiplex assay as the reverse primers, while a nucleotide string, common to all the surface protein genes, was used as the forward primer (table 1). these primers allowed detection of nucleotide sequences specific for single alp proteins, discriminated according to the size of pcr product, which directly indicated the presence of gene encoding specific alp protein. these primers were used to screen 152 isolates s. agalactiae of bovine origine by multiplex pcr. representative results are shown on fig. 1. results derived from the direct analysis of amplicon size were correlated with the gene sequence of the corresponding amplified product for each individual strain. by this multiplex analysis in set of bovine isolates were identified 31 isolates with gene encoding alpc protein, 50 isolates with sequence for gene encoding rib, 39 isolates with alp2/3 coding sequence, 10 isolates with gene alp4, and 6 isolates with gene alp5. also two another amplicons with significantly longer lengths 1.095 bp and 1.347 bp were found. the amplicon with 1.095 bp length was found in case of 9 strains, namely 245, 251, 252, 256, 257, 259, 264, 347 and 348, the amplicon with 1.347 bp length was detected in 2 strains, 1925/3 and 2285/1 (fig. 1.). all these amplicons were sequenced and compared by blast. pcr with dna s. agalactiae from the strain 341 resulted an amplicon with length 852 bp, that was 99.5 % identical to original alp5 from strain 27. comparison showed the differences in nucleotide sequence observed on 23rd, 25th, 29th and 81st nucleotide downstream, that resulted in change of 3 amino acids at nterminal coding region of translated protein. the amplicon sequence of alp6 showed considerable homology with alp4, bca, rib and other members of the surface protein gene family defined by bca-rib. the homologous regions were located at the 5` ends of the genes (for bca, the positions are in region between nucleotides 251 and 559, and for the corresponding amino acid sequence, they are between positions 58 and 160). the ratios of similarity to dna sequences of alp4, bca, rib, alp2 and alp3 are 89 %, 73 %, 66.9 %, 62.4 % and 62.3 %, respectively. the proteins belonging to the alpha-like protein family are of interest as models for other repetitive proteins, which are common in gram-positive bacteria (kong et al., 2002; lachenauer et al., 2000). this typical feature was not recognized in alp6, but its sequence is the most similar to that of bca, which encodes c alpha, the prototype of the surface protein family, here it is proposed, that the gene could be considered as putative alpha-like protein. in the case of two isolates, 1925/3 and 2285/1, was observed the pcr product with 1.347 bp length, and was named alp7. sequencing of this amplicon and its comparison by blast showed similarity to the members of alpha-like family; in particular alp1, alp5 and bca, the ratios of similarity nova biotechnologica 9-1 (2009) 67 were 82.8 %, 88.2 % and 77 %, respectively. the homologous regions were located at the 5` ends of the genes, whereas for alp1 and alp5 the positions are in the region between nucleotides 297 to 710. the nucleotide sequence organization in alpha-like family proteins is similar to that in many surface proteins of gram positive bacteria. an n-terminal signal sequence is followed by a nonrepeated n-terminal region, a repeated region, a wall anchoring region by which the protein is anchored to the cell wall via a structure close to the c-terminus consisting of an lpxtg sequence motif followed by a membrane-spanning hydrophobic region and a charged sequence (deswaux et al., 2006; wästfelt et al., 1996; michel et al., 1992). fig. 1. elecrophoresis of multiplex pcr for detection of presence the genes encoding alp proteins, alp2/3, rib, alphac, alp4, and alp5, discriminable by pcr product size on 0.9% agarose gel. other two new genes were also detected in the set of s. agalactiae bovine isolates, alp6 and alp7. dna ladder represents molecular size marker (finnzymes). a repetitive structure in amino acid sequence is conserved and was remarked also in nucleotide sequence of alp7, where differ in the last one base pair, which had no impact to the amino acid sequence. this tandem repeat consists of 82 amino acid residues, whereas one consists of 246 base pairs repetition. the alp7 tandem repeat motif is similar to that of alp1, alp4, bca and rib, with the ratios of similarity 93.9 %, 91.7 %, 91.6 % and 44 %, respectively (fig. 2.). it has been supposed, that the repeat region may act as a rod needed for exposure of the unique ligand-binding region (luo et al., 2000; hamburger et al., 1999). the lpxtg motif was found in both, alp6 and alp7, amino acid sequences, whereas in for this part of sequence in alp7 was observed similarity related to immunoglobulin a1 protease. because domains with the ig-like fold are often implicated in molecular recognition it could has such function (callebaut et al., 2000). like many other surface proteins of gram-positive bacteria, alpha-like protein family members, also 68 vidová, b. et al. with recently here reported, lack cysteine residues. the importance of alpha-like surface proteins consists in the facts, that these proteins play a role as serotype markers, may be important in the pathogenesis of s. agalactiae disease, and may be considered vaccine candidates (maeland et al., 2004). they play an important role as serotype markers, because were noted relationships between serotypes and surface protein genes occurrence (kong et al., 2002), eg. protein alpc is commonly expressed with serotype ib or ii, protein rib with serotype iii, and alp3 protein with serotype v (persson et al., 2008). s. agalactiae strains are typical in high variability on the gene level, and genes under high selection pressure could undergo to various mutations. fig. 2. multiple sequence alignments of the deduced amino acid sequences of the repeat regions of the selected alpha-like proteins. alignments of the repeat regions illustrated conserved motifs near the beginnings and the ends of the repeats. black boxes indicate identity and gray boxes indicate similarity. alpc, alpha c protein repeat element, rib, repeat element from protein rib, alp1, alpha-like repeat element from alp1, alp4, alpha-like repeat element from alp4, and alp7, alpha-like repeat element from alp7. the results of this study indicate that the genes encoding surface proteins were found to constitute a heterogeneous group, with no particular gene dominating. the genes studied here were found in all of the bovine isolates. in the set of bovine isolates was the most common gene encoding protein rib (34%). the overall distribution of these genes was similar to that reported previously (zeng et al., 2006; kong et al., 2002; lachenauer et al., 2000). in this study are also reported two new putative members of this family. like the alpha c and rib protein genes, these genes each contain conserved nand c-terminal encoding regions, and a tandem repeat region was recognized in amino acid sequence of alp7. it has been previously reported, that with a few exceptions, s. agalactiae strains of a given polysaccharide serotype contain the same alpha-like protein, disregarding tandem repeat number (lachenauer et al., 2000, 1999). this concordance may reflect clonal linkages within a species that is naturally incompetent and consistent with several studies showing a predominance of individual clones among s. agalactiae isolates (blumberg et al., 1996). alternatively, it is conceivable that there is a functional linkage between the alpha-like proteins and polysaccharide serotype that would require this specificity. because of a relatively low occurrence of the alp6 and alp7 within the set of bovine isolates and dependence of their presence on a geographical locality, these genes of the alpha-like nova biotechnologica 9-1 (2009) 69 protein family could not be designated for the detection of s. agalactiae, but they could be proof of the biological variability within this protein family. furthermore, characterisation of variability in expression of surface proteins is very important with the perspective of surface proteins application in vaccine development. in this context, it is extremely important to have a rapid and reliable molecular test, as here reported multiplex pcr method, 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ecochemistry and radioecology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (pipiska@ucm.sk) 2consortium for environmental biotechnology and environmental chemistry, hlavná 418, špačince, sk-919 51, slovak republic abstract: removal of cd2+ and zn2+ ions from single and binary solutions by dried activated sludge was studied in batch experiments. it was shown that the metal removal is a rapid process significantly influenced by solution ph. maximum uptake of both cd and zn was reached at ph 6.0 and negligible uptake was observed at ph 2.0. the langmuir isotherm was found to well represent the measured equilibrium sorption data in single metal systems and the maximum sorption capacities qmax of the activated sludge (d.w.), calculated from langmuir model were 540 ± 16 μmol/g for zn2+ and 510 ± 17 μmol/g for cd2+ ions. the response surface methodology (rsm) was used for investigation of interaction and competitive effects in binary metal system. it was found that dried activated sludge in binary system cd-zn has slightly higher affinity for cd2+ comparing with zn2+ ions. competitive effect of cd on zn uptake increased with increasing solution ph and cd initial concentration. maximum sorption capacities of the activated sludge were 321 μmol cd2+/g and 312 μmol zn2+/g. rsm appears to be a better tool for the evaluation of interaction and competitive effects in binary systems than both the simple extrapolation from single-component systems and experimentally difficult study of multi-component systems. key words: cadmium, zinc, biosorption, activated sludge, isotherms, rsm 1. introduction industrial effluents contain both organic and inorganic pollutants. these pollutants upset natural balance in water ecosystems, interfere with organisms, accumulate in biota and enter into the food chain with human on the top. contaminants removal by conventional treatment methods, such as chemical precipitation, membrane separation, evaporation and ion-exchange is often limited due to their low efficiency and economic viability (nayak and lahiri, 2006). therefore, there is a need for an effective and economical treatment alternative. biological processes such as biosorption and bioaccumulation represent possible interactions of toxic pollutants with biological systems in contaminated environment. current research activity in the field attempts to evaluate whether biosorption may eventually provide such an effective and economical treatment process alternative (naja et al., 2010). many researchers studied biosorption from single systems (ghodbane et al., 2008; kang et al., 2007), although the degree of removal of metal ions from wastewaters by biosorption depends mainly on the competitive interactions of co-ions when present in solution (ma and tobin, 2003). results from single component systems can provide useful data about the uptake capacity of used biosorbent, but they 118 remenárová, l. et al. do not exactly reflect the real situation in wastewaters (gönen and aksu, 2009). therefore it is necessary to study sorption process as complex process, which consists of many mechanisms and can be affected by many parameters. effect of various parameters (ph, temperature, sorbent particle size, initial concentration of sorbates) can be studied by the classical approach, when one variable is changed at a time. however this procedure represents a time consuming and less effective method. multivariate optimization is faster and overcome circumstances not explained by the traditional methods such as interactions between the variables that influence the response e.g. sorption capacity (zolgharnein et al., 2010). response surface methodology (rsm) is a collection of mathematical and statistical techniques useful for designing experiments, building models and analyzing the effects of the several independent variables (mune et al., 2008). main advantage of statistical design of experiments is the reduced number of experiments to be performed as well as it considers interactions among the variables and can be used for optimization of the operating parameters in multivariable systems (gönen and aksu, 2009). from the literature it was found that rsm has been widely used for optimization of biosorption processes mainly in single systems. only a few studies utilized rsm methodology for statistical analysis of individual and interaction effects of parameters in binary and ternary sorption systems (fereidouni et al., 2009; pakshirajan and swaminathan, 2009). within this context the objective of present study was firstly to quantify the ability of the biosorbent prepared from dried activated sludge to sorb cd2+ and zn2+ ions. equilibrium isotherm models according to langmuir and freundlich were used for mathematical description of sorption equilibria in single systems. the second objective was to study the competitive and interaction effects of above mentioned ions in binary cd2+-zn2+ system at various solution ph values using response surface methodology (rsm). 2. material and methods 2.1 biosorbent preparation activated sludge was obtained from waste water treatment plant in enviral a.s. (leopoldov, slovak republic) producing fuel ethanol. the sludge was washed twice in deionised water, oven-dried for 72 h at 90°c. after drying activated sludge was ground to various particle sizes, from which particle size <450 μm was used in biosorption experiments. 2.2 batch sorption experiments in single systems the biosorption kinetics was determined by suspending of activated sludge (2.5 g/l, d.w.) in metal solutions (ph 6.0) containing 1000 μmol/l cdcl2 or zncl2 spiked with 109cdcl2 or 65zncl2. the content was agitated on a reciprocal shaker (120 rpm) at 20°c and in time intervals liquid samples were taken and the radioactivity was measured. nova biotechnologica 10-2 (2010) 119 the metal sorption capacity of dried activated sludge was determined by suspending of activated sludge (2.5 g/l, d.w.) in metal solutions (ph 6.0) containing cdcl2 or zncl2 in concentration range 100-4000 μmol/l spiked with 109cdcl2 or 65zncl2 and exposing for 4h at 20°c on a reciprocal shaker (120 rpm). to analyze the influence of ph, activated sludge was shaken in metal solutions containing 1000 μmol/l cdcl2 or zncl2 spiked with 109cdcl2 or 65zncl2 for 4 h on a reciprocal shaker at 120 rpm and 20°c adjusted to different ph values (2.0 – 9.0) by adding 0.5 m hcl or 0.1 m naoh. at the end biomass was filtered out, washed twice in deionised water and radioactivity of both activated sludge and liquid phase was measured. the metal uptake was calculated as mccvq eq /)( 0 −= (1) where q is the uptake (μmol/g, d.w.), c0 and ceq is the initial and the final metal concentrations in solution (μmol/l) and m is the amount of dried biosorbent (given in grams). all experiments were performed in duplicate. 2.3 experimental design of binary cd-zn system the box-behnken design under response surface methodology (rsm) was used to investigate interaction and competitive effects in binary metal system cd2+-zn2+. levels of factors (initial concentrations c0 of cd 2+ and zn2+ ions and initial ph of solution) considered for sorption in binary system are shown in table 1. the design matrix of 16 experiments is given in table 3. based on the matrix, experiments were performed in erlenmeyer flasks, activated sludge (2.5 g/l, d.w.) was added and the content was agitated on a reciprocal shaker (120 rpm) for 4 h at 20°c. table 1. levels of factors considered for sorption of cd2+ and zn2+ ions from binary system using boxbehnken design. coded levels factor unit factor code -1 0 1 c0 cd 2+ µmol/l a 1000 2000 3000 c0 zn 2+ µmol/l b 1000 2000 3000 ph c 3.0 4.5 6.0 the behavior of the binary sorption system is explained by the following empirical second-order polynomial model ∑∑∑ ≤≤== +++= k ji jiij k i iiii k i i xxxxq 11 2 10 ββββ (2) where q is the predicted response (specific sorption qeq of both cd and zn), xi, xj, .....xk are the input variables (c0 cd 2+, c0 zn 2+ and solution ph) which affect the response q, β0 is the intercept term, βi is the linear effect, βii is the quadratic effect and βij is the interaction effect (fereidouni et al., 2009). 120 remenárová, l. et al. 2.4 radiometric analysis the gamma spectrometric assembly using the well type scintillation detector 54bp54/2-x, nai(tl) (scionix, the netherlands) and the data processing software scintivision 32 (ortec, usa) were used for 109cd and 65zn determination in activated sludge and supernatant fluids at the energy of γphotons: 109cd – 88.04 kev and 65zn – 1115.52 kev. standardized 109cdcl2 solution (3.937 mbq/ml; 50 mg cdcl2/l in 3 g/l hcl) and 65zncl2 solution (0.8767 mbq/ml; 50 mg zncl2/l in 3 g/l hcl) were obtained from the czech institute of metrology, prague (czech republic). 2.5 speciation modeling prediction of the speciation of cd and zn in the aqueous systems as a function of total salt concentration and solution ph was performed using the visual minteq (version 2.52) program. visual minteq 2.52 is a chemical equilibrium computer program that has an extensive thermodynamic database for the calculation of metal speciation, solubility and equilibria (gustafson, 2004). 2.6 data analysis to calculate the qmax values and the corresponding parameters of adsorption isotherms non-linear regression analysis was performed by origin 7.0 professional (originlab corporation, northampton, usa) and graphpad prism 5.0 (graphpad software, usa). response surface graphs and regression analysis of the obtained data were performed by statgraphics centurion xv (statpoint inc., usa) and the design expert 7.0 (stat-ease, inc., usa). 3. results and discussion 3.1 cd2+ and zn2+ uptake by activated sludge in order to determine the minimum necessary time to reach the sorption equilibrium, the time-course studies on the biosorption of cadmium and zinc ions from single metal systems by biosorbent prepared from dried activated sludge were performed. fig. 1 shows that biosorption of cd2+ and zn2+ ions is a rapid process where equilibrium is reached within several tens minutes. at initial phase driving force is higher and binding sites on activated sludge with higher affinity are occupied. after that time concentration of cd2+ and zn2+ ions in solution decreases and the residual binding sites with lower affinity toward metal ions are occupied slowly. the final equilibrium was reached within 4 hours. such two-phase sorption has been also reported by yang et al. (2010) in the case of zn sorption by activated sludge. they found that zinc adsorption capacity increased obviously during the first 60 min and the final equilibrium was reached within 180 min. nova biotechnologica 10-2 (2010) 121 from fig. 2 it can be seen that maximum biosorption of both cd2+ and zn2+ occurred at ph 6.0. this curve is characteristic also for cd and zn biosorption by the moss rhytidiadelphus squarrosus (pipíška et al., 2010) and biosorption of other metal ions on activated sludge, algae and other biosorbents (wang et al., 2010; liu et al., 2009; gundogdu et al., 2009). observed slightly lower biosorption at ph 4.0 and negligible at ph 2.0 is closely related to protonation of binding sites, resulting in competition between h+ and cd2+ or zn2+ ions for occupancy of the active sites. in the case of zn2+ at ph 8.0 sharp decrease of biosorption was observed. 0 200 400 600 800 1000 1200 1400 1600 0 50 100 150 200 250 300 350 400 q eq [μ m ol /g ] t [min] cd2+ zn2+ fig. 1. effect of contact time on cd2+ (c0 = 1000 μmol/l, 100 kbq 109cdcl2) and zn2+ (c0 = 1000 μmol/l, 65 kbq 65zncl2) ions biosorption by dried activated sludge (2.5 g/l d.w.) at 20°c and ph 6.0. it must be pointed out, that the ph influenced also the level of ionization and speciation of metals in aqueous solution. as can be calculated by visual minteq speciation program (data not shown) at ph 8.0 besides the divalent cation zn2+, zinc occurs in solution also in insoluble forms such as [zn(oh)]+. therefore the decrease of zn2+ sorption at higher ph values (> ph 8.0) can be caused also by precipitation. according to calculations insoluble cadmium species occurred at ph > 9.0. it is reasonable to suppose that the dependence of metal uptake on ph is related to both the surface functional groups on the biosorbent and the metal speciation in solution. during sorption experiments ph was not regulated. from fig. 2 it is evident that the equilibrium ph increased after biosorption experiments what indicates that functional groups on the surface accumulate besides cd2+ and zn2+ ions simultaneously h+ ions. similar behaviour observed also chen et al. (2002) in cu sorption by activated sludge. several mechanisms of heavy metals uptake by activated sludge have been proposed (wang et al., 2010; laurent et al., 2010). short-term cation uptake is generally regarded as an abiotic process governed by: surface complexation of cations with exposed functional groups (carboxyl-, sulfhydryl-, amineetc.) on the biosorbent; ion exchange; coordination and chelation of metals; adsorption or by precipitation of solid phases on the cell walls. 122 remenárová, l. et al. 2 4 6 8 0 50 100 150 200 250 300 350 q eq cd2+ ph 0 q eq [μ m ol /g ] 2 4 6 8 ph eq 2 4 6 8 0 50 100 150 200 250 300 350 q eq zn2+ ph 0 q eq [μ m ol /g ] 2 4 6 8 ph eq fig. 2. effect of initial ph on cd2+ and zn2+ ions (c0 = 1000 µmol/l) sorption by dried activated sludge (2.5 g/l, d.w.) at 20°c; (-) represents shifts in ph after biosorption. 3.2 sorption equilibrium in single systems two well known adsorption isotherm models langmuir (eq. 3) and freundlich (eq. 4) were applied for the analysis of the experimental data in single sorption systems. eq eq eq bc cbq q + = 1 max (3) )/1( n eqeq kcq = (4) parameters of these models provide an insight into the sorption process, reflect the nature of the sorbent, surface properties as well as the degree of the affinity of the nova biotechnologica 10-2 (2010) 123 sorbents and can be used to compare biosorption performance (volesky, 2003). the langmuir and freundlich isotherms were fitted to the equilibrium data for cd2+ and zn2+ sorption on dried activated sludge. isotherm curves and parameters of the models determined from the experimental data using non-linear regression analysis are reported in fig. 3, 4 and table 2. 0 1000 2000 3000 0 200 400 600 langmuir a ceq [μmol/l] q eq [ μ m ol /g ] 0 1000 2000 3000 0 200 400 600 800 freundlich b ceq [μmol/l] q eq [ μ m ol /g ] fig. 3. isotherms for the sorption of zn2+ ions by dried activated sludge at 20°c and ph 6.0 according to langmuir (a) and freundlich (b). data points represent experimental results, curves represent the calculated values from isotherm models, dotted lines represent the 95 % confidence interval. the langmuir isotherm fits the data of both cd2+ and zn2+ ions sorption by dried activated sludge better than the freundlich isotherm, as is demonstrated by higher values of coefficient of determination (r2), the more homogeneous standard deviation of each observed parameter and by the lower the sum of squares (rss) values obtained as well as standard deviation of the residuals (sy,x ) (table 2). also hammaini et al. (2007) found that the sorption of cd2+ and zn2+ ions by activated sludge was well described using the langmuir isotherm. the maximum sorption capacity qmax obtained from langmuir isotherm for cd 2+ was 510 ± 17 μmol/g at ph 6.0. slightly higher value of qmax was observed in the case of zn2+ sorption, 540 ± 16 μmol/g at ph 6.0. the affinity constant b of the isotherms corresponds to the initial gradient, which indicates the biosorbent affinity at low 124 remenárová, l. et al. 0 1000 2000 3000 0 200 400 600 langmuir a ceq [μmol/l] q eq [ μ m ol /g ] 0 1000 2000 3000 0 200 400 600 800 freundlich b ceq [μmol/l] q eq [ μ m ol /g ] fig. 4. isotherms for the sorption of cd2+ ions by dried activated sludge at 20°c and ph 6.0 according to langmuir (a) and freundlich (b). data points represent experimental results, curves represent the calculated values from isotherm models, dotted lines represent the 95 % confidence interval. table 2. langmuir and freundlich parameters for the sorption of cd2+ and zn2+ ions by dried activated sludge obtained by non-linear regression analysis. model metal ion qmax [μmol/g] b [l/μmol] k [l/g] 1/n r 2 rss sy,x zn2+ 540 ± 16 0.006 ± 0.001 0.995 723 15.5 l an gm ui r cd2+ 510 ± 17 0.005 ± 0.001 0.994 769 16.0 zn2+ 50.3 ± 29.4 0.30 ± 0.08 0.877 18817 79.2 fr eu nd lic h cd2+ 49.9 ± 28.9 0.29 ± 0.08 0.878 16347 73.8 concentrations of metal ions. a greater initial gradient corresponds to a higher affinity constant (sheng et al., 2007). from fig. 3 and 4 it is evident that the cadmium and nova biotechnologica 10-2 (2010) 125 zinc isotherms have similar behavior at lower equilibrium concentrations. in the case of b, cadmium recorded 0.005 ± 0.001 l/μmol compared to 0.006 ± 0.001 l/μmol for zinc indicating slightly higher affinity of activated sludge for zinc ions. it should be realized that despite the fact that langmuir isotherm offers no insights into the mechanism of biosorption (liu and liu, 2008) it remains a convenient tool for comparing equilibrium data on a quantitative basis (determination of maximum sorption capacity qmax and affinity parameters b) and providing information on biosorption potential. 3.3 experimental design of binary cd-zn system box-behnken design under the response surface methodology (rsm) was used for investigation of interaction and competitive effects between variables in binary system cd-zn. according to preliminary experiments and our previous studies (pipíška et al., 2010; pipíška et al., 2008) the sorption capacity of biosorbent in multi-component systems mainly depends on the initial concentration of primary ion and co-ions in sorption system and on the solution ph. therefore, initial concentration of cd and zn and solution ph were used as process variables in experimental design (table 3) and two responses – qeq(cd) and qeq(zn) were studied simultaneously. table 3. box-behnken experimental design matrix and experimental and predicted values of sorption capacity (qeq) of cd2+ and zn2+ ions by dried activated sludge from binary system cd-zn. a – c0 cd (µmol/l), b – c0 zn (µmol/l), c – ph. factor qeq cd qeq zn run order a b c qeq (experimental) (µmol/g) qeq (predicted) (µmol/g) qeq (experimental) (µmol/g) qeq (predicted) (µmol/g) 1 1000 1000 4.5 205.8 206.5 178.0 179.2 2 3000 1000 4.5 321.0 321.7 113.2 122.8 3 1000 3000 4.5 130.7 130.0 312.0 302.4 4 3000 3000 4.5 261.4 260.7 226.2 226.9 5 1000 2000 3 93.77 90.64 138.1 145.5 6 3000 2000 3 211.2 208.0 118.3 115.3 7 1000 2000 6 170.8 173.9 284.9 287.8 8 3000 2000 6 299.3 302.4 193.3 185.9 9 2000 1000 3 180.9 183.4 75.86 69.18 10 2000 3000 3 132.7 136.5 154.0 156.1 11 2000 1000 6 297.8 294.0 151.1 148.9 12 2000 3000 6 206.0 203.5 282.6 289.3 13 2000 2000 4.5 242.5 245.0 213.3 214.6 14 2000 2000 4.5 242.5 245.0 213.3 214.6 15 2000 2000 4.5 254.5 245.0 227.8 214.6 16 2000 2000 4.5 241.1 245.0 198.4 214.6 126 remenárová, l. et al. the behavior of the binary sorption system cd-zn is explained by the following quadratic models determined by multiple regression analysis: ( ) phzncphcznccdcphznc cdcphznccdccdqeq ××−××+××⋅+×− ×⋅−×⋅−×+×⋅−×+−= − −−− 0007.000019.000109.303.170 103.20103.11940101.20097.0382 622 6255 (5) ( ) phzncphcznccdcph znccdcphznccdcznqeq ××+××−××⋅−×− ×⋅−×⋅+×+×+×+−= − −− 0009.00012.000108.42.16 0102.10105.51870075.00008.0394 62 2526 (6) the adequacy of the models was further justified through anova (data not shown). the model f-values of 246 (for cd2+ sorption) and 58.3 (for zn2+ sorption) and values of p<0.0001 indicate that both models are significant. good agreement between experimental and predicted values of sorption capacity qeq (table 3) confirmed high values of coefficient of determination (r2), 0.997 (for cd2+) and 0.987 (for zn2+). equations 5 and 6 represent the quantitative effect of the variables (initial concentration of cd and zn, solution ph) and their interactive effects on the qeq(cd) and qeq(zn) in binary system cd-zn. a positive sign in the equation implies a synergistic effect of the variables, while a negative sign indicates an antagonistic effect. 1000,0 c0 zn (µmol/l) 3000,0 6,0 q eq c d (µ m ol /g ) 160 190 220 250 280 310 c0 cd (µmol/l) 3000,0 1000,0 ph 3,0 fig. 5. main effects plot for cd2+ biosorption by dried activated sludge from binary system cd-zn. fig. 7a, b, c illustrate the three-dimensional response surface plots of the quadratic model (eq. 5) for cd2+ sorption from binary system cd-zn. the combined effect of initial cd2+ concentration and ph on cd2+ sorption by dried activated sludge at various concentrations of zinc as co-ion is shown. it is evident that cd2+ uptake increased with increasing of initial solution ph as well as with initial cd2+ concentration. on the contrary, increasing zn2+ concentration from 1000 to 3000 μmol/l diminished cd2+ sorption as a result of competition between metal ions (fig. 5). increase in solution ph and initial zn2+ sorption caused increase in zn2+ sorption as can be seen from main effects plot (fig. 6). similarly, increase in cd2+ concentration decreased zn2+ sorption from binary system cd-zn. nova biotechnologica 10-2 (2010) 127 1000,0 c0 zn (µmol/l) 3000,0 3,0 6,0 q eq z n (µ m ol /g ) 120 150 180 210 240 270 c0 cd (µmol/l) 3000,0 1000,0 ph fig. 6. main effects plot for zn2+ biosorption by dried activated sludge from binary system cd-zn. 3.00 3.75 4.50 5.25 6.00 1000.00 1500.00 2000.00 2500.00 3000.00 0 87.5 175 262.5 350 q eq c d µm ol /g c: ph c0 cd µmol/l 3.00 3.75 4.50 5.25 6.00 1000.00 1500.00 2000.00 2500.00 3000.00 0 87.5 175 262.5 350 q eq c d µm ol /g c: ph c0 cd µmol/l b a 128 remenárová, l. et al. 3.00 3.75 4.50 5.25 6.00 1000.00 1500.00 2000.00 2500.00 3000.00 0 87.5 175 262.5 350 q eq c d µm ol /g c: ph c0 cd µmol/l fig. 7. response surface plots showing the effect of ph and initial cd2+ concentration on cd2+ biosorption by dried activated sludge (2.5 g/l d.w.) at 20°c and different concentrations of zn2+ as competing ion (a) 1000 µmol/l, (b) 2000 µmol/l, (c) 3000 µmol/l. maximum sorption capacities of the activated sludge were 321 μmol cd2+/g and 312 μmol zn2+/g. it was found that dried activated sludge in binary system cd-zn has higher affinity for cd2+ ions when cd2+ and zn2+ are present in equimolar ratio 1:1 (table 3) comparing with higher affinity to zn2+ in single systems. this is consistent with the hypothesis that variance in affinity in multi-component systems could be attributed to different ionic characteristics of metal ions (pakshirajan and swaminathan, 2009). 4. conclusions biosorption of cd2+ and zn2+ from aqueous solution by dried activated sludge is a rapid and ph dependent process. maximum uptake of metals was found to occur at ph 6. the experimental equilibrium data of the single-component systems for cd2+ and zn2+ ions have been well described by the langmuir isotherm and the maximum sorption capacity qmax were 540 ± 16 μmol/g for zn 2+ and 510 ± 17 μmol/g for cd2+ ions. the use of rsm revealed the existence of interaction and competitive effects between variables in binary system cd-zn. cd2+ uptake increased with increasing of initial solution ph as well as with initial cd2+ concentration. on the contrary, increasing zn2+ concentration from 1000 to 3000 μmol/l diminished cd2+ sorption as a result of competition between metal ions. activated sludge in binary system cd-zn has higher affinity for cd2+ comparing with zn2+ ions when cd2+ and zn2+ are present in equimolar ratio 1:1. rsm appears to be a useful tool for obtaining interaction and competitive effects in binary systems since it requires less reagents and experimentation time. references chen, j.p., lie, d., wang, l., wu, s., zhang, b.: dried waste activated sludge as biosorbents for metal removal: adsorptive characterization and prevention of organic leaching. j. chem. technol. biotechnol., 77, 2002, 657-662. c nova biotechnologica 10-2 (2010) 129 fereidouni, m., daneshi, a., younesi, h.: biosorption equilibria of binary cd(ii) and ni (ii) systems 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shahmoradi, a., mousavi, s.n.: optimization of removal of methylene blue by platanus tree leaves using response surface methodology. anal. sci., 26, 2010, 111-116. nova biotechnol chim (2022) 21(1): e1089 doi: 10.36547/nbc.1089 1 nova biotechnologica et chimica in vitro culture initiation and regeneration of two highly productive clones of poplar yuliia khoma, lidiia khudolieieva, namik rashydov, nataliia kutsokon department of biophysics and radiobiology, institute of cell biology and genetic engineering, national academy of sciences of ukraine, acad. zabolotnogo, 148, 03143, kyiv, ukraine  corresponding author: kutsokon@nas.gov.ua article info article history: received: 6th august 2021 accepted: 9th january 2022 keywords: growth media leaf and petiole explants microclonal propagation of trees plant regeneration populus spp. abstract in vitro shoot regeneration is a fast and reliable method for propagation of valuable tree genotypes and important step in genetic manipulations. this study aimed to establish an optimal in vitro culture initiation and regeneration protocol for highly productive poplar clones ‘novoberlinska-3’ (populus pyramidalis × p. laurifolia) and ‘volosystoplidna’ (p. trichocarpa). in vitro culture was initiated either on ms (im-ms) or wpm (im-wpm) medium both containing bap (0.2 mg.l-1) and iba (0.1 mg.l-1). results demonstrated that the best initiation medium for the clone ‘volosystoplidna’ was im-wpm on which 93.3 % of explants survived, while explants from ‘novoberlinska-3’ better survived on im-ms. after establishing both genotypes into aseptic culture, regeneration experiments were started using two types of explants, leaf and petioles, planted on callus induction medium containing 2ip (1.02 mg.l-1) and naa (1.86 mg.l-1). furthermore, microshoots were placed on shoot induction medium supplemented with 0.04 mg.l-1 thidiazuron, and then transferred on shoot elongation medium with 0.2 mg.l-1 bap. regeneration protocol showed better performance of poplar ‘novoberlinska-3’, but it was also efficient for ‘volosystoplidna’. plant regeneration from leaf explants in the clone ‘novoberlinska-3’ showed the highest regeneration percentage (92.3 %) and the number of shoots per explant (3.1), which significantly exceeded other explants. our results showed a significant difference between the survivability of two clones during culture initiation. the differences in regeneration rates between the clones as well as leaf and petiole explants were also determined. obtained aseptic cultures of highly productive poplar hybrids will be used in our further studies, especially for genetic transformation. introduction poplars (populus spp.) belong to the plants of the salicaceae family, which are ecologically and economically important tree species. these fastgrowing trees are widely used for biofuel production, wood, pulp, and paper industries, environmental protection, and restoration of degraded soils (gordon 2001; pilipović et al. 2019; galović et al. 2021). poplars are also trees of great scientific importance due to ease of vegetative propagation, short rotation cycle, small genome size, and the ability to regenerate in vitro. these important features, combined with the complete sequencing of the genome of populus trichocarpa, made poplar a model plant offering molecular tools for basic research in genetics, physiology, and mailto:kutsokon@nas.gov.ua nova biotechnol chim (2022) 21(1): e1089 2 biochemistry of trees (han et al. 2000; tuskan et al. 2006; kutsokon et al. 2020). in recent decades, short-rotation plantations of fastgrowing trees for biomass production have been increasingly established worldwide (wullschleger et al. 2002; aylott et al. 2008; kutsokon et al. 2017; moravčíková et al. 2017). for the effective management of these plantations, it is important to properly select highly productive and easily propagated intensively growing clones. it is known that some valuable poplar hybrids do not reproduce vegetatively or reproduce poorly. the method of micropropagation can overcome this problem by providing genetically homogeneous planting material identical to the original form. application of in vitro culture methods makes it possible to produce high-quality plants at any time, quickly and in large quantities. in addition, micropropagation preserves the genotype of the original plant, allows to control the growth environment, monitor the physiological and biochemical parameters of the plants, as well as to work regardless of weather conditions and season (giri et al. 2004). micropropagation of trees is much more difficult than the micropropagation of herbaceous plants. it requires the development of specific cultivation techniques for different species. in addition to selection of the nutrient medium composition for macroand micronutrient content, it is also necessary to add various vitamins, phytohormones or growth regulators. it may be also necessary to add absorbents and antioxidants, as woody plant species can release phenolic compounds into the growth environment. these compounds can be rapidly oxidized and may lead to plant death (shahzad et al. 2017; abiri et al. 2020). in vitro shoot regeneration is not only a rapid and reliable method for valuable genotypes propagation but also the indispensable prerequisite for some biotechnological techniques, such as genetic transformation and gene editing. genetic manipulations are able to increase the plant growth and resistance to biotic and abiotic stresses, change the quality of wood (e.g., reduce or change lignin content), and improve phytoremediation characteristics (confalonieri et al. 2003; kutsokon 2011; song et al. 2019). however, not all tree species can be easily in vitro regenerated. their ability to morphogenesis depends on the genotype, tissue types or explants, cultivation environment, media nutritional components, and growth regulators (giri et al. 2004; musienko and panyuta 2005; ferreira et al. 2009). the ratio of auxins to cytokinin plays a crucial role in the success of plant regeneration (hill and schaller 2013; bidabadi and jain 2020). several authors have shown that the effective plant regeneration methods should be optimized for each clone individually (noël et al. 2002; thakur et al. 2012; kutsokon et al. 2013; kwon et al. 2015; pokorná et al. 2017). additionally, although many studies have been reported using the populus explants for tissue culture propagation, those studies are limited to a small number of reports on shoots regeneration from leaf and petiole explants for different species that is an important step in genetic manipulations. in our previous work, poplar clones ‘novoberlinska-3’ (populus pyramidalis × populus laurifolia) and ‘volosystoplidna’ (populus trichocarpa torr. et gray) were found to be between the most productive and energy-valuable clones in the short-rotation trials (kutsokon et al. 2017). these clones can easily be multiplied by woody cuttings; however, for regeneration, gene transformation, and other in vitro manipulations, a homogeneous quality of the plant material is needed, thus the use of micropropagation is strongly recommended (müller et al. 2013). one of the clones studied, p. trichocarpa, as described for genotype ‘nisqually-1’, is known as recalcitrant in in vitro conditions, though few protocols are known for regeneration (kang et al. 2009) and transformation (li et al. 2015) of ‘nisqually-1’ clone. a limited number of papers about in vitro cultivation and genetic transformation of hybrids p. laurifolia x nigra 'italica' (p. x berolinensis) were published, but they described only partial steps of the protocol (pavlichenko et al. 2016; zolotovskaya et al. 2018). nevertheless, the clones ‘novoberlinska-3’ and ‘volosystoplidna’ have not been used in in vitro culture in the studies before. therefore, in this paper we reported the optimal conditions for their in vitro culture initiation and regeneration which are the key steps for further genetic transformation. nova biotechnol chim (2022) 21(1): e1089 3 experimental plant material and surface sterilization buds and young softwood shoots of poplar clones ‘novoberlinska-3’ (p. pyramidalis × p. laurifolia) and ‘volosystoplidna’ (p. trichocarpa torr. et gray) were collected at the beginning of the growing season. the plants in the juvenile stage of development, maintained in a pot stock plant collection, kept at the institute of cell biology and genetic engineering nas of ukraine, were used as softwood shoot sources. explants were surface sterilized using our previously established protocol (khudolieieva et al. 2017), by treating them with the different solutions in three subsequent steps: 1) concentrated soap solution (2 min); 2) commercial bleach solution of sodium hypochlorite diluted with distilled water to 30 % (10 min); 3) 70 % ethanol solution (1 min). after each step, the decontaminated explants were rinsed three times with sterile water. culture initiation, shoot regeneration, media, and growth conditions in order to evaluate the effect of basal salt medium on plant growth during culture initiation, surfacesterilized stem explants of poplar clones ‘novoberlinska-3’ and ‘volosystoplidna’ were placed on two different types of initiation media, either on ms (im-ms) or woody plant (im-wpm) medium. both nutrient media were modified with 0.2 mg.l-1 6-benzylaminopurine (bap) and 0.1 mg.l-1 indole-3-butyric acid (iba). detailed compositions of all nutrient media used in this research are summarized in table 1. table 1. composition of the nutrient media. im-ms 2.18 g.l-1 ms, 2 ml.l-1 wpm vitamins, 0.1 g.l-1 myo-inositol, 30 g.l-1 sucrose, 0.26 g.l-1 mes, 0.2 g.l-1 l-glutamine, 7.5 g.l-1 agar, 0.2 mg. l-1 bap, 0.1 mg. l-1iba, ph 5.8 im-wpm 1.15 g.l-1 wpm, 2 ml.l-1 wpm vitamins, 20 g.l-1 sucrose, 7.5 g.l-1 agar, 0.2 mg. l-1 bap, 0.1 mg. l-1 iba, ph 5.8 cim 2.18 g.l-1 ms, 2 ml.l-1 wpm vitamins, 0.1 g.l-1 myo-inositol, 30 g.l-1 sucrose, 0.26 g.l-1 mes, 0.2 g.l-1 l-glutamine, 7.5 g.l-1 agar, 1.86 mg. l-1 naa, 1.02 mg. l-1 2-ip, ph 5.8 sim 2.18 g.l-1 ms, 2 ml.l-1 wpm vitamins, 0.1 g.l-1 myo-inositol, 30 g.l-1 sucrose, 0.26 g.l-1 mes, 0.2 g.l-1 l-glutamine, 7.5 g.l-1 agar, 0.04 mg. l-1 tdz, ph 5.8 sem 2.18 g.l-1 ms, 2 ml.l-1 wpm vitamins, 0.1 g.l-1 myo-inositol, 30 g.l-1 sucrose, 0.26 g.l-1 mes, 0.2 g.l-1 l-glutamine, 7.5 g.l-1 agar, 0.2 mg. l-1 bap, ph 5.8 pm-ms 2.18 g.l-1 ms, 2 ml.l-1 wpm vitamins, 0.1 g.l-1 myo-inositol, 30 g.l-1 sucrose, 0.26 g.l-1 mes, 0.2 g.l-1 l-glutamine, 7.5 g.l-1 agar, ph 5.8 pm-wpm 1.15 g.l-1 wpm, 2 ml.l-1 wpm vitamins, 20 g.l-1 sucrose, 7.5 g.l-1 agar, ph 5.8 im-ms – initiation media contained ms (murashige and skoog) salts; im-wpm – initiation media contained wpm (mccown woody plant medium) salts; cim – callus induction medium; sim – shoot induction medium; sem – shoot elongation medium; pm-ms – propagation media contained ms (murashige and skoog) salts, pm-wpm – propagation media contained wpm (mccown woody plant medium) salts; bap – 6-benzylaminopurine; iba – indole-3-butyric acid; mes – 2-(nmorpholino) ethanesulfonic acid; naa – α-naphthaleneacetic acid; tdz – thidiazuron. all chemicals were purchased from duchefa (the netherlands) unless otherwise stated. fifteen explants per variant were planted, each in a separated glass tube. the explants were maintained in a tissue culture growth room at 24 °c under a 16-h photoperiod. the percentage of surviving stem explants was determined after 30 days of cultivation on induction media. successfully initiated into in vitro culture poplars ‘novoberlinska-3’ and ‘volosуstoplidna’ were micropropagated on pm-ms and pm-wpm respectively (table 1) and maintained by subculturing at 4-week intervals. well-developed onemonth plants were used for indirect shoot regeneration according to the protocol of meilan and ma (2006). this protocol was successfully performed in our previous studies for the regeneration of several poplar clones (khudolieieva et al. 2014; 2017). therefore, it was tested in the current study. leaf and petiole explants were cultivated on petri dishes with callus-inducing medium (cim) supplemented with growth regulators 1.02 mg.l-1 n6-(2-isopentenyl) adenine (2-ip) and 1.86 mg.l-1 α-naphthaleneacetic acid nova biotechnol chim (2022) 21(1): e1089 2 (naa) (table 1) at 21 °c in the dark for 3 weeks. the explants produced calli were transferred on petri dishes with shoot induction medium (sim) supplemented with 0.04 mg.l-1 thidiazuron (tdz) (table 1). plants were cultured in the tissue culture growth room at 24 °c under a 16-h photoperiod for 2 weeks. then the explants with microshoots (1 – 1.5 cm) were transferred to shoot elongation medium (sem) containing 0.2 mg.l-1 bap (see table 1). at least 15 leaf and 15 petiole explants were collected per clone at the initial stage of regeneration. the efficiency of regeneration was evaluated after six weeks since beginning the experiment by the percentage of explants producing at least one shoot to the total number of explants used as well as by the number of shoots per regenerated explant. statistical analysis statistical processing of the results was performed according to standard methods. data are expressed as % ± sp or as mean ± standard error of the mean. data normality and variance homogeneity were checked using shapiro-wilk and levene’s tests, respectively. because the data were not normally distributed, nonparametric tests were applied. significant differences in the survivability of explants and percentages of explants producing the shoots were tested with the chi-square test. the differences in the number of shoots per regenerated explant were tested using the mann-whitney u test. all tests were declared significant at p ≤ 0.05. all statistical analyses and tests were performed using originpro9.0 software (originlab corporation). results and discussion survivability of stem explants initiated into in vitro culture in this study, two poplar clones ‘novoberlinska-3’ and ‘volosystoplidna’ were established in aseptic cultures (fig. 1), and the optimal composition of the im for the first stage of cultivation was selected. as shown in fig. 2, the best initiation nutrient medium for the poplar clone ‘volosystoplidna’ was the im-wpm on which the survival rate was 93.3 %. for poplar clone ‘novoberlinska-3’, a higher survival rate (73.3 %) was observed on im-ms compared to im-wpm (46.7 %), but in this case, the difference between the rates was not significant. fig. 1. establishment of aseptic cultures of poplars from stem explants: clone ‘novoberlinska-3’ on the im-ms (a) and clone ‘volosystoplidna’ on the im-wpm (b). fig. 2. survivability of stem explants of the poplar clones ‘novoberlinska-3’ and ‘volosystoplidna’ while initiated into in vitro culture. im-wpm and im-ms were both supplemented with 0.2 mg.l-1 bap and 0.1 mg.l-1 iba. different letters above the bars indicate significant differences in survivability between the samples (% ± sp) after the chi-square test (p ≤ 0.05), n = 15 replicates for each genotype. a b 4 nova biotechnol chim (2022) 21(1): e1089 3 the survival rates of both poplar clones on the im demonstrated that initiation into in vitro culture requires an individual approach for each clone separately. in general, the use of growth regulators has different effects on plant regeneration and rooting efficiency in populus species (bannoud and bellini 2021). in particular, the study by pardhi et al. (2019) showed that the addition of 0.5 mg.l-1 bap promoted the initiation of p. nigra explants into in vitro culture on the 21-st day of the experiment. an application of 0.1 μm iba showed high efficiency in rooting of himalayan poplar shoots (p. ciliata wallich ex royle) (aggarwal et al. 2015). shoot indirect regeneration from leaf and petiole explants after establishing both poplar genotypes, and their micropropagation in aseptic cultures, regeneration experiments were started. both types of explants, the leaf and petioles, formed callus within three weeks on cim with the growth regulators 2-ip and naa (fig. 3a). the explants of both poplar clones, transferred on sim with tdz (0.04 mg.l-1), formed the microshoots after two weeks of cultivation, more actively in poplar ‘novoberlinska-3’ compared to poplar ‘volosystoplidna’(fig. 3b and 3c). later, these microshoots were transferred on sem with the addition of bap (0.2 mg.l-1) (fig. 3d) and within two weeks regenerants from leaf and petiole explants in both poplar clones were obtained (fig. 3e and 3f) with different regeneration efficiencies (table 2). fig. 3. regeneration of poplar clones ‘volosystoplidna’ (a, c, e) and ‘novoberlinska-3’ (b, d, f) from leaf and petiole explants. a – callogenesis on cim after 10 days of cultivation; b, c – formation of microshoots after two weeks of cultivation on sim; d – microshoots transferred on sem; e, f – shoots growing on sem during two weeks. table 2. shoot regeneration from leaf and petiole explants of poplar clones ‘novoberlinska-3’ and ‘volosystoplidna’. clone type of explant the number of explants planted the number of explants with shoots the number of shoots per explant ‘novoberlinska-3’ leaves 39 36 112 petioles 30 27 43 ‘volosystoplidna’ leaves 38 19 30 petioles 31 18 33 5 nova biotechnol chim (2022) 21(1): e1089 3 the poplar clone ‘novoberlinska-3’ showed higher regeneration efficiency. in particular, the percentage of regeneration of this clone from leaf (92.3 %) and petiole (90 %) explants was significantly higher than in the poplar clone ‘volosystoplidna’, where the percentage of regeneration was lower, at 50 % from both leaf and petiole explants (fig. 4). as shown in the table 2, the number of shoots obtained from leaf explants in the clone of poplar ‘novoberlinska-3’ was 112, and from petiole explants – 43, while in the clone ‘volosystoplidna’ the number of shoots obtained from leaf and petiole explants was 30 and 33, respectively. therefore, the number of shoots per regenerated explant in the clone of poplar ‘novoberlinska-3’ from leaf explants was 3.1 (fig. 4b) that significantly exceeded all other variants by 1.5 – 2 times. thus, plant regeneration from leaf explants in the clone ‘novoberlinska-3’ showed the highest efficiency, which significantly differed from all other explants. as our experiments have shown, the composition of the nutrient medium plays an important but different role in the regeneration of the shoots of individual poplar clones. many studies (khatab 2011; cai et al. 2015; kwon et al. 2015; pardhi et al. 2019) have demonstrated that the optimal composition of the nutrient medium and concentration of phytohormones should be selected separately for each species and even for each cultivar and the type of explants. in particular, studies (tsvetkov et al. 2007) have shown that the addition of thidiazuron to the main nutrient medium at both high and low concentrations has different effects on shoot regeneration in white poplar (p. alba). the greatest regeneration efficiency was observed when lower concentrations were added (0.11 – 0.56 μm) compared to higher concentrations (14 μm). similarly, thidiazuron applied to stem, leaf, and root explants of p. berolinensis in higher concentrations (0.1 – 0.5 mg.l-1), than we did (0.04 mg.l-1), was inefficient for regeneration. in those experiments only very weak efficiency was determined for petiole explants under the lowest concentration of tdz (0.1 mg.l-1) (pavlichenko et al. 2016). addition of 0.5 μm naa induced callus formation in p. euphratica oliv. (cai et al. 2015). studies by garcia-angulo et al. (2018) showed that the addition of 0.5 μm naa leaded to the highest frequency of shoot regeneration in two hybrid clones of populus (p.×euramericana) and (p.×interamericana), but the shoots formed roots better in medium without naa. all these studies suggest that the appropriate composition of the nutrient medium must be selected at each stage of organogenesis. fig. 4. the efficiency of regeneration from leaf and petiole explants of poplar clones ‘novoberlinska-3’ and ‘volosystoplidna’ in vitro. the percentage of regenerated explants (% ± sp) (a) and the number of shoots per regenerated explant (mean ± standard error of the mean) (b) are presented. different letters above the bars indicate significant differences between the samples in the percentage of regenerated explants and the number of shoots per regenerated explant after the chi-square and the mannwhitney u test, respectively (p ≤ 0.05), n = 15 replicates. our results showed a significant difference between the survival rates of two clones during the culture 6 nova biotechnol chim (2022) 21(1): e1089 2 initiation. the differences in regeneration rates were also determined between the clones as well as leaf and petiole explants at the same composition of medium and growth regulators. thus, we can assume that the regeneration of shoots in poplar clones depends on both the genotype of the plants and on the tissues to be regenerated, and therefore requires the selection of optimal conditions for each clone. conclusion in this study, two clones of poplar ‘novoberlinska3’ and ‘volosystoplidna’ were introduced into in vitro culture. an optimal initiation nutrient medium for poplar ‘volosystoplidna’ was im-wpm supplemented with bap (0.2 mg.l-1) and iba (0.1 mg.l-1), on which the survival rate was 93.3 %. it was significantly higher than the survival rate of explants cultivated on im-ms with the same hormone composition. for poplar clone ‘novoberlinska-3’, although better survival rate (73.3 %) was observed on im-ms than on imwpm (46.7 %) but without a significant difference. the protocol chosen for indirect in vitro regeneration showed better performance of poplar clone ‘novoberlinska-3’, but was also efficient for the clone ‘volosystoplidna’. the percentage of regeneration in the clone ‘novoberlinska-3’ from leaf (92.3 %) and petiole (90 %) explants was significantly higher than in the poplar clone ‘volosystoplidna’, where the percentage of regeneration in each type of explants equalled 50 %. the regeneration rate of leaf explants of clone ‘novoberlinska-3’ (3.1) was also significantly higher than that of petiole explants and exceeded the rates of explants of clone ‘volosystoplidna’. obtained aseptic cultures of two highly productive poplar hybrids will be used in our further studies, and optimal protocols for regeneration will be applied for their genetic transformation. acknowledgements this work was carried out in the frame of the national academy of sciences of ukraine program “biofuel resources and bioenergy” (2019-2021) (the state registration number 0120u000278). it was also partially supported by the national research foundation of ukraine within the research competition “support for research of leading and young scientists”, the project “effects of stress factors on prion-like proteins synthesis in plants” (registration number 2020.02/0316). conflict of interest the authors declare that they have no conflict of interest. references abiri r, atabaki n, abdul-hamid h, sanusi r, ab-shukor na, shaharuddin na, malik s (2020) the prospect of physiological events associated with the micropropagation of eucalyptus sp. forests. 11: 1211. aggarwal g, gaur a, srivastava dk (2015) establishment of high frequency shoot regeneration system in himalayan poplar (populus ciliata wall. ex royle) from petiole explants using thidiazuron cytokinin as plant growth regulator. j. forestry res. 26: 651-656. aylott m, casella e, tubby i, street n, smith p, taylor g (2008) yield and 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participation and schools of young scientists “mechanisms of resistance of plants and microorganisms to unfavourable environmental”, irkutsk, russia, july, 1015. pp. 1244-1247. 8 microsoft word vidova2008b_manuscript_phagedispnbt.doc nova biotechnologica 8-1 (2008) 23 phage display – a tool for detection and prevention against pathogens. a review barbora vidová1,2, andrej godány2, ernest šturdík1 1institute of biochemistry, nutrition and health, slovak university of technology, sk-812 37 bratislava, slovak republic (b.vidova@gmail.com) 2 institute of molecular biology, slovak academy of sciences sk-845 51, bratislava, slovak republic 3department of biotechnology, university of ss. cyril and methodius, sk-917 01 trnava, slovak republic abstract: in this article are reviewed the promising uses of phage display in the areas such as microbial pathogens detection of and vaccination. phage display is a molecular technique by which foreign proteins are expressed at the surface of phage particles. such phages thereby become vehicles for expression that not only carry within them the nucleotide sequence encoding expressed proteins, but have also the capability to replicate. recent data acquired from genome sequencing and advances in phage biology research have aided the development of phage-derived bacterial detection and treatment strategies. key words: phage display, microbial pathogens, detection 1. introduction bacteriophages (phages) are viruses that infect bacteria. phages are either virulent (lytic) or temperate and use the host bacterium as a factory for their own replication. since their independent discovery by twort and d’herelle, research on phage has enabled major fundamental and technological advances that have been essential for the emergence of modern molecular biology (mcauliffe et al., 2007). in fact, it is difficult to over-emphasize the large impact that phage research has had on molecular biology. for example, phage research was cardinal in defining the central dogma of molecular biology, in the identification of dna as the genetic material, the definition and mapping of the gene, and the discovery of mrna as an intermediate for the production of proteins. furthermore, phages were used, simply and elegantly, to demonstrate the random pre-existing nature of genetic mutations before evolutionary selection, providing clear evidence of darwin’s natural selection theory (cairns et al., 2006). our understanding of gene regulation has also profited immensely from studies using lambda phage (λ) (ptashne, 2004). in molecular biology, the impact of phage-derived technology is clearly visible: transducing phage, restriction enzymes, phage promoter-based expression systems, random mutagenesis (e.g. phage mu), phage-based cloning vectors and genomic libraries, and phage display techniques, to name but a few. the sheer abundance of phage on earth (estimated to be 1031 (wommack and colwell, 2000)) and the accumulating data from both phage and bacterial genome sequencing projects have highlighted the global significance of phage in bacterial ecosystems and their key role in the adaptive evolution of bacteria. 24 vidová, b. et al. sequence analysis indicates that up to 20% of each bacterial genome might consist of phage-related dna in the form of prophage or phage remnants (casjens, 2003). the importance of these phage-related sequences in the evolution of bacteria is most striking in the phenomenon of lysogenic conversion. for example, in vibrio cholerae, the genes encoding cholera toxin are provided by a converting phage, resulting in a virulent strain (waldor and friedman, 2005). phage are genetically diverse; approximately half of newly sequenced phage genes have no known homologues (hambly and suttle, 2005), and phage genomes vary in size by at least two orders of magnitude (kutter, 2005). this review aims to provide an insight into how an understanding of phage biology can be exploited to generate novel bacterial detection and vaccination strategies. 2. phage display principles in phage display, a heterologous peptide or protein is displayed on the surface of the phage (fig. 1) through transcriptional fusion with a coat-protein gene (smith, 1985), which is accomplished by the incorporation of the nucleotide sequence encoding the protein to be displayed into a phage or phagemid genome as a fusion to a gene encoding a phage coat protein. this fusion ensures that as phage particles are assembled, the protein to be displayed is presented at the surface of the mature phage, while the sequence encoding it is contained within the same phage particle. this physical link between the phenotype and genotype of the expressed protein and the replicative capacity of phage are the structural elements that underpin all phage display technology (fig. 1) (willats, 2002). this results in a production a novel phage particle that has a variety of potential uses. the most widely used phage display methods are based on the use of m13 and related filamentous phages of escherichia coli but others, including the e. coli phages λ and t7, have also been used (benhar, 2001, willats, 2002). because particulate phages are relatively easy and inexpensive to purify, phage display can also provide a means of purifying a particular protein or antibody. phage-display libraries can be screened in several ways to isolate displayed peptides or proteins with practical applications. for example, it is possible to isolate displayed peptides that bind target proteins with affinities similar to those of antibodies. they can then be used as therapeutics, which act either as agonists or through the inhibition of receptor–ligand interactions. the high-affinity, displayed peptides also have the potential to be used for the detection of pathogens and agents posing a biological threat in the environment (petrenko and vodyanoy, 2003). a more complete discussion of the potential of phage display is given in several recent reviews (benhar, 2001, willats, 2002, wang and yu, 2004). the simplest way of achieving the expression of a foreign protein is to simply create a fusion between the nucleotide sequence to be expressed and a coat protein gene within the viral genome (fig. 2a). using this direct approach all the copies of the chosen coat protein become fusion proteins (winter et al. 1994). this can be advantageous in terms of numbers of nova biotechnologica 8-1 (2008) 25 expressed foreign proteins but if the functionality of the chosen coat protein is compromised by the fusion then phage viability may be affected, especially since no wild type versions of the coat protein are retained. fig. 1. the principle of phage display. (a) simplified hypothetical bacteriophage with coat proteins piii, pvi, pviii, pvii, pix. (b) forein peptide or protein is dispalyed at the phage surface by the fusion of gene encoding forein protein with phage piii coat protein coding gene, or (c) by the fusion of gene encoding forein protein with phage pviii coat protein coding gene. the number of copies of forein protein displayed is related to which phage coat protein is chosen as a fusion partner (melita et al., 2001). this can be avoided if hybrid phages are produced in which some versions of a given coat protein are wild type and some are fused to a foreign protein (fig. 2b and 2c). in some hybrid phage systems, the gene fusion is an additional element of the phage genome so that a wild type copy of the coat protein gene is retained and phage particles express both wild type and fusion proteins (fig. 2b) (sidhu, 2001). alternatively, hybrid phage may be created using a phagemid-based system and this approach has been widely adopted (fig. 2c). sequences encoding fusion proteins are carried by phagemids (plasmids with a phage origin of replication) while the most of the genes required for the formation of phage particles are carried by helper phage that is co-infected together with phagemids into host bacteria (sidhu, 2001) (fig. 2c). 26 vidová, b. et al. fig. 2. strategies for the expression of proteins at the surface of a simplified hypothetical bacteriophage. the simplest format for the expression of a peptide or protein (a) is to fuse the gene (gχ) encoding the foreign protein (pχ) to one of the phage coat protein genes (e.g., g1) hybrid phage (b) may be created by incorporating the gene fusion (g1/gχ) as an additional element in the phage genome. with this arrangement, two versions of the phage coat protein chosen as the fusion partner are encoded one by the native gene (p1) and one by the fusion gene (p1/pχ). as phage particles are assembled both p1 and p1/pχ are incorporated into the phage coat. phagemid based systems (c) are also widely used to construct hybrid phage. however, instead of being present on a single genome, the genes encoding wild type coat protein and fused protein are carried by helper phage and phagemid respectively. host bacteria contain both phagemid and helper phage dna and both genomes contribute to the synthesis of hybrid phage particles (willats, 2002). 3. phages for the detection and typing of bacteria many methods of detecting specific bacteria are available, but the low-cost and ready production of large numbers of phage, added to their specificity for a target nova biotechnologica 8-1 (2008) 27 bacterial species, makes them ideal for bacterial detection. although other biomolecules can confer specificity of molecular recognition, they have their own limitations. for example, antibodies are expensive to produce and sensitive to harsh environmental conditions. another important advantage of phage-based systems is that they usually only detect living bacteria, thereby reducing the number of false positives that arise from the use of approaches such as pcr. furthermore, dead cells present after processes such as pasteurization or disinfection might be no hazardous but still can be detected by antibodies (petty et al., 2007). phages bounded to bacteria can be detected by specific labeled antibodies, thereby increasing the sensitivity of detection (watson and eveland, 1965). for specific typing, different species of phage can be plated out onto a lawn comprising an unidentified bacterial strain, and the presence of clear areas (plaques) where an individual phage particle has grown and lysed the surrounding cells enables identification of the specific bacteria. recombinant phages are often used in phagebased detection systems (benhar, 2001; willats, 2002). the methods that have been used to detect pathogenic bacteria include (i) using phages specifically to deliver reporter genes, e.g. lux (kodikara et al., 1991) or green fluorescent protein (funatsu et al., 2002), which are expressed after infection of target bacteria; (ii) using phages that have a fluorescent dye covalently attached to the phage coat, and detecting the specific adsorption (hennes et al., 1995; goodridge et al., 1999); (iii) the detection of released cellular components, such as adenylate kinase (corbitt et al., 2000), after specific lysis; and (iv) using phages displaying peptides or antibody fragments that will bind specific bacterial pathogens or toxins. other means of improving the sensitivity of detection are phage amplification assays. in brief, phage infection of the pathogenic bacterium results in the release of many phage particles that can be detected by a second (non-pathogenic), sensitive bacterial strain. success with this approach has been seen with the detection of mycobacterium tuberculosis (petrenko and vodyanoy, 2003). a more detailed account of these and other techniques is provided elsewhere (kutter, 2005) and a selection that emphasizes the range and sensitivity of the technologies used and the diversity of detectable bacterial species is summarized in table 1 (petty et al., 2007). 4. phages as vaccine delivery vehicles phages have been used as potential vaccine delivery vehicles in two different ways: by directly vaccinating with phages carrying vaccine antigens on their surface or by using the phage particle to deliver a dna vaccine expression cassette that has been incorporated into the phage genome (clark and march, 2004). in phage-display vaccination, phages can be designed to display a specific antigenic peptide or protein on their surface. alternatively, phages displaying peptide libraries can be screened with a specific antiserum to isolate novel protective antigens or mimetopes – peptides that mimic the secondary structure and antigenic properties of a protective carbohydrate, protein or lipid, despite having a different primary structure (folgori et al., 1994; phalipon et al., 1997). the serum of convalescents can also be used to screen phage-display libraries to identify potential vaccines against 28 vidová, b. et al. a specific disease, without prior knowledge of protective antigens (meola et al., 1995). in a few cases, whole phage particles displaying antigenic proteins have been used as vaccines in animal models (wang and yu, 2004; irving et al., 2001). specific antibody responses (di marzo veronese et al., 1994) and protection against an immune challenge have been shown (bastien et al., 1997). tab. 1. applications of the different phage-based antibacterial strategies target species type of assay matrix assay sensitivity refs time (hours) listeria monocytogenes luxab reporter phage and single-tube culture 2 102–103 cfu/ml loessner et al., 1996 luminometer spiked salad 22–24 1 cfu/g e. coli 0157:h7 fluorescently labeled phage and flow ground beef 6 2.2 cfu/g goodridge et al., 1999 cytometry milk 10 10 1–102 cfu/ml e. coli xl1blue quorum sensing and bioluminescence culture 1.5 10 8 cfu/ml ripp et al., 2006 (luxcdabe) 10.3 1 cfu/ml spiked lettuce 12.1 105 cfu/ml leaves 22.4 102 cfu/ml e. coli bl-21 biotin-tagged phage and quantum dots culture 1 10 cfu/ml edgar et al., 2006 nanocomplexes, fluorescence microscopy and flow cytometry pseudomonas aeruginosa phage amplification culture 4 40 cfu/ml stewart et al., 1998 e. coli phage-mediated release of adenylate culture 1 10 4 cfu/ml blasco et al., 1998 kinase mycobacteriu m tuberculosis phage replication assay sputum 2 days 100 cfu/ml mcnerney et al., 2004 microlunatus phosphovorus fluorescently labelled phage and activated sludge 1 10 2 cfu/ml lee et al., 2006 epifluorescent microscopy e. coli k-12, mg1655 b-d-galactosidase reporter phage and culture 6–8 0.01 cfu/ml neufeld et al., 2003 amperometric detection e. coli k-12 w3110 sensing of phagetriggered ion cascade culture 10 min 1 cfu/ml dobozi-king et al., 2005 (septic) staphylococcu s aureus spreeta surface plasmon resonance culture not stated 10 4 cfu/ml balasubra manian et al., 2006 nova biotechnologica 8-1 (2008) 29 rather than generating a transcriptional fusion to a coat protein, substances could also be artificially conjugated to the surface of phages after growth, which further increases the range of antigens that can be displayed (molenaar et al., 2002). because phage particles are naturally immunostimulatory (clark and march, 2004; kleinschmidt et al., 1970), an antigen presented on the phage coat would come ‘ready conjugated’ with a natural adjuvant activity, without the need for separate protein purification and subsequent conjugation to a carrier molecule before immunization. more recently, it has also been shown that unmodified phages can be used to deliver dna vaccines more efficiently than standard plasmid dna vaccination (clark and march, 2004; march et al., 2004; jepson and march, 2004). when compared with standard dna vaccination, superior antibody responses have been shown in mice (clark and march, 2004) and rabbits (march et al., 2004). 5. conclusion according to here reviewed information about phage display, this technique/tool has a multipurpose utilization in molecular evolution, analysis of protein/ligand interactions and the generation of antibodies. taking into account a prevention against pathogenic microorganisms, phage display could be used as a potential vaccine against streptococcus agalactiae, a well-known bovine mastitis pathogen of particular importance because it is highly infectious (spreading from cow to cow by machine milking, unless care is taken to disinfect the teat cups), and causes mainly subclinical infections. as a result, s. agalactiae can spread widely within a herd, causing immediate loss due to reduced milk yield, and eventual large losses, when it is finally recognized. additionally, this pathogen can be transmitted through dairy product to consumers. therefore, useful vaccine against this bacterium is of great interest. all clinical isolates of s. agalactiae express a polysaccharide capsule, with nine capsular serotypes identified so far. the major invasive disease causing serotypes are ia, ib, ii, and iii (schuchat, 1998). indeed, it was observed that the protection conferred by capsular polysaccharides is type specific (kasper et al., 1996). noncapsular surface antigens are being investigated as potential vaccine candidates or carrier proteins (gravekamp et al., 1999; stålhammar-carlemalm et al., 1993). one of these noncapsular antigens, the sip protein, was highly conserved and produced by every s. agalactiae strain examined to date, which included representative isolates of all serotypes (brodeur et al., 2000; chotár et al., 2006). it was also established that sip-specific antibodies recognized their epitopes at the cell surfaces of different s. agalactiae strains (rioux et al., 2001). more importantly, the immune response to purified recombinant sip protein efficiently protected adult mice against experimental infection with s. agalactiae strains representing serotypes ia/c, ib, ii/r, iii, v, and vi (brodeur et al., 2000). however, the direct use of a peptide presenting by a recombinant phage displaying as a vaccine to examine its protective potential would be simpler and much less expensive than the conventional method of peptide synthesis or recombinant protein preparation and purification for immunization. the recombinant phage displaying a pathogen specific peptide, in s. agalactiae the displaying of mentioned sip protein or its part, representing an immunogenic mimic of 30 vidová, b. et al. natural antigen. this could lead to production of antibodies specific to pathogen. recombinant bacteriophage displaying a disease specific epitope can be use as a vaccine to examine the potential of an epitope of interest to prevent disease. phage display has also the overwhelming advantage that it is has cheap and easy. it uses standard microbiological techniques that are familiar to all molecular biologists, and its key resources phage libraries and clones are replicable and therefore nearly costfree after their initial construction or selection. it is astonishing to contemplate that within a single tube could be fit a few hundred trillion phage particles displaying billions of different peptide structures an abundance and diversity from which hundreds of different users with altogether different purposes in mind can select clones of great value. and when that supply has nearly run out, only simple propagation is needed to replenish what is left to satisfy the needs. where a safe and efficacious vaccine available, screening and administration of antibiotics would be unnecessary, policies for the prevention of s. agalactiae infections would be simplified and importantly, prevention would be more successful, whereby this approach is applicable to many other pathogenic microorganisms. additionally, cost-effectiveness analyses invariably demonstrate that vaccination is the most cost-effective of all potential prevention. acknowledgements: figure 1 has been reprinted from current opinion in 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ecosystems. microbiol. mol. biol. rev., 64, 2000, 69–114. nova biotechnol chim (2022) 21(2): e1228 doi: 10.36547/nbc.1228 1 nova biotechnologica et chimica phenolic contents, antioxidant, and enzyme inhibitory effects of crude extracts of polycarpon polycarpoides fiori subsp. catalaunicum o. bolòs & vigo madiha arioua1, asma cheribet1, lynda gali2, 1department of biochemistry and cellular and molecular biology, faculty of natural and life sciences, frères mentouri constantine 1 university, constantine, algeria 2biotechnology research center (crbt), ali mendjli, nouvelle ville, uv 03, bp e73 constantine, algeria  corresponding author: lyndag652@gmail.com article info article history: received: 14th october 2021 accepted: 11th march 2022 keywords: α-amylase acetylcholinesterase antioxidant activity caryophyllaceae polycarpon polycarpoides urease abstract the present work aimed to study some pharmacological effects of extracts of the aerial parts of polycarpon polycarpoides fiori subsp. catalaunicum o. bolòs & vigo (caryophyllaceae) widely distributed in the mediterranean basin. the crude extracts were prepared using four solvents (petroleum ether, ethyl acetate, methanol, and methanol: water 1 : 1, v/v), and then examined for their phenolic contents using spectrophotometric methods and for their in vitro antioxidant activity and ability to inhibit urease, acetylcholinesterase, and α-amylase. low levels of total phenolic were recorded ranging from 14.39 ± 3.40 to 101.84 μg.gae.mg-1 of extract corresponding to the petroleum ether and ethyl acetate extracts, respectively. as for the flavonoids, very low values varying from 0.37 ± 0.13 to 4.22 ± 0.83 μg.gae.mg1 of extract were obtained. a moderate antioxidant effect was exerted by the extracts most often the methanol and ethyl acetate extracts were the most potent probably due to their polyphenolic content. remarkable inhibitory effect has been exhibited by the extracts against α-amylase and more specifically, the petroleum ether extract displayed the strongest capacity with a percentage inhibition of 48.19 ± 2.99 % at 400 µg.ml-1. however, all extracts were inactive against urease and acetylcholinesterase. these results could constitute a starting point for carrying out more studies on the plant in order to assess the possibility of valuing it as a source of bioactive compounds. introduction recently, the awareness of the harmful effects of the excessive use of synthetic products incorporated in pharmaceutical or food formulations for therapeutic, nutritional, organoleptic purposes has led to increased interest in natural products. in addition, the interest of natural products has also arisen from the fact that these compounds can exert multiple effects by their action on various targets providing more beneficial effects (ayaz et al. 2019). therefore, it is important to research new therapeutic substances, nutraceuticals, or food additives as alternatives to synthetic products (moldes et al. 2017). in this context, plants remain the main source of new therapeutic molecules with high healing potential and less toxic effects (christaki et al. 2012; gali et al. 2020). mailto:lyndag652@gmail.com nova biotechnol chim (2022) 21(2): e1228 2 natural antioxidants have received a lot of attention in recent decades for their incorporation into foods as additives to delay the oxidation reactions responsible for the loss of their nutritional and organoleptic qualities (lourenço et al. 2019). in fact, the use of synthetic antioxidants such as butylhydroxyanisol (bha) and butylhydroxytoluene (bht) in the food industry has raised concerns about their possible involvement in certain carcinogenesis processes and therefore their replacement by natural compounds with less harmful effects constituted a primary need (dolatabadi and kashanian 2010). antioxidants-based supplements have also been the subject of recent years as there is growing evidence for the involvement of free radicals and other reactive species in the development of certain chronic diseases such as cancer, inflammatory, metabolic, cardiovascular and neurological diseases (moure et al. 2001). among phytochemicals, polyphenols are recognized as the most potent natural antioxidants found in both edible and nonedible plants due to their structure with hydroxyl groups giving them potential redox properties (shahidi and ambigaipalan 2015). plants are also considered an important source of therapeutics to treat many diseases due to the presence of various phytochemicals targeting cellular constituents such as enzymes that are central to biochemical reactions. in fact, many therapies used in the management of certain diseases are based on the inhibition of key enzymes as in the case of alzheimer's disease and type 2 diabetes. the need for new molecules and especially of natural origin comes from the toxicity and/or non-specificity of the available treatments and therefore lower effectiveness of these therapies (kekuda and rao 2019). caryophyllaceae also known as the rose family is one of the largest families of around 3,000 species generally distributed in the northern hemisphere, particularly in the mediterranean basin (derici et al. 2021). this family is known for its richness in active metabolites, in particular flavonoids, alkaloids, triterpene saponins, phytoecdysteroids and sterols (jakimiuk et al. 2021). the presence of various active constituents makes the species of this family of medical importance and can be interesting sources of new bioactive constituents of therapeutic or food interests. caryophyllaceae plants have been reported to exert various biological effects such as antioxidant, antiviral, anticancer, anti-inflammatory and antimicrobial effects. (chandra and rawat 2015). polycarpon polycarpoides (biv.) fiori subsp. catalaunicum o. bolòs & vigo is a species of the caryophyllaceae family that grows in northern algeria. however, data on its pharmacological effects are poorly provided. thus, this study is devoted to determining some of the biological effects of this species for its valuation as medicinal or food plant. experimental chemicals folin-ciocalteu, sodium carbonate, vanillin, 2,2-diphenyl-1-picrylhydrazyl (dpph), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (abts), potassium persulfate (k2s2o8), neocuproine, ache type vi-s from electric eel (<1000 u.mg-1 solid), urease from canavalia ensiformis (jack bean, ≥5 u.mg-solid), α-amylase from aspergillus oryzae (≥150 u/protein) trolox, gallic acid, quercetin, catechin, and other chemicals were purchased from sigma-aldrich (saint-louis, usa) acetylthiocholine-iodide was obtained from applichem gmbh, (darmstadt, germany. all organic solvents and chemicals used in this study were of analytical grade. all the assays were carried out on a 96-well microplate, and the absorbance was recorded using a enspire multimode plate reader (perkinelmer, waltham, usa). plant material the aerial part of polycarpon polycarpoides was collected in annaba (northern algeria) in december 2017. the plant was identified by dr. tarek hamel (department of biology, badji mokhtar university, annaba, algeria). the samples were cleaned and air dried in the dark, and then ground to a fine powder using an electric grinder. nova biotechnol chim (2022) 21(2): e1228 3 preparation of crude extracts the extraction procedure was carried out by soaking 25 g of vegetable powder in 100 ml of the extraction solvent (petroleum ether, ethyl acetate, ethanol and methanol: water (1:1, v/v) for 2 h with continuous stirring (300 rpm) at room temperature, the solution obtained was filtered through a whatman filter (whatman filter paper n°4). the marc is extracted twice more to extract the maximum of soluble compounds. the various solvents were then eliminated under reduced pressure at 40 °c using the rotary evaporator buchi r-215 (büchi ag, uster, switzerland). the residues were stored at 4 °c for further analysis. total phenolic contents total phenolic content in polycarpon polycarpoides extracts was determined by the folin-ciocalteu method on a 96-well microplate as reported by müller et al. (2010). the reaction consisted of mixing 20 µl of samples (1 mg.ml-1), 100 µl of folin-ciocalteu reagent (diluted in distilled water, 1 : 9, v/v), and 75 µl of sodium carbonate (7.5 %) for 2 h in darkness at room temperature. the absorbance was then read at 765 nm in a microplate reader (perkin elmer enspire, singapore). total phenolic contents were expressed as µg gallic acid equivalents per mg of extract (µg.gae.mg-1) using the equation of linear regression obtained from the gallic acid standard curve (y = 0.0034 gallic acid + 0.1044, r2 = 0.9972). total flavonoids content total flavonoids were quantified by the aluminum chloride (alcl3) method according to terkmane et al. (2018) with some modifications. a volume of 100 µl of plant extract (1 mg.ml-1) and an equal volume (100 µl) of a methanolic solution of aluminum chloride hexahydrate (alcl3·6h2o, 2 % prepared in methanol) were mixed. the reaction was left for 15 min and the absorbance was then recorded at 415 nm against a blank containing the sample and methanol instead of alcl3. the results were reported in µg of quercetin equivalent per mg of extract (µg.qe.mg-1) calculated from the standard curve of quercetin (y = 0.027 quercetin, r² = 0.9989). condensed tannins content condensed tannins contents were investigated following the method reported by bakhouche et al. (2021). in a 96-well microplate, each extract was reacted with hydrochloric acid (30 %) and a methanolic solution of vanillin (4 % in methanol). the absorbance of the red complex flavonolsvanillin was measured at 500 nm. the results were expressed as µg of catechin equivalents (µg ce) per mg of extract calculated from the standard curve of catechin (y = 0.0026 catechin + 0.0301). dpph scavenging activity the capacity of the polycarpon polycarpoides extracts to trap the dpph radical was determined according to the method reported by el aanachi et al. (2020). briefly, 40 µl of extracts or standards (trolox and ascorbic acid) at different concentrations (6.25 – 800 µg.ml-1) were reacted with a 160 µl of a freshly prepared solution of dpph (1 mm in methanol). the residual purple color of the dpph radical was followed at 517 nm after 15 min of incubation. the results were expressed as percentages of inhibition calculated according to the formula below (eq. 1): inhibition (%) = ((ac – as)/ ac) × 100 (1) where ac and as are the absorbances of the control (methanol and dpph solution) and of the sample, respectively. the concentration of the extract inhibiting 50 % of the radical (ic50) was determined from the curve drawn by the percentages of inhibition at different concentrations. scavenging activity of abts•+ the ability of extracts to trap radicals abts has been evaluated using the method described by dudonné et al. (2009) with some modifications. the abts•+ cation solution has been previously prepared by reacting a volume of 7 mm of abts with a same volume of 2.5 mm potassium persulfate for 16 hours at room temperature and in nova biotechnol chim (2022) 21(2): e1228 2 the dark. the resulting solution is diluted with distilled water to obtain a solution having an absorbance of 0.7 at 734 nm. then, samples (40 μl) at different concentrations were pipetted in a microplate and then 160 μl of a diluted solution of abts•+ were added and the mixture was allowed to react for 10 min. the absorbance was recorded at 734 nm. the inhibition rates of the radical were determined using the previous formula (1). ic50 values were calculated from the equation of the regression curves. phenanthroline assay the method reported by szydłowska-czerniak et al. (2008) was used to measure the ability of extracts to reduce ferric iron in the phenanthroline-fe (iii) complex. in a 96-well microplate, 10 µl of extract, 50 µl fecl3 (0.2 %), 30 µl phenanthroline (0.5 % in methanol), and 110 µl methanol were mixed. the reaction was left for 20 min at room temperature and the absorbance was then measured at 510 nm. the results are reported in the form of absorbances. trolox and ascorbic acid were used as standards. cupric reducing antioxidant capacity (cuprac) the cuprac test was performed by the method described by apak et al. (2004). in a 96-well microplate, 40 μl of extract, 60 μl of ammonium acetate (1 m), and 50 μl of neocuproine (7.5 mm), and 50 μl of copper (ii) chloride (10 mm) are mixed. the mixture is incubated for 1 h, and the absorbance was read at 450 nm. results were reported as absorbances at different concentrations and were compared to the standards trolox and ascorbic acid. α-amylase inhibition the determination of the inhibitory activity of α-amylase is carried out according to the method of yang et al. (2012) with modifications. in a 96 wellmicroplate, 25 μl of each extract at different concentrations was mixed with 50 μl of the αamylase solution (1 u.ml-1 in the phosphate buffer saline, 100 mm, ph, 6.9, 6 mm nacl) for 10 min at 37 °c. the enzymatic reaction was initiated by adding 50 μl of starch (0.1 %). a second incubation for 10 min at 37 °c is carried out and the reaction was stopped by the addition of 25 μl hcl (1 m), and the residual starch is revealed by 100 μl of iodine-potassium iodide solution (iki). the reading is carried out at 630 nm against a negative control containing all reagents without the extracts. in parallel, controls containing only the starch solution and iki, or the sample solutions and iki were performed. results are reported as percentages of inhibition at different concentrations using the formula below (eq. 2): inhibition (%) = [1-((as-ab)/(ae-ac))]*100 (2) where: ac is the absorbance of the reaction containing only starch, iki, and hcl without enzyme and inhibitor solutions, ae is the absorbance of the control containing all the reagents without inhibitor, as is the absorbance of the reaction containing all the reagents in presence of the test sample, ab is the absorbance of the blank containing only the test sample and iki. urease inhibition the inhibitory effect of the extracts on urease was examined using the protocol described by nabati et al. (2012). the extracts (10 μl) were mixed with 25 μl of the urease solution (1 mg.ml-1 in 100 mm of phosphate buffer, ph 8.2) and 50 μl of urea (30 mm). the amount of ammonia in the medium was quantified by adding 45 µl of phenol reagent (0.4 g of phenol and 2 mg of sodium nitroprusside in 40 ml of deionized water) and 70 µl of the basic reagent (0.3 g of naoh and 0.5 ml of hypochlorite in 60 ml of deionized water). the absorbance of the blue color of the complex ammonia-phenol-hypochlorite was recorded at 630 nm. results are reported in terms of percent inhibition calculated using the following formula (eq. 3): % inhibition = (e-s/e)× 100 (3) where e represents the absorbance of the control containing the enzyme solution without the tested samples, whereas s is the absorbance of the 4 nova biotechnol chim (2022) 21(2): e1228 3 reaction medium in the presence of the tested sample. acetylcholinesterase inhibition the inhibition of acetylcholinesterase was examined by the method previously reported by (cetin cakmak and gülçin 2019). briefly, a volume sample (10 µl) were put in contact with 20 µl of acetylcholinesterase (5.32.10-3 u in 0.1 m phosphate buffer, ph 8) at 25 °c for 15 min. the reaction was started by adding acetylthiocholine iodide (0.71 mm) and the resulting thiocholine was quantified by dtnb (10 µl, 0.5 mm). the absorbance as monitored at 415 nm. the rate of inhibition was determined using the preceding eq. 3. statistical analysis data were presented as mean ± standard deviation of three measurements. one-way anova analysis followed by the tukey's multiple comparison test was adapted to analyse the difference between samples using graphpad software version 5 (graphpad software inc, california, usa). the p-value was set at 0.05. results phenolic compounds contents the results of the quantification of the main classes of polyphenols are shown in table 1. as shown, the total phenolic and flavonoids contents are influenced by the extraction solvent. ethyl acetate and methanol extracts gave the highest total phenolic contents with values of 108.12 ± 8.15 and 101.84 ± 2.21 µg.gae.mg-1 of extract, respectively (the values are not significantly different, p>0.05). in another hand, the contents of flavonoids recorded were very weak ranging from 0.37 ± 0.13 (petroleum ether) to 4.22 ± 0.83 µg.gae.mg-1 of extract observed in the ethyl acetate extract. table 1. extraction yield, total phenolic, total flavonoids, and condensed tannins contents of polycarpon polycarpoides extracts. total phenolic [µg.gae.mg-1] total flavonoids [µg.gae.mg-1] condensed tannins [µg.ce.mg-1] petroleum ether 14.39 ± 3.40c 0.37 ± 0.13c nd ethyl acetate 108.12 ± 8.15a 4.22 ± 0.83a nd methanol 101.84 ± 2.21a 1.43 ± 0.27b nd methanol: water 43.12 ± 1.02b 1.04 ± 0.26b nd gae – gallic acid equivalents, qe – quercetin equivalents, ce – catechin equivalents. values bearing different letters in the column are statistically different (p < 0.05). antioxidant activity the antioxidant activity of the extracts was assessed using five different methods and the results were reported as percentage of inhibition or absorbances at different concentrations (fig. 1). the values of ic50 obtained in the dpph and abts assays were also reported in table 2. anti-free radical scavenging activity has been studied using free radicals dpph and abts, which are widely used to determine in vitro the ability of extracts or compounds to scavenge free radicals. the extracts showed dose-dependent scavenging activity against both dpph and abts free radicals, as shown in fig. 1a, b. comparing the extracts on the basis of their ic50 values, the methanol extract showed the most potent scavenging activity against dpph and abts radicals with ic50 values of 249.46 ± 11.22 and 73.86 ± 1.89 µg.ml-1, respectively, followed by ethyl acetate extract, methanol: water and petroleum ether showing the weakest effect against these radicals (ic50>800 µg.ml -1). in addition, the antioxidant activity of polycarpon polycarpoides extracts was measured by methods based on the reduction of a metal ion associated with a chromophore, which undergoes a color change upon receipt of an electron. the ability of the extracts to reduce iron ions was assessed using the phenanthroline test (fig. 1c). the extracts can 5 nova biotechnol chim (2022) 21(2): e1228 2 be classified according to their ability to reduce fe (iii) to fe (ii) reported in terms of absorbances at 200 µg.ml-1in descending order as follows: methanol: water > ethyl acetate > methanol > ether from petroleum giving the corresponding absorbances 0.65 ± 0.02, 0.58 ± 0.03, 0.50 ± 0.00, 0.35 ± 0.04, respectively. these results were considered low compared to trolox and ascorbic acid standards giving absorbances of 0.56 ± 0.02 and 0.80 ± 0.0, respectively using a very low concentration (6.25 µg.ml-1). in the reduction of copper ions evaluated by the cuprac assay (fig. 1d), ethyl acetate extract was the most potent providing the highest absorbance at the concentration of 800 µg.ml-1 (1.85 ± 0.09). methanol extract also showed a remarkable ability to reduce copper ions with the absorbance of 1.85 ± 0.09 at the highest concentration used (800 µg.ml-1), followed by ether petroleum extract (1.11 ± 0.02), and finally, aqueous methanol has the lowest reducing capacity with an absorbance of 0.8 ± 0.03. trolox and ascorbic acid exerted a very potent ability to reduce copper ions with absorbances of 1.34 ± 0.13 and 1.38 ± 0.11, respectively at very low concentrations (25 µg.ml-1). 0 200 400 600 800 0 20 40 60 80 100 petroleum ether ethyl acetate methanol methanol: water concentration µg/ml % i n h ib it io n o f d p p h 0 200 400 600 800 0 20 40 60 80 100 petroleum ether ethyl acetate methanol methanol: water concentration µg/ml % i n h ib it io n o f a b t s 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 petroleum ether ethyl acetate methanol methanol: water concentration µg/ml a b s o rb a n c e a t 5 1 0 n m 0 200 400 600 800 0.0 0.5 1.0 1.5 2.0 2.5 petroleum ether ethyl acetate methanol methanol: water concentration µg/ml a b s o rb a n c e a t 4 5 0 n m a b c d fig. 1. dose-dependent effect of the extracts showed in the antioxidant activity determined by different assays. scavenging effect of the radical dpph (a), abts (b), reduction of fe (iii)-phenanthroline (c), and reduction of copper (d). each point presents the mean ± sd (n = 3). table 2. ic50 values of polycarpon polycarpoides obtained with dpph and abts. dpph ic50 [µg.ml-1] abts ic50 [µg.ml-1] petroleum ether >800 >800 ethyl acetate 333.76 ± 1.08b 90.51 ± 2.82a methanol 249.46 ± 11.22a 73.86 ± 1.89b methanol: water 500,06 ± 3.19c 92.15 ± 1.99a trolox 5.12 ± 0.21 3.21 ± 0.06 ascorbic acid 4.39 ± 0.01 3.04 ± 0.05 values are reported as means ± sd of three measurements. values bearing different letters in the same column are significantly different (p < 0.05). 6 nova biotechnol chim (2022) 21(2): e1228 3 enzyme inhibitory effect p. polycarpoides extracts have been tested for their ability to inhibit certain enzymes, including αamylase, urease and acetylcholinesterase and the results were shown in fig. 2. the results indicated that all the plant extracts exhibited remarkable inhibition of alpha-amylase at a dose-dependent manner (results not shown) and was significantly (p < 0.05) influenced by the extraction solvent (fig. 2a). in this context, the petroleum ether extract was the most potent with a percentage inhibition at 400 µg.ml-1 of 48.19 ± 2.99 %.the other extracts also exerted an interesting inhibitory effect and can be classified in ascending order as follows: ethyl acetate > methanol > methanol : water showing percentages of inhibition at the highest concentration (400 µg.ml-1) of 38.89 ± 0.82, 31.74 ± 2.27, and 15.31 ± 2.75 %, respectively. all the extracts were more potent than acarbose, which moderately inhibited the enzyme using high concentrations (62.5 – 4,000 µg.ml-1). at the highest concentration (4,000 µg.ml-1), acarbose inhibited the enzyme with a rate of 53.05 ± 1.59 %. all the extracts were inactive against urease and acetylcholinesterase (fig. 2b, c, respectively). p et ro le um e th er e th yl a ce ta te m et ha no l m et ha no l:w at er a ca rb os e 0 10 20 30 40 50 60 400 µg/ml (4 00 0 µg /m l) in h ib it io n o f a lp h a -a m y la se ( % ) p et ro le um e th er e th yl a ce ta te m et ha no l m et ha no l:w at er t hi ou re a 0 20 40 60 80 100 200 µg/ml in h ib it io n o f u r e a se ( % ) p et ro le um e th er e th yl a ce ta te m et ha no l m et ha no l:w at er g al an ta m in e 0 20 40 60 80 100 200 µg/ml in h ib it io n o f a c h e ( % ) a b c a b c d fig. 2. enzyme inhibitory effect of polycarpon polycarpoides extracts. inhibition pecentages of the extracts on α-amylase at 400 µg.ml-1 in comparison with acarbose at 4,000 µg.ml-1 (a). activity of the extracts and thiourea on urease at 200 µg.ml-1 (b). effect of the extracts and galantamine on acetylcholinesterase at 200 µg.ml-1 (c). each column presents the mean ± sd of three measurements. columns bearing different letters are significantly different (p < 0.05). 7 nova biotechnol chim (2022) 21(2): e1228 3 discussion natural bioactive compounds in plants have gained a lot of attention in recent decades as alternatives to synthetics widely used in the pharmaceutical and food industries. local plants can constitute potential reserves of new bioactive molecules and the study of their bioactivity, and their phytochemical composition represents the starting point of their valuation. polyphenols are an important class of molecules of plant origin exhibiting a wide range of pharmacological effects and exerting beneficial effects on human health (quiñones et al. 2013). the phenolic structure of these compounds makes them the most potent antioxidants among other natural phytoconstituents and therefore it is essential to quantify them in plant extracts (shahidi and ambigaipalan 2015). the quantity as well as the nature of the compounds extracted strongly depend on the extraction solvent. the highest yield is observed in methanol: water (1 : 1) and this observation could be explained by the fact that caryophyllaceae plants are particularly rich in glycosylated saponins with high solubility in water. however, phenolic compounds are more concentrated in ethyl acetate and methanol extracts, which is in agreement with the reported literature supporting that these compounds are more soluble in the polar organic solvent than water alone or weakly polar solvents (babbar et al. 2014; gali and bedjou 2019; yakoubi et al. 2021) the antioxidant properties of plant extracts or natural ingredients have prompted manufacturers to incorporate them as natural alternative additives in foodstuffs to preserve their nutritional and organoleptic quality or for their use for therapeutic purposes in the form of supplements in order to prevent the deleterious actions of reactive species on biological molecules (moure et al. 2001). polycarpon polycarpoides extracts have shown moderate antioxidant activity as studied by free radical scavenging methods (dpph and abts) and by the reduction of iron and copper ions indicating that the various constituents of the extracts can act as free radical scavengers or reducing agents. although various phytoconstituents have an antioxidant effect, phenolic compounds are to date the most powerful natural antioxidant due to their structure bearing hydroxyl groups allowing them to transfer electrons and/or hydrogen (shahidi and ambigaipalan 2015). this fact can explain the high antioxidant effect exhibited by the ethyl acetate, methanol, and methanol: water extracts with the highest phenolic levels in comparison with petroleum ether extract having the lowest contents. indeed, several studies have reported a tight relationship between the phenolic content of different materials (fruits, vegetables, and medicinal plants, and algae) and their antioxidant capacity (abdullah et al. 2013; odabasoglu et al. 2004; ye et al. 2015) besides the antioxidant action, p. polycarpoides showed an interesting effect of inhibiting α-amylase, a key enzyme involved in the degradation of polysaccharides and therefore preventing their absorption. this approach is widely adopted to control postprandial hyperglycemia and prevent the complications characteristic of type 2 diabetes (bourebaba et al. 2016). indeed, herbal extracts have been reported to exert a better effect than the most widely used drugs like acarbose and miglitol and therefore can be used as alternatives. especially, the species belonging to the caryophyllaceae family are known to exert antidiabetic effects through their ability to inhibit carbohydrates hydrolysing enzymes including α-glucosidase and α-amylase (patra et al. 2020; derici et al. 2021).in contrast to the antioxidant effect of p. polycarpoides, the petroleum ether extract with low content of phenolic compounds exhibited the highest inhibitory effect on α-amylase compared to other extracts which suggests that lipophilic molecules can exert a more effective action than hydrophilic molecules. the effect of extraction solvent on decreasing α-amylase activity was also observed in extracts of swertia chirata where the inhibitory effect diminished with increasing solvent polarity to be totally inactive using water (dutta et al. 2012). on another hand, p. polycarpoides was inactive against urease and acetylcholinesterase. urease is involved in the survival mechanisms of helicobacter pylori in the acid environment of the stomach by producing ammonia. the action of plants or drugs on urease may be of therapeutic interest in combating the development of h. pylori 8 nova biotechnol chim (2022) 21(2): e1228 2 since it is believed that most gastric and duodenal ulcers are due to this bacterium (amtul et al. 2012). while acetylcholinesterase is the principal target for alzheimer's disease treatments that aim to increase the concentrations of acetylcholine in the synapsis clefts to replace the lack of the secretion of this neurotransmitter due to the degeneration of neurons and therefore restore the cholinergic transmission directly associated with the cognitive capabilities (gali and bedjou 2019). by referring to literature, only a small number of species belonging to the caryophyllaceae family have been reported for their inhibitory effect on urease. saponaria officinalis l. has been reported to inhibit jack bean urease with an ic50 value of 607.80 µg/ml, which is very low activity compared to standard hydroxyurea (ic50 = 37 µg.ml -1) (mahernia et al. 2015). similarly, the anticholinesterase effect of caryophyllaceae species has been rarely studied. recently, mamadalieva et al. (2019) reported slight activity against acetylcholinesterase and butyrylcholinesterase from a methanolic extract as well as some ecdysteroids isolated from silene viridiflora. since ethyl acetate extract gave significant antioxidant activity and inhibitory effect on α-amylase, one would conclude that ethyl acetate is the best solvent to extract bioactives of therapeutic interest from p. polycarpoides. this solvent exhibited some degree of hydrophobicity to extract low polar molecules with inhibitory effects on α-amylase and was able to extract aglycone phenolic compounds with antioxidant properties. conclusion throughout the paper, some biological effects (antioxidant activity and the inhibition of α-amylase, urease, and acetylcholinesterase) of the endemic plant polycarpon polycarpoides were examined to investigate its possible therapeutic potential or its use as a source of bioactive compounds. following the various results showing the low contents of polyphenols explaining the moderate antioxidant activity of the plant, and no activity against urease and acetylcholinesterase but remarkably active against alpha-amylase, the plant can be valued in the management of type 2 diabetes. however, as this work represents the first study on the biological actions of p. polycarpoides, other biological effects must be studied to take into account the possible presence of compounds that may exert other effects to conclude on the usefulness of the plant as a remedy or as a source of bioactive ingredients. acknowledgements we gratefully thank wissam mazouz (larbi ben mhidi university, oum el bouaghi, algeria) for providing us with the plant material. we also thank the biotechnology research center of constantine (algeria) and the dgrsdt for their contribution to the realization of this work. conflict of interest the authors declare that they have no conflict of interest. references abdullah ar, bakhari na, osman h (2013) study on the relationship of the phenolic, flavonoid 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(2021) phenolic contents and in vitro antioxidant, antityrosinase, and anti-inflammatory effects of leaves and roots extracts of the halophyte limonium delicatulum. s. afr. j. bot. 139: 42-49. bourebaba l, saci s, touguit d, gali l, terkmane s, oukil n, bedjou f (2016) evaluation of antidiabetic effect of total calystegines extracted from hyoscyamus albus. biomed. pharmacother. 82, 337-344. cetin cakmak k, gülçin i̇ (2019) anticholinergic and antioxidant activities of usnic acid-an activity-structure insight. toxicol. rep. 6: 1273-1280. chandra s, rawat ds (2015) medicinal plants of the family caryophyllaceae: a review of ethno-medicinal uses and pharmacological properties. integr. med. res. 4: 123-131. 9 nova biotechnol chim (2022) 21(2): e1228 3 derici ge, özdaş s, canatar i̇, koç m (2021) antidiabetic activities of bolanthus spergulifolius (caryophyllaceae) extracts on insulin-resistant 3t3-l1 adipocytes. plos one 16: e0252707. dolatabadia jen, kashanian s (2010) a review on dna 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phytotherapie 16 (s1): s225-s236. yakoubi r, megateli s, hadj sadok t, gali l (2021) photoprotective, antioxidant, anticholinesterase activities and phenolic contents of different algerian mentha pulegium extracts. biocatal. agric. biotechnol. 34: 102038. yang xw, huang mz, jin ys, sun ln, song y, chen hs (2012) phenolics from bidens bipinnata and their amylase inhibitory properties. fitoterapia 83: 1169–1175 ye f, liang q, li h, zhao g (2015) solvent effects on phenolic content, composition, and antioxidant activity of extracts from florets of sunflower (helianthus annuus l.). ind. crops prod. 76: 574-581. 10 microsoft word brodnjak2 nb 9-2.doc nova biotechnologica 9-2 (2009) 211 chemometrics in analytical chemistry darinka brodnjak vončina faculty of chemistry and chemical engineering, university of maribor, smetanova 17, 2000 maribor, slovenia (darinka.brodnjak@uni-mb.si) abstract: chemometrics is a scientific discipline closely connected with statistics and mathematics. it has an important role in analytical chemistry. modern analytical methods provide opportunity to collect large amounts of data for various samples. for handling analytical results different chemometric methods are employed, such as basic statistical methods for the determination of mean and median values, standard deviations, minimal and maximal values of measured parameters and their mutual correlation coefficients, the principal component analysis (pca), cluster analysis (ca), and linear discriminant analysis (lda). the objectives of chemometrics in analytical chemistry are focused on characterization and chemometrical classification of different samples. the quality of environmental samples such as water, sediment, soil, air samples etc. can be determined according to measured physical and chemical parameters, which represent the individual samples. chemometric methods give information regarding measured parameters about similarity between sampling locations, sources of pollution, seasonal behavior and time trends. monitoring of general pollution of environmental samples and following measuring parameters which are above permitted level given by legislation can be used for searching of pollution source and for planning prevention measures from pollution. food samples can also be characterized by chemometrical methods. chemometrics can be used for fast and efficient determination of food sample categories, such as edible oils, wines, fruits and fruit juices etc. classification can also be performed according to the origin, source or season. from all these facts it is evident that the aim of chemometrics in analytical chemistry is high and extensive. key words: analytical chemistry, chemometrics, principal component analysis, classification, characterization 1. introduction chemometrics is a scientific discipline between measurement oriented chemistry and applied statistics. analytical chemistry is the most significant discipline where chemometrics plays an important role (adams 1990; wilkins 1990). chemometrics uses mathematical and statistical methods to choose optimal procedures and experiments and to provide chemical information by analysing chemical data (willet 1987; nilsson 1965). in many fields of chemistry there is a need for solving practical oriented problems. chemists usually can not measure directly the parameters they want to determine, but they are measuring signals on the instruments which then should be calculated into individual parameters, like concentration of components in samples, or different physical parameters. modern analytical methods provide large amounts of data. in environmental chemistry monitoring of pollutants in waters, soil and air, provides different multidimensional data, which comprise measurements of parameters for the individual sample, named also an object. each sample is revealed by many measurements, named also variables. using multivariate analysis can give us hidden information about quality of environmental samples or about similarity or dissimilarity between different environmental samples (šnuderl et al., 2007; vončina et al., 2007). one use of 212 brodnjak vončina, d. multivariate data in chemistry is discrimination, another is classification. each object is characterised by a set of measurements. it can be presented as a vector in a multidimensional space. multivariate methods can help in visualising different objects. chemometrics includes exploratory data analysis (massart et al., 1997; brereton 1990), experimental design (varmuza 1980; van der waterbeemd 1995; demming et al., 1987) and modelling. chemometrics methods are used to find hidden information present in multivariate data, coming from analytical instruments. it can also be used for instrument control, or for multivariate calibration (adams 1990). there is also possibility to use chemometrics for determining different properties of individual samples in biology, food chemistry, forensic chemistry, geochemistry, archaeology etc. (lankmayr et al., 2004; šnuderl et al., 2005; brodnjakvončina et al., 2005). it has to be stressed that measurements should be of good quality, accurate and reliable, as this is necessary for obtaining high quality information from multivariate data. chemometrics methods are divided mainly into two groups. first group are so called “unsupervised learning methods“ where problems are solved by finding similarities in the data. the second group are “supervised learning methods”, where also quantitative information about known classes of different samples is available. unsupervised learning methods group analytical data using clustering methods, or by projecting high dimensional data onto lower dimensional space, usually onto one plane (fig. 1). supervised learning is a technique for providing a mathematical function from training data. training consists from pairs of input, objects (vectors) and desired outputs (class label). the most used techniques for exploratory data analysis are principal component analysis, cluster analysis (massart et al., 1997; brereton 1990), and kohonen maps (kohonen 1998). training set with known class memberships is used to calculate a classifier. a prediction set, containing objects not used in the training and also with known class memberships, serves to test the performance of the classifier. the most used techniques for classification are linear discriminant analysis, k nearest neighbour classification and artificial neural networks, (hecht-nielsen 1987; kohonen 1998; dayhof 1990; zupan 1989; zupan et al., 1993). the application of supervised learning methods in chemistry is: recognition of compound classes, determination of origin of samples, detection of low/high quality products, etc. multivariate calibration is the multivariate technique, used in chemical laboratories (adams 1990). traditional techniques are multiple linear regression and non linear neural networks. the goal in chemometrics methods is to find a mathematical function of the multivariate data for defining new latent variables, possessing maximum problem relevant information. methods can be grouped in linear and nonlinear methods. 2. cluster analysis (ca) clustering techniques have been applied to a wide variety of research problems, usually for classification or characterisation. cluster analysis belongs to exploratory nova biotechnologica 9-2 (2009) 213 data analysis. it allows grouping of samples on the basis of their similarities or differences. cluster analysis uses all the variance or information contained in original data set. the starting point is a distant matrix containing the distances between all possible pairs of objects. the pairs with the smallest distance are merged and thus number of object is reduced. result is a hierarchical tree, named “dendrogram”. 3. principal component analysis (pca) principal component analysis pca is by far the most important method in multivariate data analysis. it has two main applications: visualisation of the multivariate data and data reduction and transformation. mathematical pre-processing of the variables, features may have a great influence on the result of data interpretation. usually data transformation should be performed for variables, i.e. columns in data matrices. best known are two methods, column centering and column standardisation. column centering data means that the mean value of each column is subtracted from individual elements of all objects. column standardisation or autoscaling is performed in such way, that the mean of the column elements is subtracted from individual elements and divided by the column standard deviation. consequently, each column has zero mean and unit variance. the column standardisation method is used when units of individual variables are incomparable. principal component analysis pca is also the most frequently used concept for defining a latent variable. it tries to represent the multidimensional data structure optimally. direction which best describes the relative distance between the objects is the direction with maximum variance. this direction is called first principal component pc1. the second principal component (pc2) is orthogonal to pc1 and has the second maximum possible variance. pca can transform data from multidimensional space into two dimensions without losing considerable amount of information. pca estimates the correlation structure of the variables and hence the possible structure of latent variables, (principal component influencing data structure). detection of outliers is also possible since they do not belong to a certain class of objects. the projection of the data on the plain of the new latent variables (principal components) is called score of the objects. the scores of the principal component are weighed sum of original variables and weights are called loadings. the scores contain information about objects, and loadings about variables. similar object are placed closely together in the pc1/pc2 plane. in fig. 1 the classification of different vegetable oils according to the content of fatty acids is presented. it is evident that the clusters are formed, corresponding to seven different oil classes. the samples of mixed or unknown oil type labeled with “0” are distributed into seven classes according to their composition of oils.. 4. linear discriminant analysis (lda) linear discriminant analysis belongs to supervised pattern recognition methods (jurs et al., 1975, varmuza 1980, massart et al., 1997, brereton 1990) 214 brodnjak vončina, d. and has the aim to assign object to several predetermined classes. lda uses latent variables (discriminant variables), that maximally separate different classes of objects between each other. using data from training set linear functions named discriminant functions are calculated that can be used to predict a class for samples with unknown class label. fig. 1. 132 oil samples: pumpkin (1), sunflower (2), peanut (3), olive (4), soybean (5), rapeseed (6), corn (7), mixed or unknown type (0) in the pc1-pc2 co-ordinate system. 5. conclusions it is evident that importance of chemometrics methods in analytical chemistry is exceptional. it helps in solving problems and in obtaining hidden information from complex data and in evaluating different analytical results. it can be applied to develop and implement automated methods for classification of different samples in routine control laboratories. it is used for classification and characterisation of different nova biotechnologica 9-2 (2009) 215 samples from environmental chemistry, food chemistry, biology, forensic chemistry, geochemistry, archaeology and many other scientific disciplines. references adams, m.j.: chemometrics in analytical spectroscopy. the royal society of chemistry, cambridge 1990. brereton, r.g.: chemometrics. application of mathematics and statistics to laboratory systems, ellis horword, new york, 1990. brereton, r.g.: multivariate pattern recognition in chemometrics. elsevier, amsterdam 1990. brodnjak-vončina, d., cencič-kodba, z., novič, m.: multivariate data analysis in classification of vegetable oils characterized by the content of fatty acids. chemometr. intell. lab. syst., 75, 2005, 31-43. brodnjak-vončina, d., dobčnik, d., novič, m., zupan, j.: determination of concentrations at hydrolytic potentiometric titrations with models made by artificial neural networks. chemometr. intell. lab. syst., 47, 1999, 79-88. dayhof, j.: neural network architectures, an introduction. van nostrand reinhold, new york, 1990, p.192. demming, s. n., morgan, s.l.: experimental design: a chemometric approach. elsevier, amsterdam, 1987. hecht-nielsen, r.: counterpropagation networks. appl. optics, 26, 1987, 49794984. kohonen, t.: self-organization and associative memory. springer-verlag, berlin, 1988. jurs, p.c., isenhour, t.l.: chemical application to pattern recognition. wiley, new york, 1975. lankmayr, e., mocak, j., šnuderl, k., balla, b., wenzl, t., bandoinene, d., gfrerer, m., wagner, s.: chemometrical classification of pumpkin seed oils using uv-vis, nir and ftir spectra. j. biochem. biophys. methods, 61, 2004, 95-106. massart, d.l., vandeginste, b.g.m., buydens, l.m.c., de jong, s. lewi, p.j., verbeke, j.s.: handbook of chemometrics and qualimetrics: part a. elsevier, amsterdam, 1997. nilsson, n.j.: linear learning machines. mc.graw-hill, newyork, 1965 sharaf, m.a., illman, d. l., kowalski, b.r.: chemometrics. wiley, new york, 1986. šnuderl, k., netriová, j., mocak, j., lehotay, j., brodnjakvončina, d.: chemometric analysis of biochemical laboratory data of oncology patients after morphine treatment. sci. pap. univ. pardubice. ser. a, fac. chem. technol., 11, 2005, 315-330. šnuderl, k., simonič, m., mocak, j., brodnjak-vončina, d.: multivariate data analysis of natural mineral waters. acta chim. slov., 54, 2007, 33-39. teach/me, sdl software development lohninger. teach/me datalab 2.002 © 1999 springer, berlin, developed by h. lohninger and the teach/me people. 216 brodnjak vončina, d. van der waterbeemd, h.: chemometric methods in molecular design. methods and principles in medical chemistry. vch, weinheim, 1995. varmuza, k.: pattern recognition in chemistry. springer, berlin, 1980. vončina, e., brodnjak-vončina, d., sovič, n., novič, m.: chemometric characterisation of the quality of ground waters from different wells in slovenia. acta chim. slov., 54, 2007, 119-125. wilkins c.l, computerenhanced analytical spectroscopy. the royal society of chemistry, cambridge, 1990. zupan, j.: algoritms for chemists. wiley , chichestert, 1989. zupan, j.: gasteiger, j.: neural networks for chemists. vch, weinheim, 1993. willet, p.: similarity and clustering in chemical information. wiley, new york, 1987. microsoft word el baz et al. nb 2010-2-corr.doc 79 ) 2010(2 -10nova biotechnologica callus formation, phenolics content and related antioxidant activities in tissue culture of a medicinal plant colocynth (citrullus colocynthis) farouk k. el-baz, amal a. mohamed, sami i. ali plant biochemistry department, national research centre, dokki, cairo, egypt (amin_amal@yahoo.com) abstract: callus cultures from stems, leaves and roots of colocynth were initiated on ms media supplemented with various combinations of 2,4 dichlorophenoxyacetic acid (2,4-d) with kinetin (kin) and benzyladenine (ba) with α-naphthaleneacetic acid (naa). the highest percentage of callus formation frequency (98.9%) was obtained from stem explants grown on ms media supplemented with (1.0 mg/l) 2,4-d + (1.0 mg/l) kin. the total phenolics and flavonoid content of the colocynth callus cultures were measured. the results showed that the ms medium supplemented with 6.0 mg/l 2,4-d + 2.0 mg/l kin (md3) gave the highest content of total phenolics (19.2 mg/100g d.w.) in leaf-derived calli. the highest content of flavonoids (47.3 mg/100g d.w.) was obtained in stem derived calli grown on the same medium (md3). antioxidant activities of extracts were determined using different assays, including dpph radical scavenging activity, hydrogen peroxide (h2o2) scavenging activity and ferric reducing power. leaf-derived calli cultured on ms medium + 2.0 mg/l 2,4-d + 1.0 mg/l kin (md1) showed the highest dpph radical scavenging activity (85.3%). the highest percentage of h2o2 scavenging activity (61.4%) was detected in leaf explant-derived calli growing on md1. the leaf-derived calli growing on (md3) gave the highest ferric reducing power (22.3 µg/g d.w.), compared to the activities of stems, leaves and roots of in vitro grown seedlings (3.28, 12.9 and 2.85 µg/g d.w.), which were used as controls. on the basis of the current findings, we conclude that ms media supplemented with different combinations of 2,4-d and kin yields higher phenolics, flavonoids contents and antioxidant activities than ms media supplemented with ba and naa. key words: callus culture, phenolics, flavonoids, h2o2 scavenging activity, plant growth regulators 1. introduction plant cell cultures are an attractive alternative source to whole plant for the production of high value secondary metabolites (alfermann and petersen, 1995; dornenburg and knorr, 1997). however, a considerable progress has been made to stimulate production and accumulation of secondary metabolites using plant cell cultures (kalidass et al., 2010; abouzid et al., 2010). several strategies have been adopted for the enhancement of bioactive metabolite production in in vitro cultures; one of them is using growth regulators which are often a crucial factor in secondary product accumulation (duangporn and siripong, 2009). the type and concentration of auxin or cytokinin or the auxin/cytokinin ratio may alter dramatically both the growth and the product formation in cultured plant cells (mantell and smith, 1984). auxin appears to be the primary factor controlling growth and morphology of roots, while the effects of cytokinin vary depending on secondary metabolites and species concerned (roa and ravishankar, 2002). for example kinetin stimulated the production of anthocyanins in haplopappus gracilus but inhibited the formation of anthocyanins in populus cell cultures (seitz and hinderer, 1988). .et al. k.f, baz el80 in recent years, with enhanced awareness of the importance of antioxidant compounds in health and disease, considerable attention has been devoted to medicinal plants with high antioxidant properties (kong et al., 2010; mohamed et al., 2010). colocynth or bitter melon (citrullus colocynthis) is a medicinal plant species of cucurbitaceae family. it grows fast in sandy soils and is widespread in different parts of south eastern desert of egypt (hassanane et al., 2001). a number of secondary metabolites have previously been reported in this plant including cucurbitacins, flavonoids, caffeic acid derivatives and terpenoids in addition to flavonoid glycosides and cucurbitacin glucosides (seger et al., 2005; delazar et al., 2006). moreover, higher content of total cucurbitacins and cucurbitacin-e have been attained in colocynth callus culture as a result of the impact of different combinations of growth regulators (hegazy et al., 2010). colocynth fruit extracts demonstrated activity against some bacteria and fungi prevalent in dermatology, so it can be considered as an effective antimicrobial agent to treat numerous diseases (ziyyat et al., 1997). no information is available about the stimulation effects of plant growth regulators on antioxidant activity of colocynth callus cultures. the aim of this study was to develop conditions for callus cultures of c. colocynthis manipulating the concentrations and combinations of plant growth regulators, and determination of the effect of media composition on total phenolics and flavonoids content together with the related antioxidant activity of induced calli. 2. materials and methods 2.1 plant material mature seeds of c. colocynthis (l.) schrad. were collected from naturally growing plant populations in wadi soule, sinai, egypt. the collected seed material was botanically authenticated by the herbarium of the botany dept., faculty of science, cairo university. dehusked sterilized seeds were in vitro germinated on hormone-free ms medium (murashige and skoog, 1962). 2.2 in vitro culture and growth regulators treatments two weeks old in vitro germinated seedlings of c. colocynthis were used as a source of explants for initiation of callus cultures. stems, leaves and roots were cut into small pieces and cultured on ms medium supplemented with different combinations of 2,4-dichlorophenoxyacetic acid (2,4-d) and kinetin (kin) as well as ba and naa in different concentrations as presented in table 1. the cultures were incubated for callus induction in the growth chamber at 24 ± 2ºc under a fluorescent light (80 µe m-2 s-1) at a 16-h photoperiod for three weeks. after that callus samples were collected for determination of callusing frequency as well as determination of total phenolics, total flavonoid and antioxidant activity by different methods. 2.3 callusing frequency the frequency of callus induction was calculated according to the following formula: callus induction frequency (%) = [no. of seeds produced calli / no. of seeds cultured] x 100. 81 ) 2010(2 -10nova biotechnologica table 1. list of ms media supplemented with different growth regulators used for colocynth callus cultures. no. media codes growth regulators concentration (mg/l) 1 md1 ms + 2.0 2,4-d + 1.0 kin 2 md2 ms + 1.0 2,4-d + 1.0 kin 3 md3 ms + 6.0 2,4-d + 2.0 kin 4 md4 ms + 2.0 2,4-d + 4.0 kin 5 mb1 ms + 0.0 ba + 5.0 naa 6 mb2 ms + 0.01 ba + 1.0 naa 7 mb3 ms + 0.1 ba + 5.0 naa 8 mb4 ms + 1.0 ba + 0.1 naa 2.4 preparation of plant extracts callus cultures derived from stems, leaves and roots as well as in vitro raised seedling materials (used as control) were air dried at room temperature and ground in a mortar. the extracts were prepared using the modified method of matkowski and piotrowska (2006). briefly, 0.5 g of the dried powder from each sample was refluxed with methanol in a water bath at 45°c for 3 h. the extracts were filtered through whatman filter paper no. 4. the collected filtrates were dried under vacuum at 40°c. the extraction was repeated twice. the resulting residue was re-dissolved in methanol and used for the determination of phenolics, flavonoid contents and antioxidant activities. 2.5 determination of total phenolics content the total phenolics content of the extracts was determined using the folinciocalteu reagent (kaur and kapoor, 2002). each extract solution (200 µl) was completed to 3 ml with distilled water then mixed thoroughly with 0.5 ml of folinciocalteu reagent. after mixing for 3 min, 2 ml of 20% (w/v) sodium carbonate was added and allowed to stand for a further 60 min in the dark. the absorbance of the reaction mixtures were measured at 650 nm. the total phenolics content was expressed as mg pyrogallol equivalents / 100 g dry weight (d.w.) of the extract. 2.6 determination of total flavonoid content the total flavonoid content was determined according to the method of kim et al. (2003). in brief, 0.5 ml of sample solution was completed to 1 ml with methanol then mixed with 4 ml of distilled water and subsequently with 0.3 ml of 5% nano2 solution. after 6 min of incubation, 0.3 ml of 10% alcl3 solution was added and then allowed to stand for 5 min, followed by adding 2 ml of 1 m naoh solution to the mixture. immediately after water was added to the sample to bring the final volume to 10 ml and the mixture was thoroughly mixed. the absorbance was determined at 510 nm. the total flavonoid content was expressed as mg rutin equivalents / 100 g d.w. of the extract. .et al. k.f, baz el82 2.7 determination of antioxidant activity 2.7.1 dpph free radical scavenging activity quantitative measurement of radical scavenging properties of colocynth was carried out according to the method of blois (1958). briefly, 0.1 mm solution of 1,1diphenyl-2-picryl-hydrazyl (dpph·) in methanol was prepared and 1 ml of this solution was added to 3 ml of each methanolic extract at one concentration (500 μg/ml). butylated hydroxytoluene (bht) was used as a positive control. discoloration was measured at 517 nm after incubation for 30 min. measurements were taken at least in triplicate. the capacity to scavenge the dpph· radical was calculated using the following equation: dpph· scavenging effect (%) = [adpph − as / adpph] x100 where, adpph is the absorbance of the dpph· solution and as is the absorbance of the solution when the sample extract is added. the extract concentration providing 50% inhibition of radical-scavenging activity (ic50) was calculated and expressed as mg/ml, d.w. 2.7.2 ferric reducing power determination ferric reducing power was determined following the method reported by zhao et al. (2008). extracts at concentration of 500 µg/ml were mixed with phosphate buffer (2.5 ml, 200 mm, ph 6.6) and 1% potassium ferricyanide (2.5 ml). then the mixtures were incubated at 50 °c for 20 min. the quantity 2.5 ml of 10% trichloroacetic acid was added and the mixture was centrifuged at 10000 rpm for 10 min. the upper layer of the solution (5 ml) was mixed with distilled water (5 ml) and 0.1% ferric chloride (1 ml). the absorbance of the reaction mixtures were measured at 700 nm. the final results were expressed as µg ascorbic acid equivalents / g based on dry weight of the extract. 2.7.3 hydrogen peroxide scavenging activity the hydrogen peroxide scavenging ability of methanolic extracts was determined according to the method of shon et al. (2007). a solution of h2o2 (43 mm) was prepared in phosphate buffer (0.1 m, ph 7.4). extracts at concentration 50 μg/ml dissolved in 3.4 ml phosphate buffer were added to a h2o2 solution (600µl). the absorbance of the reaction mixture was recorded at 230 nm. the percentage of h2o2 scavenging of each extract and bht at concentration 50µg/ml used as positive control was calculated as: h2o2 scavenging effect (%) = [acontrol – asample / acontrol] x 100 where acontrol is the absorbance of the control (blank, without extract), and asample is the absorbance in the presence of the sample extract. 83 ) 2010(2 -10nova biotechnologica the extract concentration providing 50% of h2o2 scavenging activity (ic50) was calculated and expressed as µg/ml based on sample dry weight. 2.8 statistical analysis the experiment was conducted using completely randomized design (crd). all tests were conducted in triplicate. data are reported as means ± standard deviation (sd). analysis of variance and significant differences among means were tested by one-way anova using the costat computer package (cohort software, 1989). the least significant difference (lsd) at p ≤ 0.05 level was calculated. correlation coefficients (r2) from regression analysis between total phenolic or total flavonoid contents and antioxidant activities were also calculated. 3. results and discussion calli of colocynth began to appear on stems, leaves and roots grown on ms media supplemented with different concentrations and combinations of growth regulators (2,4-d/kin and ba/naa) within one weak. the results presented in table 2 shows that the stem explants grown on md2 gave the highest frequency of callus formation (98.9 %). generally, among all 2,4-d and kin combination treatments, the medium md2 showed significant superiority in frequencies of callus formation for the three studied explants. these results are in accord with hegazy et al. (2010) who reported that high concentration of kin more than 2,4-d in the ms-media had a positive effects on the callus induction (fresh and dry weight) in both stem and root explants of colocynth. on the other hand, dabauza et al. (1997) found that the highest frequency (81.8 %) of citrullus colocynthis cotyledon explants derived calli was obtained on a medium containing 25 µm 6-benzylaminopurine (6-ba). table 2. callusing frequency of different explants derived from seedlings of colocynth cultured on ms media supplemented with different combinations of growth regulators. callus formation frequency (%, mean ± sd) origin of callus (explants) growth regulators treatments media codes stems leaves roots md1 94.9 ± 1.80e 68.6 ± 1.81b 26.8 ± 1.41a md2 98.9 ± 1.10g 86.4 ± 1.38f 61.1 ± 0.95d md3 83.2 ± 2.17b 70.5 ± 1.78c 44.2 ± 0.96b 2, 4d + k in md4 98.8 ± 1.20g 47.8 ± 0.82a 45.2 ± 1.29c mb1 85.1 ± 1.73c 85.0 ± 1.30e 68.6 ± 1.58e mb2 81.8 ± 1.42a 74.3 ± 1.48d 77.8 ± 1.62f mb3 97.9 ± 1.38f 98.2 ± 1.17h 91.7 ± 1.80g b a + n a a mb4 90.9 ± 1.90d 92.6 ± 1.53g 92.9 ± 1.50h lsd ≤ 0.05 0.699 0.324 0.281 data represents mean of 10 replicates per treatment in three repeated experiments. data with different superscript letters in the same column were significantly different (p ≤ 0.05). .et al. k.f, baz el84 fig. 1 and 2 show the total phenolics content in different colocynth callus cultures. one-way anova analysis showed significant differences (p ≤ 0.05) in total phenolics content among the eight studied media formulations. a wide range of total phenolics content was found in 2,4-d and kin treatments as shown in fig. 1. the high concentration of 2,4-d over kin in md3 media yielded the highest phenolics content (19.2 mg/100g d.w.) in the leaf derived calli. this content was higher than the content of phenolic compounds in stems, leaves and roots organs of seedlings (3.32, 17.1 and 4.49 mg/100g d.w.) which were used as controls. fig. 1. total phenolics content of colocynth callus cultures grown on ms media supplemented with different combinations and concentrations of 2,4-d and kin. data with different superscript letters in the same column were significantly different (p ≤ 0.05). bars on the columns represent the standard deviation. fig. 2 shows the total phenolics content in calli derived on ba and naa containing media. overall, the higher concentration of naa over ba (mb2) gave the maximum amount of total phenolics (7.54 and 6.52 mg/100g d.w.) in the stem and root derived calli respectively. unlike, the lowest value of phenolics content (2.71 mg/100g d.w.) was detected in the leaf-derived calli cultured on medium mb3. the effect of plant growth regulators on biosynthesis of phenolic compounds were studied in hairy roots of panax ginseng by jeong et al. (2007). they found that addition of benzylaminopurine and kinetin to the culture media led to increasing the phenolic compound biosynthesis. in addition, nikolaeva et al. (2009) reported that growing transgenic tea callus tissue (strain ifr chs-2) on nutrient medium containing naa at concentration of 2 × 10–5 m stimulated total soluble phenolics more than total phenolics which presented in intact plants. the total flavonoid content showed significant (p≤ 0.05) differences among the explant types and the different combinations of growth regulators as shown in fig. 3. dealing with 2,4-d and kin media treatments, md3 gave the maximum values of total flavonoid content (47.3 mg/100g d.w.) in the stem derived calli. this content is higher than those contents (11.8, 46.4 and 15.6 mg/100g d.w.) of in vitro raised 85 ) 2010(2 -10nova biotechnologica seedlings stems, leaves and roots respectively, which were used as a control. it is clear that 2,4-d was the favorable enhancer of flavonoids accumulation in colocynth callus cultures. these results are in agreement with matkowski (2004), who reported that medium containing 2,4-d was found to be optimum for isoflavone accumulation in callus cultures of pueraria lobata and psoralea sp. similarly, shinde et al. (2009) confirmed that the content of isoflavones in root and leaf-derived callus cultures of psoralea corylifolia was higher on medium containing 2,4-d and iaa than in intact plant roots and leaves. fig. 2. total phenolics content of colocynth callus cultures grown on ms media supplemented with different combinations of ba and naa. data with different superscript letters in the same column were significantly different (p ≤ 0.05). bars on the columns represent the standard deviation. fig. 3. total flavonoid content of colocynth callus cultures grown on ms media supplemented with different combinations of 2,4-d and kin. data with different superscript letters in the same column were significantly different (p ≤ 0.05). bars on the columns represent the standard deviation. .et al. k.f, baz el86 the effect of ba and naa treatments on stimulation of total flavonoid content is presented in fig. 4. the high concentration of ba over naa (mb4 media) gave the highest flavonoid content (34.0 mg/100g d.w.) in the stem derived calli. however, this content is lower than the flavonoid content (46.4 mg/100g d.w.) detected in the leaves of the in vitro raised seedlings. the present results are in agreement with findings of agarwal and kamal (2007). the authors confirmed that the maximum flavonoid content was observed in 2 weeks old callus culture of momordica charantia cultured on ms medium supplemented with naa (2 mg/l) and bap (0.5 mg/l). fig. 4. total flavonoid content of colocynth callus cultures grown on ms media supplemented with different combinations of ba and naa. data with different superscript letters in the same column were significantly different (p ≤0.05). bars on the columns represent the standard deviation. it is well known that, the concentrations of phenolics content usually higher than the concentrations of flavonoid in most cases, but in this study we found an inverse trend. it can be explained by the fact that differences in the polarity of the extracting solvents could result in a wide variation in polyphenolic contents of the extract. so, the low content of phenolics in our extracts may be possibly due to the fact that extraction with methanol does not release bound phenolics from the callus cells. on the other side, the folin-ciocalteu method is a rapid and widely-used assay, to detect the total phenolic content but it is known that different phenolic compounds have different responses in the folin-ciocalteu method (kähkonen et al., 1999). moreover, different authors found a higher concentrations of flavonoid more than phenolics such as sultana et al. (2009) found a higher concentration of total flavonoids (1.68 g/100g, d.w.) in absolute methanolic extract of moringa oleifera root more than the total phenolics (0.22 g/100g, d.w.). also, the aqueous extract of momordica charantia yielded higher content of total flavonoid (62 mg/g, d.w.) than total phenolics (51.6 mg/g, d.w.) as reported by (wu and ng, 2008). the variation in the total flavonoid production may be attributed to the activation of key enzyme phenylalanine ammonialyase (pal) which is involved in the flavonoid biosynthesis pathway (guo et al., 87 ) 2010(2 -10nova biotechnologica 2007). the knowledge of biosynthetic pathway and mechanisms for stimulation of flavonoids production in callus culture remains unclear. whether phytohormones induce different biosynthetic enzymes responsible for the biosynthesis of flavonoids is yet to be clarified. the antioxidant activities of methanolic extracts of colocynth callus cultures and of the standard antioxidant bht were determined using the dpph method. this method is based on the reduction of alcoholic dpph solution in the presence of a hydrogendonating antioxidant due to the formation of the non-radical form dpph-h by the reaction (gülçin, 2006). the dpph radical scavenging activity of methanolic extracts of colocynth callus cultures at concentration 500 µg/ml is presented in table 3. the leaf-derived calli cultured on md1 showed relatively higher radical scavenging activity (85.3 %) with ic50 value (0.209 mg/ml) as presented in table 4. this activity was higher than the activity of stem, leaf and root organs of seedling which were used as control (51.6, 80.9 and 27.9 %) with ic50 values (0.485, 0.241 and 0.915 mg/ml) respectively. the scavenging activity of bht which was used as positive control showed higher activity (87.5 %) with ic50 value (0.086 mg/ml) than our methanolic extracts of colocynth callus cultures. these results are consistent with those of tadhani et al. (2007) who found a relatively higher dpph radical scavenging activity (56.8%) with ic50 value (0.528 mg/ml) of extract of stevia rebaudiana leafderived callus cultured on ms media supplemented with 2.0 mg/l naa and 0.3 mg/l ba than the activity (33.2 %) with ic50 value (0.904 mg/ml) of intact plant leaves. table 3. dpph radical scavenging activity of colocynth callus cultures grown on ms media supplemented with different combinations of growth regulators. dpph scavenging activity (%, mean ± sd ) origin of callus (explants) growth regulators treatments media codes stems leaves roots md1 15.23 ± 0.046g 85.31 ± 0.685i 38.73 ± 0.158h md2 11.62 ± 0.016c 56.32 ± 0.213f 27.55 ± 0.110d md3 8.45 ± 0.028a 70.89 ± 0.223g 22.80 ± 0.073c 2, 4d + k in md4 10.02 ± 0.016b 23.01 ± 0.084a 28.17 ± 0.081f mb1 14.70 ± 0.770f 44.63 ± 0.276c 32.13 ± 0.097g mb2 13.83 ± 0.012e 46.78 ± 0.014d 19.56 ± 0.035b mb3 12.59 ± 0.013d 41.47 ± 0.066b 25.44 ± 0.047a b a + n a a mb4 16.60 ± 0.073h 49.32 ± 0.110e 59.42 ± 0.204i control 51.60 ± 0.445i 80.90 ± 0.748h 27.91 ± 0.081e lsd≤ 0.05 0.442 0.705 0.299 bht (150µg/ml) 87.50± 0.099 note: media formulations are presented in table 1. each value is expressed as mean ± standard deviation (n =3). data with different superscript letters in the same column were significantly different (p ≤ 0.05). in addition, grzegorczyk et al. (2007) reported that the methanolic extracts (50 µg/ml) of salvia officinalis shoot cultures grown on ms media supplemented with .et al. k.f, baz el88 0.1 mg/l iaa and 0.45 mg/l bap possessed a high dpph radical scavenging activity (81.4 %) comparable to that (72.3 %) of shoots from field collected plants. with respect to these results, it is possible to consider the callus cultures as a potential source of natural antioxidants. although, which constituents of colocynth callus methanolic extracts, show the free radical scavenging action is still unclear. it is possible that the antioxidative properties of colocynth extracts are caused, at least in part, by the presence of polyphenols and flavonoids and other yet to be discovered antioxidant compounds. table 4. ic50 for inhibition of dpph radical formation of colocynth callus cultures grown on ms media supplemented with different combinations of growth regulators ic50 values (mg/ml, d.w.) of dpph scavenging activity origin of callus (explants) growth regulators treatments media codes stems leaves roots md1 1.670 0.209 0.654 md2 2.190 0.449 0.919 md3 2.970 0.354 1.118 2, 4d + k in md4 2.500 1.090 0.898 mb1 1.730 0.569 0.794 mb2 1.860 0.540 1.325 mb3 2.030 0.613 1.000 b a + n a a mb4 1.520 0.511 0.428 control 0.485 0.241 0.915 bht (150µg/ml) 0.086 the presence of reductants such as antioxidant substances in samples causes the reduction of the fe3+ ferricyanide complex to the ferrous form. the transformation of iron (iii) to iron (ii)-reducing activity in the colocynth callus methanolic extracts was expressed as µg ascorbic acid equivalent/g sample based on dry weight. the results showed statistically significant (p≤0.05) differences among the explant types and growth regulator treatments. fig. 5 shows that the highest reducing power activity (22.3 µg/g d.w.) was found in leaf-derived calli cultured on md3 comparing to the activities (3.28, 12.9 and 2.85 µg/g d.w.) of stems, leaves and roots of the in vitro seedlings which were used as controls, respectively. among the ba and naa treatments, fig. 6 indicated that high concentration of naa in the medium (mb1) has notable effect on total reductive activity. it gave the highest activity (7.52 and 6.61 µg/g d.w.) in the callus cultures of stem and leaf explants respectively. however, these activities are lower than those of leaves of in vitro seedlings (12.9 µg/g d.w.). in this concern, parsaeimehr et al. (2010) reported that ephedra strobilacea stem callus cultures grown on ms media supplemented with 1.5 mg/l naa and 1 mg/l kin gave ferric reducing antioxidant power (0.28 mmol quercetin/g). this value was lower than in intact plants (1.61 mmol quercetin /g). 89 ) 2010(2 -10nova biotechnologica fig. 5. ferric reducing power of colocynth callus cultures grown on ms media supplemented with different combinations of 2,4-d and kin. data with different superscript letters in the same column were significantly different (p ≤ 0.05). bars on the columns represent the standard deviation. fig 6. ferric reducing power of colocynth callus cultures grown on ms media supplemented with different combinations of ba and naa. data with different superscript letters in the same column were significantly different (p ≤ 0.05). bars on the columns represent the standard deviation. hydrogen peroxide can cross membranes and may slowly oxidize a number of compounds. thus, removing of hydrogen peroxide as well as superoxide anion is very important for protection of food systems (gülçin et al., 2007). table 5 points out that h2o2 scavenging activity of colocynth callus methanolic extracts at concentration 150 µg/ml differed significantly (p≤0.05) among the different concentrations of .et al. k.f, baz el90 growth regulators. generally, high ratio of 2,4-d over kin in md1 exhibited remarkable scavenging activity on hydrogen peroxide, it gave the highest h2o2 scavenging activity (61.4 %) in leaf-derived calli with ic50 value 40.74 µg/ml as presented in table 6. this activity value was higher than the activity of the in vitro grown seedlings stems, leaves and roots organs (used as controls) which showed h2o2 scavenging activities of 26.2, 27.3 and 26.4 % with ic50 values 95.4, 91.7 and 94.7 µg/ml, respectively. the bht which was used as a positive control gave h2o2 scavenging activity of 90.5 % with ic50 value 27.64 µg/ml, this value was higher than in colocynth callus cultures. moreover, high concentration of ba in mb4 media gave the highest hydrogen peroxide scavenging activity in the root derived calli (51.8 %). table 5. hydrogen peroxide (h2o2) scavenging activity of colocynth callus cultures grown on ms media supplemented with different combinations of growth regulators h2o2 scavenging activity (%, mean ±sd) origin of callus (explants) growth regulators treatments media codes stems leaves roots md1 23.59 ± 0.117b 61.37 ± 0.089g 35.97 ± 0.056h md2 24.26 ± 0.139bc 33.44 ± 0.062d 15.19 ± 0.023a md3 21.36 ± 0.110a 39.22 ± 0.407e 17.47 ± 0.027d 2, 4d + k in md4 39.37 ± 0.121e 24.42 ± 0.048a 18.83 ± 0.051e mb1 25.83 ± 0.094cd 40.38 ± 0.455f 33.03 ± 0.053g mb2 22.32 ± 0.049ab 30.00 ± 0.070c 16.72 ± 0.026b mb3 27.66 ± 0.091d 33.48 ± 0.039d 16.75 ± 0.053c b a + n a a mb4 21.48 ± 0.058ab 38.44 ± 0.062e 51.77 ± 0.079i control 26.21 ± 0.235d 27.27 ± 0.623b 26.39 ± 0.051f lsd≤0.05 1.572 1.088 0.022 bht (50µg/ml) 90.46±0.011 each value is expressed as mean ± standard deviation (n =3). data with different superscript letters in the same column were significantly different (p ≤ 0.05). in this concern, rakotoarison et al. (1997) found that both callus and cell suspension cultures of crataegus monogyna showed high scavenging activities against h2o2. the decomposition of hydrogen peroxide into water may occur according to the presence and character of antioxidant compounds. since antioxidant compounds present in the extract are good electron donors, they may accelerate the conversion of h2o2 into h2o. phenolic and flavonoid compounds have been reported to be responsible for the antioxidant activities of medicinal plants and other botanical materials (mohamed et al., 2010). table 7 shows that the total phenolic compounds gave a positive correlation (r2 = 0.511 and r2 = 0.667) with dpph radical scavenging activity and ferric reducing power respectively. these data could indicate that phenolic compounds in colocynth calli were the major contributors to dpph radical scavenging activity and ferric reducing power. polyphenolic compounds in extracts of colocynth 91 ) 2010(2 -10nova biotechnologica callus cultures might play a role as electron and hydrogen donors. the antiradical activity of flavonoids and phenolics is principally based on the redox properties of their hydroxy groups and the structural relationships between different parts of their chemical structure (burda and oleszek, 2001). in this concern, shinde et al. (2009) indicated that the content of phenolics is partially correlated with dpph radical scavenging (r2 = 0.466) in callus cultures of psoralea corylifolia. table 6. ic50 values of hydrogen peroxide (h2o2) scavenging activity of colocynth callus cultures grown on ms media supplemented with different combinations of growth regulators. ic50 values (µg/ml, d.w.) of h2o2 scavenging activity origin of callus (explants) growth regulators treatments media codes stems leaves roots md1 105.98 40.74 69.5 md2 103.05 74.76 164.58 md3 117.04 63.43 143.1 2, 4d + k in md4 63.50 102.38 132.77 mb1 96.79 61.91 75.69 mb2 112.01 83.33 149.52 mb3 90.38 74.67 149.7 b a + n a a mb4 116.39 65.04 48.29 control 95.38 91.68 94.73 bht (50µg/ml) 27.64 table 7. correlation analysis (r2)* between antioxidant activities and antioxidant content (total phenolics and total flavonoids). antioxidant contents antioxidant activities total phenolics total flavonoids dpph radical scavenging activity 0.511 0.048 h2o2 scavenging activity 0.017 0.026 ferric reducing power 0.667 0.358 * correlation coefficient (r2) however, the poor correlations of h2o2 scavenging activity (r 2 = 0.017) with the total phenolic compounds as well as the poor correlations of dpph radical scavenging activity, h2o2 scavenging activity and ferric reducing power (r 2 = 0.048, r2 = 0.026 and r2 = 0.358) with total flavonoids respectively in this study suggested that flavonoid compounds in colocynth calli might be a weak scavenger of dpph radical and h2o2 and they do not contribute to ferric reducing power. in conclusion, the development of in vitro cell lines with high antioxidant capacities provides a possibility of generating uniform material cultured under conditions independent of environmental factors. the results of the present study concluded that ms media supplemented with 2,4-d and kinetin produced higher contents of total phenolics, total flavonoids and antioxidant activities more than ms .et al. k.f, baz el92 media supplemented with ba and naa in colocynth callus cultures. stimulation of antioxidant activities in c. colocynthis will help to select callus culture type as a source of natural antioxidants and nutraceuticals to enhance health benefits. moreover, more scientific work needs to be done regarding the characterization of effective antioxidant compounds contained in this plant species in order to further verify their antioxidant effects in in vitro conditions. acknowledgments: we are grateful to dr. mahmoud m. saker, professor of plant biotechnology, national research centre, where the practical part of tissue culture was carried out in his lab. we are also grateful to dr. ahmed k. hegazy, professor of plant ecology, faculty of science, cairo university, for his critical reading of the manuscript. references abouzid, s.f., el-bassuony, a.a., nasib, a., khan, s., qureshi, j., choudhary, m.i.: withaferin a production by root cultures of withania coagulans. int. j. appl. res. nat. prod., 3, 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(2017), administering probiotics increases the activity of natural killer (nk) cells in humans. furthermore, probiotics heighten the activity of phagocytes from macrophages and levels of immunoglobulin a (iga) in mice model diarrhea (rocha-ramírez et al. 2017). jatmiko et al. (2017) stated that lactic acid bacteria (lab) from the genus lactobacillus and bifidobacterium also act as probiotics. studies on lab probiotics from fermented products, digestive system, feces, milk, and human breast milk have been widely published. some labs found in human breast milk include b. longum, l. gasseri, l. rhamnosus, l. acidophilus, l. pentosus, l. plantarum, l. reuteri, l. fermentum, l. brevis, l. casei, l. gastricus, and l. salivarius (sinkiewicz and ljunggren 2008; jamyuang et al. 2019; łubiech and twarużek 2020). the isolation of lab from different humans can affect the differences in species obtained and their abilities. in algeria and china, lab isolates, including l. casei, l. paracasei, l. rhamnosus, l. fermentum, and l. plantarum, and, namely, l. plantarum were found in human breast milk. according to jiang et al. (2016), lab isolated from human breast milk is probiotic and contains various health benefits. the lab of human breast milk as a probiotic potential was assessed by following specific criteria and tested to determine low ph and bile salts resistance. furthermore, it was analyzed to determine the antimicrobial properties, hydrophobicity, and aggregation, correlating with the ability to attach to the digestive tract (li et al. 2020). during initial selection, an in vitro assessment of hydrophobicity and auto-aggregation properties was performed to assess lab's capacity to bind the digestive tract. one of the important functions for inhibiting pathogenic bacteria colonization is its ability to co-aggregate with lab. a partial sequence of the 16s rrna gene was used to identify the lab with the best ability as an indigenous probiotic substance. the standard identification method and phylogenetic relationships in a bacteria is 16s rrna gene sequence analysis, consisting of highly converted and hypervariable regions (tilahun et al. 2018). therefore, it can be used to determine phylogenetic relationships and the levels of kinship between microbes. moreover, the 16s rrna gene is constant and does not change for long, though there is a minute mutation rate (johnson et al. 2019). furthermore, its amplification and sequencing processes identify a bacterial isolate to the species level. this study used universal primers to partially amplify lab isolates from human breast milk to a base sequence of 1,500 bp in the v1-v9 regions of the 16s rrna gene. according to bukin et al. (2019) and michel et al. (2016), the v1-v9 regions can differentiate between species. regional sequences v1 and v3 were used to identify lab up to strain level (bin masalam et al. 2018). variations in subjects used to collect human breast milk samples affected the lab skill obtained and the results of species recognition. this study aimed to obtain lab species that can serve as an indigenous probiotic from human breast milk for health benefits. experimental sampling breast milk samples were collected from 20 mothers (aged 27 – 30) breastfeeding babies of 0 – 6 months. participants were recruited from malang regional unit hospital, east java, indonesia, between september-october 2020. the sampling process involved using a sterilized breast pump. however, the initial 10 – 50 ml was discarded, while the subsequent 50 – 100 ml was put into a sterile bottle for utilization. the sample was kept frozen at 4 oc. the lab isolation process was conducted (kaškonienė et al. 2017). this study was approved for ethical clearance from lppt ugm no 00154/08/lppt/ii/2020. the isolation and purification of lab the isolation and purification of lab from human breast milk were effected using the kaškonienė et al. (2017) method. a sample of 10 ml was put into nova biotechnol chim (2022) 21(2): e1053 3 a test tube and diluted to 10-5 by inserting 1 ml spacemen into 99 ml of equates. the inoculum was streaked using de man rogosa and sharpe agar (mrsa) medium alongside1 % calcium carbonate. thereafter, samples were incubated for 48 h at 37 oc with a clear zone around a single bacterial colony, indicating a positive result. the cultures were further purified using the streak plate method and incubated at 37 oc for 48 h on mrsa. notably, the pure cultures were stored on mrsa slants at 4 – 10 oc. analysis of lab resistance to acid ph and bile salts plab resistance test from human breast milk to acidic ph 2.0 and bile salts was carried out by shehata et al. (2016) with the same method used on the de man rogosa and sharpe broth (mrsb), which rejuvenated lab culture for 24 h. a culture of 0.1 ml was inoculated into 10 ml of mrsb media (control) with ph 2.0 by adding 37 % hcl into the medium and incubated at 37 °c for 5 h. a total culture of 0.1 ml was inoculated into 10 ml mrsb (control) and added 0.3% bile salt, then incubated at 37 °c for 5 h. afterward, calculations on the number of colonies on mrsa media using the plate count method were conducted. the resistance of lab isolates to acidic ph bile salts was calculated based on the difference in log units of the number of colonies that grew in control. the smaller the difference grew, the more resistant the lab culture was to acid ph and bile salts. hydrophobicity analysis of lab from human breast milk activities from hydrophobicity analysis of bacteria were determined using the microbial adhesion to solvent (mats) (krausova et al. 2019). the analysis was conducted using solvents with different polarities, namely: xylene, ethyl acetate, and chloroform (all from merck kgaa, darmstadt, germany). adhesion of bacteria cells to xylene solvents describes hydrophobicity, while chloroform and ethyl acetate analyze cell surface properties. bacteria were grown in mrsb at 37 °c for 16 – 18 h. the cells were harvested using the centrifugation method (hermle z 383 k, hermle ag, gosheim, germany) at a speed of 3,500 rpm for 20 min. the precipitates were washed using urea magnesium phosphate buffer (pum) and suspended in the pum buffer until the bacterial cell was collected at 108 cfu.ml-1. afterward, the original value was calculated at 600 nm wavelength (x0) with a total cell suspension of 5 ml in the pum buffer transferred to a new tube. furthermore, 1 ml of solvents like xylene, ethyl acetate, and chloroform were applied and combined with the vortex in a test tube rapidly for a minute and left still for an hour at 37 °c for disillusion. the aqueous was carefully transferred using a sterile pipette alongside a spectrophotometer (uv1800 uv-vis, shimadzu corp., kyoto, japan) to measure its absorbance at 600 nm (x1). absorbance decrease value in the aqueous phase is expressed as adhesion value to the solvent (%) using eq. 1. (1) x0 = initial absorbance value at 600 nm x1 = final absorbance value at 600 nm autoaggregation analysis of lab from human breast milk the autoaggregation analysis of lab was determined using the krausova et al. (2019) method, with the isolates grown on mrsb for 18 h at 37 °c. the cells were then harvested using centrifugation at 3,500 rpm for 20 min. the residue was washed twice with phosphate buffer saline (pbs) and later suspended in pbs up to 108 cfu.ml-1. furthermore, autoaggregation was determined by measuring the initial (0 hours) alongside the final absorbance (5 h) after incubation at room temperature. this process was implemented by putting 0.1 ml of the upper suspension into 3.9 ml of pbs with a spectrophotometer at 600 nm, measuring the absorbance. the autoaggregation percentage is expressed in eq. 2. (2) at = absorbance at time t = 5 a0 = absorbance at time t = 0 nova biotechnol chim (2022) 21(2): e1053 2 coaggregation analysis of lab from human breast milk the coaggregation analysis conducted by krausova et al. (2019) proposed this method. lab isolates were grown on the mrsb and pathogenic bacteria on tsb for 18 h at 37 °c. furthermore, the lab and pathogenic cells were later harvested by centrifugation at 3,500 rpm for 20 min. notably, the residue obtained was washed twice and suspended in pbs up to 108 cfu ml-1. the volume of 2 ml of each suspension was then mixed in a control tube containing 4 ml per bacteria. the coaggregation was determined by measuring the initial (0 h) and the final absorbance (5 h) after room temperature incubation. this measurement was determined by putting 0.1 ml of the upper suspension into 3.9 ml of pbs and measuring the absorbance using a spectrophotometer at 600 nm. the percentage of co-aggregation is expressed in eq. 3. (3) mx = absorbance of lab isolate suspension my = absorbance of pathogenic isolate suspension mx+y = absorbance of the mixture of lab and pathogenic isolate suspensions antimicrobial activity of lab from human breast milk antimicrobial activity was qualitatively analyzed using the proper diffusion method proposed by le et al. (2019). the test microbes used were e. coli atcc 25922, s. aureus atcc 25923, and c. albicans atcc 11778. also, 1 ml of test microbes were piped into a petri dish and poured in a sterilized medium before cooling to about 40 oc. the tryptone soy agar (tsa) is left to solidify for 1 h after cooling, with a diameter of 0.5 cm at room temperature. the 1 ml test isolate was inoculated in the well and incubated at 37 oc for 2 d. after that, the inhibition zone diameter was measured from three different sides. molecular identification direct pcr is the initial stage for 16s rrna gene analysis. 16s rrna gene is a common feature for best acid ph and bile salts, antimicrobial, and autoaggregation. the process of amplifying 16s rrna gene partial sequence from lactic acid bacteria was conducted using direct pcr amplification as proposed by woodman et al. (2008). one lab colony with the potential of an indigenous probiotic was withdrawn from a petri dish to a tube. thereafter, the pcr mix (50 μl) contained 2 μl dna templates (100 ng/μl), 5 μl 10x buffer (mg2+), 4 μl dntp (10 mmol/l), 1.5 μl primer fa-27f (10 pmol/μl), 1.5 μl primer ra-1495r (10 pmol/μl), 0.5 μl taq dna polymerase (5 u/μl) and 35.5 μl tri-distilled water were added. the primers used to amplify lactic acid bacteria were 27f (5’agagtttgatcctggctcag-3’) and 1495r (5’-ctacggctaccttgttacga-3’). afterward, pcr amplification was carried out by denaturation of 94 oc for 5 minutes, to 30 denaturation cycles at 94 oc for 1 min, annealing at 58 oc for 1 min, to 72 oc for 2 min, and the final extension of 72 oc for 10 min and 4 °c for heat preservation (wang et al. 2016). the pcr products were separated on 1 % gel agarose by electrophoresis process in 100 volts for 30 min and visualized using gel documentation (gel doc xr+ system, bio-rad laboratories, inc., hercules, usa). the dna marker benchtop 1kb (promega corp., madison, usa) was used as the molecular weight standard. moreover, dna sequencing was achieved using an automated dna sequencer. the results were analyzed by comparing the dna sequences database from ncbi (http://www.blast.ncbi.nlm.nih.gov) with blast (basic local alignment search tool). mega 5.0 used for phylogenetic tree analysis was achieved using the neighbor-joining (nj) algorithm method with 1,000 bootstrap repetitions based on pdistance. data analysis the quantitative data group was analyzed by spss 16.0 and tested for normality and homogeneity using the shapiro-wilk and levene test. when the 4 http://www.blast.ncbi.nlm.nih.gov/ nova biotechnol chim (2022) 21(2): e1053 3 results are normal and homogenous, the analysis of variance process is carried out by further dmrt test to determine the effect of differences between isolates. results the ability of lab at acidic ph and bile salts a total of eight lab isolates were collected with different macro and microscopic characters and later purified with human breast milk. all isolates were analyzed and proved to function as probiotics, including low ph resistors. the lab analysis with low ph and bile salts indicated that the l19a isolate had better resistance. however, l19e and l19h isolates performed better than l19b, l19c, l19d, l19f, and l19g, as shown in fig. 1. the tolerance for bile acids and salts was characterized by survival in media containing a ph of 2.0 and a bile salt of 0.5 % with a difference below 2 logs. fig. 1. analysis of the resistance of lab isolates to low ph and bile salts; asterisks indicate mean comparisons that differ significantly (p < 0.05). hydrophobicity analysis of lab from human breast milk the hydrophobicity nature affects its autoaggregate and adhesiveness to various surfaces. some isolates indicated a higher affiliation to chloroform than for ethyl acetate, as shown in table 1. l19a had the strongest hydrophobicity properties based on xylene solvent affinity and was significantly unique. the high xylene affinity is indicated by l19e and l19h, and those above 50 % are declared strong hydrophobic. table 1. adhesion (%) of lab isolates from human breast milk with various solvents. bal isolate adhesion [%] xylene eethyl acetate chloroform l19a 71.01 ± 3.17e 77.21 ± 3.54f 52.01 ± 2.20d l19b 28.21 ± 2.50a 10.68 ± 5.47a 16.23 ± 1.33a l19c 37.05 ± 0.67b 17.47 ± 2.13b 13.89 ± 2.22a l19d 38.18 ± 4.80b 22.32 ± 3.81b 18.05 ± 2.82a l19e 66.02 ± 1.85de 65.33 ± 6.06e 45.56 ± 2.50c l19f 38.45 ± 3.70b 32.44 ± 4.40c 19.44 ± 3.80ab l19g 48.13 ± 3.63c 44.52 ± 4.66d 20.69 ± 2.95b l19h 55.09 ± 0.71de 69.41 ± 1.45e 47.26 ± 1.44c note: asterisks indicate mean comparisons that differ significantly (p < 0.05). 5 nova biotechnol chim (2022) 21(2): e1053 2 autoaggregation analysis of lab from human breast milk lab isolates from human breast milk showed autoaggregation of over 10 %. however, six exceeded 40 %, as shown in fig. 2. l19a, l19e, and l19h isolates had better auto-aggregation than others, while l19a performed best. each lab isolate has a unique probiotic auto-aggregation ability determining its colonization and survival in the digestive tract. based on the analyses, l19a, l19e, and l19h isolates have the best values, despite the statistical analysis indicating varying analysis tests. therefore, the three isolates ought to analyze the 16s rrna gene to determine the species' name to be used as a candidate for probiotics. fig. 2. the ability of lactic acid bacteria to adhere to the small intestinal mucosa; different superscripts showed a different mean comparison significantly (p < 0.05). coaggregation analysis of lab from human breast milk this study conducted co-aggregation testing between lab isolates and two pathogenic bacteria with gram-positive and negative characteristics, as shown in table 2. the co-aggregation activity against s. aureus exceeded e. coli because the cell wall arrangement of gram-positive bacteria contains more peptidoglycan and polysaccharides, with less lipid. the co-aggregation results indicated that isolate l19a had the highest ability with the five pathogenic bacteria. in the following table, the auto-aggregation test correlates with antimicrobial activity, where l19a has the strongest antimicrobial properties. notably, the differences in species and strains strongly influence the coaggregation ability. out of eight isolates tests, only three exceeded 30 % co-aggregation values. meanwhile, the results of this study showed that l19a, l19e, and l19h had a co-aggregation value of 31.05 – 38.08 %, while the rest were below 30 %. table 2. coaggregation ability (%) of lab isolates from human breast milk on gram-positive and negative pathogenic bacteria. bal isolate coaggregation [%] e. coli atcc 25922 s. aureus atcc 25923 l19a 19.00 ± 1.02e 38.08 ± 1.17e l19b 2.86 ± 1.29a 11.46 ± 2.11a l19c 7.76 ± 4.26ab 11.70 ± 1.11a l19d 6.06 ± 1.06ab 16.36 ± 0.29b l19e 15.69 ± 2.72c 31.05 ± 0.20d l19f 12.85 ± 1.04c 17.83 ± 0.48b l19g 1.62 ± 1.80a 19.16 ± 1.27c l19h 17.31 ± 2.74cd 31.82 ± 0.61d note: asterisks indicate mean comparisons that differ significantly (p < 0.05). 6 nova biotechnol chim (2022) 21(2): e1053 3 antimicrobial activity of lab from human breast milk the ability of lab to inhibit pathogenic microorganisms was analyzed based on the presence of hindrance of the zone marked. a clear part appeared around the isolated colonies after 48 hours combination, both in the cultures of s. aureus atcc 25923, e. coli atcc 25922, and c. albicans atcc 11778s. this indicated that lab isolated from human breast milk inhibited pathogenic microorganisms. however, the diameter of the inhibition zone formed by lab isolates against s. aureus, e. coli, and c. albicans varied, as shown in fig. 3. quantitative analysis of the inhibition zone of the antimicrobial test of lab isolates against pathogenic microbes showed that l19a, l19e, and l19h had higher values than l19b, l19c, l19d, l19f, and l19g. however, the l19h isolate had the best ability. fig. 3. inhibition zone of lab isolates antimicrobial test against pathogenic microbes; asterisks indicate mean comparisons that differ significantly (p < 0.05). direct pcr product analyzing based on resistance at low ph and bile salts, hydrophobicity, autoaggregation, co-aggregation, and inhibiting pathogenic microorganism’s properties, l19a, l19e, and l19h were the best indigenous probiotic candidates. however, the 16s rrna gene sequence amplified in direct pcr lab products obtained amplicons with a size of ±1,585 bp, as shown in fig. 4. molecular analysis of lab isolates from human breast milk was carried out using partial sequences of the 16s rrna. the partial sequences were suitable for analysis of bacteria down to the species level. fig. 4. the amplicon of partial sequences of 16s rrna bal gene from human breast milk; m – marker. 7 nova biotechnol chim (2022) 21(2): e1053 2 data from sequences of l19a, l19e, and l19h isolates alongside those in the ncbi were analyzed using the blast program, as shown in table 3. therefore, this analysis indicates that the l19a isolate is similar to l. casei nwafu1544, while l19e and l19h are equivalent to l. rhamnosus jcm 8649 and l. paracasei 2281. table 3. blast results from isolates l19a, l19e, and l19h from human breast milk. isolate blast species similarity [%] seq. id. l19a l. casei nwafu1544 99 mg551218.1 l19e l. rhamnosus jcm 8649 99 ab690234.1 l19h l. paracasei 2281 99 mt604755.1 phylogenetic analysis phylogenetic tree reconstruction based on 16s rrna gene sequences indicated that l19a, l19e, and l19h isolates formes two clades that can be separated from the out-group bacillus subtilis (fig. 5). l19a isolate has similarities with l. casei nwafu1544 up to 99 % with a genetic distance of 0.021. also, the l19e isolate has a similarity value with l. rhamnosus jcm 8649 with a genetic distance of 0.00, meaning both are identical. similarly, the l19h isolate has 99 % resemblance with l. paracasei 2281 with a genetic distance of 0.011. the bootstrap value for branching between isolates is 99 %, indicating stability. fig. 5. phylogenetic tree reconstruction of l19a, l19e, and l19h isolates with reference species based on the neighborjoining method, bootstrap 1,000x. discussion the study aimed to investigate whether human breast milk can be the best indigenous probiotics. several supporting methods were considered, including the ability of lab at acidic ph and bile salts resistance, hydrophobic analysis, autoaggregation, coaggregation analysis, antimicrobial, and gene analyses based on 16s rrna. the initial analysis was conducted to determine whether the lab isolates obtained could survive and grow in acid and bile salt conditions. the eight isolates showed positive results following li et al. (2020) and shehata et al. (2016). some of the main requirements for microbes that function as probiotics include the resistance to low ph, ability 8 nova biotechnol chim (2022) 21(2): e1053 3 to grow on bile salts, the capability to colonize and possess antimicrobial activity. tolerance of acidic ph and bile salts is an important characteristic of probiotic bacteria since its a requirement to pass through the digestive tract to reach the colon. lab isolates adapt to low ph due to their ability to balance the internal cell ph, carried out by removing a proton (h+) from the cell through the hydrolysis process of atp (h+-atpase). also, the lab has histidine decarboxylase and arginine deaminase, an important condition for acidic survival (chen et al. 2019). some lactic acid bacteria comprise of enzyme bile salt hydrolase (bsh) to hydrolyze, which changes physicochemical properties to non-toxic. hydrophobicity is a significant supporting characteristic for probiotics. the nature of hydrophobicity determines the ability to adhere and auto-aggregate to various surfaces. according to krausova et al. (2019), the hydrophobic nature of the lab cell surface is demonstrated by its adhesion or affinity to xylene. specifically, the hydrophobic tests help comprehend the surface properties of the bacterial cells critical for adhesion. in an acidic solvent, an affinity for chloroform indicates that the cell surface is an electron donor. aggressiveness and hydrophobicity are among the components responsible for adhesion because the attachment mechanism is complex (li et al. 2020). based on the outcomes, a few isolates have a high affinity for xylene exceeding 50 %. however, it is considered moderate between 20 – 30 %, while below 20 % is indicated as negative (guan et al. 2020). moreover, a few isolates had a poor affinity for xylene, indicating a more hydrophilic surface with cell wall components such as phospholipids and polysaccharides (krausova et al. 2019). the bacterial cell wall component is attached to the host, forming hydrophobic interactions. according to li et al. (2020), hydrophobic properties result from protein on the cell surface and lipoteichoic acid, while polysaccharides produce hydrophilic properties. bacteria are hydrophobic because their cell surface is negatively charged and associated with bacterial adhesion properties, which vary depending on the strain. also, it is influenced by the plant medium, the age of the bacteria, and the bacterial cell's surface structure. the hydrophobicity of probiotics depends on autoaggregation. all lab isolates samples from human breast milk indicated autoaggregation ability, shown by the results exceeding 10 %. according to li et al. (2020), bacteria with autoaggregation ability below 10% are non-autoaggregation, important for probiotics in intestinal epithelial cells attachment. subsequently, the autoaggregation nature of probiotic strains is important for attaching the intestinal epithelial cells (krausova et al. 2019). li et al. (2020) stated that cellular aggregation depends on species and the environment because aggregates stick to the mucosal surface, supporting probiotic bacteria survival in the intestine. aggregates also hinder pathogenic bacteria from sticking to the intestine's surface, hence it is easy to extract from the digestive tract. the cellular aggregation mechanism is a complex interaction between cell surface components and secreted factors, such as protein, glycoprotein, teichoic acid, and lipoteichoic acid on the bacterial cell's surface, affecting the adhesion autoaggregation hydrophobicity (krausova et al. 2019). however, the mechanism of these compounds in influencing adherence, autoaggregation, and hydrophobicity has not been clearly described. according to krausova et al. (2019), the autoaggregation nature of a bacterium is influenced by the cell surface hydrophobicity. the co-aggregation analysis of a probiotic bacterium correlates with its ability to form a barrier capable of preventing the colonization of pathogenic bacteria in the digestive tract (li et al. 2020). therefore, lab that aggregates with the pathogenic bacteria is a beneficial trait as a probiotic (campana et al. 2017). the coaggregation activity against s. aureus was higher than e. coli because the cell wall arrangement of gram-positive bacteria contains peptidoglycan, less lipid, and more polysaccharides. teichoic acid is a water-soluble polymer that serves as positive ion transport, which demonstrating-positive bacterium that makes it easier to penetrate polar peptidoglycan than non-polar ones (septiani et al. 2017). co-aggregation testing is a suitable method to evaluate the interaction between lab and pathogenic bacteria. lab aggregates with pathogens expose antimicrobial compounds directly to pathogenic bacteria (li et al. 2020). therefore, lab controls the surrounding pathogens 9 nova biotechnol chim (2022) 21(2): e1053 2 (gao et al. 2019). to produce antimicrobial compounds, a mechanism for inhibiting the colonialization of pathogens in the digestive tract involves co-aggregation (campana et al. 2017). antimicrobial tests supported the co-aggregation properties of inhibiting pathogenic microbial colonies based on the clear zone diameter formed. according to li et al. (2015), differences in lab antimicrobial activity against several tested microbes are based on differences in the structure of the cell walls of assessed microbes and the ability of concentrations variation of antimicrobial compounds to produce different inhibition zones. this inhibitory activity occurs due to the accumulation of primary metabolites in lactic acid, ethanol, and carbon dioxide. according to kumar et al. (2016), this also occurred due to the presence of secondary metabolites in the form of hydrogen peroxide and bacteriocins. amarantini et al. (2019) added that clear zones around lab isolates are formed due to the activity of bactericidal antimicrobial compounds in the form of organic acids., which affect the cytoplasm of pathogenic microbial cells to become acidic and inhibit the transmembrane potential along with their substrate transport. according to le et al. (2019), the antibacterial activity was determined based on the diameter of the bacterial inhibition zone against pathogenic microorganisms. inhibition zone diameters ranging from 0 mm to 3 mm have weak antibacterial activity. the ones from 3 mm to 6 mm and over 6 mm contain good and strong antibacterial activities. these results are in line with ren et al. (2018), which stated that lab isolates inhibit escherichia coli bacteria with an inhibition zone diameter of 7 – 14.3 mm. species analysis was performed for the top 3 isolates based on previous tests. furthermore, molecular analysis of lab isolates from human breast milk was carried out using partial sequences of the 16s rrna. the partial sequences were suitable for analysis of bacteria down to the species level. this is in line with bukin et al. (2019), which stated that the 16s rrna gene's partial sequences could be used as a universal barcode of lab. isolates with the same 16s rrna gene sequence above 97 % represent similar species (edgar 2018). wullur et al. (2020) stated that isolates are considered the same species when they have a maximum identity of 99 %. the bootstrap value for branching between isolates is 99 %, indicating stability. according to (tilahun et al. 2018), a branching value of 70 – 100 indicates that phylogenetic trees remain unchanged. comparative sequencing analysis of the 16s rrna gene is currently a common pathway for identifying and classifying bacteria. since 1994, there have been strains with more than 97 % similarity of the 16s rrna gene sequence, considered the same species. in 2018, the percentage level boundary for species was evaluated to be 98.65 % (beye et al. 2018). however, in some cases, there were several adjacents in species, including the lactobacillus buchneri group (l. buchneri, l. kefiri, l. parabuchneri, and l. parakefiri), l. casei group (l. casei, l. paracasei, and l. rhamnosus), lactobacillus plantarum group (l. fabifermentans, l. plantarum, l. paraplantarum, and l. pentosus) and lactobacillus sakei group (l. curvatus, l. graminis, and l. sakei). all these groups were indistinguishable using 16s rrna gene sequencing due to a high degree of similarity up to 99 % between species (fontana et al. 2018). lactobacillus casei, paracasei, and rhamnosus have close phylogenetic and phenotypic relationships considered to be in the l. casei group. according to hill et al. (2018), members of this group are facultative heterofermentative, have a g+c dna content of 45 – 47 %, with identical peptidoglycan types (l-lys-dasp). the widely recognized probiotic strains belonging to this group, such as l. casei strain shirota and l. rhamnosus gg are used worldwide in fermented milk products or dietary supplements to improve the host's health (orlando et al. 2016). although this group comprises many strains of commercial value, their taxonomic status is still debatable. also, strains of commercial value as probiotics, the taxonomic status, and nomenclature of the l. casei are still debatable due to the difficulty in identifying these three species using the most frequently used genotypic methodology of 16s rrna gene sequencing (huang et al. 2018). strain l. casei and l. paracasei are widely used as probiotics or symbiotic supplementation to improve clinical outcomes. the results of several clinical trials indicate that l. casei and l. paracasei improve patients' condition from various digestive diseases, chronic infections, obesity, and depressive disorders. oral administration of probiotics 10 nova biotechnol chim (2022) 21(2): e1053 3 shortens the duration of acute diarrheal disease in children by about 1 day. the prevention of acute diarrhea in adults and children, using l. rhamnosus gg and l. casei, has proven effective in several specific doses (lai et al. 2019; li et al. 2019). according to andriani et al. (2020), l. casei and l. rhamnosus probiotics are effective as a substitute for antibiotic growth promoter (agp) against total cholesterol, low-density lipoprotein (ldl), and high-density lipoprotein (hdl). the l. casei group remains in demand as a probiotic and is widely used. in recent years, many studies have focused on its application to health promotion or prevention of several digestiverelated ailments and disorders. lcg could be used prophylactically or therapeutically in diseases associated with the gut microbiota. this group has been researched extensively on the stress response, essential to their survival, therefore applied as a probiotic. lcg from human breast milk is an indigenous probiotic candidate that needs further investigation to determine other abilities, physiology, and metabolite products. conclusion lactic acid bacteria from human breast milk have the ability of an indigenous probiotic. subsequently, isolates l19a, l19e, and l19h had the best ability to withstand acidic ph and bile salts, hydrophobicity, autoaggregation, coaggregation, and inhibit pathogenic microorganisms. the 16s rrna gene analysis showed that the three isolates belonged to the l. casei (lcg) group. l19a isolate was similar to l. casei, l19e was similar to l. rhamnosus, and l19h was similar to l. paracasei. these isolates can be used as indigenous probiotics. conflict of interest the authors declare that they have no conflict of interest. references amarantini c, satwika d, budiarso ty, yunita er, laheba ea (2019) screening of antimicrobial-producing lactic acid bacteria isolated from traditional fish fermentation against pathogenic bacteria. j. phys. conf. ser. 1397: 012045. andriani ad, lokapirnasari wp, karimah b, hidanah s, alarif ma, soeharsono s, harijani n (2020) efektifitas probiotik lactobacillus casei dan lactobacillus rhamnosus sebagai pengganti antibiotic growth promoter terhadap total kolesterol, low density lipoprotein dan high density lipoprotein ayam broiler. 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biodivers. 21: 2735-2740. yeoh yk, zuo t, lui gcy, zhang f, liu q, li ayl, chung ack, cheung cp, tso eyk, fung ksc, chan v, ling l, joynt g, hui dsc, chow km, ng sss, li tcm, ng rwy, yip tcf, ng sc (2021) gut microbiota composition reflects disease severity and dysfunctional immune responses in patients with covid-19. gut 70: 698-706. 12 microsoft word puterova nb 9-2.doc nova biotechnologica 9-2 (2009) 167 applications substituted 2-aminothiophenes in drug design zita puterová1, alžbeta krutošíková2, daniel végh3 1department of chemical theory of drugs, faculty of pharmacy, comenius university, kalinčiakova 8, bratislava, sk-832 3, slovak republic (puterova@fpharm.uniba.sk) 2department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (alzbeta.krutosikova@ucm.sk) 3institute of organic chemistry, catalysis and petrochemistry, faculty of chemical and food technology, slovak university of technology, radlinského 9,bratislava, sk-812 37, slovak republic (daniel.vegh@stuba.sk) abstract. highly substituted thiophene derivatives are important heterocycles found in numerous biologically active compounds. title compounds are attractive derivatives because their applications in pharmaceuticals, agriculture and pesticides. they exhibit antimicrobial activity against various gram(+) and gram(-) bacteria and fungi. many of these molecules act as allosteric enhancers of a1-adenosine receptor, glucagon antagonists as well as antioxidant and anti-inflammatory agents. key words: substituted 2-aminothiophenes, gewald reaction, thieno[2,3-d][1,3]oxazin-4-ones, allosteric enhancers, 3-deazathiadiamine, drug design 1. introduction the chemistry of 2-aminothiophenes has received much attention upon their convenient availability through the most versatile synthetic method developed by gewald and his co-workers (gewald et al., 1988,). there are four basic variations originally described by gewald and co-workers and about up to fifteen modifications to accomplish the synthesis of highly functionalized 2-aminothiophenes (sabnis et al., 1999; gronowitz and hörnfeldt, 2004). the improvements of the gewald synthesis are based in diminution of the reaction time using microwave technology (huang et al., 2005, hesse et al., 2007). the recent information of synthesis of title compounds is reviewed in our recent chapter (puterová and krutošíková, 2009). this article details about the use of substituted 2aminothiophenes in the synthesis of thienotype heterocycles as a group of precursors applied in pharmaceuticals and in drug design. 2. synthesis of pharmaceuticals and drugs the ultimate positions of substituted 2-aminothiophenes in the field of drug design and synthesis of pharmaceuticals comes from their advantageous properties – the thiophene ring as is bioisosteric replacement for phenyl group broadly present in an active drugs, the thiophene core exists in many natural and synthetic pharmaceuticals. 168 puterová, z. et al. moreover, they act as active precursors in broad range of synthetic pathways towards compounds used in therapy and biodiagnostic (jarvest et al., 1999; doré et al., 2004). 2.1 5-substituted 2-aminothiophenes as a1 adenosine receptor allosteric enhancers adenosine is an important endogenous tissue-protective compound released during ischemia, hypoxia or inflammation. four receptor subtypes (a1, a2a, a2b, a3) have been defined based on pharmacological properties (linden, 1997). considerable effort has been directed towards developing therapeutic agents targeting these receptors (bruns and fergus, 1990). substituted 2-aminothiophenes of structure 1 – 4, with alkyl, aryl and cycloalkyl substituents in c-4 and c-5 position and aroyl substituent in c-3 position (fig. 1), maintained the best allosteric enhancer activity (nikolakopoulos et al., 2006; fergusson et al., 2008). fig. 1. structure of some aminothiophene-based allosteric enhancers. the significant effort in the area of synthetic aminothiophene-based allosteric enhancer is directed to development and synthesis of adenosine receptor agonists with limited side-effects. since the active compounds with potential and utility are substituted 2-aminothiophenes their synthesis in principal based on the gewald reaction. 2.2 synthesis thieno[2,3-d][1,3]oxazin-4-ones as inhibitors of human leukocyte elastase a series of thieno[2,3-d][1,3]oxazin-4-ones 8a – h was synthesized and evaluated in vitro for inhibitory activity toward human leukocyte elastaze (hle). the strategy presented by authors (gütschow and neuman, 1998; gütschow et al., 1999) is base on the replacement of the benzene ring in benzoxazinones by thiophene. the study demonstrates the versatility of 2-aminothiophenes prepared by gewald reaction as a synthetic entry to serine protease-inhibiting, fused 1,3-oxazin-4-ones. the synthetic route to novel thieno[2,3-d][1,3]oxazin-4-ones 8a – h using alkyl 2aminothiophenecarboxylates 5a,b as substrates exhibits a facile three step synthesis, as is presented on scheme 21. aminothiophenes 5a,b were converted to isothiocyanatonova biotechnologica 9-2 (2009) 169 thiophenes 6a,b by the action of thiophosgene. deprotection of tert-butoxycarbonyl group resulted directly to ring closure of the intermediates isothiocyanatothiophenecarboxylic acids 7a,b. these key intermediates were alkylated with appropriate alkyl halides to furnish the final derivatives 8a – h (scheme 1). scheme 1. synthesis of substituted thieno[2,3-d][1,3]oxazin-4-ones 8a–h extracellurar hle is a serine protease contained in the azurophilic granules of human neutrophils and has been shown to contribute to the pathogenesis of destructive lung diseases, such as pulmonary emphysema, cystic fibrosis, adult respiratory distress syndrome and inflammatory disorders such as rheumatoid arthritis. for that reason, much attention is focused on the inhibition of hle by low-molecular-weight inhibitors that might serve as therapeutic agents. 2.3 synthesis of 3-deazathiamine authors (hawskey et al., 2001) outlined the synthesis of 3-deazathiamine (13) in ten chemical steps, through key intermediate substituted 2-aminothiophene 9. on the scheme 2 we have outlined the synthesis of target compound 13 starting from appropriate aminothiophene 9. deamination of aminothiophene 9 via the bromide and following cleavage with zn in acid media to afford derivative 10 was very efficient, displaying none of side reactions. conversion of formed ester 10 to final 3deazathiamine (12) was accomplished in four subsequent steps isolating the crucial intermediates – aldehyde 11 and nitrile 12. the readily available and inexpensive starting materials and reagents, and the lack of protection and de-protection steps make this synthesis very fashionable (scheme 2). deazathiamine diphosphate (deaza-tdp) is an analogue of thiamine diphosphate (tdp), the biologically active for of thiamin (vitamin b1), with a neutral thiophene replacing positively charged thiazolium ring. tdp is co-enzyme present in a number of enzymes, including pyruvate decarboxylase, transketolase, pyruvate oxidase. 170 puterová, z. et al. scheme 2. reaction sequence towards 3-deazathiamine 13. 2.4 other important pharmaceuticals developed from2-aminothiophenes the synthesis and antitumor a potent thieno[2,3-b]azepin-4-one antineoplastic agents was reported (koebel et al., 1975). the meaningful structure-activity relationships have been established in monocarbonyl and dicarbonyl series of thieno[2, 3-b]azepin-4-one 14, 15 (fig. 2) prepared by dieckmann ring closure reaction in multi step reaction from substituted 2-aminothiophenes. cinnamyl derivatives of thieno[2, 3-d]oxazinones 16 (fig. 3) inhibits herpes protease processing in hsv-2 infected cells. the synthesis and pharmacology of this series of derivatives was presented by authors (jarvest et al., 1997 and 1999) from ethyl 2amino-4-methylthiophene-3-carboxylate. fig. 2. structure of potential antineoplastics thieno (2,3-b) azepin-4-ones 14,15 fig. 3. the family of herpes proteases hsv-2 thieno (2,3-d) oxazinones 16 nova biotechnologica 9-2 (2009) 171 transglutaminases (tgases) are a family of ca2+ dependent enzymes which are normally expressed at low levels in many different tissues and serve vital roles, such as blood clothing and epithelia formation. some tgase isoenzymes are involved in diverse pathological conditions like celiac disease, atherosclerosis and neurodegenerative disorders. thieno[2,3-d]pyrimidin-4-hydrazide derivatives related to structure 17 (fig. 4) were discovered as a moderately potent inhibitors of tgase-2 (tissue transglutaminase) (duval et al., 2000). the rna polymerase holoenzyme is a proven target for antibacterial agents. a high-throughput screening program based on this enzyme from staphylococcus aureus had identified a 2-ureido-thiophene-3-carboxylate 18 (fig. 5) as a low micromolar inhibitor. it displayed good antibacterial activity against s. aureus and s. epidermidis. based on these observations the authors (ahrin et al., 2006) reported a facile synthesis of the number of analogs of 18 via the gewald reaction and evaluated for cytotoxic activity against rifampicin-resistant s. aureus. fig. 4. thieno (2,3-d) pyrimidin-4-hydrazide 17 lead structure in inhibition of tgase-2. fig. 5. 2-ureido-thiophene-3-carboxylate 18 antibacterial agent against s. aureus. a novel class of thiophene-derived antagonists of the human hepatic glucagon receptor (hgcrg) has been discovered (duffy et al., 2005). the synthesis of derivatives based on the lead structure 19 (fig. 6) was accomplished using the gewald reaction. the further investigations of such structures are challenging in development of therapeutics of the diabetes mellitus. diabetes mellitus is a condition characterized by chronically elevated levels of blood glucose caused by incorrect function of the hormone responsible for the hgcrg activation. fig. 6. thiophene-based antagonist of hgcrg 19 172 puterová, z. et al. because the structure-based drug design program through substituted 2aminothiophenes has been investigated broadly, up to this date there are many other research works dealing with the synthesis, pharmacology and application of thiophenebased structures in medicinal chemistry (kourounakis et al., 2000). it is no doubt, that this area of gewald-like thiophene derivatives exhibits the highest progress in a scope and utilization. 3. conclusions in this short review the importance of substituted 2-aminothiophenes in the medicinal chemistry was extended in terms of drug design and synthesis of pharmaceuticals. the scope of this work does not include all of the publications in this field, but the most interesting studies in the subject areas are considered. the further detailed information in the latter aspects can be found within the list of the references. acknowledgements: the support of this work by grants vega 1/1005/09, vega 1/4453/07, vega 1/4300/07, vvce 0004-07 and uk/102/2009 is gratefully acknowledged references arhin, f., bélanger, o., ciblat, s., dehbi, m., delorme, d., dietrich, e., dixit, d., lafontaine, y., lehoux, d., liu, j., mckay, g. a., moeck, g., reddy, r., rose, y., srikumar, r., tanaka, k.s.e., williams, d.m., gros, p., pelletier, j., parr, t.r., rafai far, a.: triaminotriazine dna helicase inhibitors with antibacterial activity. bioor. med. chem. lett., 16, 2006, 1286-1290. bruns, r.f., fergus, j.h.: allosteric enhancement of adenosine a1 receptor binding and function by 2-amino-3-benzoylthiophenes. mol. pharmacol., 38, 1990, 939-949. doré, k., dubus, s., ho, h.-a., lévesque, i., brunette, m., corbeil, g., boissinot, m., boivin, g., bergeron, m., boudreau, d., leclerc, m.: fluorescent polymeric transducers for the rapid, simple and specific detection of nucleic acids at the zeptomole level. j. am. chem. soc., 126, 2004, 263-287. duffy, j.l., kirk, b.a., konteatis, z., campbell, e.l., liang, r., brady, e. j., candelore, m.r., ding, v.d.h., jiang, g., liu, f., qureshi, s.a., saperstein, r., szalkowski, d., tong, s., tota, l.m. xie, d., yang, x., zafian, p., zheng, s., chapman, k.t., zhang, b.b., tata, j.r.: discovery and investigation of a novel class of thiophene-derived antagonists of the human glucagon receptor. bioor. med. chem. lett., 15, 2005, 1401-1405. duval, e., case, a., stein, r., cuny, g.d.: structure-activity relationship study of novel tissue transglutaminaze inhibitors. bioor. med. chem. lett., 15, 2005, 1885-1889. fergusson, g.n., valant, c., horne, j., figler, m., flynn, b.l., linden, j., chalmers, d.k., sexton, p.m., christopoulos, a., nova biotechnologica 9-2 (2009) 173 scammells, p.: 2-aminothienopyridazines as novel adenosine a1 receptor allosteric modulators and antagonists. j. med. chem., 51, 2008, 6165-6172. hawskey, d., griffin, d.a., leeper, f.j.: synthesis of 3-deazathiamine. j. chem. soc. perkin trans., 1, 2001, 144-148. hesse, s., perspicace, e., kirsch, g.: microwave 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(eds.), elsevier academic press, kidlington, oxford, uk, 2004, 1-986. gütschow, m., kuerschner, l., neumann, u., pietsch, m., löser, r., koglin, n., eger, k.: 2-(diethylamino)thieno[1,3]oxazin-4-ones as stable inhibitors of human leukocyte elastaze. j. med. chem., 42, 1999, 5437-5447. gütschow, m., neumann, u.: novel thieno[2,3-d][1,3]oxazin-4-ones as inhibitors of human leukocyte elastaze. j. med. chem., 41, 1998, 1729-1740. jarvest, r.l., connor, s.c., gorniak, j.g., jennings, l.j., serafinovska, h.t., west, a.: potent selective thienoxazinone inhibitors of herpes proteases. bioor. med. chem. lett., 7, 1997, 1733-1738. jarvest, r.l., pinto, i.l., ashman, s.m., dabrowski, c.e., fernandez, a.v., jennings, l.j., lavery, p., taw, d.g.: inhibition of herpes proteases and antiviral activity of 2-substituted thieno[2,3-d]oxazinones. bioor. med. chem. lett., 9, 1999, 443448. koebel, r.f., needham, l.l., de witt blanton, c.: synthesis of thieno[2,3b]azepines-4-ones. j. med. chem.,18, 1975, 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doi: 10.36547/nbc.1258 1 nova biotechnologica et chimica municipal sewage sludge as a source of microelements in sustainable plant production: a laboratory lysimeter study vanda adamcová 1 , martin valica 1 , jozef gubiš 2 , marcela gubišová 2 , katarína ondreičková 2 , miroslav horník 1, , silvia dulanská 3,4 1 department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava nám. j. herdu 2, trnava 91701, slovak republic 2 plant production research institute, national agricultural and food centre, bratislavská cesta 122, piešťany 92168, slovak republic 3 institute of medical physics, biophysics, informatics and telemedicine, faculty of medicine, comenius university, sasinkova 2, bratislava 81372, slovak republic 4 faculty of public health, slovak medical university, limbová 12, bratislava 83303, slovak republic  corresponding author: miroslav.hornik@ucm.sk article info article history: received: 5 th november 2021 accepted: 18 th december 2021 keywords: agriculturally used soils heavy metals lysimeters nicotiana tabacum plant growth sewage sludge application abstract the aim of the work was to characterize the samples of sewage sludge (ssl) originated from the wastewater treatment plant piešťany (tavos, a.s., trnava, slovakia) in terms of their potential application into the soils. within the physicochemical characterization of ssl, the samples were analysed in terms of the values of ph, cation exchange capacity (cec), total organic carbon (toc), water holding capacity (whc), as well as the presence of heavy metals. it was found that ssl contained significant amounts of microelements zn (1,269 mg.kg -1 , d.w.) and cu (224 mg.kg -1 , d.w.). laboratory lysimeter experiments involving the application of ssl into the top layer of agriculturally used soil (0 – 10 cm) forming a soil column, in which seedlings of tobacco (nicotiana tabacum l.) were cultivated, showed that in the case of ssl application in the highest permitted amounts (15 t.ha -1 ) only 0.11 % zn and 0.07 % cu were released into the soil eluate during a 28 day of exposure, while in tobacco plants 0.13 % zn and 0.05 % cu were accumulated from the total amount of zn and cu originated from the application of ssl (zn – 133 mg and cu – 23.5 mg). when applying ssl in amount 30 t.ha -1 i.e., in the dose exceeding the permitted limits, only 0.02 % zn and 0.04 % cu were released into the soil eluate and 0.16 % zn and 0.09 % cu were accumulated in tobacco plants from the applied amount of ssl (zn – 267 mg and cu – 47.0 mg).  university of ss. cyril and methodius in trnava introduction sewage sludge (ssl) represent large-volume wastes annually produced in the whole world. the current global ssl rate is about 45 million dry tons of sludge per year. approx. 6 million tonnes of dry ssl were produced in china in 2013, while approx. 14 million tons of dry ssl per year were released during wastewater treatment in the united states (seiple et al. 2017; teoh and li 2020). currently, it is estimated that at least 10 million tons of dry ssl will be produced within the european union annually (wang et al. 2019). the composition of ssl is dependent on its origin in mailto:miroslav.hornik@ucm.sk nova biotechnol chim (2021) 20(2): e1258 2 terms of the type of wastewater treatment, and therefore is variable and unpredictable as well. in this way the public concern and definition of regulations or directives are primarily associated with toxic metals, organic xenobiotics or pathogens occurrence in this type of wastes (zhang et al. 2017). all these factors play an important role in decision with regard to the ultimate ssl disposal, which can involve: composting, landfilling, agricultural application, as well as sea or surface waters disposal and incineration (donatello and cheeseman 2013). in the slovak republic, ssl production represents more than 50,000 tons/year and ssl is predominantly used for compost production (62.7 %), reclamation (16.4 %), landfilling (13.5 %), incineration (5.5 %) and direct application into the soil (1.9 %) (according to data of mžp sr, 2012). ssl have increasingly been used in the last decades on farmland, mostly for their potentially appropriate fertilizing properties, due to its high content of organic matter as well as of macroand micronutrients of plant nutrition (usman et al. 2012). the application of ssl into the agriculturally used soils partially solves the problem of ssl disposal but on the other hand, may pose risks related to the contamination of soil and groundwater by inorganic contaminants (especially metals), organic xenobiotics (especially pharmaceuticals or chlorinated compounds) or microbial pathogens (coliform and faecal coliform bacteria) (santoro et al. 2017). in particular, toxic metals (cd, as, pb, hg) entering into agriculturally used soils through the application of ssl may contaminate the food chain through plants and the surface or ground water. however, high amounts of heavy metals belonging to microelements (zn, cu, mn) in ssl may also represent risks associated with the contamination of soils and ground water. in the eu, the application of ssl in agriculture is regulated by the eu council directive 86/278/eec. also, this directive determines the limit values for the content of heavy metals in ssl in this way. in slovak republic, the handling of ssl as a large-volume waste produced is regulated by the act no. 79/2015. in terms of the application of ssl into the soils, these activities are regulated by the act no. 188/2003. this act also determines the characteristics of the soil which must be evaluated, especially in terms of soil ph. the mentioned act no. 188/2003 indicates the value of the amount of ssl that can be applied into the soil, whereby the permitted amount of ssl applied shall not exceed 15 tons of dry matter per 1 ha over a period of five years. in many studies, the application of ssl in agriculture is studied and evaluated mainly from the point of view of ssl utilization as a source of important macroand microelements for plant nutrition, plant production, risks of pathogens transfer present in the ssl, and translocation of toxic elements or xenobiotics to the soils and plants (bettiol and ghini 2011; marguí et al. 2016; mossa et al. 2017; borba et al. 2018). the availability and mobility of macroand microelements or toxic metals in the systems ssl/soil have been examined in short-term laboratory studies using both leaching columns and incubation (toribio and romanyà 2006), lysimeters (borba et al. 2018), and under conditions of experimental fields (wolejko et al. 2013). lysimeter devices currently represent important tools for studying and predicting the behaviour of a wide range of substances in terms of their movement and overall balance within the soil biosystems defined by soil – soil solution – plant, as well as in the solution of various issues in agriculture, forestry, pedology or hydrology. lysimeter systems also offer the possibility to assess and analyse the impacts of climate change, the hydrodynamic changes associated with the construction of water structures or other large structures, the transport of a wide range of pesticides, xenobiotics, toxic metals, and radionuclides into the food chain or subsurface water, as well as in the analysis of microbial activity, whether from the point of view of agricultural production or microbial degradation of organic substances present in the soil. lysimeters also allow to analyse the impacts of the use of fertilizers and various chemicals on soil, groundwater and plant physiological status. in this way, it is possible to optimize the doses not only of the substances mentioned in such a way that they bring maximum benefit to the soil, but also the irrigation can be optimized for the given location. the latest lysimeter systems allow evaluate movement of water in large monolithic soil nova biotechnol chim (2021) 20(2): e1258 3 columns with a diameter of 1 m and a height of 1.5 m with high accuracy at a level of ± 20 g (meissner et al. 2014). in previous papers, samples of municipal ssl were characterized as a potential soil additive (šuňovská et al. 2013) or metals sorbent (frišták et al. 2013) and evaluated from the point of view of responses of rhizosphere bacterial (ondreičková et al. 2019a) or fungal (ondreičková et al. 2019b; 2021) communities to the sewage sludge application into soil as well. the aim of this work was to characterize samples of ssl originated from the wastewater treatment plant of company tavos, a.s. (municipal wastewater treatment plant, piešťany, slovak republic) in terms of their potential application into the soils using a laboratory lysimeter system. in the first part of the work, a physico-chemical characterization of obtained ssl samples as well as samples of reference soil – an agriculturally used soil (aus) was carried out. subsequent laboratory lysimeter experiments were focused on the total balance of the movement of metals – microelements zn and cu occurring in significant amounts in the obtained samples of ssl within the soil biosystem defined by ssl/aus – soil solution – root system and aboveground parts of tobacco (nicotiana tabacum l.) plants as a model of fast-growing crops. experimental sewage sludge and soil samples the sewage sludge (ssl) was sampled from the wastewater treatment plant of company tavos, a.s. (municipal wastewater treatment plant piešťany, slovakia). obtained samples of ssl represented activated sludge treated into the form of solid particles using a mechanical drainage system. in the experiments, ssl dried under laboratory conditions, mechanically homogenized and sieved to achieve a particle size < 0.150 mm was used. a reference soil – an agriculturally used soil (aus) was obtained from experimental fields of plant production research institute (location borovce, slovakia, 4834n, 1744e, altitude 160 m). the individual soil samples were taken gradually in layers with the aim to keep the natural soil stratification: 1. layer 0 – 10 cm; 2. layer 10 – 20 cm; 3. layer 20 – 30 cm, and 4. layer 30 – 40 cm. plant seedlings as a model for fast-growing plants, tobacco (nicotiana tabacum l.) seedlings were chosen. tobacco plants were obtained by germination of seeds and cultivation under hydroponic conditions in the inorganic carrier perlite watered by a nutrient solution according to hoagland (1920) diluted with deionized water in the volume ratio of 1 : 3 (25 % concentration strength). plant cultivation was carried out in a plant growth chamber kbwf 720 (binder, germany) under the following conditions: photoperiod 16 h light/8 h dark (max. photosynthetic photon flux density ppfd = 200 μmol.m 2 .s -1 with gradual reaching of maximum and minimum in the steps of 0 %, 40 %, 60 %, and 100 % light intensity), temperature 28/18 ºc and relative humidity 60 – 80 %. for experimental purposes, uniform plants with slight variations in the growth phase, height (10 – 12 cm), mass (wet material, 2 – 4 g), number of leaves (5 – 6 leaves) after four weeks of cultivation were obtained. characterization of studied matrices the determination of ph or cation-exchange capacity (cec) of ssl and aus were realized according to iso standard method no. 10390 or no. 11260, respectively. the value of ph was measured in the suspension of solutions 0.01 mol.dm -3 cacl2, 1 mol.dm -3 kcl, or deionized water in the volume ratio 1 : 5 (sample : solution) after 1 h of sedimentation. cec determination was carried out using bacl2 solutions. water holding capacity (whc) of individual samples of ssl and aus or mixture ssl : aus in different weight ratio: 1 : 76, 1 : 38, 1 : 19, and 1 : 9 were evaluated using glass columns with an inner diameter of 2.5 cm and length of 30 cm. the elemental analysis of mechanically homogenized ssl (particle size of < 0.063 mm) was performed by xray fluorescence spectrometry using the highperformance x-ray fluorescence spectrometer xlab 2000 (spectro, germany). total organic carbon (toc) was determined using toc analyzer (toc-vcpn, shimadzu, japan). nova biotechnol chim (2021) 20(2): e1258 2 the determination of bioavailable amount of zn and cu in studied samples of ssl and individual layers of aus was realized by one-step extractions using solutions according to mehlich (1984) – mehlich ii (in mol.dm -3 : nh4cl – 0.2; ch3cooh – 0.2; nh4f – 0.015; hcl – 0.12) and mehlich iii (in mol.dm -3 : edta – 0.001; hno3 – 0.001; nh4f – 0.015; nh4no3 – 0.25; ch3cooh – 0.2). the suspension of ssl or aus in mehlich ii or mehlich iii solution (10 g in 100 cm 3 of solution) was incubated for 10 min under agitation (250 rpm) at 25 °c. after exposure, the mixture was filtered through a filter paper and then through a syringe filter (13 mm diameter; 0.45 µm permeability; chs filterpure ptfe hydrophobic) and the content of zn and cu obtained in the filtrate was determined by atomic absorption spectrometry. laboratory lysimeter experiments the laboratory lysimeter system (ecotech, germany) was self-designed and used to perform the lysimeter experiments. this system consisted of two parts (fig. 1a), a vessel to create a soil column (diameter 30 cm and height 40 cm) and a cultivation part for growing plants in a soil column supplemented by a watering head. in this system, it is possible to install the soil monolith or create a soil column with a dry weight of 30 to 40 kg. the lysimeter contains on each side of the lysimetric vessel 3 inputs for sensors recording soil moisture tension, humidity, or temperature. bottom probe input is located at the interface of the lowest fourth and third layer of the soil, the middle probe input at the interface of the third and second layer of the soil, and the upper probe input at the interface of the second and top layer of the soil. data from individual tensiometers and temperature sensors are analysed and collected by a datalogger (fig. 1b), from which they can be wirelessly transferred to a computer. before, during and at the end of the experiment, the whole lysimetric system can be weighed using a weighing device with the aim to evaluate the total water balance (fig. 1c). the lysimeter system also includes apparatus for watering the soil column via a peristaltic pump and watering head, as well as apparatus for suction of soil eluate by a vacuum pump and through the drainage system at the bottom of the lysimeter vessel (fig. 1d). within the preparation of the lysimeter experiment, 8 kg of the soil was transferred from each obtained layer of aus (1. layer 0 – 10 cm; 2. layer 10 – 20 cm; 3. layer 20 – 30 cm, and 4. layer 30 – 40 cm) into the lysimeter vessel (dry weight, particle size < 2 mm, humidity < 5 %). the individual soil layers were inserted into the lysimeter sequentially in the order in which they occurred naturally at the sampling site, thus respecting their natural vertical distribution. in the first lysimeter experiment a, a homogeneous mixture of ssl : aus in the weight ratio 1 : 76 (105 g of the studied ssl) was added to the top (first) layer of the prepared soil column. in the second experiment b, a mixture of ssl : aus with double amount of ssl (210 g of ssl; ssl : aus = 1 : 38) was applied. the first addition of ssl represents the amount of 15 tons of ssl per hectare, which corresponds with the limits permitted by act no. 188/2003. in the case of the second lysimeter experiment, this amount was increased to 30 t.ha -1 thus exceeding the permitted limit value of 15 t.ha -1 . fig. 1. the design of the laboratory lysimeter system used in experiments. due to the fact that dry soil samples were placed into the lysimeter vessel, prior to planting fast4 nova biotechnol chim (2021) 20(2): e1258 3 growing tobacco (n. tabacum l.) plants, it was necessary to water the soil column using 3.5 dm 3 of artificial rainwater prepared according to the authors do lago et al. (2013). after equilibration (24 h) in terms of soil moisture tension distribution analysed by installed tensiometers, the soil eluate was drained from the drainage bottom of the lysimeter vessel before planting the plants of tobacco. subsequently, 3 seedlings of tobacco plants were planted into the topsoil layer of the soil column and the whole lysimeter system was placed in the growth chamber, where plants were cultivated under the following conditions: photoperiod 16 h light/8 h dark (max. photosynthetic photon flux density ppfd = 200 μmol.m -2 .s with gradual reaching of maximum and minimum in the steps of 0 %, 40 %, 60 %, and 100 % light intensity), temperature 28/18 ºc and relative humidity 60 – 80 %. during the experiments, the soil column was subjected to watering by a watering head with the application of 1.5 – 2.0 dm 3 of artificial rainwater at precisely defined intervals. after each watering and equilibrium reaching (after 24 h), the soil eluate was drained from the drainage bottom of the lysimeter vessel by a vacuum pump. the total water balance in the lysimeter system was also evaluated during the experiment by weighing the whole system. a continuous analysis (every 5 min) of soil moisture tension and temperature, as well as air humidity and temperature was carried out using installed probes. data was collected and stored by a datalogger. atomic absorption spectrometry the presence of zn and cu as microelements in the samples of soil layers, soil eluates, or individual parts of tobacco plants at the end of the lysimeter experiments and the bioavailability of zn and cu in aus and/or ssl samples were determined by atomic absorption spectrometry (aas) using atomic absorption spectrometer ice 3000 (thermo scientific, usa). in the case of soil and plant biomass, before the analysis the samples were mineralized in concentrated hno3 at 210 °c and 20 bars using a microwave mineralization system multiwave 3000 (anton paar, austria). based on the estimated amount of studied metals in the given samples, the determination was carried out either by flame atomization (acetylene/air; ppm amounts) or by electrothermal atomization (argon as an inert gas; ppb amounts). in the measurement of absorbance of samples, hollow cathode lamps (heraeus, germany) as radiation sources were used. deuterium background compensation was applied for correction of background effect. results and discussion characterization of studied matrices from the comparison of the values of physicochemical parameters ph, cation-exchange capacity (cec) and total organic carbon (toc) (table 1) it is clear that the samples of sewage sludge (ssl) and samples of agriculturally used soil (aus) differed significantly in these evaluated parameters. in the case of samples of individual soil layers, such significant differences in the mentioned values were not found. soil samples showed ph values within the range from 7.29 to 7.87, while for ssl ph values from 6.04 to 6.11 were determined. the slightly acidic character of the studied ssl samples after their application to soils similar to the soil studied in terms of their ph reaction may have a positive effect on reducing the ph values of typical alkaline soils to values ph of neutral soils. a similar observation was confirmed by žaltauskaitė et al. (2022). however, the reduction in ph is variable depending on the type of soil, sludge dosage, and characteristics, incubation time etc. (dhanker et al. 2021). it is generally known that soil ph value plays an important role in the mobility and bioavailability of some elements, e.g. significant microelements in the soil (see e.g. hechmi et al. 2021). at alkaline and neutral ph, metals tend to form insoluble minerals, whereas at acidic ph, metals are in more soluble and bioavailable forms (alloway 2012). for typical microelements (zn, mn, cu), their mobility and bioavailability for plant uptake in the soil increased with decreasing of ph value (below ph 7.0) of the soil. from the point of view of agricultural production, it can have a positive effect. on the other hand, from an ecotoxicological point of view, it can be evaluated rather negatively due to: (i) risk of heavy metals entering the groundwaters and 5 nova biotechnol chim (2021) 20(2): e1258 2 food chain, and (ii) risk of increased heavy (toxic) metals uptake by plants and their transport into the food chain. in the case of cec values, the studied ssl showed a 1.5-fold higher cec value and in the case of toc values up to 40-fold higher value in comparison with aus samples (cec = 15.1 – 16.8 meq/100 g and toc = 0.21 – 0.76 %) (table 1). for individual samples of vertical soil layers, the cec and toc values decreased slightly from the topsoil layer (0 – 10 cm) to the lowest soil layer (30 – 40 cm). from the literature it is evident that soils showing cec values in the range of 11 – 50 meq/100 g can be characterized as soils with low sand content, high clay content, medium or high organic matter content, and high whc. soils showing low cec values in the range of 0 – 10 meq/100 g are negative in this respect (daniels and haering 2006). based on this, it can be assumed that the addition of the studied ssl to soils can have a positive effect in terms of increasing the cec and toc values of soils, as well as improving the characteristics of the soils important, especially from the point of view of agricultural production. table 1. the values of ph (phh2o, phcacl2, and phkcl), cation exchange capacity (cec) and total organic carbon (toc) determined for ssl and individual layers of aus. sample phh2o phcacl2 phkcl cec [meq/100 g] toc [%] ssl 6.11 6.04 6.04 24.6 27.7 aus1 0 – 10 cm 7.87 7.29 7.42 16.6 0.76 aus2 10 – 20 cm 7.84 7.34 7.53 16.8 0.53 aus3 20 – 30 cm 7.80 7.41 7.47 15.7 0.32 aus4 30 – 40 cm 7.87 7.40 7.51 15.1 0.21 according to performed analyses determining the values of water holding capacity (whc), it was found that ssl samples had more than 4-times higher whc compared to aus samples (whc in the range of 44 to 48 g h2o/100 g, d.w.) (fig. 2). fig. 2. water holding capacity (whc) obtained for studied matrices – sewage sludge (ssl) and individual layers of agriculturally used soil (aus) (aus1: topsoil layer 0 – 10 cm; aus2: 10 – 20 cm; aus3: 20 – 30 cm; aus4: 30 – 40 cm) or for mixtures ssl : aus prepared in different weight ratio. individual experiments also showed that the addition of ssl to soil had a positive effect in terms of gradual increase in whc values, when with increasing ssl : aus weight ratio whc values increased from 73 g h2o/100 g (d.w.) found for ssl : aus = 1 : 76 to the value of 97 g h2o/100 g (d.w.) found for ssl : aus = 1: 9. boudjabi and chenchouni (2021) found that the application of ssl at the soil surface significantly increased soil moisture (25.95 %) compared to ssl mixed treatment, which was 24.46 % and the controls. elemental analysis of ssl samples carried out by x-ray fluorescence spectrometry confirmed that these samples did not exceed the limits for heavy metals concentrations permitted by act no. 188/2003 in terms of their direct application to soil (table 2). however, an interesting finding was that the samples of ssl contained significant amounts of heavy metals – microelements zn (1,269 mg.kg -1 , d.w.) and cu (224 mg.kg -1 , d.w.). table 2. metal concentrations in the studied sewage sludge. total concentration of metal [mg.kg -1 ; d.w.]; (loq) as cd cr cu fe mn ni pb se zn 3 (1) < 5 (1) 36 (5) 224 (5) 2.69 (0.05) 0.21* (0.05) 22 (4) 46 (5) 6 (1) 1,269 (5) * determined in %. 6 nova biotechnol chim (2021) 20(2): e1258 3 it is generally known that higher total amounts of important elements in a given substrate (soil) may not automatically mean a more favourable environment for plant growth. in addition to the total amounts of elements, their chemical form in which they occur in the given environment (free ionic forms, inorganic or organic complexes, and others) and the portion of their bioavailable amounts also play an important role. this results in the fact that the plant is not able to absorb the total amount of the element present in the given environment, but only its bioavailable part. the literature shows that the bioavailability of many heavy metals is strongly dependent on several factors, such as ph, organic matter content, clay content and other components (kabata-pendias 2011). following the above mentioned results regarding the total zn and cu content in the studied matrices, analyses with the aim to evaluate the bioavailability of these metals were carried out. for this purpose, one-step extraction procedures using mehlich ii and mehlich iii solutions were used. the choice of both extraction procedures was made in order to compare the obtained results, despite the fact that the mehlich ii solution is recommended for determination of the extractability of macroelements, e.g. mg, k, ca, and p. one-step extractions using mehlich iii solution showed that the percentage of the extractable zn to the total amount of zn occurring before extraction in the ssl sample was slightly higher compared to the aus samples and reached the value 8.7 % (fig. 3). fig. 3. extractability of zn (a) and cu (b) from the samples of ssl and individual layers of aus determined using mehlich ii and mehlich iii extractions. extractability of metals expressed as the amount of metal released form 1 kg of the given matri x (in mg/kg), the percentages represent the ratio of the extractable amount of metal to the total amount of the metal present before extraction in the matrix (see table 3). error bars represent the standard deviations of the mean (± sd; n = 3). table 3. total concentration of zn and cu in the studied matrices. total concentration of metal [mg.kg -1 ; d.w.] sample ssl aus1 aus2 aus3 aus4 zn 1,269 71 70 70 73 cu 224 24 22 21 23 markowicz et al. (2021) found that the bioavailable fraction of zn in the control soil was only 0.14 % of its total content and was 2.8 ± 0.7 mg.kg -1 . for ssl, the bioavailable fraction of zn was only 7.7 ± 0.5 mg.kg -1 . in contrast, the percentage of extractable cu in ssl (only 1.8 %), was approx. 6times lower compared to aus samples. thus, it can be stated that despite the significant presence of zn and cu in the studied sample of ssl, their bioavailability is relatively low. this result can be evaluated positively because high values of the bioavailability of zn and cu would mean the immediate availability of these metals for plants, and it could be the case that the root system would not be able to absorb this amount effectively at a given moment. it follows that the studied ssl can represent an interesting source of zn and cu as important microelements for plants, but relatively tightly bound, which can be released from this matrix in smaller proportions controlled by the gradual concentration equilibria [m]solution : [m]ssl a b 7 nova biotechnol chim (2021) 20(2): e1258 2 reaching or decomposition of this matrix in the soil environment. laboratory lysimeter experiments in this part of the work, the laboratory lysimetric experiments involving the application of ssl (15 t.ha -1 – experiment a or 30 t.ha -1 – experiment b) into the topsoil layer of aus (0 – 10 cm) forming part of a 40 cm soil column in which seedlings of tobacco (n. tabacum l.) plants were cultivated, allowed us to evaluate the overall balance of zn and cu in terms of their amount applied into the soil in the form of ssl. these experiments were focused on the overall balance of metal movement – zn and cu microelements occurring in significant amounts in the obtained samples of ssl within the designed soil biosystem: ssl/aus – soil solution – root system and aboveground parts of tobacco as a model fast-growing plant. the first addition of ssl represents the amount of 15 tons of ssl per hectare, which corresponds with the limits permitted by act no. 188/2003. in the case of the second lysimeter experiment, this amount was increased to 30 t.ha -1 , thus exceeding the permitted limit value of 15 t.ha -1 . it follows that the first experiment involving the application of ssl within the permitted limits focuses primarily on the evaluation of the positive effects of ssl as potential effective carriers of important microelements for plant growth. on the other hand, the second experiment with a double dose of ssl application (30 t.ha -1 ) makes it possible to assess their negative effects, especially in terms of the risks of heavy metals release from ssl into groundwater or the food chain through the plant uptake. fig. 4 graphically shows the changes in the values of water pressure potential (soil moisture tension) measured by tensiometers placed in individual layers of the prepared soil column caused by watering, suction of the soil eluate or evapotranspiration of water under given conditions. the value of pressure potential near 0 hpa represents saturated soil with water and values up to -600 to -700 hpa represent soil with minimal water content. during a 28 day experiment a, the analysis of zn fig. 4. soil moisture tension changes analysed by tensiometers placed in individual layers of the prepared soil column caused by watering, suction of the soil eluate or evapotranspiration of water under conditions given by the growth chamber during 28 days of the lysimeter experiment a and b. and cu amount in the samples of soil eluates drained from bottom of the lysimeter vessel confirmed a gradual increase in the concentration of zn and cu (0.21 mg.dm -3 for zn and 0.03 mg.dm -3 for cu to 0.96 mg.dm -3 and 0.19 mg.dm -3 , respectively) in the soil eluates collected. a similar phenomenon was also observed in the case of the experiment b. the gradual increase of concentrations of zn and cu in the soil eluates is probably related to the gradual release of these metals from the ssl applied into the topsoil layer (0 – 10 cm). at the end of the experiments, the grown tobacco plants and the individual soil layers were removed from the lysimeter vessel and subjected to quantitative analysis of the content of zn and cu. in the first step, the extractability of these microelements was evaluated by a one-step extraction procedure using a mehlich iii solution in order to determine the bioavailability of zn and cu within the individual layers of the soil column. the highest extractability of zn and cu was determined in the topsoil layer (0 – 10 cm) involving the application of the studied ssl (fig. 5a), when 13.8 % of zn of the total amount of zn in the analysed topsoil occurred in the extractable – bioavailable form. in the case of cu and the topsoil layer, the bioavailable form of this metal represented only 8.4 % of the total amount of cu under the same 8 nova biotechnol chim (2021) 20(2): e1258 3 conditions. from a quantitative point of view, it can also be said that the bioavailability of zn and cu in the topsoil layer was up to 3-times and about 30 % higher than in the case of lower soil layers without the direct application of ssl, respectively. also in the second experiment b (fig. 5b), it was found that the highest extractability of zn and cu was in the topsoil layer involving the application of the studied ssl, when up to 14.6 % of zn and 9.2 % of cu of the total amount of zn and cu in the analysed topsoil were occurred in the bioavailable forms. similarly, the bioavailability of zn and cu in the topsoil layer was up to 2-times and about 40 % higher than in the lower soil layers without the direct application of ssl, respectively. fig. 5. extractability of zn and cu from individual aus layers (aus1: topsoil layer 0 – 10 cm; aus2: 10 – 20 cm; aus3: 20 – 30 cm; aus4: 30 – 40 cm) at the end of laboratory lysimeter experiments a and b expressed as the amount of metal released form 1 kg of the given soil layer. error bars represent the standard deviations of the mean (± sd; n = 3) from the analysis of the samples of tobacco plants, it was observed that during experiment a, the plants increased their quantitative parameters, such as height of plants, length of above-ground parts or weight of plants, at least 3-times. from the point of view of the presence of zn and cu in the tissues of tobacco plants, it can be said that zn was accumulated in the concentration of 84.4 mg.kg -1 (d.w.) and cu in the amount 21.9 mg.kg -1 (d.w.). the ratio between accumulated amounts of zn and cu corresponds to the initial concentration ratio of these metals in the topsoil layer involving the application of ssl (86.8 mg.kg -1 for zn and 26.6 mg.kg -1 for cu). within this analysis, it was also evaluated the transfer factor (tf) defined as [m]plant : [m]soil and the portion of these metals in the tissues of individual parts of plants – root, stem and leaves in order to determine the concentration factor (cf) defined as [m]aboveground parts : [m]root. the calculated tf values were 0.97 for zn and 0.82 for cu and cf reached the same value of 0.74 for both metals. from the results of experiment b, it can also be concluded that during the experiment the plants increased their quantitative parameters at least 3times. due to the double dose of ssl (30 t.ha -1 ), in terms of the amounts permitted by act no. 188/2003 (limit 15 t.ha -1 ), the toxic effects of this increased addition of ssl on the growth of tobacco plants were not observed. based on the portion of zn and cu in the tissues of tobacco plants, it was found that in this case zn was present in a concentration of 96.2 mg.kg -1 (d.w.) and cu in the amount 15.7 mg.kg -1 (d.w.). thus, these amounts represent similar concentrations as in the case of an experiment involving the application of half amount of ssl. when calculating the values of concentration factors (cf) and transfer factors (tf), we found slightly different data than in previous experiment a. in both parameters, higher values were obtained for zn (cf = 0.74; tf = 0.94) in the comparison with the data for cu (cf = 0.36; tf = 0.54). a b 9 nova biotechnol chim (2021) 20(2): e1258 2 fig. 6. percentage distribution of the total amount of zn and cu occurring in the applied ssl (experiment a: zn – 133 mg and cu – 23.5 mg; experiment b: zn – 267 mg and cu – 47.0 mg) within the soil biosystem ssl/aus – soil solution – 3 tobacco plants (n. tabacum l.) at the end of laboratory lysimeter experiments. details are described in fig. 4. the use of a laboratory lysimeter system allowed to assess the total balance of zn and cu, based on the dose of ssl applied into the soil at the beginning of the experiment (experiment a: zn – 133 mg and cu – 23.5 mg; experiment b: zn – 267 mg and cu – 47.0 mg), the amount of zn and cu occurring in the collected soil eluates during the experiment and based on the amount of these metals accumulated in tobacco plants. in the case of the application of ssl in the highest dose permitted (15 t.ha -1 ), the total balance of zn and cu originating from the applied amount of ssl into the soil at the beginning of the experiment showed that only 0.11 % zn and 0.07 % cu were released in the form of soil eluate during a 28 day of exposure, while in tobacco plants 0.13 % zn and 0.05 % cu were accumulated (fig. 6). when applying ssl in the amount of 30 t/ha, i.e. in the amount exceeding the permitted limits, it was found that from the applied amount of ssl into the soil at the beginning of the experiment only 0.02 % zn and 0.04 % cu were released in the form of soil eluate during a 28 day exposure, while in tobacco plants 0.16 % zn and 0.09 % cu were accumulated. the remaining percentages of cu and zn stayed bound in the prepared soil column. similar minimal amounts of zn and cu released in the soil eluate or accumulated in giant reed (arundo donax l.) plants were also observed in the work of šuňovská et al. (2015), despite the increased bioavailability of these metals in the topsoil layer. from the above mentioned results, it is clear that double dose of ssl applied into the topsoil layer had the effect of increasing the accumulated amounts of zn and co in plant tissues rather than releasing them into the lower soil layers or soil eluate at the bottom of the lysimeter vessel. conclusion from the point of view of evaluating the possibility of application of the studied sewage sludge (ssl) to agricultural soils, it can be stated that the applied ssl represents an interesting source of zn and cu as important microelements in plant nutrition. these metals occur in the studied ssl experiment a 0.16 % zn 0.09 % cu 99.82 % zn 99.87 % cu 0.02 % zn 0.04 % cu experiment b plants: soil column: soil eluate: 0.13 % zn 0.05 % cu 99.76 % zn 99.88 % cu 0.11 % zn 0.07 % cu 10 nova biotechnol chim (2021) 20(2): e1258 2 in a bioavailable form, while their bioavailable portion is higher than in the case of agriculturally used soil (aus), but not enough to represent the risk of significant transport of these heavy metals into the lower soil layers or groundwater. in this regard, it was also shown that even exceeding the permitted limits in terms of application of ssl into the soil (30 t.ha -1 ) did not have negative effects in terms of significant vertical transport of zn and cu to lower layers of the soil column or in terms of the growth of tobacco (n. tabacum l.) as a model of a fast-growing plant. however, it must be taken into account that long-term and especially repeated application of higher amounts of ssl into the soils can lead to a negative accumulation of heavy metals originating from this application and to soil contamination. in this sense, relevant legislation of the slovak republic is also drafted (act no. 188/2003). an interesting scientific challenge, as well as a challenge for practice, is the application of ssl as a large-volume waste in terms of their application to 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metals in soil and in mixtures of lawn grasses. environ. prot. eng. 39: 67-76. zhang q, hu j, lee d-j, chang y, lee y-j (2017) sludge treatment: current research trends. bioresour. technol. 243: 1159-1172. žaltauskaitė j, kniuipytė i, praspaliauskas m (2022) earthworm eisenia fetida potential for sewage sludge amended soil valorization by heavy metal remediation and soil quality improvement. j. hazard. mater. 424: 127316. 12 microsoft word urik nb 9-2.doc nova biotechnologica 9-2 (2009) 141 basic interactions of aspergillus niger with se(iv) martin urík1, jaroslav ševc1, pavol littera1, marek kolenčík1, slavomír čerňanský2 1geological institute, faculty of natural sciences, comenius university in bratislava, mlynská dolina, bratislava, sk-842 15, slovak republic (urik@fns.uniba.sk) 2department of ecosozology and physiotactics, faculty of natural sciences, comenius university in bratislava, mlynská dolina, bratislava, sk-842 15, slovak republic abstract: filamentous fungus aspergillus niger is commonly found on decaying vegetation or in indoor environment and has a number of uses, including application in bioremediation. hence, the basic interactions of this common mould with selenite were studied, including biovolatilization, bioaccumulation and toxicity effects of selenite on fungal growth. the fungal strain, originally isolated from noncontaminated soil, was cultivated under aerobic conditions on liquid cultivation media with concentration of se(iv) 19 or 27 mg.l-1 during 25 days. the fungal growth in the presence of selenite was not inhibited when compared to control, only the sporulation was reduced. the concentration of se(iv) in liquid medium decreased rapidly within first ten days to 1 mg.l-1. however, according to results from the 25th day of cultivation, the concentration of total selenium in medium did not change significantly and only negligible amount of selenium (less then 1%) was bioaccumulated. that indicates some biotransformation of selenite into other selenium species. during the cultivation, up to 21% of total amount of selenium was transformed into volatile derivatives (biovolatilization) by filamentous fungus a. niger. key words: selenite, filamentous fungi, biovolatilization, bioaccumulation 1. introduction selenium is an essential micronutrient in animals. however, its elevated concentrations may result in toxic effects (eisler, 2000). hence, environmental pollution with selenium mobilized from geological sources as a result of industrial or agricultural activities may be serious threat for ecosystems (davis et al., 1988). bioremediation, including biosorption, bioaccumulation and biovolatilization is an alternative promising technology for removal of toxic metals from soils and waters (hiller et al., 2008; pipíška et al., 2008). biovolatilization of selenium by microorganisms, including fungi, has received much attention because of its application in bioremediation of selenium contaminated soils or waters (thompsoneagle and frankenberger, 1992). this process is realized via methylation pathway, where the selenium oxyanions are reduced, and the elemental selenium can be produced, a process which also results in detoxification (gadd, 1993). despite fungal capability to biovolatilize selenium, it is need to be known how does the fungi react on elevated concentrations of bioavailable selenium species, especially selenite, which is scavenged from waters to a greater extent then selenate (hamilton, 2004). 142 urík, m. et al. the objective of this study was to evaluate selenite uptake and transformation into volatile derivatives by aspergillus niger and the fungal growth responses to elevated selenite concentrations during cultivation. 2. materials and methods the strain of aspergillus niger used in this study was originally isolated from non-contaminated soil and was maintained on agar medium under laboratory conditions. fungal inocula were prepared from 14-day old cultures of this strain. the stock solutions of selenite were prepared in deionized water from na2seo3.5h2o (merck, germany). the cultivation system for experimental purposes included 15 ml of sabouraud cultivation media (himedia, mumbai, india), 5 ml of prepared fungal inocula and 5 ml of stock solutions to reach concentration 19 or 27 mg.l-1of se(iv) in cultivation system. selenite free control was also set up and monitored for 25 day. the ph of cultivation media, weight of dried biomass and concentration of se(iv) in medium were determined on 5th, 10th, 15th, 20th and 25th day of cultivation. on 25th day the concentration of total selenium in biomass and cultivation media was determined. all experiments were preformed in triplicate. 3. results and discussion growth of aspergillus niger in absence of selenite is illustrated in fig. 1. there is a linear growth phase in first 5 day of cultivation, followed by decline phase. on the 5th day the dry weight of biomass reached its maximum (0.36 g). surprisingly, the declination phase in presence of selenium (fig. 1) is not so evident comparing to the control. fig. 1. changes in weight of dry biomass during cultivation of aspergillus niger in absence (control) or presence of selenite with different initial concentration (19 or 27 mg.l-1). the first phase is followed by relative stationary phase until the 25th day of cultivation. the maximum dry weight of biomass is almost the same for the control nova biotechnologica 9-2 (2009) 143 and growth of a. niger in presence of selenite. however, the presence of selenite significantly reduced sporulation of the fungus. these observations suggest the relative resistance of a. niger to high selenium concentrations. generally, filamentous fungi are resistant to high levels of metals and metalloids, which indicates the potential of fungi as bioremeditation agents (iram et al., 2009). fig. 2. changes in ph of cultivation media during cultivation of aspergillus niger in absence (control) or presence of selenite with different initial concentration (19 or 27 mg.l-1). the initial ph of the cultivation media in absence of selenite was around 5.8 and decreased to 4.03 in first days of cultivation (fig. 2), followed by rapid increase to 7.92 determined on the 10th day of cultivation. the ph of the liquid medium did not significantly change until the end of experiment. the changes in ph value of cultivation media in presence of selenite (fig. 2) were significantly different compared to control. after initial decrease (3.6 and 3.4 for cultivation systems with initial se(iv) concentration of 19 and 27 mg.l-1 , respectively), the ph value only slightly increased with time up to approximately 4.6 for both initial concentrations of selenite. the decrease in ph of liquid medium during cultivation of white-rot fungus phanerochaete chrysosporium in presence of heavy metals was published by falih (1997), who claims, the decrease in ph is unfavorable condition for the fungus, probable because of increasing solubility of metals in the medium. in recent experiment, the ph and metabolic activity of fungus should significantly affect selenium toxicity, its ionic forms and mobility. during the initial growth rate decreased the concentration of se(iv) rapidly by more than 50% and later to 1.07 and 1.26 mg.l-1 at the end of the 10th day of cultivation for systems with initial concentration of se(iv) 19 and 27 mg.l-1, respectively (fig. 3). the concentration of selenite in cultivation media steadily decreased with time. however, the concentration of total selenium should not be changed during the cultivation. the table 1 shows the results of total selenium in media and fungal biomass at the end of cultivation, on the 25th day. the concentration of total selenium in cultivation medium decreased only slightly suggesting possible transformation of se(iv) into different chemical species. biomass-associated transformation of selenite (e.g. reduction to amorphous elemental selenium) has been 144 urík, m. et al. published previously (tomei et al., 1992). according to differences in amount of added se(iv) and the sum of se(iv) in biomass and medium, it is evident that the total selenium mass balance has change during the cultivation period. fig. 3. changes in se(iv) concentration in cultivation media during cultivation of aspergillus niger in presence of selenite with different initial concentration (19 or 27 mg.l-1). table 1. the amount of total selenium in biomass and cultivation media on the 25th day of cultivation with calculated amount of biovolatilized selenium during cultivation. initial concentrations of se(iv) (mg.l-1) concentration of total selenium in medium (mg.l-1) concnetration of total selenium in biomass (mg.kg-1) amount of biovolatilized selenium (%) 19 17.790 1.351 8.89 27 21.355 1.309 21.58 the loss of selenium in cultivation system represents approximately 8.9% and 21.6% for system with initial concentration of se(iv) 19 and 27 mg.l-1, respectively. the amount of accumulated selenium in biomass was negligible, less than 1% of total selenium present in cultivation system. previously, it was demonstrated that penicillium sp. is capable of selenite transformation in aqueous medium by biomass associated process as well as the formation of volatile organic derivatives (brady et al., 1996). 4. conclusions 1. the weight of dry biomass was not significantly different during the cultivation in presence and absence of se(iv) (19 or 27 mg.l-1) in cultivation media. 2. when comparing to control, the ph value of cultivation media in presence of selenite maintained in acidic region of ph (around 4), while the ph value of media in absence of selenite rapidly increased up to ph 8. 3. the concentration of se(iv) decreased rapidly within 10 days of cultivation. however, the concentration of total selenium decreased only slightly suggesting nova biotechnologica 9-2 (2009) 145 possible transformation of se(iv) into different chemical species. 4. while the accumulation of selenium after 25-day cultivation was negligible, the almost all of uptaken selenium was transformed into volatile derivatives (up to 21%) or chemical species other than selenite (according to previous statement). acknowledgements: this research was supported by vega 1/4361/07. references brady, j.m., tobin, j.m., gadd, g.m.: volatilization of selnite in aqueous medium by a penicillium species. mycol. res., 100, 1996, 955-961. davis, e.a., maier, k.j., knight, a.v.: the biological consequences of selenium in aquatic ecosystems. calif. agr., 42, 1988, 29. eisler, r. selenium. in: handbook of chemical risk assasment: health hazards to humans, plant and animals. lewis publishers, crc press, boca raton, 2000, 1649-1705. falih, a.m.: influence of heavy-metals toxicity on the growth of phanerochaete chrysosporium. bioresour. technol., 60, 1997, 87-90. gadd, g.m.: interactions of fungi with toxic metals. new phytol., 124, 1993, 25-60. hamilton, s.j.: review of selenium toxicity in aquatic food chain. sci. total. environ., 326, 2004, 1-31. hiller, e., jurkovič, ľ, bartál, m.: effect of temperature on the distribution of the polycyclic aromatic hydrocarbons in soil and sediments. soil wat. res., 3, 2008, 231-240. iram, s., ahmad, i., stuben, d.: analysis of mines and contaminated agricultural soil samples for fungal diversity and tolerance to heavy metals. pak. j. bot., 41, 2009, 885-889. pipíška, m., horník, m., vrtoch, l., augustín, j., lesný, j.: biosorption of zn and co ions by evernia prunastri. chem. ecol., 24, 2008, 181-190. thompson-eagle, e.t., frankenberger, w.t.: bioremediation of soils contaminated with selenium. adv. soil sci., 17, 1992, 262-310. tomei, f.a., barton, l.l., lemanski, c.l., zocco, t.g.: reduction of selenate and selenite to elemental selenium by wolinella succinogenes. can. j. microbiol., 38, 1992, 1328-1333. microsoft word brodnjak nb 9-2.doc nova biotechnologica 9-2 (2009) 113 high-performance liquid chromatographic determination of selected phenolic acids in wine darinka brodnjak vončina, maša islamčević razboršek, marjana simonič university of maribor, faculty of chemistry and chemical engineering, smetanova 17, maribor, slovenia (darinka.brodnjak@uni-mb.si) abstract: the aim of this study was to develop a method for identification and quantification of phenolic acids in different wine samples. the simple reversed-phase hplc-uv method for simultaneous determination of p-coumaric and ferulic acid was developed. the method was validated and working range, linearity, repeatability, accuracy, limit of quantitation loq and limit of detection lod were determined. the linearity of the method was tested in concentration ranges 0.1-1 mg l-1 and 1-10 mg l-1. the correlation coefficients (r2) were greater than 0.996 and quality coefficients (qc) ≤ 5%. detection limit for both compounds was 0.01 mg l-1. the method is precise (rsd<10%) and accurate. two different wine sorts were used for analysis, slovene red wine refošk and slovene white wine laški rizling. the wine samples were ultrasonically extracted with ethyl acetate, centrifuged, evaporated, re-dissolved in mobile phase and filtered. the solution was analyzed directly by hplc-uv. phenolic compounds were separated with a c18 reversed-phase column by isocratic elution using 2 % acetic acid in water-methanol (85:12, v/v) as the mobile phase at a flow rate of 0.7 ml min-1. acids were detected and quantitation was performed at wavelength of 320 nm. extraction procedure was optimized and yields were calculated from internal standard recovery. o-coumaric acid was used as internal standard. from the results it is evident that the red wine refošk contains higher concentrations of investigated compounds then the white wine laški rizling. stability tests were performed with standard compounds of p-coumaric and ferulic acid. it was established that both standards occur as trans-isomers, but when they were dissolved in methanol and stored exposed to daylight and room-temperature they were gradually transformed to cis-isomers. all closely related geometrical cisand trans-isomers were successfully separated under the same conditions as described before. the described method is simple and rapid as it involves liquid extraction with no other pretreatments of samples. it is suitable for simultaneous determination of cis and trans isomers of p-coumaric and ferulic acid in different liquid samples. key words: p-coumaric acid, ferulic acid, o-coumaric acid, hplc, wine. 1. introduction in recent years lots of studies were focused on phenolic acids due to their antioxidant antiviral, antibacterial and anti-inflammatory properties (zgórka and kawka, 2001). a variety of phenolic acids were separated using high-performance liquid chromatography (hplc) as reported in literature (kallithraka et al., 2006; robbins and bean, 2004; tarnawski et al., 2006; urakova et al., 2008; zgórka and kawka, 2001). in most studies reversed-phase hplc method was applied using non-polar c18 column. isocratic elution was applied (tarnawski et al., 2006; urakova et al., 2008; zgórka and kawka, 2001; yi et al., 2009) as well as a gradient method (kallithraka et al., 2006; robbins and bean, 2004; sladkovský et al., 2004). different mobile phases were prepared, using methanol–aqueous or acetonitrile–aqueous acidified mixtures. it 114 brodnjak vončina, d. et al. was determined that ph value is very important due to protonation of the acids. therefore the best ph was determined to be from 1-3. garciá et al. (2007) paid a lot of attention to sample pre-treatment. they used liquid-liquid separation with diethyl ether, followed by centrifugation, and vacuum evaporation. a variety of different wines was analysed by kallithraka et al. (2006). samples were only filtered and no other pre-treatment methods were used. hplc-dad method was applied for determination of phenolic acids at different wavelengths (265, 280, 320, and 365 nm, respectively, at 1 ml min-1 flow). the concentration of phenolic acids varied a lot depending on weather conditions, geographic sites, the way of wine storage and aging. these discoveries agree also with other studies (özkan et al., 2006). the aim of our work was to develop the analytical method to separate and determine two target components in wine samples, namely p-coumaric and ferulic acid. attention was paid to the most appropriate sample pre-treatment technique. 2. materials and methods all reagents and solvents used were of analytical grade. milli-q water was used. methanol (meoh) was purchased from sigma-aldrich (germany), acetic acid (ch3cooh), ethyl-acetate (c4h8o2), 98% trans ferulic acid (fa), 98 % trans pcoumaric acid (p-ca) and 97% ocoumaric acid (o-ca) were supplied from merck (germany). 2.1 preparation of calibration solutions and calibration curves standard stock solution of trans fa, trans p-ca and o-ca was prepared by dissolving each of them with meoh in 10 ml volumetric glass flasks to obtain concentration of 1 g l-1. o-ca was used as an internal standard (istd). this solution was degassed for 20 minutes by sonication. five working calibration solutions were prepared from standard stock solutions by further dilution with mobile phase consisting of 2% acetic acid in water-methanol mixture (82:18, v/v). concentration ranges of the final standard solutions used for calibration curves were 0.1-1 mg l-1 and 1-10 mg l-1. calibration solutions were injected into the hplc-uv system in five replicates. in addition, three replicate analyses of the calibration solutions were performed. curves were constructed by linear regression of the peak-area ratio (y) of individual acids to the istd, versus the concentration (x). all standard solutions were kept at -18 ° c for a maximum one week. prior to injection the solutions were filtered through 0.45 μm filter. 2.2 sample preparation wine samples were refošk (red wine) and laški risling (white wine) from slovenia. wine samples undergo a liquid-liquid extraction prior to their analysis by hplc. 5 ml of wine sample was transferred into a 50-ml round-bottomed glassstoppered centrifuge tube, spiked with 50 µl of istd (0.5 mg l-1) and extracted with 5 ml of ethyl acetate by sonication (ultrasonic bath model sonis 4, iskra, slovenia) for nova biotechnologica 9-2 (2009) 115 20 minutes. the extract was centrifuged (centrifuge model lc 321, tehnica železniki, slovenia) at 1000 rpm for 10 min and the supernatant was accurately transferred into 200-ml conical glass flask using a glass pasteur pipette. this extraction procedure was repeated three times. supernatants were combined and concentrated to dryness by rotary evaporation (büchi rotavapor r-205, switzerland). the residue was redissolved in 5 ml of mobile phase consisting of 2% acetic acid in water-methanol mixture (82:18, v/v). finally, the pre-treated sample was filtered through 0.45 µm filter and analysed. the compounds in wine extracts were quantified from the corresponding calibration curves. 2.3 stability tests trans p-ca and trans fa standard compounds were dissolved in meoh separately, to obtain concentrations of 1 mg l-1. the contents of trans p-ca and trans fa were determined from the corresponding calibration curves and they were used for the control. then each solution was divided into more equal parts and transferred into graduated glass-stoppered test tubes. four parts were placed under the daylight and room temperature for varied lengths of time (5 h, 24 h, 48 h and 1 week); another four parts of each solution were stored in the refrigerator in the darkness at -18 °c for varied lengths of time (5 h, 24 h, 48 h and 1 week). after exposure to the mentioned conditions, the content of trans p-ca and trans fa were determined from the corresponding calibration curves and compared to the control. equipment and chromatographic analyses a simple and rapid chromatographic separation was carried out using reversephase hplc. chromatographic analyses were performed on varian, prostar (usa) hplc system, which consisted of varian prostar 210 hplc pump, varian prostar injection valve, and uv-vis detector. chromatographic system was connected to the pc. separation was carried out on 250 mm x 3mm i.d., 5 µm non-polar chromspher 5, c18 column. elution was performed isocratically with the mobile phase consisting of 2% acetic acid in water-methanol mixture (82:18, v/v). the flow rate was 0.7 ml min-1, the pressure 180×105 pa and the injection volume was 100 µl. peaks were detected at 320 nm. 3. results and discussion based on linear regression analysis, the responses for investigated compounds were linear over concentration ranges tested. the equations of calibration curves and their correlation coefficients are presented in table 1. the reproducibility of chromatographic analyses was evaluated by the relative standard deviation (rsd) of five replicate analyses of five calibration solutions. rsd was between 2.91 and 3.81 %. the extraction procedure was optimized regarding extraction solvent and recovery. ethyl-acetate was used for extraction of the compounds from wine. the best recoveries (over 89%) were obtained by using three equal volumes of mentioned solvent, while using only two equal volumes gave lower recoveries. the recovery was evaluated using o–ca which was added into the wine sample before the extraction. 116 brodnjak vončina, d. et al. this compound is structurally related to the investigated compounds and behaves similar on the column as analytes. it does not occur in wine. therefore it is suitable for quantitative analysis of investigated compounds. table 1. regression equations and correlation coefficients for investigated compounds investigated compound correlation coefficient (r2) regression equation trans p–ca 0.9969 y = 0.4088 x + 0.1021 trans fa 0.9968 y = 0.3403 x + 0.0705 chromatographic separation was optimized with respect to the mobile phase, eluent composition, and flow rate. several experiments were carried out to resolve the acid mixture using a different mobile phases. to increase the retention behaviour acetic acid was used. optimal peak resolution was achieved using 2% acetic acid in water-methanol mixture (82:18, v/v). in fig. 1 chromatograms of investigated compounds, present red wine extract is presented. the separation with good resolution has been achieved for phenolic acids within 32 min. their average contents in different wine extracts (mg l-1) are presented in table 2. concentrations of trans p-ca and trans fa are higher in red wine. very high concentration of trans fa was determined in refošk, which was even 100-times higher compared with white wine. contents of phenolic acids are comparable to those reported in literature (robbins and bean, 2004; kallithraka et al., 2006; garciá et al., 2007; sladkovský et al., 2004; ozkan et al., 2006; castellari et al., 2002). trans p-ca in different brands of red wine were reported in concentrations between 0.9 to 7 mg l-1 and trans fa from 0.14 to 6 mg l-1l, respectively. concerning white wine brands lower concentrations of both acids were fig. 1. chromatogram of investigated compounds present in extract of red wine-refošk. nova biotechnologica 9-2 (2009) 117 found in literature: trans p-ca varied from 0.2 to 1.4 mg l-1and trans fa from 0.1 to 0.7 mg l-1, respectively. table 2. contents of trans p–ca and trans fa in extracts of red wine-refošk and white wine-laški rizling (mg l-1). each content value is the mean of five replications. compound red wine refošk (n=5) white wine laški rizling (n=5) trans p–ca 2.95 1.82 trans fa 7.75 0.07 the results of stability tests showed that trans p–ca and trans fa gradually converted to their cis forms when they were dissolved in meoh and stored in daylight at room temperature. the trans-cis isomerization started already after a few hours. the content of trans p–ca and trans fa has lowered during the time of storing, while the content of cis p–ca and cis fa has increased. after being stored for 1 week in the refrigerator in darkness at -18 °c, trans p–ca and trans fa dissolved in meoh were found to be stable, and no isomer conversion occurred. although cis and trans forms of phenolic acids are geometric isomers with very similar structures, they can be well distinguished by order of elution during hplc (cis isomers are retained longer than trans isomers) (fig. 2) fig. 2. chromatogram of compounds present in mixture of trans p–ca and trans fa in meoh after being stored in daylight and room temperature for 24 hours. 4. conclusion the developed isocratic hplc method is a rapid and simple one compared with other hplc methods. the selection of proper mobile phase is crucial for good separation of phenolic compounds. proper storing conditions are important to ensure 118 brodnjak vončina, d. et al. stability of the compounds. the described method can be routinely used for separation and determination of closely related cisand trans-isomers of phenolic acids in different wine extracts and other liquid samples with complex natural matrices, even if they are present in the minor contents. references buiarelli, f., cartoni, g., coccioli, f., levetsovitou, z.: determination of phenolic acids in wine by hplc with a microbore column. j. chromatogr., a, 695, 1995, 229-235. castellari, m., sartini, e., fabiani, a., arfelli, g., amati, a.: analysis of wine phenolics by high-performance liquid chromatography using a monolithic type column. j. chromatogr., 973, 2002, 221-227. chamkha, m., cathala, b., cheynier, v., douillard, r.: phenolic composition of champagnes from chardonnay and pinot noir vintages. j. agric. food chem., 51, 2003, 3179-3184. garcía, falcón, m.s., pérez, lamela, c., martínez, carballo, e., simal, gándara, j.: determination of phenolic compounds in wines: influence of bottle storage of young red wines on their evolution. food chem., 105, 2007, 248-259. kallithraka, s., tsoutsouras, e., tzourou, e., lanaridis, p.: principal phenolic compounds in greek red wines. food chem., 99, 2006, 784793. özkan, g., göktürk, baydar, n.: a direct rp-hplc determination of phenolic compounds in turkish red wines. j. fac. agriculture akdeníz university, 19, 2006, 229-234. robbins, r. j., bean, s. r.: development of a quantitative high-performance liquid chromatography-photodiode array detection measurement system for phenolic acids. j. chromatogr., a, 1038, 2004, 97-105. sladkovský, r., solich, p., urbánek, m.: high-performance liquid chromatography determination of phenolic components in wine using off-line isotachophoretic pre-treatment. j. chromatogr., 1040, 2004, 179-184. tarnawski, m., depta, k., grejciun, d., szelepin, b.: hplc determination of phenolic acids and antioxidant activity in concentrated peat extract-a natural immunomodulator. j. pharm. biomed. anal., 41, 2006, 182-188. urakova i. n., pozharitskaya, o. n., shikov, a. n., kosman, v. m.: development and validation of an lc method for simultaneous determination of ascorbic acid and tree phenolic acids in sustained release tablets at single wavelength. j. chromatogr., 67, 2008, 709-713. yi, c., shi, j., kramer, j., xue, s., jiang, y., zhang, m., ma, y., pohorly, j.: fatty acid composition and phenolic antioxidants of winemaking pomace powder. food chem., 114, 2009, 570-576. zgórka, g., kawka, s.: application of conventional uv, photodiode array (pda) and fluorescence (fl) detection to analysis of phenolic acids in plant material and pharmaceutical preparation. j. pharm. biomed. anal., 24, 2001, 10651072. microsoft word miklovic nbc 12-2.doc nova biotechnologica et chimica 12-2 (2013) 75 doi 10.2478/nbec-2013-0009 © university of ss. cyril and methodius in trnava synthesis, crystal structure and spectral properties of copper(ii) and cobalt(ii) 3-methylthiophene-2carboxylato complexes with furopyridines jozef miklovič1, miroslava bašnárová1, peter segľa2, marián koman2 1department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, j. herdu 2, trnava, sk-917 01, slovak republic (jozef.miklovic@ucm.sk) 2institute of inorganic chemistry, faculty of chemical and food technology, slovak university of technology in bratislava, radlinskeho 9, bratislava, sk-812 37, slovak republic abstract: the synthesis and characterization of eleven new cu(ii) and co(ii) complexes is reported. the complexes were characterized by elemental analyses, infrared and electronic spectra. copper(ii) with 3-methylthiophene-2-carboxylic acid (hmtk) forms a dinuclear complex of the acetate type [cu2(mtk)4(h2o)2]. by reaction of this complex with 2-metylfuro[3,2-c]pyridine (mefp), not only acetate type complexes [cu2(mtk)4l2] (l= fp, mefp) were obtained, but also monomeric complex [cu(mtk)2(fp)2]. in the cases of [1]benzofuro[3,2-c]pyridine (bfp) and 2-(3-trifluoromethylphenyl)furo[3,2-c]pyridine (cf3fp) only monomeric complexes [cu(mtk)2l2] (l = bfp, cf3fp) were obtained. it is possible to observe, that with increasing amount of the ligand, the yield of monomeric complexes increases too. in monomeric complexes, the carboxylic group of anionic mtk binds to atom cu(ii) by asymmetrically chelating o,o-coordination. the crystal structure of the complex [cu(mtk)2(mefp)2] was determined by x-ray single crystal structure analysis. the copper(ii) atom lies in the crystallographic centre of symmetry in an distorted tetragonal-bipyramidal arrangement. the structure of this complex confirms an asymmetric chelate coordination of the carboxylic group. hmtk and cobalt(ii) form coordination compound [co(h2o)6](mtk)2 with assumed ionic mode of coordination of anionic mtk. with furopyridines monomeric complexes [co(mtk)2l2] (l= fp, mefp, bfp, cf3fp) with distorted octahedral coordination polyhedron around co(ii), were formed. key words: complex, copper(ii), cobalt(ii), furopyridine, carboxylate 1. introduction derivatives of thiophene-2-carboxylic acid alone and their coordination compounds are interesting from biological as well as structural point of view (panagoulis, et al., 2007). the thiophene-2-carboxylic acid is an hypocalcemic agent and is effective in lowering the level of serum-glucose and fatty acids (ribeiro and santos, 2008). in this paper, we describe synthesis, spectral properties and crystal structure of 3-methylthiophnenecarboxylatecopper(ii) and cobalt(ii) complexes and their adducts with n-heterocyclic ligands: [cu2(mtk)4l2] (l=h2o, fp, mefp); [cu(mtk)2l2] (l=fp, mefp, bfp, cf3fp); [co(h2o)6](mtk)2, [co(mtk2)l2] (l=fp, mefp, bfp, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:02 utc 76 miklovič, j. et al. cf3fp). the crystal and molecular structure of one of the complexes under study [cu(mtk)2(mefp)2] has also been studied by x-ray structure analyses. topological structures and abbreviations of heterocyclic ligands under study are given in fig. 1. s ch 3 co 2 o n o n h3c o n o n f 3c 3-m ethyltiophene-2-carboxylate mtk furo[3,2-c]pyridine fp 2-m etylfuro[3,2-c]pyridine mefp 2-(3-trifluorom ethylphenyl)furo[3,2-c]pyrid in e cf3fp [1]benzofuro[3,2-c]pyridine bfp fig. 1. structures and abbreviations of heterocyclic ligands. 2. material and methods 2.1 chemical reagents, analysis and physical measurements all used chemicals were of reagent grade and used without further purifications. derivatives of furopyridine (fp, mefp, bfp and cf3fp) have been prepared using eloy-derickere procedure (eloy and deryckere, 1971). cobalt and copper were determined by chelatometry after mineralization of the complexes. carbon, hydrogen, nitrogen and sulfur were determined by microanalytical methods (thermo electron flash ea 1112). analytical data for the complexes are given in table 1. electronic spectra of the powdered samples were recorded on a specord 200. ir spectra were recorded on a nicolet 5700 ft-ir spectrometer (thermo scientific). 2.2 crystallography data collection for [cu(mtk)2(mefp)2] (complex 4) was obtained using siemens p4 diffractometer (siemens, xemp. version 4.2, 1990) with graphite monochromated mokα , radiation at 293 k. the diffraction intensities were corrected for lorenz and polarization factors. the structures were solved by the heavy atom method with shelxs-86 (g. m. sheldrick, 1985), subsequent fourier synthesis using shelxl-97 (g. m. sheldrick, 1997) and refined by the fullmatrix least-square method on all f2 data using the program shelxl-97 (g. m. sheldrick, 1997). geometrical analysis was performed using shelxl-97 [10]. crystal data and conditions of data collection and refinement are reported in table 2. 2.3 preparation of the complexes [cu2(mtk)4(h2o)2] (1): naoh (0.6 g; 9 mmol) was dissolved in water (10 cm 3) and hmtk was added (1.28 g; 9 mmol). ph was adjusted to 7. solution of cuso4 (1.12 g; bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:02 utc nova biotechnologica et chimica 12-2 (2013) 77 4.5 mmol) in water (5 cm3) was then added. the mixture was stirred for 1 h and precipitate was filtered off. the precipitate was washed with water and dried at room temperature. yield: 0.9 g. table 1. cu(ii) a co(ii) analytical data for the complexes. compounds empirical formula formula weight calcd / found (%) (g mol-1) m c h n s [cu2(mtk)4(h2o)2] 1 c12h12cuo5s2 363.9 17.46 17.98 39.61 39.65 3.32 3.23 17.62 17.78 [cu2(mtk)4(fp)2] 2 c19h15cuno5s2 465.00 13.66 14.15 49.08 48.60 3.25 3.12 3.01 2.67 13.79 13.44 [cu2(mtk)4(mefp)2] 3 c20h17cuno5s2 479.03 13.26 13.74 50.15 49.87 3.57 3.48 2.92 2.70 13.39 13.24 [cu(mtk)2(mefp)2] 4 c28h24cun2o6s2 612.18 10.38 10.84 54.93 54.44 3.95 3.91 4.57 4.34 10.47 10.05 [cu(mtk)2(bfp)2] 5 c34h24cun2o6s2 684.24 9.28 9.66 59.68 59.65 3.53 3.51 4.09 4.08 9.37 8.87 [cu(mtk)2(cf3fp)2] 6 c40h26cuf6n2o6s2 872.32 7.28 7.65 55.07 54.72 3.00 2.86 3.21 3.11 7.35 6.92 [co(h2o)6](mtk)2 7 c12h22coo10s2 449.36 13.11 12.63 32.07 31.99 4.93 4.43 14.27 14.53 [co(mtk)2(fp)2] 8 c26h20con2o6s2 579.51 10.17 9.88 53.89 54.38 3.48 3.80 4.83 4.67 11.06 10.59 [co(mtk)2(mefp)2] 9 c28h24con2o6s2 607.57 9.70 9.36 55.35 54.87 3.98 4.08 4.61 4.51 10.55 10.56 [co(mtk)2(bfp)2] 10 c34h24con2o6s2 679.63 8.67 8.50 60.09 60.58 3.56 3.70 4.12 4.46 9.43 8.84 [co(mtk)2(cf3fp)2] 11 c40h26cof6n2o6s2 867.71 6.79 7.12 55.37 54.93 3.02 3.12 3.23 3.35 7.39 6.98 [cu2(mtk)4(fp)2] (2); [cu2(mtk)4(mefp)2] (3); [cu(mtk)2(mefp)2] (4): complex 1 (0.382 mg; 0.525mmol) was dissolved in methanol (9 cm3) and ligand (fp, mefp; 5 mmol) in methanol (3 cm3) was added. solution was then heated to 50°c and stirred for 1 h. the precipitate formed during the reaction was filtered off, washed with methanol and dried at r. t. blue filtrate evaporated at r. t. and crystals were formed and separated. yield: complex 2 0.5 g; complex 3 0.4 g; complex 4 0.2 g. [cu(mtk)2(bfp)2] (5); [cu(mtk)2(cf3fp)2] (6): complex 1 (0.382 mg; 0.525mmol) was dissolved in methanol (9 cm3) and ligand (bfp, cf3fp, 5 mmol) in methanol (9 cm3) was added. solution was then heated to 50°c and stirred for 1 h. the precipitate formed during the reaction was filtered off, washed with methanol and dried at r. t. yield: complex 5 1.5 g; complex 6 0,7 g. [co(h2o)6](mtk)2 (7): naoh (0.14 g; 3.5 mmol) was dissolved in water (10 cm 3) and hmtk was added (0.5 g; 3.5 mmol). ph was adjust to 7. solution of coso4 (0.49 g; 1.75 mmol) in water (5 cm3) was then added. mixture then evaporated at r. t. and subsequent crystals were separated. yield: 1.1 g. [co(mtk)2(fp)2] (8); [co(mtk)2(bfp)2] (10); [co(mtk)2(cf3fp)2] (11); the complex 7 (0.449 g; 1 mmol) was dissolved in methanol (5 cm3) and ligand (fp, bfp, cf3fp; 5 mmol) in methanol (3 cm 3) was added. solution was then heated to 50°c and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:02 utc 78 miklovič, j. et al. stirred for 1 h. mixture then evaporated at r. t. and subsequent crystals were separated. yield: complex 7 2.1 g; complex 10 2.1 g; complex 11 0.9 g. [co(mtk)2(mefp)2] (9): the complex 7 (0.449 g; 1 mmol) was dissolved in dmf (15 cm3) and ligand (mefp; 5 mmol) in dmf (5 cm3) was added and stirred for 1 h. mixture then evaporated at r. t. yielded crystals were separated and dried at r. t. yield: 0.4 g. table 2. crystal data and structure refinement for [cu(mtk)2(mefp)2] (complex 4). identification code mk75 empirical formula c28h24cun2o6s2 formula weight 612.19 temperature 293(2) k wavelength 0.71073 å crystal system, space group triclinic, p-1 unit cell dimensions a = 6.352(1) å α = 82.53(1)o b = 10.489(1) å β = 80.95(1)o c = 10.501(1) å γ = 87.09(1)o volume 684.74(14) å3 z, calculated density 1, 1.485 mg/m3 absorption coefficient 0.995 mm-1 f(000) 315 crystal size 0.12 x 0.4 x 0.5 mm θ range for data collection 4.17 to 26.35o. limiting indices -1<=h<=1, -13<=k<=13, -13<=l<=13 reflections collected / unique 1384 / 692 [r(int) = 0.0754] completeness to θ = 26.35 24.8 % absorption correction none refinement method full-matrix least-squares on f2 data / restraints / parameters 692 / 0 / 179 goodness-of-fit on f2 0.901 final r indices [i > 2σ(i)] r1 = 0.0383, wr2 = 0.0886 r indices (all data) r1 = 0.0491, wr2 = 0.0957 extinction coefficient 0.000(5) largest diff. peak and hole 0.165 and -0.128 e. å -3 3. results and discussion 3.1 description of crystal structure of [cu(mtk)2(mefp)2] the structure of [cu(mtk)2(mefp)2] (complex 4) is shown in fig. 2. the copper(ii) atom is bonded in trans square-planar arrangement to two nitrogen atoms of two methylfuropyridine molecules [cu – n1 = 2.050 å] and one carboxylate oxygen atom from each of two 3-methyl-2-thiophene carboxylate anions [cu – o1 = 1.980 å]. the remaining two carboxylate oxygen atoms of [cu(mtk)2(mefp)2] which are bonded to the copper [cu – o2 = 2.560 å] bonds, lie at 57.43o from the plane of cuo2n2 and create tetragonal-bipyramidal coordination. the copper(ii) atom lies in the crystallographic center of symmetry. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:02 utc nova biotechnologica et chimica 12-2 (2013) 79 fig. 2. the molecular structure of [cu(mtk)2(mefp)2] (complex 4). 3.2 ir an electronic data all the typical features of ir spectra are clearly compatible with the structural characteristics of the complexes under study. some characteristic ir bands of the sodium salt namtk.h2o as well as of cu(ii) complexes are given in table 3. the ir spectrum of complex 1 shows absorption bands in the region from 3200 to 3500 cm-1. these bands correspond to the antisymmetric and symmetric oh stretch and confirm the presence of water. the difference between the antisymmetric stretch and symmetric stretch (δ) gives information on carboxylic bonding mode for the complexes after comparison with δ values of compounds with ionic carboxylic groups (nakamoto, 1997). the δ values for the complexes 1 (152 – 211 cm-1), 2 (175 – 234 cm-1) and 3 (181 – 237 cm−1) are comparable with δ value for sodium salt (156 – 211 cm-1) and suggested bridging mode of coordination carboxyl group. the considerable splitting of bands assigned to νs(coo −) for complexes 1, 2 and 3, confirms that the mtk anion as a ligand is most probably coordinated via a bridging carboxyl group (nakamoto, 1997). in these compounds, dimeric structure of acetate type known in other cu(ii) heterocyclic carboxylate (jašková, et al., 2007) is predicted. the δ values for the monomeric complexes 4 (188 – 261 cm-1), 5 (174 – 247 cm-1) and 6 (178 cm-1 – 261 cm-1) are greater than for namtk·h2o (156 – 211 cm -1). that is rather typical for asymmetrically chelating carboxylate groups. however, in these cases, δ values are comparable to those of unidentate complexes (nakamoto, 1997). the suggested asymmetrically chelating o,o’-coordination of carboxylate groups for complexes under study is in agreement with the structure determined by xray analysis for complex 4. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:02 utc 80 miklovič, j. et al. table 3. spectroscopic dataa (in cm-1) of cu(ii) complexes. a vs – very strong; s – strong; m – medium; br – broad; sh – shoulder, bδ = νas(coo-) νs(coo-). the solid state electronic spectra of complexes 1 – 3 show a broad absorption band (band i) in the visible region with a maximum from 13 450 cm−1 to 14 400 cm−1 (table 3), which is assigned to a dxy,yz → dx 2 −y 2 transition (kato, 1988). moreover, the spectrum of complexes 1 – 3 displays a shoulder at about 28 500 cm−1 (band ii). band ii has been assigned to a charge transfer absorption and is believed to be indicative of a dimeric complex, finally, complexes 1 – 3 displays bands i and ii in the usual range for cu(ii) compounds in a square-pyramidal cuo4o’ (complex 1) or cuo4n environment. the solid state electronic spectra of all other copper(ii) complexes under study exhibit a asymmetrical broad ligand field band with a maximum at about 17 500 cm−1. this type of d–d spectra for complexes 4 – 6 is typical for tetragonally distorted octahedral copper(ii) complexes (lever, 1984). some characteristic ir bands of the complexes co(ii) are given in table 4. the ir spectrum for complex 7 shows absorption bands in the region from 3200 to 3500 cm-1 and confirms the presence of water. these bands are not present in other co(ii) complexes and confirm the absence of water. the δ values for the complex 7 (151– 214 cm-1) are comparable with δ values of namtk·h2o (156 – 211 cm -1) which suggests ionic mode of coordination of carboxylate group with hexaaquacobalt(ii) cation and non-coordinated mtk anion. this manner of coordination is known (zhang and ng, 2005). on the other hand, the δ values from 169 to 241 cm-1 for complexes 8 – 11, greater than for namtk·h2o (156 – 211 cm -1) are typical for an asymmetric chelating coordination of the carboxyl group of mtk anion. infrared data carboxyl group electronic data compound νas(coo-) νs(coo-) δb band i band ii namtk.h2o 1573vs,br 1417vs 1364vs,br 156 209 [cu2(mtk)4(h2o)2] 1 1574vs 1422vs 1373vs 1363vs 152 211 14 400 28 600sh [cu2(mtk)4(fp)2] 2 1597vs 1422vs 1375vs 1363vs 175 234 13 700 28 000sh [cu2(mtk)4(mefp)2] 3 1600vs,br 1419vs 1375vs 1363vs 181 237 13 450 29 000sh [cu(mtk)2(mefp)2] 4 1603s 1415vs,br 1342vs,br 188 261 17 500 13 500sh [cu(mtk)2(bfp)2] 5 1592vs 1418vs 1379vs 1345vs 174 247 17 400 13 900sh [cu(mtk)2(cf3fp)2] 6 1601vs 1423vs 1380vs 1340vs 178 261 17500 13 800sh bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:02 utc nova biotechnologica et chimica 12-2 (2013) 81 table 4. spectroscopic dataa (in cm-1) of co(ii) complexes. infrared data carboxyl group electronic data compound νas(coo-) νs(coo-) δb 4t1g(f)→4t1g(p) 4t1g(f)→4t2g namtk·h2o 1573vs,br 1417vs 1364vs,br 156 209 [co(h2o)6](mtk)2 7 1566vs 1415vs 1352s,br 151 214 20 000 21 300 sh 8 600 [co(mtk)2(fp)2] 8 1589s 1420vs 1359vs 169 230 20 900sh 20 100 19 800sh 9400 [co(mtk)2(mefp)2] 9 1605vs 1420vs 1362vs 185 243 20 900sh 20 100 9300 [co(mtk)2(bfp)2] 10 1593vs 1421vs 1358vs 172 235 20 950 20 100sh 9 500 [co(mtk)2(cf3fp)2] 11 1591 1422vs 1350 169 241 21 100 9 300 a vs – very strong; s – strong; m – medium; br – broad; sh – shoulder, bδ = νas(coo-) νs(coo-). the electronic spectra (table 4) of all the cobalt complexes are clearly consistent with tetragonally distorted octahedral structure. the electronic spectra consist of a band ν1 assigned to the lowest energy transition 4t1g(f)→4t2g in the near infrared and the band ν3 in the visible near 20,000 cm -1, which is assigned to the 4t1g(f)→4t1g(p) transition. acknowledgements: this work was financially supported by the grants apvv-0014-11, vega 1/0233/12, vega 1/0472/13 of the slovak grant agency for science. this article was created with the support of the ministry of education, science, research and sport of the slovak republic within the research and development operational programme for the project "university science park of stu bratislava", itms 26240220084, co-funded by the european regional development fund. references eloy, f., deryckere, a.: sur la synthése des furo[3,2-c]pyridines. j. heterocycl. chem. 8, 1971, 57-60. g. m. sheldrick: shelxs-86, oxford university press, oxford, 1985, 175. g. m. sheldrick: shelxl-97, university of göttingen, germany, 1997. jašková, j., mikloš, d., korabik, m., jorík, v., segľa, p., kaliňáková, b., hudecová, d., švorec, j., fischer, a., mrozinsky, j., lis, t., melník, m.: synthesis, spectral and magnetic properties and crystal structures of copper(ii) pyridinecarboxylates as potential antimicrobial agents. inorg. chim. acta 360, 2007, 2711-2720. kato, m., muto, y.: factors affecting the magnetic properties of dimeric copper(ii) complexes. coord. chem. rev. 92 1988 45-83. lever, a,b.p.: inorganic electronic spectroscopy, 2nd ed., elsevier, amsterdam, 1984, p. 480. nakamoto, k.: infrared and raman spectra of inorg. and coord. compounds, part b, 5th edition, new york, 1997, 384-387. panagoulis, d., pontiki, e., skeva, e., raptopoulou, c., girousi, s., hadjipavlou-litina, d., dendrinou-samara, c.: synthesis and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:02 utc 82 miklovič, j. et al. pharmacochemical study of new cu(ii) complexes with thiophen-2-yl and α,βunsaturated substituted carboxylic acids. j. inorg. biochem. 101, 2007, 623-634. ribeiro da silva, m. a. v., santos, a. f. l. o. m.: thermochemical properties of three 2-thiophene carboxylic acid derivatives. j. chem. therm. 40, 2008, 14511457. siemens, xemp. version 4.2 analytical x-ray instruments inc., madison, wisconsin, usa (1990). zhang, x.l., ng, s. w.: hexaaquacobalt(ii) bis(6-hydroxypyridine-3-carboxylate). acta cryst. e61, 2005, m1140-m1141. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:02 utc microsoft word kodba nb 9-3.doc nova biotechnologica 9-3 (2009) 249 validation of an improved european standard method for the determination of pcbs in oil samples zdenka cencič kodba1, darinka brodnjak vončina2 1institute of public health maribor, environmental protection institute, prvomajska 1, 2000 maribor, slovenia (zdenka.kodba@zzv-mb.si) 2faculty of chemistry and chemical engineering, university of maribor, smetanova 17, 2000 maribor, slovenia (darinka.brodnjak@uni-mb.si) abstract: two european standard methods (en 12766 and en 61619) are currently used for the determination of pcbs in specific oil matrix. however, apolar matrix compounds (e.g. hydrocarbons) elute from the adsorbent together with the pcbs and are injected into the analytical system where their presence contaminates the inlet, detectors and columns; and decreases system performances. insufficient cleanup causes delay of elution of pcbs from gc columns. by using new sulfoxide-bonded silica, pcbs are better separated from aliphatic hydrocarbons because the specificity of the stationary phase for these compounds is much higher that that used in both standard methods. a gas chromatograph at6890 with two capillary columns of different polarities (hp-5ms and db-1701) coupled to two μecds is used. oven temperature program is as followed: 90°c (1 min), 70°c/min to 180°c, 5°c/min to 230°c (0.1 min), 1.5°c/min to 280°c. run time is 46 min. the procedure was validated through regular analysis of blanks, fortified samples (transformer oil, motor used and unused oil) and certified materials (bcr-449 and bcr-420, waste mineral oils, high and low pcb levels). two internal standards were used (pcb 30 and pcb 209). an average recovery ± rsd of 82.8 ± 5.4 % was achieved for all six pcbs in different matrices. the loq per single pcb congener is 0.2 mg kg-1. the average recovery ± rsd for the bcr-420 is 92.0 ± 4.6 % and for the bcr-449 is 105 ± 2.5 % for all certified pcbs in waste oils. key words: pcbs, sulfoxide-bonded silica, validation, oil samples 1. introduction the election of standards to quantify pcbs is based on a number of factors among which the type of sample, the availability of reference compounds and the aim of the determination are included. two european standard methods (en 12766 and en 61619) are currently used for the determination of pcbs in specific oil matrix. the first one is applicable to unused, used and treated petroleum products including synthetic lubricating oils and the second one is applicable to unused, reclaimed or used insulating liquids where it is specifically emphasized that the presence of mineral oil may reduce the sensitivity of the ecd. in addition to the official methods (where a combination of acidified silica/anion exchange (sioh-h2so4/sa) and silica adsorbent is proposed) numerous sample preparation techniques are available for pcb analysis in oil samples ranging from extraction with aprotic solvents (e.g. dimethylsulfoxide, dimethylformamide) (larsen et.al., 1991; lawn and toffel, 1987), enzyme immunoassay (lambert et.al., 1997), hs spme (criado et.al., 2004), sulphuric 250 kodba, z.c. and brodnjak vončina, d. acid and alkali treatment (shin and kim, 2006; shu et.al., 1995), gel permeation chromatography (sheridan et.al., 1995), normal-phase (florisil, alumina) (lulek, 1998; nerin and domeno, 1999; storr-hansen et.al., 1992), reversed-phase chromatography and many surface modified silica for spe may be in use (nh2, nh2cl, nh2-so, scx-2, scx-2-nh4, sax, sax-so4, cn, diol and psa) for the separation of pcbs from mineral oils (takada et.al. 2001; na et.al., 2008; numata et.al., 2006; numata et.al., 2007; numata numata et.al., 2008). pcbs are not easily separated from oil matrices because their physical and chemical characteristics are very similar. the remaining oil during the analytical procedure was not an immediate analytical problem, because when the sample fraction was analyzed by selective detectors like ecd, the co-extracted compounds are not directly detected. also, lower limits of quantification are required. that is why a new modification of silica was used for sample preparation. supelclean sulfoxide spe (psa-so) consists of a patent pending sulfoxide-bonded silica where pcbs are preferentially retained on the spe phase whereas sample interferences (e.g. long chain hydrocarbons) are eluted in the early fraction. the procedure was validated through regular analysis of blanks, fortified samples (transformer oil, motor used and unused oil) and certified materials (bcr-449 and bcr-420, waste mineral oils, high and low pcb levels). two internal standards were used (pcb 30 and pcb 209). the working range, limit of quantification, repeatability, reproducibility and accuracy were determined. 2. materials and methods a surrogate standard solution was prepared by dissolving six pcb congeners (pcb 28, pcb 52, pcb 101, pcb 138, pcb 153 and pcb 180) from dr. ehrenstorfer and two pcb congeners (pcb 30 and pcb 209) in hexane. reference materials bcr crm-420 and bcr crm-449 with a certified concentration of several pcbs were purchased from the institute for reference materials and measurements (geel, belgium). j.t.baker supplied acetone and hexane. individual solutions of internal standards were prepared in acetone followed by dilution with hexane. three different matrices of oil samples were taken for analysis. waste oils were classified according to their origin into transformer oil, motor used and unused oil. 2.1 sample preparation the following spe method is based on a modified supelco procedure. spiked and non-spiked oil samples (0.25 g) were dried with anhydrous sodium sulphate and diluted to 10 ml with hexane. samples were homogenized and placed into the spe station. supelclean sulfoxide pp spe (3g/6ml) from supelco and sioh pp spe (500mg/3ml) from chromabond were used for the sample cleanup. the schematic spe process is shown in table 1. a gas chromatograph at6890 with two capillary columns of different polarities (hp-5ms and db-1701) coupled to two μecds was used. nova biotechnologica 9-3 (2009) 251 oven temperature program was 90°c (1 min), 70°c/min to 180°c, 5°c/min to 230°c (0.1 min), 1.5°c/min to 280°c. four calibration standards were prepared to cover the optimum range with the lowest calibration level of 0.2 mg kg-1 per single pcb congener. table 1. sample cleanup preparation by spe. i. supelclean sulfoxide spe (3g/6ml) 1. condition and equilibration the spe with 10 ml of acetone and 20 ml of hexane 2. add 0.5 ml of pre-treated sample in hexane 3. wash and remove the oil fraction with 0.5 ml and 4.5 ml hexane 4. elute the analytes with 13 ml hexane collect and concentrate an eluent to approximately 0.5 ml ii. sioh spe (500mg/3ml) 1. condition and equilibration the spe with one volume of acetone and one volume of hexane 2. add 0.5 ml of pcb fraction 3. wash and elute the pcb fraction with 2 x 1 ml and one volume of hexane collect and concentrate to 0.2 ml 3. results and discussion as shown in the chromatogram (fig. 1) good peak shape and sufficient separation of the critical pairs was obtained (resolution was 0.5 or greater). the working range of individual pcb congener was from 0.2 mg kg-1 to 100 mg kg-1. the accuracy was also very good. repeatability was less than 10% for all spiked samples, for all congeners over the whole calibration range with some exceptions for the transformer oil. fig. 1. gc-ecd-ecd chromatograms of the bcr standard, waste mineral oil. an average recovery ± rsd of 82.8 ± 5.4 % was achieved for all six pcbs in different matrices (fig. 2). the average recovery ± rsd for the bcr-420 was 92.0 ± 4.6 % and for the bcr449 105 ± 2.5 % for all certified pcbs in waste oils (fig. 3 and fig. 4). 252 kodba, z.c. and brodnjak vončina, d. fig. 2. results of the repeatability and recovery studies on fortified different matrices. fig. 3. results of the repeatability and recovery studies on the bcr-420. fig. 4. results of the repeatability and recovery studies on the bcr-449. 4. conclusions by using new sulfoxide-bonded silica, pcbs are better separated from aliphatic hydrocarbons because the specificity of the stationary phase for these compounds is much higher than that used in both standard methods. this previously developed sulfoxide-bonded silica and ammonium-salt bonded silica stationary phase (psa-so) together with the additional silica one, offer a simple, fast and efficient sample preparation for the extraction of pcbs from waste, transformer and mineral oil samples (especially paraffin-based), which can replace that mentioned in both european standards without any possible damage to the gc system. nova biotechnologica 9-3 (2009) 253 references criado, m.r., pereiro, i.r., torrijos, r.c.: selective determination of polychlorinated biphenyls in waste oils using liquid-liquid partition followed by headspace solid-phase microextraction and gas chromatography with atomic emission detection. j. chromatogr. a, 1056, 2004, 263-266. canals, a., forteza, r., cerda, v.: the routine determination of pcbs in waste automotive engine oils. chromatographia, 34, 1992, 35-40. lambert, n., fan, t.s., pilette, j.: analysis of pcbs in waste oil by enzyme immunoassay. sci. total environ., 196, 1997, 57-61. lulek, j.: levels of polychlorinated biphenyls in some waste motor and transformer oils from poland. chemosphere, 37, 1998, 2021-2030. larsen, b., tilio, r., kapila, s.: a dmso-based cleanup procedure for determination of pcbs in waste oil. chemosphere, 23, 1991, 8-10. lawn, r.e., toffel, s.a.: determination of polychlorinated biphenyls in waste oil by gas-liquid chromatography. analyst, 112, 1987, 53-56. nerin, c., domeno, c.: determination of polyaromatic hydrocarbons and some related compounds in industrial waste oils by gpc-hplc-uv. analyst, 124, 1999, 67-70. numata, m., aoyagi, y., tsuda, y., yarita, t., takatsu, a.: separation of polychlorinated biphenyls from mineral oil using alkylammonium ion-bonded silica stationary phases. anal. sci., 22, 2006, 785-788. numata, m., aoyagi, y., tsuda, y., yarita, t., takatsu, a.: preparation of sulfoxide residue bonded silica stationary phase for separation of polychlorinated biphenyls from mineral oils. anal. chem., 79, 2007, 9211-9217. numata, m., kaneko, t., mi, q., ye, m., kawamata, s., matsuo, m., yarita, t.: preparation of a sulfoxide group and ammonium-salt bonded silica stationary phase for separation of polychlorinated biphenyls from mineral oils. j. chromatogr. a, 1210, 2008, 68-75. na, y., kim, k., hong, j., seo, j.: determination of polychlorinated biphenyls in transformer oil using various adsorbent for solid phase extraction. chemosphere, 73, 2008, s7-s12. shin, s.k., kim, t.s.: levels of polychlorinated biphenyls (pcbs) in transformer oils from korea. j. hazard. mat., 137, 2006, 1514-1522. shu, y.y., dowdall, j.e., chiu, c., lao, r.c.: interference of transformer oil matrices to the internal standards on pcb quantification. intern. j. anal. chem., 60, 1995, 185-194. storr-hansen, e., cleemann, m., cederberg, t., jansson, b.: selective retention of non-ortho substituted coplanar chlorinated biphenyl congener on adsorbents for column chromatography. chemosphere, 24, 1992, 323-333. sheridan, b.r., poole, g., dawdall, e., chiu, c.: the effect of temperature on gpc for the separation of pcbs from transformer oil and subsequent analysis by gc-msd. int. j. environ. anal. chem., 60, 1995, 195-202. takada, m.i., toda, h., uchida, r.: a new rapid method for quantification of pcbs in transformer oil. chemosphere, 43, 2001, 455-459. microsoft word illková nb 9-3.doc nova biotechnologica 9-3 (2009) 225 characterization of preparation for fat separators kateřina illková, jiřina omelková, živan gojković, jana pavlačková faculty of chemistry, brno university of technology, purkyňova 118, 612 00 brno, czech republic, (xcillkova@fch.vutbr.cz;omelkova@fch.vutbr.cz; xcgojkovic@fch.vutbr.cz; xcpavlackova@fch.vutbr.cz) abstract: fats, oils and greases in waste water frequently cause serious environmental problems. a commercial preparation was tested for its ability to degrade oil and grease in wastewater. as substrates, two fats (lard and beef fat) and four oils (rape seed oil, sunflower oil, palm oil and olive oil) were chosen. the degradation ability of commercial preparation for different substrates was investigated by the determination of lipid degradation at ph 7, temperature 25 °c, aerobic condition and agitation at 160 rev/min for 14 days in erlenmeyer flasks. simultaneously, the lipolytic activity was spectrophotometrically determined at 420 nm. all tested substrates were degraded by the different rate to basic units (fatty acids). rape seed oil and lard were decomposed by bacterial lipase from commercial preparation. key words: fats, oils, waste water, lipid degradation, preparation, lipolytic activity 1. introduction a wide variety of industries (e.g. the dairy industry and food processing) produce the effluents rich in fats, oils and greases (fogs). high concentrations of fats, oils and greases in wastewater often initiate problems in wastewater treatment processes (becker et al., 1999; stoll and gupta, 1997). fogs often cause foul odours, the blockage of pipes and sewer lines. these problems are solved by the preliminary refining equipment, so-called grease traps. grease traps may sometimes fail to retain dissolved and emulsified fogs, allowing them to enter the water treatment system. an interesting strategy in present time is the use of lipase producing microorganisms in wastewater treatment system. lipases are hydrolytic enzymes that catalyze the cleavage of ester bonds in triglycerides, resulting in production of glycerol and free fatty acids. microbial lipases are produced by yeasts, fungi and bacteria, as extracellular and intracellular enzymes. the extracellular lipases from bacteria are interesting for their abilities to degrade fogs. in the present study, the degradation ability of commercial microbial product to degrade fogs under laboratory conditions was investigated. as substrates, four oils and two fats were used. 2. materials and methods the degradation ability of the commercial microbial products for grease separators was investigated. as substrates, four plant oils (palm oil, sunflower oil, olive oil and rape seed oil) and two animal fats (lard and beef fat) were used. 226 illková, k. et al. 2.1 commercial microbial product this commercial product is the mixture of bacteria, enzymes and detergents (illková et al., 2008). on the basis of previous study, it was found out that the product in the grease traps contain mixture of spore gram-positive bacteria, obviously bacillus sp. (gojković, 2009). 2.2 medium and growth conditions surface cultivation proceeded on spirit blue agar plates, to which tributyrin and tween 20 were added as a lipase substrate in ratio 1:1. incubation time was three days by 27 °c. this agar was used for the verification of the presence of spores of the lipolytic bacteria. medium for submerged cultivation contain per litre of distilled water 1.12 g k2hpo4; 0.48 g kh2po4; 5 g nacl; 0.1 g mgso4. 7h2o; 2 g (nh4)2so4 and 0.002 g edta. medium was autoclaved at 121 °c for 20 min. then the medium was supplemented with 1 ml plant oil and 1 g animal fats, as the natural substrate. medium was inoculated by commercial microbial product and was incubated under aerobic conditions at 25 °c, agitated at 160 rev/min for 14 days in erlenmeyer flasks in a shaker (el-bestawy et al., 2005). 2.3 verification of the presence of spores of lipolytic bacteria this verification was carried out on spirit blue agar plates. commercial product was diluted to the concentration 10-3, and then was filtered through bacteriological filter. filter was placed on the agar plates (gojković, 2009). a presence of spores of the lipolytic bacteria shows itself as a transparent halo around the colonies. 2.4 growth of the biomass growth of the biomass on various fats and oils substrates was examined spectrophotometrically at 650 nm. samples were taken away in definite time interval for four days and absorbencies were measured against blank (gojković, 2009). 2.5 enzyme assay the lipolytic activity was determined by using 0.5 ml culture in 0.65 ml 0.05 m phosphate buffer (ph 7.2) a 0.1 ml 0.0025 m p-nitrophenyllaurate in ethanol. the hydrolytic reaction was carried out at 37 °c for 30 min, after which 0.25 ml 0.1 m na2co3 was added. the mixture was centrifuged and the activity determined at 420 nm. one unit of lipase activity is defined as the amount of enzyme which liberates 1 µg p-nitrophenol from p-nitrophenyllaurate, as a substrate in 30 min under assay condition (nawani et al., 1998; sigurgísladóttir et. al., 1993). lipolytic activity was calculated on 1 mg of protein and expressed in the form of the specific activity. nova biotechnologica 9-3 (2009) 227 2.6 lipid degradation from each erlenmeyer flasks 20 ml culture medium was aseptically drawn and transferred to a separating funnel, where it was mixed with 20 ml of hexane. the mixture was agitated for 2 min and the upper layer was put into the clean and weighted beaker. the lower layer was re-extracted by a fresh 20 ml of hexane and the upper layer after the extraction was collected to the beaker again. the extract in the beaker was evaporated by heating at 100 °c. then the dry extracted lipids were weighted and dissolved in 50 ml of alcohol in the presence of phenolphthalein indicator. the solution was titrated with 0, 1 m koh until the developing of pink colour. the same procedure was repeated within 2, 6, 8, 10 and 14 days. the free fatty acids (%) in the sample, which indicates the lipid degradation and fatty acids utilization, was calculated according to this equation: m mcv acidsfattyfree kohkoh .1000 100... % =  here, vkoh is the volume of 0.1 m koh at the end point, ckoh actual concentration of the 0.1 m koh, m is the molecular weight of the oleic acid and m is the weight of the dry extract (illková et al., 2008; el-bestawy et al., 1998). 3. results and discussion the ability of commercial product to degrade various lipid substrates, occurring in wastewater, was tested. lipolytic activities, growth of the biomass, presence of spore and lipid degradation were investigated. 3.1 presence of spore for this test spirit blue agar was used. after three days of incubation it was found out that all bacteriological filters caught spore from commercial product. this effect was detected as a transparent halo around the colonies, fig. 1. fig. 1. appearance of colonies at the bacteriological filter. 3.2 growth of the biomass growth of the biomass was investigated by four plant oils (olive, palm and sunflower oil and rape seed oil) and two fats (lard and beef) during four days (fig. 2,3). 228 illková, k. et al. growth of the biomass for oil substrates 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 0 10 20 30 40 50 60 70 80 90 time (hours) to ta l s ol id (m g/ m l) sunflower oil olive oil palm oil rape seed oil fig. 2. growth of the biomass for various plant oil substrates. growth of the biomass for fat substrates 0 0,2 0,4 0,6 0,8 1 1,2 1,4 0 10 20 30 40 50 60 70 80 90 time (hours) to al s ol id (m g/ m l) lard beef fat fig. 3. growth of the biomass for various animal fat substrates. 3.3 lipolytic activity as substrate for lipase 0, 0025 m p-nitrophenyllaurate in ethanol was used, activity was measured spectrophotometrically at 420 nm. activity of lipases was determined as a dependence of absorbance on the time in the definite time interval at 37 °c. results for plant oils were expressed at mg of protein at table 1 and fig. 4 and for animal fats at table 2 and fig. 5. fig. 4. specific activity of lipases (mmol/min/mg protein). nova biotechnologica 9-3 (2009) 229 fig. 5. specific activity of lipases (mmol/min/mg protein). table 1. specific activity for plant oils (mmol/min/mg protein). table 2. specific activity for fats (mmol/min/mg protein). fats/hours 24 48 168 192 lard 0.084 0.032 0.387 0.005 beef fat 0.023 0.021 0.064 0.056 3.4 lipid degradation from the determination of lipid degradation and fatty acid utilization follows that commercial product has the ability to degrade the lipids and then utilize the fatty acids the highest increase of the free fatty acids (ffa) percentage for olive (47.6 %), palm (58.5 %) and rape seed oil (49.8 %) was observed after 72 hours. for sunflower oil (52.2 %) after 120 hours and for lard (88.6 %) and beef fat (86.5 %) after 96 hours, table 3. table 3. percent of free fatty acids during lipid degradation. quantity of the free fatty acids (%) during lipid degradation oils or fats/hours 48 72 96 120 192 216 olive 14.8 47.6 16.8 18.8 13.9 9.9 palm 14.2 58.5 49.8 26.1 12.4 10.9 rape seed 21.3 49.8 20.3 29.3 12.8 6.9 sunflower 18.2 36.6 12.5 52.5 10.3 7.9 lard 40.5 63.7 88.6 12.7 66.5 33.9 beef fat 74.6 83.4 86.5 70.9 33.5 27.3 oil /time (h) 24 48 72 168 192 216 olive 0.222 0.043 0.055 0.042 0.015 0.012 palm 0.111 0.114 0.042 0.008 0.012 rape seed 0.039 0.032 0.023 0.012 0.023 sunflower 0.043 0.042 0.053 0.027 0.030 230 illková, k. et al. 4. conclusions from the experimental results follows that the studied commercial microbial product for grease traps has the ability to degrade various plant oils and animal fats with different efficiency. best lipolytic activity was observed at palm oil, olive oil and lard, but best degrading substrates are rape seed oil and lard. growth of biomass was found out by all used substrates. total concentration of the biomass during cultivation differs, according to the sort of the substrate. it depends on the emulsifier properties of oils or fats. verification of the presence will be investigated in the other study. acknowledgement: this work has been supported by ministry of education, youth and sports under project msm 021630501 references becker, p., koster, d., popov, m.n., markossian, s., antranikian, g., markl, h.: the biodegradation of olive oil and treatment of lipid – rich wool wastewater under aerobic thermophilic condition. water res., 33, 1999, 653-660. el-bestawy, e., el-masry, m.h., el-adl, n.e.: the potentially of free gram – negative bacteria for removing oil and grease from contamined industrial effluents. world j. microbiol. biotechnol., 21, 2005, 815-822. gojković, ž.: studium přípravků do odlučovačů tuků. diplomová práce, vysoké učení technické v brně, fakulta chemická, 2009, 59 p. illková, k., omelková, j., vlčková, b.: lipid degradation and its application in lipid – containing wastewater. chem. listy, 99, 2008, 1234-2345. nawani, n., dosanjh, n. s., kaur, j.: a novel thermostable lipase from a thermophilic bacillus sp.: characterization and esterification studies. biotechnol. lett., 20, 1998, 997-1000. sigurgísladóttir, s., konráösdóttir, m., jónson, á., kristjánsson, j.k., matthiasson, e.: lipase activity of thermophilic bacteria from icelandic hot springs. biotechnol. lett., 15, 1993, 361-366. stoll, u., gupta, h.: management strategies for oil and grease residues. waste manage. res., 15, 1997, 23-32. instructions to authors submitting papers for the nova biotechnologica 9-3 (2009) 271 effect of chelating agents on phytoxicity and bioaccumulation of heavy metals in vascular plants miroslav horník1,2, jana guldanová1, martin pipíška1,2, jana marešová1, jozef augustín1,2 1department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (hornikm@ucm.sk) 2 consortium for environmental biotechnology and environmental chemistry, 418 špačince, 919 51, slovak republic abstract: in this work the effect of chelating agents ethylenediaminetetraacetate (edta) and nitrilotriacetate (nta) on phytotoxicity and bioaccumulation of cd and co in tobacco plants (nicotiana tabacum l.) hydroponically grown in diluted hoagland media (hm) spiked with 109cd and 60co was studied. speciation analysis using a program visual minteq showed, that the portion of bioavailable ionic me2+ forms significantly decreased in the presence of edta or nta in 25% hm for account of [me-edta]2or [me-nta]complexes. we found that the equimolar addition of edta or nta to 50 μmol/dm3 cdcl2 or cocl2 in hm positively diminished phytotoxicity of cd or co on tobacco plants. bioaccumulation of cd by tobacco roots during 8 d cultivation was minimally affected in the presence of equimolar concentrations of edta or nta to 10 μmol/dm3 cdcl2 in media. on the contrary, equimolar concentration of edta or nta added into hm caused considerable decrease of co uptake by tobacco roots. cadmium showed higher mobility in conductive tissues of tobacco plants than cobalt and the transport ratio in the presence of edta or nta increased 2-times or 3-times in comparison with control experiments (without addition of chelates), respectively. in the case of cobalt this effect was observed in a less extent. obtained data suggest the possibilities and constraints in the use of chelating agents in phytoextraction technologies in term of phytotoxicity, uptake and translocation of metals in plant tissues. key words: cadmium, cobalt, bioaccumulation, phytotoxicity, nicotiana tabacum, speciation 1. introduction heavy metals soil pollution is a serious worldwide problem and can be potentially harmful to human health via the food chain. in most cases, metals soil contamination results from anthropogenic activities such as mining, smelting, fertilizer application, and in the case of radionuclides from typical operations of nuclear fuel cycle, nuclear weapon testing and occasional nuclear disasters. the stricter implementation of environmental laws urges the development of costeffective soil remediation methods. traditional techniques of remediation are expensive and may cause secondary pollution. phytoremediation, the use of green plants to remove pollutants from soil, is one cost-effective method to remediate metal and radionuclide contaminated soils. the technical aspects of phytoremediation are described in numbers of review papers (see latest works in this area e.g. pilonsmits and leduc, 2009; vangronsveld et al., 2009) and monographs (see e.g. mackova et al., 2006; willey, 2007). phytoextraction as one of the most perspective phytoremediation method depends mainly on the bioavailability of toxic metals and radionuclides in the soil and the plant 272 horník, m. et al. capacity to accumulate these contaminants. therefore, some researchers have found that complexing ligands (e.g. edta, nta, edds and others) can solubilize the heavy metals with the purpose of making them available for uptake (salt et al., 1998), but they are not necessarily available for plant uptake (tandy et al., 2006). they redistribute surface contamination down the soil profile, causing a reduction in the concentration near the soil surface (robinson et al., 2003) and distribute the heavy metals within the entire root zone for uptake. in addition, some authors reported that complexes of me-chelate were less phytotoxic than free me2+ forms or protonated chelates (hernández-allica et al., 2007). our previous works studied the bioaccumulation of zn, co, cd or cs from nutrient solutions by roots of hydroponically cultivated sunflower (helianthus annuus l.), tobacco (nicotiana tabacum l.) and celery (apium graveolens l.) plants (horník et al., 2005; horník et al., 2007; horník et al., 2008) and radiocesium accumulation from contaminated soil by autochthonous vegetation of plants (pipíška et al., 2005). the aim of the current work was to investigate the influence of chelating agents ethylenediaminetetraacetic acid (edta) and nitrilotriacetic acid (nta) on cd and co phytotoxicity and bioaccumulation in tissues of hydroponically cultivated tobacco plants (nicotiana tabacum l.). bioaccumulation of cd and co ions in plants was analyzed by gamma-spectrometry using 109cd and 60co as radiotracers. 2. material and methods 2.1 plant material seeds of tobacco (n. tabacum l.) were germinated and grown in pots filled with granulated perlite as an inert carrier and watered with 25% strength hoagland nutrient medium (hoagland, 1920) at light/dark photoperiod 12/12 h (2 000 lx) and 22°c. the composition of the full strength (100%) nutrient solution was (in mg/dm3) mgso4.7h2o – 370; kno3 – 404; cacl2 – 444; nah2po4.2h2o – 292; na2hpo4.12h2o – 46.5; feso4.7h2o – 17.9; nano3 – 340; nh4cl – 214; nh4no3 – 160; h3bo3 – 8.5; na2moo4.2h2o – 0.06; mnso4.5h2o – 5.0; znso4.7h2o – 0.66; cuso4.5h2o – 0.8 (ph 6). after 8 weeks pre-cultivation seedlings were gently removed from perlite and roots were washed free of any adhering perlite granules by deionized water and used in bioaccumulation experiments. 2.2 bioaccumulation experiments and cultivation conditions plants from pre-cultivation phase were transferred into vessels with a cover to protect plant roots against lights and cultivated for 8 days in 25% strength hoagland medium containing cdcl2 or cocl2 spiked with 109cd or 60co. there were two treatments without or with chelating agents edta or nta. the ph of nutrient solutions was adjusted to 6.0 using 1 m naoh. bioaccumulation experiments were carried out in triplicate series at photoperiod 12/12 h (2000 lx) and 22°c. in time intervals samples of nutrient solution were taken and 109cd or 60co radioactivity was measured by gamma-spectrometry. at the end of the experiments plants were nova biotechnologica 9-3 (2009) 273 harvested, roots were carefully rinsed in deionized water and incorporated radioactivity in roots, stems and leaves was measured. plant parts were then oven dried (at 60°c for 24 hours) and dry weights were determined. growth value (gv) was calculated during the experiments as a ratio between m(i) – m(t) and m(i), where m(i) or m(t) are fresh weight of plants at the start or end of experiments, respectively. 2.3 speciation modelling for in silico estimations of cd and co speciation in nutrient solutions as a function of total salt concentration, the presence or absence of chelating agents (edta or nta), solution ph and temperature were carried out with visual minteq (version 2.53). this speciation modelling program allows the calculation of complexes [me-edta] or [me-nta] portion in cultivation media for specified conditions. 2.4 radiometric analysis gamma spectrometric scintillation detectors 54bp54/2-x and 76bp76/3 with well type crystal nai(tl) (scionix, netherlands) and data processing software scintivision32 (ortec, usa) were used for 109cd and 60co determination in plant parts and cultivation media. a library of radionuclides was built by selecting characteristic γ-ray peaks (88.04 kev for 109cd and 1173.24 kev or 1332.50 kev for 60co) for energy and efficiency calibration. standardized 109cdcl2 and 60cocl2 solutions were obtained from czech metrological institute (czech republic). 3. results and discussion 3.1 effect of chelating agents on cd and co phytotoxicity in our work for calculation of cd and co ionic forms in nutrient media the program visual minteq ver. 2.53 we used. however, this evaluation has to be considered with caution, because the modelling program does not include the participation of the plant. moreover, the high concentrations of edta and nta used here likely also mask the effects of the exudates from the roots. plant exudates can contribute to changing the ph, the eh, and the predominant chemical species of the toxins of concern (niu et al., 2007). even so, we used the simulation results as a first approximation to the chemical mechanisms in the near-root zone. we found that in used hoagland media (hm) at ph 6.0 and 22°c more than 95% of co was found as bioavailable me2+ ions. only 78% of cd was found in the form of cd2+ ions, whereby also significant proportion represented ionic cdcl+ form (12.7%) and cdhpo4 form (6.4%). complexing ligands significantly decreased the concentration of cationic co2+ or cd2+ forms and the concentration of [me-edta]2or [me-nta]complex forms increased. for evaluation of cd and co phytotoxic effect on tobacco plants the calculation of growth value (the ratio of fresh weight of plants at the start or end of experiments 274 horník, m. et al. difference to fresh weight of plants at the start of experiments) and macro and microscopy observation of phytotoxic symptoms on leaves were used. 0,0 0,2 0,4 0,6 0,8 1,0 w ith 5 0 μ m n t a 50 μm50 μm50 μm20 μm10 μm w ith 5 0 μ m e d t a g v concentration of cdcl2 in medium 0 μm without chelating agents 0,0 0,2 0,4 0,6 0,8 1,0 w ith 5 0 μ m n t a 50 μμ50 μμ50 μμ20 μμ10 μμ g v concentration of cocl2 in medium 0 μμ w ith 5 0 μ m e d t a without chelating agents fig. 1. the evaluation of phytotoxicity effect of cd (a) and co (b) on the basis of growth value (gv) of tobacco plants (n. tabacum l.) after 8 d cultivation in 25% hm without or with addition of edta or nta at illumination 12/12 day/night period (2 000 lx), ph 6.0 and 22°c. fig. 2. phytotoxicity effect of cadmium (die-back of leaves) in tobacco plants (n. tabacum l.) after 8 d cultivation at illumination 12/12 day/night period (2 000 lx) in 25% hm, ph 6 containing: a. 0 μmol/dm3 cdcl2; b. 10 μmol/dm3 cdcl2; c. 20 μmol/dm3 cdcl2; d. 50 μmol/dm3 cdcl2; e. 50 μmol/dm3 cdcl2 + 50 μmol/dm3 edta; f. 50 μmol/dm3 cdcl2 + 50 μmol/dm3 nta. gv (growth value): a. 0.70; b. 0.69; c. 0.47; d. 0.39; e. 0.59; f. 0.56. fig. 1 shows, that the equimolar addition of edta or nta to 50 μmol/dm3 cdcl2 or cocl2 in hoagland media positively diminished phytotoxicity effect of cd or co on tobacco plants growth evaluated on the basis of growth values (gv) calculation. a b c a b d e f nova biotechnologica 9-3 (2009) 275 fig. 2 and 3 depict the die-back of plant leaves in the presence of cd or chlorosis of leaves in the presence of co in media as well as the effect of chelating agents on these processes. phytotoxicity symptoms (die-back of leaves) and lower growth value were observed at 20 or 50 μmol/dm3 cdcl2 concentrations in media (fig. 1a, 2c, 2d). after 50 μmol/dm3 co-treatment, plants yellowed with typical chlorotic ringspot symptoms and lower growth value than the control were found (fig. 1b, 3d). moreover, we found that this co-treatment used in our experiment had impact on trichomes size and density (fig. 4a). lefèvre et al. (2009) found that more than 30% of accumulated cd was found at the leaf surface and accumulated in trichomes. on the other side bakkaus et al. (2005) reported that cobalt was mainly located around the leaf veins. at the end of the 50 μmol/dm3 cd-treatment or co-treatment with addition of edta or nta in media, no sign of phytotoxicity was observed (fig. 2e, 2f, 3e, 3f and 4b). a b c d e f fig. 3. phytotoxicity effect of cobalt (chlorosis of leaves) in tobacco (n. tabacum l.) plants after 8 d cultivation at illumination 12/12 day/night period (2 000 lx) in 25% hm, ph 6 containing: a. 0 μmol/dm3 cocl2; b. 10 μmol/dm3 cocl2; c. 20 μmol/dm3 cocl2; d. 50 μmol/dm3 cocl2; e. 50 μmol/dm3 cocl2 + 50 μmol/dm3 edta; f. 50 μmol/dm3 cocl2 + 50 μmol/dm3 nta. gv (growth value): a. 0.70; b. 0.72; c. 0.59; d. 0.41; e. 0.57; f. 0.63. we suggest that the decrease of metal concentration in me2+ form caused by chelating agents in nutrient media and the formation of [me-ligand] complexes in media could diminish phytotoxic effect. similar effect was also found by others authors (see e.g. hernández-allica et al., 2007). this fact could be helpful in phytoremediation of contaminated soils with high concentration of toxic metals, where most plants can not grow under normal conditions. 276 horník, m. et al. a1 a2 a3 fig. 4. chlorosis of tobacco leaves (n. tabacum l.) after 8 d cultivation at illumination 12/12 day/night period (2 000 lx) in 25% hm, ph 6 containing: a. 50 μmol/dm3 cocl2; b. 50 μmol/dm3 cocl2 + 50 μmol/dm3 edta. macroand microscopic photos: 1. second the youngest tobacco leaf; 2. surface of adaxial leaf section (magnification 50x); 3. adaxial and abaxial leaf trichomes (magnification 100x). b1 b2 b3 3.2 effect of chelating agents on cd and co bioaccumulation bioaccumulation of cd by tobacco roots during 8 d cultivation was minimally affected in the presence of edta or nta equimolar concentrations to 10 μmol/dm3 cdcl2 in media. on the contrary, equimolar concentration of edta or nta added into hoagland media caused considerable decrease of co uptake by tobacco roots (fig. 5a). calculated speciation of cd and co in used hoagland medium (hm) is depicted in fig. 5b. for evaluation of metals mobility in conductive tissues of plants in the term of metal translocation efficiency we established non-dimensional transport ratio (tr), which represent the ratio of metal concentration in aboveground part of plants [me]shoot to metal concentration in root system of plants [me]root. cadmium showed higher mobility in conductive tissues of tobacco plants than cobalt and the transport ratio in the presence of edta or nta increased 2-times or 3-times in comparison with control experiments (without addition of chelates), respectively (fig. 6). in the case of cobalt this effect was observed in a less extent. some authors reported that metal uptake is related to the availability of metals in soils and the addition of natural and/or synthetic chelators into soil could increase uptake and translocation of metals (see e.g. quartacci et al. (2007). however, the amendment with non-biodegradable chelants of high metal-binding capacity, the high persistence of chelates and their nova biotechnologica 9-3 (2009) 277 potential leaching to groundwater pose severe environmental concerns connected with the use of synthetic chelating agents such as edta and its derivatives. quartacci et al. (2007) found that nta and mainly edds were high effective in enhancing the concentrations of cd, cu, pb and zn in brassica carinata shoots. 0 20 40 60 80 100 co b io ac cu m ul at io n [% ] control edta nta cd 0 20 40 60 80 100 [co-chelate]co2+[cd-chelate] p or tio n in m ed ia [% ] speciation form control edta nta cd2+ a b fig. 5. a. effect of edta and nta on cd and co uptake by tobacco roots (n. tabacum l.). data after 8 d cultivation at 22°c and illumination 12/12 day/night period (2 000 lx) in 25% hm ph 6.0 containing 10 μmol/dm3 cocl2 or 10 μmol/dm3 cdcl2 and equimolar edta or nta. b. portion of me2+ (cd2+ or co2+) and [me-chelate] ([me-edta]2or [me-nta]-) forms in cultivation media at 22°c and ph 6.0. speciation of cd and co was calculated by program visual minteq ver. 2.53. 0,0 0,1 0,2 0,3 0,4 0,5 co [m e] sh oo t/[ m e] ro ot control edta nta cd fig. 6. effect of edta and nta on translocation of cd and co from roots to shoots (tr) of tobacco (n. tabacum l.) after 8 d plant cultivation in 25% hm containing 10 μmol/dm3 mecl2 (me = cd or co) without or with equimolar addition of edta or nta, ph 6.0 at illumination 12/12 day/night period (2 000 lx) and 22°c. 4. conclusions the results from hydroponic cultivation of tobacco plants (n. tabacum) indicate that chelating agents (edta and nta) are able to decrease of cd and co phytotoxicity, and on the other side these agents can increase of metal translocation from roots to shoots of tobacco plants. this fact can positively affect all processes involved in phytoremediation of contaminated environment with toxic metals or radionuclides. 278 horník, m. et al. references bakkaus, e., gouget, b., gallien, j.-p., khodja, h., carrot, f., morel, j.l., collins, r.: concentration and distribution of cobalt in higher plants: the use of micro-pixe spectroscopy. nucl. instrum. methods phys. res. b, 231, 2005, 350-356. hernández-allica, j., garbisu, c., barrutia, o., becerril, j.m.: edta-induced heavy metal accumulation and phytotoxicity in cardoon plants. environ. exp. bot., 60, 2007, s. 26-32. hoagland, d.r.: optimum nutrient solution for plants. science, 52, 1920, 562564. horník, m., pipíška, m., sekáčová, j., augustín, j.: determination of long distance transport of cs+, co2+ and zn2+ ions in vascular plants by autoradiography and gamma-spectrometry. nova biotechnol., vii-1, 2007, 33-40. horník, m., pipíška, m., vrtoch, ľ., sekáčová, j., augustín, j., lesný, j.: influence of complexing ligands on zn uptake and translocation in tobacco and celery plants. acta agronomica ovariensis, 2008, 50, 65-71. horník, m., pipíška, m., vrtoch, ľ., augustín, j., lesný, j.: bioaccumulation of 137cs and 60co by helianthus annuus. nukleonika, 50, 2005, s49-s52. lefèvre, i., marchal, g., meerts, p., corréal, e., lutts, s.: chloride salinity reduces cadmium accumulation by the mediterranean halophyte species atriplex halimus l. environ. exp. bot., 65, 2009, 142-152. mackova, m., dowling, d., macek, t.: phytoremediation rhizoremediation. springer, dordrecht, the netherlands, 2006, 300 pp. niu, z.x., sun, l.n., sun, t.h., li, y.s., wang, h.: evaluation of phytoextracting cadmium and lead by sunflower, ricinus, alfalfa and mustard in hydroponic culture. j. environ. sci., 19, 2007, 961-967. pilon-smits, e.a.h., leduc, d.l.: phytoremediation of selenium using transgenic plants. curr. opin. biotechnol., 20, 2009, 207-212. pipíška, m., horník, m, lesný, j., augustín, j.: transfer rádiocézia 137cs z kontaminovanej neobrábanej pôdy do voľne rastúcej vegetácie. agriculture (poľnohospodárstvo), 51, 2005, 470-478. quartacci, m.f., irtelli, b., baker, a.j.m., navari-izzo, f.: the use of nta and edds for enhanced phytoextraction of metals from a multiply contaminated soil by brassica carinata. chemosphere, 68, 2007, 1920-1928. salt, d.e., smith, r.d., raskin, i.: phytoremediation. annu. rev. plant physiol. plant mol. biol., 49, 1998, 643-668. vangronsveld, j., herzig, r., weyens, n., boulet, j., adriaensen, k., ruttens, a., thewys, t., vassilev, a., meers, e., nehnevajova, e., van der lelie, d., mench, m.: phytoremediation of contaminated soils and groundwater: lessons from the field. environ. sci. poll. res., (in press). willey, n.: phytoremediation. methods and reviews. humana press, new jersey, usa, 2007, 478 pp. microsoft word gasparova nb 9-3.doc nova biotechnologica 9-3 (2009) 349 reactions of 4-hydroxycoumarin with heterocyclic aldehydes renata gašparová1, katarína kotlebová1, margita lácová2 1department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (gasparor@ucm.sk) 2department of organic chemistry, comenius university, mlynská dolina ch-2, bratislava, sk-842 15, slovak republic (lacova@fns.uniba.sk) abstract: reactions of 4-hydroxycoumarin 1 with heterocyclic aldehydes 2-4 led to bis-4-hydroxycoumarin derivatives 5-7 under microwave irradiation as well as under the classical heating. the subsequent reactions of products 5-7 are described. 4,4’-epoxydicoumarins 8, 9 were prepared by the reaction of 5-7 in acetic acid / p-toluenesulfonic acid medium. compound 10 was prepared by the reaction of 5 in acetic anhydride in the presence sodium acetate. dioxocine-1,15-dione 11 was prepared by the reaction of 6 with dichloromethane in sodium hydroxide-toluene. key words: coumarin, 4-oxo-4h-chromene, furan, furo[3,2-b]pyrrole, microwave 1. introduction coumarins are biologicaly active compounds with antifungal, antineoplastic, antibacterial, spasmolytic or cytotoxic activity (stanchev et al., 2007; jung et al., 2004). 4-hydroxycoumarin 1 and its derivatives, e.g. warfarin and dicoumarol are known as anticoagulants. 4-hydroxycoumarin 1 reacts with aromatic or aliphatic aldehydes to give bis-4hydroxycoumarin derivatives (giovani et al., 1991; manolov et al., 2006; mao et al., 2002), which are interesting for their biological activity and they can serve as intermediates for synthesis of various heterocycles (hamdi et al., 2008; yamashita et al., 1987). 2. experimental melting points of products were determined on a kofler hot plate apparatus and are uncorrected. all solvents were predistilled and dried appropriately prior to use. 1h nmr spectra were obtained on a 300 mhz spectrometer varian gemini 2000 in cdcl3 or dmso-d6 or cf3cood with tetramethylsilane as an internal standard. all microwave experiments were performed in a panasonic nn-e205 type microwave oven. the apparatus was adapted for laboratory applications; n-hexane was used as coolant for the condenser. 2.1 general procedure for 5 and 6 a) classical heating. a mixture of 4-hydroxycoumarin 1 (0.002 mol) and 6-r-4-oxo4h-chromen-3-carbaldehyde 2 (or 5-arylfuran-2-carbaldehyde 3) (0.001 mol) in acetic 350 gašparová, r. et al. anhydride (15 cm3), in the presence of a catalytic amount of k2co3 (or acok) was heated at 80 ºc for 3.5 h. after cooling the solid product was filtered off and crystallized from ethanol. o o oh r-cho r oo o o oh hoac2o, acok (k2co3) r 5 6-r1-4-oxo-4h-chromen-3-yl 6 5-arylfuran-2-yl 7 5-methoxycarbonylfuro[3,2-b]pyrrol-2-yl 1 2-4 r oo o o o o o o o oac oac o o oac br r 8 6-r1-4-oxo-4h-chromen-3-yl 9 5-arylfuran-2-yl 10 ac2o acoh p-tsoh scheme 1 acona r oo o o o r 11 5-(4-iodophenyl)lfuran-2-yl o naoh toluene b) microwave method. a mixture of 4-hydroxycoumarin 1 (0.002 mol) and 6-r-4oxo-4h-chromen-3-carbaldehyde 2 (or 5-arylfuran-2-carbaldehyde 3) (0.001 mol) in acetic anhydride (4 cm3), in the presence of a catalytic amount of k2co3 (or acok) was irradiated in microwave oven for 4 min. the work-up was the same as described above. 2.1.1 3,3'-[(4-oxo-4h-chromen-3-yl)methylene]bis(4-hydroxy-2-oxo-2h-chromen-2one) 5a for c28h16o8 (480.4): mp: 275-280 °c; react. time 3.5h (a), 4min (b); yield: 81% (a), 82% (b); 1h nmr (cdcl3): 6.01 (d, 1h, ch j = 1.5 hz); 7.34-7.36 (m, 1h, h-6); 7.39 (dd, 4h, h-8´, h-8´´, h-6´, h-6´´, 3j = 7.97 hz); 7.46 (d, 1h, h-8, j = 8.25 hz); 7.59-7.61 (m, 2h, h-7´, h-7´´); 7.67 (m, 1h, h-7); 7.92 (d, 1h, h-2, j = 1.7 hz); 8.04 (dd, 2h, h-5´, h-5´´, j = 7.9 hz); 8.11 (dd, 1h, h-5, j = 7.9 hz); 11.50 (bs, 2h, oh) 2.1.2 3,3'-[(6-methyl-4-oxo-4h-chromen-3-yl)methylene]bis(4-hydroxy-2-oxo-2hchromen-2-one) 5b for c29h19o8 (495.5): mp: 276-278 °c; react. time 3.5h (a); yield: 79% (a); 1h nmr (dmso-d6): 2.38 (s, 3h, ch3); 6.07 (d, 1h, ch, j = 1.5 hz); 7.19 (dd, 2h, h8´, h-8´´, j = 7.5, 1.1 hz); 7.24 (dd, 2h, h-6´, h-6´´, j = 7.5, 0.9 hz); 7.46 (ddd, 2h, nova biotechnologica 9-3 (2009) 351 h-7´, h-7´´, j = 8.2 , 1.5 hz); 7.47-7.50 (m, 1h, h-7); 7.55 (dd, 1h, h-8, j = 8.6, 1.6 hz); 7.69 (d, 1h, h-5, j = 1.5 hz); 7.78 (dd, 2h, h-5´, h-5´´, j = 7.9, 1.5 hz); 7.83 (d, 1h, h-2, j = 1.5 hz); 17,2 (bs, 2h, oh) 2.1.3 3,3´-[(5-(4-chlorophenyl)furan-2-yl)methylene]bis(4-hydroxy-2h-chromen-2one) 6a for c29h17clo7 (512.9): mp: 265-269 °c; react. time 93h (a), 1.5h (b); yield: 68% (a), 70% (b); 1h nmr (dmso-d6): 5.15 (s, 1h, ch); 6.52 (d, 1h, h – 4, j = 3.3 hz); 6.91 (d, 1h, h – 3, j = 3.3 hz); 7.41 (d, 2h, ar h, j = 8.5 hz); 7.54 – 7.57 (m, 6h, ar – h); 7.77 (ddd, 2h, h – 7´, j = 8.7, 1.2 hz); 8.43 (dd, 2h, h – 5´, j = 8.5, 1.3 hz). 2.1.4 3,3´-[(5-(4-nitrophenyl)furan-2-yl)methylene]bis(4-hydroxy-2h-chromen-2one) 6b for c29h17no9 (457.5): mp: 317-319 °c; react. time 51h (a), 1.5h (b); yield: 60% (a), 62% (b); 1h nmr (dmso-d6): 5.19 (s, 1h, ch); 6.65 (d, 1h, h – 4, j = 3.3 hz); 7.23 (d, 1h, h – 3, j = 3.3 hz); 7.54 – 7.57 (m, 8h, ar h); 7.82 – 7.84 (m, 5h, ar h); 8.24 (d, 2h, h –8´´, j = 8.3 hz). 2.1.5 3,3´-({5-[4-(3-trifluoromethyl)phenyl]furan-2-yl}methylene)bis(4-hydroxy-2hchromen-2-one) 6c for c30h17f3o7 (546.5): mp: 257-260 °c; react. time 70h (a), 1.5h (b); yield: 50% (a), 51% (b); 1h nmr (dmso-d6): 5.17 (s, 1h, ch); 6.65 (d, 1h, h – 4, j = 3.3 hz); 7.11 (d, 1h, h – 3, j = 3.3 hz); 7.58 – 7.61 (m, 5h, ar h); 7.77 – 7.83 (m, 5h, ar h); 8.44 (d, 2h, h –8´´, j = 8.4 hz). 2.1.6 3,3´-[(5-(4-iodophenyl)furan-2-yl)methylene]bis(4-hydroxy-2h-chromen-2-one) 6d for c29h17io7 (604.4): mp: 248-252 °c; react. time 70h (a), 1.5h (b); yield: 51% (a), 53% (b); 1h nmr (dmso-d6): 5.14 (s, 1h, ch); 6.52 (d, 1h, h – 4, j = 3.3 hz); 6.92 (d, 1h, h – 3, j = 3.3 hz); 7.35 (d, 2h, ar h, j = 8,7 hz); 7.51 – 7.59 (m, 4h, h-6´,h-8´); 7.73 (d, 2h, ar-h j = 8.7 hz); 7.83 (ddd, 2h, h – 7´, j = 8.7, 1.5 hz); 8.42 (dd, 2h, h – 5´, j = 8.4, 1.2 hz). 2.2 synthesis of methyl 2-[bis(4-hydroxy-2h-chromen-3-yl)methyl]-4hfuro[3,2-b]pyrrole-5-carboxylate 7 a) classical heating. a mixture of 4-hydroxycoumarin 1 (0.002 mol) and methyl 2formyl-4h-furo[3,2-b]pyrrole-5-carboxylate 4 (0.001 mol) in pyridine (15 cm3) was heated at 105 ºc for 3 h. after cooling the solid product was filtered off and crystallized from ethanol. b) microwave method. a mixture of 4-hydroxycoumarin 1 (0.002 mol) and 2-formyl4h-furo[3,2-b]pyrrole-5-carboxylate 4 (0.001 mol) in pyridine (4 cm3) was irradiated in microwave oven for 3 min. the work-up was the same as described above. 352 gašparová, r. et al. for c27h15o9n (497.4): mp: 275-280 °c; react. time 3h (a), 3 min (b); yield: 52% (a), 56% (b); 1h nmr (dmso-d6): 3.89 (s, 3h, ch3); 6.90 (s, 1h, ch); 7.33-7.40 (m, 4h, ar-h); 7.71-7.79 (m, 2h, ar-h); 7.97 (m, 2h, ar-h); 8.28 (s, 1h, h-3); 8.66 (s, 1h, h-6); 9.05 (s, 1h, nh); 12.46 (d, 2h, 2 oh). 2.3 general procedure for 8 and 9 compound 5 (or 6) (0.2 mmol) in acetic acid (5 cm3) and catalytic amount of ptoluenesulfonic acid was heated at 80°c. after cooling the solid was filtered off and crystallized in ethanol. 2.3.1 3,3´-[(4-oxo-4h-chromen-3-yl)methylene]-4,4´-epoxydicoumarin 8 for c28h14o7 (462.4): mp: 330-335 °c; react. time 2.5h; yield: 92%; 1h nmr (dmso-d6): 4.77 (s, 1h, ch; 7.41-7.42 (m, 1h, h-6); 7.51-7.78 (m, 4h, h-8´, h-8´´, h-6´, h-6´´); 7.66 (d, 1h, h-8, j = 8,4 hz); 7.76-7.78 (m, 3h, h-7´, h-7´´); 7.88 (d, 1h, h-5, j = 7,8 hz); 8.45 (d, 2h, h-5´, h-5´´, j = 7.8 hz); 8.71 (s, 1h, h-2). 2.3.2 3,3´-[(5-(4-nitrophenyl)furan-2-yl)methylene]-4,4´-epoxydicoumarin 9 for c28h14o7 (439.4): mp: 322-325 °c; react. time 6h; yield: 62%; 1h nmr (dmso-d6): 5.41 (s, 1h, ch); 6.58 (d, 1h, h – 4, j = 3.4 hz); 7.10 (d, 1h, h – 3, j = 3.4 hz); 7.35 – 7.50 (m, 6h, ar h); 7.62 – 7.74 (m, 4h, ar h); 8.10 – 8.13 (m, 2h, h – 3´, 5´). 2.4 synthesis of 2,4´,4´´-triacethyl-3-[bis(4-hydroxy-2-oxo-2hchromen-3-yl)methyl]-chromen-4-one 10 a mixture of 4-hydroxycoumarin 1 (0.002 mol) and 6-bromo-4-oxo-4h-chromen3-carbaldehyde 2 in acetic anhydride (5 cm3) and catalytic amount of sodium acetate was heated at 80 ºc for 4.5 h. after cooling the solid product was filtered off and crystallized from ethanol. for c34h21bro12 (701.4): mp: 158-163 °c; yield: 66%; 1h nmr (cdcl3): 2.09 (s, 3h, ch3); 2 x 2.18 (s, 3h, ch3); 7.12 (d, 1h, h-8, j = 8.5 hz); 7.35-7.41 (m, 4h, h6´, h-6´´, h-8´, h-8´´); 7.63-7.67 (m, 2h, h-7´, h-7´´); 7.66 (d, 1h, h-5, j = 2.4 hz); 7.78 (s, 1h, h-2); 7.96-8.07 (m, 3h, h-5´, h-5´´, h-7). 2.5 synthesis of 16-[5-(4-iodophenyl)furan-2-yl]-1h,15h,16hdibenzopyrano[3,4-g:4'3'-d]dioxocine-1,15-dione 11 a mixture of compound 6d (0.4 mmol), dichloromethane (0.8 mmol) and sodium hydroxide (0.8 mmol) in toluene (3 cm3) was heated at 60°c for 117 h. after cooling the solid was filtered off and crystallized in ethanol. for c30h17io7 (616.4): mp: ›350 °c; yield: 47%; 1h nmr (cf3cood): 4.36 (s, 2h, ch2); 5.32 (s, 1h, ch); 6.57 (d, 1h, h – 3, j = 3.3 hz); 7.26 –7.51 (m, 9h, h – 4, ar h); 7.64 – 7.70 (m, 2h, h -3´,5´); 8.10 – 8.13 (m, 2h, h 2´´, 12´´). nova biotechnologica 9-3 (2009) 353 3. results and discussion reaction of 1 with 6-substituted 4-oxo-4h-chromene-3-carbaldehydes 2 led to bis(4-hydroxy-2-oxo-2h-chromen-2-ones 5 in 79-81% yields after 3.5h of heating. microwave-assisted method gave the comparable yield of 5a (82%), but the reaction time was remarkably shortened (4min). the use of 5-arylfuran-2-carbaldehydes 3 in reaction with 1 led to derivatives 6 in 50-68 % yields.. the effect of microwave irradiation was manifested in the remarkable shortening of reaction time from 51-93 h under classical heating to 1.5h in microwave oven. when 4-hydroxycoumarin 1 was treated with methyl 2-formyl-4h-furo[3,2b]pyrrole-5-carboxylate 4 in pyridine either by classical heating for 3h or in microwave oven for 4 min, product 7 was obtained in comparable yields (52% and 56%, respectively). epoxycoumarins 8 and 9 were obtained in 92% and 62 % yields, respectively by the dehydratation of 5 or 6 in acetic acid / p-toluenesulfonic acid medium. the heating of 4-hydroxycoumarin 1 and 6-bromo-4-oxo-4h-chromen-3carbaldehyde in acetic anhydride led to 3-acetyloxy derivative 10 in 66% yield. dioxocine-1,15-dione 11 was prepared in 47% yield by reaction of 6d with dichloromethane and sodium hydroxide in toluene. the reaction required long reaction time (117 h). the pursuit to shorter the reaction time by the influence of microwave irradiation was not successful and only decomposition polymeric product was isolated. all structures were confirmed by 1h nmr spectra. 4. conclusions 4-hydroxycoumarin 1 reacted with heterocyclic aldehydes 2-4 to give bisderivatives 5-7, which served as intermediates for synthesis of epoxycoumarins 8, 9, 2,4´,4´´-triacethylderivative 10 and dioxocine-1,15-dione 11, respectively. microwave irradiation has proved to be a suitable method for enhancement of reactions of 1 with aldehydes. acknowledgements: this work was supported by the slovak research agency under the contracts no. vega 1/1005/09 and apvv-0006-07 references giovani, a., cravotto, g., tagliapietra, s., ferraro, s., nano, g. m., palisano, g.: synthesis of 3-alkyl-4-hydroxycoumarins by reductive fragmentation of 3,3´-alkylidene-4,4´-dihydroxybis[coumarins]. helv. chim. acta, 74, 1991, 1451-1458. hamdi, n., puerta, m. c., valerga, p.: synthesis, structure, antimicrobial and antioxidant investigations of dikumarol and related compounds. eur. j. med. chem., 43, 2008, 2541-2548. jung, j. ch., lee, j. h., oh, s., leed, j. g., parkb, o. s.: synthesis and antitumor activity of 4-hydroxycoumarin derivatives. bioorg. med. chem. lett., 14, 2004, 5527–5531. 354 gašparová, r. et al. manolov, i., maichle-moessmer, c., danchev, n.: synthesis, structure, toxicological and pharmacological investigations of 4-hydroxycoumarin derivatives. eur. j. med. chem., 41, 2006, 882-890. mao, p.ch.m, mouscadet, j. f., leh, h., auclair, ch., hsu, l.y.: chemical modification of coumarin dimer and hiv-1 integrase inhibitory activity. chem. pharm. bull., 50, 2002, 1634-1637. stanchev, s., momekov, g., jensen, f., manolov, i.: synthesis, computational study and cytotoxic activity of new 4-hydroxycoumarin derivatives. eur. j. med. chem., 20, 2007, 1-13. yamashita, j., takeda, s., matsumoto, h., unemi, n., yasumoto, m.: studies on antitumor agents. vii. antitumor activities of o-alkoxyalkyl derivatives of 2´-deoxy-5-trifluoromethyluridine. chem. pharm. bull., 35, 1987, 2373-2381. microsoft word urgeova nb 9-3.doc nova biotechnologica 9-3 (2009) 327 secondary metabolites with antibacterial effects from leaves of different hop cultivars during vegetal periods eva ürgeová, ľudovít polívka department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (eva.urgeova@ucm.sk) abstract: according to the latest eu regulations it is necessary to cut down the consumption of chemically based pesticides. several research programmes are focussed on the study of secondary metabolites of plants, which have antimicrobial or antifungal activity against microbial phytopathogens. other reasons for using of natural biocides are the growth of resistance of phytopathogens against several pesticides; broaden microbial attacks to significant agricultural product for food industry due to the climate changes. the presentation is focussed on the determination of the contents of secondary metabolitesleaves of several cultivars of hop – polyphenols and flavonoids. methanol crude leaves extracts were tested on biocide effects against selected microbial phytopathogens. key words: hop, extracts, polyphenols, flavonoids, antibacterial effect 1. introduction according to the latest eu regulations it is necessary to cut down the consumption of chemically based pesticides. several research programmes are focussed on the study of secondary metabolites of plants which have antibacterial or antifungal activity against microbial phytopathogens. the plants synthesise and accumulate various secondary metabolites which are characterised by the diversity of their chemical structures, restricted distribution and protective function for an organism. in this perspective, increasing attention is being paid to the plant called common hop (humulus lupulus l) (moir, 2000). hop compounds are effective inhibitors of gram-positive bacteria (battacharya et al., 2003), mycobacteria (stavri et al., 2004) and protozoa (srinivasan et al., 2005). in addition, xanthohumol has been reported to be a broad spectrum anti-infective agent working against many bacteria, viruses, fungi, and protozoa (gerhäuser, 2005). in the study of antimicrobial activity of essential oils from the hop plant, lagezaal et al. (1992) identified their inhibitive influence on gram-positive bacteria bacillus subtilis and staphylococcus aureus, as well as on the fungus trichophyton mentagrophyles. one of the big groups of substances contained in the hop plant is the group of polyphenol compounds, chalkones, flavanols, flavonols, and antocyanides (hofta et. al., 2004). content of polyphenols in the leaves can be of potential benefit when speaking of using these as raw material for extraction of these substances (čeh et al., 2007). the most interesting of them are flavonoids, belonging to the most widespread compounds in nature. 328 ürgeová, e. and polívka, ľ. the aim of these experiments was to test the influence of the vegetation period on the content of some secondary metabolites of hop, polyphenol compounds, and flavonoids, which display a wide range of biological and pharmacological properties. biocide activity of extracts from hop leaves was tested, too. 2. material and methods 2.1 plant material the samples of hop taken from the gene bank of the slovak republic in the institute of plant production, piešťany (cvrv – vúrv) were tested. we used the following cultivars of hop: osvald’s clones 31 (k-31) and 72 (k-72), bor, sládek, zlatan, and premiant. we collected the samples at the beginning of vegetal period (april 08), before (june 08) and during the flowering time (july 08) and at the end of the vegetal period (september 08). 2.2 the tested phytopathogene bacteria for the purpose of this study we used phytopathogenic microorganisms erwinia mallotivora ccm 2890, obtained from the czech collection of microorganisms of the masaryk university, faculty of science, brno, clavibacter michiganensis subsp. sepedonicus cppb a098, erwinia amylovora cppb a203, pectobacterium carotovorum pv. carotovorum cppb ao86, pseudomonas syringae pv. syringae cppb a171, and xanthomonas vesicatoria cppb a085, obtained from the collection of phytopathogenic bacteria and referential antidotes (cppb) at the crop research institute in prague-ruzyně, czech republic. bacteria were kept as a stock culture in the refrigerator at the temperature of 8° c. 2.3 extraction the extracts were prepared by methanol extraction in the ratio of 1:20. after extraction it was evaporated on the vacuum rotary evaporator. we dissolved this matter in ethyl acetate and water in ratio 1:1. the organic phase was evaporated. dry matter was dissolved in methanol (4:3 w/v) and stored in a cold store. 2.4 method for testing biocide effect of extracts the antibacterial activity of extracts was determined in vitro against a variety of phytopathogene bacteria. antimicrobial activities were tested by the standard plate diffusion method (piddock, 1989) and zones of inhibition were measured in mm. the biocide effect was compared with the effect of 1.2 % w/v solution of tmtd (tetramethylthiuram disulfide), active substance of commercial pesticides. plant extracts (15 μl) were placed in metal cylinders on the surface of a media plate seeded with the organism being examined. the microorganism grows during incubation at 30° c; clear zones develop around cylinders, indicating the inhibition of growth. the size nova biotechnologica 9-3 (2009) 329 of the zone of inhibition caused by diffusion of agent into agar is directly related to the degree of susceptibility of an organism. 2.5 determination of phenol compounds the content of phenol substances was determined by singelton’s method (singelton et al., 1965). the amount of 0.2 ml of methanol extract was mixed with l ml of folin-ciocalteu's reagent and 0.8 ml of 7.5 % (w/v) na2c03. absorbance was measured at 750 nm after 30 minutes of incubation in dark. the content of polyphenols was compared with the absorbance of galic acid. 2.6 determination of flavonoids flavonoids were determined by rakotoarison’s method (rakotoarison et al., 1997). one ml of methanol extract was added to 1 ml of methanol solution of alcl3.6h20, 2 % (w/v), and absorbance was measured after 10 minutes at 394 nm. quercetin was taken as the standard. 3. results and discussion the first samples were tested before the flowering and the second samples were tested at the end of vegetal period. all cultivars showed different secondary metabolites during the vegetation period, their content in leaves decreased from the end of june to september (table 1). table 1. polyphenols content in hop leaves during the vegetation period. polyphenols [mg g-1 dm] cultivars 6/2008 9/2008 k-31 6.99 ± 0.14 4.53 ± 0.12 k-72 12.48 ± 0.36 3.03 ± 0.09 bor 14.34 ± 0.15 3.77 ± 0.20 sládek 12.32 ± 0.22 3.30 ± 0.12 zlatan 13.38 ± 0.14 3.57 ± 0.11 premiant 9.50 ± 0.33 4.68 ± 0.06 the maximum of content of polyphenols was found in cultivar bor, the cultivar with a high content of bitter acids, at the beginning of the vegetal period; it was more than twice of the content of cultivar k-31. the content of polyphenols at the end of the vegetation period was similar in all the tested hop cultivars. the experiment took place in 2008. some relevant data from 2007 were included for comparison (fig. 1). phenol substances were found in the interval between 5.00 – 8.83 mg g-1 of dry matter in 2007 compared to 9.50 to 14.34 mg g-1 of dry matter in 2008 in leaves before flowering, and from 0.90 to 2.25 mg g-1 of dry matter in 2007 compared to 3.03 – 4.68 mg g-1 of dry matter in 2008 in leaves at the end of the vegetal period. in the year 330 ürgeová, e. and polívka, ľ. 2008, there was less sunshine than in 2007 and there were more showers at the beginning and also during the time of flowering than in 2007. the quantity of phenol substances was higher in 2008 than in 2007. the highest content of polyphenols was detected in cultivar bor at the beginning of the vegetation period in 2008. 0 2 4 6 8 10 12 14 16 k-31 k-72 bor sládek zlatan premiant cultivars of hop po ly ph en ol s [m g g1 d m ] june 07 june 08 september 07 september 08 fig. 1. polyphenol content in hop leaves in 2007 and 2008. flavonoid content in leaves was similar, but different. we noted a decline during the vegetal period in both cases, as well as in phenol substances (table 2). table 2. content of flavonoids in hop leaves in 2007 and 2008. flavonoids [mg g-1 dm] cultivars 6/07 6/08 9/07 9/08 k-31 6.37 ± 0.51 0.24 ± 0.02 0.65 ± 0.02 0.10 ± 0.01 k-72 6.31 ± 0.49 1.43 ± 0.05 0.48 ± 0.02 0.20 ± 0.01 bor 3.45 ± 0.12 0.15 ± 0.01 0.71 ± 0.01 0.16 ± 0.02 sládek 3.24 ± 0.21 0.25 ± 0.01 0.76 ± 0.01 0.15 ± 0.01 zlatan 5.03 ± 0.22 0.14 ± 0.01 0.61 ± 0.01 0.13 ± 0.01 premiant 2.13 ± 0.09 0.19 ± 0.01 0.72 ± 0.01 0.18 ± 0.01 the quantity of flavonoids was higher in 2007. the content of flavonoids was within the range of 2.13 – 6.37 mg g-1 of dry matter in the leaves before flowering in 2007, and 0.14 to 1.43 mg g-1 of dry matter in 2008. the ranges decreased to 0.48 – 0.76 mg g-1 of dry matter of flavonoids at the end of vegetation in 2007. similarly, the interval of flavonoid content at the end of the vegetal period was 0.10 to 0.20 mg g-1 of dry matter in 2008. antibacterial activity was tested during the vegetation of hop plants. the marked inhibition effect of the extract of cultivar bor on erwinia mallotivora, 89% of inhibition effect of the standard tmtd, was noticed. the extracts from cultivars k31a had a similar effect on these bacteria, 67% of inhibition effect of the standard tmtd (fig. 2.). nova biotechnologica 9-3 (2009) 331 0 10 20 30 40 50 60 70 80 90 100 bor k-31 zlatan sládek premiant k-72 h op c ul ti va rs % of biocide effect of tmtd standard sept 08 july 08 june 08 april 08 fig. 2. biocide effect of hop extracts on the growth of erwinia malotivora. the average size of inhibition zones of extracts from leaves at the beginning of vegetation (april 08) was lower than the average size of extracts before the flowering time (june 08) (table 3). tab. 3. antibacterial effect of hop extracts during the vegetal period described as zones without the growth of bacteria on the agar plate in mm. hop cultivars bacteria vegetal period k-31 k-72 bor sládek zlatan premiant april 08 3 2 <1 2 2 2 june 08 4 3 2 4 2 2 e. amylovora sept 08 <1 1 2 <1 <1 1 april 08 3 3 3 2 2 <1 june 08 4 3 4 2 3 3 x. vesicatoria sept 08 4 2 <1 <1 <1 4 april 08 4 <1 2 <1 <1 2 june 08 4 5 3 1 6 4 p. carotovorum subsp. carotovorum sept 08 2 2 <1 4 <1 4 april 08 2 <1 <1 <1 2 4 june 08 4 3 3 2 3 4 p. syringae pv. syringae sept 08 <1 2 1 2 <1 2 april 08 <1 <1 <1 2 2 <1 june 08 2 3 1 3 2 3 c. michiganensis subsp. sepedonicus sept 08 1 1 <1 1 2 2 the inhibition effect of the extracts from hop leaves at the end of the vegetation period was minor. 332 ürgeová, e. and polívka, ľ. 4. conclusions the comparison of the results obtained during two years proved that the concentration of secondary metabolites, phenol substances and flavonoids depended mainly on the vegetal period. the influence of climatic conditions on the contents of these secondary metabolites of hop leaves was verified. the contents of these constituents were different in identical cultivars, compared to the years 2007 and 2008. the results of antibacterial activity suggested that this effect related mainly with sensibility of the bacterial strain and cultivar of hop. we observed the influence of the vegetation period, too. acknowledgement: this work was financially supported within the grants vega 1/0436/08 and av 4/2024/08. references bhattcharya, s., virani, s., zavro, m., haas, g., j.: inhibition of streptococcus mutans and other oral streptococci by hop (humulus lupulus l.) constituents. econ. bot., 57, 2003, 118-125. čeh, b., kač, m., iztok, j., košir, l., abram, v.: relationships between xanthohumol and polyphenol content in hop leaves and hop cones with regard to water supply and cultivar. int. j. mol. sci., 8, 2007, 989-1000. gerhauser, c.: broad spectrum anti-infective potential of xanthohumol from hop (humulus lupulus l.) in comparison with activities of other hop constituents and xanthohumol metabolites. mol. nutr. food res., 49, 2005, 827-31. hofta, p., dostálek p., basařová, g.: xantohumol − chmelová pryskyřice nebo polyfenol? chem. listy, 2004, 98, 825-830. langezaal, c.r.; chandra, a.; scheffer, j.c.: antimicrobial screening of essential oils and extracts of some humulus lupulus l. cultivars. pharm. weekbl., 1992, 14, 353-356. moir, m.: hops a millenium review. j. am. soc. brew. chem., 58, 2000, 131-146. piddock, j. v. l.: techniques used for the determination of antimicrobial resistance and sensitivity in bacteria. j. appl. bacteriol., 68, 1989, 307-318. rakotoarison, d., a., gressier, b., trotin, f., brunet, c., dine, t., luyckx, m., vasseur, j., cazin, m., cazin, j., c., pinkas, m.: antioxidant activites of polyphenolic extracts from flowers, in vitro callus and cell suspension cultures of crataegus monogyna. pharmazia, 52, 1997, 60-63. singleton, v., l., rossi, j., a.: colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. am. j. enol. vitic, 16, 1965, 144-158. srinivasan, v., goldberg, d., haas, g.j.: contributions to the antimicrobial spectrum of hop constituents. econ. bot., 58, 2004, s230-s238. stavri, m., schneider, r., o‘donnell, g., lechner, d., bucar, f., gibbons, s.: the antimycobacterial components of hops (humulus lupulus l.) and their dereplication. phytother. res., 18, 2004, 774-776. microsoft word závodská et al nb 9-3.doc nova biotechnologica 9-3 (2009) 303 physical-chemical characterization of uranium containing sediments lucia závodská1, eva kosorínová2, juraj lesný2, dušan bodiš3 1department of nuclear chemistry, comenius university in bratislava, mlynská dolina, bratislava, sk842 15, bratislava 4, slovak republic (zavodskalucia@centrum.sk) 2department of biotechnology, university of ss cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (lesnyj@ucm.sk) 3state geological institute of dionýz štúr, mlynská dolina 1, bratislava 11, sk-817 04, slovak republic (dusan.bodis@geology.sk) abstract: the presented paper is intended to study the chemical behaviour of combined geogenicanthropogenic uranium content in specific stream sediments. the sampling points have been chosen with respect to the natural conditions in the locality of groundwater outflow from a former uranium mine adit in považský inovec mountain range, near kálnica village. besides the total uranium determination and physical-chemical characterization of the relevant waterand sediment samples we carried out modified tessier type sequential fractionation extractions of naturaland artificially contaminated sediment samples after time dependent agitation in air and in the atmosphere of argon. the obtained results have been compared with those fulfilled with montmorillonite k-10. the total uranium concentrations of the relevant groundwater samples as well as of stream sediments have been determined by icp-ms using hp 4500. the determinations of uranium in extracts have been accomplished according stn757614, utilizing arsenazo iii as a selective complex forming reagent for spectrophotometric determination of uranyl-ions at 650 nm. the total uranium concentration of the groundwater outflow and in the sediment taken in its immediate vicinity has been 31.75±0.35 μg dm-3 and 38.0±2.7 μg g-1 respectively. unlike montmorillonit k-10, in which the carbonate-bound fraction of uranium after 1 week aeration and agitation in argon atmosphere represents 22.8% and 18.6% respectively, uranium in investigated sediments has been present predominantly in carbonate-bound fraction-reaching under similar conditions 38.6% and 26.6%, respectively. key words: uranium, sediments, groundwater, sequential fractionation 1. introduction uranium is an element of highly specific chemical, physical and biological properties. mostly because of its essentiality for nuclear energy production and its toxicity, the interest in its environmental mobility attracts constantly increasing attention. among basic features of uranium the following ones are crucial: it is a typical lithophilic natural radioactive component of the environment and contrary to its notoriety its radiotoxicity is negligible in comparison with chemical toxicity. the substantial chemical properties of uranium decisively influencing its transport in the environment as well as its determination possibilities are outlined in our earlier papers (závodská et al., 2008; szabó-nagy et al., 2009). uranium in natural waters may be present in four different oxidation states (u(iii), u(iv), u(v) and u(vi). in general, the tetravalent and hexavalent uranium species are stable in aqueous media, but the u(iv) –ion is only stable under reducing condition. a few solid and semi-metalis compounds such as uo and us exist in the formal oxidation 304 závodská, l. et al. state u(ii), although no simple ions are known to exist in solution for that state. the most prevalent form of uranium in aqueous solution is the light yellow, fluorescent uranyl ion ( uo +22 ). the u 4+ cation (green in solution) can be obtained by strong reduction of u(vi), but in the air it oxidizes back to uo +22 . the pentavalent ion uo+2 can be reversibly formed by reduction of uo +22 , but it readily disproportionates into u(iv) and u(vi). the trivalent u3+ can be formed by reduction of u(iv) but it is unstable as it oxidizes in aqueous solution (greenwood and earnshaw, 1997). 2. pe and ph dependence of u oxidation states in natural waters the thermodynamic principles of advanced aquatic chemistry including modelling methods are fairly discussed in a series of recent monographs (benjamin, 2002; morel and hering, 1993; grenthe and puigdomenech, 1997). putting to data set the updated thermodynamic values concerning uranium hydroand carbonate species (guillaumont et al., 2003) and utilizing the freeware phreeqc version 2.15.0 (usgs, 2008) we calculated the proportions of u(iii), u(iv), u(v) and u(vi) concentrations in dependence of pe and ph introducing the following model conditions (fig. 1-4): total uranium: 0.00005 mmol dm-3; alcalinity: 3.0 mmol dm-3; calcium: 1.5 mmol dm-3; temperature: 298.15 k; thermodynamic data: u4+ + e = u3+ (log k = -9.353; δh = 102.098 kj) u4+ + 4 h2o = u(oh)4 + 4 h + (log k = -8.538; δh = 103.596 kj) u4+ + 5 h2o = u(oh)5 + 5 h + (log k = -13.147; δh = 115.395 kj) u4+ + 2 h2o = uo+2 + 4 h + + e (log k = -6.432; δh = 130.248 kj) u4+ + 2 h2o = uo +22 + 4 h + + 2e (log k = -9.217; δh = 144.055 kj) uo +22 + h2o = ou2oh + + h+ (log k = -5.250; δh = 46.087 kj) 2 uo +22 + 2 h2o = (uo2)2(oh) +2 2 + 2 h + (log k = -5.620; δh = 150.791 kj) 3 uo +22 + 5 h2o = (uo2)3(oh) + 5 + 5 h + (log k = -15.550; δh = -185.226 kj) uo +22 + co _23 = uo2co3 (log k = 9.94; δh = 5.000 kj) uo +22 + 2 co _23 = uo2 (co3) _2 2 (log k = 16.61; δh = 8.500 kj) uo +22 + 3 co _23 = uo2 (co3) _4 3 (log k = 21.840; δh = -39.200 kj) fig. 1. proportion of u(iii) species in model natural waters in dependence of pe and ph. nova biotechnologica 9-3 (2009) 305 fig. 2. proportion of u(iv) species in model natural waters in dependence of pe and ph. fig. 3. proportion of u(v) species in model natural waters in dependence of pe and ph. fig. 4. proportion of u(vi) species in model natural waters in dependence of pe and ph. 306 závodská, l. et al. 3. materials and methods 3.1 sediment and water sampling, sample site and sample points the sampling of the investigated sediments and water has been performed on november 20, 2008 according to iso 5667-12 (guidance on sampling of bottom sediments) and iso 5667-15 (guidance on the preservation and handling of sludge and sediment samples). the sample site has been chosen in close proximity of a former uranium mine adit in považský inovec mountain range (fig. 5). uranium mineralization in perm of považský inovec mts. (kálnica, selec) is stratiform type (rojkovič, 1980; ivanička et al., 2007). major elements of mineralization act in psammitic and psefitic rocks. increased accumulation of uranium and accompanying minerals arose due to clastic material acquisition and by precipitation from aqueous solution. an important source of uranium was volcanisms of quartz porphyry and partly exposed crystalline rocks. during sedimentation and diagenesis in the productive complex of strata occurred the accumulation of uranium and accompanying minerals and during the alpine orogeny their concentration in the form of lenses. the most common ore minerals are pyrite, uraninite and rutile. the main elements of mineralization are u, pb, cu, mo nova biotechnologica 9-3 (2009) 307 and coincident elements ni, co and partly y and v. sampling point 3 (n 48°44.81'; e 17°57.68) has been chosen directly at the groundwater outflow from the uranium mine, which flows to the stream prostredný potok. sampling points 2 (n 48°44.80'; e 17°57.68) and 1 (n 48°44.22'; e 17°57.32) have been chosen at the above mentioned stream, approx. 25 and 50 m respectively north-west from the outflow. 3.2 physical-chemical characterization of the sediment-water system the primary characterization of the investigated system – the measuring of the water column height, the ph and eh of water and sediment, the thickness of the sediment and the definition of the sediment colour have been accomplished in situ. the additional assays – the ph determination of the wateras well as the 0.01 mol dm-3 cacl2 extracts of the sediments, the sediments dry matter determination, the measuring of the water conductivity, the sedimentation test (for the sample taken at sampling point 3) as well as the granulometric analysis (for the sample taken at sampling point 1) – have been accomplished in the laboratory. the sedimentation test has been realized with 20 g air dry sediment in 100 cm3 graduated cylinder. for the wet granulometric analysis (utilizing water taken at the identical sampling point) 2.0 mm, 1.0 mm, 0.25 mm, 0.125 mm and 0.06 sieves have been applied and the fractions have been weighed after drying at laboratory temperature. the mineralization of the samples (sediments from sampling points 2 and 3 as well as the reference sediment gbw 07306) has been executed using the microwave sample digestion system multiwave 3000 (anton paar, austria). 0.25 g air dry sediments have been treated with 5 cm3 conc. hno3 and 2 cm3 conc. h2so4 at tmax= 496 k and p = 2.6 . 10 6 pa (sediment 2 and gbw 07306) as well as tmax= 449 k and p = 1.36 . 10 6 pa (sediment 3). the total uranium concentrations in mineralized sediments have been determined by icp-ms (hp – 4500, hewlett packard, usa) utilizing icp-ms vi (merck) calibration solutions and certified reference sediment tmda 62 (national water research institute, canada). the uranium concentrations in particular fractionation extracts have been determined by spectrophotometry according stn 75 7614 (water quality. determination of uranium), using uv-vis spectrophotometer cary 50 bio, varian, australia. the extraction fractionations of the sediments have been accomplished applying a slightly modified tessier type sequential extraction procedure. in order to study the influence of oxygen to the uranium mobility in the watersediment system we realized a time dependent aeration of investigated materials in contact with uranium contaminated water. 1 g of air dry sediment 3 has been placed in a test-tube and 8 cm3 of water has been added. to 1 g of sediment 2 and montmorillonite k-10 placed in the same type of test-tubes 8 cm3 of uranium acetate solution (uranium concentration 12 mg dm-3) has been added. using a simple laboratory aeration apparatus a time dependent aeration (1h, 1 day, 1 week) has been accomplished and after centrifugation (4000 rpm, 300 s) the modified tessier type sequential extraction has been carried out. the same experiment has been fulfilled, using argon atmosphere. 308 závodská, l. et al. 4. results and discussion the obtained results of the primary characterization of the investigated system are shown in table 1 (in situ determinations) and in table 2 (laboratory determinations). table 1. in situ determinations. h: water column height, th: sediment thickness, col: sediment colour, * water sampling points identical with 1, 2, 3 sediment sampling points. h [cm] th [cm] col ph (25°c) pe sediment 1 2 7.5 grey ––– –––– sediment 2 2 7.5 brown ––– –––– sediment 3 2 7.5 orange ––– 0.76 (8.7°c) water 1* ––– ––– –––– 8.15 + 2.96 (7.4°c) water 2* ––– ––– –––– 7.86 + 1.59 (7.5°c) water 3* ––– ––– –––– 7.40 + 0.35 (8.7°c) table 2. laboratory determinations. d: dry substance. extract ph d (1h, 105°c) h2o 0.01mcacl2 χ [μs cm-1] sediment 1 74.95% 7.33 6.93 ––– sediment 2 72.69% 7.90 7.52 ––– sediment 3 13.26% 7.85 7.42 ––– water 1* ––– ––– –––– 773 (17.9°c) water 2* ––– ––– –––– 887 (18.3°c) water 3* ––– ––– –––– 873 (18.7°c) moving away from the outflow we observed moderately increasing ph values of water, slightly decreasing ph values of 0.01 m cacl2 sediment extracts, as well as increasing oxidation-reduction potential. the mentioned facts are obviously connected with decreasing dissolved co2 and increasing dissolved o2 concentrations as well as with the subsequent oxidation of several metal ions causing production of their precipitates. sedimentation curves of sediment 3 (not shown) confirm the apparent and simply distinguishable homogeneity of the clayey sediment, which even after 3 days did not show any tendency to swell. the granulometric analysis of sediment 1 showed a substantial fraction, namely 56.8% of grains sized >2 mm, 30.5% of the grain fraction 0.25 – 1.0 mm, ~4.7% of the grain fraction 0.25 – 0.125 mm and ~4.5% of the grain fraction 0.125 – 0.06%. according the recent knowledge (breitner et al., 2008) uranium is preferentially bound to the finest sediment grains and therefore the experiments connected with the sequential extractions have been fulfilled with samples of the grain fraction 0.125 – 0.06 mm. nova biotechnologica 9-3 (2009) 309 icp-ms determinations of total uranium have been performed for groundwater outflow at sampling point 3, for sediment 2 and for sediment 3 in duplicate samples. we obtained the following results: 31.75±0.35 μg dm-3 for groundwater outflow; 38.0±2.71 μg g-1 for sediment 3 and 9.0±0.59 μg g-1 for sediment 2. uranium concentrations in particular fractions of sediment 2 and sediment 3 using the applied sequential extractions (modified tessier type procedure) are shown in table 3. table 3. uranium concentrations in the investigated sediment fractions using modified tessier type procedure. f0: water soluble fraction, f1: ion-exchangeable fraction, f2: carbonate-bound fraction, f3: fe/mn oxides-bound fraction, f4: organic matter-bound fraction. uranium concentration [μg g-1] fraction sediment 2 sediment 3 f0 –––– 1.550±0.533 f1 –––– 1.620±0.533 f2 0.789±0.725 12.935±0.559 f3 0.893±0.722 1.113±0.658 f4 –––– 1.742±0.532 1 2 3 4 5 6 0 10 20 30 40 50 60 70 m on tm or illo ni t se di m en t 3 se di m en t 2 u ra ni um p or tio n in p ar tic ul ar fr ac tio ns [% ] m on tm or illo ni t se di m en t 3 se di m en t 2 argono 2 + co 2 f0 f1 f2 f3 fr fig. 6. comparison of uranium fractionation in sediment samples and montmorillonite k-10 after 1 week aeration and argonation. f0: water soluble fraction, f1: ion-exchangeable fraction, f2: carbonate-bound fraction, f3: fe/mn oxides-bound fraction fr: residual fraction. considering the obtained results it is straightforward, that for sediment 3 the prevailing amount of uranium is in the carbonate-bound fraction. this finding is in accordance with the published papers (bunzl et al., 1998 and martin et al., 1998). for sediment 2 the same fraction markedly drops and in the same time the fe/mn oxides-bound fraction become the most uranium containing one. the mentioned facts are with high probability connected to the quick aqueous co2concentration fall in the water as well as with a consequent uranium species sorption on the stream sediments. 310 závodská, l. et al. via aeration/argonation experiments (outlined in section 3.2) we gained a valuable comparison of time dependent uranium fractionation in investigated sediments vs. in montmorrillonite k-10 with and without contact with air. fig. 6 illustrates the obtained results after 1 week of aeration and argonation respectively. 5. conclusions while the proportion of u(iii) species in uranium contaminated natural waters at pe -5 and ph 4 hardly reach 1% and u(v) species exist only in relatively narrow pe and ph range, a considerable portion of uranium may exist in u(iv) oxidation state, namely at relatively low oxidation-reduction potential (pe values from -5 to 0) and the main portion in u(vi) oxidation state at positive pe values. the uranium concentration of the investigated water outflow from the former uranium mine adit reached as much as ~31 μg dm-3. in the investigated conditions the uranium in water is present predominantly in u(vi) oxidation state. with raising distance from the spring the water has a tendency of increasing pe and decreasing ph values. in montmorillonite k-10 the carbonate-bound uranium fraction after 1 week of aeration reached ~22%. in aerated conditions uranium has been present in carbonate-bound fraction of investigated sediments in much higher concentration reaching ~40%. the presence of air in all cases gave rise to a measurable increase of uranium proportion in biologically available fractions. compared to montmorillonite k-10, where the influence of air has been smaller, in similar conditions the biologically available uranium fractions unambiguously increased reaching 30-35% after 1 week aeration. acknowledgements: we would like to express our sincere thanks to dr. adriana shearman for accomplishing the icp-ms determinations. references závodská, l., kosorínová, e., ščerbáková, l., lesný, j.: environmental chemistry of uranium. hej, env-081221-a, 2008, 1-19. szabó-nagy, a., závodská, l., mátel, ľ., lesný, j.: geochemistry and determination possibilities of uranium in natural waters. acta tech. jaurin., 2009, in press. greenwood, n.n., earnshaw, a.: chemistry of the elements. second edition. butterworth-heinemann, elsevier science, linacre house, jordan hill, oxford ox2 8dp, 1997. united states geological survay: phreeqc version 2.15.0, 2008. benjamin, m.m.: water chemistry. mcgraw-hill, singapore, 2002. morel, f.m.m., hering, j.g.: principles and applications of aquatic chemistry. john wiley & sons, usa, 1993. grenthe, i., piugdomenech, i.: modelling in aquatic chemistry. nea&oecd, france, 1997. guillaumont, r., fanghänel, t., fuger, j., grenthe, i., neck, v., palmer, d.a., rand, m.h.: update on the chemical thermodynamics of nova biotechnologica 9-3 (2009) 311 uranium, neptunium, plutonium, americium and technetium. chemical thermodynamics, vol. 5, elsevier, the netherlands, 2003. rojkovič, i.: geochemická charakteristika uránového zrudnenia v perme považského inovca. manuskript, geofond bratislava, 1980. ivanička, m., havrila, m., kohút, m. (editors): geologická mapa regiónu považský inovec a jv. časti trenčianskej kotliny. 1:50 000, štátny geologický ústav dionýza štúra, bratislava, 2007. breintner, d., turtiainen, t., arvela, h., vesterbacka, p., johanson, b., lehtonen, m., hellmuth, k.h., szabó, c.: multidisciplinary analysis of finnish esker sediment in radon source identification. sci. total environ., 405, 2008, 129-139. bunzl, k., kracke, w., schimmack, w., zelles, l.: forms of fallout 137cs and 239+240pu in successive horizons of forest soil. j. environ. radioact., 39, 1998, 55-68. martin, r., sanchez, d.m:, gutierrez, a.m.: sequential extraction of u, th, ce, la and some heavy metals in sediments from ortigas river, spain. talanta, 46, 1998, 1115-1121. microsoft word vaverkova nb 9-2.doc nova biotechnologica 9-2 (2009) 191 the possibility to enhance flavonoids production in rubia tinctorum l. callus cultures daniela kákoniová1, štefánia vaverková2 , desana lišková1, eva urgeová3, zuzana juráková1 1institute of chemistry slovak academy of sciences, dúbravská cesta 9, sk-845 38 bratislava, slovak republic (chemdaka@savba.sk) 2department of botany and pharmacognosy, faculty of pharmacy comenius university, kalinčiakova 8, sk-832 32 bratislava, slovak republic (vaverkova@fpharm.uniba.sk) 3faculty of natural sciences, university of ss. cyril and methodius, trnava, slovak republic abstract: production of flavonoids in madder callus culture (rubia tinctorum l.) was dependent on culture conditions and culture media composition. the content of flavonoids increased in calli maintained on media supplemented with naa (4 mg.l-1) or naa:bap (4 mg.l-1 a 1 mg.l-1) in 16 h photoperiod. flavonoids represented 2.08 – 2.25 % of callus dry mass. the presence of cd(no3)2 (3.1 or 31.0 mg.l-1 concentrations) negatively influenced callus growth, but enhaced the percentage of dry mass in callus cells. during 42 days of culture an increase of cadmium accumulation and even of flavonoids has been observed. the most considerable influence of cdcl2 or cd(no3)2 on flavonoids content has been shown in short-term experiments after 48 h of callus culture. more distinct influence has been observed under the treatment with cdcl2 (0.005 mg.l-1) in comparison with cd(no3)2. key words: cadmium, callus, growth, madder, secondary metabolites 1. introduction the effort to use natural resources of vegetal and animal origin supports detailed investigation of familiar plant species for discovery of their still unknown impact against negative factors of the environment. cultivation of medicinal plants is in the focus worldwide. this is evoked not only by the appearance of numerous civilization deseases, but also by the new look on prophylaxis and food health. prominent plant species contain secondary metabolites usable in food industry, cosmetics, and mainly in pharmaceutical industry. besides standard application and isolation of secondary compounds from natural resources, increasing significance win biotechnologies aimed on bioactive metabolites production and enhancement of their production abilities, predominantly plant cell cultures in vitro (pšenáková et al., 2003; vanisree and tsay, 2004; vanisree et al., 2004). cell and callus cultures represent a continual renewable source of plant biomass and unlimited resource of desired pharmaceutical products. madder (rubia tinctorum l.) is a medicinal plant (thurzová et al., 1983). secondary compounds isolated from its root have diuretic effect and come in use by the treatment of kidney stones and as an auxilliary means in medication of rachitis and 192 kákoniová, d. et al. anaemia. this plant, coming from the south of europe, was grown for red dye, alizarine. this drug contains also flavonoids, compounds with antioxidative effect. commercial importance of flavonoids and a need for renewable resources of valuable chemicals has lead to attempts in developing alternative systems for their production. different in vitro systems have been developed for flavonoids production, e.g., callus, cell suspension cultures, root and shoot cultures (jedinák et al., 2004). biotic and abiotic elicitors can enhance secondary metabolites production. the stimuli are perceived by receptors activating secondary messengers. these transmit signals into the cell through signal transduction pathways leading to gene expression and biochemical changes (sudha and ravishankar, 2002) resulting in compounds formation. the basis for successful elicitation of secondary metabolites is the choice of suitable elicitor, its concentration, and optimal time of treatment. many plant secondary metabolites are involved in the interaction of the plant with the environment. increased contamination of the environment by toxic metals has negative consequence for all kinds of organisms, including higher plants. toxic metals in high concentrations inhibit growth and development of plants and disturb or change their biochemical and physiological processes. cadmium is one of common industrial pollutants, harmful to plants already at low concentrations (nehnevajová, 2002; šupalová, 2004). the aim of this study was to detect the potential of madder (rubia tinctorum l.) callus cultures to produce flavonoids in dependency on culture media composition, physical conditions, combination and concentration of growth regulators, and the effect of cd-salts cd(no3)2 and cdcl2. 2. material and methods 2.1 cultivation of callus cultures callus cultures of madder (rubia tinctorum l.) belong to the collection of in vitro cultures at the institute of chemistry sas in bratislava. callus cultures were isolated from leaf segments of madder seedlings and cultured on modified murashigeskoog (ms) medium (1962) or on z medium (čierna et al., 1991) at 25 ± 1 °c, under 60 % relative air humidity, 16 h photoperiod, irradiance of 45-60 µmol m-2.s-1, or in the dark. the callus cultures were subcultured every four weeks. 2.2 growth parameters and statistics the growth dynamics (∆ri(j+7) = ri(j+7) rij, ri = ∆m/m0, ∆m = m – m0, m = fresh mass, m0 = initial mass of inoculum, j = day of culture) was statistically evaluated by student`s t-test and anova. in all experiments 10 samples were used. the experiments were repeated twice. 2.3 elicitation of flavonoids the possibility to increase the content of flavonoids has been examined on media supplemented with naa:bap in various ratios 2:0, 2:1, 4:0, 4:1 (mg.l-1) under 16 h nova biotechnologica 9-2 (2009) 193 photoperiod or in the dark, in the presence of cadmium salts: cd(no3)2 or cdcl2 in different concentrations (0.005 mg.l-1, 0.05 mg.l-1, 0.5 mg.l-1, 3.1 mg.l-1, and 31.0 mg.l1) during a short-term culture (24, 48, and 168 hours), during 42 days on ms solid media, or with liquid media on paper bridges. 2.4 determination of flavonoids content flavonoids were determined colorimetrically from lyophilized dry mass. callus cultures were collected during the cultivation period, lyophilized, homogenized, and pulverized. the drug was subsequently extracted with acetone, shaked with etoac and then the samples were evaluated at 425 nm on spekol carl zeiss – jena 2 by modified method of tůmová and rusková (1998). the average content of flavonoids was calculated from standard curve of quercetin (europ. formulary 1). 2.5 determination of cadmium content the content of cadmium in madder cells was analysed at the institute of geology, faculty of natural sciences, comenius university in bratislava, bratislava, slovakia by nuclear absorption spectrophotometry. 3. results and discussion callus cultures of madder (rubia tinctorum l.) isolated from leaf segments and cultivated on two different media (ms or z) showed similarity in growth and growth dynamics (fig. 1a, 1b). the content of flavonoids in cells altered as an answer on growth hormones in culture media and light period (16 h photoperiod or in the dark). their values oscilated between 1.65 and 2.25 % of callus dry mass (fig. 2). the highest values of flavonoids were determined in calli grown at 16 h photoperiod on media containing naa:bap in the ratio 4:0 or 4:1 (mg.l-1). in these cases flavonoids made up 2.08 up to 2.25 % of dry mass (fig. 2). plant growth hormones represent important and in many cases essential compounds of culture media as for callus cultures growth, so for secondary metabolites production (siatka, 1998). type and concentration of growth hormones are specific for every culture grown in vitro. their mutual combinations are determined experimentally. the highest amounts of flavonoids in bellis perennis l. callus culture were determined on media supplemented with 2,4-d in 0.1 and 1 mg.l-1 concentration, or with iaa in 0.1 mg.l-1, possibly also with naa (1 mg.l-1). lower or higher concentrations of growth hormones reduced the production of flavonoids (siatka, 1998). the content of flavonoids in rubia tinctorum l. callus cells depended on the photoperiod length and naa concentration, and the combination with bap in culture media. the highest values of flavonoids were detected in calli growing on media with naa (4 mg.l-1) or when naa (4 mg.l-1) was combined with bap (1 mg.l-1) and grown in 16 h photoperiod (fig. 2). it is known that some metals may positively affect the production of secondary metabolites (zheng and wu, 2004; rai et al., 2005). cadmium e.g. enhances the 194 kákoniová, d. et al. 0 0,5 1 1,5 2 2,5 3 0 7 14 21 28 35 42 day m /m 0 z ms a 0 0,2 0,4 0,6 0,8 1 0 7 14 21 28 35 42 day δ r i z ms b biosynthesis of ajmalicine in suspension cultures of catharanthus roseus during 24 – 48 h of cultivation (zheng and wu, 2004). fig. 1. rubia tinctorum l. callus culture growth (a), growth dynamics of rubia tinctorum l. callus culture (b). 0 0,5 1 1,5 2 2,5 1 2 3 4 5 6 7 8 media % o f f la vo n o id s ms z fig. 2. effect of ms and z modified media and physical culture conditions (16 h photoperiod/dark) on flavonoids content in callus cultures of rubia tinctorum l. naa:bap (mg.l-1) 1 – 2:0; 2 – 2:1; 3 – 4:0; 4 – 4:1 – dark; 5 – 2:0; 6 – 2:1; 7 – 4:0; 8 – 4:1 – 16 h photoperiod noticeable increase of rosmarinic acid in suspension cultures of melissa officinalis l. was observed at the end of the second subculture (4 weeks of culture) by šupalová (2004) when cultivated on media supplemented with 31.0 mg.l-1 cd(no3)2. in this case the content of rosmarinic acid was comparable with its content in intact plants. the presence of cd(no3)2 in 3.1 and 31.0 mg.l -1 concentrations influenced negatively madder callus growth (fig. 3a), but increased the % of dry mass after 42 days of culture (fig. 3b). the treatment of phyllanthus amarus with a cd-salt (higher than 50 ppm and up to 100 ppm) significanly inhibited their growth. decrease of dry mass, content of proteins, chlorophyll, and saccharides was evident. in contrast the content of starch nova biotechnologica 9-2 (2009) 195 -1 -0,5 0 0,5 1 1,5 2 0 7 14 21 28 35 42 day δ r i c cd1 cd2 a 0 2 4 6 8 7 14 21 28 35 42 day % o f d .m . c cd1 cd2 b 0 100 200 300 400 500 7 14 21 28 35 42 day c d μ g .g 1 cd1 cd2 c 0 0,5 1 1,5 2 7 14 21 28 35 42 day % o f f la vo no id s c cd1 cd2 d increased, similarly as therapeutically active compounds – phyllantine and hypophyllantine (rai et al., 2005). in madder cells cadmium accumulation corresponded with its concentration in culture media and duration of the treatment (fig. 3c). fig. 3. growth dynamics (a), percentage of dry mass (b), accumulation of cadmium (c) and content of flavonoids (d) of rubia tinctorum l. callus culture on ms medium supplemented with cd(no3)2. c – control; cd1 – 3.1 mg.l-1; cd2 – 31.0 mg.l-1 these results are supported also by the findings of nehnevajová (2002) in ginkgo biloba l. callus culture. the highest concentration of cd(no3)2 during 4 subcultures enhanced the toxic effect of this salt on callus growth parameters, callus pigmentation, cells plasmolysis, and cell wall irregular thickenings. in madder callus, at the 31.0 mg.l-1 cd(no3)2 concentration, the content of cd increased already on the 7th day of culture and this trend continued till the day 42 of culture on agar media (479 µg.g-1 d.m.) (fig. 3c). the production of flavonoids was time-shifted at both concentrations of the cd-salt compared with the control. the presence of 3.1 mg.l-1 cd(no3)2 caused moderate stimulation already after 21 days with the maximum value on the 35th day of culture. the highest values of flavonoids on media supplemented with the higher concentration (31.0 mg.l-1) of cd(no3)2 on 28 th day were determined (fig. 3d). production of flavonoids in madder callus cells cultured on paper bridges was positively affected by all concentrations of cdcl2 and cd(no3)2 tested, mainly after 24 and 48 h of culture. most significantly was this process affected by the concentration 0.005 mg.l-1 of cdcl2, when the content of flavonoids increased by 57-64 % in comparison with the control (fig. 4). 196 kákoniová, d. et al. 0 0,4 0,8 1,2 1,6 2 c 1 2 3 4 5 6 media % fl av o n o id s 24 h 48 h 168 h fig. 4. content of flavonoids in rubia tinctorum l. callus cultures grown on paper bridges. c control; 1 0.005 mg.l-1 cdcl2; 2 0.05 mg.l-1 cdcl2; 3 0.5 mg.l-1 cdcl2; 4 0.005 mg.l-1 cd(no3)2; 5 0.05 mg.l-1 cd(no3)2; 6 0.5 mg.l-1 cd(no3)2. during this short-term culture no growth or uptake of cd-salts by cells growing on paper bridges were observed. very diminutive uptake was determined at the 0.005 mg.l-1 concentration of cdcl2. this effect could be the result of ions interaction in liquid medium and of the short-term cd-salt treatment. tůmová and rusková (1998) have also ascertained a positive effect of cdcl2 as well as cuso4 on the production of flavonoids in ononis arvensis l. callus culture. they have observed that cdcl2 in 0.05 mg.l -1 or 0.5 mg.l-1 concentrations significantly increased flavonoids content after 48 h, but the lowest concentration (0.005 mg.l-1) affected it already after 24 h of culture. it can be concluded that plant growth hormones, and also cd-salts (in suitable combination and concentration) in connection with physical conditions and length of the treatment, enhance the production of secondary metabolites in madder (rubia tinctorum l.) cells cultured in vitro. acknowledgement: this work was supported by slovak grant agency vega, grant no. 1/4354/07, apvv-cost -0004-06, and cost action 859. references čierna, m., kákoniová, d., lišková, d.: a medium for rapid plant callus growth. biologia, 46, 1991, 271-272. european pharmacopoea (ph.eur.) 4-nd. ed. maissoneueve, france, 2000, 375-376 pp. nova biotechnologica 9-2 (2009) 197 jedinák, a., faragó, j., pšenáková, i., maliar, t.: approaches to flavonoid production in plant tissue cultures. biologia, 59, 2004, 697-710. murashige, t., skoog, f.: a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiol. plant., 15, 1962, 473-497. nehnevajová, e.: effect of cadmium on growth parameters of ginkgo biloba l., thesis, comenius university, bratislava, 2002, 89 pp. [in slovak] pšenáková, i., faragó, j., vašková, l.: application possibilities of in vitro plant systems for the production of secondary metabolites. nova biotechnol. iii-2, 2003, 103-113. [in slovak] rai, v., khatoon, s., bisht, s.s., mehrotra, s.: effect of cadmium on growth, ultramorphology of leaf and secondary metabolites of phyllanthus amarus schum. and thonn. chemosphere, 61, 2005, 1644-1650. siatka, t.: effect of auxins on bellis perennis l. callus cultures growth and flavonoids production. čes. slov. farm., 47, 1998, 273-275. [in czech] sudha, g., ravishankar, g.a.: involvement and interaction of various signaling compounds on the plant metabolic events during defense response, resistance to stress factors, formation of secondary metabolites and their molecular aspects. plant cell tiss. org. cult., 71, 2002, 181-212. šupalová, a.: the influence of cadmium on growth parameters and content of rosmarinic acid in callus and suspension cultures of lemon balm (melissa officinalis l.), thesis, comenius university, bratislava, 2004, 113 pp. [in slovak] thurzová, ľ., kresánek, j., mareček, š., mika, k.: small atlas of medicinal plants, osveta, bratislava, 1983, 324-325 pp. [in slovak] tůmová, l., rusková, r.: effect of cdcl2 and cuso4 on flavonoids production in ononis arvensis l. cultured in vitro. čes. slov. farm., 47, 1998, 261-263. [in czech] vanisree, m., tsay, h.-s.: plant cell cultures – an alternative and efficient source for the production of biologically important secondary metabolites. int. j. appl. sci. eng., 2, 2004, 29-48. vanisree, m., lee, ch.-y., lo, s.-f., nalawade, s.m., lin, ch.y, tsay, h.-s.: studies on the production of some important secondary metabolites. bot. bull. acad. sin., 45, 2004, 1-22. zheng, z.u., wu, m.: cadmium treatment enhances the production of alkaloid secondary metabolites in catharanthus roseus. plant sci., 166, 2004, 507-514. microsoft word janecek 9-1 2009.doc nova biotechnologica 9-1 (2009) 5 amylolytic enzymes focus on the alpha-amylases from archaea and plants štefan janeček1,2 1department of biotechnology, university of ss. cyril and methodius, j. herdu 2, sk-917 01 trnava, slovak republic (stefan.janecek@ucm.sk) 2institute of molecular biology, slovak academy of sciences, dúbravská cesta 21, sk-845 51 bratislava, slovakia (stefan.janecek@savba.sk) abstract: amylolytic enzymes represent a group of starch hydrolases and related enzymes that are active towards the α-glycosidic bonds in starch and related polyand oligosaccharides. the three best known amylolytic enzymes are α-amylase, β-amylase and glucoamylase that, however, differ from each other by their amino acid sequences, three-dimensional structures, reaction mechanisms and catalytic machineries. in the sequence-based classification of all glycoside hydrolases (ghs) they have therefore been classified into the three independent families: gh13 (α-amylases), gh14 (β-amylases) and gh15 (glucoamylases). some amylolytic enzymes have been placed to the families gh31 and gh57. the family gh13 together with the families gh70 and gh77 constitutes the clan gh-h, well-known as the α-amylase family. it contains more than 6,000 sequences and covers 30 various enzyme specificities sharing the conserved sequence regions, catalytic tim-barrel fold, retaining reaction mechanism and catalytic triad. among the gh13 α-amylases, those produced by plants and archaebacteria exhibit common sequence similarities that distinguish them from the α-amylases of the remaining taxonomic sources. despite the close evolutionary relatedness between the plant and archaeal α-amylases, there are also specific differences that discriminate them from each other. these specific differences could be used in an effort to reveal the sequence-structural features responsible for the high thermostability of the α-amylases from archaea. key words: α-amylase, glycoside hydrolase families, sequence-structural features, archaebacteria, plants, evolutionary relatedness. 1. introduction starch is an important source of energy for a wide spectrum of animals (including humans), plants and microorganisms. it consists exclusively from glucose monomers that are linked by α-1,4and α-1,6-glycosidic linkages. amylose (15-25% of starch) is formed by α-1,4-linearly bound glucoses, whereas amylopectin (75-85% of starch) contains also the branching points with the α-1,6-linked glucoses (leveque et al., 2000b; bertoldo and antranikian, 2002). starch industry covers many well-developed and also recently established sophisticated technologies that utilize amylolytic enzymes. these amylases represent approximately 30% of the worldwide industrial enzyme production, the starch hydrolysis being considered to be the main way of their use (van der maarel et al., 2002). 2. amylolytic enzymes with regard to a complex structure of starch and related oligoand polysaccharides the starch-degrading organisms have to dispose by relevant 6 janeček, š. combination of starch hydrolases and related enzymes (legin et al., 1998; bertoldo and antranikian, 2002). these enzymes are in general called amylases. the amylolytic enzymes form a large group of starch hydrolases and related enzymes that are active towards starch, pullulan, glycogen and other related oligoand polysaccharides (vihinen and mantsala, 1989; pandey et al., 2000; janecek, 2009). it is a common way of binding of a glucose residue of the substrate in the enzyme active centre, termed conventionally as a substrate-binding subsite (davies et al., 1997), that is responsible for the activity of amylolytic enzymes. most of them belong to glycoside hydrolases (ghs) that constitute the individual gh enzyme families without mutual sequence similarities (henrissat, 1991). now the gh families are part of the cazy web-server (cantarel et al., 2009) that covers also other carbohydrate-active enzymes (fig. 1). fig. 1. carbohydrate-active enzyme (cazy) classification (http://www.cazy.org/). the individual proteins and enzymes are within the cazy server classified into four main groups of sequence-based families: (i) gh, glycoside hydrolases; (ii) gt, glycosyl transferases; (iii) pl, polysaccharide lyases; and (iv) ce, carbohydrate esterases. the cbm stands for the family classification of carbohydrate-binding modules. for details, see cantarel et al. (2009). the most known amylolytic enzymes are α-amylase (ec 3.2.1.1), β-amylase (ec 3.2.1.2) and glucoamylase (ec 3.2.1.3) that are, however, quite different from each other. they differ not only in their primary and tertiary structures, but also in their catalytic machineries and reaction mechanisms employed (janecek, 1994a; pujadas et al., 1996; coutinho and reilly, 1997). they have therefore been classified into different gh families: gh13 α-amylases, gh14 β-amylases, and gh15 glucoamylases (henrissat, 1991). nova biotechnologica 9-1 (2009) 7 the enzymatic hydrolysis of a glycosidic bond can be characterized by a general acid catalysis that requires two essential components: a proton donor (an acid) and a nucleophile (a base). according to the anomeric configuration of the resulting hydroxyl group with regard to conformation of the cleaved o-glycosidic linkage, two basic mechanisms exist for this hydrolysis (fig. 2): retaining or inverting (mccarter and withers, 1994). whereas α-amylase employs retaining mechanism (i.e. the products of its action are α-glucans), both β-amylase and glucoamylase are inverting hydrolases (i.e. they produce β-glucans). fig. 2. (a) retaining reaction mechanism of glycoside hydrolases (macgregor et al., 2001). the proton donor protonates the glycosidic oxygen and the catalytic nucleophile attacks at c1 leading to formation of the first transition state. the catalytic base promotes the attack of the incoming molecule roh (water in hydrolysis or another sugar molecule in trasnglycosylation) on the formation of the covalent intermediate resulting in a second transition state, leading to hydrolysis or transglycosylation product. (b) inverting reaction mechanism of glycoside hydrolases (sauer et al., 2000). the catalytic base (top) and acid (bottom) in the water-assisted hydrolysis of substrate leading to inversion of the configuration of the anomeric carbon. from the structural point of view (fig. 3), both α-amylase and β-amylase rank among the tim-barrel enzymes, i.e. they possess the (β/α)8-barrel catalytic domain, while glucoamylase adopts a helical version of catalytic tim-barrel, the so-called (α/α)6-barrel. within the cazy classification the α-amylases from the family gh13 with closely related families gh70 and gh77 constitute the clan gh-h that is wellknown as the α-amylase family (macgregor et al., 2001; cantarel et al. 2009). it is worth mentioning that some α-amylases with sequences and structures different from the main gh13 α-amylases have been placed to the family gh57 (janecek, 2005) and some amylolytic enzymes are present also in the family gh31 (nakai et al. 2005; kang et al., 2008). (b) (a) 8 janeček, š. fig. 3. three-dimensional structures of amylases. (a) gh13 α-amylase from aspergillus oryzae (pdb code: 2taa; matsuura et al., 1984); (b) gh14 β-amylase from soybean (1bya; mikami et al., 1993) and (c) gh15 glucoamylase from aspergillus awamori (1agm; aleshin et al., 1992). the catalytic machineries of gh13, gh14 and gh15 α-amylases, β-amylase and glucoamylases, respectively, are also different: whereas the enzymes from the family gh13 possess a catalytic triad formed by two aspartates and one glutamate (uitdehaag et al., 1999), both β-amylases (mikami et al., 1993) and glucoamylases (aleshin et al., 1992) have their catalytic machineries formed by two glutamic acid residues that are, however, not alignable due to mutual amino acid sequence differences (pujadas et al., 1996; coutinho and reilly, 1997). it thus could be summarised that amylases and related enzymes classified into the families gh13 (forming with gh70 and gh77 the clan gh-h), gh14, gh15 as well as gh31 and gh57 differ from each other by their amino acid sequences, threedimensional structures, catalytic machineries and reaction mechanism (janecek, 2009). 3. α-amylase enzyme family most of amylolytic enzymes are grouped in the α-amylase family (macgregor et al., 2001). it was originally recognised as a group of starch hydrolases and related enzymes (such as α-amylase, cyclodextrin glucanotransferase, neopullulanase, etc.) that exhibited sequence similarities and commonly predicted tim-barrel fold (macgregor and svensson, 1989; takata et al., 1992). within the sequence-based classification of ghs, it was originally established as the family gh13 (henrissat, 1991), but later the families gh70 and gh77 were added to form the presently well-accepted gh-h clan (macgregor, 2005; janecek, 2009). (a) domain b domain c (β/α)8-barrel (b) (β/α)8-barrel (c) (α/α)6-barrel nova biotechnologica 9-1 (2009) 9 3.1. clan gh-h the above-mentioned families gh13, gh70 and gh77 form the clan gh-h, i.e. the α-amylase family, which at present consists of 30 various enzyme specificities (table 1) and contains more than 6,000 sequences (cantarel et al., 2009). the members of the α-amylase family are not only hydrolases, but also transferases and isomerases. based on amino acid sequence similarities, even some heteromeric amino acid transporter proteins may be considered to be the non-amylolytic members of the clan gh-h (janecek et al., 1997) (fig. 4). not all family enzymes attack the glycosidic bonds in starch; they are active towards the analogous bonds in glycogen, pullulan and other related polyand oligosaccharides, like trehalose, sucrose, etc. (macgregor et al., 2001). whereas the family gh77 is a monospecific family, i.e. it contains only one enzyme specificity amylomaltase (alternative names 4-α-glucanotransferase or disproportionating enzyme; ec 2.4.1.25), the family gh70 consists of two specificties glucosyltransferase (glucansucrase; ec 2.4.1.5) and alternansucrase (ec 2.4.1.140), and the family gh13 is formed by all the remaining enzyme specificies (amylomaltase being also present). gh13 is thus taken as the main α-amylase family (macgregor et al., 2001). enzymes that are members of the α-amylase family have to obey the following four criteria (kuriki and imanaka, 1999; macgregor et al., 2001; janecek, 2002; van der maarel et al., 2002): (i) they act on α-glucosidic bonds (not only the α-1,4and α-1,6-linkages); (ii) they employ the retaining reaction mechanism; (iii) they contain from 4 up to 7 conserved sequence regions; and (iv) they possess the same catalytic machinery within the catalytic tim-barrel fold consisting of the aspartate residue near the end of the strand β4 (catalytic nucleophile), glutamate residue near the end of the strand β5 (proton donor) and aspartate residue near the end of the strand β7 (transition-state stabiliser). the conserved sequence regions (fig. 4) represent the short stretches of amino acid sequence that can be found in every α-amylase family member in equivalent positions and that contain the catalytic triad (asp206, glu230 and asp297; aspergillus oryzae α-amylase numbering; matsuura et al., 1984) and other functionally important residues (nakajima et al., 1986; janecek, 2002). these conserved sequence regions common for the entire clan gh-h may also be used as the sequence “fingerprints” since they contain amino acid residues exclusively specific for the individual enzyme specificities (janecek, 2008). 3.2. glycoside hydrolase family gh13 α-amylase is the most known and widely used enzyme of the gh-h clan. in general, α-amylases are endo-enzymes specific towards the α-1,4-glucosidic bonds, but there are also related gh13 exo-amylases, the so-called maltooligosaccharideproducing amylases (maltogenic α-amylase, maltotriohydrolase, maltotetraohydrolase, etc.), preferentially active at one side of the polysaccharide chain producing small oligosaccharides, such as maltose, maltotriose, maltotetraose, etc. (macgregor et al., 2001). 10 janeček, š. the α-amylase family members are multidomain proteins (fig. 3a) containing the main catalytic domain in the form of a parallel (β/α)8-barrel (domain a) that is interrupted by a usually small domain in the place of the loop 3 connecting the strand β3 with the helix α3 (domain b) and succeeded by the antiparallel β-sandwich domain (domain c). the α-amylase-type of the barrel was confirmed in all members of the αamylase family whose three-dimensional structure has already been determined (fig. 4). the (β/α)8-barrel of α-amylases was first revealed in the structure of taka-amylase a (matsuura et al., 1984), i.e. in the structure of the α-amylase from aspergillus oryzae. since this type of fold was first identified in triose-phosphate isomerase (tim), the (β/α)8-barrel is often simply called tim-barrel (farber and petsko, 1990). it is a barrel of eight inner parallel β-strands surrounded outside by eight αhelices (fig. 3a,b). table 1. the members of the α-amylase family (clan gh-h). enzyme class enzyme ec gh hydrolases α-amylase 3.2.1.1 13 oligo-1,6-glucosidase 3.2.1.10 13 α-glucosidase 3.2.1.20 13 pullulanase 3.2.1.41 13 amylopullulanase 3.2.1.1/41 13 cyclomaltodextrinase 3.2.1.54 13 maltotetraohydrolase 3.2.1.60 13 isoamylase 3.2.1.68 13 dextran glucosidase 3.2.1.70 13 trehalose-6-phosphate hydrolase 3.2.1.93 13 maltohexaohydrolase 3.2.1.98 13 maltotriohydrolase 3.2.1.116 13 maltogenic α-amylase 3.2.1.133 13 maltogenic amylase 3.2.1.133 13 neopullulanase 3.2.1.135 13 maltooligosyltrehalose hydrolase 3.2.1.141 13 maltopentaohydrolase 3.2.1.13 sucrose hydrolase 3.2.1.13 transferases amylosucrase 2.4.1.4 13 glucansucrase 2.4.1.5 70 sucrose phosphorylase 2.4.1.7 13 glucan branching enzyme 2.4.1.18 13 cyclodextrin glucanotransferase 2.4.1.19 13 4-α-glucanotransferase (amylomaltase) 2.4.1.25 13, 77 glucan debranching enzyme 2.4.1.25/3.2.1.33 13 alternansucrase 2.4.1.140 70 maltosyltransferase 2.4.1.13 isomerases isomaltulose synthase 5.4.99.11 13 maltooligosyltrehalose synthase 5.4.99.15 13 trehalose synthase 5.4.99.16 13 hatsa rbat protein --13 4f2hc antigen --13 a hats means the heteromeric amino acid transporter proteins. adapted from janecek (2009). nova biotechnologica 9-1 (2009) 11 fi g. 4 . s eq ue nc e fi ng er pr in ts o f th e αam yl as e fa m ily m em be rs . o ne r ep re se nt at iv e of e ac h en zy m e sp ec if ic ity is p re se nt ed . t he c at al yt ic tr ia d is h ig hl ig ht ed i n ye llo w a nd s ig ni fi ed b y as te ri sk s. t he o th er f un ct io na lly i m po rt an t re si du es c or re sp on di ng w ith h is 12 2, a rg 20 4, an d h is 29 6 of α -a m yl as e ar e al so c ol ou re d. t he w el lco ns er ve d as pa rt at e (b eg in ni ng o f th e st ra nd β 3) i s si gn if ie d by b la ck -a nd -w hi te in ve rs io n. t he r es id ue s co ns er ve d in a t le as t 50 % o f se qu en ce s ar e co lo ur ed w ith g re y ba ck gr ou nd . t he r ep re se nt at iv es o f he te ro m er ic am in o ac id tr an sp or te r pr ot ei ns ( h a t s) a re a ls o sh ow n. t he ‘ y ea r’ d en ot es th e ye ar o f th re edi m en si on al s tr uc tu re d et er m in at io n (i f an y) . a da pt ed fr om j a n e c e k (2 00 2) . 12 janeček, š. the active site of these enzymes is localised at the c-terminal end of the timbarrel (matsuura et al., 1984, qian et al., 1993; kadziola et al., 1994; linden et al., 2003). comparison of known tertiary structures of various α-amylase family members with sequence alignments have shown that differences in specificity result from different variation of substrate binding at the β->α loops (svensson, 1994; janecek, 1997). also the active-site cleft is not of the same shape in each case (kamitori et al., 1999; przylas et al., 2000), despite the fact it always contains the same catalytic triad accompanied, however, by several additional residues depending on a given enzyme specificity (matsuura, 2002). differences especially in the length, sequence and secondary structure have also been seen within the domain b protruding out of the catalytic tim-barrel in the place of the loop 3 (jespersen et al., 1991, 1993). it was pointed out that these differences may be directly related to enzyme specificity (janecek et al., 1997). with regard to domain c succeeding the catalytic tim-barrel, this domain could contribute to the overall catalytic domain stability by shielding the hydrophobic residues of the barrel (katsuya et al., 1998). as far as the conserved sequence regions of the α-amylase family are concerned (fig. 4), four of them (the regions i, ii, iii and iv) belong to the best known regions established more than 20 years ago, whereas the three additional ones (the regions v, vi and vii) were identified more recently. the former regions (friedberg, 1983; nakajima et al., 1986; macgregor et al., 2001), positioned near the c-termini of the β-strands β3, β4, β5 and β7 of the catalytic tim-barrel, contain most of the functionally important residues including the catalytic triad (fig. 4). the latter regions (janecek, 1992, 1994a,b, 1995, 2002), located near the c-terminal end of domain b and of β-strands β2 and β8, cover the features distinguishing the individual enzyme specificities from each other. even the absence of the fifth conserved sequence region, for example, may be used as a feature characteristic of a given specificity (janecek, 2000). although the basic arrangement of the α-amylase family members is the same counting the three domains a, b and c (fig. 3a), it should be taken into account that there are some family members that contain additional cand/or n-terminal domains, for example cyclodextrin glucanotransferase (klein and schulz, 1991) and neopullulanase (hondoh et al., 2003). they may play various and still not completely recognised functions, but most of them have been anticipated to be involved in binding starch (glycogen, pullulan) and related substrate analogues. these non-catalytic domains were in many cases confirmed to have this property and thus have been called starch-binding domains (penninga et al., 1995; sorimachi et al., 1997). it was found that starch-binding domain disrupts the starch surface and thus increases the effect of the amylolytic hydrolysis (southall et al., 1999). within the cazy server (fig. 1), these motifs have been classified into the cbm (carbohydratebinding module) families (cantarel et al., 2009). at present, nine families of starch-binding domains are known: cbm20, cbm21, cbm25, cbm26, cbm34, cbm41, cbm45, cbm48, and cbm53. the motifs from the family cbm20 belong to most intensively studied starch-binding domains (svensson et al., 1989; janecek and sevcik, 1999; rodriguez-sanoja et al., 2005; machovic and janecek, 2006a). based on a detailed bioinformatics analysis it was suggested nova biotechnologica 9-1 (2009) 13 to establish a common cbm clan from the families cbm20 and cbm21 (machovic et al., 2005) and the motifs classified recently into the families cbm48 and cbm53 could also join the proposed cbm clan (machovic and janecek, 2006b, 2008). 3.3. glycoside hydrolase families gh70 and gh77 the family gh70 contains the sucrose-utilising glucosyltransferases (glucansucrase and alternansucrase) that possess a circularly permuted version of the α-amylase-type catalytic tim-barrel (macgregor et al., 1996). the first element of the gh70-type barrel is the α-helix equivalent to helix α3 of the α-amylase-type tim-barrel, whereas the last element is the β-strand equivalent to strand β3 of αamylases (fig. 5). this means that instead of e1-h1-e2-h2….e8-h8 present in αamylases (and overall in both the families gh13 and gh77), in gh70 glucosyltransferases there is h3-e4-h4-e5….h2-e3, where e and h stand for βstrand and α-helix, respectively (macgregor et al., 1996). the glucansucrases are usually large multidomain proteins occurring exclusively in lactic acid bacteria (van hijum et al., 2006). fig. 5. the arrangement of the secondary structure elements in gh70 with respect to gh13 α-amylase type tim-barrel. (a) typical “ordinary” tim-barrel present in the members of the family gh13 (and also gh77); (b) circularly permuted version of the family gh70. the helices are represented by black rectangles and the strands are shown as arrows. the order of the helices in the gh13 (and gh77) is 12345678 from the nterminal end of the protein, whereas in the gh70 the order is 34567812. adapted from macgregor (2005). the structure/function relationships within the family gh70 and its relatedness to the main α-amylase family gh13 were recently elucidated by determining the tertiary structure of the gh70 glucansucrase from leuconostoc mesenteroides (pijning et (a) (b) 14 janeček, š. al., 2008) that has confirmed the previous predictions concerning the circular permutation (fig. 5). the solved structure interestingly revealed that the enzyme adopts the so-called “u-fold” domain arrangement so that 4 of the 5 domains are formed by combining an nand a c-terminal part of the polypeptide chain (dijkstra et al., 2007). the family gh77 contains only one enzyme specificity, the amylomaltase (table 1), known also as 4-α-glucanotransferase in bacteria (terada et al., 1999) and archaeons (kaper et al., 2005) or disproportionating enzyme (d-enzyme) in plants (takaha et al., 1993). they exhibit a lower degree of sequence similarity to the family gh13 (fig. 4) and the main feature characteristic for the gh77 members is the lack of domain c (przylas et al., 2000) succeeding typically the catalytic timbarrel in gh13 (fig. 3). the gh77 structure contains several, mainly α-helical insertions that can be divided into three subdomains (fig. 6): (i) subdomain b1 corresponds with gh13 domain b; (ii) subdomain b2 is unique for the gh77 amylomaltases; and (iii) subdomain b3 is equivalent to gh13 domain c (strater et al., 2002). fig. 6. three-dimensional structure of gh77 amylomaltase from thermus aquaticus (1cwy; przylas et al., 2000). the interest in the family gh77 was recently increased by revealing the putative amylomaltases from a few borreliae that exhibited in their amino acid sequences the non-gh77 features (godany et al., 2008). it was especially the arginine positioned two residues before the catalytic nucleophile in the conserved sequence region ii (fig. 4) that was recognized to be replaced naturally by a lysine in the gh77 amylomaltaselike protein from borrelia burgdorferi (machovic and janecek, 2003). this arginine was otherwise considered to belong to the four residues conserved invariantly throughout the α-amylase family, i.e. the entire clan gh-h (janecek, 2002). the exclusive (i.e. the non-gh77) sequence features present in gh77-like proteins from borreliae have already been confirmed as well as it was determined that the b. (β/α)8-barrel subdomain b2 subdomain b1 subdomain b3 nova biotechnologica 9-1 (2009) 15 burgdorferi gh77 amylomaltase-like protein exhibits a typical amylomaltase activity, i.e. the enzyme catalyzes both the hydrolysis of maltooligosaccharides and formation of their transglycosylation products (godany et al., 2008). based on the bioinformatics analysis of various gh77 real and hypothetical amylomaltases, some of the borrelial gh77-like proteins were suggested to exhibit an intermediary character within this family (janecek, 2008). 3.4. glycoside hydrolase families gh31 and gh57 the families gh31 and gh57 are not the members of the clan gh-h, i.e. they do not belong to the α-amylase family in terms as it is widely accepted (macgregor et al., 2001), but they both deserve some attention here since they contain similar enzyme specificities (α-amylase, α-glucosidase, amylopullulanase, 4-αglucanotransferase, branching enzyme, etc.). the family gh31 contains, in addition to the above-mentioned α-glucosidases (ec 3.2.1.20 similar to gh13), also α-xylosidases and α-glucan lyases (frandsen and svensson, 1998; lee et al., 2005; kang et al., 2008). although it employs the retaining mechanism (fig. 2a) and its members adopt the catalytic tim-barrel domain (fig. 7a) similar to that adopting in the α-amylase family (lovering et al., 2005; fig. 7. three-dimensional structure of (a) gh31 α-xylosidase from escherichia coli (1xsi; lovering et al., 2005) and (b) gh57 4-α-glucanotransferase from thermococcus litoralis (1k1w; imamura et al., 2003). ernst et al., 2006; sim et al., 2008) with even the corresponding catalytic nucleophile (rigden, 2002), the family gh31 has not joined the clan gh-h. one of the reasons is the difference in the proton donors used in gh31 and gh-h (b) (β/α)7-barrel domain ii (β/α)8-barrel domain b domain c (proximal) domain n domain c (distal) (a) 16 janeček, š. (matsuura et al., 1984; uitdehaag et al., 1999; lovering et al., 2005). based on a detailed bioinformatics study, an idea on the so-called remote homologies between the family gh31 and clan gh-h was proposed recently (janecek et al., 2007) indicating a possibility to create a level of evolutionary hierarchy higher than a clan. as far as the family gh57 is concerned, it contains several enzyme specificities that are also members of the main α-amylase family, only the α-galactosidase (ec 3.2.1.22) being different (janecek, 2005; murakami et al., 2006). it also employs the retaining mechanism, but due to a different catalytic domain an incomplete version of a tim-barrel, i.e. a (β/α)7-barrel (fig. 7b) and catalytic machinery (imamura et al., 2003; dickmanns et al., 2006) it should be evolutionarily more distantly related to gh13 than is the family gh31 (janecek, 1998). moreover, gh57 exhibits its own conserved sequence regions (zona et al., 2004) that are different from those characteristic for the clan gh-h (janecek, 2002). 4. α-amylases from archaebacteria and plants at present it is well-known and accepted that plant and archaeal α-amylases from the family gh13 are sequentially similar and evolutionarily related. this remarkable finding was first observed ten years ago (janecek et al., 1999; jones et al., 1999). before the first gh13 α-amylases from archaea became available, the plant αamylases were positioned in the evolutionary tree (fig. 8) on a branch next to the cluster of bacterial liquefying and intracellular α-amylases represented by bacilli and enterobacteria, respectively (janecek, 1994b). 4.1. similarities and differences the first detailed bioinformatics study focused on the archaeal α-amylases and their counterparts from a wide spectrum of remaining living organisms from bacteria and eucarya revealed (janecek et al., 1999) that the sequence features exclusive for the α-amylases from hyperthermophilic archaeons are present also and almost only in the plant α-amylases (fig. 9). these features are as follows (janecek et al., 1999): (i) ile107 (thermococcus hydrothermalis α-amylase numbering; leveque et al., 2000a) succeeding the conserved aspartate in the conserved sequence region region i (strand β3); (ii) (ala194)-trp195 at the beginning, tyr199 in the middle and gly202 at the end of the region ii (strand β4); (iii) ala219 succeeding the conserved tryptophane and tyr223-trp224 succeeding the catalytic proton donor (glu222) in the region iii (strand β5); (iv) ala286 in the region iv (strand β7); (v) ile196 in the region v (located within the loop3, i.e. domain b); (vi) ile42 succeeding the conserved glycine at the beginning and dipeptide pro48-pro49 at the end of the region vi (strand β2); and (vii) gln309 succeeding the conserved glycine at the beginning, tripeptide ile312phe313-tyr314 in the middle and asp316 at the end of the region vii (strand β8). it is worth mentioning that some of the above-mentioned residues have already been recognised as functionally important residues (kadziola et al., 1998; linden et nova biotechnologica 9-1 (2009) 17 fig. 8. the evolutionary tree of microbial (including fungi and yeasts), plant and animal α-amylases. the bacterial sources are abbreviated as follows: dicth, dictyoglomus thermophilum; micsp, micrococcus sp.; bacme, bacillus megaterium; escco, escherichia coli; salty, salmonella typhimurium; bacst, bacillus stearothermophilus; bacam, bacillus amyloliquefaciens; bacli, bacillus licheniformis; bacsu, bacillus subtilis; butfi, butyrivibrio fibrisolvens, xanca, xanthomonas campestris; aerhy, aeromonas hydrophila. the tree does not contain any archaeal α-amylase since at the beginning of 90s of the previous century no sequence of an archaeal α-amylase was available. the red arrow indicates the the cluster of plant αamylases. adapted from janecek (1994b). fig. 9. sequence fingerprints of α-amylases. the enzymes represent the individual taxonomic sources with focus on archaea and plants. the sequence features characteristic of the archaeal α-amylases are highlighted in black-and-white inversion. the catalytic triad is signified by asterisks and yellow highlighting. adapted from janecek (2008). 18 janeček, š. al., 2003). thus for example the glycine from the region ii (gly202 of the archaeal αamylase) serves as a specific ligand for calcium ion and the tryptophane from the region iii (trp224 of the archaeal α-amylase) forms a stacking interaction with one of the acarbose rings bound in the active site in the complex structure of barley αamylase with acarbose (kadziola et al., 1998). these residues should play the same roles in the structure of the archaeal α-amylase from pyrococcus woesei (linden et al., 2003). the close sequence similarity between the α-amylases from archaea and plants has evoked the idea on a possibility to reveal the factors responsible for the high thermostability of the archaeal α-amylases that exhibit the temperature optima around and above 80 oc (leveque et al., 2000b; bertoldo and antranikian, 2002). the plant enzymes are generally substantially less thermostable. it is worth mentioning that on the one side the archaeal and plant α-amylases contain the common sequence features that discriminate them from the remaining sources, but on the other side they have to possess the additional sequence features that should enable one to distinguish them from each other, e.g., the alanine from the region iv (ala286 of the archaeal α-amylase) that has no correspondence in the plant counterparts (fig. 4). such specific differences could be utilized in an effort to identify the molecular basis of high thermostability of the archaeal α-amylases via the approaches of site-directed mutagenesis and protein design. fig. 10. the evolutionary tree of α-amylases. the tree reflects the conserved sequence fingerprints of αamylases (fig. 9). adapted from janecek (2008). nova biotechnologica 9-1 (2009) 19 4.2. evolutionary relatedness the close evolutionary relatedness of the α-amylases from archaea and plants from the family gh13 is shown in figure 10. the gh13 as one of the largest gh families (cantarel et al., 2009) has recently been divided into the subfamilies (stam et al., 2006), the plant and archaeal α-amylases being placed into the subfamilies gh13_6 and gh13_7, respectively. with regard to the α-amylases most closely related to those from plants and archaea (fig. 10), these are the bacterial enzymes from bacillus licheniformis (yuuki et al., 1985) and escherichia coli (raha et al., 1992) that represent the liquefying and intracellular α-amylases, respectively, as observed originally (janecek, 1994b). it should be noted, however, that the close evolutionary relationships between the α-amylases from archaea and plants illustrated here only for a limited sample of living organisms (fig. 10) has been confirmed also in the more recent evolutionary trees comparing a wider spectrum of taxonomic sources including novel groups of α-amylases from bacteria (da lage et al., 2004) and fungi (van der kaaij et al., 2007). acknowledgements: this work was supported in part by the grants no. 1/4363/07 and 2/0114/08 from the slovak grant agency vega and the project av-4/2023/08 from the ministry of education of the slovak republic. references aleshin, a.e., golubev, a., firsov, l.m., honzatko, r.b.: crystal structure of glucoamylase from aspergillus awamori var. x100 to 2.2 å resolution. j. biol. chem., 267, 1992, 19291-19298. bertoldo, c., antranikian, g.: starch-hydrolyzing enzymes from thermophilic archaea and bacteria. curr. opin. chem. biol., 6, 2002, 151-160. cantarel, b.l., coutinho, p.m., rancurel, c., bernard, t., lombard, v., henrissat, b.: the carbohydrate-active enzymes database (cazy): an expert resource for glycogenomics. nucleic acids res., 37, 2009, 233238. coutinho, p.m., reilly, p.j.: glucoamylase, structural, functional, and evolutionary relationships. proteins, 29, 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university yahia farès of médéa, médéa 26000, algeria 3interdepartmental center for industrial agri-food research, university of bologna, cesena, italy 4department of agricultural and food sciences, university of bologna, bologna 33-40126, italy 5centro de referencia para lactobacilos (cerela), conicet, san miguel de tucumán, tucumán 4000, argentina  corresponding author: boubakri.kamel@univ-medea.dz article info article history: received: 20th november 2021 accepted: 7th january 2022 keywords: kaddid starter culture traditional fermentation weissella abstract kaddid is a dry-fermented meat product traditionally produced in north africa by spontaneous fermentation. as a reservoir of natural biodiversity, the identification and relevant traits of lactic acid bacteria (lab) were carried out from south-western algeria homemade samples. after a preliminary physiological and biochemical screening, 19 presumptive lab isolates were selected on the basis of antimicrobial compounds production. the isolates were identified by 16s rrna gene sequencing as weissella cibaria, w. confusa, w. paramesenteroides, pediococcusacidilactici, and enterococcus hirae. as predominant, the safety and technological characterization of weissella strains were performed. the production of antimicrobial and antifungal compounds was observed, while neither h2s, biogenic amines nor hemolytic activity were detected; antibiotic resistance was exhibited, however several strains were more susceptible to assayed antibiotics. technological characterization of weissella strains showed high acidification rates even in the presence of up to 10 % nacl, autolytic and proteolytic capacity however, no eps production and lipolytic activity were observed. strains characterization led to the selection of w. cibaria bk2, w. confusa bk6 and bk11 as well as w. paramesenteroides bk8 to be considered as possible candidates for use as starter culture for kaddid fermentation to improve and standardize this traditional meat product. introduction even today, traditional meat products are generally homemade as a mean to preserve meat to be consumed in times of scarcity. among traditional products, the appreciation of fermented meats is probably related to their unique and specific sensory properties and alleged rootedness in a socio-cultural context. meat products prepared in north african countries are usually dried or cooked because of the weather and are rarely smoked; they are produced from different meat (beef, lamb, goat and camel) depending on the geographic area. dried kaddid has been traditionally used as mailto:boubakri.kamel@univ-medea.dz nova biotechnol chim (2022) 21(2): e1269 2 ingredient to prepare different winter dishes as stews and soups (gagaoua and boudechicha 2018). in algeria, it is prepared by adding salt and spices to lamb meat cutting into thin strips, which are then hang on a string and exposed to the air until thoroughly dried and stored at room temperature; salt content in the final product ranges from 7 % to 12 % (bennani et al. 1995; benlacheheb et al. 2018). microbiological survey of dry-salted kaddid showed the presence of lactic acid bacteria (lab) in addition to microorganisms related to the product hygiene (bennani et al. 2000; ben belgacem et al. 2008, 2010; essid et al. 2009; benlacheheb et al. 2018). physicochemical features of kaddid during ripening such as ph, moisture, salt content and water activity are main modulators of microbiological evolution. it is known that the presence of lab induces desirable attributes to traditional fermented food products, enhancing their typical characteristics while conferring additional safety and health benefits. distinctive features derived from lab metabolic activities in salted meat will determine final quality of fermented products. in view to design novel functional starter cultures, it would be necessary to exploit the autochthonous lab with technological, functional and safety characteristics. a quick growth and high acidification rate in the presence of high salt content are the main criteria for strains selection by which a safe initial environment can be created and food pathogen and spoilage reduced (fusco 2015; fessard and remize 2017). from a safety point of view, the production of antibacterial and antifungal compounds during fermentation are desired features as these metabolites inhibit pathogen and spoilage proliferation. the lack of antibiotic resistance, virulence factors and aminogenesis among other traits, are also required (castellano et al. 2017). in addition, the united nations food and agriculture organization (fao)/world health organization (who) stated the importance to conduct a minimum safety assessment including several specific metabolite productions, toxin production, and potential hemolysis, even for microbial populations classified as gras (fao/who 2002). moreover, changes in meat and fat are responsible for the flavour in fermented meat. proteolysis and lipolysis by meat enzymes and lab will impact on the development of typical sensory characteristics by peptides and amino acids generation (fadda et al. 2010; vignolo et al. 2019). thus, the aim of this study was the identification of the lab population from algerian kaddid samples and traits related to technological and safety features of autochthonous isolated lab were investigated. experimental samples collection and lab isolation samples of homemade kaddid were collected from the southwestern region of algeria, namely tindouf (one sample from camel meat) and béchar (6 samples from lamp meat) provinces. approximately 400 g of ready to consume kaddid pieces from each producer were placed in sterile plastic bags and transported refrigerated to the laboratory for analyses. each sample (10 g) was suspended in 90 ml of sterile tryptone-salt diluents (tryptone 1 g.l-1; nacl 0.85 g.l-1; tween 80 1 ml.l-1), homogenized for 3 min (stomacher 400, seward, worthing, uk) and serially diluted. dilutions were then plated in duplicate on mrs and m17 (merck, darmstadt-germany) media, and microaerobically incubated at 30 °c during 72 h. an average of 20 – 25 colonies per kaddid sample were randomly picked from both media plates containing 100 – 300 colonies. gram-positive and catalase-negative bacteria (presumptive lab) were sub-cultured on the corresponding medium and stored in 25 % of glycerol at -80 °c for further use. physiological and biochemical characterization of lab sixty-three presumptive lab isolates were preliminarily identified according to von wright and axelsson (2012) and the scheme described by carr et al. (2002). growth at 10 ºc and 45 ºc during 7 d and 24 h, respectively, at ph 4 to 9.6 and in the presence of 4 to 10 % nacl were evaluated in mrs (bacilli) and m17 (cocci). production of gas (co2) from glucose (gibson and abdelmalek 1945) and arginine hydrolysis (møller 1955) were analyzed. for carbohydrate nova biotechnol chim (2022) 21(2): e1269 3 fermentation mevag medium (biokar diagnostic, allonne, france) supplemented with different sugars (table 1) was used, lab suspensions being inoculated by a central puncture and covered by a vaseline layer to promote anaerobiosis; after incubation (30 °c; 24 h) color changes due to the sugars utilization by bacteria were recorded. molecular identification genomic dna was extracted from pure lab cultures using instagene matrix (bio-rad laboratories inc., hercules, usa). molecular identification was performed as described by montanari et al. (2015). the partial 16s rrna gene sequence was amplified using the primers lpigf/lpigr (5’-tacgggaggcagcagtag3’ and 5’-catggtgtgacgggcggt-3’; eurofins genomics germany gmbh, ebersberg, germany). pcr amplifications were performed using taq dna polymerase kit from thermo fisher scientific (waltham, usa). reaction mixtures consisted of buffer 5x, 2 mm mgcl2, 50 mm each of the 4 deoxynucleoside triphosphates (dntp), 1.25 u of taq-polymerase, 0.5 mm of each primer, and 0.5 μl of appropriately diluted template dna in a final volume of 50 μl. amplification was performed on a biometra t3000 thermal cycler (analytik jena gmbh, jena, germany) with initial denaturation at 94 °c for 5 min, then 34 cycles at 94 °c for 1 min, 55.5 °c for 2 min, and 72 °c for 2 min, followed by final extension at 72 °c for 10 min. pcr products were separated by electrophoresis on 1.5 % (w/v) agarose gel (lonza, italy) stained with ethidium bromide (0.5 μg.ml-1). the 600 bp amplicons were eluted from an agarose gel, purified with the qiaquick pcr purification kit (qiagen, hilden, germany) and sequenced at the bmr genomics srl. sequencing facility (padova, italy) using the same primers as for amplification. sequence similarity searches were performed using blast network service (http://blast.ncbi.nlm.nih.gov/) and ez-taxon server (http://147.47.212.35:8080/). antimicrobial activity lab (63 strains) inter-species inhibitory capacity was first investigated by using the disc method described by tadesse et al. (2004) with modifications. overnight lab cultures (0.5 ml) used as indicators were mixed with 12 ml of melted and cooled mrs agar media, poured into plates, let solidify and dried. lab cultures were centrifuged (7,000 × g; 15 min) and the obtained cell free supernatants were used to impregnate sterile filter paper discs that were deposited on the seeded agar. on the other hand, the antibacterial activity of identified weissella isolates was evaluated by a semi-quantitative agar-spot-test as described by fontana et al. (2015) against gram positive and gram-negative bacteria (table 3). overnight lab cultures were centrifuged (7,000 × g; 15 min), and supernatants (5 µl) were spotted onto 10 ml of bhi agar (britania, argentina) plates (0.7 %) previously inoculated with 50 µl of each indicator strain. plates were incubated at 30 °c for 48 h and the presence of a clear inhibition zone around the spots was considered as a positive antagonistic effect. inhibitory activity was expressed as + (halos presence) or – (no halos) around the spot. positive antibacterial activity lab supernatants were neutralized (4n naoh) and treated with catalase (1,000 u.ml-1) (sigmaaldrich, st louis, usa) to determine the chemical nature of the inhibitory substance. antifungal activity against aspergillus flavus, penicillium expansum, and fusarium oxysporum albedinis was investigated by a modified agar diffusion assay (magnusson et al. 2003). petri plates containing potato dextrose agar (pda) were inoculated with the fungus and incubated at 25 °c during 48 h. after visible formation of conidiospores, they were collected and adjusted to 105 spores/ml of sterile saline solution. lab selected strains were streakinoculated on mrs agar plates and after incubation (30 °c; 48 h), plates were overlaid with 10 ml of pda soft agar (0.7 % agar) containing fungal spore suspensions (104.ml-1) and incubated aerobically (30 °c; 48h). plates were then examined for clear inhibition zones around the lab streaks and scored as – (no growth inhibition), + (1 – 5 mm growth inhibition), ++ (5 – 10 mm growth inhibition) and +++ (˃ 10mm growth inhibition). 3 http://blast.ncbi.nlm.nih.gov/ http://147.47.212.35:8080/ nova biotechnol chim (2022) 21(2): e1269 2 biogenic amines and h2s production the ability to decarboxylate amino acids used as precursor was tested according to bover-cid and holzapfel (1999). briefly, the plates with the agar medium, supplemented with histidine, tyrosine, ornithine, and lysine 1 % (w/v) were spotted with the active weissella (7 strains) and incubated anaerobically (30 °c; 2 – 5 d). growth of decarboxylating strains was easily recognizable because of a purple halo in the yellow medium. production of h2swas investigated on tsi (triple sugar iron) agar medium by a central puncture inoculation. after incubation (30 °c; 48 – 72 h), h2s production was confirmed by the blackening of the medium and gas bubbles in the agar (guiraud 2003). antibiotic resistance antibiotics recommended for the european food safety authority (efsa 2012) to identify bacterial strains with potential acquired resistance were used. ampicillin (amp; 0.032 – 16 g.ml-1), vancomycin (van; 0.25 – 128 g.ml-1), chloramphenicol (chl; 0.125 – 64 g.ml-1), gentamycin (gen; 0.5 – 256 g.ml-1), streptomycin (str; 0.5 – 256 g.ml-1), kanamycin (kan; 2 – 1024 g.ml-1), tetracycline (tet; 0.125 – 64 g.ml-1), erythromycin (ery; 0.016 – 8 g.ml-1) and clindamycin (cli; 0.032 – 16 g.ml1) were tested. the minimum inhibitory concentration (mic) of antibiotics was determined by the broth micro-dilution method reported by the iso 10932/idf 233 standard. the strains were classified as susceptible or resistant according to the cut-off values proposed by efsa (2012). a bacterial strain was defined as susceptible or resistant when it was inhibited or not, at a specific antimicrobial concentration equal or lower than the established cut-off value. hemolysin and gelatinase activity hemolysin activity was determined on columbia blood agar (oxoid) containing 5 % defibrinized horse blood after 48 h of incubation at 37 °c, both under aerobic and anaerobic conditions. the type of hemolysis (α, β or ɣ) was determined. staphylococcus aureus atcc25923 and escherichia coli atcc25922 were used as positive control for βand α–hemolysis, respectively. zones of clearing around colonies indicated -hemolysin production. gelatinase production was detected by inoculating lab onto freshly prepared peptone yeast extract agar containing gelatin (30 g.l-1; difco). plates were incubated overnight at 37 °c and cooled at room temperature for 2 h. the presence of turbid zone around the colonies was considered as positive result. growth and acidification selected weissella strains were inoculated (1 %) into mrs broth, incubated at 10, 30, 37 and 44 °c for 48 h and growth at od620 was measured. similarly, the growth of each strain was evaluated in mrs with ph adjusted to ph 4.5, 5, 6, 7, 8 and 9.6 and supplemented with 4, 6.5 and 10 % nacl. the od620 was measured at 0, 2, 4, 6, 24 and 48 h. acidification ability was evaluated as reported by ammor et al. (2005), using sausage-broth (sb) medium. eighty (80) ml of sb medium was inoculated with an overnight culture of each strain. the ph values and od620 were recorded after 0, 3, 6, 24, 48, 72 and 96 h of incubation at 30 °c using a ph meter and a spectrophotometer. autolytic activity and thermoresistance each strain was suspended in pbs buffer (ph 7) at a do620 : 0.2 and subjected to a freeze cycle (-20 °c for 24 h) and after thawing, strains were incubated at 30 °c for 24 h. autolytic activity was determined by the decrease percentage in absorbance at d620 after time interval (piraino et al. 2008) as %aa: (ai-at) × 100/ai, where aa: autolytic activity, ai: initial absorbance and at: absorbance after 24 h of incubation. autolysis was classified according to lactobacilli genus (ayad et al. 2004), ranged from 70 – 96 % (good), 40 – 69 % (low) and 0 – 39 % (poor). thermo-resistance was evaluated as reported by badis et al. (2004); lab strains inoculated in mrs broth were heated at 60.5 °c during 30 min, and then incubated at 30 °c for 24 to 48 h and the colonies in mrs agar were enumerated. 4 nova biotechnol chim (2022) 21(2): e1269 3 proteolytic and lipolytic activities five microliters (5 μl) of each lab strain suspended in pbs buffer (ph 7) were spot inoculated onto tryptone soy agar (tsa) supplemented with sterile skim milk (10 %) as described by guiraud (2003). after incubation at 30 °c for 5 d, the caseinolytic activity (measured in mm) was determined by the presence of a clear area around the spot. for lipolytic activity, the technique reported by mauriello et al. (2004) with minor modifications was used. one ml of lab overnight cultures was inoculated into 10 ml of a broth containing tryptone (1 % (w/v)), yeast extract (0.5 % (w/v)), nacl (3 % (w/v)), ph 7.0, supplemented with 4 % (w/v) lamb fat previously homogenized by vigorous shaking. after incubation at 30 °c for 7 d, free fatty acids were then determined. the lipids were extracted into 10 ml of petroleum ether by shaking for 1 min. the fatty acids of the upper phase were titrated with naoh (0.1 m) in ethanol using 1 % phenolphthalein-ethanol solution as indicator. results were expressed as % of oleic acid by a × n × 28.2/g, where a: ml naoh used for titration, n: naoh normality, 28.2: % of oleic acid equivalent weight and g: amount of lamb fat used. exopolysaccharides (eps) production active cultures of lab strains were spotted on mrs agar in which glucose was replaced by sucrose and incubated at 30 °c for 2 – 7 d. ropiness was examined by the presence of a ropy condition after touching the colony with a loop. statistical analysis agar assays were performed by duplicate and growth curves by triplicate. in the case of antibiotic resistance, media values were compared with cutoff points. the media and sd were calculated for growth data, results (means od ± sd) being evaluated by the application of anova to define differences and statistical significances were determined by the tukey test. results and discussion physiological and biochemical characterization of lab isolates sixty-three presumptive lab isolates (42 cocci and 21 bacilli) were subjected to a preliminary characterization. results showed that 10 % of the isolates produced gas from glucose (table 1). among homofermentative cocci/coccobacilli isolates, those tetrads-forming cocci (23.8 %) that grew at 10 °c and 40 – 45 °c, up to 10 % nacl but not at ph > 8.0 were presumed as pediococcus. however, chains-forming homofermentative cocci (47.7 %) that developed at the same temperatures, in a wider ph range (4.0 to 9.6) and up to 6.5 % nacl were assigned to enterococcus genus, differing from lactococci in that the latter are not able to grow at 40 – 45 °c. on the other hand, heterofermentative cocci/coccobacilli isolates (28.6 %) able to grow at 10 °c, up to 6.5 % nacl and ph between 4.5 and 8.0 (arginine mostly positive and variable growth at ph > 6.0, between 40 – 45 °c and nacl > 6.5 %) have been characterized as presumptive leuconostoc or weissella. concerning bacilli (21 %) involving homo and heterofermentative isolates would be assigned to lactobacillus or weissella genus with variable arginine hydrolysis, ability to grow between 40 – 45 °c in a wide range of ph and resistant to nacl and sugar fermentation capacity except for xylose. sugars fermentation for cocci/coccobacilli isolates exhibited variable carbohydrates fermentation; heterofermentative coccobacilli fermented cellobiose, fructose, maltose, mannose, sucrose, and xylose but not melibiose, raffinose, sorbitol and trehalose. based on phenotypic, morphologic and biochemical characterization, ph, temperature, salt tolerance and sugars fermentation, it can be preliminary suggested that cocci/coccobacilli isolates belong to pediococcus, enterococcus, leuconostoc, weissella and lactobacillus genera. these results agree with the lab from high saltcontaining fish and meat products (najjari et al. 2008; ben belgacem et al. 2010; belfiore et al. 2013). 5 nova biotechnol chim (2022) 21(2): e1269 2 molecular identification of isolates exhibiting antimicrobial activity after the first inter-lab species inhibition assay, selected isolates (19) were subjected to molecular identification. table 1. physiological and biochemical characterization of lab isolated from kaddid. physiological traits cocci/coccobacilli (42) bacilli (21) homofermentative heterofermentative homo/hetero microscopy cocci/ tetrads cocci/ chains coccobacilli single/pairs chains/ pairs co2 from glucose – – + +/– arginine hydrolysis +(v) +/– – +/– growth at 10 °c + + + + 45 °c + + +/– v nacl 4% + + + + 6.5% + + +/– + (v) 10% + +/– – (v) + (v) ph 4.5 + + + + 6.5 + + + (v) + 8.0 – + +/– v 9.6 – + – – d-glucose + + + + d-arabinose +/– +/– +/– +/– d-cellobiose – +/– + + d-fructose +/– + + + d-galactose +/– + +/– + d-lactose + + + + d-maltose – – + + d-mannose – + + + d-mannitol + – +/– + d-melibiose – +/– – + d-ribose + +/– + + d-raffinose +/– – – +/– d-sorbitol +/– +/– – +/– d-sucrose +/– + + +/– d-trehalose – + – + d-xylose +/– +/– + – + = positive; – = negative; +/– = more or less positive; v = variable. in parallel with the physiological/biochemical characterization, amplification of partial 16s rrna gene sequence allowed the identification of 15 isolates as weissella (w.) cibaria/confusa with a similarity level of 99 to 100 %, one isolate as w. paramesenteroides (similarity 99.87 %), two isolates as p. acidilactici (similarity > 99 %) and one isolate as e. hirae (similarity 99.77 %), (table 2). this result indicated that most of labs were heterofermentative, while only three strains were homofermentative. because weissella species constituted the major population among identified lab, they were used for further studies. high relatedness of w. cibaria and w. confusa observed in this study agrees with that described by lynch et al. (2015). however, based on differential capacity to ferment carbohydrates, w. cibaria was described to ferment arabinose but not galactose and ribose, while these latter were utilized by w. confusa (björkroth et al. 2002; quattrini et al. 2019). results from carbohydrates fermentation were used to complement molecular identification (table 1). thus, strains were distinguished as w. cibaria (bk1, bk2, bk3, bk9, bk10, bk13, bk18, bk19), w. confusa (bk4, bk5, bk6, bk7, bk11, bk12, bk16), w. paramesenteroides (bk8), p. acidilactici (bk14, bk17) and e. hirae (bk15) as shown in table 2. species of this genus have been isolated from a wide range of ecological niches including soil, plants, breast milk, oral cavity, urogenital and git tract of humans and animals as well as a huge variety of fermented foods (abriouel 6 nova biotechnol chim (2022) 21(2): e1269 3 et al. 2015; fusco et al. 2015; maldonado et al. 2018). from a technological point of view, weissella plays a key role in food fermentation based on vegetables and to a lesser extent in meat. however, the identification of w. cibaria, w. confusa and w. paramesenteroides from dry-salted algerian kaddid agrees with those reported during dry-cured sausages fermentation (fusco et al. 2015). indeed, w. cibaria and w. confusa were previously retrieved from portuguese fermented sausages, thai pork sausages (nham), morcilla de burgos and fermented fish (santos et al. 2005; srionnual et al. 2007; albano et al. 2009; kopermsub and yunchalard 2010; wongsuphachat et al. 2010). likewise, w. paramesenteroides was identified from italian fermented sausages (urso et al. 2006; papagianni and papamichael 2011). although w. cibariahas been first isolated from thai fermented meat (björkroth et al. 2002), together with w. confusa have been associated with a wide range of vegetable fermented products and their ability to use plant carbohydrates was reported (fusco et al. 2015). besides plant sugars utilization, w. confusa and w. paramesenteroides showed to use ribose, suggesting they also may grow in meat. table 2. lactic acid bacteria (lab) strains identified from dried salted algerian kaddid. lab isolates isolation source closest relative identity [%] accession no.* bk1 sk1 weissella cibaria 99.41 % mt158598.1 bk2 sk1 weissella cibaria 99.87 % mt012260.1 bk3 sk1 weissella cibaria 99.87 % mt012260.1 bk4 sk2 weissella confusa 100 % mk503640.1 bk5 sk2 weissella confusa 100 % mk503640.1 bk6 sk3 weissella confusa 99.76 % mk503640.1 bk7 sk3 weissella confusa 100 % mk503640.1 bk8 sk4 weissella paramesenteroides 99.87 % mn994365.1 bk9 sk4 weissella cibaria 99.75 % mt012260.1 bk10 sk5 weissella cibaria 99.55 % mt012260.1 bk11 sk5 weissella confusa 99.50 % mk503640.1 bk12 sk6 weissella confusa 99.88 % mk503640.1 bk13 sk6 weissella cibaria 99.65 % mt012260.1 bk14 sk6 pediococcus acidilactici 99.74 % cp050079.1 bk15 sk7 enterococcus hirae 99.77 % mt197246.1 bk16 sk7 weissella confusa 99.05 % mk503640.1 bk17 sk7 pediococcus acidilactici 99.88 % cp050079.1 bk18 sk7 weissella cibaria 100 % mt012260.1 bk19 sk7 weissella cibaria 99.75 % mt012260.1 sk – sample of kaddid. kaddid southwestern algerian samples were from: sk1, sk2, sk3 (béchar city), sk5 (beniounif), sk6 and sk7(igli) from béchar province, and sk4 from tindouf province. *sequence similarity searches were performed using blast networkservice (http://blast.ncbi.nlm.nih.gov/). antimicrobial activity in a first attempt to distinguish those isolates exhibiting inhibitory activity, from 63 lab isolates preliminarily assigned to different lab genera, 19 of them showed inter-species antagonistic activity (result not shown) suggesting the presence of antibacterial metabolite/s in the supernatant. indeed, when neutralized supernatants and catalase addition were evaluated, the inhibitory activity was suppressed indicating that organic acids or some compound of protein nature would be responsible for the antibacterial effect. the production of inhibitory metabolites active against food pathogens could be an important improvement for starter cultures and might be of interest in controlling meat fermentation, which naturally contain competing food-borne pathogens. therefore, as a dominant population, sixteen labs assigned to weissella genus were investigated for their antibacterial and antifungal activity against a range of pathogens and contaminants (table 3). results showed weissella isolates bk2, bk3 and bk19 as the highest inhibitory strains against the assayed pathogens. bacteriocin production by weissella species was widely reported, especially 7 https://www.ncbi.nlm.nih.gov/nucleotide/mt158598.1?report=genbank&log$=nucltop&blast_rank=2&rid=89bpjf6n01r https://www.ncbi.nlm.nih.gov/nucleotide/mt012260.1?report=genbank&log$=nucltop&blast_rank=1&rid=89dnr0xf016 https://www.ncbi.nlm.nih.gov/nucleotide/mt012260.1?report=genbank&log$=nucltop&blast_rank=1&rid=89efxj7z014 https://www.ncbi.nlm.nih.gov/nucleotide/mk503640.1?report=genbank&log$=nucltop&blast_rank=2&rid=89epeghv014 https://www.ncbi.nlm.nih.gov/nucleotide/mk503640.1?report=genbank&log$=nucltop&blast_rank=2&rid=89f7yzyh014 https://www.ncbi.nlm.nih.gov/nucleotide/mk503640.1?report=genbank&log$=nucltop&blast_rank=2&rid=89fp23gy01r https://www.ncbi.nlm.nih.gov/nucleotide/mk503640.1?report=genbank&log$=nucltop&blast_rank=2&rid=89fxm8by014 https://www.ncbi.nlm.nih.gov/nucleotide/mn994365.1?report=genbank&log$=nucltop&blast_rank=1&rid=89gnjhaw01r https://www.ncbi.nlm.nih.gov/nucleotide/mt012260.1?report=genbank&log$=nucltop&blast_rank=1&rid=89mtemfx014 https://www.ncbi.nlm.nih.gov/nucleotide/mt012260.1?report=genbank&log$=nucltop&blast_rank=1&rid=89n8wemv014 https://www.ncbi.nlm.nih.gov/nucleotide/mk503640.1?report=genbank&log$=nucltop&blast_rank=2&rid=89njnt5g016 https://www.ncbi.nlm.nih.gov/nucleotide/mk503640.1?report=genbank&log$=nucltop&blast_rank=2&rid=89pjr8fz014 https://www.ncbi.nlm.nih.gov/nucleotide/mt012260.1?report=genbank&log$=nucltop&blast_rank=1&rid=89rv1pyc01r https://www.ncbi.nlm.nih.gov/nucleotide/cp050079.1?report=genbank&log$=nucltop&blast_rank=1&rid=89st2gre014 https://www.ncbi.nlm.nih.gov/nucleotide/mt197246.1?report=genbank&log$=nucltop&blast_rank=1&rid=89t640s2016 https://www.ncbi.nlm.nih.gov/nucleotide/mk503640.1?report=genbank&log$=nucltop&blast_rank=2&rid=89tj232x014 https://www.ncbi.nlm.nih.gov/nucleotide/cp050079.1?report=genbank&log$=nucltop&blast_rank=1&rid=89tsx2w701r https://www.ncbi.nlm.nih.gov/nucleotide/mt012260.1?report=genbank&log$=nucltop&blast_rank=1&rid=89u1554w016 https://www.ncbi.nlm.nih.gov/nucleotide/mt012260.1?report=genbank&log$=nucltop&blast_rank=1&rid=89u7cp5h016 http://blast.ncbi.nlm.nih.gov/ nova biotechnol chim (2022) 21(2): e1269 2 with activity against other lab species (fusco et al. 2015). the exceptional inhibitory ability against e. coli, s. typhimurium and pseudomonas was in coincidence with that reported for weissella species (woraprayote et al. 2015; fessard and remize 2017). in addition, supernatants of the examined presumptive weissella exhibited a high inhibitory activity against l. monocytogenes scott a, gm1 and gm2 and l. innocua atcc51742 and dsm20649 as well as s. aureus atcc 29213 indicator strains, with inhibition zones between 8 to 20 mm, whereas enterococcus and pediococcus were not inhibited. the ability of weissella isolates to prevent listeria and staphylococcus growth agree with those reported for w. paramesenteroides, w. hellenicaand w. viridescens from pickles, sea foods and meat fermented products (papagianni and papamichael 2011; masuda et al. 2012; leong et al. 2013; chen et al. 2014; castilho et al. 2019). moreover, the antifungal activity of 10 out of 16 weissella isolates against fungal indicators (table 3) coincide with that reported for w. cibaria, w. confusa and w. paramesenteroides against other phytopathogenic or food fungal strains (trias et al. 2008; valerio et al. 2009; ndagano et al. 2011; bianchini et al. 2015; quattrini et al. 2019). the production of lactic and acetic acids by heterofermentative weissella may account for their great inhibitory activity, in agreement to fungal inhibitory compounds production reported by gerez et al. (2013). due to their antimicrobial activity, weissella have been found to act as foodbiopreservatives and probiotics in humans and animals (abriouel et al. 2015; fusco et al. 2015). the production of antimicrobial compounds is desired, thus the proliferation of pathogens or spoilage microorganisms can be controlled during fermentation. based on these antimicrobial features, w. cibaria bk2, bk3 and bk19, w. confusa bk4, bk6 and bk11, and w. paramesenteroides bk8 were selected to investigate their major safety and technological properties. safety evaluation although many lab species have been recognized as gras organisms by fda (1999) or have attained the qps status by efsa (2004), no weissella species were included. studies on antibiotic resistance profile of this genus are limited, and mic cut-off has not still defined by efsa. when weissella strains resistance/sensitivity to clinical antibiotics was investigated (table 4), a multiresistance pattern was found, this being in correlation with that described by abriouel et al. (2015). similar to other lab, w. cibaria, w. confusa and w. paramesenteroides exhibited intrinsic resistance to van (ouoba et al. 2008). however, additional high resistance to kan, gen and tet was exhibited for all weissella strains, strains bk3 and bk19 were also resistant to str and chl, this being in coincidence with those reported for strains isolated from chinese dry fermented meat product (wang et al. 2018) and fermented salted squid (le and yang 2018). similar resistance to gen and kan of w. cibaria strain from goat milk was described (elavarsi et al. 2014), and str resistance of w. cibaria and w. confusa from fruit/juices was also reported (xu et al. 2018). however, the investigated strains were sensitive to ery, which disagree with that found for w. cibariaof vegetable origin (xu et al. 2018; dentice maidana et al. 2019). antibiotic resistance patterns found here are related to the controversial nature of weissella genus reported by abriouel et al. (2015), and the lack of use as commercial starter so far (fessard and remize 2017). despite the resistance to aminoglycosides kan and gen, w. cibaria bk2 was the more sensitive among assayed strains. within the framework of food safety, weissella strains were also investigated for their hemolytic and gelatinase activity as well as biogenic amines production. results showed neither gelatinase nor -hemolytic activity was exhibited by weissella strains, only -hemolysis being observed. similarly, biogenic amines were not produced by the analyzed strains (data not shown). due to their controversial status, determination of these safety traits for weissella strains help in carefully selecting strains lacking pathogenic potential. technological characterization in view to select weissella strain/s to be used for kaddid fermetnation, several technological traits 8 nova biotechnol chim (2022) 21(2): e1269 3 were evaluated. the strains tested showed a good adaptation towards cultural stresses, such as temperature, nacl concentration and ph (table 4). acidification showed ph values between 2.10 and 2.37 units after four days, showing a decrease to 4.03 – 4.27 after 6 h of incubation at 30 °c, reaching final values (96 h) in the range of 3.80 – 4.10 (data not shown). the ph reduction is in correlation with the acid production by lab strains, w. paramesenteroides bk8 being the most acidogenic. the average acidification rate of assayed strains resulted in 0.55 units/day, which was higher than that of traditional kaddid (benlacheheb et al. 2018). as expected, optimal temperature for weissella strains was 30 °c reaching od620 between 1.96 and 2.11 at 48 h with an average growth rate of 0.47/h. when nacl concentration increased from 4 to 10 % a decrease in weissella growth from od620 of 1.68 to 0.06 was produced, w. paramesenteroides bk8 being the most resistant to 10 % of nacl (table 4). osmotic adaptation of strains correlated with their growth under the high salt concentration of kaddid. to prevent spoilage/pathogens proliferation, quick growth/acidification capacity is an important criterion for the selection of lab starter. in addition, the evaluation of enzymatic activities that could play a role in the flavor development, such as the release of intracellular enzymes by cell lysis (autolysis) showed values in the range of 4.23 – 8.08 %, while proteolytic activity was only exhibited by w. confusa bk11.autolysis of weissella strains at 24 h here obtained showed lower values compared to that reported for w. confusa strain isolated from indian fermented foods (sharma et al. 2018). all weissella strains showed to be thermoresistant when heated at 60.5 ºc during 30 min while no lipolytic activity was detected (data not shown). similarly, a lack of lipolytic activity was also reported for lactobacillus plantarum isolated from tunisian kaddid (essid et al. 2009), but were able to hydrolyze casein, contrarily to the results for w. cibaria strains in this study. w. cibaria has been reported to have an extensive peptidolytic activity (lynch et al. 2015); perhaps the use of casein as a protein source did not allow evidencing this activity. furthermore, the lack of h2s and eps production favor their use as starter culture in meat fermentation; even when the ability to produce eps is a common trait for w. cibaria and w. confusa (fusco et al. 2015; lynch et al. 2015; quattrini et al. 2019), formation of these compounds in meat products would lead to an indication of sensory spoilage. therefore, based on antimicrobial activity, antibiotic resistance patterns, growth, and acidification, nacl tolerance and moderate protein hydrolysis as well as the lack of virulence factors and adverse sensory traits, the strains w. cibaria bk2, w. confusa bk6 and bk11 as well as w. paramesenteroides bk8 may be selected as candidates to be used in the fermentation of algerian kaddid. conclusion microbiological examination of southwestern algeria dried and salted kaddid samples, was performed. antimicrobial activity and other safety traits of lab isolates were used to select those inhibitory against food pathogens and contaminants, with low antibiotic resistance, unable to produce virulence factors and not aminogenic. molecular identification showed weissella species as dominant population and in a lesser extent p. acidilactici and e. hirae. in view to use these strains as autochthonous starter and functional culture, weissella strains showing technological and safety traits allowed selecting w. cibaria bk2, w. confusa bk6 and bk11 as well as w. paramesenteroides bk8 as valuable candidates which may contribute not only to improve overall quality but also preserve typicality, which benefits for both producers and consumers. acknowledgements the authors thank the university yahia-fares in médéaalgeria, as well as the fellowship grant in italy, to perform some trials as molecular identification of strains. also, a particular thanks to algerian ministry of hight education and scientific researche (mhesr) for the scholarship longstay (pne 2018/2019, n° 256) at cerela-conicet, tucumán, argentina. conflict of interest the authors declare that they have no conflict of interest. 9 nova biotechnol chim (2022) 21(2): e1269 3 table 3. antimicrobial activity of weissella strains isolated from algerian kaddid. cfs – cell free supernatant. kaddid southwestern algerian samples sk1, sk2, sk3 (béchar city), sk5 (beniounif), sk6 and sk7 (igli) from béchar province, and sk4 from tindouf province. *antibacterial activity is expressed by inhibition zone diameter (mm); ** antifungal activity expressed as: – no growth inhibition), + (1 – 5 mm growth inhibition), ++ (5 – 10 mm growth inhibition) and +++ (˃ 10 mm growth inhibition). indicator microorganism isolated bacteria antibacterial and antifungal activity of bacterial cfs bk1 bk2 bk3 bk4 bk5 bk6 bk7 bk8 bk9 bk10 bk11 bk12 bk13 bk16 bk18 bk19 samples source sk1 sk1 sk1 sk2 sk2 sk3 sk3 sk4 sk4 sk5 sk5 sk6 sk6 sk7 sk7 sk7 gram (-)bacteria* escherichia (e.) coli algerian meat 10.5 2.5 6 e. coli atcc25922 salmonella (s.) bongori algerian meat 2.5 5.5 7.5 6.5 3 s. typhimurium atcc2572 11.5 5.5 7.5 7.5 6.5 8 7 4.5 12 klebsiella pneumoniae algerian kaddid (sk4) 10.5 4.5 2.5 10 citrobacter farmeri algerian kaddid (sk1) 5.5 4 pseudomonas (p). frederiksbergensis algerian kaddid (sk2) 11.5 4.5 6 7 14 p. aeruginosa atcc27853 9.5 8.5 4 5 acenitobacter baumanii atcc 19606 5 4 6 gram (+) bacteria* listeria (l.) monocytogenes scott a 8 9 11 14 8 9 11 15 10 9 12 19 18 15 16 16 l. monocytogenes atcc13932 l. monocytogenesgm1 italian chicken meat 9 15 14 15 15 13 10 12 13 14 9 8 12 15 12 14 l. monocytogenes gm2 italian chicken meat 6 7 11 14 13 10 13 14 14 14 12 8 10 7 13 15 l. monocytogenes atcc 15313 7.5 10 l. innocua atcc51742 10 16 13 11 12 13 10 13 13 12 13 13 13 10 15 15 l.innocua dsm20649 13 8 12 16 15 15 9 16 20 13 15 11 14 19 bacillus cereus atcc 10876 enterococcus (en.) faecalis atcc 49452 9 7 en. faecalis atcc 29212 en. hirae bk15 algerian kaddid (sk5) staphylococcus (s.) aureus atcc 25923 10 7 s. aureus atcc 29213 9 18 16 16 15 15 13 13 15 14 14 14 14 13 13 14 staphylococcus sp. algerian kaddid (sk3) 5 pediococcus acidilactici bk14 algerian kaddid (sk6) molds** penicillium expansum algerian wheat aspergillus flavus algerian wheat + +++ ++ +++ + + ++ fusarium oxysporum albedinis algerian dates + +++ +++ +++ +++ ++ ++ +++ 10 nova biotechnol chim (2022) 21(2): e1269 3 table 4. safety and technological characterization of weissella strains from algerian kaddid. *ph was determined in sb as ph (96 h) – ph (0 h). van – vancomycin; chl – chloramphenicol; gen – gentamycin; str – streptomycin; kan – kanamycin; tet – tetracycline. weissella strains antibiotic resistance ph* growth (od620) at 48 h autolytic activity [%] temperature [°c] nacl [%] 10 30 44 4 6.5 10 w. cibaria bk2 van/kan/gen 2.10 ± 0.02 0.13 ± 0.01 2.10 ± 0.40 1.10 ± 0.3 1.63 ± 0.12 1.23 ± 0.14 0.04 ± 0.00 6.03 ± 0.05 bk3 van/kan/chl/str 2.10 ± 0.01 0.15 ± 0.02 2.10 ± 0.08 1.87 ± 0.33 1.63 ± 0.32 1.23 ± 0.00 0.02 ± 0.01 6.06 ± 0.17 bk19 van/kan/gen/tet/chl 2.10 ± 0.00 0.15 ± 0.02 2.08 ± 0.11 1.85 ± 0.26 1.63 ± 0.17 1.23 ± 0.11 0.02 ± 0.0 8.08 ± 0.2 w. confusa bk4 van/kan/gen/tet 2.10 ± 0.03 0.15 ± 0.00 1.96 ± 0.23 1.87 ± 0.70 1.57 ± 0.09 1.23 ± 0.03 0.06 ± 0.01 4.23 ± 0.37 bk6 van/kan/gen 2.10 ± 0.11 0.19 ± 0.03 2.11 ± 0.07 1.85 ± 0.29 1.63 ± 0.44 1.23 ± 0.10 0.05 ± 0.00 4.23 ± 0.10 bk11 van/kan/gen 2.15 ± 0.07 0.13 ± 0.01 2.10 ± 0.17 1.68 ± 0.03 1.70 ± 0.05 1.35 ± 0.12 0.04 ± 0.01 5.75 ± 0.44 w. paramesenteroides bk8 van/kan/gen 2.37 ± 0.03 0.07 ± 0.00 2.10 ± 0.04 1.68 ± 0.15 1.99 ± 0.12 1.40 ± 0.14 0.18 ± 0.01 5.33 ± 0.21 11 nova biotechnol chim (2022) 21(2): e1269 3 references abriouel h, lavilla lerma l, casado muñoz m delc, pérez montoro b, kabisch j, pichner r, cho g-s, neve h, fusco v, franz chmap, gálvez a, benomar n (2015) the controversial nature of the weissella genus: technological 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tibor roháčik2, jana sokolovičová2 1department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (filip.kraic@ucm.sk) 2selekt, research and breeding institute, bučany 591, sk-919 28 slovak republic abstract: the final goal of this work is development of new genotypes of wheat with better properties compared to the standard set. prediction of optimal descriptors and properties related to the production characteristics is performed using several statistical and chemometrical tools, like correlation analysis, principal component analysis, cluster analysis and linear discriminant analysis. optimisation of wheat genotypes is directed towards high food quality. key words: wheat genotypes, crop, correlation analysis, principal component analysis, cluster analysis. 1. introduction in the last two years a large number of data concerning various wheat genotypes, their properties and observations were prepared in a form of spreadsheets. they stemmed the databases, which are periodically complemented by several slovak breeding stations, state and private research institutions. the main part of the data in this work is from selekt, research and breeding institute, bučany. the evaluated wheat descriptors are of different character; they concern the plant itself (plant height, mass of thousand grains), possible diseases (rust, powdery mildew, fusariosis, septoriosis) as well as some laboratory results (content of gluten, swellingness, sedimentation test, etc.). diversity studies have already been performed on wheat using grain quality (högy et al., 2008; every et al., 2008), fysiological factors (reynolds et al., 2002; reynolds et al., 2004; goggin et al., 2004), and resistance against fungal diseases (hamzehzarghani et al., 2005; snijders, 2004). our previous chemometrical study revealed interesting latent relations among the soil quality (with regard to nutrition elements), content of nutrition elements in the vine leaves, vine crop and quality if the produced wine (kraic et al., 2008). success of that chemometrical data treatment encouraged us to investigate the present agruicultural problem by means of several statistical/chemometrical tools. 2. material and methods 2.1 description of the studied data seventy wheat samples were investigated; among them 16 samples from bučany (designated bu-108 to bu-124 but without bu-122); 13 was from trebišov (bu-108 to bu-124 but without bu-109, bu-110, bu-116, bu-119), 16 from malý šariš (bu-108 to 102 kraic, f. et al. bu-124 but without bu-117) and 17 was from pstruša (bu-108 to bu-124). in addition, the data from all mentioned agricultural locations contained 2 standard wheat strains named ilona and bardotka. the samples omitted from individual locations did not contain all measured (or observed) quantities therefore these samples with the missing values were not further statistically processed. all investigated samples were characterized by the following 6 variables – wet and dry gluten (denominated as wgluten, dgluten, resp.), prugar’s number (prugar) – which represents a relationship between wet gluten and swell gluten, swell (swell), sedimentation (sedimen) and viscosity (viscos). it should be noted that the names starting with capital letter and stressed by italics fonts denote the variables selected for further chemometrical processing. the wheat samples were splitted into two categories according to the value of two categorical variables representing two different classification criteria: (1) sample quality, by which the samples are distributed to four classes designated as a1 (best), a2, b1 and b2 (worst); this way of categorization was made by the agricultural experts in bučany research and breeding institute. (2) sample origin respecting four locations in slovakia, namely bučany, trebišov, malý šariš and pstruša. 2.2 multidimensional data analysis statistical calculations were performed using following techniques: correlation analysis, principal component analysis (pca), cluster analysis (ca) and linear discriminant analysis (lda). in calculations two contemporary software commercial packages were used: stagraphics plus 5.1 and sas jmp 7.0. 3. results and discussion 3.1 correlation analysis the output of correlation analysis is the correlation table, which contains pair (pearson) correlation coefficients expressing the strength of correlation between all possible pairs of variables. the entries of this table are symmetrical according to diagonal. the correlation table comprising mutual dependence of all pairs of six chemical or physico-chemical variables relevant to the wheat quality is summarized in table 1. the following conclusions may be drawn from the correlation table: (a) the highest correlation is between wgluten and dgluten. (b) very high correlations were found between wgluten and prugar as well as dgluten and prugar. (c) very significant correlations (rcrit ≥ 0.306 at p ≤ 0.01) are between the following pairs of variables: prugar and sedimen, swell and viscos, wgluten and sedimen, dgluten and sedimen, viscos and prugar, wgluten and swell (inverse dependence), and sedimen and viscos. (d) a significant correlation (at the 95 % or higher probability level, p ≤ 0.05) is between dgluten and swell (ireverse dependence), and prugar and swell. all hitherto mentioned correlation coefficients are larger or equal than the critical value of the correlation coefficient, rcrit = 0.184, and are marked by bold faces. (e) no significant correlation was proved in all other pairs of variables. nova biotechnologica 9(1) (2009) 103 table 1. pearson correlation coefficients exhibiting the strength of correlation between individual pairs of variables for 70 studied wheat samples. wgluten dgluten prugar swell sedimen viscos wgluten 1 dgluten 0.988 1 prugar 0.762 0.837 1 swell -0.376 -0.256 0.293 1 sedimen 0.452 0.468 0.497 0.073 1 viscos 0.024 0.069 0.389 0.474 0.305 1 critical values of the correlation coefficient (absolute values) for n = 70 are: rcrit = 0.198 (p=0.10), rcrit = 0.235 (p=0.05), rcrit = 0.306 (p=0.01); significant correlations are marked bold. 3.2 cluster analysis generally, the agglomeration process in cluster analysis may be performed either with the studied objects or variables (khattree and naik, 2000). in this work, the clustering was made for the studied six chemical/physico-chemical variables. the result of the performed cluster analysis is a dendrogram depicted in fig. 1. the basis for the performed calculations were the data of 6 selected variables characterizing 70 samples of the wheat strains at 4 quality levels and 4 research locations. ward’s method of clustering and squared euclidean distance was used in all calculations. two main clusters of the progressively agglomerated variables can be seen in fig. 1. the first cluster, connected at the lowest distance level, is formed by wgluten, dgluten, which are most similar, gradually connected to prugar and further to sedimen. the second main cluster is formed by swell and viscos. the variables forming the same cluster are most similar; the measure of mutual similarity is given by the distance on the vertical axis of dendrogram. the results of cluster analysis are in agreement with the outputs of correlation analysis. 3.3 principal component analysis in principal component analysis, pca, some natural grouping of the objects (the wheat samples in this work) and the studied variables (the sample characteristics) may be seen. the principal components, pcs, are calculated as the linear combinations of original variables (sharma, 1996; khattree and naik, 2000). according to the computed eigenvalues only three out of six principal components (pcs) were found important as their value was larger than 1, which is usually considered as the criterion of significancy. three kinds of graphical outputs are used in the pca, namely scatterplot showing the objects, the loadings plot showing the variables, and the biplot where both, the objects and variables are depicted together. the advantage of first two graphical representations is a possibility to obtain the 2d graph as well as the 3d illustration where usually the first two or three most important pcs are used as the axes. on the other hand, the biplot, even though plotted in two dimensions, provides some additional information about the studied problem. 104 kraic, f. et al. fig. 1. cluster analysis of 6 variables characterizing quality of 70 wheat samples obtained from all four research locations. software statgraphics plus 5.1. fig. 2. biplot pc2 vs. pc1 for 6 measured chemical descriptors and 70 samples from 4 localities (1 – bučany, 2 trebišov, 3 – malý šariš, 4 – pstruša). software spss 15. fig. 2 exhibits the biplot, which simultaneously represents the wheat samples (depicted here by the numbers) and six original quality descriptors, depicted by the rays starting from the origin and ending at the point determining the position of the corresponding variable. the samples are here categorized by four locations, namely bučany, trebišov, malý šariš and pstruša. even though pca method is not used for nova biotechnologica 9(1) (2009) 105 classification, high values of the second principal component, pc2, are observable for the samples from trebišov (2), which at the same time means that they are characterized by the highest swelling and viscosity factors. the factors mostly influencing the first principal component, pc1, are wet and dry gluten and prugar number (with relatively smaller contribution of sedimentation). 3.4 linear discriminant analysis linear discriminant analysis (lda) is a multivariate technique focused on separating distinct sets of objects into two or more populations and then allocating the objects, not readily known where they belong to, into one of the considered populations (sharma, 1996). its main goal is to ensure a maximal discrimination among the classes of objects. taking this into account, the original variables are linearly combined into discriminant functions, the number of which is equal to the number of the classes minus one (lankmayr et al., 2004). classification performance depends on the selected categorical target variable. if quality was used as the target variable (the categorization of wheat samples is described in part 2.1) then 94.3% of the originally grouped objects were correctly classified when the discrimination model was calculated and 92.9% of the objects were correctly classified using leave-one-out cross-validation method. it means that 5 objects out of 70 ones were categorized into a different category then supposed). classification of the wheat samples by locality was also successful when considering that four localities were used for categorization. leave-one-out cross-validation showed 82.9 % correct classifications. it is worth noting that in this case 25 % success corresponds to random categorization so that the classification success is clearly pronounced. 4. conclusions principal component analysis and cluster analysis allow display a natural grouping of the wheat samples. both methods revealed that six utilized variables can be divided into two groups: (1) wet and dry gluten, prugar number and sediment, (2) swelling and viscosity factors. the obtained results demonstrate a good applicability of the used multivariate statistical methods for graphical representation of the wheat samples in two dimensional visual display. wheat classification may be conveniently illustrated by linear discriminate analysis, which was proved as an appropriate multidimensional classification technique. further study on the investigated wheat samples using different measurement techniques and observations is under progress. references every, d., motoi, l., rao, p.s., shotner, s.c., simmons, l.d.: predicting wheat quality – consequences of the ascorbic acid improver effect. j. cereal sci., 48, 2008, 339-348. goggin, d.e., colmer, t.d.: wheat genotypes show contrasting abilities to recover from anoxia in spite of similar anoxic carbohydrate metabolism. j. plant physiol., 164, 2007, 1605-1611. 106 kraic, f. et al. hamzehzarghani, h., kushalappa a.c., dion, y., rioux, s., comeau, a., yaylayan, v., marshall, w.d., mathee, d.e.: metabolic profiling and factor analysis to discriminate quantitative resistance in wheat cultivars against fusarium head blight. physiol. mol. plant pathol., 66, 2005, 119-133. högy, p., fangmeier, a.: effects of elevated atmospheric co2 on grain quality of wheat. j. cereal sci., 48, 2008, 580-591. khattree, r., naik, d.n.: multivariate data reduction and discrimination. sas institute, cary, north carolina, usa, 2000. kraic, f., mocak, j., argay, m.: influence of nutrients in soil and vine leaves and meteorological factors upon vine crop and must. nova biotechnol., 8, 2008, 71-77. lankmayr, e., mocak, j., serdt, k., balla, b., wenzl, t., bandoniene, d., gfrerer, m., wagner, s.: chemometrical classification of pumpkin seed oils using uv–vis, nir and ftir spectra. j. biochem. biophys. meth., 61, 2004, 95-106. reynolds, m.p., trethowan, r., crossa, j., vargas, m., sayre, k.d.: erratum to “physiological factors associated with genotype by environment interaction in wheat“. field crop res., 85, 2004, 253-274. reynolds, m.p., trethowan, r., crossa, j., vargas, m., sayre, k.d.: physiological factors associated with genotype by environment interaction in wheat. field crop res., 75, 2002, 139-160. sharma, s.: applied multivariate techniques. wiley, new york, 1996. snijders, c.h.a.: resistance in wheat to fusarium infection and trichothecene formation. toxicol. lett., 153, 2004, 37-46. microsoft word mrazova_nb.doc nova biotechnologica 8-1 (2008) 65 computer-aided diagnosis of lung malignity using multidimensional analysis of tumour marker data viera mrázová1, ján mocák1, elena varmusová2, denisa kavková2 1department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (viera.mrazova@ucm.sk) 2institute for tuberculosis and respiratory diseases, department of clinical chemistry, kvetnica, poprad, sk-058 87 slovak republic abstract: the aim of this work is assessing diagnostic performance of lung tumour markers. three clinical laboratory tests were used for indicating lung malignancy in order to verify or predict the patient’s diagnosis. the data set of 182 patients was examined and two main groups of the patient samples were created – 86 with diagnosed malignancy (confirmed by histology) and 96 with diagnosed benign tumours or tuberculosis. the following tumour markers were analyzed: carcinoembryonic antigen and cytokeratin 19 fragment, which were sampled in the pleural exudates, and the same tumour markers in serum. in addition, the patient’s age and the gender of the corresponding individual were used as further variables in the original data matrix. three laboratory tests were used for indicating lung malignancy in order to verify or predict the patient’s diagnosis not only by using the results of the chosen individual laboratory test but also applying multivariate statistical approach, which jointly utilizes all performed tests in the form of their optimal linear combination. key words: lung malignity, multidimensional analysis, tumour marker. 1. introduction diagnosis of any disease can be confirmed or predicted not only using appropriate laboratory tests but also using multivariate statistical analysis, which uses simultaneously all performed tests in the form of their optimal (usually linear) combination. this new way of enhancing the diagnostic effectiveness, advocated by us, is applied here to the results of laboratory analysis of lung tumour markers in serum as well as pleural effusion (exudate). pleural effusion is common for several kinds of lung illnesses in clinical practice. malignancy is one of the main causes of pleural effusion. greater than 90 % of malignant pleural effusions are due to metastatic disease, mainly from lung or primary breast malignancies. the initial diagnostic approach includes examinations: thoracocentesis, cytology, and biochemical laboratory tests. however, the sensitivity of these non-invasive techniques is considered to be only 40 %–70 %. to improve upon these rates, a number of tumour markers (tm) in the pleural fluid have been intensively evaluated. the most common markers found to be of diagnostic significance were carcinoembryonic antigen (cea), cancer antigen 15-3 (ca), cancer antigen 19-9, and cytokeratin 19 fragment (cyfra 21-1). cea was identified in 1965 and has been widely used during the follow up of various tumours i.e.: colorectal cancers (mroczko et al., 2007; duffy et al., 2007; 66 mrázová, v. et al. yamamoto et al., 2005), breast cancer (nicolini et al., 2008; chen et al., 2006; sölétormos et al., 2004). cyfra 2l-1 assay measures cytokeratin 19 fragment and his concentration is increased with the extent of the malignant disease in non-small cell lung cancer. the serum cyfra 21-1 distribution differs significantly according to histology, disease stage and performance status. lung cancer is the leading cause of cancer deaths in europe. levels of tumour markers, especially cea and cyfra 21-1, may help to establish the diagnosis of pleural malignancy (matsuoka et al., 2007; shitrit et al., 2005; okamoto et al., 2005; fuhrman et al., 2000). it should be added that the most effective positive test is histology of the appropriate tissue sample but this way is invasive and takes a long time. therefore the use of tm may prevent the loss of time necessary for medical treatment in urgent cases. 2. material and methods 2.1 description of the studied data tumour markers were determined at the institute for tuberculosis and respiratory diseases (itrd) in poprad kvetnica, slovakia. the data set of 182 patients was examined; two main groups of the patient samples were created – 86 malignant (with malignancy confirmed by histology) and 96 benign tumours or tuberculosis. the following tumour markers were analyzed in pleural effusion: cea (coded as excea), cyfra 21-1 (excyf) as well as in serum: (scea and scyf, respectively). in addition, the patient’s age (coded as age) and the gender of the corresponding individual (coded as sexn) were used as the variables in the original data matrix. when using classification multivariate statistical techniques, two values of the categorical classification variable dg (diagnosis) were used: 1 indicating malignant diseases and 2 for others. this categorization was made on the basis of known histology results. 2.2 multidimensional data analysis statistical calculations were performed using the principal components analysis (pca), cluster analysis (ca), the linear discriminant analysis (lda), and logistic regression (lr). several software commercial packages were used: stagraphics plus 5.1, spss 15 and jmp 6.0.2. 2.3 analytical procedures tumour markers were analysed by automatic analysers elecsys 1010 and elecsys 2010, which use immunoanalysis with electrochemically generated chemiluminiscent detection. for determinations in pleural effusion an original procedure was used developed at the itrd. nova biotechnologica 8-1 (2008) 67 3. results and discussion 3.1 principal component analysis (pca) the data set of the patients characterized by four variables, namely scea, excea, excyf and scyf was used for a preliminary study. in the way, described in detail in the part 3.1 it was found that the variable scyf is of the least importance. considering this as well as economical aspects the variable scyf was exclude from further studies. instead, the variable age, always accessible, was used in the detailed pca study. fig. 1. pca biplot showing 4 selected variables (excyf, excea, scea, age) and 182 objects – patient’s samples. software spss 15. pca reveals a natural grouping of the studied objects as well as the used variables in a reduced dimensional space (fig. 1). the first principal component (pc1) performs a linear combination of four original variables, optimized with respect to preserving maximal variance of the data. the variables are demonstrated in the exhibited pca biplot by the rays (connecting the variable position in the pc2 – pc1 plane with the origin). the numbers 1 and 2 represent the category where the investigated sample belongs (1 – malignant, 2 – non-malignant). the inspection of the biplot depicted in fig. 1 reveals that the pc1 axis represents malignancy. all tumour markers are positively correlated with the pc1, which is the proof that all patient samples with a high pc1 value are malignant and is in accordance with the observation that the malignant cases are located at high pc1 values. 3.2 cluster analysis (ca) among the clustering techniques, ward´s method with squared euclidean distance metrics was selected for variable clustering. the obtained results are in agreement with 68 mrázová, v. et al. clinical expectations: excea and scea are clustered with excyf so that all tumour markers indicating positive diagnosis result are together. variable age is clustered with sexn since it simply reflects the fact that the average age of women is higher than that of men. d is ta nc e 0 200 400 600 800 s c e a e x c e a e x c y f a g e s e x n fig. 2. cluster analysis of variables using ward`s method, squared euclidean. 182 patient samples with lung diseases. software statgraphics 5.1. 3.3 discriminant analysis with regard to the solved problem the main goals of the applied classification multivariate methods, namely the linear discriminant analysis (lda), quadratic discriminant analysis (qda), and logistic regression (lr) is: (1) to create diagnostic categories and the training data set using the entries of the individual samples with known diagnosis, (2) to elaborate a classification model using the categorized patient samples in the training set, (3) to perform the categorization of the not yet classified samples (belonging to the test set of data) into the selected classes. fig. 3. linear discriminant analysis of the samples denoted by the numbers on the vertical axis. df1 denotes the only discriminant function. 86 samples corresponding to malignancy confirmed by histology (dg=1) and 96 samples regarding benign tumours or tuberculosis (dg=2). software statgraphics 5.1. df1 n um be r dg 2 1 -0,6 1,4 3,4 5,4 7,4 9,4 0 40 80 120 160 200 nova biotechnologica 8-1 (2008) 69 figure 3 represents the lda graphical output, which shows that the non-malignant patient samples (numbers 1-96) are located in a narrow cluster at very negative values of the first discriminate function (df1) whilst the malignant samples (97-182) form a wide tailing cluster at higher df1 values, which is a typical behaviour in many clinical studies. the classification performance for different software packages providing the classification outputs is collected in table 1. the exhibited results of the classification performance regard three types of samples: (1) the training set samples (used for calculating the classification model), (2) the samples omitted from the training set in a step-by-step manner according to the leave-one-out procedure (which was applicable only using the spss software), (3) the samples creating a special test set, which were not included into the training set. tab. 1. classification results for various multivariate methods and software. spss jmp statgraphics classification method training set leave-1out test set training set test set training set test set lda true/all 139/182 137/182 26/30 139/182 26/30 139/182 26/30 % true 76.4 75.3 86.7 76.4 86.7 76.4 86.7 qda true/all 150/182 n/a 25/30 n/a n/a n/a n/a % true 82.4 – 83.3 – – – – lr true/all 163/182 n/a 27/30 163/182 27/30 n/a n/a % true 89.6 – 90.0 89.6 90.0 – – note: the number of the patient samples is given by denominator in the “true/all” ratio. decision upon malignity was predicted using four variables scea, excea, excyf, age except lr where also sexn was used. n/a means that the calculation was not possible when using the cited software. the predictive ability of the used multivariate methods is expressed by the results referring to the last two types of the samples. it is better for the lda (over 86 %) than the qda. however, the best results (90 %) were achieved by logistic regression, where in addition to the variables used in other techniques, the patient’s gender (woman/man) was used (in the form of the binary variable sexn). 4. conclusions principal component analysis and cluster analysis allow display a natural grouping of the samples belonging to the individuals treated for lung diseases. the obtained results demonstrate very good applicability of the used multivariate statistical methods for graphical representation and the samples classification in a reduced number of dimensions. patient’s diagnosis may be predicted or verified not only using the results of the selected individual laboratory test but also utilizing all performed laboratory tests jointly in the form of their optimal combination ensured by an appropriate multidimensional statistical technique. 70 mrázová, v. et al. references chen, ch.ch., hou, m.f., wang, j.y., chang, t.w., lai, d.y., chen, y.f., hung, s.y., lin, s.r.: simultaneous detection of multiple mrna markers ck19, cea, c-met, her2/neu and hmam with membrane array, an innovative technique with a great potential for breast cancer diagnosis. cancer lett., 240, 2006, 279-288. duffy, m.j., van dalen, a., haglund, c., hansson, l., holinski-feder, e., klapdor, r., lamerz, r., peltomaki, p., sturgeon, c., topolcan, o.: tumour markers in colorectal cancer: european group on tumour markers (egtm) guidelines for clinical use. eur. j. cancer, 43, 2007, 1348-1360. fuhrman, c., duche, j.c., chouaid, c., abd alsamad, i., atassi, k., monnet, i., tillement, j.p., housset, b.: use of tumor markers for differential diagnosis of mesothelioma and secondary pleural malignancies. clin. biochem., 33, 2000, 405-410. matsuoka, k., sumitomo, s., nakashima, n., nakajima, d., misaki, n.: prognostic value of carcinoembryonic antigen and cyfra 21-1 in patients with pathological stage i non-small cell lung cancer. eur. j. cardio-thorac., 32, 2007, 435-439. mroczko, b., groblewska, m., wereszczynska-siemiatkowska, u., okulczyk, b., kedra, b., laszewicz, w., dabrowski, a., szmitkowski, m.: serum macrophage-colony stimulating factor levels in colorectal cancer patients correlate with lymph node metastasis and poor prognosis. clin. chim. acta, 380, 2007, 208-212. nicolini, a., carpi, a., ferrari, p., rossi, g.: immunotherapy prolongs the serum cea-tpa-ca 15.3 lead time at the metastatic progression in endocrinedependent breast cancer patients: a retrospective longitudinal study. cancer lett., 263, 2008, 122-129. okamoto, t., nakamura, t., ikeda, j., maruyama, r., shoji, f., miyake, t., wataya, h., ichinose, y.: serum carcinoembryonic antigen as a predictive marker for sensitivity to gefitinibhis in advanced non-small cell lung cancer. eur. j. cancer, 41, 2005, 1286-1290. shitrit, d., zingerman, b., shitrit, a. b., shlomi, d., kramer, m. k.: diagnosis value of cyfra 21-1, cea, ca 19-9, ca 15-3, and ca 125 assay in pleural effusions: analysis of 116 cases and review of the literature. oncologist, 10, 2005, 501-207. sölétormos, g., nielsen, d., schioler, v., mouridsen, h., dombernowsky, p.: monitoring different stages of breast cancer using tumour markers ca 15-3, cea and tpa. eur. j. cancer, 40, 2004, 481-486. yamamoto, y., hirakawa, e., mori, s., hamada, y., kawaguchi, n., matsuura, n.: cleavage of carcinoembryonic antigen indeces metastatic potential in colorectal carcinoma. biochem. bioph. res. commun., 333, 2005, 223-229. zemanova nbc 12-2(1 nova biotechnologica et chimica 12-2 (2013) 100 doi 10.2478/nbec-2013-0012  university of ss. cyril and methodius in trnava synthesis and reactions of new derivatives of furo[3,2-b]pyrrole ivana zemanová, renata gašparová department of chemistry, faculty of natural sciences, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (ivanazemanova@zoznam.sk) abstract: synthesis of methyl 4h-furo[3,2-b]pyrrole-5-carboxylate is taken place in two step synthesis. vilsmeier-haack reaction of 4h-furo[3,2-b]pyrrole-5-carboxylate 1 led to methyl 2-formyl-4h-furo[3,2b]pyrrole-5-carboxylate 4, which served as starting compound for synthesis of furo[3,2-b]pyrrole-2aldoxime 5 and methyl 2-[(4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-4h-furo[3,2-b]pyrrole-5carboxylate 7. the hydrazinolysis of 1 was prepared by carbohydrazide 2, which subsequently reacted with aldehydes to form of derivatives 3. pyrazole derivatives 11 were prepared by the reaction of azlactone 9 with derivatives 10. reactions were carried out under microwave irradiation or at classical heating. key words: aldoxime, carbohydrazide, furo[3,2-b]pyrrole, microwave irradiation, hippuric acid 1. introduction during the past few decades many results have been published in the area of the synthesis of heterocyclic compounds containing furo[3,2-b]pyrrole skeleton (puterová et al., 2004) due to their biological activity (krutošíková et al., 1994; gorugantula et al., 2010). carboxhydrazides and their derivatives are interesting class of compounds, which exhibits antitubercular (jordão et al., 2011), antimicrobial (piaczonka et al., 2013), antifungal (telvekar et al., 2012), anticonvulsant and anti-inflammatory (ulloora et al., 2013) activities. the present paper is a continuation of previous research, which dealt with the synthesis and reactions of furo[3,2-b]pyrrole system (gašparová et al., 2005; gašparová et al., 2007). 2. material and methods melting points of products were determined on a kofler hot place and are uncorrected. 1h nmr spectra were obtained on a 300 mhz spectrometer varian gemini 2000 in cdcl3 or dmso-d6 with tetramethylsilane as the internal standard. the infrared spectra (ir) were taken on a ftir iraffinity-1 spectrophotometer using kbr technique. all microwave experiments were performed in a panasonic nn-e205 type microwave oven. the apparatus was adapted for laboratory applications; nhexane was used as coolant for the condenser. methyl 4h-furo[3,2-b]pyrrole-5carboxylate (4), 4h-furo[3,2-b]pyrrole-5-carbohydrazide (5) and methyl 2-formyl-4hfuro[3,2-b]pyrrole-5-carboxylate (7) were synthesized following the published procedures (krutošíková et al., 1994; gajdoš et al., 2005). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:11 utc nova biotechnologica et chimica 12-2 (2013) 101 2.1. synthesis of compounds 3a-d the mixture of 4h-furo[3,2-b]pyrrole-5-carbohydrazide 2 (1.21 mmol), 5substituted furan-2-karbaldehyde (1.21 mmol) in ethanol (5 ml), and catalytic amount of 4-methylbenzenesulfonic acid was refluxed at 70-80°c for 0.5-1 h (scheme 1). after cooling, the solid product was filtered off, washed with ethanol and recrystallized from ethanol. 2.1.1 n'-{[5-(4-nitrophenyl)furan-2-yl]methylidene}-4h-furo[3,2-b]pyrrole-5 carbohydrazide (3a) yield 48 %; m. p 279-282 °c. calcd. for c18h12n4o5 (364.31) c, 59.34; h, 3.32; n, 15.38. found: c, 58.84; h, 3.24; n, 14.76 %. ir (kbr): 3350, 3140, 1650, 1594, 1510, 1330, 1275, 1137, 1075, 1022, 915, 852, 753, 656 cm-1. 1h nmr (dmso-d6): 11.71 (s,1h, nh); 11.54 (s, 1h, nh); 8.75 (d, 2h, j = 7.8 hz, h-11', h-7'); 7.78 (d, 1h, j = 4.5 hz, h-2); 7.73 (d, 2h, j = 8.4 hz, h-10', h-8'); 7.5 (s, 1h, h-7); 6.98 (s, 1h, h-6); 6.28 (d, 1h, j = 3 hz, h-3'); 5.88 (s, 1h, h-3). 2.1.2 n'-{[5-(4-methylphenyl)furan-2-yl]methylidene}-4h-furo[3,2-b]pyrrole-5carbohydrazide (3b) yield 53 %; m. p 207-210 °c. calcd. for c19h15n3o3 (333.34) c, 68.46; h, 4.54; n, 12.61. found: c, 67.80; h, 4.39; n, 12.40 %. ir (kbr): 3453, 3235, 3113, 1634, 1616, 1571, 1490, 1431, 1251, 1137, 1072, 1028, 986, 819, 724 cm-1. 1h nmr (dmso-d6): 11.58 (s,1h, nh); 11.59 (s, 1h, nh); 8.28 (s, 1h, h-7); 7.77 (d, 1h, j = 4.5 hz, h-2); 7.70 (d, 2h, j = 7.8 hz, h-11', h-7'); 7.31 (d, 2h, j = 8.4 hz, h-10', h8'); 7.07 (d, 1h, j = 3.3 hz, h-4'); 7.02 (m, 2h, h-6, h-3'); 6.60 (d, 1h, j = 3.9 hz, h3); 2.36 (s, 3h, ch3). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:11 utc 102 zemanová, i. and gašparová, r. 2.1.3 n'-[(5-phenylfuran-2-yl)methylidene]-4h-furo[3,2-b]pyrrole-5-carbohydrazide (3c) yield 35 %; m. p 198-200 °c. calcd. for c18h13n3o3 (319.31) c, 67.71; h, 4.10; n, 13.16. found: c, 67.38; h, 3.96; n, 13.62 %. ir (kbr): 3331, 3124, 1631, 1471, 1441, 1358, 1305, 1273, 1135, 1075, 1022, 914, 806, 762, 734, 688, 646 cm-1. 1h nmr (dmso-d6): 11.53 (br, 2h, nh); 8.28 (s, 1h, h-7); 7.70 (m, 3h, h-2, h-7', h11'); 7.49 (m, 3h, h-10', h-8'); 7.37 (m, 1h, h-9'); 7.02 (d, 1h, j = 3 hz, h-4'); 7.04 (d, 1h, j = 3 hz, h-3'); 6.99 (s, 1h, h-6); 6.60 (d, 1h, j = 3.9 hz, h-3). 2.1.4 4-methyl-2-{[2-({2-[3-(trifluoromethyl)phenyl]-4h-furo[3,2-b]pyrrol-5yl}carbonyl)hydrazinylidene]methyl}-4h-furo[3,2-b]pyrrole-5-carboxylic acid (3d) yield 40 %; m. p 244-246 °c. ir (kbr): 3257, 2962, 2596, 1674, 1614, 1533, 1448, 1332, 1261, 1222, 1161, 1072, 957, 796, 694 cm-1. 1h nmr (dmso-d6): 11.84 (s,1h, nh); 11.66 (s, 1h, nh); 8.27 (s, 1h, h-7); 8.12 (m, 2h, h-7˝, h-9˝); 7.67 (m, 2h, h-10˝, h-11˝); 7.43 (s, 1h, h-6'); 7.25 (s, 1h, h-6); 7.04 (s, 1h, h-3'); 7.03(s, 1h, h-3); 6.82 (s, 1h, h-5'); 3.96 (s, 3h, ch3). 2.2 synthesis of methyl 2-[(hydroxyimino)methyl]-4h-furo[3,2-b] pyrrole-5-carboxylate (5) classical heating (a). to mixture of ethanol (26 ml) and water (8 ml) was added hydroxylamine hydrochloride (0.6 g, 9 mmol), sodium hydroxide (2 g, 50 mmol) and methyl 2-formyl-4h-furo[3,2-b]pyrrole-5-carboxylate 4 (0.38 g, 2 mmol) (scheme 1). the reaction mixture was stirred at room temperature for 24 h. the precipitate was filtered off, washed with water and recrystallized from methanol. microwave method (b). to mixture of ethanol (26 ml) and water (8 ml) was added hydroxylamine hydrochloride (0.6 g, 9 mmol) and methyl 2-formyl-4hfuro[3,2-b]pyrrole-5-carboxylate 4 (0.38 g, 2 mmol) (scheme 1). the reaction mixture was irradiated at 90 w for 13 min. after cooling, the solid product was filtered off, washed with water and recrystallized from methanol. yield: 50 % (a), 70 % (b); m. p 236-241 °c. calcd. for c9h8n2o4 (208.17): c, 51.93; h, 3.87; n, 13.46. found: c, 51.88; h, 3.89; n, 13.61 %. ir (kbr): 3290, 3145, 3084, 2960, 1678, 1458, 1315, 1273, 1220, 1124, 985, 920, 769 cm-1. 1h nmr (dmso-d6): 12.09 (d, 1h, j = 0.6 hz, oh); 11.88 (s, 1h, nh); 7.59 (s, 1h, h-6); 7.26 (d, 1h, j = 0.9 hz, h-3); 6.79 (d, 1h, j = 0.7 hz, ch); 3.81 (s, 3h, och3). 2.3 methyl 2-{[(benzyloxy)imino]methyl}-4h-furo[3,2-b]pyrrole-5carboxylate (6) the mixture of methyl 2-[(hydroxyimino)methyl]-furo[3,2-b]pyrrole-5-carboxylate 5 (3.3 g, 1.6 mmol), benzyl chloride (3.8 g, 30 mmol) and catalytic amount of sodium carbonate in acetone (200 ml) was refluxed for 48 h (scheme 1). sodium carbonate bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:11 utc nova biotechnologica et chimica 12-2 (2013) 103 was filtered off and the excess of benzylchloride was distilled under reduced pressure. ice water (100 ml) was added and the mixture was extracted with diethylether (2×50 ml). the organic layer was dried with sodium sulphate and the solvent was evaporated. yield: 20 %; m. p 224-227 °c. calcd. for c16h14n2o4 (298.29): c, 64.42; h, 4.73; n, 9.39. found: c, 64.01; h, 4.69; n, 9.12 %. 1h nmr (cdcl3): 11.72 (s, 1h, nh); 7.77 (s, 1h, h-6); 7.42 (m, 6h, ph); 6.77 (s, 1h, ch); 5.01 (s, 2h, ch2); 3.88 (s, 3h, och3). 2.4 synthesis of methyl 2-[(4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene) methyl]-4h-furo[3,2-b]pyrrole-5-carboxylate (7) the mixture of methyl 2-formyl-4h-furo[3,2-b]pyrrole-5-carboxylate 4 (5 g, 26 mmol), rhodanine (7 g, 53 mmol) and sodium acetate (13 g) in glacial acetic acid (45 ml) was refluxed for 30 min. (scheme 2). the reaction mixture was poured into water (260 ml). the separated precipitate was washed with water (140 ml), ethanol (50 ml), diethylether (20 ml), and the solid product was recrystallized from acetone. yield: 70 %; m. p 296-298 °c. calcd. for c12h8n2o4s2 (308.33): c, 46.74; h, 2.62; n, 9.09. found: c, 46.44; h, 2.60; n, 8.88 %. 1h nmr δh (dmso-d6): 13.69 (brs, 1h, nh); 12.12 (s, 1h, nh); 7.51 (s, 1h, ch); 7.29 (s, 1h, h-6); 6.87 (s, 1h, h3); 3.83 (s, 3h, och3). 2.5 synthesis of 2-[(e)-2-carboxy-2-sulfanylethenyl]-4h-furo[3,2-b] pyrrole-5-carboxylic acid (8) the mixture of 2-[(4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-furo[3,2b]pyrrole-5-carboxylate 7 (4 g, 1.5 mmol) and aqueous sodium hydroxide (7 g naoh in 18 ml h2o) was refluxed for 30 min. (scheme 2). after cooling, 20 m hydrochloric acid (24 ml) was added into the reaction mixture. the formed solid product was filtered off and recrystallized from methanol. yield: 80 %; m. p >350 °c for c10h7no5s (253.23). 1h nmr (dmso-d6): 11.74 (s, 1h, nh); 7.56 (s, 1h, h-6); 6.99 (d, 1h, h-3); 6.74 (dd, 1h, j = 2.7 hz, 1.1 hz, h7); 3.64 (brs, 1h, sh). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:11 utc 104 zemanová, i. and gašparová, r. 2.6 synthesis of methyl 2-[(5-oxo-2-phenyl-1,3-oxazol-4(5h)-ylidene) methyl]-4h-furo[3,2-b] pyrrole-5-carboxylate (9) classical heating (a). the mixture of methyl 2-formyl-4h-furo[3,2-b]pyrrole-5carboxylate 4 (0.5 g, 2.6 mmol), hippuric acid (0.46 g, 2.6 mmol) and catalytic amount of fused potassium acetate (0.38 g, 38 mmol) in acetanhydride (12.5 ml) was refluxed for 1 h (scheme 3). after cooling, the reaction mixture was poured into ice water. the formed solid product was filtered off, washed with water and recrystallized from ethanol. microwave method (b). the mixture methyl 2-formyl-furo[3,2-b]pyrrole-5carboxylate 4 (0.5 g, 2.6 mmol), hippuric acid (0.46 g, 2.6 mmol) and catalytic amount of fused potassium acetate (0.38 g, 38 mmol) in acetanhydride (12.5 ml) was irradiated at 90 w for 12 min (scheme 3). the work-up was the same as above method a. yield: 80 % (a), 92 % (b); m. p 324-326 °c. calcd. for c18h12n2o5 (336.30): c, 64.29; h, 3.60; n, 8.33. found: c, 63.96; h, 3.55; n, 8.21 %. ir (kbr): 3286, 3140, 1786, 1768, 1689, 1631, 1500, 1450, 1396, 1288, 1247, 1219, 1132, 977, 854, 761, 702 cm-1. 1h nmr (cdcl3): 11.76 (s, 1h, nh); 7.61 (m, 5h, ph); 7.18 (s, 1h, h-6); 7.01 (s, 1h, h-3); 6.82 (s, 1h, h-7); 3.93 (s, 3h, och3). 2.7 synthesis of compounds (11a-b) the mixture of methyl 2-[(5-oxo-2-phenyl-1,3-oxazol-5(4h)-ylidene)methyl]-furo [3,2-b]pyrrole-5-carboxylate 9 (1.4 mmol) and corresponding carbohydrazide (1.4 mmol), acetic acid (10 ml) and catalytic amount of fused potassium acetate (0.4 g) was irradiated at 90 w for 17 min (scheme 3). after cooling, the solid product was filtered off, washed with water and recrystallized from ethanol. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:11 utc nova biotechnologica et chimica 12-2 (2013) 105 2.7.1 methyl 2-{[1-{[(2,3-dimethyl-4h-furo[3,2-b]pyrrole-5-yl)carbonyl]imino}-5-oxo -2-phenyl-4h-imidazol-4-ylidene]methyl}-4h-furo[3,2-b]pyrrole-5-carboxylate (11a) yield: 65 %; m. p 285-287 °c. calcd. for c27h21n5o6 (511.49): c, 63.40; h, 4.14; n, 13.69. found: c, 63.19; h, 4.35; n, 12.82 %. 1h nmr (dmso-d6): 11.78 (s, 1h, nh); 11.30 (s, 1h, nh); 10.14 (s, 1h, nh); 8.09 (m, 2h, h-2', h-6'); 7.62 (m, 3h, h3', h-4', h-5'); 7.26 (s, 1h, h-7); 6.87 (s, 1h, h-6˝); 6.86 (d, 1h, j = 1.9 hz, h-6); 6.68 (d, 1h, j = 1.8 hz, h-3); 3.78 (s, 3h, och3); 2.28 (s, 3h, ch3); 2.04 (s, 3h, ch3). 2.7.2 methyl 2-{[1-(phenylcarbonylimino)-5-oxo-2-phenyl-4h-imidazol-4-ylidene] methyl}-4h-furo[3,2-b]pyrrole-5-carboxylate (11b) yield: 74 %; m. p 310-314 °c. calcd. for c25h18n4o5 (454.43): c, 66.07; h, 3.99; n, 12.33. found: c, 65.78; h, 3.94; n, 11.78 %. ir (kbr): 3296, 1685, 1629, 1512, 1450, 1286, 1219, 1143, 817, 758, 698 cm-1. 1h nmr (dmso-d6): 12.07 (s, 1h, nh); 11.71 (s, 1h, nh); 8.05 (dd, 2h, j = 8.1 hz. j = 1.5 hz, h-2',h-6'); 7.91 (dd, 2h, j = 8.2 hz, j = 1.5 hz, h-3', h-5'); 7.66 (m, 6h, ar); 7.53 (s, 1h, h-6); 7.26 (s, 1h, h-7); 6.88 (s, 1h, h-3); 3.85 (s, 3h, och3). 3. results and discussion methyl 2-formyl-4h-furo[3,2-b]pyrrole-5-carboxylate 7 was routinely prepared by condensation of furan-2-carboxaldehyde with methyl azidoacetate in methanol in the presence of sodium methoxide giving methyl 2-azido-3-(furan-2-yl)propenoate, which underwent the cyclization in boiling toluene to give methyl 4h-furo[3,2-b]pyrrole-2carboxylate 4 (krutošíková et al., 1994). compounds 3a-d were formed by reaction with carbohydrazide 2 using corresponding aldehydes in 35-53% yield. the structures of compounds 3a-d were corroborated by the presence two singlets of nh protons at 11.84 and 11.53 ppm. ir spectra of 3a-d exhibit absorption bands of carbonyl group at 1631-1674 cm-1 and absorption bands of nh group at 3258-3453 cm-1. one of the most interesting results in the study is synthesis of methyl 2[(hydroxyimino)methyl]-4h-furo[3,2-b]pyrrole-5-carboxylate 5, which was prepared by reaction of methyl 2-formyl-4h-furo[3,2-b]pyrrole-5-carboxylate 4 with hydroxylamine hydrochloride in sodium hydroxide either by classical heating for 24 h in 50% yield or in microwave oven for 13 min. in 70% yield. the only one preparation of 4h-furo[3,2-b]pyrrole-2-aldoxime 5 has been described by gorugantula et al., 2010. in their study there was described the synthesis of 4h-furo[3,2-b]pyrrole-2aldoxime via 4-nitro-5-(2-phenylethenyl)furan-2-aldoxime palladium-catalysed reductive cyclization. reaction of aldehyde 4 with hydroxylamine has been described as useful method of nitrile synthesis (krutošíková et al., 1993), but in that case aldoxime 5 has not been isolated. 1h nmr spectrum displayed doublet signal of =noh proton at 12.09 ppm. doublet signal of ch proton occur at 6.79 ppm. ir spectrum of 5 shows the absorption bands of carbonyl group at 1678 cm-1 and of nh group at 3290 cm-1. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:11 utc 106 zemanová, i. and gašparová, r. reaction of 5 with benzyl chloride in acetone led to methyl 2{[(benzyloxy)imino]methyl}-4h-furo[3,2-b]pyrrole-5-carboxylate 6 in yield 20 % after 48 h of refluxed. 1h nmr spectra display singlet signal of nh proton at 11.72 ppm, multiplet signal of ph group at 7.42 ppm, singlet signal of ch2 bond at 5.01 ppm. the reaction of methyl 2-formyl-4h-furo[3,2-b]-pyrrole-5-carboxylate 4 with rhodanine afforded compound 7, which subsequently underwent hydrolysis to provide derivative 8 in 80 % yield. compound 8 display singlet signal of nh group at 1.74 ppm and broad signal of sh group at 3.64 ppm. signal cooh group is not occurring in the spectrum as a result of the rapid exchange hydrogen for deuterium. the reactions of azlactone 9 with carbohydrazides 10 were carried in acetic acid under microwave irradiation in the presence of potassium acetate to give compounds 11a-b in yields 65-74 %. compounds 11a and 11b display two or three singlets at 12.01–10.14 ppm in their 1h nmr spectra due to nh group. three singlets of nh groups of compound 11a appear at 11.78, 11.30 and 10.14 ppm. the characteristic bands observed at 1680 cm-1 and 3296 cm-1 in ir spectra correspond to the c=o and nh group. the structures of the prepared compounds were confirmed by 1h nmr and ir spectra. 4. conclusions derivatives 3 were obtained by the reaction of carbohydrazide 2 with heterocyclic aldehydes. methyl 2-formyl-4h-furo[3,2-b]pyrrole-5-carboxylate 4 served as the substrate for synthesis of furo[3,2-b]pyrrole-2-aldoxime 5 as well as derivatives 7, 9. the prepared furo[3,2-b]pyrrole-2-aldoxime 5 was used as starting compound for the condensation reaction with benzyl chloride to give a compound 6. the hydrolysis of compound 7 gave 2-[2-carboxy-2-sulfanylethenyl]-4h-furo[3,2-b]pyrrole-5-carboxylic acid 8. pyrazole derivatives 11 were prepared by the reaction of azlactone 9 with derivatives 10. acknowledgement: this work was supported by the slovak research agency under the contract no. vega 1/0233/12 references gajdoš, p., pavlíková, s., bureš, f., krutošíková, a.: 2-[3(trifluoromethyl)phenyl]furo[3,2-b]pyrroles: synthesis and reactions. cent. eur. j. chem., 3, 2005, 311-325. gašparová, r., moncman, m., horváth, b.: microwave assisted reactions of 2-[3-(trifluoromethyl)phenyl]-4-r1-furo[3,2-b]pyrrole-5-carboxhydrazides. cent. eur. j. chem., 6, 2007, 180-187. gašparová, r., zbojek, d., lácová, m., kráľová, k., gatial, a., horváth, b., krutošíková, a.: reactions of substituted furo[3,2b]pyrrole-5-carboxhydrazides and their biological activity. cent. eur. j. chem., 3(4), 2005, 622-646. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:11 utc nova biotechnologica et chimica 12-2 (2013) 107 gorugantula, p. s., martínez-carrero, m. g., dantale, w. s., söderberg, c. g. b.: palladium-catalyzed reductive n-heterocyclization of alkenyl-substituted nitroarenes as a viable method for the preparation of bicyclic pyrrolofused heteroaromatic compounds. tetrahedron, 66, 2010, 1800-1805. jordão, k.a., sathler, c. p., ferreira, f.v., campos, r. v., de souza, c.b.v.m., castro, c.h., lannes, a., lourenco, a., rodrigues, r. c., bello, l. m., lourenco, c.s.m., calvalho, s.l.g., almeida, c.b.m, cunha, c.a.: synthesis, antitubercular activity, and sar study of n-substitutedphenylamino-5-methyl-1h-1,2,3-triazole-4-carbohydrazides. bioorg. med. chem., 19, 2011, 5605–5611. krutošíková, a., dandárová, m., alföldi, j.: synthesis and reactions of furo[3,2-b]pyrrole type aldehydes. coll. czech. chem. commun., 58, 1993, 21392149. krutošíková, a., dandárová, m., alföldi, j.: substituted vinyl azides in the synthesis of condensed nitrogen heterocycles. chem. papers, 48, 1994, 268273. pieczonka, m. a., strzelczyk, a., sadowska, b., mlostoń, g., staczek, p.: synthesis and evaluation of antimicrobial activity of hydrazones derived from 3-oxido-1h-imidazole-4-carbohydrazides. eur. j. med. chem., 64, 2013, 389–395. puterová, z., sterk, h., krutošíková, a.: reaction of substituted furan-2carboxaldehydes and furo[b]pyrrole type aldehydes with hippuric acid. molecules, 9, 2004, 11-21. telvekar, n. v., belubbi, a., bairwa, k.v., satardekar. k.: novel n′benzylidene benzofuran-3-carbohydrazide derivatives as antitubercular and antifungal agents. bioorg. med. chem. lett., 22, 2012, 2343–2346. ulloora, s., shabaraya, r., ranganathan, r., adhikari, v.a.: synthesis, anticonvulsant and anti-inflammatory studies of new 1,4dihydropyridin-4-yl-phenoxyacetohydrazones. eur. j. med. chem., 70, 2013, 341– 349. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:11 utc microsoft word fecskeova et al. nb 2010.doc nova biotechnologica 10-2 (2010) 131 cloning and characterization of coba, one of vitamin b12 biosynthesis pathway genes from selenomonas ruminantium lívia fecskeová, peter pristaš, peter javorský institute of animal physiology, slovak academy of sciences, šoltésovej 4-6, košice, slovak republic (fecskeova@saske.sk) abstract: bacterial biosynthesis of vitamin b12 can occur via either aerobic or anaerobic route. while the aerobic pathway has been fully elucidated and understood, less is known about the anaerobic pathway. selenomonas ruminantium is thought to be the main producer of this vitamin in rumen environment and must use the anaerobic pathway. in our work we found one of the genes of vitamin b12 biosynthetic pathway of s. ruminantium, encoding for the cobalamin adenosyltransferase, enzyme taking part at the last steps of the synthesis process. deduced amino acid sequence showed the highest similarity to cobalamin adenosyltransferases of other ruminal anaerobic bacteria and that of species selenomonas. phylogenetic comparisons of coba protein sequences of several anaerobic bacteria of clostridiale order indicate possible horizontal transfer of this gene. key words: vitamin b12, cobalamin biosynthesis, selenomonas ruminantium 1. introduction one of nature’s structurally most complex small molecule, vitamin b12 or cobalamin, is an essential nutrient for humans and animals. it is used to treat pernicious anaemia and peripheral neuritis, and is used as a dietary supplement. while it is required in both prokaryotic and eukaryotic metabolism, only some bacterial and archaeal species are able to synthesize it de novo (rodionov et al., 2003). some prominent b12-dependent reactions in bacteria and archaea include the process of acetate formation in acetogenic bacteria, transfer of methyl group in methaneproducing archaea and anaerobic fermentation of 1,2-propanediol, ethanolamine and glycerol in enteric bacteria (marten et al., 2002). animals and humans require b12 as coenzyme for two enzymes only: methionine synthase for homocysteine methylation and methylmalonyl-coa mutase involved in oxidation of fatty acids and amino acids, respectively. plants and fungi are thought to neither synthesize, nor need it (duda et al., 1967). two different cobalamin synthetic pathways have been found an evolutionally older anaerobic pathway found in salmonella enterica or propionibacterium shermanii (roessner and scott, 2006) and a more recent aerobic route in pseudomonas denitrificans (battersby, 1994), which differ at the point of cobalt insertion. as the industrial production by chemical synthesis would be too expensive and technically demanding, vitamin b12 is produced industrially by biosynthetic fermentation processes, employing almost exclusively p. denitrificans and propionibacterium species (martens et al., 2002). selenomonas ruminantium, a gram-positive obligate anaerobe originally isolated from rumen of cattle, is one of the most numerous bacteria of the ruminal microbial 132 fecskeová, l. et al. population. considerable metabolic role it plays in rumen is given by its capability to convert succinate to propionate and to utilize lactate and many amino acids (ricke et al., 1996). previous research has showed that this bacterium is the main b12 contributor to the rumen environment (dryden et al., 1962), where its role is in the assimilation of propionic acid, a major fermentation product of the ruminal microflora, by b12-dependent methylmalonyl-coa mutase. 2. materials and methods 2.1 bacterial strain and growth conditions bacterial strain s. ruminantium s8 was cultivated anaerobically in the atmosphere of pure co2 in selective m10 broth medium (caldwell and bryant, 1966) with an addition of 10 % clarified rumen fluid, 0.1 % mineral solution and 0.1 % vitamin solution (clark and holms, 1976). fructose (4g/l) was used as the sole carbon source. 2.2 dna isolation and pcr reaction total dna from bacterial cells was isolated by sodium dodecylsulphate lysis and subsequent phenol extractions (pospiech and neumann, 1995). total dna was amplified in techne tc 3000 (usa) thermal cycler in 50 μl reaction mix containing 40 μmol/l each dntp, 20 pmol of each primer, 1x reaction buffer and 0.5 u of taq dna polymerase (invitrogen, netherland). pcr primers used were srdrecf (5´tctcgaaaatggggcgcagc3´) and srdrecr (5´tttgagamactcataa gtgcgcattc3´). initial denaturation step (95 °c 5 min) was followed by 35 cycles (94 °c 1 min, 58 °c 1 min, 72 °c 1 min) and a final incubation at 72 °c for 10 min. 2.3 molecular cloning and sequence analysis pcr amplicon was ligated into plasmid ptz57r/t (fermentas, lithuania) and escherichia coli er2267 competent cells were transformed with the ligation mixture. recombinant plasmid was extracted using the genejet plasmid miniprep kit (fermentas, lithuania) and the dna insert was sequenced. using the blast algorithm (altschul et al., 1990) at ncbi the obtained nucleotide sequence was subjected to homology search against nucleotide and protein database. for phylogenetic comparisons mega 4 software (tamura et al., 2007) was used. 3. results and discussion small plasmids of s. ruminantium were suggested to undergo recombination events during the rolling circle replication, when highly recombinogenic single-stranded plasmid intermediates form. recombination is proposed to rely on highly conserved, short dna sequences characteristic for many small rcr plasmids of s. ruminantium, nova biotechnologica 10-2 (2010) 133 such as the srsr elements (selenomonas ruminantium sequence repeats) (nakamura et al., 1999). during experiments on the possible role of srsr mediated recombination in the plasmid evolution and spreading in s. ruminantium using pcr and specifically designed primers amplifying interchangeable plasmid rep cassettes multiple amplicons were obtained. while most of the obtained amplicons contained plasmid derived sequences encoding for replication protein (not published yet), surprisingly in one case the complete coba gene encoding for cobalamin adenosyltransferase was present. after pcr reaction we obtained an approximately 800 bp amplicon, which we cloned and sequenced. while no significant similarity was observed at nucleotide level, the 780 bp dna fragment contained single orf encoding for a 178 amino acid protein with significant similarity to cob(i)alamine adenosyltransferase or cob(i)yrinic acid a,c-diamide adenosyltransferase, one of many enzymes of the b12 biosynthetic pathway. the sequence was submitted to genbank database under the accession number hq383925. comparison of the deduced amino acid sequence with the generalized structure of conserved domains of cobalamin adenosyltransferases (fig. 1) shows the presence of all conserved domains, within which each amino acid is conserved according to the generalized structure. we found the presence of two walker motifs type a and b, hydroxycobalamin binding site, atp binding site and homodimer interface. derived protein sequence showed the highest similarity to mitsuokella multiacida (65 % identity, 78 % similarity), megamonas hypermegale art12/1 (64 % identity, 79 % similarity), and s. flueggei atcc 43531 (62 % identity, 79 % similarity) coba sequences. phylogenetic comparison of several available coba genes of anaerobic bacteria (fig. 2) within the clostridiales order showed that s. ruminantium coba sequence groups together with other selenomonas spp. and related sequences. surprisingly this group of coba sequences is more related to the coba sequences of peptococcaceae family than to other members of veillonellaceae family, where s. ruminantium belongs to, indicating possible horizontal gene transfer of coba gene. fig. 1. schematic representation of selected motifs in s. ruminantium coba gene. the position of conserved amino acids characteristic for coba protein domains is indicated by blafl triangles. importance of bacterial production of vitamin b12 is given by its role in the elimination of some volatile fatty acids produced in the rumen. despite it very limited information is available on the genetic background of its production by ruminal selenomonads, which are the predominant suppliers of this vitamin to the rumen 134 fecskeová, l. et al. environment. so far, only one study has been performed investigating the b12 synthesis in s. ruminantium (anderson et al., 2001), in which the authors described five genes organized as an operon, encoding for enzymes acting in the first steps of b12 synthesis. a bifunctional gene, coba + hemd, coding for a protein with two catalylic functions – uroporphyrinogen iii synthase and uroporphyrinogen iii 2,7methyltransferase – was found and protein phylogenetic comparisons showed that this form of the enzyme is probably restricted to strictly anaerobic bacteria. the gene we describe here encodes for cobalamine adenosyltransferase, enzyme catalyzing one of the last steps of b12 biosynthesis. fig. 2. phylogenetic tree showing relatedness of s. ruminantium coba sequence (shown in bold). the tree was constructed using neighbour-joining algorithm implemented in mega 4 software. the sequence of alphaproteobacterium caulobacter segnis coba was used as an outgroup. considering the fact that the coba gene is located between highly conserved sequences found on s. ruminantium plasmids, which might carry out a recombinationmediated exchange of the gene or complete gene cassettes; and that protein phylogenetic comparisons also indicate possible horizontal transfer of this gene between different species, it is possible that a certain part encoding b12 pathway enzymes could have been gained in mechanisms of horizontal transfer. acknowledgment: this publication is the result of the projects no. 26220120001 and 26220120043 implementation supported by the research & development operational programme funded by the erdf. references altschul, s.f., gish, w., myers, e.w., lipman, d.j.: basic local alignment search tool. j. mol. biol., 215, 1990, 403-410. nova biotechnologica 10-2 (2010) 135 anderson, p.j., entsch, b., mckay, d.b.: a gene, coba + hemd, from selenomonas ruminantium encodes a bifunctional enzyme involved in the synthesis of vitamin b12. gene, 281, 2001, 63-70. battersby, a.r.: how nature builds the pigments of life: the conquest of vitamin b12. science, 264, 1994, 1551-1557. caldwell, d.r., bryant, m.p.: medium without rumen fluid for nonselective enumeration and isolation of rumen bacteria. appl. microbiol., 14, 1966, 794-801. clark, b., holms, w.h.: control of the sequential utilization of glucose and fructose by escherichia coli. j. gen. microbiol., 95, 1976, 191-201. duda, j., pedziwilk, z., zodrow, k.: studies on the vitamin b12 content of the leguminous plants. acta microbiol pol, 6, 1967, 233-238. dryden, l.p., hartman, a.m., bryant, m.p., robinson, i.m., moore, l.a.: production of vitamin b12 and vitamin b12 analogues by pure cultures of ruminal bacteria. nature, 14, 1962, 201-202. martens, j.-h., barg, h., warren, m.j., jahn, d.: microbial production of vitamin b12. appl. microbiol. biotechnol, 58, 2002, 275-285. nakamura, m., nagamine, t., ogata, k., tajima, k., aminov, r.i., benno, y.: sequence analysis of small cryptic plasmids isolated from selenomonas ruminantium s20. current microbiol., 38, 1999, 107-112. pospiech, a., neumann, b.: a versatile quick/prep of genomic dna from grampositive bacteria. trends genet., 11, 1995, 217-218. ricke, s.c., martin, s.a., nisbet, d.j.: ecology, metabolism, and genetics of ruminal selenomonads. crit. rev. microbiol., 22, 1996, 27-65. rodionov, d.a., vitreschak, a.g., mironov, a.a., gelfand, m.s.: comparative genomics of the vitamin b12 metabolism and regulation in prokaryotes. j biol. chem., 278, 2003, 41148-41159. roessner, c.a., scott, a.i.: fine-tuning our knowledge of the anaerobic route to cobalamin (vitamin b12). j bacteriol., 188, 2006, 7331-7334. tamura, k., dudley, j., nei, m., kumar, s.: mega4: molecular evolutionary genetics analysis (mega) software version 4.0. mol. biol. evol., 24, 2007, 1596-1599. nova biotechnol chim (2021) 20(2): e1010 doi: 10.36547/nbc.1010 1 nova biotechnologica et chimica formation of carnosine an ab initio study roman boča, beáta vranovičová department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava 91701, slovakia  corresponding author: roman.boca@ucm.sk article info article history: received: 11th june 2021 accepted: 8th september 2021 keywords: carnosine histidine beta-alanine ab initio calculations molecular properties equilibrium constant note: used non-si units kcal mol-1 = 4.184 kj.mol-1, debye, d = 3.336 × 10-30 ams, bohr, a0 = 5.292 ×10 −11 m, angstrom, å = 10-10 m, atm = 101,325 pa abstract formation of carnosine from histidine and -alanine is studied by ab initio molcao-scf method followed by the perturbative configuration interaction (mp2) in vacuo. after the full geometry optimization at the scf level, the molecular properties were evaluated and followed by the vibrational-rotational analysis. consequently, the energy, entropy and free energy were evaluated for the reactants and products of the reaction histidine + -alanine → carnosine + h2o and finally, the equilibrium constant was enumerated. © university of ss. cyril and methodius in trnava introduction carnosine (beta-alanyl-l-histidine) is a simple peptide formed by the condensation reaction of histidine and -alanine (eq. 1). histidine + -alanine → carnosine + h2o (1) the molecular structures are viewed in fig. 1. carnosine is a substance with multi-beneficial effects (boldyrev et al. 2013). it is synthesized in organisms by the carnosine-synthase (eq. 2) atp + l-histidine + -alanine → adp + phosphate + carnosine (2) fig. 1. from top to bottom: structure of the histidine, alanine, and carnosine (zwitterionic forms) as extracted from the ccdc (cambridge crystallographic data centre). ccdc codes: 1889705, 1105698, and 1105666. colours: c – dark grey, n – blue, o – red, h – white. mailto:roman.boca@ucm.sk nova biotechnol chim (2021) 20(2): e1010 2 and it is metabolized by the canosinase. beneficial effects of the carnosine were proven in many areas and the most important of them are: prevention of neurodegeneration, improvement of memory, antidepresive action, and suppressing of cancer (schön et al. 2019). as carnosine penetrates the blood-brain barrier (bbb) and causes only little side effects, it possesses high utility potential. it serves also as a reservoir of histidine which is transformed to histamine by l-histidinedecarboxylase; the histamine itself does not penetrate bbb. experimental x-ray structure data for the reactants and products of the reaction (eq. 1) have been retrieved from the ccdc database (cambridge crystallographic data centre). these solid-state structures were used as an initial guess for the full geometry optimization in vacuo. for such a purpose the ab initio molcao-scf method was utilized using the 631g** basis set (hyperchem 2008). the dilemma of the zwitterionic versus amino forms in vacuo was solved by considering both of them. at the scf level a number of molecular properties were evaluated: the energies of the homo (the highest occupied molecular orbital) and lumo (the lowest unoccupied molecular orbital), the adiabatic ionization energy ei and electron affinity eeg based upon the positively and/or negatively charged open-shell system after geometry optimization, followed by the evaluation of the mulliken electronegativity  = (ei eeg)/2 and the pearson hardness  = (ei + eeg)/2 (pearson 1977; sen 1993). for the electroneutral molecule also the dipole moment p and the polarizability volume  (one-third trace of the polarizability tensor) were calculated at the scf level. the molecular electrostatic potential (politzer et al. 1985; politzer and murray 2002) was displayed as a 3d contour map on the isovalue surface of charge density; this shows the acidic/basic sites along the molecular skeleton suitable for nucleophilic and/or electrophilic interactions. the energetic properties were improved by a partial inclusion of the correlation energy by employing the 2nd-order perturbative configuration interaction using the moller-plesset partitioning (mp2). the above results have been compared with those obtained by the enlarged basis set 6-311g(d,p) after the full geometry optimization at the mp2 level. the optimized geometry has been a starting point for the full vibrational-rotational analysis yielding the corresponding energy levels. they enter the partition function allowing the evaluation of the thermodynamic functions, such as the internal energy u, entropy s, the free energy a and the gibbs energy g at the room temperature. results and discussion the mo-lcao-scf calculations started from the experimental solid-state geometry that is a zwitterionic form for histidine, -alanine and also carnosine. the full geometry optimization converged to the zwitterionic form (hereafter z) as displayed in fig. 2 for histidine. there exists a fivemembered ring h(nh2)cco with the short contact nh…o forming a bent hydrogen bond. the molecular electrostatic potential shows that the (h2n)h site is positively charged as opposite to the –coo residue. zwitterionic form amino acid form optimum geometry at the scf level start form the ccdc geometry (zwitterion), final zwitterionic form with the nh…o contact start from the reallocated hydrogen atom molecular electrostatic potential; contour 0.03 ea0 -1 fig. 2. optimized structure and molecular electrostatic potential for histidine. (a0 – bohr). in addition, the calculations were done for the amino-acid form (hereafter a) in which the nova biotechnol chim (2021) 20(2): e1010 2 hydrogen atom from the ammonium site was reallocated to the distant oxygen atom. for histidine this canonical form is a bit more stable by 17 kcal.mol-1 at the scf level and 10 kcal.mol-1 at the mp2 level (table 1). the molecular properties of the zand a-forms are almost analogous; the only marked difference exhibits the dipole moment that for the z-form is very high. the pearson hardness reflects the resistance of the molecule against the electron transfer and for the histidine it is high (155, 161 kcal.mol-1) relative to the series of other amino acids (vranovičová and boča 2021). table 1. calculated properties of histidine. a a all energy quantities in units of kcal mol-1. -alanine belongs to the simplest amino acids and it crystallizes in the zwitterionic form. however, the geometry optimization resulted in travelling of the hydrogen atom from the nh3 + site to the –coo– residue where a six-membered ring (h2n)cccoh is formed with the bent n…ho hydrogen bond (fig. 3). the ground state molecular energy for the zand aforms are almost identical, favouring the a-form only by 0.6 kcal.mol-1 (table 2). the calculated molecular properties are very similar, again except the dipole moment. the molecular electrostatic potential is rather unique with a positive tail at the ho– edge pointing to the negatively charged nh2– group. the hardness is high: 160 and 167 kcal.mol-1. for this small molecule the geometry optimization was performed also at the mp2 level. the final geometry is almost identical with that fixed at the scf level and it is stabilized by only 2 kcal.mol-1. zwitterionic form amino acid form optimum geometry at the scf level start from the ccdc geometry (zwitterion), final amino acid form with the n…ho contact start from the reallocated hydrogen atom molecular electrostatic potential; contour 0.03 ea0 -1 fig. 3. optimized structure and molecular electrostatic potential of -alanine. table 2. calculated properties of -alanine. a a all energy quantities in units of kcal.mol-1. zwitterion amino acid homo -215 -201 lumo 101 116 e+ -342,138 -342,136 e0 (optimized geometry) -342,323 -342,340 e− -342,220 -342,252 ei(scf) 185 204 eeg(scf) 103 88 m(scf) 41 58 p(scf) 144 146 e+(mp2) -343,183 -343,177 e0(mp2) -343,399 -343,409 e−(mp2) -343,305 -343,318 ei(mp2) 216 232 eeg(mp2) 94 91 m(mp2) 61 70 p(mp2) 155 161 dipole moment p/debye 12.99 6.20 polarizability /å3 75.6 75.6 surface area s /å2 321 325 volume v/å3 475 482 zwitterion amino acid homo -261 -247 lumo 116 124 e+ -201,788 -201,778 e0 (optimized geometry) -201,985.2 -201,985.8 e− -201,878 -201,890 ei(scf) 197 208 eeg(scf) 107 96 m(scf) 45 56 p(scf) 152 152 e+(mp2) -202,361 -202,351 e0(mp2) -202,593 -202,591 e−(mp2) -202,505 -202,496 ei(mp2) 232 240 eeg(mp2) 88 95 m(mp2) 72 72 p(mp2) 160 167 dipole moment p/debye 6.75 2.97 polarizability /å3 40.7 40.5 surface area s/å2 242 242 volume v/å3 331 331 3 nova biotechnol chim (2021) 20(2): e1010 2 the molecule of carnosine during the geometry optimization becomes folded as shown in fig. 4. zwitterionic form amino acid form optimum geometry at the scf level start from the ccdc geometry (zwitterion) with spatially separated nh3 + and coo groups start from the reallocated hydrogen atom molecular electrostatic potential; contour 0.03 ea0 -1 fig. 4. optimized structure and molecular electrostatic potential of carnosine. table 3. calculated properties of carnosine. zwitterion [kcal.mol-1] amino acid [kcal.mol-1] homo -162 -200 lumo 38 115 e+ -496,423 -496,451 e0 (optimized geometry) -496,552 -496,619 e− -496,526 -496,540 ei(scf) 129 168 eeg(scf) 26 79 m(scf) 51 44 p(scf) 77 123 e+(mp2) -496,846 -496,870 e0(mp2) -498,113 -498,171 e−(mp2) -496,959 -496,967 ei(mp2) 1,267 1,301 eeg(mp2) 1,154 1,204 m(mp2) 56 48 p(mp2) 1,210 1,252 dipole moment p/debye 25.24 4.95 polarizability /å3 113.8 112.3 surface area s/å2 438 445 volume v/å3 677 689 the a-form is preferred against the z-form by 67 and 58 kcal.mol-1, respectively. the inclusion of the correlation energy via the mp2 method causes a dramatic increase of the ionization energy, electron affinity and consequently the pearson hardness. the effect to the mulliken electronegativity is small (table 3). the molecule of water possesses rather high ionization energy and electron affinity (table 4). the calculated ionization energy ei(mp2) = 283 matches the experimental value of 291 kcal.mol-1. the pearson hardness p(mp2) = 202 kcal.mol -1 refers to the class of hard molecules. table 4. calculated properties of water.a homo -312 lumo 135 e+ -47,455 e0 (optimized geometry) -47,706 e− -47,577 ei(scf) 251 eeg(scf) 129 m(scf) 61 p(scf) 190 e+(mp2) -47,547 e0(mp2) -47,830 e−(mp2) -47,708 ei(mp2) 283 eeg(mp2) 122 m(mp2) 80 p(mp2) 202 dipole moment p/debye 2.15 polarizability /å3 4.87 surface area s/å2 116 volume v/å3 117 a experiment: ei (vertical) = 12.62 ev = 291 kcal.mol -1. all energy quantities in units of kcal.mol-1. the free energies of the reactants and products allow determining the overall change a(t) for the reaction (eq. 1) which enters the equilibrium constant lnk = -a/rt. as evident from table 5, for the amino-acid forms at the scf level there is a ~ 0 at 300 k which yields k ~ 1. this value leads to the conclusion that the formation of carnosine and its hydrolysis via eq. 1 are equally probable processes. this result gives a thermodynamic (not kinetic) predisposition to the carnosine hydrolysis. hydrolysis of carnosine has been observed as an enzyme-assisted process (pegova et al. 2000). the results obtained in an analogous way but using the enlarged basis set 6-311g(d,p) and mp2optimized geometry gave almost the same results as evident from table 5. with expectations, all 4 nova biotechnol chim (2021) 20(2): e1010 2 molecular energies are a bit lower. amino acid and zwitterionic forms of histidine, -alanine, and carnosine are close in energy. with g = -0.32 kcal mol-1 the equilibrium constant reads k = exp (-g/rt) = 1.14. table 5. energies, entropies, and free energies of the reactants and products at 300 k.a histidine -alanine carnosine h2o reaction (eq.1) scf level, 6-31g** e0, z-form -342,323 -201,985.2 -496,552 -47,706 50.2 e0, a-form -342,340 -201,985.8 -496,619 -47,706 0.8 u, 300 k -342,225 -201,908 -496,444 -47,689 0 s, 300 k 0.0984 0.0785 0.1283 0.0450 -0.0036 a, 300 k -342,254 -201,932 -496,483 -47,703 0 evib 113.4 75.8 172.9 14.6 -1.7 erot 0.89 0.89 0.89 0.89 0 etrans 0.89 0.89 0.89 0.89 0 svib 0.0283 0.0138 0.0544 0 0.0123 srot 0.0290 0.0253 0.0317 0.0103 -0.0122 strans 0.0411 0.0394 0.0422 0.0347 -0.0036 mp2 level, 6-31g** e0, z-form -343,399 -202,593 -498,113 -47,830 49 e0, a-form -343,409 -202,591 -498,171 -47,830 -1 mp2 level, 6-311g(d,p) (5d, 7f) e0, z-form -344,886 -203,472 -500,247 -48,046 65.4 e0, a-form -344,885 -203,470 -500,307 -48,046 1.57 u, 300 k -344,782 -203,396 -500,141 -48,031 6.73 s, 300 k 0.1084 0.0804 0.1365 0.0465 -0.0058 g, 300 k, 1 atm -344,805 -203,420 -500,181 -48,044 -0.32 a energies in kcal mol-1, entropies in kcal k-1 mol-1. z – zwitterion, a – amino acid form. conclusions ab initio mo-lcao-scf calculations show that the amino acid forms of the -alanine, histidine, and carnosine are more stable than the zwitterionic form in vacuo. the large pearson hardness of the carnosine means that it is very resistant against the electron transfer. the formation of the carnosine from the -alanine and histidine via eq. 1 is an equally probable process as its hydrolysis, k ~ 1. acknowledgments grant agencies of slovakia are acknowledged for the financial support (projects vega 1/0013/18, apvv 16-0039 and itms no 313011asn4). conflict of interest the authors declare that they have no conflict of interest. references boldyrev aa, aldini g, derave w (2013) physiology and pathphysiology of carnosine. physiol. rev. 93: 18031845. cambridge crystallographic data centre, https://www.ccdc.cam.ac.uk/. hyperchem (2008) molecular modeling system, ver. 8.0.6. hypercube inc. pearson rg (1977) chemical hardness, wiley-vch, weinheim, 210 p. pegova a, abe h, boldyrev a (2000) hydrolysis of carnosine and related compounds by mammalian carnosinases. compar. biochem. phys. 127: 443-446. politzer p, murray js (2002) the fundamental nature and role of the electrostatic potential in atoms and molecules. theor. chem. acc. 108: 134-142. 5 https://www.ccdc.cam.ac.uk/ nova biotechnol chim (2021) 20(2): e1010 2 politzer p, laurence pr, jayasuriya k (1985) molecular electrostatic potentials: an effective tool for the elucidation of biochemical phenomena. envir. health persp. 61: 191-202. sen, kd (1993) chemical hardness. in clarke mj et al. (eds.), structure and bonding, springer-verlag, berlin heidelberg, pp. 268. schön m, mousa a, berk m, chia wl, ukropec j, majid a, ukropcová b, de courten b (2019) the potential of carnosine in brain-related disorders: a comprehensive review of current evidence. nutrients. 11: 1196. vranovičová b, boča r (2021) ab initio study of the biogenic amino acids. j. mol. model. 27: 355. 6 microsoft word luptakova et al 2010-1.doc nova biotechnologica 10-1 (2010) 23 metals recovery from acid mine drainage alena luptakova1, magdalena balintova2, jana jencarova1, eva macingova1, maria prascakova1 1department of mineral biotechnologies, institute of geotechnics of the slovak academy of sciences, watsonova 45, košice, sk-043 53, slovak republic (luptakal@saske.sk, jencarova@saske.sk, macingova@saske.sk, prascak@saske.sk) 2institute of building and environmental engineering, civil engineering faculty, technical university of košice, vysokoškolská 4, košice, sk-042 00, slovak republic (magdalena.balintova@tuke.sk) abstract: the objectives of the present work give the results view of some physicochemical, chemical and biological-chemical methods for the heavy metals removal from acid mine drainage (amd). the background of the studied physicochemical methods was the adsorption by turf, chemical methods the heavy metals precipitation by the neutralization with naoh. the principles of the biological-chemical methods were the bioprecipitation by the applications of sulphate-reducing bacteria (srb), the sorption by the bacterially produced iron sulphides and sorption by brown coal bio-modified by micromycetes. key words: acid mine drainage, neutralization, bioprecipitation, sorption, biosorption 1. introduction acid mine drainage (amd) is unique among industrial contaminants on the subject of mainly the mining industry of the sulphide minerals. amd generation proceeds increased considerably after the closure of mines. acid mine waters have low ph-values, and typically high concentrations of sulphates, iron and non-ferrous metals (kontopoulos, 1988), which serve as buffering systems. development of cost-effective and sustainable remediation solutions for the mine water problem has been the subject of extensive review. in addition to monitored natural attenuation, the two broad philosophies which have been pursued in the treatment and abatement of mine water pollution include measures directed towards prevention at source, usually involving physical intervention of one form or another, and measures directed at the resulting effluent, including active or passive remedial systems. both active and passive systems may be implemented using physicochemical, chemical or biological-chemical treatment technologies (skousen et al., 1998; kadukova and stofko, 2006a). nowadays is pay attention to physical, chemical and biological methods for the selective recovery of metals from amd. these methods constitute the possibility of the recovery metals in the suitable forms for commercial value. current research of heavy metals removal in water environment is aimed to application of natural materials as well as industrial wastes that are regarded as cost effective sorbents 24 luptakova, a . et al. (garcia sanchez et al. 1999). among the most often tested sorbents of heavy metals belong: zeolite, carbonate, clays, turf, oxide and hydroxide of iron. deorkar and tavlarides (1998) development of amd physicochemical treatment by application of an adsorption process comprised of inorganic chemically active adsorbents to selectively recover of fe, cu, zn, cd and pb from amd without neutralization. jacke and diebold (1983) evaluated the chemical treatment of amd on the ground of metal recovery from amd by addition of sulphides followed by oxidation and selective titration. cu and zn precipitated as sulphides and fe, al, mn and mg were recovered as hydroxides. tabak et al. (2003) conducted the biotreatment of amd by selective sequential precipitation to recover metals as hydroxides and sulphides. for the metals removal from amd can be used different biosorbents on the base of sawdust, algae, microscopic fungi, biogenous ferrous sulphides biomass and etc. (kadukova and stofko, 2006b; kadukova and vircikova, 2003). in slovak republic there are some localities with existing amd generation conditions. our previous research demonstrates the amd critical values in the abandoned cu – fe ore deposit smolnik (luptakova et al., 2006). it is necessary to develop methods for their treatment. that was the reason for starting a systematic monitoring of geochemical development in acid mine drainage in 2004 in order to prepare a prognosis in terms of environmental risk and use of these waters as an atypical source of a wide range of elements. on this account was our research oriented on the development suitable methods or their combination for the amd effluent treatment from deposit smolnik. we studied some physicochemical, chemical and biological-chemical methods. heavy metals model solutions and amd from smolnik were used for experiments. the background of the used physicochemical methods was the adsorption by turf brush peatsorb. experiments of the chemical methods were oriented on to study of the metals selective precipitation in the hydroxides form by the neutralization with naoh. the principle was the endpoint titration. the backgrounds of the biological-chemical methods were the bioprecipitation by the applications of sulphate-reducing bacteria (srb), the sorption by the bacterially produced iron sulphides and sorption by brown coal modified by different micromycetes strains. the metabolic process of srb is the anaerobic reduction of sulphates by the formation of hydrogen sulphide reacting in the water with cations of metals forming little soluble sulphides. investigated was the process of the heavy metals precipitation by bacterially produced hydrogen sulphide with the combination of the metals precipitation by sodium hydroxide at the various amd ph values. in the literature this methods is named as the selective sequential precipitation (ssp) (tabak et al., 2003). besides srb application gives rise to fe sulphides (fexsy) with the magnetic properties, which are suitable bio-sorbents of heavy metals and are utilizable for amd treatment in combination with the magnetic separation. for the heavy metals removal from solutions also unconventional sorbents prepared from brown coal by micromycetes were used. it was studied bio-modification of brown coal sorption materials with the aim to enhance its sorption properties. the micromycetes (aspergillus niger, aspergillus clavatus, nova biotechnologica 10-1 (2010) 25 penicillium glabrum and trichoderma viride) have been selected for biological activation of coal samples. 2. material and methods 2.1 model solutions stock solutions of each metal: cu(ii) containing 30-300 mg/l, zn(ii) containing 30-300 mg/l and cd(ii) containing 50-100 mg/l were prepared by dissolving cuso4.5h2o, znso4.h2o and 3cdso4.8h2o of analytical grade in distilled water. 2.2 acid mine drainage the experiments were conducted with amd coming from the abandoned and flooded deposit of smolnik (slovak republic). the average values of ph and the major metals composition of acid mine drainage was following: ph 3.9, so4 2 2 938 mg/l, fe 307 mg/l, mn 26 mg/l, cu 5 mg/l, zn 11 mg/l, al 77 mg/l. 2.3 adsorption metals by turf based on our previous results, where various adsorbents were tested (balintova and kovalikova, 2008) for our study of cu, fe, al, zn ions removal from acid mine drainage by adsorption, turf brush peatsorb (reo amos slovakia) was used. the dependence of cu, fe, al, zn concentration decreasing on time (1, 3, 5 and 10 min), was investigated under dynamic conditions using turf brush peatsorb. to intensify the adsorption process, sample of amd was continuously stirred with 5 g of turf brush peatsorb. in filtrate was determined ph (mettler toledo) and cu, fe, al, zn by colorimeter dr 890 (hach lange). 2.4 metals precipitation by naoh the precipitation by 1m naoh solutions was used for the removal of metals as hydroxides from amd. experiments were carried out by raw amd samples of 100 ml and each were titrated to ph end points ranging from 5 to 9 using 1m naoh. when the preset ph end point was reached, the titrated solution was filtered to precipitated metals removing. during titration the amd solution was continuously stirred, the ph was monitored and the concentration of metals was determined too. 2.5 metals precipitation by biogenic h2s and naoh for the production of the bacterially h2s the cultures of srb (genera desulfovibrio) were used. these bacteria were isolated from a mixed culture obtained from the potable mineral water (gajdovka spring, the locality kosice-north, slovakia). 26 luptakova, a . et al. this biological-chemical method contains several process steps and can be divided in to these main steps, as well as: the bacterially h2s production by srb; the heavy metals precipitation by the bacterially produced h2s; the heavy metal sulphides separation by the filtration; the setting ph of the filtrate from previous steps by 1m naoh (the precipitation of metals as hydroxides); the heavy metal hydroxides separation by the filtration; the subsequent precipitation of the heavy metals by bacterially produced h2s. values of ph for the heavy metals precipitation were assigned on the ground of the study of the orientation conditions for the selected metal removal from amd by precipitation using naoh and our previous works and enumerations (luptakova et al., 2003). the concentration of metals was determined by atomic absorption spectrometry using spectrometer aa-30 varian instrument. a glass ph electrode combined with the reference ag/agcl electrode was used to measure ph. digital phmeter gprt 144 agl was used. 2.6 sorption of metals by bacterially produced iron sulphides the preparation of biogenic sulphides was realized in the bioreactor filled with 400 ml of modified nutrient medium dsm-63 and inoculated with 100 ml of a culture of srb during 21 days at 30°c under anaerobic conditions. these conditions were generated by introducing an inert gas (n2) and chemically with sodium thioglycolate. the ph of the medium was adjusted to the value 6.8 with sodium hydroxide. preparation was realized under 2 different modes. during discontinuous mode the bioreactor worked without addition of fresh nutrient medium. during semicontinuous process of preparation the bioreactor worked 4 days in batch mode and then 3 days in continuous mode (i.e. fresh medium was supplied into reactor). batch sorption experiments were performed in 100 ml erlenmeyer flasks with the sorbent dose 1 g/l. sampling was conducted during 90 minutes. the concentration of zinc and cadmium was determined by atomic absorption spectrometry. 2.7 sorption by bio-modified brown coal the cultures of selected micromycetes were grown in sabouraud agar medium. suspension was injected (5 ml) of every 14-day-old culture spores in sab to the 10 g of brown coal mixed with 10 ml of sab medium. the cultivation was executed in the dark at ambient temperature. the leaching process took 7 weeks. after leaching, the suspensions were filtered, washed with distilled water, dried and prepared for adsorption experiments. all the adsorption experiments were conducted at ambient temperature in a laboratory shaker. metal solutions of known concentrations were introduced into the glass erlenmeyer bottles containing defined amounts (10 g/l) of the adsorbent. the bottles were shaken horizontally and the adsorbent was removed by filtration after 1 hour adsorption. the equilibrium concentrations of heavy metals were determined by atomic adsorption spectroscopy and the metal uptake was calculated from the difference. the langmuir adsorption isotherms have been constructed and the maximum adsorption capacity of the adsorbents has been nova biotechnologica 10-1 (2010) 27 determined. for cu(ii) sorption the ph reached 5, as cu(ii) ions undergo hydrolysis reactions in water and form insoluble aqueous complexes with increasing ph. zn(ii) sorption experiments were performed at ph 7 for the same reason as in case of cu(ii) adsorption. 3. results and discussion 3.1 adsorption metals by turf table 1 documents the physical-chemical method studies and there are also given the results of ph measuring in amd turf brush mixture depending on time and influence of adsorption processes on cu, fe, al, zn concentrations in individual samples. from the results follows, that it is possible to decrease the cu, fe, al and zn concentrations in polluted surface water by physical adsorption. the highest turf brush efficiency was observed for zinc removal, where the decrease of concentration in solution was 95.71%. then follows copper (decreasing in amd about 55.88%), iron (21.19%) and aluminium (15.6%). based on experimental results we can also state that chosen adsorbent have not influenced the ph increasing above 4 that is connected with precipitation of metals. table 1. dependence of cu, fe, al and zn removal from amd versus adsorption time. cu fe al zn adsorption time ph [mg/l] amd 3.95 1.36 358.2 54.5 17.5 1 min 3.10 0.86 347.8 52.7 1.5 3 min 2.95 0.61 353.3 46.4 1.2 5 min 2.97 0.61 333.5 46.2 1.0 10 min 2.93 0.60 282.3 46.0 0.75 3.2 metals precipitation by naoh the results of the titration test amd by 0.2m naoh documents fig. 1. it is a documentation of the titration curve and elemental amd analysis. the titration curve of amd can be divided into a number of by its different slopes. it shows that the optimum ph for metals precipitation is different for each metal: for al – ph 5; for cu – ph 6; for zn – ph 7; for fe – ph 8; for mn – ph 9. table 3 demonstrates results of metals precipitation by naoh that is titration of amd to ph end points for the individual metals ranging from 5 to 9 using 1m naoh. as it is seen from table 2 was removed 97% of al at ph 5; 99.9% of cu at ph 6; 99.9% of zn at ph 7; 99.9% of fe at ph 8 and 99.9% of mn at ph 9. initial assumption about precipitation of followed metals up to ph 9 has been confirmed. 28 luptakova, a . et al. 3.3 metals precipitation by biogenic h2s and naoh the selective sequential precipitation of heavy metals form amd sample was performed in two interconnected bioreactors with a capacity 1000 ml (the first bioreactor) and 250 ml (the second bioreactor), which operated at the semi-continual conditions. using the operating conditions and obtained results of the selective sequential precipitation of heavy metals form amd sample illustrated in table 3. the bacterially produced hydrogen sulphide by srb at ph 3.9 (initial ph of amd), 4.5 and 6.0 realized the selective sequential precipitation of cu, zn and fe, respectively (i.e. steps 1, 3 and 5). al and mn were precipitated as aluminium and manganese hydroxide at ph 6.0 and 9.0, respectively (i.e. steps 4 and 6). fe was precipitated predominantly as hydroxide (steps 2, 4 and 6). fig. 1. titration curve and metal amd analysis of amd from the deposit of smolnik. table 2. precipitation of al, cu, zn, fe and mn in ph dependence (initial ph of amd – 3.9). ph al (mg/l) cu (mg/l) zn (mg/l) fe (mg/l) mn (mg/l) 3.9 58.8 1.38 6.88 338.6 22.88 5 0.4 0.65 6.75 268.8 22.88 6 0.4 <0.02 5.38 9.8 22.88 7 0.4 <0.02 <0.03 2.5 20.38 8 0.4 <0.02 <0.03 <0.03 12.25 9 0.4 <0.02 <0.03 <0.03 0.03 table 3. metals precipitation by bacterially produced h2s and naoh (initial amd ph – 3.9). step 1. step 2. step 3. step 4. step 5. step 6. ph 3.9 4.5 4.5 6.0 6.0 9.0 precipitating agent h2s naoh h2s naoh h2s naoh removed metals cu fe, al zn al, fe fe mn, fe form of removed metals (solid phase) cus fe(oh)3 al(oh)3 zns al(oh)3 fe(oh)3 fes mn(oh)2 fe(oh)2 filtrate composition concerning of metals presence (liquid phase) fe, al, zn, mn fe, al, zn, mn fe, al, mn fe, mn fe, mn ----- nova biotechnologica 10-1 (2010) 29 3.4 sorption of metals by bacterially produced iron sulphides fig. 2 shows sorption of cadmium and zinc ions from model solutions by iron sulphides during 90 minutes by semi-continuous and discontinuous sorbents, when initial concentration of metal ions in model solutions was 50 mg/l. 0 5 10 15 20 25 0 10 20 30 40 50 60 70 80 90 time [min] so rp ti on [ m g/ g ] cd-ss (50) cd-ds (50) zn-ds (50) zn-ss (50) fig. 2. sorption of cadmium and zinc ions from model solutions. the quantities of metal ions that iron sulphides captured from 100 ml of solution are in calculation on 1 g weights of dry the sorbent. we can see that the sorption value for cadmium after 90 minutes for semi-continuous sorbent (cd-ss) is 21.96 mg/g and 21.83 mg/g for discontinuous sorbent (cd-ds). values of zinc sorption are 9.49 mg/g for semicontinuous sorbent (zn-ss) and 5.22 mg/g for discontinuous sorbent (zn-ds). 0 5 10 15 20 25 30 35 40 45 50 0 10 20 30 40 50 60 70 80 90 time [min] so rp ti o n [ m g /g ] cd-ss (100) cd-ds (100) zn-ds (100) zn-ss (100) fig. 3. sorption of cadmium and zinc ions from model solutions. 30 luptakova, a . et al. fig. 3 compares sorption of zinc and cadmium ions during 90 minutes by semicontinuous and discontinuous sorbents, when initial concentration of metal ions in model solutions was 100mg/l. in this case the highest value was obtained for zinc sorption by semi-continuous sorbent (zn-ss) 43.13 mg/g and the lower value belong to cadmium sorption by semicontinuous sorbent (cd-ss) 22.75 mg/g. 3.5 sorption by bio-modified brown coal fig. 4 presents adsorption isotherms of copper and zinc adsorption by biomodified coal adsorbents. experimental data were fitted by langmuir equation. the correlation coefficient ranged from 0.96 to 0.99. experimental results showed that maximum increase of metals uptake for biologically activated brown coal was achieved by the activation by penicillium glabrum (sample s3), i.e. 8.8 mg cu(ii)/g of sorbent and 14.3 mg zn(ii)/g of sorbent. 0 50 100 150 200 0 2 4 6 8 10 cu adsorption q [m g/ g] c e [mg/l] coal + a. niger coal + a. clavatus coal + p. glabrum coal + t. viride coal 0 50 100 150 200 0 2 4 6 8 10 coal + a. niger coal + a. clavatus coal + p. glabrum coal + t. viride coal zn adsorption q [m g/ g] c e [mg/l] fig. 4. langmuir adsorption isotherms of cu (a) and zn (b) adsorption on brown coal treated by different microorganisms. the results of adsorption experiments showed that selected type of sorbent preparation had positive influence on sorption properties of activated material and prepared sorbents had good affinity to selected metals. obtained results confirmed that there is a possibility to prepare the sorbents by new non-conventional methods and the obtained results appear as promising for the research and development in utilisation of non-energetic coal. 4. conclusions the major metal ions in the amd from the abandoned and flooded deposit of smolnik (slovak republic) were fe, al, ca and mg, among which fe and al were potentially valuable, while others such as cu, zn and mn were present as minor metals at significantly low concentrations. the value of ph, contend of fe, al, zn, cu, mn nova biotechnologica 10-1 (2010) 31 and mg of amd did not meet the effluent limitation of nv sr no. 296/2005 z.z. up to now amd was not treated at the site. results of the amd titration test document that the titration curve can be divided into four ranges (i to iv). its show the different optimum ph for metals precipitation of each metal: for al – ph 5; for cu – ph 6; for zn – ph 7; for fe – ph 8; for mn – ph 9. the metals precipitation using 1m naoh confirms that was removed 97% of al at ph 5; 99.9% of cu at ph 6; 99.9% of zn at ph 7; 99.9% of fe at ph 8 and 99.9% of mn at ph 9. metals removal by precipitation is possible up to ph 9. the study of the selective sequential precipitation and bio recovery of metals from aforementioned amd was realized by the combination of the metals precipitation by the bacterially produced hydrogen sulphide and the precipitation of metals by sodium hydroxide at the various values of ph amd. for the removal of cu and zn in the form of sulphides were received excellent results. was not come to good results point of view of the fe, al and mn the selective precipitation, because was determined the co-precipitation of fe and al or fe and mn. obtained results can be used for suggestion of technology for selective metal recovery from acid mine drainage from smolnik. the base this technology will be the combination of chemical and biological agent application. acknowledgements: this work was supported by the slovak research and development agency under the contract no. apvv-51-027705 and bilateral project apvv sk-cz-0105-07. references balintova, m., kovalikova, n.: removal of heavy metals from acid mine drainage using adsorption methods. proceedings of the 8th international scientific conference modern management of mine producing, geology and environmental protection sgem 2008, albena, 2008, 155-160. boonstra, j., van lier, r., janssen, g., dikman, h., buisman, c.j.n.: biological treatment of acid mine drainage. proceedings of the 13th international biohydrometallurgy symposium, biohydrometallurgy and the environment toward the mining of the 21st century ii., ibs, madrid, 1999, 559-566. deorkar, n.v., tavlarides, l.l.: adsorption process for metal recovery from acid mine waste: the berkeley pit problem. environ. prog., 17, 1998, 120-125. foucher, s., battaglia-brunet, f., ignatiadis, i., morin, d.: treatment by sulfate-reducing bacteria of chessy acid-mine drainage and metals recovery. chem. eng. sci., 56, 2001, 1639-1645. garcia sanchez, a., alavarez ayuso, e., jimenez de blas, o.: sorption of heavy metals from industrial waste water by low-cost mineral silicates. clay miner., 34, 1999, 469-477. janke, d.r., diebold, f.e.: recovery of valuable metals from acid mine drainage by selective titration. water res., 17, 1983, 1639-1645. kadukova, j., vircikova, e.: minerálne biotechnológie iii., biosorpcia kovov z roztokov, všb tu ostrava, 2003, isbn 80-248-0244-9. 32 luptakova, a . et al. kadukova, j., stofko, m.: environmentálne biotechnológie pre hutníkov, equilibria, košice, 2006a, isbn 80-8073-496-8. kadukova, j., stofko, m.: biosorption of heavy metals ions from aqueous solutions. environmental research trends, nova publishers, 2006b, isbn 160021-556-4. kalin, m., fyson, a., wheeler, n.w.: the chemistry of conventional and alternative treatment systems for the neutralization of acid mine drainage. sci. tot. environ., 366, 2006, 395-408. kontopouls, a.: acid mine drainage control. in: castro, s.h., vergara, f., sánches, m.a. (eds.) effluent treatment in the mining industry. university of concepcion, chile, 1988, 57-118. luptakova, a., kusnierova, m., bezovska, m., fecko, p.: the selective precipitation of heavy metals by sulphate-reducing bacteria. proceedings of the 15th international biohydrometallurgy symposium, nereus congress and conferences, athens, greece, 2003, 665-672. luptakova, a., kusnierova, m., slesarova, a.: environmental risks and impact of old mine loads in the slovak republic. proceedings of 2nd international conference on environmental research and assessment, university of bucharest, centre for environmental research and impact studies, bucharest, 2006, 254-257. skousen, j., rose, a., geidel, g., foreman, j., evans, r., hellier, w.: a handbook of technologies for avoidance and reclamation of acid mine drainage, morgantown, wv: national mine land reclamation center, west virginia university, 1998. tabak, h.h., scharp, r., burckle, j., kawaharai, f.k., govind, r.: advances in biotreatment of acid mine drainage and biorecovery of metals: 1. metal precipitation for recovery and recycle. biodegradation, 14, 2003, 423436. veeken, a.h.m., rulkens, w.h.: innovative developments in the selective removal and reuse of heavy metals from waste-waters. water sci. technol., 47, 2003, 9-16. microsoft word ubaldini_luptakova 2010-1.doc nova biotechnologica 10-1 (2010) 15 application of biohydrometallurgical processes for heavy metals removal from acid mine drainage stefano ubaldini1, alena luptakova2, eva macingova2, roberto massidda1, pietro fornari1 1instituto di geologia ambientale e geoingegneria, cnr, rome, italy, (stefano.ubaldini@igag.cnr.it) 2institute of geotechnics, slovak academy of sciences, slovakia (luptakal@saske.sk) abstract: the main scope of this study was to remediate acid mine drainage (amd) by application of biohydrometallurgical processes, environmentally friendly, to remove heavy metals such as zn, cu, mn, cd, al and fe. the processes studied have been electrowinning and bioprecipitation. the samples utilised were collected from the zinc mine located in italy and from a cooper – iron ore deposit in slovakia. by electrochemical experiments, high metals removal, with a low energetic consumption, has been achieved: in particular, by zn electrodeposition, it was possible to achieve 95-99% zn removal. culture of sulphatereducing bacteria (srb) of genera desulfovibrio sp. was used for the bioprecipitation tests. the precipitation kinetic of metals at the original ph of aforementioned amd by srb has been investigated. this method has been performed in two interconnected reactors. achieved results indicate the 98-99% selective elimination of cd from amd italian mine, and the 98-99% selective elimination of cu from amd slovak mine by bacterially produced h2s. both the electrowinning and bioprecipitation processes have been demonstrated the technical feasibility to decrease the heavy metals concentration. the experimental work has been carried out in the framework of the agreement of scientific cooperation between the institute of environmental geology and geoengineering of the cnr, italy and the institute of geotechnics of slovak academy of sciences, slovakia (years 2007-2009). key words: heavy metals, biohydrometallurgical processes, acid mine drainage, desulfovibrio sp. 1. introduction in contrast to most organic pollutants, heavy metals are never degraded. the only ways to remedy heavy metals-polluted lands are stabilization or extraction using the suitable methods (veglió et al., 2003; kadukova and stofko, 2006); beolchini et al., 2007). various methods are used for redevelopment of soils and waters in the world (apha, 1989), but any of them are universal (pagnanelli et al., 2003; bálintova and kovaliková, 2008; ubaldini et al., 2009). classical treatments for the removal of heavy metals from contaminated waters are precipitation with lime or more expensive chemicals (epstein, 2003). however, these methods present negative drawbacks the production of secondary wastes (e.g. lime precipitation generates high volumes of solid wastes) (ubaldini et al., 2009). there is a need for new and low-cost technologies in the field of elimination metals from environment. with respect to involved proposition various authors have studied, at laboratory scale, the application of physicochemical and biological-chemical processes. between the innovative and unconventional technologies belong for example the electrowinning and the bioprecipitation. electrowinning process is 16 ubaldini, s. et al. currently used at large scale to purify process solutions and to recover precious metals. microorganisms play important roles in the environment fate of heavy metals (ronald, 1995) with a multiplicity of physical, chemical and biological mechanisms effecting transformations between soluble and insoluble phases (beolchini et al., 2009a; beolchini et al., 2009b). the biggest environmental problems relating to mining and processing activities in the worldwide is the formation and treatment of acid mine drainage (dikman and buisman, 1999). the source of acid mine drainage (amd) is the residues of mining activity mainly after the mining of deposits containing of sulphide minerals. amd contains sulphuric acid, metals in the soluble form and its ph can be very low (ubaldini et al., 2006a). in italy and slovakia there are some localities with existing amd generation conditions (ubaldini et al., 2007a; ubaldini et al., 2007b). 2. materials and methods 2.1 samples the investigation has been carried out at laboratory scale by synthetic solutions, starting from amd from the zinc mine located in montevecchio mine (italy) and slovak mine located in smolník. the amd characterisation is reported in tables 1 and 2. table 1. composition of the amd italian sample in mg/l. zn cd cu ni as sb pb mn fe so4 2 1600 3.50 0.50 4.00 0.006 0.005 0.076 86 190 1800 table 2. composition of amd slovak sample in mg/l. zn cd cu ni as al pb mn fe so4 2 10.13 0.1 4.31 0.32 0.042 79.50 0.019 20 270 2938 2.2 physical-chemical process: electrowinning nitric acid (hno3) has been added to the synthetic solution, with the aim to oxidise fe2+ to fe3+. in a subsequent step, sodium hydroxide (naoh) was added to reach ph 4.0. successively, the deposit has been separated by filtration. electrowinning tests have been performed in a cylindrical glass laboratory cell of 200 cm3 volume according to ubaldini et al., 2008. the cell was connected to a potentiostat-galvanostat. with the scope to study the electrodeposition kinetic, liquid samples of 3 cm3 have been whit drawn and submitted to chemical analysis by icpms, while the purity of the solid deposit was determined by x-ray diffraction technique (xrd). metallic content of the deposit was analysed by icp-ms. nova biotechnologica 10-1 (2010) 17 2.3 biological-chemical processes: bioprecipitation the cultures of srb (genera desulfovibrio sp.) were used which were isolated from a mixed culture obtained from the potable mineral water (luptakova et al., 2002; luptakova et al., 2008). scanning electron micrographs of sulphatereducing bacteria is reported in figure 1. the precipitation of heavy metals form amd sample was performed in two interconnected bioreactors with a capacity 1000 ml (the first bioreactor) and 250 ml (the second bioreactor) (ubaldini et al., 2006a; luptakova et al., 2008a). the heavy metals concentration in the liquid samples taken from the bioreactor was determined by atomic absorption spectrophotometry. the qualitative analysis of precipitates obtained by bacterial produced hydrogen sulphide was realized by energy dispersive spectrometry (eds) analysis. samples of precipitates were dried and coated with gold prior to the eds analysis. fig. 1. scanning electron micrographs of sulphate-reducing bacteria (desulfovibrio sp.) (luptakova et al., 2002). 3. results and discussion 3.1 physical-chemical process: electrowinning preliminary precipitation step has been carried out before electrowinning. during this phase, also al deposition has been achieved. the liquor prepared was treated by using an electrowinning lab-scale operation, to verify the technical feasibility of the metals deposition. the average results of the electrowinning tests on montevecchio (initial ph 4.6) and smolnik samples (initial ph 3.5), show that, after 30’, zn deposit was of low quality. after 1 h, zn deposit was uniform, while on counter electrode surface mno2 deposited. manganese deposited to the anode as mno2 and to the cathode as mn +. after 2 hours, 90-95% of the metals have been removed by a quantitative cathodic deposition. the high grade purity of the metallic deposit has been achieved, such as it was demonstrated from the results of analysis conducted by xrd. at the end of the processes, all the metals concentrations decrease under the recommended limit suggested from ec (data not shown here) (ubaldini et al., 2006a; ubaldini et al., 2006b; ubaldini et al., 2006c). table 3 and table 4 show the main results of zn electrowinning from synthetic solution (amd italian and slovak 18 ubaldini, s. et al. samples, respectively), while table 5 shows the results attained after chemical precipitation by naoh (amd from slovak sample). table 3. main results of zn electrowinning of synthetic solution (amd italian sample). time (min) r (%) η* (%) e* (kwh/kg) 90 97.98 21.65 16.23 120 99.85 13.05 30.21 * η faradic current efficiency, e energetic consumption table 4. main results of zn electrowinning of synthetic solution without chemical precipitation (amd slovak sample). time (min) r (%) η* (%) e* (kwh/kg) 90 92.18 20.10 17.13 120 96.89 11.92 31.21 * η faradic current efficiency, e energetic consumption table 5. main results of zn electrowinning of synthetic solution after chemical precipitation (amd slovak sample). time (min) r (%) η* (%) e* (kwh/kg) 90 97.07 25.94 12.00 120 99.71 14.99 24.91 * η faradic current efficiency, e energetic consumption 3.2 biological-chemical processes: bioprecipitation during the metals bioprecipitation at the original ph of aforementioned amd, only the precipitation of cd (in the case of the amd sample from montevecchio mine) and cu (in the case of amd sample from smolnik adit pech) were observed. 0 1 2 3 4 5 6 0 100 200 300 0 50 100 150 200 cd ni cu zn fe m n c on ce nt ra tio n of c u, n i a nd c d [m g/ l] c on ce nt ra tio n of f e an d m n [m g/ l]; zn [m g/ l]. 1 01 time of biop recip itation [min] fig. 2. precipitation of heavy metals by biologically produced h2s by srb from amd italian mine at original ph value of amd. nova biotechnologica 10-1 (2010) 19 fig. 2 presents that at ph 4.6 cd was effectively recovered from amd of montevecchio mine using biologically produced h2s. after 30 minutes the concentration of cd was 0.03 mg/l. on the basis of the results of eds qualitative analysis, cd was precipitated in the form cadmium sulphides. in the event of the amd smolnik at ph 3.5, the initial copper concentration 4.31 mg/l was decreased to less than 0.05 mg/l after 4 hours (fig. 3). eds qualitative analysis of precipitates demonstrates that cu was precipitated in the form sulphides cus. concentration changes of other metals (zn, fe, ni, mn), were not significant or remained without changes in case of both aforementioned amd. 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 0 100 200 300 t ime of bioprecipit at ion [min] 0 5 10 15 20 25 30 cu ni cd zn fe m n c on ce nt ra tio n of z n an d m n [m g/ l]; fe [m g/ l]. 1 01 c on ce nt ra tio n of c u, n i a nd c d [m g/ l] fig. 3. precipitation of heavy metals by biologically produced h2s by srb from amd – slovak mine at original ph value of amd. 4. conclusions by application of biohydrometallurgical processes, the technical feasibility to decrease the heavy metals concentration on the amd studied has been determined. in particular, through electrowinning, it was possible to achieve high zn removal with a low energetic consumption, while bioprecipitation process demonstrates the selective precipitation of heavy metals by srb from the amd samples. at original ph value of amd from italian mine was achieved only the precipitation of cd. from amd of slovak mine was achieved only the precipitation of cu at original ph value. obtained results indicate the 98-99% elimination of cd or cu by bacterially produced h2s. acknowledgements: this work was supported by italian national council researches under cnr project n. 6.1.132.48.2, under project cnr rstl n. 0042.0005 and by slovak research and development agency under the contract n. apvv-51-027705. moreover, the authors are grateful to ms. emanuela tempesta for helpful collaboration during the experimental work. 20 ubaldini, s. et al. references apha: standard methods for the examination of water and wastewater. 17th edition, american public health association, usa, washington d. c., 1989. 1. balintova, m., kovalikova, n.: testing of various sorbents for copper removal from acid mine drainage. chem. listy, 102, (2008), 343-344. beolchini, f., ubaldini, s., passariello, b., gul, n., ture, d., veglió, f., danovaro, r., dell’anno, a.: bioremediation of dredged sediments polluted by heavy metals. adv. mat. res., 20-21, 2007, 307-310. beolchini, f., dell’anno, a., de propris, l., ubaldini, s., cerrone, f., danovaro, r.: autoand heterotrophic acidophilic bacteria enhance the bioremediation efficiency of sediments contaminated by heavy metals. chemosphere, 74, 2009a, 1321-1326. beolchini, f., fonti, v., ferella, f., ubaldini, s., vegliò, f.: nickel, vanadium and molybdenum extraction from exhaust lc-finer catalysts by biohydrometallurgical technologies. proceedings of the 1st international conference biotechnologies and metals 2009. kosice, slovak republic, september 24th – 25th, 2009b, 11-14. dikman, h., buisman, c.j.n.: biological treatment of acid mine drainage. in: biohydrometallurgy and the environment toward the mining of the 21st century. amils r., ballester a. 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6.78 for garden soil), ec (ss 7.80 ds.m.-1; gs 0.32) and ex na+ (ss 8.81 cmol.kg-1; gs 0.4181 cmol.kg-1) was observed in the saline soil while there was a decrease in organic carbon, total nitrogen and available phosphorus in saline soil. proximate analysis of c. maxima and t. occidentalis leaves revealed that fiber, carbohydrate, and caloric value were slightly reduced in saline soil treatments while ash, protein and lipids contents were slightly increased. amf inoculation and pm application had significant effect on proximate composition of these plant leaves but caloric value which was significantly (p ≤ 0.05) increased in non-saline soil treatments. physiological parameters such as leaf turgid weight (ltw), leaf relative water content (lrwc), vigor index (vi), and salt tolerance index (sti) were significantly reduced by salinity, while electrolyte leakage (el) was higher in saline soil treatments than nonsaline soil treatments. however, inoculation with amf in combination with pm amendment significantly increased ltw, lrwc, vi, and sti in both saline and non-saline soil treatments above single treatment with mycorrhizal species and poultry manure. however, el reduced with mycorrhizal inoculation. the results of this work have shown that amf and pm can enhance plants ability to tolerate salinity possibly through some morphological and physiological changes which improved water and nutrients uptake. introduction soil salinity is a term used to describe the amount of mineral salts present in soil (shan 2009). the mineral salts constitute a mixture of electrolytes. the major cations in saline soils include na+, ca2+, mg2+, and k+. the major anions include clˉ, so4 2-, hco3-, co3 2-, and no3-. these constituents are mailto:abdelhak.rhouma@gmail.com nova biotechnol chim (2022) 21(1): e1170 2 usually reported in units of mg/l (ppm), mmol.l-1 or mmol charge.l-1 (meq.l-1) in solution extracted from a soil saturated with water (tanji 2002). salinity is often measured as electrical conductivity (ec), a measure of the ability of a substance to conduct electricity. the natural factors contributing to salinization of soils include weathering of rocks, deposition of oceanic salts, topographical factor, groundwater table fluctuation, salt lake, scald, fallow period, and flood water. however, the major contributors to soil salinization are weathering of parental rocks, oceanic salt deposition, and groundwater table fluctuation. rocks are mostly rich in sodium and other salts (saxena et al. 2017). the most common man-made factors contributing to salinization are the irrigational water and the use of chemical fertilizers and pesticides. irrigation is an important factor in the crop productivity. therefore, the use of high salt-containing water for crop irrigation and poor management practices for appropriate leaching of salts lead to the accumulation of salts and deteriorate the crop fields (zhu 2007). salinity not only decreases the agricultural production of most crops, but also, as a result of its effect on soil physicochemical properties, adversely affects the associated ecological balance of the area. the harmful impacts of salinity include: low agricultural production, low economic returns due to high cost of cultivation, reclamation management, soil erosion due to high dispersibility of soil, ecological imbalance due to halophytes and marine life forms from fresh water to brackish water, poor human health due to toxic effects of elements such as b, f, and se (hu and schmidhalter 2002; ashok et al. 2012). crop species show a spectrum of responses to salt, although all have their growth, and eventually, their yield reduced by salt. salt effects are the combined result of the complex interaction among different morphological, physiological, and biochemical processes (ashok et al. 2012). salinity may directly or indirectly inhibit cell division and enlargement and finally the growth of the whole plant. in addition to these factors, some other factors like water deficit (drought stress), ion toxicity, ion imbalance and soil compaction may cause growth reduction, injury of foliage, nutrient deficiencies, destruction of soil structure which ultimately hampers the growth of the plant. some above ground visible morphological symptoms of plants are marginal yellowing/browning of foliage, premature fall of leaves, twig and branch die back, loss of vigor and stunted growth (ashok et al. 2012). the high concentration of salts in soil may induce three types of stresses: osmotic, ionic or oxidative, which drastically affect plant growth and productivity (saxena et al. 2017). osmotic stress leads to altered water potential, thereby reducing the water use efficiency of plant due to induction of physiological drought conditions (saxena et al. 2017). ionic stress causes disruption of ion homoeostasis at both cellular and whole-plant levels, oxidative stress elicits release of reactive oxygen species, which inhibit cell growth and plant metabolism. experiments carried out to understand amf-salinity interaction revealed that mycorrhizal fungi reduce negative effects of these stresses and promote plant growth (wu et al. 2006; zuccarini and okurowska 2008; evelin et al. 2009; wu et al. 2010a, 2010b). plants encounter a variety of biotic and abiotic stresses, which cause significant structural and functional changes that will result in decreased yield and vegetative traits (plant height, shoot length, number of branches, fresh and dry biomass, etc.) (agha et al. 2021; matrood and rhouma 2021; sofy et al. 2021a). in the other hand, salinity is among the most significant threats hindering global food security. arbuscular mycorrhizal fungi and poultry manure (practice alone or in combination) were commonly used for agricultural production for their abilities to improve plant health to salinity stress (solaiman et al. 2020; zhang et al. 2020; sallam et al. 2021; sofy et al. 2021a, 2021b). arbuscular mycorrhizal (am) fungi are among the most common soil fungi and the majority of plant species have associations with am fungal species (selvaraj and chellappan 2006). it is thought that about 80 % of vascular plants form am associations (hodge 2000). mycorrhizal fungi can help plants to survive and grow under different environmental conditions, and also help plants increase their reproductive output (bolandnazar et al. 2007). the basic elements of the symbiosis are that the plant provides the mycorrhiza with carbohydrates while the fungi enhance the plant’s uptake of certain nutrients needed for growth nova biotechnol chim (2022) 21(1): e1170 3 (selvaraj and chellappan 2006). it has been estimated that in compensation for the additional nutrients and water provided by mycorrhizas, a plant must provide 20 % of its fixed carbon to the roots for mycorrhizal establishment and maintenance of the association (ebrahim 2014). little information is available about the influence of combination poultry manure/arbuscular mycorrhizal fungi on yield and quality of cucurbits. elmer and pignatello (2011) and blackwell et al. (2015) pointed out that poultry manure application and arbuscular mycorrhizal fungi could serve as a sustainable nutrient alternative to chemical fertilizer. this is due to the potential of the fungi to utilize unavailable organic nutrients and increase the efficiency use of nutrients through improved nutrients uptake, which might enhanced plant growth and yield (elmer and pignatello 2011; blackwell et al. 2015). earlier researchers have clearly documented the positive effect of organic amendments in combination with arbuscular mycorrhizal fungi (hammer et al. 2014, 2015) on plant growth and improve nutrients uptake (abdullahi et al. 2015; solaiman et al. 2019). solaiman et al. (2020) pointed out that the cucumber growth responses to poultry manure application and arbuscular mycorrhizal symbiosis showed better plant growth and yield of cucumber and reduced the negative impact of stress salinity. this combination also improved the nutrient uptake and soil fertility of sandy soils (mickan et al. 2016; paymaneh et al. 2018; solaiman et al. 2020). this research was undertaken to investigate the effect of salt stress on the proximate composition and physiological parameters of two different cucurbits (telfairia occidentalis and cucurbita maxima) and determine ways of ameliorating its effect using arbuscular mycorrhizal species and poultry manure. experimental study area saline soil and salt water were collected from the saline ecosystem of iwuochang, ibeno local government area (latitude 4.56°n and longitude 7.57°e), akwa ibom state, nigeria, with an annual rainfall of about 4,021 mm and mean temperature variation of 22 – 31 ºc. the experiment was set up in a safe and secured environment at mbioto 1, etinan local government area (latitude 4.51°n and longitude 7.50°e), akwa ibom state, nigeria, with an annual rainfall of about 4,000 mm and mean temperature variation of 26 – 36 ºc (aksg 2008). non-saline soil for the control and nonsaline treatments was obtained from a farmland in mbioto 1, etinan local government area; fresh water was used for watering the non-saline and control treatments. a map showing the saline water/soil collection and experimental set-up locations is presented in fig. 1. fig. 1. map showing saline water/soil collection and experimental set-up locations (source: field data). experimental design this experiment was set up in a randomized complete block design and arranged in three blocks of 108 pots each for each species of cucurbits (c. maxima and t. occidentalis). this gave a total of 12 treatments (table 1) for each species with nine replicates totaling 108 combinations for each nova biotechnol chim (2022) 21(1): e1170 2 species of cucurbits. this experiment was carried out in 1 march 2020 in a greenhouse. table 1. experimental design. treatments meaning smpsalinity, mycorrhiza, poultry s+ mp+ salinity, mycorrhiza, poultry s+ m+ p(gg) + salinity, + mycorrhiza (g. geosporum), poultry s+ m+ p(ri) + salinity, + mycorrhiza (r. irregularis), poultry s+ mp+ + salinity, mycorrhiza, + poultry s+ m+ p+ (gg) + salinity, + mycorrhiza (g. geosporum), + poultry s+ m+ p+ (ri) + salinity, + mycorrhiza (r. irregularis), + poultry sm+ p(gg) salinity, + mycorrhiza (g. geosporum), poultry sm+ p(ri) salinity, + mycorrhiza (r. irregularis), poultry sm+ p+ (gg) salinity, + mycorrhiza (g. geosporum), + poultry sm+ p+ (ri) salinity, + mycorrhiza (r. irregularis), + poultry smp+ salinity, mycorrhiza, + poultry planting the experimental soils were steam sterilized in the oven in bits for 2 h at 100 ºc to kill weed seeds and soil microorganisms and sieved through a 2 mm mesh to remove pebbles. the poultry manure used in this research consists of moisture (39.69 %), n (2.47 %), p2o5 (2.84 %), k2o (1.84 %). treatments consisted of 1 kg of poultry manure per pot. am fungi glomus geosporum and rhizophagus irregularis (60 – 65 spores per 5 g) were purchased from international institute of tropical agriculture (iita) ibadan, nigeria. matured seeds of cucurbita maxima and telfairia occidentalis were obtained from akwa ibom state agricultural development project (akadep) in etinan. the obtained seeds were selected to eliminate infected seeds. five seeds each of c. maxima and t. occidentalis were sown in each of the pots filled with about 10 kg of sterilized soils. arbuscular mycorrhiza fungi were inoculated by placing 25 g of soil/root fragments containing 60 – 65 spores per 5 g in planting hole at 15 cm depth, before planting the c. maxima and t. occidentalis. following seedling emergence, the plants inoculated were allowed to establish for up to 15 d before being treated with the first dose of saline water. this was to ensure the establishment of am colonization and avoid sudden plant death due to salinity shock. the roots treatment with saline water (100 ml) using an erlenmeyer flask (250 ml) on the two cucurbits species was performed 1 d after the establishment of the mycorrhizal symbiosis and the treatments were spaced every 3 d until the end of the trial (30 june 2020). assessments were conducted 90 d after the treatment (with poultry manure and am fungi). at the end of trial (30 june 2020), 27 plants (3 plants × 9 replicates) per treatment and per block were assessed for each cucurbit species (c. maxima and t. occidentalis). physico-chemical properties of experimental soils soil samples were taken using a 7-cm-diameter soil auger. a total of 108 samples per block were collected in sterile polythene bags. for each block, samples were mixed together into a single one. nine soil samples (500 g) per block (3 blocks) and per replicate (3 replicates) were collected from each treatment after and before the trials and brought to the laboratory. the soil samples were taken and air-dried at room temperature and ground in a wooden mortar to pass through a 2 mm mesh sieve and stored in labelled bags. sub-samples were taken from each soil sample and analysed for physico-chemical properties of the soil. soil samples were analysed following the standard procedures outlined by the association of official analytical chemist (aoac 2005) procedure for wet acid digestions (rhouma et al. 2019). soil physicochemical properties (ph, total nitrogen (%), available phosphorus (mg.kg-1), silt (%), clay (%), sand (%), ex. ca (cmol.kg-1), ex. mg (cmol.kg-1), ex. na. (cmol.kg-1), ex. k. (cmol.kg-1), organic carbon (%), exchangeable acidity (meq/100 g), ecec (cmol.kg-1), base saturation 4 nova biotechnol chim (2022) 21(1): e1170 2 (%) and electrical conductivity (ec) (ds.m-1) were determined for each samples (rhouma et al. 2019). analysis of water samples nine water samples (1,000 ml) per replicate (3 replicates) were collected from saline (water used in treatment) and freshwater). a total of 27 samples were collected in sterile glass bottles (2,000 ml) and brought to the laboratory. water ph, electrical conductivity, and total dissolved solid (tds was measured using portable ph/ec/tds/temperature combined (hi 991301, hanna instruments ltd., leighton, uk). dissolved oxygen was measured using digital portable analyzer jpb-607a portable dissolved oxygen analyzer (tech instrumentation inc., elizabeth, usa). biological oxygen demand (bod5), total alkalinity, acidity, chloride, calcium, nitrate, sulphate, phosphate, magnesium, sodium and potassium concentration was determined by the conventional method of the association of official analytical chemists (aoac 2005). salinity was determined using digital salt meter, which measures the salinity of seawater in parts per thousand (rhouma et al. 2017). determination of proximate content the determination of ash, lipid, carbohydrate and crude fibre was carried out using the standard methods described by cella and watson (2000) and adeola et al. (2010). caloric value was determined using a bomb calorimeter. physiological parameters electrolyte leakage was calculated using the formula (shi et al. 2006): ec1/ec2. values of fw, tw, and dw were used to calculate lrwc using the formula (eq. 1; kaya et al. 2003): (1) to determine the turgid weight (tw), leaves were soaked in distilled water inside a closed petri dish. leaf samples were weighed periodically after gently wiping the water from the leaf surface with tissue paper until a steady state was achieved. the turgid weight of the leaves was determined by weighing the soaked leaves on a weighing balance and weight recorded (kaya et al. 2003). plant vigor index in each treatment was calculated using the formula (eq. 2; maisuria and patel 2009): vigor index = root length + shoot length × percentage emergence )%( (2) plant salt tolerance index (psti) was calculated using the formula of jaarsma et al. (2013) (eq. 3): (3) statistical analysis all data in the present study were subjected to analysis of variance (anova) using statistical package for social sciences (spss) and data are presented as standard error of mean (± sem) of triplicate experiments. the student’s t-test was used to determine the significant difference between means of the soil and water parameters analyzed. the differences between the means were separated and compared using the duncan’s multiple range tests. however, a probability level of p ≤ 0.05 was considered statistically significant. results and discussion analysis of the saline and garden soils used in this study revealed significant variations in their soil physico-chemical parameters (p ≤ 0.05). significant increase in parameters such as ph (7.75), ec (7.80 ds.m-1) and ex na+ (8.81 cmol.kg-1) was observed in the saline soil while there was a decrease in organic carbon (1.61 %), total nitrogen (0.49 %) and available phosphorus (24.66 mg.kg-1) in saline soil (p ≤ 0.05) (table 2 and 3). this observation is in line with the work of miller and gardiner (2007) who reported an increase in ph and ec in saline soils in new jersey due to salt stress. deleke and akomolafe (2013) also made similar findings as they observed an increase in ph, ec and ex na+ in saline soils and a decrease in organic carbon, organic matter, total nitrogen and phosphorus in salinity influenced soils in nigeria. soil organic 5 nova biotechnol chim (2022) 21(1): e1170 3 carbon content is influenced by two opposing factors: reduced plant inputs and reduced rates of decomposition (garg and manchanda 2008). this might be responsible for the significant decrease in the soil oc, total n and p observed in this work. high concentrations of salts in soil, especially na+ ions drastically affect the basic structure of soil (garg and manchanda 2008). the presence of na+ ions in the cation exchange complex makes the soil compact and subsequently decreases soil porosity and aeration, which hampers plant growth and hinders their productivity (garg and manchanda 2008). the presence of the salts of calcium and magnesium forms a white crust on the soil surface that changes soil water osmotic potential; therefore, plants growing in saline soils face salt-induced physiological drought conditions. salinity impairs plant’s major processes such as photosynthesis, protein and lipid metabolism, nutrient acquisition, and ion homeostasis. indeed, water moves out of the plant due to salt-induced osmotic stress, which makes the plant dehydrated and eventually leads to the death of the plant (evelin et al. 2011). table 2. physicochemical properties of the experimental soils. s/no. parameters soil before treatment with saline water soil after treatment with saline water 1. ph 6.78b 7.75a 2. total nitrogen [%] 2.27a 0.49b 3. available p. [mg.kg-1] 36.31a 24.66b 4. silt [%] 4.00b 5.60a 5. clay [%] 4.20b 12.00a 6. sand [%] 92.04a 82.40b 7. ex. ca [cmol.kg-1] 5.25a 2.97b 8. ex. mg [cmol.kg-1] 4.36a 3.80b 9. ex. na [cmol.kg-1] 0.41b 8.81a 10. ex. k. [cmol.kg-1] 6.98a 1.48b 11. organic carbon (%) 5.61a 1.61b 12. exchangeable acidity (meq/100g) 3.56a 3.20b 13. ecec [cmol.kg-1] 20.56a 20.26b 14. base saturation [%] 82.68b 84.20a 15. ec. (ds.m-1) 0.32b 7.80a * significant at p ≤ 0.05, ex – exchange, ecec – effective cation exchange capacity, ec – electrical conductivity. table 3. water analysis of the experimental irrigation water. s/no. parameters saline water (water used in treatment) fresh water 1. ph 7.70a 6.70b 2. ec [µs.cm-1] 3080.00a 27.70b 3. tds 1021.00a 11.00b 4. acidity [mg.l-1 as caco3] 80.00 b 95.40a 5. alkalinity [mg.l-1 as caco3] 138.00 a 53.20b 6. do [mg.l-1] 6.40b 7.60a 7. bod [mg.l-1] 3.20b 2.80a 7. sulphate [mg.l-1] 102.31a 1.91b 9. phosphate [mg.l-1] 0.09a 0.04b 10. nitrate [mg.l-1] 0.06b 2.82a 11. cl[mg.l-1] 2560.13a 55.23b 12. ca2+ [mg.l-1] 55.71b 106.20a 13. mg2+ [mg.l-1] 120.20b 232.81a 14. na+ [mg.l-1] 1027.00a 0.11b 15. k+ [mg.l-1] 6.42b 8.40a 16. salinity [ppt] 33.21a 0.32b * significant at p ≤ 0.05, ec – electrical conductivity, do – dissolved oxygen, bod – biological oxygen demand, tds – total dissolved solids. 6 nova biotechnol chim (2022) 21(1): e1170 3 proximate analysis of c. maxima and t. occidentalis leaves revealed that fibre, carbohydrate and caloric value were slightly reduced in saline soil treatments while ash, protein and lipids contents were slightly increased (table 4 and 5). amf inoculation of c. maxima and t. occidentalis and poultry manure application had some significant effect on proximate composition of these plant leaves but caloric value which was significantly increased in non-saline soil treatments (p ≤ 0.05) (table 4 and 5). proximate composition of c. maxima and t. occidentalis such as carbohydrate, caloric value and fibre were significantly reduced with salinity, while ash, protein and lipids increased in saline soil treatments compared to non-saline soil treatments. however, inoculation of c. maxima and t. occidentalis with arbuscular mycorrhizal fungi (amf) (r. irregularis and g. geosporum) in conjunction with soil amelioration with poultry manure (pm) increased some proximate composition of c. maxima and t. occidentalis. proximate analysis of c. maxima and t. occidentalis leaves increased dramatically for the plants treated with g. geosporum+manure poultry (ash 18.48 – 17.09 %, respectively; fibre 15.20 – 20.11 %, respectively; protein 8.81 – 8.21 %, respectively; lipid 8.38 – 6.88 %, respectively; cho 49.12 – 47.71 %, respectively; caloric value 297.60 – 281.41 kcal, respectively) and r. irregularis + manure poultry (ash 17.54 – 17.02 %, respectively; fibre 14.67 – 19.82 %, respectively; protein 8.22 – 8.41 %, respectively; lipid 6.94 – 6.66 %, respectively; cho 50.03 – 48.09 %, respectively; caloric value 318.71 – 289.00 kcal, respectively) even in the presence of salinity (table 4 and 5). it can be concluded from the results of this study that c. maxima plants tolerate better the salinity when treated with g. geosporum+manure poultry. we concluded that the addition of r. irregularis in combination with poultry manure to soil optimally improved the studied parameters of t. occidentalis in water stress conditions (table 4 and 5). increase in ash, protein and lipids have also been reported by uddin et al. (2017) in clinacanthus nutans with increasing salinity. the findings in this study agree with the results of ali et al. (2014) on portulaca oleracea, hibiscus sabdariffa and sorghum bicolor, kekere (2014) in a. hypogea, uzun et al. (2013) and kapoor and srivastava (2010) who reported that increasing salinity levels tended to enhance crude protein synthesis. the increase in crude lipid content of c. maxima and t. occidentalis in response to salinity may be because plants have some level of tolerance at these salinity levels or due to accumulation of compatible solutes (uddin et al. 2017). these results agree with those previously reported on p. oleracea by teixeira and carvalho (2009) and oenothera biennis by heuer et al. (2002). they observed increased total fat content in plants exposed to moderate saline environment. table 4. effects of arbuscular mycorrhizal fungi (amf) inoculation on the proximate composition of c. maxima grown in saline soil and ameliorated with poultry manure. treatments ash [%] fibre [%] protein [%] lipid [%] cho [%] caloric value [kcal] smp*16.06 ± 0.66b 16.11 ± 0.38a 8.95 ± 0.21b 6.54 ± 0.15b 52.33 ± 4.55a 314.73 ± 10.51c s+ mp0.00 ± 0.00c 0.00 ± 0.00b 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00g s+ m+ p(gg) 18.65 ± 1.24a 16.21 ± 0.33a 10.71 ± 1.61a 8.22 ± 0.25a 46.21 ± 3.42b 242.71 ± 8.41f s+ m+ p(ri) 18.40 ± 0.71a 15.72 ± 0.61a 9.20 ± 0.75a 8.61 ± 0.34a 48.07 ± 3.11b 257.10 ± 8.11e s+ mp+ 19.06 ± 1.62a 15.68 ± 0.46a 11.24 ± 1.22a 8.68 ± 0.29a 45.34 ± 2.05b 239.12 ± 6.34f s+ m+ p+ (gg) 18.48 ± 1.44a 15.20 ± 0.51a 8.81 ± 0.61b 8.38 ± 0.41a 49.12 ± 4.12a 297.60 ± 6.81d s+ m+ p+ (ri) 17.54 ± 1.34a 14.67 ± 0.25a 8.22 ± 0.57b 6.94 ± 0.38b 50.03 ± 4.48a 318.71 ± 9.66c sm+ p(gg) 16.76 ± 0.84b 15.49 ± 0.35a 7.97 ± 0.43b 6.61 ± 0.31b 53.17 ± 4.33a 320.12 ± 10.67c sm+ p(ri) 16.24 ± 0.69b 16.01 ± 0.42a 7.77 ± 0.64b 6.58 ± 0.50b 53.40 ± 4.37a 322.50 ± 10.81c sm+ p+ (gg) 15.45 ± 0.51b 14.81 ± 0.29a 8.96 ± 0.72b 6.62 ± 0.34b 54.16 ± 4.71a 332.13 ± 12.12b sm+ p+ (ri) 16.28 ± 0.75b 14.77 ± 0.32a 8.06 ± 0.76b 6.48 ± 0.34b 54.41 ± 4.21a 351.00 ± 14.32a smp+ 16.79 ± 0.65b 14.58 ± 0.45a 8.09 ± 0.59b 7.92 ± 0.51b 51.02 ± 3.55a 298.30 ± 9.42d *mean of three replicates ± sem. means within of each column followed by different letters are significantly different at p < 0.05 according to duncan’s multiple range test. s(no salinity), m(no mycorrhiza), p(no poultry manure), s+ (plus salinity), m+ (plus mycorrhiza), p+ (plus poultry droppings), (gg) – glomus geosporum, (ri) – rhizophagus irregularis, 0.00 (means the plants were dead). 7 nova biotechnol chim (2022) 21(1): e1170 2 table 5. effect of arbuscular mycorrhizal fungi (amf) inoculation on the proximate composition of t. occidentalis grown in saline soil and ameliorated with poultry manure. treatments ash [%] fibre [%] protein [%] lipid [%] cho [%] caloric value [kcal] smp*17.20 ±1.51b 20.17 ± 2.78a 8.74 ± 0.15a 6.81 ± 0.57a 47.08 ± 3.58a 298.16 ± 6.45b s+ mp21.43 ± 1.91a 23.11 ± 2.88a 8.67 ± 0.31a 8.27 ± 0.66a 38.52 ± 2.94b 197.14 ± 4.88f s+ m+ p(gg) 18.96 ± 0.52b 21.22 ± 1.94a 8.21 ± 0.25a 7.08 ± 0.4a 45.45 ± 3.42a 246.30 ± 6.12d s+ m+ p(ri) 17.47 ± 0.78b 21.43 ± 1.81a 8.87 ± 0.38a 6.71 ± 0.49a 45.52 ± 3.64a 275.28 ± 5.14c s+ mp+ 22.00 ± 1.24a 21.77 ± 1.57a 8.77 ± 0.42a 8.25 ± 0.67a 39.21 ± 3.11b 202.11 ± 3.88e s+ m+ p+ (gg) 17.09 ± 0.64b 20.11 ± 1.45a 8.21 ± 0.33a 6.88 ± 0.35a 47.71 ± 3.31a 281.41 ± 5.74c s+ m+ p+ (ri) 17.02 ± 0.45b 19.82 ± 1.39a 8.41 ± 0.47a 6.66 ± 0.42a 48.09 ± 4.01a 289.00 ± 6.14c sm+ p(gg) 16.42 ± 0.64b 18.76 ± 1.43a 7.91 ± 0.51a 6.21 ± 0.29a 50.70 ± 3.69a 301.10 ± 7.22b sm+ p(ri) 16.53 ± 0.51b 18.64 ± 0.84a 7.61 ± 0.39a 6.11 ± 0.43a 51.11 ± 4.12a 307.42 ± 7.31a sm+ p+ (gg) 17.06 ± 0.48b 18.65 ± 1.44a 7.66 ± 0.45a 6.06 ± 0.28a 51.57 ± 4.24a 309.40 ± 6.48a sm+ p+ (ri) 16.22 ± 0.33b 18.70 ± 0.87a 7.25 ± 0.42a 6.21 ± 0.57a 51.62 ± 4.1a 311.10 ± 7.24a smp+ 17.03 ± 0.66b 19.76 ±1.46a 7.84 ± 0.37a 5.29 ± 0.61a 50.08 ± 3.82a 299.90 ± 6.75b *mean of three replicates ± sem. ameans within of each column followed by different letters are significantly different at p < 0.05 according to duncan’s multiple range test. s(no salinity), m(no mycorrhiza), p(no poultry manure), s+ (plus salinity), m+ (plus mycorrhiza), p+ (plus poultry droppings), (gg) – glomus geosporum, (ri) – rhizophagus irregularis, 0.00 (means the plants were dead). measured physiological parameters of c. maxima and t. occidentalis such as leaf turgid weight (ltw), leaf relative water content (lrwc), salt tolerance index (sti) and vigor index (vi) were significantly reduced in saline soil treatments when compared to the control, while electrolyte leakage increased in saline soil treatments (p ≤ 0.05) (table 6 and 7). amelioration of the saline soil with poultry manure alone also enhanced the physiological parameters of c. maxima and t. occidentalis. inoculation with amf alone or together with poultry manure amelioration significantly increased performance of the physiological parameters in the two test plants both in saline and non-saline soil treatments during the first and second cropping seasons (p ≤ 0.05) (table 6 and 7). ltw, lrwc, sti and vi were all significantly higher in amf inoculated and poultry manure ameliorated c. maxima plants grown in non-saline soil (p ≤ 0.05) (table 6 and 7). table 6. effects of arbuscular mycorrhizal fungi (amf) on the physiological parameters of c. maxima grown in saline soil ameliorated with poultry manure. treatments leaf turgid weight (ltw) [g] leaf relative water content (lrwc) [%] salt tolerance index [sti] vigour index (vi) [%] electrolyte leakage (el) [ds.m-1] smp*0.67 ± 0.06b 38.00 ± 2.51c 1.00 ± 0.01a 7481.00 ± 7.04h 0.81 ± 0.24c s+ mp0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00l 0.00 ± 0.00d s+ m+ p(gg) 0.26 ± 0.11c 36.00 ± 1.24c 0.36 ± 0.08c 1,303.00 ± 6.06i 2.22 ± 0.67a s+ m+ p(ri) 0.23 ± 0.20c 36.36 ± 1.66c 0.29 ± 0.06c 956.00 ± 8.13j 2.31 ± 0.38a s+ mp+ 0.21 ± 0.09c 30.00 ± 1.02c 0.28 ± 0.12c 856.00 ± 4.90k 2.52 ± 0.44a s+ m+ p+ (gg) 0.68 ± 0.12 b 44.78 ± 2.42b 0.71 ± 0.33b 8,037.00 ± 9.02f 1.66 ± 0.14b s+ m+ p+ (ri) 0.69 ± 0.34 b 42.65 ± 1.57b 0.71 ± 0.24b 7,897.00 ± 8.37g 1.51 ± 0.16b sm+ p(gg) 0.70 ± 0.30b 44.93 ± 3.10b 1.04 ± 0.67a 9,465.00 ± 9.53c 0.72 ± 0.20c sm+ p(ri) 0.69 ± 0.27b 45.59 ± 1.67b 1.03 ± 0.75a 8,378.00 ± 8.86d 0.91 ± 0.48c sm+ p+ (gg) 0.74 ± 0.61b 45.21 ± 2.01b 1.06 ± 0.54a 10,088.00 ± 10.75b 0.85 ± 0.44c sm+ p+ (ri) 0.93 ± 0.84a 51.09 ± 3.44a 1.14 ± 0.68a 10,583.00 ± 12.15a 0.67 ± 0.28c smp+ 0.68 ± 0.22b 44.78 ± 1.97b 1.01 ± 0.24a 8,099.00 ± 7.14e 0.83 ± 0.41c *mean of three replicates ± sem. ameans within of each column followed by different letters are significantly different at p < 0.05 according to duncan’s multiple range test. s(no salinity), m(no mycorrhiza), p(no poultry manure), s+ (plus salinity), m+ (plus mycorrhiza), p+ (plus poultry droppings), (gg) – glomus geosporum, (ri) – rhizophagus irregularis, 0.00 (means the plants were dead). 8 nova biotechnol chim (2022) 21(1): e1170 2 table 7. effects of arbuscular mycorrhizal fungi (amf) on the physiological parameters of t. occidentalis grown in saline soil ameliorated with poultry manure. treatments leaf turgid weight (ltw) [g] leaf relative water content (lrwc) [%] salt tolerance index [sti] vigour index (vi) [%] electrolyte leakage (el) [ds.m-1] smp*0.89 ± 0.51a 56.58 ± 3.11b 1.00 ± 0.01c 11,984.00 ±3.68f 0.51 ± 0.24c s+ mp0.51 ± 0.20c 37.78 ± 1.05d 0.28 ± 0.08g 662.00 ± 3.80k 2.06 ± 0.97a s+ m+ p(gg) 0.66 ± 0.32b 49.09 ± 1.57c 0.48 ± 0.10f 8,784.00 ± 2.09i 1.91 ± 0.11b s+ m+ p(ri) 0.71 ± 0.55b 47.37 ± 1.22c 0.50 ± 0.25f 9,265.00 ± 6.00h 1.76 ± 0.21b s+ mp+ 0.63 ± 0.19b 52.73 ± 1.67b 0.61 ± 0.33e 8,265.00 ± 4.33j 2.01 ± 0.40a s+ m+ p+ (gg) 0.83 ± 0.22a 57.75 ± 2.15b 0.88 ± 0.51d 10,602.00 ± 7.09g 1.27 ± 0.22b s+ m+ p+ (ri) 0.85 ± 0.46a 60.27± 1.97a 1.02 ± 0.66c 12,169.00 ± 2.64e 1.14 ± 0.31b sm+ p(gg) 0.88 ± 0.75a 63.38 ± 2.28a 1.21 ± 0.58b 13,248.00 ± 4.64h 0.47 ± 0.26c sm+ p(ri) 0.91 ± 0.69a 66.67± 2.11a 1.23 ± 0.64b 13,665.00 ± 1.95c 0.49 ± 0.34c sm+ p+ (gg) 0.95 ± 0.56a 68.49 ± 3.15a 1.37 ± 0.69a 13,901.00 ± 2.84b 0.52 ± 0.58c sm+ p+ (ri) 0.99 ± 0.87a 70.67 ± 3.47a 1.40 ± 0.81a 14,183.00 ± 8.28a 0.48 ± 0.61c smp+ 0.89 ± 0.61a 57.89 ± 2.30b 1.07 ± 0.55c 12,201.00 ± 7.38l 0.53 ± 0.42c *mean of three replicates ± sem. ameans within of each column followed by different letters are significantly different at p < 0.05 according to duncan’s multiple range test. s(no salinity), m(no mycorrhiza), p(no poultry manure), s+ (plus salinity), m+ (plus mycorrhiza), p+ (plus poultry droppings), (gg) – glomus geosporum, (ri) – rhizophagus irregularis, 0.00 (means the plants were dead). el was however, highest in sole poultry manure ameliorated saline soil treatments. a similar trend was observed in t. occidentalis, except for el which was highest in pure saline soil non-fertilized with poultry manure and uninoculated with amf in the first cropping (table 6 and 7). the symbiotic association of g. geosporum+manure poultry applied on c. maxima and t. occidentalis plants allows better leaf turgid weight (0.68 – 0.83 g, respectively), leaf relative water content (44.78 – 57.75 %, respectively), salt tolerance index (0.71 – 0.88, respectively), vigour index (8,037 – 10,602 %, respectively), and electrolyte leakage (1.66 – 1.27 ds.m-1, respectively) underwater stress conditions; whose t. occidentalis plants are more tolerant to water salinity. also, the highest value of leaf turgid weight (0.69 – 0.85 g, respectively), leaf relative water content (42.65 – 60.27 %, respectively), salt tolerance index (0.71 – 1.02, respectively), vigour index (7,897 – 12,169 %, respectively), and electrolyte leakage (1.51 – 1.14 ds.m-1, respectively) was obtained when the c. maxima and t. occidentalis plants are treated with r. irregularis+manure poultry under water stress conditions; whose t. occidentalis plants are more tolerant to water salinity (table 6 and 7). foliar na+ accumulation in of c. maxima and t. occidentalis had corresponding significant reduction on some physiological parameters such as leaf turgid weight (ltw), leaf relative water content (lrwc), salt tolerance index (sti), vigour index (vi) and electrolyte leakage (el) of c. maxima and t. occidentalis (p ≤ 0.05) (fig. 2 and 3). amelioration with poultry manure showed some improvements in these physiological parameters in the two test plants. inoculation of c. maxima and t. occidentalis with amf in non-saline soil treatments showed significant increase in physiological parameters when compared to the control and saline treatments (p ≤ 0.05) (fig. 2 and 3). ltw, lrwc, sti, vi and el were improved when the plants were treated with g. geosporum+manure poultry and r. irregularis+manure poultry underwater stress conditions. we can conclude that these treatments enhanced the studied parameters of t. occidentalis in water stress conditions (fig. 2 and 3). physiological parameters of c. maxima and t. occidentalis such as leaf turgid weight (ltw), leaf relative water content (lrwc), vigour index (vi) and salt tolerance index (sti) were significantly reduced by salinity, while electrolyte leakage (el) was higher in saline soil treatments than non-saline soil treatments. however, inoculation of c. maxima and t. occidentalis with amf (r. irregularis and g. geosporum) in conjunction with poultry manure amendment significantly increased their leaf turgid weight 9 nova biotechnol chim (2022) 21(1): e1170 2 (ltw), leaf relative water content (lrwc), vigor index (vi) and salt tolerance index (sti) in both saline and non-saline soil treatments above single treatment with mycorrhizal species and poultry manure (p ≤ 0.05). however, electrolyte leakage (el) reduced with mycorrhizal inoculation. fig. 2. comparative assessment of the influence of foliar na+ accumulation on leaf turgid weight, leaf relative water content, salt tolerance index, vigour index and electrolyte leakage of c. maxima. 10 nova biotechnol chim (2022) 21(1): e1170 2 fig. 3. comparative assessment of the influence of foliar na+ accumulation on leaf turgid weight, leaf relative water content, salt tolerance index, vigour index and electrolyte leakage of t. occidentalis. the increasing of the studied parameters in the cucurbit plants with application of arbuscular mycorrhizal fungi and poultry manure could be attributed to two processes: (i) through increased 11 nova biotechnol chim (2022) 21(1): e1170 2 nutrient transport found in the poultry manure through the arbuscular mycorrhizal fungi and (ii) direct connection between cucurbits plant and mycorrhizal hyphal network. arbuscular mycorrhizal fungi and poultry manure application are widely claimed to be able to aid plants under soil salinity conditions. cyril et al. (2014), and adebisi and adewole (2019) showed that organic manures particularly poultry manure, mixed with arbuscular mycorrhizal fungi had positive effects in increasing red amaranth yield and improving crop quality under saline conditions. abusuwar (2018) reported that the arbuscular mycorrhizal fungi coupled with poultry manure enhanced leaf area, number of leaves per plant, plant height, fresh and dry biomass, yield and nutritive value of forage crops grown in saline soils and irrigated with saline water. mickan et al. (2016) poultry manure applied to the agricultural soils with the presence of am fungi stimulated growth of extra-radical hyphae in soil and increased mycorrhizal colonization of roots. based on the understanding of these interactions, it has been claimed that am fungi can be more important to plant growth and yield than poultry manure (mickan et al. 2016; solaiman et al. 2019, 2020; zhang et al. 2020). over the last couple of decades, the universal symbiosis between arbuscular mycorrhizal fungi and cucurbits is such an old tie that, perhaps, enabled the establishment of plants in land (rouphael et al. 2015; chen et al. 2017) by boosting photosynthesis, plant growth, nutrient acquisition and decreasing membrane leakage and reactive oxygen species under condition of salinity stress (cavagnaro et al. 2015; liu et al. 2016; sofy et al. 2021a). this symbiosis had been reported 400 million years ago (selosse et al. 2015). several research studies have reported the efficiency of am fungi to impart growth and yield enhancement in plants under salinity stress (abdel latef and chaoxing 2014; talaat and shawky 2014). wang et al. (2018) have noted considerable improvement in fresh and dry biomass, and n concentration of root and shoot due to mycorrhizal inoculation under saline conditions. chen et al. (2017) revealed that the stem diameter, plant height, dry weight, root to shoot ratio of cucumber seedlings inoculated with glomus sp., and rhizophagus sp. were improved greatly compared with the non-inoculated control. the same authors showed that glomus sp., and rhizophagus sp. increase the nutrient concentration, net photosynthetic rate, chlorophyll content, root activity, light saturated rate of the co2 assimilation, maximum carboxylation rate and maximum ribulose-1,5-bis-phosphate regeneration rate. moreover, arbuscular mycorrhizal fungi can significantly improve plant nutrient uptake and resistance to several abiotic stress factors particularity salinity stress (sun et al. 2018; begum et al. 2019). liu et al. (2016) documented that the am fungi inoculation could effectively enhance the cucumber growth and other cucurbit crops, which is closely associated with the secondary metabolism in plants. notably, yang et al. (2014) and he et al. (2017) showed a positive stimulatory effect of glomus spp., and rhizophagus spp. on peanut and tomato in terms of photosynthetic characteristics, growth and hormone status. yadav et al. (2013) found that the inoculation of gloriosa superba with glomus sp. can interact synergistically and maximize benefits, resulting in root length, higher leaf area and colchicine content. it is widely believed that arbuscular mycorrhizal fungi have been considered as an alternative to inorganic fertilizers in the near future (ortas 2012), which is probably why am fungi enhanced plant tolerance to abiotic factors (plassard and dell 2010). qiu et al. (2019) documented that the soil application of arbuscular mycorrhizal fungi could improve the chemical and biological properties of soil and enhance their nutrient levels and enzymatic activities. a prominent role of such am fungi application is to transfer nutrients, (organic carbon in the form of sugars and lipids) (jiang et al. 2017; luginbuehl et al. 2017). amiri et al. (2017) revealed that am fungi-pelargonium graveolens symbiosis increased the concentrations of n, p, and fe under drought stress. gomez-bellot et al. (2015) reported the levels of p, ca, and k in euonymus japonica have been enhanced under salinity stress due to instant am fungi attachment. in addition, am fungi application improved p and n contents in plant tissues of chrysanthemum morifolium (wang et al. 2018) and increased seedling biomass by enhancing intercellular co2, p, and n contents and water content in leymus chinensis (jixiang et al. 2017). furthermore, hashem et al. (2018) documented that the synthesis of jasmonic acid, salicylic acid, and several important inorganic nutrients and the total concentrations of p, ca2+, n, 12 nova biotechnol chim (2022) 21(1): e1170 3 mg2+, and k+ were higher in the arbuscular mycorrhizal fungi-treated cucumis sativus plants compared with those in the un-inoculated plants under salt stress conditions. ali and hassan (2014) reported that under salt stress because of inadequate water uptake, rwc was significantly decreased in relation to salinity in chamomile herb. shou-jun et al. (2014) also reported that under no saline condition, leaf relative turgidities in nonmycorrhizal and mycorrhizal plants remained at comparatively steady-state level from 53.75 % to 54.56 % throughout the experiment. mycorrhizal inoculation led to relatively higher leaf turgidity compared to non-mycorrhizal plants in this study. the phenomenon is ascribed to improved hydraulic conductivity of plants with a longer root and an altered root system morphology induced by am fungi (shou-jun et al. 2014). sheng et al. (2008) reported that plants inoculated with amf maintain relatively higher water content compared with uninoculated plants. inoculation with amf often results in increased nutrient uptake, accumulation of an osmo-regulator, an increase in photosynthetic rate and water use efficiency, suggesting that salt stress alleviation by amf results from a combination of nutritional, biochemical and physiological effects (evelin et al. 2009). sofy et al. (2021a) proved that the combination of arbuscular mycorrhizal and organic amendment is the most effective in decreasing the damaging impacts of salt on spinach plants by increasing the up-regulation of antioxidants, morphological and physiological parameters (shoot and root length, fresh and dry biomass, membrane stability index, relative water content, mineral contents, chlorophyll content, total soluble protein content and endogenous phytohormones (auxin, abscisic acid, gibberellins, salicylic acid and jasmonic acid)) and decreasing membrane leakage and reactive oxygen species. mumtaz et al. (2013) reported that electrolyte leakage was enhanced with increasing salinity levels as compared to the control in salt sensitive cucumber plants as compared to the salt tolerant cultivar. this observation has been reported by other investigators in cucumber (kaya et al. 2001), rice (lutts et al. 1996), tomato (atilla 2014) and sugar beet (ghoulam et al. 2002). agha et al. (2021) and sofy et al. (2021b) documented that the bacteria combination was the most efficient in reducing the harmful effects of salt on soybean and pea plants by boosting antioxidant upregulation and lowering membrane leakage and reactive oxygen species. a major effect of environmental stress (i.e., salt, drought) on plant is membrane modification, which results in cell membrane perturbed function or total dysfunction. changes in membrane leakage and injury can be measured by the extent of el (electrolyte leakage) in tissues (atilla 2014). the positive effects of am fungi inoculation may result in improving integrity, vigour and stability of the membrane since the membrane permeability has been found to be reduced by am fungi inoculation. plants inoculated with amf have been shown to maintain a lower electrolyte concentration than the non-mycorrhizal ones and hence maintain membrane stability (garg and manchanda 2008; ali and hassan 2014). poultry manure has been widely used as fertilizers for centuries. this manure contains not only the basic nutrients required by crops, but also trace elements. it is rich in mineral elements, essential nutrients n, p, and k, and other nutrients that can stimulate microbial activity and healthy plants, ameliorate seed germination, increase vegetative traits, reduce the negative impact of salinity, increase the percent root colonization of arbuscular mycorrhizal fungi, improve the physical and chemical properties of the soil (structure, texture, porosity, etc.), maintain the balance of soil nutrient, improve nutrient absorption, increase root vitality, increase fertilizer retention, enhance yields, and participate in the addition of organic matter to organically-deficient soils (mufwanzala and dikinya 2010; revell et al. 2012; awad 2016; pandian et al. 2016; sikder et al. 2019; sistani et al. 2019; solaiman et al. 2020; zhang et al. 2020; sallam et al. 2021). hirzel et al. (2018) showed that the highest values of available p and exchangeable k, s, ca and mg were obtained using poultry manure in saline soil, whereas ph and salinity (electrical conductivity) reported the lowest values. adeleye et al. (2010) documented that poultry manure application enhanced soil physicochemical properties by decreasing soil bulk density and temperature, and increasing exchangeable ca, mg, k, total porosity, soil moisture retention capacity, total n, soil organic matter, available p 13 nova biotechnol chim (2022) 21(1): e1170 2 and lowered exchange acidity under salinity conditions. conclusion soil salinity is one of the most severe abiotic stresses affecting plant establishment, growth and production worldwide as observed in this study. results of this study revealed that salt stress negatively affected physicochemical properties of the saline soil when compared to the garden soil, thus resulting in negative effects on growth parameters, proximate composition and physiological parameters of c. maxima and t. occidentalis. the effects of mycorrhizal symbiotic association on c. maxima and t. occidentalis showed improvements on the growth of the test plants. using different mechanisms c. maxima and t. occidentalis by itself or in association with arbuscular mycorrhizal fungi and poultry manure can tolerate or survive soil salinity. however, in the presence of the fungi, plant ability to resist the stress increases as a result of morphological and physiological changes and improved vigour, extensive network of the mycorrhizal plant roots and enhanced nutrient uptake are all among the processes that made the mycorrhizal inoculated plants to survive under salt stress. t. occidentalis showed better salt tolerance indices on individual parameters and overall salt tolerance index. given the importance of these strategies, arbuscular mycorrhizal fungi/poultry manure application has important implications under pot experiments. hence, field and greenhouse trials aimed at understanding the effectiveness, efficiency and durability of these strategies are suggested for future studies. conflict of interest the authors declare that they have no conflict of interest. references abdel latef aa, 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institute of science and technology, ulsan 44919, republic of korea 2department of chemistry, m. r. degree college, fort, andhra pradesh 535002, india 3department of chemistry, s.v.r.m college nagaram, guntur, andhra pradesh 522268, india 4chemistry of natural and microbial products department, pharmaceutical industries division, national research centre, dokki, giza 12622, egypt  corresponding author: bodduramakrishna87@gmail.com article info article history: received: 1st august 2021 accepted: 8th november 2021 keywords: lichens natural products secondary metabolites abstract lichens have attracted considerable interest since ancient time due to their medicinal properties. lichen produce a variety of orcinol-based compounds such as xanthones, anthraquinones, dibenzofurans, depsides, and depsidones. several related compounds have shown potent bioactivities as antiviral, antioxidant, anti-herbivore, insecticidal, antifungal, and anticancer. lichens have been employed as traditional medicines, and these are continuing to be of great interest for their biotechnological potential. the purpose of this review was to systematically evaluate the literature on the orcinol based biologically active secondary metabolites of lichen. introduction lichens are extraordinary self-supporting symbiotic microorganisms, formed by both alga (cyanobacteria) and fungi. this association is very useful for both organisms. the alga carries chlorophyll and is called photobiont. the fungi build the structure of the lichen thalli, the partner may be referred to as the mycobiont (de priest 2004). alga produces primary metabolites by photosynthesis (sugars, amino acids, carotenoids, and starch) for fungus growth and, which in turn gives a surface to the algae for the protection against dehydration (podterob 2008). the fungus and the alga are in a mutualistic relation and they are capable of dealing with ecological conditions, that neither of them would be able to survive on its own (margulis and barreno 2003; bates et al. 2011). some lichenized fungi synthesized secondary metabolites without photobiont under certain conditions, for example the lichen lecanora dispersa contains 2,7-dichlorolichexanthone as the main secondary metabolite, but cultured spore isolates, growing in absence of the alga, produced from lichen genus pannarin (leuckert et al. 1990). a lichen thalli consist of cortex layers, algal layer and medulla. naturally, lichens grow in a very slow and radial manner that is measured in millimeters per year. lichen produces different secondary metabolites (sms), and most of them occur exclusively in this symbiotic organism (boustie and grube 2005; jayanthi et al. 2012; xu et al. 2016). mailto:bodduramakrishna87@gmail.com nova biotechnol chim (2022) 21(1): e1075 2 during the complex metabolic interaction between mycobiont and photobiont, the mycobiont produce important secondary metabolites and accumulate as extracellular tiny crystals on the external sides of the hyphae. lichen-derived secondary metabolites are small and complex molecules, which represent about 20 % of lichens total dry weight (muggia et al. 2009). they act as protectors of the thalli against attacks by pathogens, herbivores, competitors, and different external abiotic conditions including high uv irradiation. approximately 1,050 lichen secondary metabolites have been extracted and identified to date. most well-known lichen substances are members of phenolic compounds such as dibenzofurans, anthraquinones, depsides, depsidones, depsidones, triterpenes, gamma lactones, and pulvinic acid derivatives. lichens synthesized sms, mainly acetylpolymalonate pathway (app), shikimic acid pathway (sap) and mevalonic acid pathway (map) (culberson and armaleo 1992; çobanoğlu et al. 2016). the polyketide biosynthetic pathway seems to be in charge of majority of the phenolic lichen compounds, while pulvinic acid derivatives derived from shikimic pathway and the abundance of di and triterpenoids, carotenoids, steroids found in lichens are synthesized via the mevalonate pathway (stocker-wörgötter 2008). on the other hand, secondary metabolites originated from the fungal partner of the lichen, are placed on the surface of the fungal hyphae. most of these fungal secondary metabolites are phenolics, crystalline in nature and soluble in organic solvents. however, the carbon is essential for lichen secondary substances biosynthesis and is obtained by the algal partner during photosynthesis. the carbohydrates involved in this process depend mainly on the algae itself and are mostly glucose and sugar alcohols (polyols) (el-garawani et al. 2020). orcinol based lichen secondary metabolites orcinol and its derivatives have been considered the most common mono-hydric phenolic compounds found in lichens (stepanenko et al. 2002; férnandez-morano et al. 2015; moreira et al. 2015). as shown in fig. 1, the major orcinol based compounds of lichens are produced by the polyketide route. in this pathway, two coenzymes such as acetyl-coa and malonyl-coa are utilized in lichen substances biosynthesis. particularly, orcinol-based secondary metabolites produced by orsellinate type of cyclization. orsellinic acid and its homologues are the most existing mononuclear structural units of polyketide-related lichen substances. orcinol type of secondary metabolites (orcinol and orsenillic acid based derivatives, e.g. lecanoric acid, montagnetol, trivaricacid, gyrophoric acid, evernic acid, methyl orsenillate, and erythrin) (subba rao and seshadri 1941; boustie and grube 2005; thadhani et al. 2011). β-orcinol type of secondary metabolites (β-orcinol and β-orsenillic acid based derivatives, (e.g. diffractic acid, pannarin, stictic acid, atranorin and vicanicin acid (kathirgamanathar et al. 2004; papadopoulou et al. 2007). fig. 1. biosynthesis of orcinol based lichen secondary metabolites. nova biotechnol chim (2022) 21(1): e1075 5 depsides depsides are phenolic compounds, constructed through intermolecular esterification of two or more orsenillic acid units. for example, the carboxylic acids of one unit are joined to the hydroxyl para to the carboxylic acid of the second unit, such esterification leads to para-depside. if the esterification occurs through meta hydroxyl with respect to the carboxylic acid called meta-depside (seshadri 1944; elix and wardlaw 1986). some of the important examples of this class are lecanoric acid, gyrophoricacid, erythrin, etc. depsidones depsidones are the important class of oxygenated heterocyclic compounds derived from lichen species. depsidones consist of both ester and ether linkage synthesized from the depsides. the depsidones biosynthesis involves oxidative coupling of depsides, which are normally biosynthesized from the condensation of orsellinic acid and orcinol derivatives. however, depsidones biosynthesis may actually operate through a depside intermediate pathway (hirayama et al. 1974; llah et al. 1993). some of the important examples of this class are diploicin, psoromic acid, virensic acid, and norstictic acid. dibenzofurans dibenzofurans are essentially unique to lichens, produced via the polyketide pathway. dibenzofurans are formed by carbon-carbon coupling and cyclodehydration of two phenolic units (orcinol and orsenillic acid derivatives). notable examples of this class are didymic acid, pannaric acid, schizopeltic acid, subbidymic acid, and usnic acid (millot et al. 2016). xanthones lichens produces poly-substituted aromatic xanthones, formed through internal cyclization of a single folded polyketide chain. majority of the lichen xanthones are synthesized via folding of a polyketide intermediate, which results in formation of structures having a methyl group in position 8. both, aldol condensation and claisen-type cyclization yield a benzophenone intermediate that might then dehydrate to form the central pyrone core. two different series of xanthones are resulting according to this folding pattern. this biosynthetic scheme gives rise to the common oxygen substitution pattern of lichexanthone and norlichexanthone (rezanka et al. 2003; masters and bräse 2012; lepogam and boustie 2016). some of the important examples of this class are lichexanthone, nor lichexanthone and arthothelin. biphenyls in lichens, biphenyls form via phenolic couplings of two methyl pholoroacetate to phoneunits, followed by o-methylation of hydroxyl groups. since the mechanism for carbon bond formation between two mono-aromatic units is common in lichens, as exemplified in the usnic acids, dibenzofurans, and depsidones formation. it is interesting to note that contortin is the only lichen biphenyl known so far. another compound also formed from two mono aromatic units bound by a carbon-carbon linkage is the diphenyl methane, bis(2,4-dihydroxy-6-n-propylphenyl) methane, which are found in protousnea sp., eg. contortin (rezanka et al. 2003). biological activity of orcinol based lichen secondary metabolites in nature, lichen fungi grow under the most extremely different ecological conditions. lichenized fungi produce variety of low molecular weight orcinol based secondary metabolites possess several biological activities, including antibacterial, anti-fungal, anti-oxidant, anti-diabetic, anti-cancer, and anti-inflammatory activities (lawrey 1986; boustie and grube 2005; shukla et al. 2010; shrestha and clair 2013). however, lichens are pharmaceutically unexplored secondary metabolites. extracts and isolated compounds from different lichen species antimicrobial activities. from literature surveys, it was found that lichen genus such as usnea, parmelia, parmotrema, cetraria, pseudevernia and hypogymnia showed potent antimicrobial and antifungal activities (türk et al. 2006; kamal et al. 2015; özyiğitoğlu et al. 3 nova biotechnol chim (2022) 21(1): e1075 2 2017). the major metabolites gyrophoric acid (1), stenosporic acid (2) isolated from the lichen, xanthoparmelia pokornyi showed antimicrobial activity towards some known food borne bacteria and fungi. these acid derivatives showed significant anti-microbial activity again sleight organisms (candan et al. 2006). methyl-β-orcinol carboxylate is an orcinol derivative isolated from the lichen, everniastrum irrhatum, tested against polyene and azole-resistant strains of candida albicans and capable of inhibiting drug-resistant strains in a dose dependant manner (candan et al. 2006). new dibenzofuran derivatives (3 – 5) obtained from the cladonia incrassate showed anti-bacterial activity against s. aureus organism. the major metabolites, (-)usnic acids (3), didymic acid (4) and condidymic acid (5) (fig. 2), were found to be potent molecules (mic : 7.5 µg.ml-1). phenonip was used as a positive control (ic50: 150 µg.ml-1) in this assay (dieu et al. 2014). fig. 2. structures of 1-5. hirtusneanoside a (6) was first extracted from the usnea hirta by rezanka and sigler (2007) and found to be an l-rhamnose-o-deoxyglycoside of an unsymmetrical dimeric tetra-hydro xanthone. it is potent when tested against staphylococcus aureus and bacillus subtilis (rezanka and sigler 2007). depside anziaic acid (7) was discovered from lichen (hypotrachyna sp.) as an inhibitor for both yersinia pestis and escherichia coli topoisomerase i and it was also found to act as an inhibitor of human topoisomerase ii (with little effect on human topoisomerase i), an important enzyme in cancer processes (lin et al. 2013). some lichen compounds such as (+) usnic acid (8), vicanicin (9), and diffractic acid (10) were identified as anticancer molecules (fig. 3). usnic acid, a di-benzo furan derivative, isolated from cladonia lepidophora lichen species. it is one of the well-studied lichen secondary metabolite, showed potent anti-cancer activity against hct116 and hela cancer cell lines with ic50 values of 17.7 µm and 23.7 µm respectively, and was found to be more active than the standard etoposide (ic50 = 40.3 µm). vicanicin (9) showed moderate anticancer activities against hct-116, hela and mcf7 cell lines (cocchietto et al. 2002). evernic acid (11) and physodic acid (12) were isolated from the acetone extract of evernia prunastri and pseudevernia furfuracea showed cytotoxicity against femx (human melanoma) and ls174 (human colon carcinoma) cell lines. particularly, physodic acid (12) exhibited the better cytotoxic activity (ic50 values of 19.52 for fem xcell line and 17.89 µg.ml-1 for ls 174) (kosanić et al. 2013). fig. 3. structures of 6-12. a new di-phenyl ether (13) and secalonic acid d (14) were isolated from the lichen, diploicia canescens and evaluated for their anti-cancer activity against the b16 murine melanoma cells. the di-phenyl ether (ic50 = 0.25 µm) and secalonic acid (ic50 = 0.28 µm) showed anti-cancer activity similar to standard etoposide (ic50 0.28 µm) against b16 cell line (millot et al. 2009). (-)usnic acid (15), a dibenzofuran derivative 4 nova biotechnol chim (2022) 21(1): e1075 5 isolated from different lichens genera such as cladonia, usnea, hypotrachyna, ramalina, evernia, lecanora, parmelia and most extensively studied lichen compound in search of anticancer agents (lumbsch et al. 1995; cocchietto et al. 2002; cansaran-duman et al. 2008). it has been shown moderate to potent proliferative activities against multiple cell lines, such as l-929 mouse fibroblast cells, k-562 human leukemia cells, hela human cervix carcinoma, femx human melanoma, ls174 human colon carcinoma 37 and uacc-62 human melanoma cells (schinkovitz et al. 2014). (-)usnic acid and m-scrobiculin were extracted from the lichen lobaria scrobiculata and exhibited cytotoxicity against hl-60 cells. both the isolated compounds showed ic50 values of 7.6 and 1.7 µm for (15 and 16 in fig. 4). doxorubicin was positive control in this assay (ic50 = 0.04µm). fig. 4. structures of 13-16. anti-oxidants inhibit undesirable oxidation processes by reacting with free radicals (reactive oxygen species) and their ability to protect the body from damage caused by oxidative stress. most of the secondary metabolites have been identified as phenolic compounds in lichens, which are strong antioxidants. some orcinol based secondary metabolites such aslobariellin and methyl haematommate isolated from l. pallida and s. strictum var. compressum exhibited promising antioxidants, able to prevent skin and neurodegenerative damage caused by oxidative stress. depsidones such as norstictic acid (17), fumarprotocetraric acid (18) and methyl haematommate (19) isolated from the lichen usnea articulata showed better superoxide anion scavenging activity with ic50 566 and 580 µm, respectively, than standard quercetin (ic50 = 754 µm) (lohézic-le dévéhat et al. 2007). orsellinic acid (20) and methyl orsellinate (21) were isolated from parmotrema stuppeum and heterodermia obscurata together with methyl-β-orcinol carboxylate (22). significant activity was observed for both the compound in the β-carotene-linoleate model system and in the nor assay (jayaprakasha and rao 2000; thadhani et al. 2011). the paradepsides lecanoric acid (23), erythrin (24) and the meta-depside sekikaic acid (25), along with the depsidone lobaric acid (26) showed exceptionally potent radical scavenging activity in the sor assay (fig. 5). interestingly, the ic50 values of the sekikaic acid (81.9 µm) lecanoric acid (91.4 µm) and lobaric acid (97.9 µm) were found lower than standard propylgallate standard (106 µm) (fig. 5). fig. 5. structures of 17-26. taborga et al. (2016) reported the new prenylated orcinol derivatives (27-29) from the direct reaction between orcinol and geraniol in the presence of bf3oet as catalyst. the cytotoxic activity of synthesized compounds was in vitro investigated against various cancer cell lines including (ht-29, pc-3, mdamb231, du-145). all the tested compounds were found to exhibit anti-cancer activity. orsellinates were synthesized by alcoholysis of lecanoric acid, a natural product isolated from the lichen parmotrema tinctorum. increasing the aliphatic alkyl chain at the ester functionality of orsellinic acid causes enhancement nova biotechnol chim (2022) 21(1): e1075 2 in the cytotoxic activity. the ethyl, propyl, butyl and pentanyl orsellinates (30 – 33) showed lc50 values range from 495 – 31 µm against brine shrimp (artemia salina) (gomesa et al. 2006). among tested ones, pentanyl orsellinate (33) possess potent inhibitory activity, whereas standard podophyllotoxin showed cytotoxicity15 µm. lichen genus parmelia is a rich source for depsides (aravind et al. 2014). atranorin (34) and diffractaic acid (35) isolated from the parmelia nepalensis and parmelia tinctorum, showed highly potent inhibitory activity against leukotriene b4 biosynthesis by a non-redox mechanism with ic50 values 6 and 8 µm respectively (fig. 6). nordihydroguaiaretic acid and anthralin were used as standard inhibitors (ic50 = 0.4 and 37 µm respectively) (kumar and müller 1999). fig. 6. structures of 27-35. conclusion from the above review it is evident that the orcinol form is an important building block for several phenolic compounds isolated from lichens. orcinol has very useful functionalities like phenolic, hydroxyl, and one methyl group, which are enable for a wide range of chemical transformations. chemical modification of orcinol is expected to elaborate wide range of diverse analogs and hybrid molecules. due to their structural diversity these compounds are expected to exhibit newer and enhanced biological 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(a) 13: 199-202. taborga l, espinoza l, moller a, carrasco h, cuellar m, villena j (2016) antiproliferative effect and apoptotic activity of linear geranyl phenol derivatives from phloroglucinol and orcinol. chem biol interact. 247: 2229. thadhani vm, choudhary m, ali s, omar i, siddique h, karunaratne v (2011) antioxidant activity of some lichen metabolites. nat. prod. res. 25: 1827-1837. türk h, yilmaz m, tay t, türk a, kivanç m (2006) antimicrobial activity of extracts of chemical races of the lichen pseudevernia furfuracea and theirphysodic acid, chloro atranorin, atranorin, and olivetoric acid constituents. z naturforsch. c. 61: 499-507. xu, m, heidmarsson s, olafsdottir e, buonfiglio r, kogej t, omarsdottir s (2016) secondary metabolites from cetrarioid lichens: chemotaxonomy, biological activities and pharmaceutical potential. phytomed. 23: 41-59. 7 microsoft word maresova 9-1 2009.doc nova biotechnologica 9-1 (2009) 73 zinc uptake and distribution in ivy (hedera helix l.) leaves jana marešová1, miroslav horník1,2, martin pipíška1,2, jozef augustín1,2 1department of biotechnology, university of ss. cyril and methodius, nám. j. herdu 2, trnava, sk-917 01, slovak republic (hornikm@ucm.sk) 2consortium for environmental biotechnology and environmental chemistry, hlavná 418, špačince, sk-919 51, slovak republic abstract: detached leaves of ivy (hedera helix l.) were used as a model for the study of zinc uptake and transport in vascular plants. by the uptake via the surface of fully immersed leaves in 25 % hoagland nutrient media (hm) spiked with 65zncl2 (50 µmol/dm3 zncl2), concentration in leaves 4.98 µg zn/g (dry wt.), i. e. 2.6 µg zn/dm2 leaf area after 7d exposition were obtained. by the uptake via immersed stalks of not immersed (transpiring) leaves concentrations up to 370 µg zn/g (dry wt.) were obtained. when zn enters into detached leaves via the surface of immersed leaf blades, zinc is uniformly distributed in leaf blades and leaf stalks. when zinc enters detached leaves via immersed stalks of non-immersed transpiring leaves, only small part of zinc is transported to leaf blades and the prevailing part remains in leaf stalks. stalks act as a trap, able to prevent other leaf tissues against inhibitory effects of high zn concentrations. mineral nutrient salts in solutions mobilize zn trapped in leaf stalks and facilitate zn transport by transpiration stream to leaf blades, what means that zn in stalks is bound in ion-exchageable forms. keywords: zinc, 65zn, foliar uptake, distribution, hedera helix. 1. introduction foliar uptake of mineral nutrients is of practical importance in agriculture and the problem was well described in numerous patents, periodicals and reviews (marschner, 1995). zinc is an essential nutrient as a trace element for animals, plants, and microorganisms. studies with wheat showed good transport of zn from stalks and leaves to developing grain (pearson et al., 1995; 1996), as well as from one root to another (pearson and rengel, 1995), indicating involvement of phloem transport. the movement of foliar applied zn to plant roots was demonstrated in small number of studies (haslett et al., 2001). radiotoxic 65zn is produced, sometimes in copious quantities, by neutron activation of stable zn in nuclear reactors. due to its half-life (τ = 244 d) and biological mobility, 65zn can be transported through food chains to man. 65zn may be taken up through leaves and roots by plants and its uptake is strongly influenced by nuclide form and soil properties (brambilla et al., 2002). english ivy (hedera helix l.) is a common, easily available plant species, possesing a number of advatages and its leaf ultrastructure is well described (canet et al., 1996; gilly et al., 1997). ivy leaf cuticle was used as a suitable model to investigate cuticular permeability. chamel (1986) described the fine structure and permeability of ivy leaf cuticles in relation to foliar development and after selective chemical treatments and determined the relationship between structure and permeability. 74 marešová j. et al. our previous papers were oriented on the bioaccumulation of 137cs, 60co and 65zn from nutrient solutions and translocation in vascular plants (horník et al., 2007; barátová et al., 2006). the objective of this study was to quantify the uptake of zinc by detached leaves of ivy (h. helix l.) submerged in solutions spiked with 65zncl2 in short-time laboratory experiments. two pathways of zn uptake and translocation, i.e. from stalks to leaf blades and from leaf blades to stalks were compared under the same experimental conditions. we used detached ivy leaves fully immersed in zncl2 solutions, instead of spraying, in order to eliminate differences in uptake values caused by different droplet volumes and contact surface areas, described by mercer (2007) and by other researchers. 2. materials and methods 2.1 plant material ivy branches (h. helix l.) were collected during spring months from freely grown garden vegetation. the upper part of branches were cut from a wild ivy plant, washed repeatedly in deionized water and used for experiments. leaves of about 0.2 – 0.3 g fresh weight and of leaf area 11.5 12.5 cm2 were used in experiments. fig. 1. photo of ivy leaves in experiments. characteristic signs: short stalks; shallow sinus; well developed veins; terminal, lateral and basal lobes. a. leaf stalks immersed in nutrient media in petri dishes; b. only leaf blades immersed in nutrient media in petri dishes. 2.2 bioaccumulation experiments 2.2.1 uptake via stalks of detached transpiring leaves leaf stalks (1.5 cm) were immersed in 10 ml 25% hoagland medium (hm, hoagland, 1920) supplemented with 5 µmol/dm3 zncl2 spiked with 65zncl2 and incubated in a cultivation room, at 22±2°c under illumination with artificial light (2 000 lx) in 12h/12h light/dark period (fig. 1a). the full-strength hm medium a b nova biotechnologica 9-1 (2009) 75 consisted of following molar concentrations of salts (in mm): mgso4.7h2o – 1.5; kno3 – 4.0; cacl2 – 4.0; nah2po4.2h2o – 1.87; na2hpo4.12h2o – 0.13; feso4.7h2o – 0.06; nano3 – 4.0; nh4cl – 3.17; nh4no3 – 2.0; h3bo3 – 0.14; na2moo4.2h2o – 0.0025; mnso4.5h2o – 0.21; znso4.7h2o – 0.023; cuso4.5h2o – 0.033. 2.2.2 uptake via surface area of immersed detached leaves leaf blades of detached leaves were fully immersed in 10 ml of 25% hm supplemented with 5µmol/dm3 zncl2 spiked with 65zncl2 in plastic petri dishes covered with lids (fig. 1b), under the conditions described in the previous paragraph. non-immersed leaf surfaces provided transpiration and respiration. at the end of experiments, leaves were carefully rinsed in deionized water and dried at 60°c to a constant weight and the incorporated radioactivity was measured by gammaspectrometry. bioaccumulation experiments were carried out in duplicate series. 2.3 radiometric analysis a gamma spectrometric assembly using the well type scintillation detector 54bp54/2-x, nai(tl) (scionix, the netherlands) and data processing software scintivision 32 (ortec, usa) were used for 65zn determination in plants and solutions. counting time of 600 s allowed to obtain data with measurement errors <2%, that do not reflect other sources of errors. a standardized solution of 65zncl2 (50 mg/dm3 65zncl2 in 3 g/dm 3 hcl) with specific radioactivity 4,901 mbq/cm3 was obtained from the czech institute of metrology (prague, czech republic). 2.4 speciation modeling the prediction of zn speciation in the aqueous systems was performed using the visual minteq (version 2.53) program. this speciation model allows the calculation of the composition of solutions for specified conditions (ph, ionic strength, concentration, temperature). 3. results and discussion leaf water repellency of adaxial or abaxial surfaces is a main limiting factor in spray application processes (watanabe and yamaguchi, 1991; holder, 2007). the permeability of the cuticle to water and to lipophilic organic molecules increases with mobility (diffusion coefficients) and solubility (partition coefficients) of these compounds within the transport-limiting barrier of the cuticles and this process is well described. significantly less is known about the permeability of the cuticle to ionic compounds. however, this is also of major significance in agriculture since, in foliar nutrition, elements such as zinc and copper can be sprayed as foliar fertilizers on leaf surfaces. in order to be effective, these ions have to penetrate the lipophilic cuticle. recently, significant progress has been achieved in measuring cuticular penetration (lin et al., 2007; schlegel et al., 2005; franke et al., 2005). 76 marešová j. et al. the aerial surfaces of higher plants are covered with a cuticle, with the exception of stomata openings. the cuticle is an extracellular, lipidic covering layer, forming the interface between the plant and environment. the cuticle represents the main barrier to the penetration of foliar-applied water soluble compounds such as mineral foliar fertilizers. the waxy leaf surface is hydrophobic and therefore surfactants are a common component of foliar formulations used for increasing the leaf wettability and to prolong the contact time of spray solutions with leaf surfaces. since charged molecules carry hydration shells (stein, 1967), they are not soluble in the lipophilic cutin and wax domains of the cuticles. according to viougeas et al. (1995) the cuticle mass of ivy leaves increases with increasing age from 234 to 539 µg/cm2. waxes increases from 12.3 to 18.6% of cuticle mass from young to old leaves. percentages of cutin and non-lipid constituents do not vary significantly with leaf age. they represent approximately 58 and 26% of the cuticle mass, respectively. cuticle thickness increases 12-fold during leaf growth to reach 4.25 µm for mature leaves. an outer lamellate zone gradually merging from an inner reticulate zone the thickness of which increases with leaf growth is characterized by a constant thickness of 0.2 µm. intracuticular wax is localized in lamellae. electron-dense fibrillae observed in the reticulate zone are made of non-lipid components (viougeas et al., 1995). we can suppose, that zn uptake data via leaf surface will be strongly dependent on the age of leaves used in experiments, due to the fact, that cuticle is the major barrier for nutrient leaf uptake. 3.1 zn uptake via stalks detached leaves are frequently used for studies of long distance transport in vascular systems of higher plants. the main advantage of such studies is in the elimination of selective effect of root system on different compounds, which takes place in transport studies via root systems of hydroponically grown plants. in our study, stalks of detached leaves of ivy (h. helix) were immersed in zncl2 solutions and both, the adaxial and abaxial leaf surfaces were in contact with atmosphere. in this case the only driving force of zn uptake was water transport driven by transpiration. we found that under given conditions, the uptake of zn was proportional to the initial zn2+ concentration c0 within the range ≤ 50 µmol/dm 3. at c0 = 50 µmol/dm 3 zncl2, concentrations of zn2+ 370 µg/g and 3.1 µg/g of dry weight of leaf stalks and leaf blades, respectively, were obtained. it means that zinc entering the plant tissues via stalks is distributed by vascular system into leaf blades only in limited extent, reaching a specific concentration ratio of [zn]stalks /[zn]blades = 120 : 1 (fig. 2a). which chemical components of stalk tissues are responsible for immobilization of zn2+ ions via xylem vessels will require more detailed study. 3.2 zn uptake via leaf surfaces for the zn foliar uptake study in our experiments we used detached ivy leaves, fully immersed in zncl2 solutions. this approach eliminates the differences in foliar nova biotechnologica 9-1 (2009) 77 uptake from sprayed solutions caused by different droplet volumes and contact surface areas of sprayed solutions, described by mercer (2007). under experimental conditions both adaxial and abaxial leaf surfaces were in contact with zncl2 solution and transpiration as the main driving force does not participate on zn influx into leaf tissues. influx of nutrients via leaf surfaces is a characteristic route for nutrient uptake by plants without efficient root systems, such as mosses and submerged aquatic plants. under such conditions the amount of zinc taken up by ivy leaves increased with increasing zn concentration in solutions, however the amount of zinc in leaf tissues represented only 33.7% of that accumulated via leaf stalks. data in fig. 2b demonstrate, that zinc entering plant tissues via leaf surfaces is distributed by vascular system into stalks, reaching the concentration ratios of [zn]stalks /[zn]blades from approximately 1 : 1 to 2 : 1. however this ratio will depend on the zinc concentration in solution. according to kramer and kozlowski (1979) and lin et al. (1995) the mobility of absorbed elements depends on their concentration in plants. 0 10 20 30 40 50 0 100 200 300 400 z n [μ g/ g] c 0 zncl 2 [μmol/dm3] leaf blades leaf stalks a 0 10 20 30 40 50 0 100 200 300 400 leaf blades leaf stalks z n [μ g/ g] c 0 zncl 2 [μmol/dm3] b fig. 2. zn concentration (µg/g, dry wt.) in leaf stalks (......) and leaf blades (____) of ivy leaves (h. helix l.) after 5 d exposition in 2.4; 5.0 and 50 µmol/dm3 zncl2 in 25% hm spiked with 65zncl2 (310 kbq/dm3). a. uptake via immersed stalks of transpiring leaves. b. uptake via surface of fully immersed leaves. cultivation at 12h/12h light/dark period (2 000 lx), ph 6.0 and 22±2°c. wet weight of leaves [g/10 ml]: a. 0.29±0.01 (±sd); b. 0.27±0.01 (±sd). leaf area [cm2]: a. 14.6±0.75 (±sd); b. 13.7±0.99 (±sd). b. transpiration 3.32 ± 0.74 cm3.dm-2.d-1 (±sd). total zn uptake via leaf surfaces was by one order lower comparing with zn uptake via xylem path of stalks of detached leaves. this is a consequence of the existence of diffusion barriers on the leaf surfaces, mainly cuticular membrane and waxes and due to the absence of transpiration stream. the driving force for zn uptake across the leaf surface is a concentration gradient on the water/leaf interface. similarly as in the case of zn uptake via leaf stalks (fig. 2a), also in the case of zn uptake via leaf blade surfaces of fully immersed leaves (fig. 2b), zn uptake increased with increasing initial zn concentration c0. however total zn uptake expressed as specific concentration in µmol/g was 9.4 times lower in stalks and 2.8 times higher in leaf blades. 78 marešová j. et al. 3.3 effect of nutrient salts distribution of zinc entering the leaves of ivy via vascular system of stalk of detached leaves immersed in nutrient medium spiked with 65zncl2 was dependent on inorganic nutrient concentration (fig. 3). when zn is taken up from low nutrient salt media, i.e. < 50 % hm, substantial part of zinc remains immobilized in stalks. 0 25 50 75 100 0 10 20 30 40 50 z n [n m ol /g ] hoagland concentration [%] a 0 25 50 75 100 0,0 0,2 0,4 0,6 0,8 1,0 x m ol ar fr ac tio n hoagland concentration [%] b fig. 3. influence of nutrient salt concentration on zn uptake and distribution in leaf stalks and leaf blades of ivy (h. helix l.). uptake via immersed stalks of transpiring leaves. data after 6 d exposition in diluted hm spiked with 65zncl2 (310 kbq/dm3) at initial zn concentration c0: 1.1; 1.7; 2.3 and 3.4 µmol/dm3. cultivation at 12h/12h light/dark period (2 000 lx), ph 5.5 and 22±2°c. data expressed as a: zn concentration in ivy leaves [nmol/g, dry wt.]; b: zn molar fraction in leaf blades (x = [zn]blades/ [zn]total ; -■-■-■-) and leaf stalks (x = [zn]stalk/ [zn]total ; -●-●-●-). biomass of the whole ivy leaves (stalks + blades) in experiments (g/10 ml, wet wt.): 0.64±0.06 g (±sd). leaf blade area of transpiring leaves: 20.03±1.26 cm2 (±sd). transpiration rate: 3.33 ± 0.74 cm3.dm-2.d-1 (±sd). when zn is taken up from solutions of high nutrient concentrations (> 50 % hm), zinc is nearly completely transported from stalks to the leaf blades (fig. 4). we can conclude that zinc entering the leaves via stalk xylem is bound on components of xylem vascular system by reversible ionic bond. zinc can be replaced from binding sites by ions showing higher activity for binding sites, present in the vascular system. to answer the question which binding sites in xylem vessels play a decisive role in zn binding in xylem of individual plant species will require more detailed study. our hypothesis is supported by published data describing zinc interaction in other plant tissues. straczek et al. (2008) used chemical extractions and exafs spectroscopy for the study of zn distribution in whole roots and isolated root cell walls of tobacco plants. their results showed that the cell walls of roots exhibited a distribution of zn from water soluble to non-exchangeable zn form. in whole roots, zn was bound with oxalate and other cooh/oh groups: the first species was probably intracellular while the second was attributed to zn bound to the cell walls and, to a lesser extent, to intracellular organic acids. zn-phosphate was also identified, and this species was cuso4 extractable. it probably resulted from chemical precipitation in the apoplast, and explained the steady increase in exchangeable root zn observed in tobacco roots during the culture. nova biotechnologica 9-1 (2009) 79 0 20 40 60 80 100 0 2 4 6 8 10 12 14 16 18 [z n] st al ks : [z n] le af b la de s hoagland concentration [%] fig.4. distribution of zn in stalks and blades of ivy leaves after 6 d uptake via immersed stalks of transpiring leaves, expressed as the [zn]stalks : [zn]blades ratio. calculated from data in fig. 2. zinc, in optimal concentration, is a typical micronutrient which is necessary for normal metabolism of all plant cells. on the other side, in supra-optimal concentrations, zinc can cause serious damages in metabolically active cells, mainly in leaves. our data support the idea, that leaf stalk can act as a barrier able to retard transport of zn ions entering the leaf via xylem path. this barrier may be less efficient in the presence of higher salt concentrations moving in the root to shoot direction. 3.4 the effect of ph the membrane permeability can be affected by solution ph, mainly by influencing the driving forces via electrical potential and by change of the properties of the solutes by dissociation. the cuticles are polyelectrolytes and their isoelectric point is around ph 3 (schönherr and huber, 1977). above this point, when ph increases, the cuticles carry fixed negative charges. these charges are also an important characteristic affecting the water content of the polymer matrix via swelling. the ph dependence of zn uptake via ivy leaf surface is shown in fig. 5. below ph 3.0, i.e. below the isoelectric point of cuticle, zn uptake was minimal, increasing up to ph 4 and showing plateau up to ph 6. the next increase of zn uptake is evident at ph > 6 what coincides with decrease of zinc concentration in bioavailable zn2+ form and formation of zinc ionic forms. 3.5 the effect of temperature our experiments did not confirm the dependence of zinc uptake by ivy leaf surface on temperature within the range from 4 to 37°c (results not shown). this problem will require more detailed, mainly kinetics studies. experiments with isolated cuticular membranes described by schönherr and luber (2001) showed that different forms of water and lipophilic substances, inorganic ions and charged organic 80 marešová j. et al. molecules penetrate the isolated cuticular membranes independently of temperature. schreiber (2005) concluded that ionic compounds use aqueous polar path of diffusion, whereas lipophilic molecules move along the lipophilic wax and cutin domains. water, as a small but uncharged molecule, can use both paths. 3 4 5 6 7 8 0 0,2 0,4 0,6 0,8 1 0 2 4 6 8 m ol ar fr ac tio n ph z n up ta ke [% ] zn uptake [%] zn2+ znso 4 znhpo 4 zn(oh)+ zn(oh) 2 10 fig. 5. the ph dependence of zn uptake via the surface of fully immersed ivy leaves (-■-■-■-) and molar fraction of zn species in solutions at given ph values. uptake data after 5 d exposition in 5.0 µmol/dm3 zncl2 in 25% hm, spiked with 65zncl2 (130 kbq/dm3). initial ph values adjusted by adding 0.05 m naoh. leaf wet weight biomass [g/10 ml]: 0.22±0.017 (±sd). leaf area [cm2]: 9.86±0.88 (±sd). presented molar fractions of zn species at given ph are calculated by the visual minteq program. 4. conclusions experiments with ivy leaves as a model plant leaves showed that total zn uptake from zncl2 solution via immersed leaf surface is by one order lower, comparing to zn uptake via xylem path of immersed stalks of detached leaves under the same conditions. zn uptake increased with increasing initial zn concentration up to c0=50 µmol/dm3 zncl2 in both cases. zinc entering the plant tissues via leaf stalks remains bound mainly in stalk and was transported by vascular system into leaf blades only in limited extent. in the last case the specific concentration ratio [zn]stalks /[zn]blades was 120-times higher, compared with the case of zinc entering plant tissues via leaf surfaces. zn uptake was minimal below ph 3, increasing at ph 3 – 5, showing plateau at ph 6 – 7 and next rapid increase at ph 7. our experiments did not confirm the dependence of zinc uptake by ivy leaf surface on temperature. more detailed study is necessary for understanding all factors influencing the efficiency of foliar uptake of mineral nutrients from foliar fertilizers. references barátová, z., sekáčová, 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of xylem transport affects zn and mn transport into developing wheat grains of cultured ears. physiol. plantarum, 98, 1996, 229-234. schlegel, t.k., schönherr, j., schreiber, l.: size selectivity of aqueous pores in stomatous cuticle of vicia faba leaves. planta, 221, 2005, 648-655. 82 marešová j. et al. schönherr, j., huber, r.: plant cuticles are polyelectrolytes with isoelectric points around three. plant physiol., 59, 1977, 145-150. schönherr, j., luber, m.: cuticular permeation of potassium salts: effects of humidity, anions and temperature. plant soil, 236, 2001, 117-122. schreiber l.: polar paths of diffusion across plant cuticles: new evidence for an old hypothesis. ann. bot., 95, 2005, 1069-1073. stein, w.d.: the movement of molecules across cell membranes. academic press, new york, 1967, 369 pp. straczek, a., sarret g., manceau, a., hinsinger, p. geoffroy, n., viougeas, m.a., rohr, r., chamel, a.: structural changes and permeability of ivy (hedera helix l.) leaf cuticles in retention to leaf development and after selective chemical treatments. new phytol., 130, 1995, 337-348. watanabe, t., yamaguchi, i.: evaluation of wettability of plant leaf surfaces. j. pestic. sci., 16, 1991, 491-498. nova biotechnol chim (2022) 21(2): e1337 doi: 10.36547/nbc.1337 1 nova biotechnologica et chimica elucidation of antibacterial activity of bacillus sp. and alcaligenes sp. metabolites against multidrug-resistant bacteria faheem ullah1, sadir zaman1, waheed ullah1,, shandana ali3, muhammed qasim1, niaz muhammad1, momina mehmood1, navid ali1, niamat khan2 1department of microbiology, kohat university of science and technology, kohat 26000, khyber pakhtunkhwa, pakistan 2department of biotechnology and genetic engineering, kohat university of science and technology, kohat 26000, khyber pakhtunkhwa, pakistan 3department of zoology, kohat university of science & technology kohat 26000, khyber pakhtunkhwa, pakistan  corresponding author: waheedwazir@gmail.com article info article history: received: 22nd december 2021 accepted: 1st july 2022 keywords: antibacterial activity cholistan desert inhibitor minimum inhibitory concentration secondary metabolites abstract different resistance mechanisms are involved in exhibiting resistance to different groups of antibiotics. researchers are searching for new therapeutic options to encounter the emerging trend of microbial resistance. bacteria were isolated from the extreme environment of cholistan desert and were screened for characterization. potential metabolites that showed broad-spectrum activity were partially purified using silica gel chromatography and determined their minimum inhibitory concentration. a collection of 50 bacterial isolates from soil samples was screened for metabolite production and among them isolate r19 of bacillus sp. and isolate a8 of alcaligenes sp. had high similarity with strong antimicrobial metabolite producers. the growth of a8 was stable at slight acidic ph while r19 was best at neutral ph. similarly, the best growth of a8 was observed at 37 °c while r19 at 35 °c. minimum inhibitory concentration of purified compounds of bacillus sp. were determined at concentration range of (3.12 – 100 %) against multidrug-resistant (mdr) strains of shigella, enterobacter aerogenes, staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, and klebsiella pneumonia and produced 10 – 25 mm zone of inhibition. metabolites of alcaligenes sp. were sufficient to inhibit the growth of all selected mdr bacteria at concentrations 12.25 – 100 % and shows 10 – 20 mm zone of inhibition. bacillus sp. and alcaligenes sp. can be used as producers of potential antibacterial metabolites. proper utilization of selected metabolites can be helpful in combating emerging drug resistant pathogenic bacteria. in addition, further proteomic analysis and structural insight should be considered to elaborate their active ingredients and its efficacy. introduction microbes are potential sources of different types of important active metabolites. in search of potential metabolites producer microbial strains, several studies have been conducted to explore different habitats for potential metabolites producer microbes. it can be deduced from different studies that microbes of diverse environments carrying strong antimicrobial potential according to andayani et al. (2015), observed by others (masand et al. 2018; rajasabapathy et al. 2020). microbial metabolites due to their increasing applications have allure the attention of scientists to mailto:waheedwazir@gmail.com nova biotechnol chim (2022) 21(2): e1337 2 explore different untouched sites for potential metabolites producer microbes kumar et al. (2018). metabolites have various therapeutic applications in the treatment and eradication of different types of bacterial, fungal and parasitic diseases demain (1999). in pre-antibiotic era, the treatment of infectious diseases was challenging due to unavailability of antibiotics adedeji (2016). the discovery and commercialization of penicillin give new hope because morbidity and mortality associated with infectious diseases were reduced in infected population. unfortunately, with passage of time resistant strains like emergence of methicillin resistant staphylococcus aureus (mrsa), vancomycin resistant enterococci and multidrug resistant (mdr) strains of gram negative bacteria were reported worldwide particularly in developing world hesse and adhya (2019). world health organization estimates that annually millions of people lost their lives due to drug-resistant bacterial strains. according to shrivastava et al. (2018) the world health organization enlist priority pathogens for which new and effective drugs need to be explored having bactericidal activity. among these pathogens, carbapenem resistant strains are global threats le thanh dong and espinoza (2020). carbapenems drugs are considered the last choice for treatment of mdr bacteria. recently, few extreme drug resistant (xdr) strains of salmonella sp. were isolated from infected patients in pakistan, which revealed resistance to most of the available drugs including ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, ciprofloxacin, and ceftriaxone while fortunately sensitive to azithromycin and carbapenems rasheed et al. (2019). in relation to the resistance, the induction of new effective drugs rate is low as compared to previously approved drugs that lose their efficacy ribeiro da cunha et al. (2019). to tackle antibiotic resistance issue, scientists are trying to explore new avenues for the isolation of potential metabolite producer microbes having strong inhibitory potential. bacillus a gram positive spore forming bacteria, having potential to cope harsh environment has been frequently isolated from extreme habitats nicholson et al. (2000). their secondary metabolites exhibit strong antibacterial activity and many of their compounds have been commercialized to use as antibiotics such as bacteriocins morikawa et al. (1992). beside bacillus, actinomycetes, fungi are also sources of antibiotics singh et al. (2019). in the current scenario, researchers demonstrate key interest in bacterial characterization from the unexplored harsh environment like deserts and marine with the hope to explore strong bioactive metabolites producer strains according to abdelkader et al. (2018); observed by cita et al. (2017). indeed, by 2008, more than 100 specialized metabolites had been isolated and identified from microorganisms isolated from such habitats. in addition, in the previous decade, 129 putative strains, producing 186 drug gable metabolites, were isolated from extreme strata sayed et al. (2020). the current study was conducted for the exploration of antibacterial metabolites producer bacterial strains from the unexplored habitat of cholistan desert (punjab, pakistan) with harsh environmental conditions like high temperature, scarcity of nutrients and water depletion. experimental collection and isolation of bacteria from soil samples samples from different locations of cholistan desert were collected in sterile bottles using standard procedure as previously described by elbendary et al. (2018). all samples were brought to the medical laboratory, department of microbiology, kust and were stored at -80 °c for further analysis. all samples were serially diluted up to 10-9 in sterilized distilled water. aliquot of 100 µl was spread from last three dilution on tryptone soya agar (tsa) and nutrient agar (na) and plates were incubated for 48 h at 37 °c. after incubation different colonies appeared on different culture media. resistant bacterial strains profile mdr bacterial strains of p. aeruginosa, e. coli, e. aerogenes, k. pneumonia, s. aureus, and shigella sp. were obtained from medical laboratory, department of microbiology, kohat university of science and technology. to assess the nova biotechnol chim (2022) 21(2): e1337 3 antibacterial potential of broth extract of metabolite producer strains, its inhibitory effects were screened against the resistant pathogens. all the selected bacterial strains revealed resistance toward different antibiotics like penicillin, cephalothin, tetracycline, amoxicillin, cefoperazone, ceftriaxone, and cefepime based on clsi guidelines. evaluation of antibacterial activity of cell-free extract isolated bacteria were grown in luria bertani broth (lb) for 5 to 7 days in shaking incubator at 170 rpm and 30 °c. after incubation, centrifugation was performed, and supernatants of each broth were filtered. the cell-free filtrates were assayed for antibacterial activity using well diffusion assay oskay (2011). growth optimization at different temperature and ph the isolates were incubated in nutrients broth at various temperatures like 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, and 75 °c. growth conditions were optimized at different ph 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. the results were recorded using spectrophotometer at 600 nm od. for proper growth, ph of the media was adjusted according to bacterial needs as previously described khalil et al. (2009). extraction of antibacterial metabolites bacterial isolates from desert soil samples (n = 50) were screened for their antibacterial metabolites production. among these isolates, a8 and r19 exhibit broad-spectrum activity against mdr bacteria and were selected for further analysis. an equal amount of ethyl acetate was added to the extracted filtrate and then metabolites were concentrated using rotary evaporator. bacterial metabolites were purified using silica gel column chromatography, chloroform/methanol in the ratio of (100 : 0, 80 : 20, 60 : 40, 50 : 50, 40 : 60, 20 : 80, 0 : 100) as an eluent. further each fraction were concentrated using rotary evaporator rao et al. (2007). minimum inhibitory concentration (mic) determination of antibacterial metabolites activity of each fraction of selected metabolites was examined on mueller hinton agar (mha) against drug resistant strains by using agar well diffusion process. fractions of best restraining were selected for mic determination against resistant pathogens. active fractions (2 ml) were considered 100 % and were serially diluted into two-fold to make 50 %, 25 %, 12.5 %, 6.25 %, and 3.125 %, and concentration respectively. further 100 μl from each concentration was used for the mic determination. biochemical and molecular characterization of metabolites producer strains strong metabolites producer strains were biochemically characterized using standard procedures of (rahimnahal et al. 2017; marathe et al. 2018). for molecular characterization, dna from the selected active antimicrobial metabolites producers strains were extracted by standard phenol chloroform method (neumann et al. 1992) using overnight culture. pcr reactions were carried out for the amplification of 16s rrna gene using universal primers forward primer 5'agagtttcctggctcag-3' and reverse primer 5'-aaggaggtgatccagcc-3' (lim et al. 2016). pcr conditions were set for amplification of 16s rrna gene. the thermal cycling reactions were performed as: initial denaturation at 94 °c for 5 min, then 25 cycles of 94 °c for 1 min, 50 °c for 40 s and 72 °c for 60 s and final extension was 72 °c for 5 min. the amplified products of pcr of the selected isolates were confirmed on 2 % agarose by adding 3.5 μl ethidium bromide as a staining dye and visualized by the digital gel doc system (analytik jena system, jena, germany). the products were subjected to sequencing and reactions were conducted at macrogene (seoul, south korea) using the universal 16s rrna forward primer. the resulted sequences were extracted using bioedit software. ncbi blast analysis was performed for sequence alignment. moreover, phylogenetic tree was constructed using mega 7 software. nova biotechnol chim (2022) 21(2): e1337 3 results antibacterial activities of a8 and r19 metabolites soil samples (n = 50) were screened for their antibacterial metabolites production and among these isolates a8 and r19 were expressed broad spectrum activity against resistant bacterial culture and were selected for further analysis. morphologically isolate a8 stained gram-negative rod shape while r19 was identified as grampositive rod shape and was also spore positive fig. 1. biochemically both isolates were catalase, oxidase, sugar fermenting, and motility recorded positive. a b fig. 1. morphological study of the selected isolates from cholistan desert. a – culture of bacteria on nutrient agar; b – pink colour shows gram negative bacteria; c – the blue colour shows gram positive bacteria. growth optimization of selected isolates on different temperature and ph best growth of selected isolate a8 was observed at 37 °c and lowest growth were observed at 15 °c. similarly isolate r19 have shown maximum growth at temperature 35 °c as shown in fig. 2a. similarly, ph also has a huge impact on the bacterial growth. a8 shows maximum growth at neutral ph while r19 showed maximum activity at ph 9.0. both isolates growth was restrained at acidic ph as shown fig. 2b. gram negative rods culture of bacteria gram positive rods c 4 nova biotechnol chim (2022) 21(2): e1337 3 0 0.2 0.4 0.6 0.8 1 1.2 1.4 o p ti c a l d e n s it y ( o d ) temp = temperature (°c) effect on growth at different temperature a8 r19 0 0.5 1 1.5 ph 3.0 ph 4.0 ph 5.0 ph 6.0 ph 7.0 ph 8.0 ph 9.0 ph 10.0 o p ti c a l d e n s it y ( o d ) ph effect on growth at different ph a8 r19 fig. 2. effects of temperature (a) and ph (b) on growth of isolates (a8 and r19). mic determination of isolate a8 collected through silica gel at different concentrations. the metabolites activities of strain a8, after silica gel purification, were observed for antibacterial activity against shigella, s. aureus, e. aerogenes, p. aeruginosa, e. coli, and k. pneumonia mdr strains respectively at different concentration as shown in table 1 and fig. 3. isolate a8 metabolites has the maximum zone of inhibition against mdr shigella and s. aureus of about 25 mm, 24 mm, respectively. similarly, it has strong inhibitory activity toward p. aeruginosa and 19 mm zone of inhibition while in case of e. aerogenes 20 mm zone of inhibition was recorded. while less inhibitory activity was observed against e. coli and k. pneumonia. a b 5 nova biotechnol chim (2022) 21(2): e1337 3 fig. 3. antibacterial activity of a8 isolate against (a) klebsiella pneumonia, (b) e. coli, (c) e. aerogenes, (d) s. aureus, (e) shigella, (f) p. aeruginosa. fig. 4. antibacterial activity of r19 isolate against (a) k. pneumonia, (b) e. coli, (c) e. aerogenes, (d) s. aureus, (e) shigella, (f) p. aeruginosa. table 1. inhibition zones of a8 metabolites against selected bacterial strains at different concentration. tested bacterial strains 3.15 % [mm] 6.25 % [mm] 12.5 % [mm] 25 % [mm] 50 % [mm] 100 % [mm] shigella --13 15 22 25 s. aureus --10 12 21 24 e. aerogenes ---11 14 20 p. aeruginosa --10 15 17 19 e. coli ---11 12 15 k. pneumonia ----14 15 antibacterial activity of r19 isolate the antibacterial activity of r19 metabolite was observed against shigella, s. aureus, e. aerogenes, p. aeruginosa, e. coli, and k. pneumonia mdr strains at different concentration as shown in (table 2, fig. 4). r19 bacterial metabolites were more effective against all selected resistant bacterial strains as compared to a8. it has shown a strong inhibitory pattern from lower to higher concentration against shigella and e. aerogenes. at lower concentration (3.12 %) it produced 10 mm zone of inhibition while at higher concentration (100 %) it generated 19 mm and 20 mm zone of inhibition against selected mdr strains respectively. similarly, it has strong inhibitory action against mdr e. coli at concentration (50, 100 %) and manifest 19 mm and 23 mm zones of inhibition toward selected strains. further their action against other tested isolates including k. pneumonia and p. aeruginosa revealed 16 – 18 mm zone of inhibition at concentration (100 %) as shown in table 2. 6 nova biotechnol chim (2022) 21(2): e1337 3 table 2. inhibition zones of r19 metabolites against selected bacterial strains at different concentration measured in diameter (mm). test bacterial strains 3.12 % [mm] 6.25 % [mm] 12.5 % [mm] 25 % [mm] 50 % [mm] 100 % [mm] shigella 10 13 15 16 18 19 s. aureus -8 9 12 16 20 e. aerogenes 10 11 12 13 14 20 p. aeruginosa -8 9 11 15 18 e. coli -5 6 10 19 23 k. pneumonia ---10 15 16 molecular characterization of a8 and r19 strains using 16s rrna gene further identification of both a8 and r19 metabolite producer strains were characterized through pcr amplification of 16s rrna gene, size 1399 bp, as shown in fig. 5. amplified product of a8 and r19 were sequenced at macrogen (seoul, south korea) using forward primer. homology analysis of both isolates were conducted at ncbi blast and there were 97.7 % identity with alcaligenes sp. for a8 isolate and 98.3 % identity with bacillus sp. for r19. based on sequencing results, phylogenetic trees were constructed using mega7 for the selected isolates (a8 and r19) as shown in fig. 6 and 7. fig. 5. gel image of amplified product of 16s rrna gene. lane l shows ladder, lanes a8 and r19 bacterial isolates a8 and r19, respectively. fig. 6. r19 phylogenetic tree using mega7. 7 nova biotechnol chim (2022) 21(2): e1337 2 fig. 7. a8 phylogenetic tree using mega. discussions to combat extending bacterial resistance, exploration of new drugs is indispensable of a current scenario. scientists are in search of new metabolites that have strong potential to address resistant strains. soil harbour diverse microbial community which has potential to cherish strong antibacterial potential. previously bacterial isolates particularly bacillus sp. have been reported from different habitats having strong antibacterial activity. in recent study efforts were made to explore microbial community for potential of strong antibacterial compounds, from diverse environmental conditions. deserts have diverse environmental conditions and have mixed microbial flora. cholistan desert of pakistan has wide range of microbial community and have potential to cope with challenging environmental conditions amin et al. (2020). in current study two isolates (a8 and r19) emerged as strong antibacterial metabolites producer strains that have been characterized based on ribotyping which revealed that a8 showed 97.74 % sequence similarity to alcaligenes sp. and r19 unveil 98.33 % sequence similarity to bacillus sp. similar active producer strains have been screened from the unexplored unique habitat of mountain himalaya which produced strong antibacterial metabolites according to hussain et al. (2018). according to das et al. (2018) study, streptomyces strains were isolated from protected forest land and most of these isolated strains carried broad spectrum antibiotic activity and were stable at 37 °c while some of the isolated strains were stable 42 °c. a8 and r19 isolate showed broad spectrum activity, against mdr clinical bacterial strains. maximum growth of a8 and r19 isolates were observed on normal nutrient media. other growth conditions like temperature and ph were also analysed, isolates that the best grown at temperature 37 ℃ and the lowest below 20 ℃ have been recorded. our result are in line with the study conducted by bharali et al. (2011) in which the alcaligenes sp. isolates growth was documented at 42 °c. a8 showed maximum growth at neutral ph while r19 was most stable at slight alkaline ph. these results confirmed previously reported by farag et al. (2019) in which metabolites of bacillus manifest promising activity was observed at slight alkaline ph. in recent development, growth of mdr strains of shigella and e. aerogenes were restricted at concentration of 3.12 % of the partially purified compound of bacillus sp. similarly, for s. aureus, p. aeruginosa and e. coli were determined 6.25 % while 25 % for k. pneumonia. the partially purified metabolites of isolated strain of bacillus sp. exhibited promising activity against gram negative bacteria and it has also excellent inhibitory activity against s. pyogenes mtcc 442, b. cereus, e. faecalis, and s. epidermidis atcc 12228. 8 nova biotechnol chim (2020) 19(2): 124-137 similarly, ramachandran et al. (2014) isolated bacillus sp. from soil samples and found a strong producer of metabolites that showed strong effective against broad-spectrum microbes. the metabolites of alcaligenes sp. manifest strong activity at lowest concentration (12.25 %) against shigella, s. aureus and p. aeruginosa while at concentration (25 %) growth of e. aerogenes and e. coli were inhibited. a thermophilic strain of alcaligene sp. was isolated from petroleum contaminated soil having potency of antibacterial activity against bacillus sp., k. pneumonia, e. coli, p. aeruginosa, and s. aureus observed by bharali et al. (2011). similarly, isolated strains of alcaligenes sp. were reported from common effluent treatment plant which demonstrated antibacterial activity against mdr enterobacter sp. and serratia sp. being supported by the production of active a nucleoside antibiotic according to kapley et al. (2016). bacillus sp. are predominantly soil bacteria and can be found in different other habitats that their existence in soil in huge numbers as a result of their capacity to form resistance endospores and develop bioactive compounds which promote their resistance to variable environmental conditions reported by onajobi et al. (2020). a study reported by abd sharad et al. (2016) of the alcaligenes sp. isolated from marine habitat has strong antibacterial activity at 3 mg.ml1 against resistant bacteria. conclusions metabolites of alcaligenes sp. (a8) and bacillus sp. (r19) have shown the maximum zone of inhibition against mdr clinical bacterial strains. the active fractions of alcaligenes sp. metabolites revealed strong inhibitory effects against all resistant pathogens. this study provides initial layout for researchers and drug developers to further characterize active ingredients. furthermore, its growth was optimized on the best ph and temperature that will be helpful in extraction and stability of metabolites. proper utilization of selected metabolites can be helpful in combating emerging drug resistance mess in pathogenic bacteria. in addition, further proteomic analysis and structural insight should be considered to elaborate their active ingredients and its efficacy. acknowledgements this study was a part of ms research project. we extend our gratitude to department of microbiology, kohat university of science and technology for providing lab facilities for conducting this research. conflict of interest the authors declare that they have no 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methodius in trnava flavonols hplc analysis, in vitro biological activities in selected humulus lupulus l. genotypes maria maliarova1, tibor maliar2, jana girmanova1, jozef lehotay1, jan kraic2 1department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, j. herdu 2, trnava, sk-917 01, slovak republic 2department of biotechnologies, faculty of natural sciences, university of ss. cyril and methodius trnava, j. herdu 2, trnava, sk-917 01, slovak republic (maria.maliarova@ucm.sk) abstract: the humulus lupulus l. is well known as necessary raw material for beer production. the main structural classes of chemical compounds identified from hop cones include terpenes, bitter acids, prenylated chalcones, and flavonol glycosides. they were subjects of presented work. the content of quercetin was found in the range 490 – 1092 µg/g and that of kaempferol from 218 to 568 µg/g of the dry hop cones. the content of isorhamnetin was very low in all varieties. from biological activities in vitro point of view, relative high level of inhibition activity was observed for six hop genotypes – zlatan, lučan, and the oswald's clones 31, 70, 71, 72, 114 on both enzymes thrombin and urokinase, but without correlation to analyzed flavonols content. in spite of this, antioxidant activity, measured by both the bclm and hpe methods, was found high and seem to be in correlation with content of analyzed flavonols. particularly the oswald's clone 114 expressed very potent biological activities. in general, obtained results indicate that hop cones are valuable material also for other application others than beer production. key words: humulus lupulus, saaz, quercetin, kaempferol, isorhamnetin, biological activity in vitro 1. introduction the hop plant (humulus lupulus l., cannabinaceae) is well-known throughout the world as the raw material for brewing industry. the female inflorescences (the hop cones, strobili lupuli) are used in the beer production especially to add bitterness (stevans et al., 1999). the main structural classes of compounds identified from the hop cones include: terpenes (compounds with sedative effect), bitter acids (prenylated derivatives of phloroglucinol, which posses antibacterial and antioxidant effects) and finally polyphenols, including flavonoids, prenylated flavonoids, and polyphenolic acids with antioxidant, antibacterial activity (krofta et al., 2008; zanoli and zavatti, 2008; nesvadba and krofta, 2009). it is well now that flavonoids exhibit a broad spectrum of pharmacological effects, especially anti-inflammatory and antioxidant activity, antimutagenic and anticarcinogenic activities but also inhibition activity on various proteolytic enzymes including enzymes involved in coagulation and fibrin clot digestion processes as thrombin and urokinase are (middleton et al., 2000; maliar et al., 2004). thrombin is key enzyme in process of fibrin clot formation of the coagulation cascade mechanism, partially different from the trypsin by the specific p1´ region bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:18 utc 130 maliarová, m. et al. cleaving peptide bond between arginine and any bulky aromatic residue (onaya et al., 1998). thrombin is pathophysiological promoter of coagulation disorder diseases and attractive target for perspective thrombolytics. urokinase (plasminogen activator of the urokinase type) is key enzyme responsible for activation of plasmin cascade leading to digestion of needless fibrin clot after wound or vessel closing and healing. urokinase is very specific serine-proteinase preferring cleaving peptide bonds beside lysine residue (schmitt et al., 1997), pathophysiological promoter of the oncological diseases with significant metastasis and attractive target for new oncolytics by the metastasis blocking mechanism. the hop is rich for flavonolglycosides, which have monoor di-saccharide bonded in position 3. segawa et al. have identified quercetin glycosides and kaempferol glycosides in the hop cones by mass spectrometry (segawa et al., 2006). the flavonolglycosides hydrolysis release free aglycons – flavonols from monoor disacharides (fig. 1). quercetin is formed from the glycosides of rutin, isoquercitrin and isoquercitrin malonate under acidic condition. kaempferol is formed from similar glycosides – kaempferol rutinosid, astragaline and astragalin malonate. isorhamnetin was also identified in the hop cones after enzyme hydrolysis (wojdylo et al., 2007). during brewing, the flavonols are extracted from hop to beer. the final content of flavonols in beer depends on both, the raw material and the brewing process conditions. however, acid hydrolysis takes place most frequently (alekseeva et al., 2004; proestos et al., 2006; stalikas, 2007). simultaneous acid hydrolysis and extraction of flavonols was firstly described by hertog for fresh and frozen vegetables (hertog et al., 1992). additional options are microwave assisted acid hydrolysis (nuutila, 2005) or enzyme hydrolysis by glycosidases (wojdylo et al., 2007). rutin quercetin isoquercitrin isoquercitrin malonate h+ h+ h+ fig. 1. structures of quercetinglycosides and their acid hydrolysis product. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:18 utc nova biotechnologica et chimica 12-2 (2013) 131 physical and chemical properties of flavonols provide important knowledge for selection of the condition for hydrolysis, separation and detection. 1-octanol/water partition coefficient – logp values and solubility in water are given in the table 1. table 1. 1-octanol/water partition coefficients and solubility in water values of selected flavonoids. log p solubility in water flavonol experimental data prediction by alogps experimental data [mg/ml] prediction by alogps [mg/ml] rutin 0.21 0.15 0.125 3.54 quercetin 1.82 ± 0.32 1.81 0.06 (16 ºc) 0.26 isorhamnetin not available 1.80 not available 0.15 kaempferol 3.11 ± 0.54 1.99 not available 0.10 despite of the fact, that these values are available by various chemical software, experimental data are most precious (rothwell et al., 2005). solubility of aglycones in water is lower than the solubility of glycosides therefore it is necessary to perform hydrolysis of glycosides in water-alcohol solutions. saaz is the former german name of the czech town žatec, known by the hop cultivation. nevertheless, it denotes also a hop variety, classified as one of the four true noble varieties used in production of pilsner style beer. this semi-early red-bine hop, belonging to the old middle european aroma group of hops, originates from the clonal selection in original hops widespread within the saaz and auscha regions. saaz variety is grown in nine clones. its typical property is fine balanced bitterness and the high level of total polyphenols. in fact, in the literature, does not existed sufficient analysis results about flavonols content, especially for these hop genotypes, typical for mentioned region, as well as any indication to biological effect is missing. the aim of this work was firstly: investigation of the content of individual flavonols in the hop clones saaz, secondly – estimation of several biological activities on in vitro level, eventually specification of any relation between flavonols content and biological activity. for the first purpose the hplc analysis was selected and three steps were necessary to accomplish this work: (1) selection of optimal conditions of the glycoside hydrolysis and extraction of flavonols. (2) selection of the optimal mobile phase and the most suitable column with the stationary phase. (3) description of the basic validation characteristics of the flavonols determination. (4) elaboration of analysis of natural samples. secondly, selected assays on in vitro level, carried out with the same hop cone extract samples, aimed to attempt to formulate any relations. 2. materials and methods 2.1 chemicals methanol (hplc gradient grade) was purchased from sigma-aldrich. hydrochloric acid and formic acid (both p.a. purity) were purchased from mikrochem. ultra pure water was prepared with simplicity 185, millipore. standards for hplc: quercetin, kaempferol and isorhamnetin were purchased from sigma-aldrich. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:18 utc 132 maliarová, m. et al. 2.2 calculation of physico-chemical properties the log p values were calculated by alogps 2.1 software accessible on-line on internet (vccl, 2011). 2.3 plant material plant material of dried hop cones was provided by the research institute of plant production piešťany, slovak republic and hop research institute žatec, czech republic. 2.4 hplc analysis the chromatographic apparatus consisted of a model 1525 binary hplc pump, a model 2487 dual λ absorbance detector, a model 2707 autosampler, a model 1500 heather column, and software empower 2 (waters, milford, ma, usa). the optimization of hplc conditions of flavonoids determination in hop extract was performed with the following colons: symmetry® c18, 4.6 x 75 mm, 3.5 μm, x terra® ms c18, 4.6 x 30 mm, 2.5 μm, x bridgetm c18, 4.6 x 50 mm, 3.5 μm (waters, milford, ma, usa). the mobile phase was composed from methanol and ultra-pure water with 0.1 % formic acid. the wavelength 360 nm was selected for detection of all investigated flavonols (quercetin, kaempferol and isorhamnetin). 2.5 enzyme inhibition assays analyses of thrombin, and urokinase inhibition activity in hop cones extracts was determined by simple photometry method using microplate reader opsys (dynex, usa), with following chromogenic substrates: n-α-benzoyl-phenylalanyl-valylarginine-paranitroanilide (bpva-pna) for thrombin, and n-glycine-arginineparanitroanilide dihydrochloride (gapna.2hcl) for urokinase, respectively. substrates were cleaved by thrombin and urokinase according to described methods (erlanger et al., 1961; geiger et al., 1991), released free paranitroaniline was detected at wavelength 410 nm. microplates were prepared manually by gradual dissolution of the substrate -inhibitor mixture. each well (three parallel wells were used for each sample) contained buffer solution with 0.6 mm substrate, 1% dmso (v/v), and tested sample diluted 200 times. control wells contained dmso. reaction was started by adding of enzyme solution (thrombin – 150 nih units.mg-1, urokinase – 500 plough units, respectively) in tris-hcl buffer, ph 7.6, without ca2+ ions and other activators. reaction temperature was 25 °c, data scanning time 1 min and 61 min. the optical density difference ∆od (od61.st.min – od1-st.min) was measured for each sample. the percentage expression of the inhibition activity was calculated in according with equation (eq. 1) and then related to standard epigallocatechin gallate (egcg) and expressed in mg of egcg equivalent (egcgeq.). % inhibition activity = ((1 – (∆od sample/∆od control))*100) (1) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:18 utc nova biotechnologica et chimica 12-2 (2013) 133 2.6 antioxidant assays bclm (β-carotene linoleate model) is simple model of antioxidant activity in vitro (ohshima et al., 1998). this method quantifies ability of the sample to prevent lipoperoxidation under lipophilic conditions. 2 mg of β-carotene were dissolved in 20 ml of chloroform. 4 ml of this solution were added to the evaporation bank with 40 mg of linoleic acid and 400 mg of tween-40. chloroform was completely evaporated using rotary vacuum evaporator at 50°c and residue was mixed with 100 ml of deionized water to produce β-carotene linoleate emulsion. 230 µl of this emulsion were mixed with 20 µl of tested extract sample in microplate wells. microplates were incubated at 50°c for 90 minutes and the reaction mixtures were measured on β-carotene content decrease at 490 nm. control wells contained pure water and standard wells contained, 90 mm solution of egcg in dmso. this method of antioxidant activity was carried out to use an endpoint photometric measurement of a dual wavelengths mode by using a microplate reader type mrx (dynex). measured data were processed by same way and related to standard epigallocatechin gallate (egcg) and expressed in mg of egcg equivalent (egcgeq.). the second method of determination of antioxidant activity was method of hydrogen peroxide elimination (hpe), based upon the catalytical effect of horse radish peroxidase with combination with fenol red in according to method described by pick and keisari (1980) modified by rakotoarison et al. (1997). 10 µl of tested extract sample was mixed with 10 µl h2o2 and immediately filled to 100 µl by phosphate buffer pbs (ph = 7.4). prepared reaction mixture was subjected to incubation for 15 minutes in thermostat under 37°c. after incubation 100 µl of fenol red solution (0.2 mg/1ml) contained horse radish peroxidase (17 u/1ml) was added and then incubated 15 minutes under room temperature. the reaction is terminated by addition 5 µl 1n naoh (1 n) and od630nm was detected by microplate reader. measured data were processed by mentioned way and related to standard epigallocatechin gallate (egcg) and expressed in mg of egcg equivalent (egcgeq.). 2.7 antimicrobial activity extract samples were tested using microplate dilution method using sterile microplates with u shape wells. microplate preparing: filling columns 2, 3, 4, 5, 6, 7, 8, 9, 10 with 100 μl of cultivation medium with inoculum. then 180 μl of this mixture was added to the column 1, together with 20 μl of tested extract samples. then is carried out the dilution by gradual transfer of 100 μl z from the wells of column 1 to the column 2, mixing then to column 3, 4 ….12. in the columns there are following titer values of tested samples (from the column 1 to column 12): 10, 20, 40, 80, 160, 320, 640, 1280, 2560, 5120, 10240. cultivation medium was nutrient broth with inoculum adapt to 0.04 – 0.06 mcfarland units, for yeast and molds was used malt broth with inoculum adapt to 105 cell per 1 ml using bürker chamber. microplates were cultivated under following conditions: bacterial species for 24 hours bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:18 utc 134 maliarová, m. et al. at 37°c, yeasts 24 – 48 hours at 22°c (24 – 48 hours), and molds 114 hours at 22°c. thirty microliters of 0.02% tetrazolium blue (ttb) solution were added after cultivation and after 30 minutes of incubation under same conditions were microplates measured and evaluate. blue formazane product released by microorganism mitochondrial dehydrogenase has been observed in wells with cell proliferation. the last well without color effect is value of mic parameter expressed in titer scale. 3. results and discussion 3.1 hplc analyze of flavonols in hop cone extracts at first, the simultaneous acid hydrolysis and extraction of flavonols was optimized with regard to concentration of hydrochloric acid, hydrolysis time, and proportion of methanol in the solution (fig. 2). the optimized conditions of hydrolysis of the hop cones are as follows: 1.2 mol/l solution of hydrochloric acid during 1 hour hydrolysis in 50 % methanol-water solution. fig. 2. optimization of the simultaneous acid hydrolysis and extraction of flavonols from hop cones conditions, (♦) quercetin, (■) kaempferol. relation of the retention factor k of flavonols to methanol portion (%) in mobile phase, (♦) quercetin, (■) kaempferol, (▲) isorhamnetin. a good separation was achieved if the composition of mobile phase was 40 – 45 % methanol in water with addition of 0.1 % formic acid. further decrease of the methanol content leads to prolongation of the total analysis time. consequently, the best conditions for hplc separation of flavonols are as follows: mobile phase 0.1% (w/v) formic acid in water and methanol (57:43), flow rate 1 ml/min, column temperature 45°c. under these conditions and using the optimized stationary phase (discussed in the next paragraph) the total analysis time is 6 min. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:18 utc nova biotechnologica et chimica 12-2 (2013) 135 different stationary phases were compared in the final step to obtain the best resolution of the analyzed compounds. when using the columns symmetry and x terra a shift of the retention time tr of the analytes was observed. the shift of the retention time did not appear when the column x bridge was used. the hplc chromatogram obtained with the mentioned best column, optimally selected mobile phase and further optimized conditions is demonstrated on fig. 3. a u 0,000 0,005 0,010 0,015 minutes 0,0 1,0 2,0 3,0 4,0 5,0 6,0 fig. 3. representative hplc chromatogram of a real extract sample: humulus lupulus oswald clone 114. denotation of peaks: i quercetin, ii kaempferol, isorhamnetin detected only in traces. column xbridge, mobile phase 0.1 % formic acid in water/methanol (57:43). basic validation characteristics of the hplc flavonols determination – calibration equation, linear range, coefficient of determination, lod and loq are summarized in table 2. table 2. selected validation characteristics for the hplc determination of three flavonol aglycones. flavonols calibration equationa linear range [μg/ml] r2 b lod c [μg/ml] loqc [μg/ml] quercetin y = 17275x – 960.3 0.134 – 8.16 0.9997 0.0726 0.090 kaempferol y = 18287x – 636.1 0.0437 – 3.18 0.9997 0.0509 0.0745 isorhamnetin y = 14955x + 43.94 0.025 – 0.100 0.9937 0.0272 0.0572 a y = peak area, x = concentration of standard in μg/ml b r2 − coefficient of determination for 8 data points in calibration curves. two parallel measurements were made for each calibration standard. c lod − limit of detection, loq − limit of quantification. before hplc analysis of the particular hop plant material it is important to verify the stability of three investigated important flavonols in the solution during hydrolysis. therefore the solutions of flavonols were subjected to model hydrolysis (reflux, 1.2 mol/l hcl in 50 % water/methanol solution for 2 hours). the model solutions were analyzed in 15 min intervals. flavonols concentration was not significantly i ii bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:18 utc 136 maliarová, m. et al. changed during 60 min, however, after 105 min a significant decrease of the concentration of all three flavonols was observed (not presented in this paper). in the application part of this study seven varieties of saaz, harvested during year 2009, were analyzed: zlatan, lučan, and the oswald clones 31, 70, 71, 72, 114 (table 3). the content of quercetin was found in the range 489 – 1092 µg/g and that of kaempferol 218 – 568 µg/g of the dry hop cones. the content of isorhamnetin was very low in all varieties, which caused a relatively high rsd values. it is worth to compare that the similar quercetin content was found in the hop cones of variety „sbornyi“ − 1000 μg/g of the dry hop matter (alekseeva et al., 2004). to discuss about obtained results it is very difficult because there are no published papers about analysis of hope cone from žatec region, moreover there are several papers aimed to identification of flavonols by ms without quantification (magalhães et al., 2010). table 3. contents of flavonols and biological activity of dry hop cones. concentration of the saaz clones of humulus lupulus l. in [μg/g] ± % sd flavonols lučan zlatan oswald clone 31 oswald clone 70 oswald clone 71 oswald clone 72 oswald clone 114 quercetin hplc [μg/g] ± sd 783.3 ± 2.9 783.3 ± 2.4 792.8 ± 0.8 771.3 ± 7.2 489.5 ± 9.7 822.9 ± 3.6 1091 ± 4.2 kaempferol hplc [μg/g] ± sd 284.8 ± 4.0 338.5 ± 2.3 396.7 ± 3.3 402.3 ± 8.4 217.7 ± 7.6 314.0 ± 5.8 568.0 ± 6.1 isoramnetin hplc [μg/g] ± sd traces traces traces 21.4 ± 13.8 16.5 ± 6.8 traces traces 3.2 biological assays of hop cone extracts in vitro the same sample of extract from hop cones were subjected to several assays in vitro, especially to two test of antioxidant activity, either test of inhibition activity on thrombin as potential pathophysiological promoter of cardiovascular coagulation diseases or test of inhibition activity to urokinase as expression of ability to suppress processes of metastasis and onco-transformed cell spreading. fig. 4 presents mentioned biological activities of six hop cones extracts (zlatan, lučan, oswald's clones 31, 70, 71, 72, 114). there is evident (fig. 4) relative high level of inhibition activity on both enzymes, higher on thrombin for lučan, zlatan and oswald's clones k-70, 71, and for urokinase in case of k-72 and k-114. these indicated certain level of ability to block undesired processes of coagulation cloth formation and extracellular matrix degradation in relation to cardio-vascular diseases or metastasis formation, respectively. these results do not correlate with amount of estimated flavonols what could be explained by the in vitro effect of other active compounds – extractives from hop cones – bitter acids, terpens and others. it was published that the 8-prenylnaringenin (8-pn) as one component of hop extract inhibits platelet aggregation induced by thrombin (di vito bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:18 utc nova biotechnologica et chimica 12-2 (2013) 137 et al., 2012) but comparison to complex hop cones extract it is not possible, not published yet. fig. 4. inhibition activities on thrombin (ia_thrombin), urokinase (ia_urokinase), expressed by egcg equivalent in mg/g of native matter, further antioxidant activities by bclm method (aox_bclm) and by hpe method (aox_hpt), expressed as trolox equivalent in mg/g of native matter for six different hop genotypes. all tested categories are on the level of statistical significance with p<0,1 to control. opposite this, antioxidant of hop, hop extract, hop cones were published relatively frequently (lermusieau et al., 2001; yamaguchi et al., 2009; tronina et al., 2013), but differences in applied methods by various labs does not allow systematic comparison. similarly, level of the antioxidant activity is relative high, but intensively varied between tested samples, the highest for genotype k-114 and the lowest level has been observed for genotype k-71, what indicates rough correlation to flavonoid content. concerning to obtained results flavonoids seem to be major compounds of hop cone extracts responsible for antioxidant effect by both used methods. image about biological activity of hop cones extract was completed by test of antimicrobial activity towards g+ bacterial species staphylococcus aureus, gbacterial species pseudomonas aeruginosa and escherichia coli, yeast candida albicans and two cereal fungal pathogens fusarium graminearum. and pyrenophora teres. the results presents following table 4. from the results it is evident high level of antimicrobial activity to cereal pathogen pyrenophora teres, and relatively low activity to clinical bacterial pathogens and yeast with myceliar form candida albicans. from the comparison point of view between tested hop cones extract samples are statistically not significant differences. in fact, several papers published antibacterial effect of hop extracts (natarajan et al., 2008; leite et al. 2013) but certain level of antibacterial effect could be explained by flavonols too (waage and hedin, 1985; sivasothy et al., 2013). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:18 utc 138 maliarová, m. et al. generally we can conclude that hop cones and preparations from them are valuable raw material with significant biological activities expressed in vitro. table 4. antimicrobial activities of six hop cones extract samples to selected microbial species. antimicrobial activity mic (titer) genotype lučan zlatan k-31 k-70 k-71 k-72 k-114 staphylococcus aureus 20 10 10 10 10 10 10 pseudomonas aeruginosa 10 10 10 10 10 10 10 escherichia coli 20 10 20 20 20 20 10 candida albicans 40 10 10 20 20 20 20 fusarium graminearum 10 10 10 10 10 10 10 pyrenophora teres 80 160 20 20 80 160 20 4. conclusions the rp-hplc method reported here represents a simple and rapid technique for simultaneous determination of important flavonol aglycones as quercetin, kaempferol and isorhamnetin contained in the hop cone extracts. for seven tested saaz clones, this is the first report regarding the conditions of the hplc determination of flavonols. the highest content of quercetin and kaempferol was determined in oswald's clone 114. in general, obtained results indicate surprisingly high degree of biological activities expressed in vitro and finally conclusion that hop cones are valuable material for other applications, others than beer production. acknowledgements: this work was realized under support of the project vega 1-0233-12 and vega 11188-12. the authors are thankful to prof. ing. jan mocak, drsc.† for this valuable discussion an encouragement in this research direction. references alekseeva, m.a., éller, k.i., arzamastsev, a.p.: determining polyphenolic components of common hop by reversed – phase hplc. pharm. chem. j. 38, 2004, 687 – 689. di vito, c., bertoni, a., nalin, m., sampietro, s., zanfa, m., sinigaglia, f.: the phytoestrogen 8-prenylnaringenin inhibits agonistdependent activation of human platelets. biochim. biophys. acta. 1820, 2012, 1724-1733. erlanger, b.f., kokowsky, m., cohen, w.: the preparation and properties of two new chromogenic substrates for 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j. ethnopharmacol. 116, 2008, 383 – 396. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:18 utc nova biotechnol chim (2022) 21(2): e1319 doi: 10.36547/nbc.1319 1 nova biotechnologica et chimica evaluation of extracellular cellulolytic potential of selected natural strains of a novel fungus sordaria fimicola isolated from evolutionary canyon under submerged fermentation wajiha zaka ansari1,2,, asifa irshad kayani3, amjid khan2, amir muhammad khan2, saman shahid1, zainab shahzadi1 and hanan mukhtar1 1department of botany, lahore college for women university, near wapda flats, jail rd, jubilee town, lahore 54000, punjab, pakistan 2department of botany, university of mianwali, mianwali, university rd mianwali 42200, punjab, pakistan 3department of botany, kinnaird college for women, 93 jail rd, g.o.r. i, lahore 54000, punjab, pakistan  corresponding author: wajihazaka44@gmail.com article info article history: received: 10th december 2021 accepted: 24th march 2022 keywords: cellulase enzyme activity evolutionary canyon optimization purification submerged fermentation abstract industrial biotechnology has a great emerging demand and sustainable expansion for mankind to utilize a variety of biodegradable material for the production of various alternative energy resources such as biogas and bioethanol. researchers are interested in exploitation of novel fungal strains for the production of extracellular cellulase from the last few decades. this study was designed to assess the extracellular cellulase production potential of novel fungal strains of sordaria fimicola first isolated from the evolution canyon, three located on south facing slope with xeric (s1, s2, s3) and other three on north facing slope with mesic (n5, n6, n7) environmental conditions. based on initial and secondary screening two hyper producer strains from each slope were selected. the best activity for s2 was 3.125 u/ml and n6 exhibited 2.829 u/ml under optimized conditions of 14 d of incubation at 30 oc, ph 6.0, 1 ml inoculum and with 2% substrate (carboxy-methyl cellulose) concentration. among the tested carbon and nitrogen sources, glucose proved to be best for both strains with s2 exhibiting maximum activity. peptone and beef extract proved to be the best nitrogen sources for s2 and n6 respectively. the cellulase after characterization for temperature and ph showed slightly thermophilic nature. the cellulase was partial purification the highest cellulase activities as surviving in more xeric conditions which contribute more resistive and productive features in those microorganisms living in the harsh environment. introduction extracellular cellulases are the most important group of enzymes that hydrolyze lignocellulosic biomass to glucose subunits. cellulases are composed of three main components: exoglucanases, endoglucanases, and βglucosidases (behera and ray 2016). hydrolytic cellulase converts lignocelluloses to sugars by the process of fermentation. cellulose is composed of long polymers of β 1-4, linked glucose units and forms a proper crystalline structure. cellulose, hemicellulose, and lignin are hydrolyzed by the complete action of extracellular cellulase (fang and xia 2015; hansen et al. 2015). enzymes isolated from microorganisms are gaining much attention due to their potential for various mailto:wajihazaka44@gmail.com nova biotechnol chim (2022) 21(2): e1319 2 industrial applications. industrially, there are in great demand as compared to intracellular enzymes because of their cost-effectiveness as well as their high stability (adrio and demain 2014). many researchers are always interested in exploring the new potential fungal strains for the enhancement of cellulase production to complete the industrial enzyme demand in future. the enzymes isolated from the fungi are generally regarded as safe (gras) as compared to bacteria because they are cheap, easy to culture and more economical (kumar 2020). the production of biofuel and bioethanol from lignocellulosic material is a potential application of cellulase which is widely used in internal combustion engines as a good substituent for gasoline (kuhad et al. 2011; gusakov and sinitsyn 2012). sordaria fimicola is a well-known homothallic coprophilous fungus that regulates the nutrients in the herbivore dung. it was first isolated from the evolutionary canyon of israel. evolutionary canyon is considered as the model microsite to study the environmental changes and the adaptive behavior of the species (pavlíček et al. 2008). a dramatic biotic contrast is displayed by the two opposite slopes which are 100 – 400 m apart from each other. african slope or south facing slope (sfs) exhibit more tropical conditions due to high solar radiation, high temperature, and more xeric conditions, more fluctuating and more heterogeneous, as they have savannah-like biota. on the contrary, the european slope or the nort facing slope (nfs) display a temperate environment, mesic conditions, lush green vegetation and maquis live-oak brushwood (nevo 2006; arif et al. 2017). s. fimicola is commonly found in animal dung and rotting vegetation, also present on wood, seed, and soil because of its slightly thermophilic nature. they are considered as the important control agents and a potential source to produce enzymes and antibiotics (lamb et al. 1998). the fermentation technology used has a significant impact on the success of cellulase production. a variety of fermentation techniques are available for cellulase production; however, the most used technologies are submerged and solid-state fermentation (sajith et al. 2016). initially enzymes were obtained from submerged fermentation due to easy handling and greater control of various physical and chemical environmental factors (ahmed et al. 2017). on the contrary, ssf decreases the cost of enzyme production and improves the yield (hareesh et al. 2016). but ssf is labor intensive and needs a longer lag time, large inoculum size due to which the current research was conducted on submerged fermentation (gowthamana et al. 2001). the aim of this report is to study the extracellular cellulase production from sordaria fimicola under smf (submerged fermentation) by optimizing various physical and chemical parameters, characterization for ph and thermo-stability and partial purification by salting out and dialysis. experimental fungal sample collection and sub-culturing six different strains (s1, s2, s3, n5, n6, and n7) of sordaria fimicola were collected from fungal culture bank, university of the punjab, lahore, pakistan. all the samples were made identified and stored at 4 oc before use. after sample collection, all the strains were sub-cultured immediately on potato dextrose agar. fungal strains were inoculated in form of small discs in triplicates. all the cultures were incubated at 25 oc in incubator. screening of fungal samples for extracellular cellulase primary screening of cellulase was carried after the growth of all strains of sordaria fimicola. the media provided for growth was prepared by adding 2 % carboxy-methyl cellulose as a substrate with potato dextrose agar and incubated with ph 5.5 at 25 oc for ten days. after ten days of incubation, all the plates were flooded with congo red stain for 15 – 30 min. the plates were washed with distilled water and then 1m nacl solution was added in all petri plates for 15 – 20 min. the zone of cellulase hydrolysis was clearly appeared around each colony of fungus. the zone of hydrolysis was measured in cm (khokhar et al. 2012; pavani et al. 2013). nova biotechnol chim (2022) 21(2): e1319 3 production of extracellular cellulase under submerged fermentation (smf) potato dextrose broth was supplemented with 2 % cmc as substrate was used for cellulase production in submerged fermentation. fungal isolates were inoculated in a 100 ml shake flask with 25 ml of cellulase production medium (pdb and 2 % cmc). all the fungal isolates were inoculated with 1 ml of fungal spore suspension containing 105 spores. all the flasks were incubated in shaking incubator for 14 – 21 d in triplicates at 30 oc with the ph 5.0. the samples were withdrawn at regular intervals for determination of enzyme activity through spectrophotometer at 540 nm (sajith et al. 2016). optimization of fermentation cultural conditions and extracellular cellulase production different parameters were taken for the production optimization of extracellular cellulase such as temperature (20 oc, 30 oc, and 40 oc), ph (4.0, 5.0, and 6.0), incubation period (7, 14, and 21 days), inoculum concentration (spore suspension of 0.5 ml, 1 ml, 1.5 ml, 2 ml, 2.5 ml, and 3 ml), substrate concentration (0.5 %, 1 %, 2 %, 3 %, and 4 % cmc), carbon sources (glucose, maltose and lactose) and nitrogen source such as peptone, beef extract and ammonium sulphate). all the experiments were done in triplicates and values are with ± standard deviation (okoye et al. 2013; saini et al. 2017). crude enzyme extraction after optimization with each cultural condition, 25 ml of filtrate from all the samples was collected and centrifugation was performed at 6,000 rpm for 15 min. the supernatant was taken as crude enzyme and further used for enzyme assay. biomass of mycelia was also calculated (karthikeyan et al. 2011). cellulase activity by carboxy-methyl cellulase (cmcase) assay dns method was used for the determination of enzyme activity (miller 1959). in this method, during the standard reaction conditions, 1 ml of crude enzyme was incubated with 1 ml of 1 % cmc as a substrate in 0.1m sodium acetate buffer for 30 min at 40 oc in water bath. the reaction was stopped by adding 3 ml dns (dinitrosalicylic acid) reagent. optical density was measured at 540 nm by spectrophotometer. one cellulase unit was determined as the amount of enzyme that releases 1 micromoles of glucose per minute (martins et al. 2008). total protein estimation bradford protein assay method was used to determine the protein concentration for all samples of fungus in the crude enzyme. 1ml of crude enzyme was reacted with 2 ml of bradford reagent and the mixture was kept at room temperature for 45 min. total protein of crude enzyme after optimization with each parameter was calculated at the absorbance of 595 nm (bradford 1976). characterization of crude enzyme the cellulase enzyme produced under optimized condition was characterized to check the optimum ranges of ph (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0) and temperature stability (30, 35, 40, 45, 50, 55, and 60 oc) (hamdan and jasim 2018). partial purification of crude cellulose the crude enzyme was partially purified by salting out with ammonium sulfate and dialysis. the enzyme collected after salting out and dialysis was assessed for protein estimation and enzyme activity to check the folds of purification. salting out was carried out by taking 20 ml of crude enzyme and brought to 80 % saturation with the solid ammonium sulfate. the mixture was placed overnight at 4 oc in a magnetic stirrer. centrifugation of the mixture was performed for 15 minutes at 6,000 – 8,000 rpm. the pellet formed was dissolved in 50 mm sodium acetate buffer having ph of 5.5. after salting out, the enzyme was subjected to determine the enzyme activity (elakkiya and nova biotechnol chim (2022) 21(2): e1319 2 muralikrishnan 2014). the enzyme collected after ammonium sulfate precipitation was dialyzed in a dialyzing bag. 8 ml of enzyme was dialyzed against 30 mm sodium acetate buffer with three different changes of buffer. the enzyme collected after dialysis was assessed for protein estimation and enzyme activity to check the folds of purification (hafiz et al. 2011). statistical analysis all the experimental work was carried out in triplicates. for statistical analyses of data statistix program, version 8.1 was used, and data were analyzed for analysis of variance (anova) using lsd p ≤ 0.05. mean values were separated (p ≤ 0.05) and represented by different letters both in tables and figures along with ± standard error. results and discussion primary screening of extracellular cellulase from fungal samples the cellulolytic potential of fungal strains was observed for the primary screening of cellulases by congo red method. a clear zone of hydrolysis was observed around each colony (fig. 1). all the fungal strains show extracellular cellulose activity. among s strains, s2 was observed as the hyper-producer and show the maximum zone of hydrolysis around its colony which was 4.81 cm. in the case of n strains, n6 shows the maximum zone of hydrolysis measured was 4.52 cm. it was concluded that both the strains have the potentiality for cellulase production. the results are similar with the findings of el-nahrawy et al. (2017), they selected twenty isolates of aspergillus tubingensis. all the isolates showed zone of hydrolysis ranges from 1.14 – 4.1 cm. the highest zone of hydrolysis was produced by f7 which was 4.1 cm. fig. 1. clear zone of hydrolysis (cm) shown by six different strains of s. fimicola in pda containing cmc. 4 nova biotechnol chim (2022) 21(2): e1319 3 secondary screening of six different fungal strains for extracellular cellulases under submerged fermentation submerged fermentation was chosen rather than solid state fermentation as the liquid medium is easy to handle and does not require extra labor work as in case of solid-state fermentation. initially, all the strains were cultured in broth for fermentation. it was carried for only confirmation of results which were achieved by the primary screening of cellulase enzyme. the results showed in (fig. 2) that among all the natural strains of s. fimicola, only s2 and n6 were observed as the hyper-producer celluloytic strains and the enzyme activities of s2 and n6 were 0.875 u/ml and 0.825 u/ml respectively and were selected for further investigations. submerged fermentation is an aerobic fermentation process that provides a long culture period. during submerged fermentation adequate oxygen, cell growth and metabolism are maintained (gomathi et al. 2012; jiang et al. 2013). fig. 2. secondary screening for extracellular cellulase production from six different strains of s. fimicola by smf. means followed by different letters are significantly different according to anova, lsd at p ≤ 0.05 (least significant difference). optimization of cultural conditions for cellulase production under submerged fermentation by two selected strains of s. fimicola the enzyme production was optimized under different parameters such as ph, temperature, time of incubation, inoculum size, substrate concentration, carbon, and nitrogen sources. effect of different temperature on enzyme activity temperature is a crucial factor for the growth of microorganisms. results showed (fig. 3a) that the optimum temperature was 30 oc for cellulase production in submerged fermentation for both the strains. s2 revealed maximum enzyme activity (0.95 u/ml) as compared to n6 (0.8 u/ml) because s2 survive in harsh conditions which will ultimately contribute to high yield. the results of the current study confirmed that s. fimicola is slightly thermophilic in nature. findings of this study agree with irfan et al. (2016) and kumar et al. (2012) who reported 30 oc optimum temperature for endoglucanase production. higher temperature ranges from 50 to 60 oc causes denaturation of the enzyme. the optimum temperature for cellulase production also depends on the strain variation (murao et al. 1988). effect of different ph on enzyme activity the effect of ph was determined for cellulase production, and the highest enzyme activity was shown by s2 (1.175 u/ml) and n6 (1.025 u/ ml) at ph 6.0 (fig. 3b). among s2 and n6, s2 revealed the highest enzyme production than that of n6. similar findings were reported that initial medium ph of 6 is suitable for maximum cellulase production (juhasz et al 2004; akinola et al 2012). extreme low and high ph cause instability of enzyme as they are protein which generally denatured at very extreme ph (nidetzky et al. 1998). effect of incubation period on enzyme activity the incubation period was determined by checking the enzyme activity from 3 to 21 d. the optimum production for both the strains showed the ideal incubation period was 14 d (fig. 3c). s2 showed more enzyme activity (1.15 u/ ml) as compared to n6 (0.975 u/ ml) after 14 days of incubation. according to el-hadi at al. (2014), the ideal incubation period for production of cmcase from aspergillus hortai was 96 h under submerged fermentation. but in case of s. fimicola, it completes its life cycle in 10 d of incubation. 5 nova biotechnol chim (2022) 21(2): e1319 3 therefore, the incubation of s. fimicola is different from a. hortai to produce extracellular cellulase enzyme. fig. 3. optimization of cultural conditions (a) effect of different temperature, (b) effect of different ph, (c) effect of different incubation period, (d) effect of different inoculum concentrations, (e) effect of different substrate (cmc) concentrations (f) effect of carbon sources (g) effect of nitrogen sources. means followed by different letters are significantly different according to anova, lsd at p ≤ 0.05 (least significant difference). 6 nova biotechnol chim (2022) 21(2): e1319 3 effect of inoculum concentration on enzyme activity inoculum of 1ml in both strains of fungus yields more enzyme production. s2 showed more enzyme activity as 1.175 u/ml as compared to n6 as 0.9 u/ml (fig. 3d). enzyme activities of both strains decrease until 3ml of inoculum due to the decrease in microbial growth. microbial growth decreases due to competition for space and nutrient among the cells which causes nutrient depletion (omojasola et al. 2008). decreased inoculum size may not be enough for the growth initiation of the species which directly affects the enzyme production. irfan et al. (2011) reported that aspergillus niger showed highest cmcase production with 3 % inoculum level. effect of substrate concentration on enzyme activity cellulase activity was observed at different substrate concentrations (fig. 3e) and it was observed that 2 % cmc act as the ideal substrate concentration for both the strains of s. fimicola for the highest production of extracellular cellulase. s2 showed maximum enzyme activity (1.174 u/ml) than that of n6 (0.983 u/ ml) at 2 % substrate concentration. similar results were reported from the findings of irfan et al. (2010). it reduces after a certain percentage of the substrate due to extra availability of nutrients and increased biomass and as a result, minimization in the metabolic activity. supplementation of excessive substrate concentration causes high viscosity and decreases the probability of the substrate to bind with the active side of the cellulase enzyme (nagah et al. 2016). effect of carbon source on enzyme activity the effect of carbon sources was studied by adding 2 % of three different carbon sources namely, sucrose, glucose, and lactose into the culture medium. among these the glucose proved to be the best carbon source (fig. 3f) resulted in producing high yield for the enzyme in s2 (1.296 u/ ml) and for n6 (1.042 u/ ml). the other two carbon sources cause a reduction in enzyme activity. similar results were obtained by the findings of nathan at al. (2014) that only the glucose results in maximum cellulase yield as compared to the other carbon sources. irfan et al. (2016) reported that fermentation medium supplemented with glucose results in high yielded cellulases from trichoderma harzianum. effect of nitrogen source on enzyme activity effect of nitrogen source on cellulase activity revealed (fig. 3g) that highest production of cellulase was attained by adding peptone in culture medium in case of s2 (1.241 u/ml) and beef extract in case of n6 (0.978 u/ml). ammonium sulfate is considered as the lowest enzyme-producing nitrogen source in case of both the fungal strains. the results are in accordance with the work of acharya et al. (2008) who reported that the maximum yield of cellulase is produced by adding organic solvents such as peptone and yeast extract in the medium. prasanna et al. (2016) and kathiresan and manivannan (2010) investigated that penicillium sp produced maximum cellulase enzyme when cultivated in the liquid broth containing yeast extract. characterization of cellulase crude cellulase enzyme produced under optimized conditions was also characterized for ph and temperature stability. the results revealed that it has the ph ranged from 4.5 to 5.5 (fig. 4a) and temperature optima ranged from 40 to 50oc (fig. 4b). several studies have reported that endoglucanases remain active at the temperature ranges from 50 – 70 oc (bai et al. 2013; boonchuay et al. 2016). for example, endoglucanase from a. niger z10 shows thermal stability at 40 oc (coral et al. 2002). karboune et al. (2008) reported the optimum temperature of 65 oc for cmcase from penicillium funiculsoum. the thermostability and ph stability of cellulase production also depend upon the strain variation of the microorganism. rahnama et al. (2016) revealed that trichoderma harzinum has maximum enzyme stability at ph of 4.5. picart et al. (2007) reported the optimum ph of 4.5 for 7 nova biotechnol chim (2022) 21(2): e1319 2 carboxymethyl cellulase from penicillium sp. 1.68 2.25 2.93 3.03 2.53 2.39 1.83 1.42 1.73 2.64 2.32 2.16 1.83 1.62 0 1 2 3 4 5 6 30 35 40 45 50 55 60 e n z y m e a c ti vi ty (u /m l) temperature oc n6 s2 b fig. 4. characterization of cellulose. (a) enzyme stability at different ph. (b) enzyme stability at different temperature. means are significantly different according to anova, lsd at p ≤ 0.05 (least significant difference). partial purification of crude cellulase cellulase produced under optimized cultural conditions was partially purified. from the results (table 1), it was observed that the purification fold increases 2.7 folds in case of s2 and 1.5 folds in n6 with yield 80.2 % and 79 % respectively after dialysis. jabbar et al. (2008) purified endoglucanases from gymnoascella citrina by gel filtration chromatography and the result revealed that purification fold increases 27.3 folds with 25.5 % yield. sulyman at al. (2020) confirmed that the purification of cellulase from aspergillus niger increases 68.1 folds when crude enzyme was treated with sephadex g-100 gel filtration. table 1. different enzyme activities and specific activities of crude enzyme, enzyme after partial purification were estimated and their folds of purification were also determined. purification step enzyme activity [u/ml] specific activity [u/mg] yield [%] purification folds s2 n6 s2 n6 s2 n6 s2 n6 crude enzyme 3.03 1.88 1.07 1.16 100 100 1 1 salting out 2.78 1.67 2.32 1.39 91 88 2.2 1.2 dialysis 2.43 1.49 2.86 1.72 80.2 79 2.7 1.5 conclusion the researchers are interested in isolating novel fungal strains and exploring their potential to produce extracellular enzymes at a low cost to complete the industrial enzyme demand in future. the conducted research study demonstrates the first-time production of cellulase enzyme from s. fimicola. it was accomplished that s2 strain resulted in maximum cellulose production under optimized conditions by using submerged fermentation technique because it developed greater resistivity due to more harsh conditions. the biomass of both strains increased up to 14 days on incubation. after that, it starts decreasing due to nutrient depletion. the enzyme shows a slightly thermophilic and acidic nature when characterized for ph stability and thermostability. the partially purified enzyme shows purification folds greater than that of crude enzyme. the results showed that s. fimicola is a good potential fungal strain and can be exploited on an industrial scale to production second8 nova biotechnol chim (2022) 21(2): e1319 3 generation biofuel by degrading agro-industrial wastes or lignocellulosic biomass at a low cost. conflict of interest the authors declare that they have no conflict of interest. references acharya pb, acharya dk, modi ha (2008) optimization for cellulase production by aspergillus niger using saw dust as substrate. afr. j. biotechnol. 7: 4147-4175. adrio jl, 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with the regeneration of protoplasts. we found that protoplasts are formed directly in cultivation media under submerged conditions in the presence of lytic enzyme. actinophage μ1/6 endolysin and lysozyme were used in this study. streptomyces strains were cultivated in several media with glycine and lytic enzyme for 24 and 48h. the highest amounts of protoplasts (about 3 x 107 cfu/ml of cultivation medium) together with the highest regeneration (95%) and transformation frequency (about 2 x 106 – 107 cfu/µg dna) were obtained reproducibly in yeme medium with high sucrose content. s. aureofaciens b96, as hardly transformable strain because of difficulties with protoplast preparation and their further regeneration, was used in this study. the same procedure was applied to s. lividans 66 tk24 and s. coelicolor a3(2), streptomycetes model strains, to confirm the general use of this method. moreover, such cultivation process was appropriate for additional quick isolation of either chromosomal as well as plasmid dna that could be further used in recombinant dna techniques. keywords: streptomyces, protoplasts, transformation, lysozyme, actinophage µ1/6 endolysin 1. introduction streptomyces are unusual among bacteria in growing as mycelial colonies with sporulating aerial hyphae. they are known for degradation of numerous macromolecules and synthesis of a wide range of antibiotics and other commercially important secondary metabolites. the complex life cycle of these bacteria includes formation of substrate mycelia, aerial hyphae and spores (pigac and schrempf 1995). the study of the genetics of streptomyces is important not only because of many antibiotics, but also because its differentiation and its regulation of secondary metabolism are of basic interest (ochi 1982). the preparation and regeneration of protoplasts are major steps following genetic manipulations such as fusion, transfection and transformation of streptomyces species. despite there have been reports of “natural” transformation and transfection of streptomyces cultures, there is no such system generally applicable to most species (kieser et al. 2000). besides transfection and transformation, there have been further means of plasmid transfer described. intergeneric conjugation, using escherichia coli as a donor, allows constructing and manipulating recombinant plasmids in e. coli and subsequently transferring them into streptomyces (paranthaman and 36 brnáková, z. et al. dharmalingam 2003; hou et al. 2008). further alternative to using chemicals for promoting an uptake of dna by cells is electroporation. protoplasts of s. venezuelae and some other streptomycetes, formerly not transformable by the standard protocol, were successfully transformed by electroporation (pigac and schrempf 1995). in addition, protoplast fusion is widely used for genome shuffling among interspecies and intergenetic microorganisms (imada et al. 2002; el-gendy et al. 2008; xu et al. 2008). though, the most of aforementioned methods require preparation of protoplasts and their successful regeneration. it has been found that the regeneration frequency of the protoplasts varies according to the species used and high regeneration frequency is limited to a narrow range of streptomyces species (shirahama et al. 1981, imada et al. 2002). for the further study of genes in various streptomyces it was necessary to develop procedures for efficient protoplast regeneration and plasmid transformation remembering also the potential presence of the restriction-modification system (matsushima and baltz 1985; matsushima et al. 1987; godány et al. 1991; muchová et al. 1991). the aim of this study was to try the effect of lytic enzymes, mainly actinophage μ1/6 endolysin, on protoplast preparation during cultivation under submerged conditions. up to now, this kind of procedure has not been reported. this work describes an easy and effective method for preparation of protoplasts from streptomyces species, mainly streptomyces aureofaciens b96, by cultivation in liquid medium containing lysozyme or actinophage μ1/6 endolysin. furthermore, cultivation with lytic enzyme facilitates the isolation of chromosomal and plasmid dna. 2. materials and methods 2.1 bacterial strains and plasmids streptomyces strains used in this study: s. aureofaciens b96 (collection of microorganisms, institute of molecular biology sas bratislava); s. coelicolor a3(2), and s. lividans 66 tk24 (hopwood et al. 1985). shuttle promoter-probe vector pkj2 (nazarov et al. 1990) was used as a control for transformation of protoplasts. 2.2 culture conditions and solutions streptomyces sp. were cultivated in tsb (tryptone soya broth powder, oxoid; containing 0.7% glycine), tssb (tsb + 10.3% sucrose, 0.7% glycine, serva), nb (nutrient broth no. 2, imuna šarišské michaľany; containing 0.7% glycine), nbs (nb + 10.3% sucrose and 0.7%glycine), yeme (prepared according to kieser et al. 2000, containing 0.7% glycine), sterile p buffer (prepared according to kieser et al. 2000) and 25% peg 1000 in t buffer (prepared according to kieser et al. 2000), lysozyme water solution (applichem, 40mg/ml, filter sterilized), actinophage μ1/6 endolysin (farkašovská et al. 2003, 5mg/ml), appropriately dried plates no. 16m (10.3% sucrose, 1.5% dextrin, 0.001% urea, 0.005% nacl, 0.005% k2hpo4, nova biotechnologica 8-1 (2008) 37 0.005% mgso4, 0.5% peptone, 0.1% beef extract, 2ml/l trace element solution (kieser et al. 2000) and 3% agar, ph 7.2). solutions used further in this study: thiostrepton (calbiochem, 50mg/ml in dmso), te buffer (10mm tris-hcl, ph 8, 1mm edta), neutral phenol-chloroform (1:1, equilibrated with te buffer, ph 8), acid phenol-chloroform (1:1, equilibrated with water), 3m sodium acetate ph 4.8. 2.3 growth of streptomyces mycelium for protoplasts preparation 10 ml of sterile media containing lysozyme (serva, final concentration 1mg/ml and 2mg/ml, respectively) or actinophage μ1/6 endolysin (final concentration 0.15mg/ml and 0.3mg/ml, respectively) in 100ml erlenmeyer flasks were inoculated with 200μl of dense spore suspension (prepared according to kieser et al. 2000). the lytic enzyme (lysozyme or endolysin) was added either immediately with inoculum or after 8h of incubation. flasks were incubated at 30°c on rotary shaker for 24 and 48 hours while testing the suitable medium for protoplast preparation. in the next step, 10 ml of tsb with glycine, but without lytic enzyme, was inoculated with 1ml of dense spore suspension. the flasks were incubated at 30°c on rotary shaker for 20 – 24 hours. after that, 1x106 of colony forming units (cfu) were used as inoculum to 25 ml of yeme medium with lytic enzyme (apart from the control flask) and incubated for 24 and 48 hours. the culture medium was poured into screw cap bottle and centrifuged. the supernatant was discarded and the pellet was suspended in 10 ml of p buffer. suspension was filtered through cotton wool (using a filter tube) and transferred into a plastic tube. the protoplasts were collected by centrifugation at 2800 rpm. pellet was resuspended in 500μl of p buffer. 2.4 transformation of protoplasts transformation of protoplasts with pkj2 plasmid dna was done according to hopwood et al. (1985) rapid small-scale procedure and plated on 16m agar plates (regeneration medium, 10.3% sucrose, 1.5% dextrin, 0.001% urea, 0.005% nacl, 0.005% k2hpo4, 0.005% mgso4, 0.5% peptone, 0.1% beef extract, 2ml/l of trace element solution (hopwood et al. 1985) and 3% agar, ph 7.2). after 20h incubation at 30°c the agar plates were overlaid with 1ml of thiostrepton (300μg/ml water solution), dried and incubated at 30°c for 2 – 3 days. 2.5 isolation of streptomyces “total” dna 1ml of medium after 24 and 48h incubation was centrifuged and neutral phenolchloroform was added to supernatant. the extraction from phenol-chloroform was repeated until the white interface was seen. the dna was precipitated from supernatant by adding 1/10 volume of 3m sodium acetate and 1 volume of isopropanol. after incubation at room temperature and centrifugation, the pellet was 38 brnáková, z. et al. washed by 96% ethanol and dried slightly. then, the pellet was dissolved in 50μl of te buffer and the presence of isolated dna was confirmed by 0.9% agarose gel electrophoresis using ethidium bromide detection. 2.6 isolation of plasmid dna for this part of work, pkj2 transformants of s. aureofaciens, s. lividans and s. coelicolor were used as inoculum. 1ml of culture medium after 24 and 48h incubation was centrifuged and acid phenol-chloroform was added to supernatant. mixture was vortex mixed and centrifuged. the supernatant was removed, leaving the white interface behind. 1/10 volume of 3m unbuffered sodium acetate and 2 volumes of 96% ethanol was added to supernatant and mixed. mixture was incubated at –20°c and then centrifuged. the pellet was dissolved in 500μl of te buffer and the solution was extracted by neutral phenol-chloroform until no white interface was seen. 1/10 volume of 3m unbuffered sodium acetate and 2 volumes of 96% ethanol was added to supernatant and mixed. mixture was incubated at –20°c and then span. the pellet was dissolved in 25μl of te buffer, visualized in 0.9% agarose gel electrophoresis using ethidium bromide detection and 5μl of such solution was used for transformation into protoplasts. 3. results and discussion 3.1 protoplasts preparation polyethylene glycol (peg)-mediated plasmid transformation of protoplasts had allowed the rapid development of gene cloning in various streptomyces species, particularly in s. lividans 66, s. ambofaciens, s. coelicolor a3(2), s. fradiae, and s. rimosus, as well as in some others (pigac and schrempf, 1995). this transformation procedure has been generally applicable to several streptomyces species, although it has been necessary to optimize growth and establish the optimal conditions for protoplast formation and regeneration. moreover, the transformation of the fragile protoplasts has been tedious and frequently not reproducible; thus, numerous streptomyces strains could not be proven to be transformable. preparation of protoplasts is essential for further genetic manipulations, such as transformation, transfection (kieser et al. 2000), electroporation (pigac and schrempf, 1995) or protoplast fusion (xu et al. 2008), known so far. furthermore, there has been considerable interest in the use of the intergeneric conjugation as a means of plasmid transfer, using e. coli as a donor (voeykova et al. 1998; hou et al. 2008). despite all possibilities, transformation stayed the most applied method, and many problems with transformation and protoplast regeneration of streptomyces species still preserved. strain development and genetic analysis of several important streptomycetes has been hindered by the apparent lack of natural fertility, the lack of transduction or transformation, and by restriction-modification systems. restriction endonuclease activities, having role in the protection of the bacterial genome, can nova biotechnologica 8-1 (2008) 39 complicate genetic manipulation of the bacterium by affecting transformation efficiency and stability of recombinant dna (apichaisataienchote et al. 2005). several methods have been designed in an attempt to improve protoplasts preparation and regeneration in streptomyces, including glycine treatment of mycelia, use of mixture of lytic enzymes, and supplementation of metal ions and osmotic stabilizers (yang and lei, 2001). our studies showed that cells could be transformed at a high frequency when the recipient streptomycetes protoplasts are formed by treatment with lytic enzyme during the cultivation. in this work, s. aureofaciens b96, hardly transformable streptomyces strain, and model strains, s. coelicolor and s. lividans, were used for setting the conditions for protoplast preparation during the cultivation process. streptomyces under submerged conditions enter the log phase before 24h, stationary phase between 24 and 96 h, and declining phase after 96h of incubation. it was reported that streptomyces protoplast formation was high in the early stationary phase and decreased in the late stationary phase (kieser et al. 2000; yang and lei, 2001). in addition, protoplast formation also increased in the decline phase, likely due to the autolysis of mycelia and partial damage of the cell wall. rodicio et al. (1978) found that protoplasts were yielded in young mycelia treated with lysozyme, even when the glycine was not added during the growth period. however, protoplast formation in old mycelia required the presence of glycine during the cultivation. therefore, mycelium grown at the late log to the middle stationary phase treated with lysozyme was effective for protoplast preparation. in our study, glycine was used in all media as a factor increasing susceptibility to the action of agents degrading the cell wall. s. aureofaciens was cultivated in various media with addition of lytic enzyme. the numbers of formed protoplasts, counted in bürker counting chamber, are shown in tables 1 and 2. table1. effect of lytic enzymes on number of s. aureofaciens b96 protoplasts in various media. lytic enzymes were added to media after 8h of incubation. protoplast concentration is in cfu/ml of medium. lysozyme endolysin 1 mg/ml 2 mg/ml 0.15 mg/ml 0.3 mg/ml medium 24h 48h 24h 48h 24h 48h 24h 48h nb + gly -------- nbs + gly 2×106 2×106 --4×106 4×106 4×106 4×106 tsb + gly 4×106 2×106 4×106 2×106 8×106 8×106 8×106 8×106 tssb + gly 12×106 8×106 14×107 12×106 16×107 14×106 16×106 14×106 there were too few protoplasts formed, when the lytic enzyme was added immediately into the cultivation media (less than100 cfu/ml). more effectual results were when the lytic enzymes were added after 8h incubation of streptomyces culture (table 1; 2×106-1×107 cfu/ml). under these conditions, the best results were acquired in tssb medium (12–14×106 cfu/ml), where additional sucrose was added to form hypertonic environment. moreover, if nb and nbs media were compared, there were no protoplasts observed in media without any osmotic stabilizer. however, the formation of protoplasts was influenced by the composition of cultivation media. nb 40 brnáková, z. et al. is a complex cultivation medium and was not suitable for streptomyces aureofaciens. optimal protoplast formation was obtained by treatment of mycelium with lytic enzyme during cultivation in yeme medium (1000mm sucrose, table 2). however, s. aureofaciens grew with much difficulties in such hypertonic medium and needed to be pre-cultivated overnight in tsb medium, where its growth was very extensive. table 2. effect of lytic enzymes on number of s. aureofaciens b96 protoplasts in yeme medium. the inoculum was 20-24h fresh culture pre-cultivated in tsb+gly medium. protoplast concentration is in cfu/ml of medium. lysozyme endolysin time of cultivation 1 mg/ml 2 mg/ml 0.15 mg/ml 0.3 mg/ml 24h 24×106 26×106 29×106 30×106 48h 26×106 29×106 31×106 31×106 the concentrations of lytic enzymes were selected according to preliminary studies in our laboratory and related to lysozyme they were consistent with the results of kieser et al. (2000) standard protocols for protoplast preparation. mycelium was grown in yeme medium with glycine for 48 hours. for comparison, protoplasts were prepared also by procedure described in kieser et al. (2000) with 1mg/ml of lysozyme for s. lividans or s. coelicolor and 2mg/ml of lysozyme s. aureofaciens (table 2), respectively. the amounts of prepared protoplasts were comparable to those formed by cultivation directly with lytic enzyme in yeme medium (approximately 50×107 cfu/ml in the case of s. lividans or s. coelicolor, and 30×107 cfu/ml in the case of s. aureofaciens). the reproducible results from liquid cultivation with lytic enzymes are summarized in tables 2 and 3. there was not a significant difference among the final protoplast amounts, when the different concentrations of lytic enzyme were used directly in media during the cultivation process. in this study, even the lower concentration (1mg/ml of lysozyme and 0.15 mg/ml of actinophage endolysin) was enough for protoplast formation. table 3. effect of lytic enzymes on number of s. lividans 66 tk24 and s. coelicolor a3(2) protoplasts in yeme medium. protoplast concentration is in cfu/ml of medium. lysozyme (2 mg/ml) endolysin (0.3 mg/ml) time of cultivation s. lividans s. coelicolor s. lividans s. coelicolor 24h 48×106 45×106 50×106 51×106 48h 50×106 49×106 51×106 51×106 following the cultivation, protoplasts were centrifuged, washed by p-buffer and filtrated to remove possible mycelia. the important fact was not to dilute cultivation medium because of osmotic instability of protoplasts. after centrifugal concentration at 2800 rpm, protoplasts were ready for transformation. in this state, acquired amounts of s. aureofaciens protoplasts were 25×109/ml and 25×1010/ml, 5×1010/ml and 4×1011/ml in the case of 1mg/ml and 2mg/ml of lysozyme; 0.25mg/ml and 0.5mg/ml of endolysin used, respectively. protoplast regeneration frequency is estimated by the ratio of cell number to protoplast number. it was reported before, that the cell regeneration from protoplasts nova biotechnologica 8-1 (2008) 41 of streptomyces depended on the medium components, age of mycelia, dehydratation of plates and culture temperature. it was also found that the presence of non-protoplast mycelium fragments could affect the cellular regeneration from protoplasts (yang and lei, 2001). regeneration medium, no. 16m was suitable for all streptomyces strains tested in this study, and protoplasts were regenerated after 2-3 days of incubation at 30°c. the regeneration frequencies were about 95% for s. aureofaciens, 94.3% and 94% for s. lividans and s. coelicolor, respectively. but in general, because of the diversity of streptomyces, the composition of the regeneration media has to be optimized for each strain. 3.2 transformation of protoplasts in past years, polyethylene glycol (peg)-mediated plasmid transformation of protoplasts had allowed the rapid development of gene cloning in various streptomyces species. although this transformation procedure is generally applicable to several streptomyces species, it is necessary to optimize growth and establish the optimal conditions for protoplast formation and regeneration. moreover, the transformation of the fragile protoplasts is tedious and frequently not reproducible; thus, numerous streptomyces strains could not be proven to be transformable (mellouli et al. 2004). peg induces plasmid transformation in streptomyces species apparently by interacting with cytoplasmic membrane structure, thus allowing plasmid dna uptake (garcia-dominguez et al. 1987). protoplasts of s. aureofaciens b96, in this study, were transformed by pkj2 plasmid dna. thiostrepton resistance marker is well expressed in s. aureofaciens and suggested good selection of transformants based on the resistance to this antibiotic. when the protoplasts of s. aureofaciens were prepared following the method developed for s. lividans and s. coelicolor by hopwood et al. (1985), the extremely low initial transformation frequency with plasmid dna was observed (less than 100 transformants per µg dna). despite, the frequencies in s. lividans and s. coelicolor were between 106–107 cfu/µg of plasmid dna. alike in the case of media without or with 300mm sucrose, there was only little transformation frequency with plasmid dna (5-10%). the highest transformation efficiency was observed when s. aureofaciens was cultivated with lytic enzyme in yeme medium (1000mm sucrose, pre-cultivated in tsb medium with glycine). protoplast numbers after concentration were 25×109 cfu/ml and 25×1010 cfu/ml in the case of 1mg/ml and 2mg/ml of lysozyme; 5×1010/ml and 4×1011/ml when 0.25mg/ml and 0.5mg/ml of endolysin was used, respectively. not only the best results in protoplast formation were in such osmotic medium, but also the regeneration of the protoplasts was more than 95% and the transformation frequency was comparable to s. lividans and s. coelicolor used in this study (in all cases the transformation frequencies were reproducibly about 2×106 – 107 cfu/μg dna). these results corresponded to the results published previously for s. lividans and s. coelicolor (kieser et al. 2000) and the other streptomyces strains (shirahama et al. 1981; matsushima and baltz 1985; and more). there was an irrelevant difference in transformation frequency regarding to concentrations of lytic enzymes used in cultivation media and 42 brnáková, z. et al. the type of plasmid. neither the size nor the origin of the plasmid dna played the role in this study. the preliminary studies in our lab showed that transformation frequency did not depend on the size of the cloning vector (data not shown). despite s. aureofaciens b96 protoplasts were stable after storing at -70°c under any condition used, they were not transformable any more. they always had to be prepared fresh. opposite, s. lividans and s. coelicolor protoplasts, prepared in the same way as aforementioned s. aureofaciens, were stable at -70°c in p buffer for at least six months. 3.3 isolation of “total” and plasmid dna there are various procedures of streptomyces dna isolation (kieser et al. 2000). in general, the first step for dna isolation is the lysis of the cell walls. they require treatment with sds or sarkosyl, then incubation with lysozyme at 30° or 37°c with periodical trituration. this step takes usually long time and sometimes not all of the mycelia are lysed. furthermore, the regeneration and transformation ability of protoplasts depends on the way of preparation in many cases. all dna isolation methods in this study were simplified procedures according to kieser et al. (2000). they were not such time-consuming, as the step of cell wall lysis was excluded. visually, except for the yeme with high sucrose content, all media were viscous after cultivation with lytic enzyme. there were only a few protoplasts after cultivation in these media, so we assumed that the most of them broke and intracellular content with dna split into the medium. after that, no lysis was needed and the whole isolation was based only on extraction from phenol, phenol-chloroform and on alcohol precipitation. if hypertonic medium was used for cultivation, like yeme, it was better to dilute the medium either with water or 10.3% sucrose to let the protoplasts break and split inner content into the medium. it was also possible to isolate plasmid dna from such cultivation media. the only difference was the extraction from acid phenol-chloroform at first. consequently, the plasmid dnas were purified like total dna. plasmid dnas isolated by this procedure were able to transform repeatedly into streptomyces protoplasts. according to one’s needs, by addition of the lytic enzyme into medium during cultivation it is possible to acquire protoplasts with high regeneration and transformation frequency or isolate chromosomal and plasmid dna. this procedure is applicable to protoplast preparation from many streptomyces species, even from hardly transformable industrial strains, when high concentration of sucrose, as osmotic stabilizer is added into liquid medium. this method is easier, more effective and not so time-consuming than developing and optimizing new procedures. additionally, chromosomal dna and plasmid dna isolations are very easy and quick, as the cell lysis step is excluded. acknowledgements this work was supported by a grant from vega no. 2/0121/08. the work has been carried out in compliance with current laws governing genetic experimentation in the slovak republic. nova biotechnologica 8-1 (2008) 43 references apichaisataienchote, b., altenbuchner, j., 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pristamycin by genome shuffling. appl. microbiol. biotechnol., doi: 10.1007/s00253-008-1540-0. yang, s.-s., lei, c.-m.: formation and regeneration of protoplasts for protease production in streptomyces rimosus. j. microbiol. immunol. infect., 34, 2001, 8– 16. microsoft word kraic nb 9-2.doc nova biotechnologica 9-2 (2009) 217 determination and classification of pollutants in waste water filip kraic1, zuzana királyová2, ján mocák1,2 1department of chemistry, university of ss. cyril & methodius in trnava, j. herdu 2, trnava, sk-917 01, slovak republic (filip.kraic@ucm.sk) 2institute of analytical chemistry, slovak university of technology, bratislava, sk-81237, slovak republic abstract: this paper deals with waste waters produced by industrial producers during recent three years. its main purpose is to evaluate the data monitored from discharges of three leather plants where eight traditional variables (cod, bod, insoluble matters, ph, and the concentrations of ammonia, total nitrogen, chromium and sulphides) were regularly analyzed and quantified. chemometrical and statistical methods were approved as very useful tools for characterization and classification of various kinds of water samples considering the environmental and metrological aspects. for this purpose, multivariate (multidimensional) techniques of data analysis and correlation analysis were especially very useful. the mentioned techniques are used in this work to (1) reveal the concealed interrelations of the monitored characteristics of waste waters, (2) compare the results of individual waste water producers and find the most important polluting source during a long time period, and (3) derive some generally valid conclusions regarding the observed results. key words: waste water, pollutants, multivariate data analysis 1. introduction pollution of the rivers and seas jeopardizes production of oxygen in water. a severe pollution source is the water waste pollution. waste water is coming into environment as a consequence of the industrial and agricultural production and the town activities. it has an unhealthy influence on human and environment. contaminations present in waste water contain various inorganic and organic matters. the most important variables, which characterize the water quality and indicate the extent of pollution are the concentrations of chromium (coded cr in further text), total nitrogen (coded ntot), ammonium (nh4), and sulphide (s2). together with chemical oxygen demand, cod, (coded chsk according to the slovak designation), biological oxygen demand, bod5, (coded bsk5 according to the slovak designation), water acidity (ph), and amount of insoluble matters (im) they were regularly monitored at the output from three industrial sites in the same slovak town during the years 2006, 2007 and (partly) 2008, which has created the measurement basis for the performed research. due to their potential environmental danger, discharges from the leather plants have to be regularly monitored. the most dangerous environmental factor is here the content of chromium, which is very toxic in the oxidation state +vi, therefore most part of cr(vi) is trapped in the technological process. also further above mentioned factors must be regularly monitored not only by the plant laboratory but also by an independent accredited analytical laboratory. when the limiting values are exceeded, the level of the respective factor must be immediately properly adjusted. 218 kraic, f. et al. characterization and classification of various kinds of water samples from the environmental and metrological aspects was effectively performed using various chemometrical and statistical methods (vončina et al., 2007; šnuderl et al., 2007; kannel et al., 2007; kraic et al., 2008). therefore in this work several multidimensional (multivariate) techniques of data analysis were used. 2. material and methods 2.1 sampling the waste water samples originated from three relatively similar industrial production sites. the sampling was done once a week by the analysts from a nearby accredited laboratory where the required measurements were made. the following 8 variables were monitored and analyzed: chsk, bsk, ph, im, ntot, nh4, cr and s2 (their codes are explained above). the sampling procedures and laboratory analyses were described in detail in our previous papers (királyová et al., 2008a; 2008b). to prevent changes in waste water samples, the analyses started two hours after sampling, which was faciliated by closeness of the analytical laboratory to the monitoring sites. 2.2 analysis cod (chsk) was measured by the spectrophotometrical dichromate method; bod5 (bsk5) was determined after treatment of analyzed water with manganese dioxide by iodometric thiosulphate determination of the present mn(iv). the ph value was measured potentiometrically using a glass electrode. insoluble matters (im), separated on an appropriate filter and filtered off, were dried at 105 °c until the constant weight and determined gravimetrically. ammonia was distilled off from the analyzed water and determined spectrophotometrically using the nessler reagent. for the total nitrogen determination, inorganic and organic nitrogen was oxidized by persulphate and mineralized in sulphuric acid. the formed nitrate reacted with 2,6dimethylphenol (in sulphuric and phosphoric acid medium) and the resulted 4-nitro2,6-dimethylphenol was determined spectrophotometrically. chromium was selectively determined by inductively coupled plasma optical emission spectrometry. sulphide anion was determined by back iodometric titration using thiosulphate standard solution. 2.3 multidimensional data analysis statistical calculations were performed using the following techniques: correlation analysis, principal component analysis (pca), cluster analysis (ca) and linear discriminant analysis (lda). in the performed calculations three contemporary software commercial packages were used: stagraphics plus 5.1, spss 15.0 and sas jmp 7.0. nova biotechnologica 9-2 (2009) 219 3. results and discussion 3.1 correlation analysis the output of correlation analysis is the correlation table, which contains the pair (or pearson) correlation coefficients expressing the strength of correlation between all possible pairs of variables. tab. 1 shows the calculated correlation table. the entries of this table are symmetrical according to diagonal. the following conclusions can be derived from the correlation table: (a) the highest correlation is between chsk and bsk5. (b) very high correlations exist between chsk and ntot, bsk5 and ntot, as well as chsk and im. (c) considerably significant correlations (rcrit ≥ 0.258 at p ≤ 0.01) exist between im and ntot, bsk5 and im, im and ph, as well as nh4 and s2. (d) significant correlations (at the 95 % or higher probability level, p ≤ 0.05) exist between bsk5 and ph, as well as im and nh4 (where both dependences are inverse). all correlation coefficients in table 1, which are larger or equal than rcrit = 0.184 are marked by bold faces. (e) no significant correlation was proved in all other pairs. table 1. pearson correlation coefficients exhibiting the strength of correlation between individual pairs of variables variable chsk bsk5 im ph nh4 ntot cr s2 chsk 1 bsk5 0.7606 1 im 0.5255 0.3195 1 ph 0.0025 -0.2352 0.2890 1 nh4 0.0058 0.0389 -0.1923 0.0786 1 ntot 0.6386 0.5766 0.4338 0.1337 0.0284 1 cr -0.0473 -0.0326 0.1056 -0.0942 -0.0982 -0.0052 1 s2 0.0986 0.0329 0.1808 -0.1102 -0.2694 0.0155 0.0973 1 note: 81 studied objects, i.e. 27 averaged month values were measured at the discharge of three leather plants. critical values of the correlation coefficient (absolute values): rcrit = 0.144 (p = 0.1), rcrit = 0.184 (p = 0.05), and rcrit = 0.258 (p = 0.01) for n = 81. significant correlations are marked bold. 3.2 principal component analysis in principal component analysis, pca, some natural grouping of the objects (the waste water samples in this work) and the studied variables might be seen. the principal components, pcs, are calculated as the linear combinations of original variables (sharma 1996; khattree and naik 2000). according to the computed eigenvalues only three principal components (pcs) were found important; their value was larger than 1, which is usually considered as the criterion. three kinds of graphical outputs are used in the pca, namely the scatterplot showing the objects, the loadings plot showing the variables, and the biplot where all objects and variables are depicted together. the advantage of first two outputs is the possibility to obtain not only the 2d graph but also the 3d one, where usually the first two and three most important pcs are used as the axes, respectively. on the other hand, the biplot, even though plotted in two dimensions, provides more complex information about the studied problem. 220 kraic, f. et al. fig. 1 exhibits the biplot, which simultaneously represents the samples, depicted here by the numbers, and eight originally utilized chemical descriptors, depicted by the rays starting from the origin and ending at the point determining the variable position. the samples are here categorized according to the sampling site located at the discharge of the plants 1, 2 and 3, respectively. from the position of variables in the plane pc2 – pc1 the following outcomes can be deduced: (a) variables chsk (cod), bsk5 (bod5) and ntot provide similar information about the sampled waste water. this is well understandable especially for the first two variables since they are highly correlated; the possibility to convert mutually their values has already been discovered. (b) similarity (interdependence) of nh4 and ph proves the role of ammonia in changing the ph value of waste water. (c) the opposite position of cr with respect to ph testifies that they are antagonists; a high level of chromium is reached at lower ph, and vice versa. the achieved pca results are in accord with the described outputs of cluster analysis introduced in further text. fig. 1. biplot pc2 vs. pc1 for 8 measured chemical descriptors and 81 waste water samples coming out from the discharges of three leather plants (coded by numbers 1, 2 and 3). software spss 15.0. when looking at the sample positions in fig. 1, it can be concluded that high sulphide and cr concentration values are characteristic for the waste coming from the second plant. on the other side, high ph and nh4 values are characteristic mainly for the third and partly for the first plant. since the samples belonging to the plant 2 are localized at low (mostly negative) values of chsk, bsk and ntot, it can be concluded that samples from plant 2 exhibit generally low values of these variables. the nova biotechnologica 9-2 (2009) 221 occurrence of negative variable values in the pca is caused by the performed variable standardization, in which the corresponding mean is subtracted from the original variable values and the result is divided by the corresponding standard deviation (e.g. the zero value is achieved for the original mean value of the variable). 3.3 cluster analysis generally, the clustering process in cluster analysis may be performed either with objects or variables (khattree and naik 2000). clustering in this work was made concerns the studied eight variables. the result of the performed cluster analysis is a dendrogram depicted in fig. 2. the basis for the performed calculations were data on 81 objects representing 27 average month data measured at 3 sampling sites (discharges of three leather production plants) for each of eight investigated variables. in these calculations ward’s method of clustering and squared euclidean distance were used. in fig. 2 three clusters of closely related variables can be seen. the first cluster is formed by chsk, bsk, connected further to ntot and im. the second cluster is formed by ph and nh4, and the third cluster contains cr and s2. the variables forming the same cluster are most similar; the measure of their mutual similarity is given by distance, which represents the vertical axis of the dendrogram. the results of cluster analysis are in a good agreement with the outputs of correlation analysis and principal component analysis. fig. 2. cluster analysis of 8 variables measured in waste water analysis of 81 samples obtained during 27 months (average values at 3 sampling sites). software statgraphics plus 5.1. chsk and bsk denote cod and bod5, respectively. 3.4 linear discriminant analysis linear discriminant analysis, lda, is a supervised learning method, in which the classification model is calculated using the data belonging to the training set where the 222 kraic, f. et al. categorization of all samples is known before the calculation starts. in the first step (training), the classification model is calculated, by which the studied objects (waste water samples in this case) are re-categorized so that the object (sample) category is either confirmed or the object is assigned to another category (sharma, 1996; vandeginste et al., 1998; khattree and naik, 2000). the success in classification is calculated by the ratio of the correctly classified objects to their total number. then, in the second step, the developed model is used for classification of the objects, which are not included into the training set. they belong either to the test data set, which is utilized for validation of the discriminant model, or the category of the investigated data is completely unknown so that the main goal of lda is prediction of the object category. table 2. evaluation of linear discriminant analysis classification of the waste water samples divided into categories given by the sampling location (plants 1, 2 and 3). predicted group membership evaluated data success by plant no. 1 2 3 total training set count 1 17 0 10 27 2 0 27 0 27 3 4 0 23 27 % 1 63.0b 0 37.0 100.0 2 0 100.0b 0 100.0 3 14.8 0 85.2b 100.0 cross-validateda count 1 14 0 13 27 2 0 26 1 27 3 6 0 21 27 % 1 51.9c 0 48.1 100.0 2 0 96.3c 3.7 100.0 3 22.2 0 77.8c 100.0 a in leave-one-out cross validation, each object was classified by the model derived from all objects other than that one. b in total 82.7 % of original grouped cases were correctly classified. c in total 75.3% of cross-validated grouped cases were correctly classified. in this work, the samples were separated into three categories, assigned by the plant releasing waste water. the classification results are summarized in table 2. the lda results testify that the waste water from plants 1 and 3 have a similar level of pollutants therefore the categorization of the samples from these two sources is often interchanged, e.g. 10 samples belonging to plant 3 were classified into group 1 (plant 1), as shown in the first line of the table. on the other hand, 100 % of the samples from plant 2 were classified correctly when the training set is considered and a 96.3 % success was obtained when performing leave-one-out validation. according to the position of the samples belonging to three plant discharges with respect to the first discriminant function expressing the overall pollutant concentration in the lda nova biotechnologica 9-2 (2009) 223 diagram (not shown here) it was proved that the worst pollutant is plant 1, the best cleaning procedures were applied in plant 2. an attempt was also made to see the seasonal effect on the waste water pollution in the lda output. therefore all data were divided into four categories according to the year seasons: spring (march, april, may), summer (june, july, august), autumn (september, october, november) and winter (december, january, february). the nominal categorical variable season was created with 4 classes spring, summer, autumn and winter and used as the discrimination criterion. the separation of the data by four seasons was not well demonstrated. however, the winter and autumn data were displayed at higher values of the first discriminant function, df1, which reflects the extent of pollution since df1 may be explained as the total concentration of the polluting substances. much more successful was seasonal categorization of the data into two parts: the warmer part of the year (spring and summer) and the colder part (autumn and winter). in this case the success of discrimination was 76.5 % for the training set and 66.7 % for the leave-one-out validation. in both cases the discrimination results are unambiguously significant. at the same time it means that in cold months the total level of pollution of the waste water from all three investigated plants is larger than in the year season when the temperature is higher. 4. conclusions the waste water characteristic parameters, namely cod (chsk), bod (bsk5), insoluble matters, ph, ammonium, total nitrogen, chromium, and sulphides were monitored at the discharge of three industrial plants of the same town during almost three years. the obtained analytical values were statistically evaluated and mutual relations and the trends of the individual waste water descriptors were discovered. in total, the most important source of pollution was the industrial plant number 1. with respect to the year seasons, more polluted waste waters are expected in cold months. acknowledgement: the support of this work by slovak grant agencies by means of grants vega 1/1005/09, vvce-0004-07 and apvv-0057-06 is highly acknowledged. references kannel, p.r., lee, s., kanel, s.r., khan, s.p.: chemometric application in classification and assessment of monitoring locations of an urban river system. anal. chim. acta, 582, 2007, 390-399. khattree, r., naik, d.n.: multivariate data reduction and discrimination. sas institute, cary, north carolina, usa, 2000. királyová, z., kraic, f., mocák, j.: study of pollutants in waste water. in: 28. proceedings of industrial toxicology 2008. tatranská štrba, slovakia, june 1820, 284-289. királyová, z., šnuderl, k., kraic, f., mocák, j.: determination and classification of pollutants in waste water. acta chim. slovaca, 1, 2008, 143-152. 224 kraic, f. et al. kraic, f., mocák, j., fiket, ž., kniewald, g.: icp ms analysis and classification of potable, spring, and mineral waters. chem. pap., 62, 2008, 445– 450. sharma, s.: applied multivariate techniques. wiley, new york, 1996. šnuderl, k., simonič, m., mocak, j., brodnjak-vončina, d.: multivariate data analysis of natural mineral waters. acta chim. slov., 54, 2007, 33-39. vandeginste, b.g.m., massart, d.l., buydens, l.m.c., de jong, s., lewi, p.j., smeyers-verbeke, j.: handbook of chemometrics and qualimetrics: part b, elsevier, amsterdam, 1998. vončina, e., brodnjak-vončina, d., mirkovič, n., novič, m.: chemometric characterisation of the quality of ground waters from different wells in slovenia. acta chim. slov., 54, 2007, 119-125. microsoft word riekkola-vanhanen 2010-1.doc nova biotechnologica 10-1 (2010) 7 talvivaara sotkamo mine – bioleaching of a polymetallic nickel ore in subarctic climate marja riekkola-vanhanen talvivaara mining company plc.,talvivaarantie 66, 88120 tuhkakylä, finland (marja.riekkola-vanhanen@talvivaara.com) abstract: the main activity of the talvivaara mining company plc. is the development and exploitation of the talvivaara deposits in sotkamo, finland using bioheapleaching. the talvivaara deposits comprise one of the largest known sulphide nickel resources in europe with 1004 million tonnes of ore, sufficient to support anticipated production for a minimum of 45 years. the mine started in late 2008 and will have an annual nickel output of approximately 50,000 tons when it reaches full production. in addition, the mine will also produce zinc (approximately 90,000 tpa), copper (approximately 15,000 tpa) and cobalt (approximately 1,800 tpa) as by-products of the process. the viability of bioheapleaching technology for the extraction of nickel has been demonstrated in a large on-site pilot trial using talvivaara ore. the three year pilot has shown that the leaching process also works well in the subarctic climatic conditions of eastern finland. keywords: bioheapleaching, nickel, zinc, copper, cobalt, talvivaara 1. introduction the talvivaara deposits are located in the southern part of the kainuu belt in eastern finland. they comprise two different polymetallic orebodies, which are located approximately three kilometres apart. the deposits are outcropping and relatively easy to mine. the mineral resources have been classified by australian jorc code with 0.07% ni cut-off at 1004 million tons, containing 0.23% of nickel, 0.51% of zinc, 0.13% of copper and 0.02% of cobalt. the black schist ore and the possible utilization of the deposits have been extensively studied for over 20 years. it was quickly apparent from the ore dressing tests that a usable nickel concentrate was not achievable due to the high graphite content of the ore. chemical and biological leaching options had to be considered as potential options (riekkola-vanhanen, 1999). talvivaara black schist ore contains pyrrhotite, pyrite, sphalerite, pentlandite, violarite, chalcopyrite and graphite. the main silica containing phases are quartz, mica, anorthite and microcline. in addition to nickel, zinc, copper and cobalt the ore contains about 0.3 % of manganese, 10 % of iron, 9 % of sulphur, 8 % of carbon and 50 % of sio2. the distribution of nickel in different sulphides is pentlandite 71 %, pyrrhotite 21 % and pyrite 8 %. the distribution of cobalt is pentlandite 11 %, pyrrhotite 26 % and pyrite 63 %. all copper is in chalcopyrite and zinc in sphalerite. 2. production process overview. the mining method at talvivaara is large scale open pit mining. materials handling covers all physical ore processing steps from the primary crusher to 8 riekkola-vanhanen, m. the heaps. after one and one-half years of bioleaching on the primary pad, the leached ore will be reclaimed, conveyed and restacked onto the secondary heap pad. after secondary leaching, the barren ore will remain permanently in the secondary heaps. in the metals recovery process, the metals will be precipitated from the pregnant leaching solution (pls) using hydrogen sulphide. the resulting products will be intermediates to be transported for further processing in refineries operated by the company’s customers. fig. 1. talvivaara process chart. open pit mining. annual ore production is approximately 22 million tons. sufficient areas to extend the pit will be prepared in subsequent years, normally a year prior to when mining is scheduled to commence. any overburden or moraine not required for road, perimeter wall or other construction, will be stockpiled for later use in rehabilitation. ore and waste are extracted using conventional large scale open pit drill and blast methods. a fleet of self-propelled diesel hydraulic track drill rigs are used. fragmentation by blasting is preferred to crushing because of lower costs. it is also beneficial to create more fracturing at blasting stage, and it increases the surface area and therefore improves leaching solution entry and consequently leaching efficiency. as a result, the drilling pattern and hole sizes are on average smaller than in mines with similar production rates. materials handling and bioheapleaching. materials handling in talvivaara cover all the physical ore processing steps from the primary crusher to the final locations on the secondary heaps. during the process, the ore is crushed and screened in four stages. after primary crushing, the ore is conveyed to the following crushing stages nova biotechnologica 10-1 (2010) 9 and screening and is agglomerated for bioheapleaching. at this stage of leaching no inoculum from a bacteria farm is added. agglomeration takes place in a rotating drum, where pls is added to the ore in order to consolidate the fine ore particles with coarser ore particles. this preconditioning step makes the ore permeable to air and water for bioheapleaching. after agglomeration, the ore is conveyed and stacked eight meters high on the primary heap pad for one and one-half years of bioheapleaching. the heap pad is equipped with piping, laid on the bottom of the pad, through which lowpressure fans supply air to the stacked ore. from the top, the heap is irrigated with leaching solution, which is collected from the bottom of the heap. a ten percent side flow is taken for metals recovery and the rest of the solution is diluted with pure water in order to keep the amount of solution constant. fig. 2. pls ponds, bioheap and metals plant after one and one-half years of leaching on the primary pad, the leached ore is expected to be reclaimed, conveyed and restacked onto the secondary heap pad, where it will be leached further in order to recover metals from those parts of the primary heaps, where leaching solution has had poor contact. such areas include, for example, the slopes of the heaps and areas between channels formed by the circulating leaching solution. at this stage the main part of copper and cobalt will be recovered, too. after secondary leaching, the barren ore is expected to remain permanently on the secondary heaps. metals recovery. in the metals recovery process, the metals are precipitated from the pls using hydrogen sulphide. the resulting products are intermediates, such as copper and zinc sulphides and a mixed nickel cobalt sulphide. these intermediates are 10 riekkola-vanhanen, m. transported for further processing in refineries operated by talvivaara’s customers. the recovery process also produces gypsum as a secondary product which will be collected and remain in a separate pond on the mining site. water management. the water management plan for the talvivaara project area includes all relevant pipelines, ponds and pump stations related to the processes outside the metals recovery plant area. it also includes surface water management, effluent treatment and other surface water construction projects. water management plays an important role at the operation. the most important component is the recycling of the leaching solution from the irrigation pond to the heap and thereafter to the pls pond. from the pls pond, approximately 90 per cent of the solution is recycled back to irrigation to increase the metal grade, while about 10 per cent is lead to metals recovery. after metals precipitation, the remaining solution goes into the raffinate pond to ph adjustment and is reused to irrigate the heaps. land and infrastructure. prior to construction of the pilot operation in 2005, the talvivaara deposits had no existing mining facilities. the development of the infrastructure and services included: • access roads, internal roads and railway; • power supply; • fuel services; • drinking water supply/water management; • metal recovery facility and • sewage and waste management. the first construction phase commenced in february 2007 with construction of roads to allow good access to the site and between the construction areas. the majority of all earthworks were completed by the end of 2008. the talvivaara sotkamo mine operational area totals 61 km2. 3. bioheapleaching at talvivaara bioleaching. bioheapleaching has been chosen for the talvivaara sotkamo mine based on its favourable capital and operational costs and the good performance data obtained with the technology in earlier trials with the talvivaara ore. talvivaara’s application of the bioheapleaching technology has its origins at outokumpu research, where it has been developed in conjunction with talvivaara ore since 1987. all the rights relating to the accumulated research and development data on bioheapleaching were transferred to talvivaara in connection with the acquisition of the mining licenses in february 2004. talvivaara has developed bioheapleaching and metals recovery from leaching solutions in collaboration with tampere, helsinki and lappeenranta universities of technology and omg kokkola chemicals ltd. the research and development work has been partly funded by the finnish funding agency for technology and innovation, through capital loans and grants since early 2004. the company has also benefited from the european union funded bioshale project. for example, gsf and talvivaara set up, as part of the bioshale project, a 110 tonne pilot trial in march 2005 nova biotechnologica 10-1 (2010) 11 to test bioheapleaching of talvivaara ore. the trial was successfully started in -20ºc conditions, thus serving as an important indicator of the feasibility of the process in subarctic conditions prior to moving to larger scale on-site trials (riekkolavanhanen, 2005). smaller scale laboratory trials have provided the company with an understanding of the key parameters of bioheapleaching e.g. particle size, ph value, temperature, oxidation and aeration rate. the ph of the bulk solution is an important parameter in bioleaching processes. the acid ph range from 1.5 to 3 is normally considered to be non-selective against any specific ironand sulphur-oxidizing acidophiles that may be potentially useful in biological leaching systems. the ph value used has to be chosen in a way to ensure maximum leaching of valuable metals but also to minimize the leaching of unwanted impurities from the ore. dissolution of silicate has to be avoided, because it can create solution flow barriers by formation of amorphous, gelatinous precipitates (dopson et al., 2008). at talvivaara the ph value has to be kept over 1.5 in order to prevent silicate precipitation. in order to validate the bioheapleaching process on a larger scale, a demonstration scale on-site pilot study was commenced at talvivaara in may 2005 (riekkolavanhanen, 2007; puhakka et al., 2006). heap irrigation was started in august 2005 and metals recovery in november 2005. the demonstration heap was constructed of 17,000 tons of talvivaara ore. similar to the laboratory scale trials, the company varied the key parameters to confirm the optimum conditions. in laboratory tests the temperature remained at the room temperature level. the size of the demonstration plant was large enough to generate heat and the rise of temperature could be observed. the rise was due to the oxidation of the large quantity of pyrrhotite and pyrite in the ore. the temperatures measured inside the heap varied from 30°c to nearly 90°c. according to the measurements there were various temperature gradients inside the heap. the heap was reclaimed during the coldest time in winter 2007 and moved to a new leaching pad. the temperature of the ore was 0 0c, when irrigation of the secondary heap was started at the end of february. the temperature started to rise immediately and was at the level of 80 0c in july and started to decrease gradually after that to the level of 20 – 40 0c, where it remained to the end of july 2008. at that time the pilot had to be stopped in order to start mining in that area. the on-site pilot trial demonstrated that the leach solution temperature and temperatures inside the heaps are practically independent of the surrounding environmental conditions. the oxidative leaching of sulphide minerals is associated with acid production (e.g. pyrite) or acid consumption (monosulphides) (ahonen and tuovinen, 1994). sulphide ores invariably contain accessory non-sulphide minerals some of which may be recalcitrant (e.g. quartz), or they may be susceptible to partial dissolution (e.g. micas, carbonate minerals) and may thereby influence the ph without the involvement of a redox reaction. associated with these reactions are the acid-consuming oxidation of fe2+ and the subsequent acid-producing hydrolysis of fe3+. thus, the net acid consumption or acid production in bioleaching processes is the sum of several concurrent dissolution, precipitation, oxidation, and reduction reactions. the acid 12 riekkola-vanhanen, m. consumption of talvivaara ore was 15 kg/t of ore in the primary leaching and 2 kg/t of ore in the secondary leaching. microbial community dynamics. the microbial inoculum to the pilot heap was obtained from local acidic metal-rich ponds that had developed naturally in areas adjacent to the exposed ore and which contain a wide range of different species of mineral oxidizing acidophiles. the enrichment was originally grown on elemental sulphur, ferrous iron and finely ground talvivaara ore. in the final stage the culture was grown on elemental sulphur and ore dust in an aerated pond on site. the ph value was adjusted to 1.8 with sulphuric acid. the enrichment culture used for inoculation as revealed by pcr-dgge-sequencing contained e.g. acidithiobacillus ferrooxidans, acidithiobacillus caldus and leptospirillum ferrooxidans. depending on the leaching conditions various chemolithotrophs became enriched during the progress of leaching. the moderate thermophiles at. caldus and sulfobacillus thermosulfidooxidans were dominant during some periods of leaching. also extreme thermophile archae were found (riekkola-vanhanen, 2007). these results demonstrate that the mine site waters and the ore harbour a microbial community consisting of several members and that depending on the leaching conditions and especially the leaching temperature the dominant biocatalysts become different. as the actual leaching heaps have gradients of temperatures due to the heat released in the exothermic reactions and the varying boreal conditions, succession of different organisms at different depths of the heap and at different times is obvious. leaching recoveries. the results from the demonstration plant were substantially better than anticipated based on laboratory and other pilot results and confirmed the viability of the bioheapleaching of the talvivaara ore. it is assumed that most of nickel and zinc will be leached in the primary heap within one and one-half years. the leaching rates were calculated from the chemical analyses of the solution taken away for metal recovery. every tenth sample was sent to an accredited laboratory in order to be sure that the results are correct. the recovery rates were higher than anticipated. nickel recovery reached 80 % within 400 days. the corresponding zinc recovery was 80 % in 480 days. the demonstration plant has shown that the assumed recoveries for nickel and zinc can be reached (puhakka et al., 2006). the recovery of cobalt remained low and the leaching rate was progressing slowly over the first 500 days. only 2.5 % of copper were recovered. copper is in chalcopyrite. the main part of cobalt is in pyrite. as sulphide minerals have semiconductor properties, galvanic interactions appear when there is an electrical contact among mineralogical phases. during dissolution of a mineral assembly of different sulphides, those minerals that have the highest rest potentials behave as cathodes which mean that they are galvanically protected and their leaching is hindered until the minerals with lower rest potentials have been leached. the electrochemical potentials of chalcopyrite and pyrite are higher than the ones of pyrrhotite, pentlandite and sphalerite (riekkola-vanhanen and heimala, 1993). the leaching of copper and cobalt proceeded well in the secondary leaching phase of the demonstration plant. nova biotechnologica 10-1 (2010) 13 0 10 20 30 40 50 60 70 80 90 100 5.9.2005 24.3.2006 10.10.2006 28.4.2007 14.11.2007 1.6.2008 18.12.2008 date m et al r ec ov er y % secondary leaching primary leaching ni zn co cu fig. 3. leaching recoveries in talvivaara pilot. 4. conclusions extensive research and pilot trials have proven bioleaching a feasible and environmentally sound technology in treating talvivaara low grade sulphide nickel deposits in subarctic conditions. talvivaara project ltd., which was established to build the mine and plant, progressed in the set timetable and budget. the mine and the plant are in industrial use since the beginning of year 2009. references riekkola-vanhanen, m., heimala, s.: electrochemical control in the biological leaching of sulfidic ores. in: a.e. torma, j.e wey, v.i. lakshmanan (eds.), proceedings of the 10th international biohydrometallurgy symposium, tms warrendale, pennsylvania, 1993, 561-570. ahonen, l., tuovinen, o.h.: solid phase alteration and iron transformation in column bioleaching of a complex sulfide ore in: c.n. alpers and d.w. blowes (eds.), environmental geochemistry of sulfide oxidation. american chemical society, washington, d.c., 1994, 79-89. riekkola-vanhanen, m., heimala, s.: study of the bioleaching of a nickel containing black-schist ore. in: r. amils, a. ballester, (eds.) proceedings of the 13th international biohydrometallurgy symposium part a, elsevier, amsterdam, 1999, 533-542. riekkola-vanhanen, m., sivelä, c., viguera, f., tuovinen, o.h.: effect of ph on the biological leaching of a black schist ore containing multiple sulphide minerals. in: v.s.t., ciminelli, o. garcia (eds.) proceedings of the 14th international biohydrometallurgy symposium part a, elsevier, amsterdam, 2001, 167-174. 14 riekkola-vanhanen, m. riekkola-vanhanen, m.: bioleaching of talvivaara black schist ore. materia, 3, 2005, 38-43. puhakka, j.a., kaksonen, a.h., riekkola-vanhanen, m.: heap leaching of talvivaara black schist ore. in: d.e. rawlings, d.b. johnson (eds.) biomining, springer-verlag, 2007, 139-152. riekkola-vanhanen, m.: talvivaara black schist bioheapleaching demonstration plant. adv. mater. res., 20-21, 2007, 30-33. dopson, m., halinen, a.k., rahunen, n., boström, d., sundqvist, j.e., riekkola-vanhanen, m., kaksonen, a.h, puhakka, j.a.: silicate mineral dissolution during heap bioleaching. biotechnol. bioeng., 99, 2008, 811-820. microsoft word dudkova nb 9-2.doc nova biotechnologica 9-2 (2009) 119 microbial dechlorination of polychlorinated biphenyls vlasta dudková1, kateřina demnerová1, donna l. bedard2 1institute of chemical technology in prague, faculty of food and biochemical technology, department of biochemistry and microbiology, technická 3, 166 28 prague 6; czech republic, (dudkovav@vscht.cz) 2rensselaer polytechnic institute, department of biology, 110 eighth street, troy, ny 12180-3590, u.s.a. abstract: polychlorinated biphenyls (pcbs) are organic xenobiotics contaminating environment for at least 50 years. they could be eventually eliminated by various organisms under different conditions. the degree of chlorine substitution per biphenyl molecule influences biodegradability which decreases with increasing chlorination. our work is focused on the pcbs biodegradation under anaerobic conditions. the suitable high chlorinated biphenyls are converted via reductive dechlorination to the chlorinated biphenyls with lower extent of chlorine, which could be eventually fully mineralized by aerobic bacteria. microbial consortium was isolated from sediment of strážský creek (located near by plant producing pcbs in the past). this consortium was able to dechlorinate polychlorinated biphenyls under anoxic conditions. the effectiveness of this process was tested under different cultivation condition – different energetic sources (aroclor 1248 or aroclor 1260 or delor 103 or delor 106), addition of potential electron donors (pyruvate, lactate or acetate with hydrogen) and further if there is necessary to add yeast extract into fresh low sulphur cultivation media. our microbial consortia so far do not need supplementation by non-contaminated sediment to maintain dechlorination activity. addition of yeast extract is non essential, but needs to be further proved in serial transfers. in all cases (except acetate without yeast extract) dechlorination proceeds at metaand flanked para position. key words: polychlorinated biphenyls, reductive dechlorination, gas chromatography 1. introduction polychlorinated biphenyls (pcbs) are a class of organic compounds with 1 to 10 chlorine atoms attached to biphenyl molecule (it is possible to derive 209 different congeners). pcbs were commercially produced as complex mixtures containing multiple isomers at different degrees of chlorination. their unique character (nonflammable; good heat transfer fluid; electroisolation properties; low vapor pressure; low water solubility and high solubility at organic solvents and fatty acids) caused their robust production and wide usage. their commercial and industrial production started at the end of 20s last century. pcbs were produced by catalytical chloration of biphenyl (at czechoslovakia they were manufactured at chemko strážské until 1985). pcbs production was banned in the 1970s (in most countries of west world) due to the high toxicity of most pcb congeners and mixtures to the organisms. 120 dudková, v. et al. listed properties caused food chain accumulation and difficulties during degradation and removing. pcbs are classified as persistent organic pollutants. they may be destroyed by chemical, thermal, and biochemical processes. it is extremely difficult to achieve full destruction, and there is the risk of creating extremely toxic dibenzodioxins and dibenzofurans through partial oxidation. chemical analogs of pcbs (as chlorophenols; chlorobenzenes or dioxines) are in nature produced during forest fires or volcano eruptions (borja et al., 2005). it was evidenced that pcbs are transformed by microorganisms (faster at laboratory conditions than in nature). it gives us possibilities how to decrease current concentration in the environment. the halftime differs through environmental kind (aerobic, anaerobic; water, soil, atmosphere, sediment), type of organisms participated on degradation (bacteria, fungi, plants, animals) and amount and character of other contaminating compounds. anaerobic bacterial degradation of pcbs is not so well-known as the aerobic degradation. but at short time were serious results achieved and it was demostrated that anaerobic degradation plays an important function through pcbs biological decontamination (maltseva et al., 1999). organisms growing on organohalides get energy for their metabolism and reproduction by reductive dechlorination (mohn and tiedje, 1992) (halogen from the organohalide molecule is used as a terminal electron acceptor). above mentioned mechanism (dehalogenation) transforms the high chlorinated biphenyls to the low chlorinated biphenyls which could be consumed during aerobic degradation. it should be very interesting but technically very difficult to try to combine anaerobic and aerobic degradation. mechanism of anaerobic degradation was first described at the end of 90s last century. it was observed at laboratory conditions that degradation ability should be primed by presence of some congeners or amended by exogenic compounds which should be used as an energy source or as a proton donor (dibromobiphenyls, lactate, malate, pyruvate, acetate, butyrate and others) (bedard et al., 1998; wu et al., 1999). adrian and coworkers (2009) finally describe pure culture (dehalococcoides sp. strain cbdb1) extensively dechlorinating commercial pcb aroclor 1260. 2. materials and methods pcb mixes and aroclors were purchased from accustandard (new haven, the u.s.a.). delor was provided by mr. kočan from the slovak republic. diethylether declared as gc grade was supplied by merck (darmstadt, germany). sediment (fractioned under 1 mm2) was resuspended in special low sulphur purely synthetic, carbonate buffered mineral medium containing ti(iii) citrate as reducing agent and vitamins (adrian et al., 2000). set of samples was amended by 4-4 dibromobiphenyl (final concentration 350 μmol/l), other was amended by 2,6 dibromobiphenyl and others were retained without addition and cultivated at two different temperatures (4 °c and 28 °c). brominated biphenyls were sorbed on silica mesh 250 nm (providing homogenic distribution of pcbs and increasing internal area). microbial activity was measured indirectly by changing concentration of every pcb. growing cultures were sampled (every 3 weeks) under anoxic condition (quensen et al., 1990). nova biotechnologica 9-2 (2009) 121 the taken media were shaked out to the diethylether (volume distribution 1:12.5). proper assesment of concentration of pcb from diethylether was provided on gas chromatograph (hp 6890n) with automatic injector (hp 7683) and micro-electrone capture detector. for separation of congeners capillary column (db-xlb, length 30m, inside diameter 0.25 mm and thickness of stationary phase 0.25 μm) was used. mobile phase was nitrogen and the final flow was 45.1 ml/min. sample (1 μl) was inject on column using splitless technique. measurment was proceded by using isobaric mode (0.993 bar). temperature program was following: the initial temperature was 50 °c and immediately increased to 160 °c (rate 30 °c/min) and then immediately increased to 300 °c (rate 2.5°c/min) and there was terminated. detector temperature was 340 °c and final flow of mobile phase was 60 ml/min (makeup gas + column flow). data were collected by using software gc chemstation. however we are still not able fully quantify the whole process (it is the reason why we show data at not very wellarranged shape – chromatograms). 3. results and discussion our first experiment should manifest presence of dehalogenating organisms and a possible way of accelerating their metabolism. after dechlorination activity was documented and extensive advance (samples cultivated at 28 °c) we transferred our microbial consortia in a fresh media (portion of primary culture was 10 vol. %). commercial mixture pcbs (aroclor 1248, or aroclor 1260, or delor 103, or delor 106 at finally concentration 50 μg/ml) sorbed on silica was added as an energy source. we added different proton sources for microbial consortia – pyruvate, or lactate, or acetate amended by hydrogen. table 1. sum of detected pcbs (%) in different time of cultivation. indicator pcbs: 28, 52, 101, 118, 138, 153, 180 sum of detected pcbs [%] sum of indicator pcbs [%] 0. day 182. day 285. day 473. day 586. day 0. day 182. day 285. day 473. day 586. day acetate 100 100 100 100 100 100 95 104 111 107 acetate with yeast extract 100 100 100 98 97 100 101 103 86 80 lactate 100 95 94 94 94 100 64 59 59 58 lactate with yeast extract 100 97 96 95 95 100 74 67 65 62 pyruvate 100 98 95 95 94 100 79 62 65 63 pyruvate with yeast extract 100 95 94 94 94 100 65 58 60 58 122 dudková, v. et al. more detailed will be discussed the microbial consortia of the 4th transfer containing only 0.04 vol. % of origin sediment cultivated on aroclor 1260 pcb mixture supplemented by pyruvate, lactate or acetate with hydrogen. we study if there is necessary to supplement fresh bacterial media for our dechlorinators by more complex carbon source yeast extract. pcb dechlorination activity was measured as described above. sum of reminder pcbs (%) as well as sum of indicator pcbs during incubation period is documented in table 1. decreasing concentration of pcbs 138, 153 and 180 which are well present at originally used pcb mixture – aroclor 1260 were mainly responsible for so huge non-expecting decreasing concentration of indicator pcbs. on the other hand we observed a bit increasing concentration of remaining indicator congeners. we also analyzed change of chlorine distribution on biphenyl molecule. documentation is at table 2. table 2: distribution of chlorines per biphenyl molecule (%). particular amount of ortho-, meta-, and parachlorines (%) acetate acetate with yeast extract lactate lactate with yeast extract pyruvate pyruvate with yeast extract 0. day 586. day 586. day 586. day 586. day 586. day 586. day ortho 39.5 39.3 41.0 43.1 42.5 42.8 43.2 meta 39.8 39.7 39.1 38.4 38.4 37.8 37.8 para 20.7 21.0 19.9 18.5 19.1 19.4 19.0 at all cases (except acetate) there is examined decreasing amount at metaand parasubstitution. dechlorination pathways were studied and it was set up our microbial communities are removing metaand flanked parachlorines from biphenyl molecule. 4. conclusions our subcultures transformed nona, octa and hepta chlorinated biphenyls through hexa to tri, tetra and penta chlorinated biphenyls. dechlorination proceeds at metaand flanked paraposition. we observed decreasing concentration of indicator pcbs 138, 153 and 180 and on the other hand increasing concentration of indicator pcbs 28, 52, 101 and 118. addition of yeast extract in fresh media seems not to be necessary for most of tested conditions (exception of acetate) – need to be further confirmed at next transfer. our consortia do not need additional supplementation with non-contaminated sediment as many others. incubation time at laboratory condition seems to be too long, but when dechlorination proceeds it is going relatively fast. we presume, that as our consortia nova biotechnologica 9-2 (2009) 123 adapt to our condition (temperature, composition of media, non-presence of sediment) the lag period would be much shorter. acknowledgement: the authors thank for the support of the grants: centrum 1m06011, frvš g4/943/2009, npv ii 2b06156 and vz msm 6046137305. references adrian, l., dudkova, v., demnerova, k., bedard, d.l.: dehalococcoides sp. strain cbdb1 extensively dechlorinates the commercial polychlorinated biphenyl (pcb) mixture aroclor 1260. appl. environ. microbiol., 2009 (in press). adrian, l., szewzyk, u., görisch, h.: bacterial growth based on reductive dechlorination of trichlorobenzenes. biodegradation, 11, 2000, 73-81. bedard, d.l., dort, h., deweerd, k.: brominated biphenyls prime extensive microbial of aroclor 1260 in housatonic river sediment. appl. environ. microbiol., 64, 1998, 1786-1795. borja, j., taleon, d.m., auresenia, j., gallardo, s.: polychlorinated biphenyls and their biodegradation. process biochem., 40, 205, 1999–2013. maltseva, o.v., tsoi, t.v., quensen, j.f., fukuda, m., james, m.: degradation of anaerobic reductive dechlorination products of aroclor 1242 by four aerobic bacteria. biodegradation, 10, 1999, 363-371. mohn, w.w., tiedje, j.m.: microbial reductive dehalogenation. microbiol. rev., 56, 1992, 482-507. quensen, j.f. iii., boyd, s.a., tiedje, j.m.: dechlorination of four commercial polychlorinated biphenyl mixtures (aroclors) by anaerobic microorganisms. appl. environ. microbiol., 56, 1990, 2360-2369. wu, q., bedard, d.l., wiegel, j.: 2,6-dibromobiphenyl primes extensive dechlorination of aroclor 1260 in contaminated sediment at 8-30 ˚c by stimulating growth of pcb-dehalogenating microorganisms. environ. sci. technol., 33, 1999, 595-602. microsoft word ondrejovic2 nb 9-2.doc nova biotechnologica 9-2 (2009) 175 optimization of rosmarinic acid extraction from lemon balm (melissa officinalis) miroslav ondrejovič1,2, hana benkovičová1, stanislav šilhár2 1department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (miroslavondrejovic@gmail.com) 2food research institute, dept. biocentre, kostolna 7, sk-900 01, modra, slovak republic abstract: the aim of this study was optimization of rosmarinic acid extraction from lemon balm (melissa officinalis). the optimal conditions for the extraction of rosmarinic acid from lemon balm were determined using response surface methodology (rsm). a center composide design (ccd) was used to investigate the effects of three independent variables, namely solid-liquid radio, solvent composition (%) and extraction temperature (°c). dependent variable was yield of rosmarinic acid. a second-order polynomial model was used for predicting the response. optimized conditions for rosmarinic acid were: peme 1:29 (w/v), temperature 66 °c a % propan-2-ol 34 %. the experimental values agreed with predicted within a 95 % confidence interval. yield of rosmarinic acid extraction by these optimized conditions was achieved 72.6 mg of rosmarinic acid / g of dry extraction matter. keywords: rosmarinic acid, optimization, melissa officinalis, extraction 1. introduction rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid (pereira et al., 2005; petersen, 1996). it has a number of interesting biological activities, e. g. antimicrobial (pal bais et al., 2002), antiallergic (ito et al., 1998; matsuno et al., 2002), antiviral and anti-inflammatory (georgiev et al., 2006; huang et al., 2006; kamatou et al., 2005). rosmarinic acid is commonly found in species of the boraginaceae and lamiaceae. the plants with rosmarinic acid content were used in traditional medicine. today, plant extracts found some application in food and cosmetic industry. the aim of the present study was to evaluate the effect of changing solvent polarity characterisation using dielectric constant, liquid to solid ratio, temperature and time of extraction on the rosmarinic acid yield in extracts. 2. material and methods 2.1 plant material adult leaves of melissa officinalis were harvested with the twigs and dried at 40°c. thereafter, the leaves were separated from the twigs and cut in small squares of 1 – 1.5 cm2. this extraction material was stored in powder flask at laboratory temperature. 176 ondrejovič, m. et al. 2.2 extraction procedures in all experiments, the extractions were done in experimental microtubes and stopped by a rapid decantation of extracts. the impact of solvent composition on the rosmarinic acid content from propanol, ethanol, methanol and their water solution (33 and 66 %; v/v) to water were explored using extraction by solid-liquid ratio 1:30 (w/v), at 40 °c during 1 hour. the influence of the solid-liquid ratios on the extraction was investigated, by considering four ratios (1:10, 1:20, 1:30, 1:40; w/v) with 33 % (v/v) water solution of propan-2-ol as extraction solvent. a kinetic study was performed to select reach an asymptotic region in the extraction. the axial (40 and 80 °c) and central (60 °c) temperatures were used in this kinetic study. the solid-liquid ratio was 1:100 and extraction solvent was again 33 % (v/v) water solution of propan2-ol. 2.3 experimental design for the response surface methodology (rsm) the rsm used a three-factor and central composite design in three blocks consisting of 17 experimental runs with three replicated at the centre point. the design variables were the solid-liquid ratio (0.019 – 0.099 g/ml; x1), the propan-2-ol proportion (propan-2-ol: water, 1 – 95 %, v/v; x2) and the temperature (20 – 80 °c; x3). the variables particle size (< 0.5 mm) and extraction time (1 hour) were kept at constant values. experimental data were fitted to the following second-order polynomial model (eq. 1) and regression coefficients were obtained. ∑∑∑∑ − < = === +++= 1 1 2 2 11 0 k ji i j jiiji k i ii k i ii xxbxbxbby (1) where x1, x2, ..., xk are the independent variables affecting the response y; b0, bi (i = 1, 2,.., k), bii (i = 1, 2, ..., k) and bij (i = 1, 2,...k – 1; j = 2, 3,..., k) are regression coefficients for intercept, linear, quadratic, and interaction terms, respectively; k is the number of variables (silva et al., 2007). the optimum conditions of rosmarinic acid extraction were looked by using statgraphic, 5.1. the verification of the validity and adequacy of the predictive extraction model was carried out in these optimum conditions of solid-liquid ratio, solvent composition and extraction temperature. three experimental replicates were performed at the optimized conditions and the experimental and predicted values were compared. 2.4 determination of rosmarinic acid content the rosmarinic acid content in prepared extracts was determined by hplc analysis using purospher star rp18e, 250 x 4 mm, 5 μm, gradient elution of 0.1 % trifluoracetic acid in water (a) and acetonitrile (b), at flow rate 1 ml/min: 0 min 15 % b, 18 min 30 % b, 25 min 80 % b, 27 min 80 %, with detection at 325 nm. final nova biotechnologica 9-2 (2009) 177 rosmarinic acid content was expressed as mg of rosmarinic acid per g of dry material (dm). 3. results and discussion 3.1 selection of optimized extraction parameters extraction efficiency of natural compounds is impacted by multiple parameters such as temperature, time and solvent polarity, among others, and their effects may be either independent or interactive. at the beginning of this study, the factors extraction solvent, solid-liquid ratio, temperature and time of extraction were investigated to determination the appropriate experimental ranges to be considered during the optimization process. as stated in the literature, for preparation of crude extracts with rosmarinic acid content, authors used methanol, ethanol, acetone and their water solutions (akowuah et al., 2005; almela et al., 2006; al et al., 1994; zelić et al., 2005). the best of rosmarinic acid yields was reached with 50 % (v/v) water solution of methanol or ethanol. yield of extracted compounds is dependent on their solubility in solvent, which dependents on polarity of solvent. for the evaluation of solvent polarity the dielectric constant is used. dependence of rosmarnic acid yields on dielectric constant (fig. 1) was developed on the base evaluation extraction yields using methanol, ethanol, propan-2-ol and their water solutions (33 and 66 %; v/v) as extraction solvents. dielectric constants were calculated according to equation 2: alcoholalcoholwaterwaterionwatersolut vv ** εεε += , (2) where ε is dielectric constant of extraction solvents and v is volume coefficient of resulting solution used as extraction solvent. 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 0 10 20 30 40 50 60 y ie ld o f r a m g/ g of d ry m at te r theoretical dielectric constant of extraction solvent fig. 1. dependence of rosmarinic acid yield on theoretical dielectric constant of extraction solvent. results indicated that the biggest rosmarinic acid yields were obtained using solvent with dielectric constant in the range from 48 to 59, which represented 33 – 47 % water solution of propan-2-ol. therefore, one of optimized parameter was concentration of propan-2-ol in water solution used as extraction solvent. dependence 178 ondrejovič, m. et al. of isolated compound yields on theoretical dielectric constant of extraction solvent is well-know from literature. similar course of this dependence was observed at various natural compounds such as antibiotics (bora and dutta, 2000) or pigments from paprika (kiss et al., 2000). the impact of the solid-to-liquid ratio on the extraction of rosmarinic acid from m. officinalis was conducted with four ratios (1:10, 1:20, 1:30 and 1:40; w/v) during 1 hour at 80 °c with 33 % (v/v) water solution of propan-2-ol as extraction solvent. the results of the one-way analysis of variance showed that there was a significant difference among the ratios studied. there was no statistically significant difference between the ratios 1:30 and 1:40 (w/v). the solid-liquid ratio optimal for rosmarinic acid extraction is between the ratios 1:20 and 1:30 (w/v). this survey is consonant with literature (akowuah et al., 2005; georgiev et al., 2006; baskan et al., 2007). 10 20 30 40 0 10 20 30 40 50 60 y ie ld o f r a m g/ g of d ry m at te r volume of extraction solvent ml/g of dry matter fig. 2. effect of the solid-liquid ratio on the extraction of rosmarinic acid from m. officinalis leaves over 1 hour with a 33 % (v/v) water solution of propan-2-ol at 80 °c. 0 20 40 60 80 100 120 36 38 40 42 44 46 48 50 52 54 56 58 60 y ie ld o f r os m ar in ic a ci d [m g/ g of d ry m at te r] extraction time [min.] fig. 3. kinetics of rosmarinic acid extraction with a 33 % (v/v) water solution of propan-2-ol at various temperatures (● – 40 °c; ▲ – 60 °c; ▼– 80 °c) by solid-liquid ratio 1:100. from kinetics of rosmarinic acid extraction represented in fig 3, it is evident, that maximum yield of rosmarinic acid was reached after 60 min of extraction for the three temperatures studied. the best bits were achieved using the exponential model, for the three temperatures and the range of time used, as expressed by the coefficients of determination (r2) (tab. 1). nova biotechnologica 9-2 (2009) 179 table 1. exponential models for the kinetic of rosmarinic acid extraction from m. officinalis leaves. y = a + b*exp(-x/c) temperature (°c) a b c r2 40 53.82 ± 0.37 -34.78 ± 3.23 16.76 ± 1.67 0.9752 60 57.34 ± 0.59 -25.75 ± 1.47 18.95 ± 2.46 0.9788 80 58.93 ± 0.54 -34.03 ± 1.06 9.22 ± 1.15 0.9556 3.2 optimization of extraction by rsm the rosmarinic acid extraction from m. officinalis was optimized through the rsm approach. a fixed extraction time (1 hour) and a fixed particle size (< 0.5 mm) were chosen. results of rosmarinic acid yields for all runs are reported in table 2. multiple linear regressions using the second-order polynomial model (eq. 1) were performed in the results of table 2. the response variability was explained by the model, the coefficient of multiple determination (r2) was 0.9253. 3.2.1 analyses of the regression coefficients and the response surface the regression coefficients of the model for rosmarinic acid yield obtained by the multiple linear regression are reported in table 3. variables in their coded form (table 2) permitted a direct interpretability of effects (linear, quadratic and interaction) of independent variables, and the surface plot (fig. 4) facilitated the visualisation of the statistically significant factors (denoted by the superscript letters on the regression coefficients of table 3) derived from the statistical analysis. table 2. experimental results for the response variable (rosmarinic acid yield) and independent variables (x1, x2 and x3) in original and coded form. factor 1 (x1) factor 2 (x2) factor 3 (x3) factor y standard order solid-liquid ratio (g/ml dm) propan-2-ol (%) temperature (°c) ra yield mg/g dm 1 0.035 (-1) 20 (-1) 32 (-1) 38.71 2 0.035 (-1) 20 (-1) 68 (1) 62.54 3 0.083 (1) 20 (-1) 32 (-1) 36.65 4 0.083 (1) 20 (-1) 68 (1) 33.52 5 0.035 (-1) 76 (1) 32 (-1) 21.04 6 0.035 (-1) 76 (1) 68 (1) 55.98 7 0.083 (1) 76 (1) 32 (-1) 17.88 8 0.083 (1) 76 (1) 68 (1) 30.59 9 0.059 (0) 48 (0) 20 (-1.68) 21.68 10 0.059 (0) 48 (0) 80 (1.68) 55.79 11 0.019 (-1.68) 48 (0) 50 (0) 62.62 12 0.099 (1.68) 48 (0) 50 (0) 17.37 13 0.059 (0) 1 (-1.68) 50 (0) 53.73 14 0.059 (0) 95 (1.68) 50 (0) 0.5 15 0.059 (0) 48 (0) 50 (0) 62.62 16 0.059 (0) 48 (0) 50 (0) 63.5 17 0.059 (0) 48 (0) 50 (0) 65.34 180 ondrejovič, m. et al. table 3. regression coefficients of second-order model for dependent variable – rosmarinic acid yield. model parameters ra yield intercept -89.391 temperature 3.52489 solid-liquid ratio 1837.34 linear propan-2-ol 0.78097 temperature x temperature -0.02493 solid-liquid ratio x solid-liquid ratio -13245.3 quadratic propan-2-ol x propan-2-ol -0.01559 temperature x solid-liquid ratio -14.2332 temperature x propan-2-ol 0.00668 interaction solid-liquid ratio x propan-2-ol 0.47061 regression coefficients with a statistically significant at p < 0.05 are printed in bold. as indicated by the p value, regarding the propan-2-ol proportion (x2), negative linear and quadratic effects were verified to be statically significant for rosmarinic acid yield. this result indicated that rosmarinic acid yield increases with decreasing of propan-2-ol proportion but quadratic effect of this variable parameter suggested forming of specific area whit maximal yield of rosmarinic acid. these results supported by results in first part of this study (an appropriate extraction solvent with dielectric constant between 48 and 55) and results of another study (durling et al., 2007). negative linear and quadratic effects of solid-liquid ratio (x1) suggested that the increase of extraction solvent volume to extraction material mass improves the rosmarinic acid yield. positive effect of temperature (x3) was detected, which confirms that the increase in temperature improves the rosmarinic acid yield. indeed, a higher temperature increases the solubility and diffusion coefficient of rosmarinic acid, allowing higher extraction rate. propan-2-ol proportion solid-liquid ratio y ie ld o f r a [m g/ g d m ] 35 45 55 65 75 85 (x 0,001) 20 40 60 80 0 10 20 30 40 fig. 4. response surface plot for yield of rosmarinic acid, in function solid-to-liquid ratio and proportion of propan-2-ol in extraction solvent at 50 °c. 3.3 determination and experimental validation of the optimal conditions in order to verify the predictive capacity of the model, an optimum condition was determined using the simplex method and the maximum desirability for the rosmarinic nova biotechnologica 9-2 (2009) 181 acid yield, and it was used for an extraction test (table 4). the measured values lay within a 95 % mean confidence interval of the predicted value for rosmarinic acid yields. these results confirm the predictability of the model for the extraction of rosmarinic acid from m. officinalis leaves in the experimental condition used. table 4. comparison of predicted and experimental values at optimal conditions of rosmarinic acid extraction. optimized conditions propan-2-ol 39 temperature [°c] 66 solid-liquid ratio 0.035 predicted value of rosmarinic acid yield [mg/g] 74.4 ± 2.59 experimental value of rosmarinic acid yield [mg/g] 72.6 ± 3.12 4. conclusions the response surface methodology was successfully employed to optimize the rosmarinic acid extraction from m. officinalis leaves. the second-order polynomial model gave a satisfactory description of the experimental data. an optimized condition for maximum extraction of rosmarinic acid was determined. the optimal conditions were – propan-2-ol proportion 39 %, extraction temperature 66 °c and solid-liquid ratio 1:29 (w/v). this study can by useful for the development of industrial extraction processes, including further studies concerning the optimal number of sequential steps to enhance the efficiency of a large-scale extraction system. references akowuah, g. a., ismail, z., norhayati, i., sadikun, a.: the effects of different extraction solvents of varying polarities on polyphenols of orthosiphon stamineus and evaluation of the free radical-scavenging activity. food chem., 93, 2005, 311-313. al, ch. b., deng, q. h., song, w. z., li, l. n.: salvianolic acid j, a depside from salvia flava. phytochemistry, 37, 1994, 907-908. almela, l., sanchez-munoz, b., fernandez-lopez, j., roca, m. j., rabe, v.: liquid chromatograpic-mass spectrometric analysis of phenolics and free radical scavenging activity of rosemary extract from different raw material. j. chromatogr. a, 1120, 2006, 221-223 baskan, s., oztekin, n., erim, f. b.: determination of carnosic acid and rosmarinic acid in sage by capillary electrophoresis. food chem., 101, 2007, 17481751 bora, m. m., dutta, n. n.: extraction equilibria of cephalosporin antibiotics with aliquat-33. j. chem. eng. data, 45, 2000, 399403 durling, n. e., catchpole, o. j., grey, j. b., webby, r. f., mitchell, k. a., foo, l. y., perry, n. b.: extraction of phenolics and essential oil from 182 ondrejovič, m. et al. dried sage (salvia officinalis) using ethanol-water mixtures. food chem., 101, 2007, 1417-1424 georgiev, m., kovatcheva, e., marcheva, n., ilieva, m.: purification rosmarinic acid extracts from lavandula vera mm cell biomass. food chem., 94, 2006, 111-114 kiss, g. a. c., forgács, e., cserháti, t., mota, t., morais, h., ramos, a.: optimisation of the microwave-assisted extraction of pigments from paprika (capsicum annuum l.) powders. j. chromatogr. a, 889, 2000, 41-49. silva, e. m., rogez, h., larondelle, y.: optimization of extraction of phenolics from inga edulis leaves using response surface methodology. separ. purif. technol., 55, 2007, 381-387 zelić, b., hadolin, m., bauman, d., vasić-rački, d.: recovery and purification of rosmarinic acid from rosemary using electrodialysis. acta chim. slov., 52, 2005, 126-130 microsoft word ondrejovic nb 9-3.doc nova biotechnologica 9-3 (2009) 313 isolation of antioxidants from alchemilla xanthochlora miroslav ondrejovič1,2, zuzana ondrigová1, janka kubincová2 1department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (miroslavondrejovic@gmail.com) 2food research institute, dept. biocentre, kostolna 7, sk-900 01, modra, slovak republic abstract: the aim of this study was to find conditions for isolation of antioxidants from alchemilla xanthochlora. the solid-liquid extraction of plant material with different solvents (n-hexane, chloroform, ethylacetate, methanol, and water) was used for preparation of crude extracts of antioxidants. for additional purification of crude extract, liquid-liquid extraction in different organic solvents (chloroform, ethylacetate, methanol) and water, and column chromatography with silica gel as stationary phase and mixture of chloroform : methanol as mobile phase were used. the content of active compounds was monitored by photometric method and special tlc analysis using dpph as basic reagent. tlc-dpph analysis of herbal extracts showed that antioxidants from alchemilla xanthochlora were separated into two groups with different polarity. finally, for the isolation of antioxidants from alchemilla xanthochlora were successfully used the following procedures: extraction of dried grounded plant material with methanol at 50°c during 1 hour, subsequent liquid-liquid extraction in isopropanol and water and fractionation of isopropanol extract by column chromatography. fraction with isolated antioxidants showed antioxidant activity 535.2 mg to g of fraction residue. keywords: antioxidants, alchemilla xanthochlora, extraction, purification 1. introduction alchemilla xanthochlora belongs to the somniferous herbs from the family rosaceae. it is commonly used for the locally treatment of various skin diseases. the antibacterial activity of this plant was related with its tannin content (djipa et al., 2000). in recent study, alchemilla xanthochlora was presented as herb with enormous antioxidant activity (trouillas et al., 2003; hamad et al., 2007) comparable with commonly used antioxidant sources such as strawberry, blackberry or raspberry (fikselová and ivanišová, 2008). various assays have been used for the analysis of natural antioxidants but the mostly widely used methods are those that involve generation of free radical species which are then neutralised by antioxidant compounds (arnao et al., 2001). the dpph method measures electron-donating activity of other compounds in the mixture and hence provides an evaluation of antioxidant activity due to free radical scavenging. any molecule that can donate an electron or hydrogen to a mixture will react with and bleach dpph. in qualitative analysis of antioxidants, the 2,2-diphenyl1-picrylhydrazyl (dpph) assay on tlc plates was used as a screening test for the radical scavenging ability of the compounds present in the different extracts (naik et al., 2003). 314 ondrejovič, m. et al. the aim of the present study was to evaluate the antioxidant activities of various solvent extracts and fractions acquired by solid-liquid and liquid-liquid extraction and column chromatography from alchemilla xanthochlora, and choose an extraction system suitable for isolation responsible compounds. 2. material and methods 2.1 plant material adult leaves of alchemilla xanthochlora were harvested with the twigs and dried at 40°c. thereafter, the leaves were separated from the twigs and cut in small squares of 1 – 1.5 cm2. this extraction material was stored in powder flask at laboratory temperature. 2.2 preparation of extracts 1 g of plant material was extracted with 20 ml of extraction solvent (hexane, chloroform, ethylacetate, methanol and distilled water) over 1 hour at 50°c. the extraction mixture was centrifuged at 3000 x g for 10 minutes and the supernatant was decanted. the solvent was removed under reduced pressure on the rotary vacuum evaporator at 50°c. the solid residue was solubilized in mixture of chloroform and water and water phase enhanced with saturated water solution of nacl was further extracted with ethylacetate and isopropanol, respectively. the isopropanol fraction (10 ml) was fractionated on a silica gel (20 g) eluted with chloroform: methanol (10:0; 9:1; 7:1; 5:1; 3:1; 1:1; 0:10). totally 21 fractions was analysed by tlc-dpph and photometric dpph analysis. 2.3 tlc-dpph analysis chemical constituents of plant extracts were analysed by thin layer chromatography (tlc) using aluminium-backed tlc plates (silica gel 60 f254). the tlc plates were developed with four following mobile phases as methanol : formic acid (10:1) (developing to ¼ of tlc plate length), chloroform : methanol (9:1) (developing to ½ of tlc plate length), toluene: acetone (7:3) (developing to ¾ of tlc plate length) and hexane: ethylacetate (5:1) (complete developing of tlc plate) respectively. between developing in mobile phases the plates were dried in the fume hood at 50 °c. to detect antioxidant compounds, chromatograms were sprayed with 0.2% 2,2-diphenyl-2-picryl-hydrazyl (dpph) in methanol, as an indicator. the presence of antioxidant compounds was detected by yellow spots against a purple background. 2.4 photometric dpph analysis the dpph assay was performed as described (paulová et al., 2004). in this assay, bht (butylhydroxytoluene) was used as positive control, and reaction mixtures nova biotechnologica 9-3 (2009) 315 contained a methanolic solution of 150 μm dpph (100 μl) and sample (25 μl) placed in a 96 well microplate and incubated at laboratory temperature for 10 minutes. after incubation, the absorbance was read at 517 nm and the radical-scavenging activity was determined by the following equation: dpph activity = [(ac as)/ac x 100] x mdpph/mrs where as is absorbance of reaction mixtures with sample, ac is absorbance of reaction mixtures with methanol instead of sample, mdpph is calculated mass of dpph in reaction mixture and mrs is calculated mass of extract/fraction residue. 3. results and discussion screening of antioxidants from alchemilla xanthochlora was made by preparation of crude extract with extraction solvents such as hexane, chloroform, ethylacetate, methanol and water. selected extraction solvents can extract a wide range of natural compounds from plant material. n-hexane chloroform ethylacetate methanol water 0,0 0,5 1,0 1,5 2,0 2,5 3,0 a nt io xi da nt a ct iv ity [g o f d p p h to g o f c ru de e xt ra ct r es id ue ] extraction solvent fig. 1. antioxidant activity of extracts from alchemilla xanthochlora prepared with various extraction solvents over 1 hour at 50°c. a antioxidant activity measured by photometric method expressed in g of dpph scavening with one g of crude extract residues; b – qualitative composition of antioxidants measured by tlc-dpph method (h – n-hexane, ch – chloroform, e – ethylacetate, m – methanol, v – distilled water). the tlc-dpph screening method (fig. 1) indicated the presence of antioxidant compounds in all tested extracts. the most prominent antioxidant activity was observed particularly in methanolic extract (fig. 1). the presence of two various groups of antioxidants from alchemilla xanthochlora were confirmed. the first group of antioxidants represents compounds with lipofilic character such as β-carotene or lycopene. compounds from this group are extracted in extraction solvents such as hexane, chloroform, ethylacetate and methanol. the second group of antioxidants comprises compounds with properties like flavonoid or coumarin glycoside. compounds from this group are extracted in methanol and water. in the literature, a b 316 ondrejovič, m. et al. phytochemical screening on the genus alchemilla revealed that there are flavonoids (e.g. quercetin-3-arabinopyranoside and quercetin-3-glucuronide) (lamaison et al., 1991; fraisse et al., 2000), tannins, polyphenols and coumarins (esculetin) (schimmer and eschelbach, 1997). these compounds may be founded in the second antioxidant active group. for the separation of these groups of natural antioxidants, the plant material can be extracted with chloroform and water. the crude extract for another analysis was prepared with methanol to obtain present antioxidants in alchemilla xanthochlora. chloroform ethylacetate isopropanol water phase 0 10 20 30 40 50 60 70 80 p ar tit io n of c ru de e xt ra ct [% ] extraction solvent % aa % sr fig. 2. effect of the extraction solvent by extraction liquid-liquid on the extraction of antioxidants from crude extract of alchemilla xanthochlora (%aa – part of crude extract antioxidant activity in the fractions; % sr – part of crude extract residue in the fractions). at first, the crude methanolic extract was purified to eliminate ballast compounds by liquid-liquid extraction. three various solvents (chloroform, ethylacetate, isopropanol) and water were used. the suitability of extraction solvent in the extraction liquid-liquid was studied by the percent evaluation of antioxidant activity and solid residue from crude extracts. these parameters were used to describe the purity and efficiency of separation step – extraction liquid-liquid. the results of these experiments are presented in the fig. 2. successive extraction was shown that more than 80 % of antioxidant activity of crude methanolic extract from alchemilla xanthochlora was cumulated in isopropanol fraction and more than 60% of crude extract residue was eliminated from isopropanol fraction by these methods. therefore, the necessity of liquid-liquid extraction of crude extract with isopropanol and water before separation by column chromatography is evident. purification of antioxidants from extracts of alchemilla xanthochlora was executed by column chromatography on the silica gel with gradient elution of chloroform : methanol mixtures. the separation of antioxidants on the column was studied by measuring of radical-scavening activity in obtained fractions (fig. 3). used separation system is suitable for isolation of antioxidants from alchemilla xanthochlora as for active compounds from first lipofilic group as for active compounds from second hydrophilic group. antioxidative active compounds were nova biotechnologica 9-3 (2009) 317 cumulated in 5 fractions (4-5; 17-19) with high content of antioxidant active compounds. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 0 100 200 300 400 500 600 a nt io xi da nt a ct iv ity [m g of d p p h /g o f f ra ct io n re si du e] fraction number fig. 3. antioxidant activity of fractions of isopropanol fraction of crude methanolic extract from alchemilla xanthochlora. (1-3 – chloroform; 4-6 – chloroform : methanol (9:1)(v/v); 7-9 – chloroform : methanol (7:1)(v/v); 10-12 – chloroform : methanol (5:1)(v/v); 13-15 – chloroform : methanol (3:1)(v/v); 16-18 – chloroform : methanol (1:1)(v/v); 19-21 – methanol). 4. conclusions for the isolation of antioxidants from alchemilla xanthochlora, extraction of the dried grounded plant material with methanol at 50°c during 1 hour with subsequent extraction liquid-liquid of crude extract residue between phases of water and isopropanol and column chromatography on silica gel with gradient elution of chloroform : methanol mixtures were successfully used. isolated fraction showed specific antioxidant activity 535.2 mg dpph to g of fraction residue. the results of this work may be used for determination of antioxidant active compounds from alchemilla xanthochlora. references arnao, m.b., cano, a., acosta, m.: the hydrophilic and lipophilic contribution to total antioxidant activity. food chem., 73, 2001, 239-244. djipa, c.d., delmee, m., quetin-leclercq, j.: antimicrobial activity of bark extracts of syzygium jambos (l.) alston (myrtaceae). j. ethnopharmacol., 71, 2000, 307-313. fikselová, m., ivanišová, e.: byliny a čaje ako potenciálne zdroje prírodných antioxidačných látok. in: aktuálne problémy riešené v agrokomplexe. spu, nitra, 2008, 334-338. fraisse, d., heitz, a., carnat, a., carnat, a.p., lamaison, j.l.: quercetin 3-arabinopyranoside, a major flavonoid compound form alchemilla xanthochlora. fitoterapia, 71, 2000, 463-464. 318 ondrejovič, m. et al. hamad, i., erol-dayi, o., pekmez, m., onay-ucar, e., arda, a.: free radical scavenging activity and protective effects of alchemilla vulgaris (l.). j. biotechnol., 131, 2007, s40-s41. lamaison, j.l., carnat, a., petitjean-freytet, c., carnat, a.p.: quercetin-3-glucuronide, main flavonoid of alchemilla, alchemilla xanthochlora rothm. (rosaceae). ann. pharm. fr., 49, 1991, 186-189. naik, g.h., priyadarsini, k. i., satav, j.g., banavalikar, m.m., sohoni, d.p., biyani, m.k., mohan, h.: comparative antioxidant activity of individual herbal components used in ayurvedic medicine. phytochemistry, 63, 2003, 97-104. paulová h., bochořáková h., táborská e.: metody stanovení antioxidační aktivity přírodních látek in vitro. chem. listy, 98, 2004, 174-179 schimmer, o., eschelbach, h.: esculetin in alchemilla speciosa: identification and antimutagenic properties. pharmazie, 52, 1997, 476-478. trouillas, p., calliste, c.a., allais, d.p., simon, a., marfak, a., delage, c., duroux, j.l.: antioxidant, anti-inflammatory and antiproliferative properties of sixteen water plant extracts used in the limousin countryside as herbal teas. food chem., 80, 2003, 399-407. microsoft word szabo nb 9-3.doc nova biotechnologica 9-3 (2009) 231 comprehensive investigation of the corrosion state and surface properties of the stainless steel tubes of steam generators andrea szabó nagy1,2, kálmán varga1, bernadett baja1, zoltán németh1, dezső oravetz3, zoltán homonnay4, ernő kuzmann4, jános schunk5, gábor patek5 1institute of radiochemistry and radioecology, university of pannonia, h-8201 veszprém, p. o. box: 158, hungary (vargakl@almos.vein.hu) 2department of physics and chemistry, university of istván széchenyi, h-9026 győr, hungary (nszaboa@sze.hu) 3institute of materials engineering, university of pannonia, h-8201 veszprém, hungary 4institute of chemistry, university of loránd eötvös, h-1117 budapest, hungary 5paks npp ltd., h-7031 paks, p. o. box: 71, hungary abstract: evaluating the water chemistry in the primary circuit and the effect of chemical decontamination of the heat exchanger tubes performed by the ap-citrox (ap: alkaline permanganate; citrox: citric and oxalic acid) procedure at paks npp (hungary), a project dealing with the comprehensive investigation of the general corrosion state of the steam generators (sgs) has been initiated. owing to the fact that there is no investigation method available for the in-situ monitoring of the inner surfaces of heat exchanger tubes, a research program based on sampling as well as on ex-situ electrochemical (voltammetric) and surface analytical measurements (sem-edx, cems, xrd, xps) was developed and elaborated. in the time period of 2000-2008 within the frame of the above project 45 stainless steel specimens, cut out from various locations of the steam generators of the paks npp were investigated. based on the measured corrosion characteristics (corrosion rate, thickness and chemical composition of the protective oxide-layer) it was found that these parameters are strongly dependent on the decontamination history of steam generators. the present work gives a brief overview on the general corrosion state of the heat exchanger tubes of sgs, concerning the long-term effects of the ap-citrox procedure on the chemical composition and structure of the protective oxide-layer. key words: stainless steel, steam generator, corrosion, decontamination 1. introduction some years ago, a systematic study of the primary and secondary circuit water chemistry data, and the corrosion effects of the chemical decontamination procedures performed at paks npp made it clear that an overall estimation of the corrosion state of the steam generators, i.e. the preparation of a so-called ‘corrosion map’ is unavoidable. this ‘corrosion map’ takes a survey of the corrosion features of the heat exchanger tubes made from austenitic stainless steel in the sgs. owing to the fact that there are no investigation methods available for the in-situ monitoring of the inner 232 szabó, n.a. et al. and outer surfaces of heat exchanger tubes, a research project based on sampling as well as on ex-situ electrochemical (voltammetric) and surface analytical measurements (sem-edx, cems, xrd, xps) was launched in the year 2000. within the frame of the above project the inner surfaces of 45 stainless steel samples, which had been in contact with the primary coolant, originating from various locations in the sgs (24 sgs altogether for the 4 reactors) in the time period of 20002008 were investigated. based on the measured corrosion characteristics (corrosion rate, thickness and chemical composition of the protective oxide-layer) it was found that these parameters are strongly dependent on the decontamination history of steam generators. during the time period of 1993-2001 chemical decontamination of 24 sgs in the blocks 1-3 of the paks npp (hungary) were carried out by a non-regenerative version of ap-citrox technology, sometimes in 2 or 3 consecutive cycles. the applied version of the ap-citrox procedure is an eight-step process, including an oxidizing pre-treatment of the surface with alkali potassium permanganate followed by a concentrated mixture of citric and oxalic acids to remove the contaminated surface layer (varga, 2004; rado et al., 2006). our previous studies have revealed that a ”hybrid” structure of the amorphous and crystalline phases is formed in the outermost surface region of the austenitic stainless steel tubes as an undesired consequence of the industrial application of the apcitrox technology (varga et al., 2006; homonnay et al., 2006; szabó et al., 2006; homonnay et al., 2007). the formation of this mobile oxide-layer increased the amount of the corrosion products in the primary circuit significantly, resulting in magnetite deposition on fuel assemblies. the primary aim of this paper is to report some new findings obtained by electrochemical (voltammetric) and sem-edx methods in order to reveal the beneficial changes in the corrosion properties, morphology and chemical composition of the inner surfaces of the decontaminated heat exchanger tubes after 4-7 years under normal operation conditions. in addition, special attention is paid to the comparative mobility studies of the surface oxide layers in boric acid solution by making use of a pilot plant model system and icp-oes method. 2. materials and methods the experiments have been performed on 45 austenitic stainless steel specimens (type: 08x18h10t (gost 5632-61) which corresponds to aisi 321 and din 1.4541; outer diameter: 16 mm, average wall thickness: 1.6 mm) originating from different sgs of the paks npp. the main characteristics of the specimens obtained from the same sgs at various sampling date are given in table 1. the surface decontamination procedure of the heat exchanger tubes was carried out at paks npp according to a nonregenerative version of the ap-citrox technology (varga et al., 2004; rado et al., 2006). the passivity of the tube samples was studied by potentiostatic polarization method. the experiments were carried out by the means of a voltalab 40 (radiometer) type electrochemical measuring system controlled by pc. the measurements were carried out in boric acid solution (c = 12 g·dm-3) in argon gas nova biotechnologica 9-3 (2009) 233 atmosphere (99.999 v/v % ar). the schematic of the measuring system, the detailed experimental procedure and the determination of the corrosion parameters have been described in our earlier papers (varga et al., 2006; szabó et al., 2006). the morphology and chemical composition of the oxide layer developed on the inner surfaces of the 45 stainless steel specimens were studied by scanning electron microscopy (sem), equipped with an energy dispersive x-ray microanalyzer (edx) (type: jeol jsm-50a, controlled with röntec edr 288 software). in addition, the metallographic cross sections of tube specimens were also studied (varga et al., 2006; szabó et al., 2006). the mobility of the surface oxide layers grown on the inner surfaces of the sg tube specimens was studied in boric acid solution, modelling the first 30 hours of reactor restarting period. a pilot plant circulation system elaborated earlier (varga, 2004) was utilized for the boric acid treatment. using icp-oes method, the concentrations of the main alloying components (fe, cr, ni) dissolved into the solutions as a function of the time were determined. additionally, the solutions removed from circulation system were filtered, and the amount of dispersed corrosion product was weighed after 1 week drying. from these findings the average thickness values of the oxide layer removed from the surface were calculated as a function of the year of sampling (baja et al., 2009). table 1. main characteristics of the stainless steel tube specimens originating from the same sgs at various sampling date. number of sg specimens* year of decontamination time period between the last decontamination and the sampling [year] i/1 1996, 1997 4 i/2 1996, 1997, 2001 4 ii/1 1996 1 ii/2 1996 4 ii/3 1996, 2001 4 iii/1 1999, 2001 1 iii/2 1999, 2001 3 iii/3 1999, 2001 5 iv/1 2001 1 iv/2 2001 5 v/1 2001 0 v/2 2001 4 vi/1 1993 7 vi/2 1993, 2001 3 vii/1 2001 1 vii/2 2001 1 vii/3 2001 6 viii/1 2001 2 viii/2 2001 6 * roman numerals refer to the same sgs. 234 szabó, n.a. et al. 3. results and discussion some illustrative polarization curves measured in boric acid solution at the inner surface of the stainless steel specimens cut out from the same sgs of the paks npp at various sampling date are shown in fig. 1. as may be seen in fig. 1, the inner surfaces of the samples have a passive character in a wide potential interval next to the corrosion potential. the average corrosion rates calculated for all samples are not higher than the literature data which were measured for the stainless steels type 08x18h10t and aisi 321 in aqueous solutions having similar composition (0.3 – 4 μm/year) (geraszimov and monahov, 1981; lister, 2003) and references therein). the electrochemical data imply that no unfavourable trend in the passivity of the inner surfaces of the heat exchanger tubes can be detected since the last sg decontaminations performed in 2000 and 2001. -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 e / v [sce] lg i / a c m -2 ii/2 -6 -7 -5 -8 -9 ii/1 ii//3 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 e / v [sce] lg i / a c m -2 iii/3 -6 -7 -5 -8 -9 iii/1 iii/2 -10 fig. 1. potentiostatic polarization curves measured at the inner surface of the samples no. ii and no iii in boric acid solution (c = 12 g·dm-3). scan rate: 10 mv·min-1. nova biotechnologica 9-3 (2009) 235 the long-term tendencies in the morphology and chemical composition of the protective oxide layers formed on the inner surfaces of the heat exchanger tubes were investigated by sem-edx method. some typical sem micrographs of the inner surface and the metallographic cross-section of steel samples studied are shown in fig. 2. as may be seen in fig. 2, on the surfaces of samples never-decontaminated, which had been in contact with the primary boric acid coolant, a thin film (thickness up to 1-2 μm) of the grown-on oxides with excellent protective character can be detected after various periods of normal operation. this grown-on layer, next to the bulk metal, is a non-stoichiometric mixed oxide of spinel structure (crxniyfe3-x-yo4, where 0≤x+y≤3) (szabó et al., 2006; baja et al., 2009). significant amounts of the crystalline magnetite (fe3o4) and occasionally some hematite (fe2o3) are deposited onto surfaces of the grown-on passive layer. a b c fig. 2. selected sem micrographs and metallographic cross sections (m = 3000 x) detected on the inner surface of a sample has never been decontaminated (a), and samples decontaminated four years before sampling (b (no. (ii/2) and c (no. i/2)). 4-7 years after the chemical decontamination treatment medium thick or thick (up to 11 µm) oxide-layer having amorphous character (“hybrid” structure) can be detected on the inner surfaces of all tube samples studied. however, the surface region is rich in chromium and nickel which refers to the presence of spinel structure (nickelferrite and chromites) in the oxide layers (homonnay et al., 2006). the increased amount of the crand ni-rich spinel constituents improve the chemical stability of the oxide-layers formed on stainless steel in the long run (baja et al., 2009). moreover, it is special importance to emphasize that the protective oxide layer of the sample no. i/2 (see fig. 2) covered with large crystals which are also rich in chromium and nickel. 236 szabó, n.a. et al. the experimental findings of the mobility studies of the surface oxide layers in boric acid solution (see fig. 3), in accordance with the sem-edx results, demonstrate without any doubt that beneficial changes in the phase and chemical compositions are accompanied with a suppressed mobility of oxide films grown on the surface of decontaminated tube specimens, and these oxide films have basically the same mechanical stability for both types of heat exchanger tubes in the time period of 2006-2007. fig. 3. average thickness of the oxide layer removed from the surfaces into the boric acid solution as a function of the sampling year. 4. conclusions in the frame of a project dealing with the comprehensive investigation of the corrosion state of the steam generators of the paks npp, hungary, the long-term tendencies in the corrosion state, chemical composition and structure of protective oxide layer of the heat exchanger tubes decontaminated by ap-citrox procedure have been studied. the ap-citrox chemical decontamination procedure does exert an undesired transformation on the stable constituents of surface layer, leading to the formation of a “hybrid” structure of amorphous and crystalline phases. as a consequence of this detrimental prompt effect, some adverse features (medium thick or thick oxide-layer with great mobility) could be detected after 1-3 years of applying the ap-citrox procedure. however, the electrochemical data imply that no unfavourable trend in the passivity of the inner surfaces of the heat exchanger tubes can be detected since the last sg decontaminations performed in 2000 and 2001. some beneficial changes (dominance of the crand ni-rich inverse spinel) take place in the phase and chemical compositions of the oxide-layers grown on decontaminated tube samples after 4-7 years under normal operation conditions. the increased amount of the crand ni-rich spinel constituents improve the chemical stability of the oxide-layers formed on stainless steel in the long run. the above nova biotechnologica 9-3 (2009) 237 changes are accompanied with a suppressed mobility of the oxide films grown on the surface of decontaminated tube specimens and yield inner surfaces covered with oxide-layers having basically the same mechanical stability for both types of heat exchanger tubes in the time period of 2006-2007. acknowledgements: this work was supported by the paks npp co. ltd (paks, hungary). references baja, b., varga, k., szabó, n.a., németh, z., kádár, p., oravetz, d, homonnay, z., kuzmann, e., schunk, j., patek, g.: long-term trends in the corrosion state and surface properties of the stainless steel tubes of steam generators decontaminated chemically in vver type nuclear reactors. corrosion sci., 2009, (in press). geraszimov, v.v., monahov, sz.: materiali jadernoj techniki. atomizdat, moskva, 1981 (hungarian translation). homonnay, z., kuzmann, e., stichleutner, s., varga, k., németh, z., szabó, a., radó, k., makó, k.é., kövér, l., cserny, i., varga, d., tóth, j., schunk, j., tilky, p., patek, g., oszvald, f.: comprehensive investigation of the corrosion state of the heat exchanger tubes of steam generators, part ii. chemical composition and structure of tube surfaces. j. nucl. mater., 348, 2006, 191-204. homonnay, z., szilágyi, p.á., kuzmann, e., varga, k., németh, z., szabó, a., radó, k., schunk, j., tilky, p., patek, g.: corrosion study of heat exchange tubes in pressurized water cooled nuclear reactors by conversion electron mössbauer spectroscopy. j. radioanal. nucl. chem., 273, 2007, 85-90. lister, d.h.: some aspects of corrosion in cooling water systems and their effects on corrosion product transport. proceedings of the eurocorr 2003, budapest, hungary, 28 september – 2 october, 2003, (on cd-rom). radó, k., németh, z., varga, k., schunk, j., kőrösi f.: fundamental issues of the effective decontamination of the steam generators of paks npp. j. radioanal. nucl. chem., 268, 2006, 313-322. szabó, a., varga, k., németh, z., radó, k., oravetz, d., makó, k.é., homonnay, z., kuzmann, e., tilky, p., schunk, j., patek, g.: effect of a chemical decontamination procedure on the corrosion state of the heat exchanger tubes of steam generators. corrosion sci., 48, 2006, 2727-2749. varga, k.: the role of interfacial phenomena in the contamination and decontamination of nuclear reactors. in: horányi, g. (ed.), radiotracer studies of interfaces, interface science and technology, vol. 3, elsevier b.v., amsterdam, 2004, 313-358. varga, k., németh, z., homonnay, z., szabó, a., radó, k., oravetz, d., schunk, j., tilky, p., oszvald, f.: comprehensive investigation of the corrosion state of the heat exchanger tubes of steam generators, part i. general corrosion state and morphology. j. nucl. mater., 348, 2006, 181-190. microsoft word luptakova nb 9-2.doc nova biotechnologica 9-2 (2009) 147 positive and negative aspects of sulphate-reducing bacteria in environment and industry alena luptakova1, eva macingova1, vlasta harbulakova2 1department of mineral biotechnology, institute of geotechnics of slovak academy of sciences, watsonova 45, kosice, sk-043 53, slovak republic (luptakal@saske.sk), (macingova@saske.sk) 2technical university of košice, civil engineering faculty, institute of building and environmental engineering, vysokoškolská 4, košice, sk-042 00, slovak republic, (vlasta.harbulakova@tuke.sk) abstract: the submitted work is oriented on the study of two aspects of the sulphate-reducing bacteria metabolism: the metals bioprecipitation and the concrete biodeterioration. the bioprecipitation of metals with the bacterially produced hydrogen sulphide by sulphate-reducing bacteria (srb) represents the positive effect of the srb existence in the environment. it allows the industrial exploitation in the area of the removal metals from industrial wastewaters. referred method involves principal stages such as: hydrogen sulphide bacterial production, metals precipitation by biologically produced hydrogen sulphide, metal sulphides separation, setting ph of the filtrate from previous steps by 1m naoh and metal hydroxides separation. the basis of the first stage i.e. the hydrogen sulphide bacterial production is the cultivation of srb. in the laboratory conditions the sodium lactate is the energetic substrate for the growth of bacteria. its price is not economic for the application in the practice and is needed investigate the alternative substitutes. therefore was studied the cultivation of sulphate-reducing bacteria to using the selected energetic substrates such as: calcium lactate, glycerol and whey. experimental studies confirm that all chosen substrates are suitable alternative substrates of sodium lactate for the bacterial sulphate-reduction. in the regard to the efficiency of bacterial sulphate reduction the calcium lactate is the best. the biodeterioration of the concrete presents the negative effect of the srb existence in the environment. the research was oriented on the simulation of the biodeterioration of concrete samples under the simultaneous influence of the sulphur-oxidising bacteria genera acidithiobacillus thiooxidans and sulphatereducing bacteria genera desulfovibrio in the environs of the waste water, the acid mine drainage, the nutrient medium and the distilled water. the observation of the surface structure changes of concrete samples confirms the highest biodeterioration influences in the case of the acid mine drainage application. key words: sulphate-reducing bacteria, sulphur-oxidising bacteria, sulphuretum, bioprecipitation, biodeterioration 1. introduction sulphur is widely distributed on the earth and occurs predominantly bound in the form of sulphides, sulphates or as elemental sulphur. in the nature sulphur circulates permanently because it is continuously oxidized or reduced by chemical or biological processes. in a biogeochemical sulphur cycle (fig. 1) the biological transformations may have either assimilatory or dissimilatory metabolic functions. with the exception of animals and humans, most plants, fungi and bacteria are capable of performing an assimilatory reduction of sulphate to sulphide, which is necessary for the biosynthesis 148 luptakova, a. et al. of sulphur containing cell compounds. on the other hand the energy producing dissimilatory sulphur metabolism is restricted to a few groups of bacteria. these groups include microorganisms such as: anaerobic dissimilatory sulphate reducers (bacteria desulfovibrio, desulfotomaculum, desulfomonas,….), anaerobic dissimilatory sulphur reducers (bacteria such as desulfuromonas,…), anaerobic phototrophic sulphur oxidisers (some cyanobacteria and most anoxygenic phototrophic bacteria), aerobic chemotrophic sulphur oxidisers (bacteria such as acidithiobacillus, sulfolobus, thiospira, thiobacterium, ….), anaerobic chemotrophic sulphur oxidisers (bacteria such as acidithiobacillus denitrificans, thiomicrospira denitrificans,..) (rehm and reed, 1981). fig. 1. the biological sulphur cycle (rehm and reed, 1981; conformed). 1 assimilatory sulphate reduction by plants, fungi and bacteria; 2 death and decomposition by fungi and bacteria; 3 sulphide assimilation by bacteria and some plants; 4 excretion of sulphate by animals; 5 dissimilatory sulphatereducing bacteria; 6 dissimilatory sulphur-reducing bacteria; 7 phototrophic and chemotrophic sulphideoxdizing bacteria; 8 phototrophic and chemotrophic sulphur-oxidizing bacteria. the microbial population of dissimilatory part is called “sulphuretum”. its activity is the fundamental of many processes in the nature and industry (e.g. biocorrosion, bioleaching, bioprecipitation, biodeterioration). the typical species of sulphate-reducing bacteria (srb) is desulfovibrio desulfuricans, which reduce sulphates according the reaction (1): srb 2 ch3chohcoo+ so42————> 2 ch3coo+ 2 hco3+ h2s (1) srb produce considerable amount of hydrogen sulphide (h2s). consequently participation of biologically produced hydrogen sulphide in next processes mentions on the positive or negative influence of the srb metabolism in the environment and industry (table 1). sulphuretum 8 4 so4 2 biologically bound sulphur s0 s2 1 5 2 3 7 6 assimilatory part dissimilatory part nova biotechnologica 9-2 (2009) 149 table 1. some influence of the srb metabolism in the environment and industry. positive influence negative influence attendance in biogenesis of oil hydrocarbons biocorrosion of pumping machinery attendance in biogenesis of sulphur deposits blackening and discoloration of products participation in the biological sulphur cycle biodeterioration of concrete removal of heavy metals from waste waters biodeterioration of building materials removal of so4 2from mine waters production of smell production of sulphur from waste waters devaluation of gasoline removal of sulphur from crude oil, petrol and coal 1.1 positive influence of the srb metabolism in the environment and industry the positive influence of srb is based on the process called bioprecipitation, when the biologically produced hydrogen sulphide precipitates of heavy metals from aqueous solution in the metal sulphides form. along this line the bacterial sulphate reduction (reaction 1) has the major application for the removal of heavy metals from industrial waste waters. it is suitable method for removal heavy metals from acid mine drainage (amd). amd generation is the most serious environmental problem created mainly by the mining industry of the sulphide minerals. the impacts of acid mine water pollution on biological systems are mostly severe. the consequence of acidity and heavy metal contamination of amd in aquatic and terrestrial ecosystems is a reduction in both species diversity and the total biomass composition of systems. nowadays is pay attention to biotreatment of amd by selective sequential precipitation to recover metals as hydroxides and sulphides. it is the process of the heavy metals precipitation by bacterially produced hydrogen sulphide with the combination of the metals precipitation by sodium hydroxide at the various values of ph amd (tabak et al., 2003). in general these methods involve principal stages such as: the biological sulphate reduction i.e. the hydrogen sulphide bacterial production, the metals precipitation by biologically produced hydrogen sulphide, the precipitate filtration, the setting ph of the filtrate by 1m naoh with simultaneously precipitation of metals as hydroxides and metal hydroxides separation. the basis of the first stage the biological sulphate reduction is the cultivation of srb. in the laboratory conditions the sodium lactate is the energetic substrate for the growth of bacteria. its price is not economic for the application in the practice and is needed investigate the alternative substitutes. various types of organic substances have been employed as electron donors and carbon sources including sewage sludge, wood chips, animal manure, vegetal compost, mushroom compost, whey and other agricultural waste (dvorak et al., 1992). in addition, synthetic organic compounds have also been used as electron donors, especially small molecular weight compounds, such as lactate, acetate, propionate, pyruvate and butyrate. ethanol and other alcohols can also be used. nearly all of these compounds are known to be fermentation products of anaerobic bacterial 150 luptakova, a. et al. degradation of carbohydrates, proteins and other constituents of dead biomass. molasses, which contains a high amount of sucrose, has also been used as electron donor. therefore was studied the cultivation of srb to using the selected energetic substrates such as: calcium lactate, glycerol and whey. 1.2 negative influence of the srb metabolism in the environment and industry on the negative influence of the srb to suggest the aspect of the biologically produced hydrogen sulphide oxidation by bacteria acidithiobacillus thiooxidans to the sulphuric acid, which corrode some materials. this process is named the concrete biocorrosion or the concrete biodeterioration. it is the term for the concrete destruction by biogenic acid (e.g. biogenic sulphuric acid) that is generated predominantly by the sulphur-oxidising bacteria genera acidithiobacillus thiooxidans and sulphate-reducing bacteria genera desulfovibrio through diverse mechanisms (okabe et al., 2007). the concrete biodeterioration has a serious economic impact worldwide, especially when the replacement or repair of municipal sewer systems is required. in sewer systems and wastewater treatment facilities where high concentrations of hydrogen sulphide, moisture, and oxygen are present in the atmosphere, the deterioration of concrete is caused mainly by biogenic sulphuric acid. roberts et al. described the general mechanism for the sulphuric acid caused corrosion of sewer systems (robertset et al., 2002). in the first step, hydrogen sulphide is produced by srb under anaerobic conditions in sewer pipes. this one enters the sewer atmosphere by volatilization and dissolves in the condensate on the sewer crown. finally, sulphur-oxidizing bacteria acidithiobacillus thiooxidans oxidize the dissolved hydrogen sulphide and other sulphur compounds to sulphuric acid, which corrodes the concrete. 2. materials and methods 2.1 positive influence of the srb metabolism in the environment and industry microorganisms the cultures of srb (desulfovibrio desulphuricans) were used, which was isolated from the potable mineral water (gajdovka spring, slovakia). bacteria desulfovibrio desulphuricans were selected from the mixed cultures by the modified dilution method (karavajko et al., 1988). cultivation of sulphate-reducing bacteria series of cultivation tests were studied in the hermetically closed glass jar at 30 °c, during 10 days under anaerobic conditions. the anaerobiosis was generated by an inert gas (n2) and chemically with sodium thioglycollate. the total volume of filling solution with ph 7.5 consisted from 50ml of the bacterial cultures and 400 ml the classical selective nutrient medium dsm-63 postgate′s c (the energetic substrate for the growth of srb was sodium lactate (dl -na)) or its the modifications. the basis of this modification was the dlnova biotechnologica 9-2 (2009) 151 na substitution by the adequate amount of chosen substrates: the calcium lactate (dlca), the glycerol (gly) and the whey (srv). sodium lactate, calcium lactate and glycerol were used in the form of chemicals with the analytical grade. whey was bought in the chemist (i.e. non analytical grade). analytical procedures a turbidimetric method was used to measure the sulphate concentration. a glass ph electrode combined with the reference ag/agcl electrode was used to measure ph by digital phmeter gprt 144 agl (germany). 2.2 negative influence of the srb metabolism in the environment and industry microorganisms for the experiments were used two types of bacterial cultures: sulphate-reducing bacteria desulfovibrio desulphuricans and sulphur-oxidising bacteria acidithiobacillus thiooxidans. bacteria desulfovibrio desulphuricans were isolated from the potable mineral water (gajdovka spring, slovakia). the srb were selected from the mixed cultures by the modified dilution method (karavajko et al., 1988). bacteria acidithiobacillus thiooxidans were isolated from the acid mine water (the shaft pech, the deposit smolník, slovakia). the isolation and following cultivation of the acidithiobacillus thiooxidans clean bacterial culture was realised using waksman´s and joffe´s selective nutrient medium by the plate dilution method (karavajko et al., 1988). concrete specimens – concrete samples in the cylinder form (25 mm diameter and 20 mm height) were taken from pipes produced using the normal industrial processes. before experiments samples were sterilized in 70% ethanol and dried in oven at 60ºc to constant weight. concrete biodeterioration – the glass desiccator was used as reactor. concrete samples were put into reactor and inserted into four containers: the first was filled by waste water (ww), the second by acid mine drainage (amd), the third by nutrient medium for acidithiobacillus thiooxidans (nm) and the fourth by distillated water (dv). the reactor was interconnected with the flask filled by bacterial cultures desulfovibrio desulphuricans and the flask filled by cadmium acetate for to capture of h2s. concrete samples were every 7 days inoculated by acidithiobacillus thiooxidans and in the same period the change of nutrient medium for desulfovibrio desulphuricans were realized during 90 days. methods – the disruption and damages of the concrete surface were investigated by stereomicroscopy, atomic force microscopy (afm) and electron microscopy. mineralogical stereomicroscope stm 723 zoom with the combination of digital camera olympus – c-770 ultra zoom was used for upper (not immersed) part concrete samples study because of noticeable changes in structure of concrete. afm (jpk instruments, germany) was used for both (immersed and not immersed) parts of concrete samples investigation. a nanowizardii contant mode afm was used to image the surface of concrete samples. compounds precipitated onto samples surface were investigated by scanning electron microscopy (sem). the qualitative analyses of precipitates from samples surface were investigated by energy dispersive spectrometry (eds) analysis. 152 luptakova, a. et al. 3. results and discussion 3.1 positive influence of the srb metabolism in the environment and industry the growth of srb in all liquid mediums (without abiotic controls) was demonstrated by the sensorial detection of h2s typical odour, the srb presence by the origin of fes according following reaction (2), fe2+ is standard compound of nutrient medium dsm-63 postgate′s c: fe2+ + h2s ⎯→fes + 2h + (2) during experiments were observed the decrease of the sulphates concentration. the formation of black precipitates, the sensorial detection of classical strong h2s smell and the decrease of sulphate concentration were not observed in the abiotic control. it confirms that aforesaid changes were caused by the bacterial metabolism of srb. fig. 2 documents, that all used substrates are suitable alternative substitutes. in the regard to the amount of reduced sulphates the calcium lactate is the best. 0 20 40 60 80 100 dl-na dl-ca gly srv a m on ut o f re du ce d su lp ha te s [% ] fig. 2. comparison of reduced sulphates amount with srb by using substrates: sodium lactate (dl-na), calcium lactate (dl-ca), glycerol (gly), whey (srv). 3.2 negative influence of the srb metabolism in the environment and industry the most considerable biodeterioration of concrete was observed on samples immerged into acid mine drainage. in the first instance the changes of concrete samples structures were observed by stereomicroscopy. fig. 3 illustrates a surface of concrete specimen before experiment. fig. 4 shows the visible surface changes after 90 days sulfuretum simulation of samples taken from amd. for detailed study of concrete samples surface changes afm method was used. in fig. 5 the surface of concrete sample before experiment is shown. the surface is quite smooth, with the low roughness. fig. 6 demonstrates the image of samples taken from amd after 90 days sulfuretum simulation. on figure is visible that the roughness increased and some aggregates were fallen out of concrete surface. nova biotechnologica 9-2 (2009) 153 fig. 3. smooth surface filling among aggregates before experiment. stereomicroscopy. magnification of image 20 x 4.5 fig. 4. considerable biodeterioration of concrete sample surface after 90 days, samples taken from amd. stereomicroscopy. magnification of image 20 x 4.5 fig. 5. surface of concrete sample before experiment. atomic force microscopy. fig. 6. amd immersed part of concrete sample atomic force microscopy. during the experiment the ph values of leachate amd were increasing up 7.03. depends on ph the lath-shaped crystals of ettringit (3cao.al2o3.3caso4.32h2o) were on the surface observed as shown on fig. 7 and 8. the ettringite is secondarily produced under alkaline conditions after exposure to sulphate (mori et al., 1992). fig. 7. image of ettringit crystals precipitated on the concrete sample surface underwater into amd (sem). fig. 8. eds qualitative ettringite analysis. 4. conclusion experimental studies of the part “positive influence of the srb metabolism in the environment and industry” confirm that all chosen substrates calcium lactate, glycerol and whey are suitable alternative substrates of sodium lactate for the bacterial 154 luptakova, a. et al. sulphate-reduction. in the regard to the efficiency of bacterial sulphate reduction the calcium lactate is the best. efficiency of bacterial sulphate reduction in the case of whey was less compared with calcium lactate. but in experiments was used reclaimed and dried whey. consequently next experiments will orient on the application of whey from dairy works. it is the dayiring industry waste with low cost. experimental studies of the part “negative influence of the srb metabolism in the environment and industry” confirmed visible changes of concrete samples surface structure by the bacteria attack. concrete samples were under biogenic sulphuric acid influence corroded. the most considerable biocorrosion was observed on samples immerged into acid mine drainage. experiment simultaneous effect of bacteria acidithiobacillus thiooxidans and desulfovibrio desulphuricans, which were the source of h2s has proven. crystals participated on concrete surface was identified as ettringite. acknowledgment: this work was supported by the slovak research and development agency under the contract no. apvv-51-027705 and grant agency vega for project no. 2/0075/08. i also thank university of duisburg-essen, biofilm centre, aquatic biotechnology, dusburg, germany especially group of prof. wolfgang sand for opportunity to work with afm. references dvorak, d.h et al.: treatment of metal-contaminated water using bacterial sulfate reduction: results from pilot-scale reactors. biotechnol. bioeng., 40, 1992, 609-16. karavajko, g.i., rossi, g., agate, a.d., groudev, s.n. and avakyan, z.a.: biogeotechnology of metals. centre of projects gknt, moscow, 1988, 350 pp. mori, t.: et al.: interactions of nutrients, moisture and ph on microbial corrosion of concrete sewer pipes. water res., 26, 1992, 29-37. okabe, s., odagiri, m., ito, t., and satoh, h.: succession of sulfur-oxidizing bacteria in the microbial community on corroding concrete in sewer systems. appl. environ. microbiol., 73, 2007, 971-980. rehm, h.j., reed, g.: biotechnology, vol. 6b, verlag chemie gmbh, weuheim, 1981, 473-475. roberts, d. j., nica, d., zuo, g., and davis, j.l.: quantifying microbially induced deterioration of concrete: initial studies. int. biodeterior. biodegrad., 49, 2002, 227-234. tabak, h.h., scharp, r., burckle, j., kawaharai, f.k., govind, r., advances in biotreatment of acid mine drainage and biorecovery of metals: 1. metal precipitation for recovery and recycle. biodegradation, 14, 2003, 423-436. microsoft word vrtoch nb 9-2.doc nova biotechnologica 9-2 (2009) 199 linear and non-linear regression analysis for the biosorption kinetics of methylene blue ľuboš vrtoch, jozef augustín department of biotechnology, university of ss cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (vrtochl@ucm.sk) abstract: the nonviable biomass of rhizopus sp. r-18, penicillium candidum and penicillium chrysogenum was studied for biosorption of methylene blue (mb). the sorption of mb was studied be performing batch kinetic experiments. kinetic measurements showed that sorption of mb reached equilibrium in 4 hours. the batch sorption models, based on a pseudo-first, pseudo-second and pseudo-nth order were applied to predict the rate constant of sorption and the equilibrium capacity. the linear and nonlinear least-square methods were used to obtain the kinetic parameters. the best-fit model was identified using statistic analysis. the results showed that both linear and nonlinear form of pseudosecond order expression could be used to fit the experimental data but nonlinear method may be a better way to obtain the desired parameters. as well the pseudo n-th order kinetic model was successfully applied to the kinetic data. the order (n) of adsorption reaction was found for all employed biosorbents: for rhizopus sp. r-18 it had value 3.1, p. candidum 3.0 and p. chrysogenum 3.8. key words: biosorption, kinetics, methylene blue, regression analysis, fungi 1. introduction environmental pollution due to technological development is one of the most important problems than men have to face. biosorption as a separation process has aroused considerable interest during recent years. in wastewater treatment, biosorption is regarded as one of the most potent techniques for the removal of dyes (aksu, 2005). two important physico-chemical aspects for evaluation of a sorption process as a unit operation are the equilibrium of the sorption and the sorption kinetics (ho et al., 2000). the nature of sorption process will depend on physical or chemical characteristics of the adsorbent systems and also on the system conditions at which sorption processes may include ion exchange, chelation, physical and chemical sorption. predicting the rate at which the pollutants removal takes place in a given solid/solution system is one of the most crucial factors for the effective sorption system design. chemical kinetics explains how fast the rate of chemical reaction occurs and also the factors affecting the reaction rate. several researchers have used different kinetic models to predict the mechanism involved in the sorption process. these models can be divided into two main types: diffusion based models and reaction based models (al-degs et al., 2006; svilović and stipišić, 2009). in the present study, we used reaction based models, namely lagergren pseudo-first order kinetic model, pseudo-second order model and pseudo-nth order kinetic model for prediction of batch sorption kinetics. in the present study, a comparison between linear and non-linear regression method has been made to predict the best sorption kinetics and also to obtain the kinetic parameters. 200 vrtoch, ľ. and augustín, j. 2. materials and methods 2.1 biosorbents and absorbate three different fungi were used as biosorbents, rhizopus sp. r-18, penicillium candidum and penicillium chrysogenum. the rhizopus sp. r-18 and p. candidum were obtained from the collection of microorganisms, ss. cyril and methodius university, trnava, slovakia. p. chrysogenum biomass in paste form was obtained from local antibiotics producing plant. the rhizopus and p. candidum biomass was obtained by cultivation in the czapek-dox broth. after 7 days the biomass was harvested, washed with distilled water and dried at 60°c for 24 h. subsequently, dried biomass was powdered by using mortar and pestle. the powder was sieved to obtain the particle size fraction below 0.31 mm. in a similar manner was utilized p. chrysogenum biomass, too. the absorbate used in all the experiments was methylene blue (basic blue 9), a cationic dye. the stock solution of mb was prepared by dissolving 0.5 g of substance in one liter of distilled water. all working solutions of desired concentrations were prepared by diluting the stock solution with distilled water. 2.2 sorption kinetic experiments kinetic experiments were conducted on the rotary shaker with constant agitation speed of 200 rpm, using 50 ml erlenmeyer flasks containing 10 ml mb solutions 200 mg.l-1, ph 6.0 and 0.03 g of each type of biosorbent for 360 min. samples were drawn from the mixture at pre-determined time intervals for analysis. the dye concentration in supernatants (3 000 rpm) were estimated spectrophotometrically at 665 nm. 2.3 regression analysis, goodness-of-fit measure and model comparison the kinetic parameters were evaluated by linear and/or non-linear regression analysis by using qc.expert® 3.1 and originpro® 7.0. in the present study, several error analysis methods were used in order to confirm the best-fitting kinetic model, namely the coefficient of determination (r2), the sum of the squares of the errors (sse), the sum of the absolute errors (sae) and chi-square analysis (reduced χ2). we used one statistical approach for comparing models: akaike’s information criterion (aic). 3. results and discussion 3.1 linear regression analysis a commonly used model for describing sorption kinetic is the pseudo-second order kinetic model. because this kinetic model is non-linear, fitting this model to measured data requires a “trial and error” approach. alternatively, a linearized version of this model (at least four different version exist -) can be used (table 1). a limitation to nova biotechnologica 9-2 (2009) 201 this approach, however, is that the transformation of data required for linearization can result in modifications of error structure, introduction of error into the independent variable, and alteration of the weight placed on each data point, often leading to difference in fitted parameter values between linear and non-linear version of the pseudo-second order kinetic model (kumar, 2006; bolster and hornberger, 2007). table 2 shows kinetic parameters obtained by using linear equations of the four pseudo-second order kinetic models. table 1. pseudo second-order kinetics and their linear forms, qt, qe-the amount of dye adsorbed at any time t and at equilibrium, respectively; k2-the rate constant of sorption (ho, 2006). kinetic model linear form plot parameters model 1 tqqkq t eet 11 2 2 += )(/ tfqt t = qe = 1/slope k2 = slope2/intercept h = 1/intercept model 2 eet qtqkq 11 ) 1 ( 1 2 2 += )/1(/1 tfqt = qe= 1/intercept k2= intercept2/slope h = 1/slope model 3 t q qk qq t e et ) 1 ( 2 −= )/( tqfq tt = qe = intercept k2 = -1/(slope*intercept) h = intercept/slope model 4 e te e t q qqk qk t q 222 2 −= )(/ tt qftq = qe= intercept/slope k2 = slope2/intercept h = intercept table 2. pseudo-second order kinetic parameters obtained by using the linear methods (qt and qe, mg.g-1; k2, g.mg-1.min-1; t, min). biosorbent model 1 k2* qe r2 model 2 k2 qe r2 model 3 k2 qe r2 model 4 k2 qe r2 rhizopus sp. r-18 -0.07 45.5 0.999 0.05 45.5 0.904 0.05 46.0 0.836 0.04 46.4 0.836 penicillium candidum 0.02 42.6 0.999 0.08 42.0 0.975 0.08 41.9 0.929 0.08 42.0 0.929 penicillium chrysogenum 0.008 40.0 0.999 0.03 37.9 0.953 0.03 38.2 0.896 0.02 38.6 0.896 *statistic non-significant parameters on the significance level α=0.05 for all biosorbents tested, the best fit was achieved by model 1. this model is statistically significant with a coefficient of determination near one for all three biosorbents. but this model gives statistically non-significant regression coefficients (in this case it is the intercepts) at a significance level of α=0.05 (statistics data are not shown). because of this, the model is not appropriate for the description of the experimental data. in the table 2 we can see that model 2 gives the best fits for all three biosorbents. it was observed that the kinetic parameters obtained from the three linear forms of pseudo-second order expressions were (models 2-4) very similar. from the aforementioned we can conclude that for description of kinetics of the sorption of methylene blue by the biomass of the fungi all three linear equations of the pseudosecond order can be used. 202 vrtoch, ľ. and augustín, j. 3.2 non-linear regression analysis in the present study we used reaction based kinetic models, namely lagergren pseudo-first order kinetic model, pseudo-second order model and pseudo-nth order kinetic model (table 3). the kinetic parameters were determined using non-linear regression analysis by using gauss-newton algorithm. table 4 shows obtained the predicted kinetic parameters. from the comparison of aic and r2 it is clear that the pseudo-nth order kinetic model gives the best fit for all biosorbents. table 3. kinetic models employed in this paper and their differential and non-linear equation forms. kinetic model differential equation non-linear equation reference pseudo-first order )(1 te t qqk dt dq −= )1( 1 tk et eqq −−= lagergren, 1898 pseudo-second order 2 2 )( te t qqk dt dq −= e e t tqk tkq q 2 2 2 1+ = ho, mckay, 1998 pseudo-nth order n ten t qqk dt dq )( −= )1/(11 ])1([ nn n eet tknqqq −− −−−= özer, 2007 table 4. kinetic model parameters obtained by using non-linear regression analysis (1-rhizopus sp. r-18; 2p. candidum; 3-p. chrysogenum; k1, k2 and kn are rate constants; n is the order of reaction; qe is the amount of dye adsorbed onto biosorbent at equilibrium (mg.g-1). bio sorbent pseudo-first order kinetic k1 qe r2 aic pseudo-second order kinetic k2 qe r2 aic pseudo-nth order kinetic kn qe n r2 aic 1. 1.7 45.2 0.962 26.1 0.05 46.1 0.984 15.4 0.001 48.0 3.1 0.991 11.0 2. 1.6 41.1 0.998 10.0 0.08 41.9 0.996 -4.9 0.004 43.1 3.0 0.999 -22.0 3. 0.5 37.2 0.927 111 0.02 38.7 0.981 14.3 5.10-7 43.0 3.8 0.997 -2.31 0 50 100 150 200 250 300 350 400 0 10 20 30 40 50 60 rhizopus sp. r-18 ( ) penicillium candidum ( ) penicillium chrysogenum ( ) q t [ m g. g1 ] t [min] a 0 50 100 150 200 250 300 350 400 0 10 20 30 40 50 60 rhizopus sp. r-18 ( ) penicillium candidum ( ) penicillium chrysogenum ( ) q t [ m g. g1 ] t [min] b fig. 1. pseudo-first (a) and pseudo-second (b) order kinetics by non-linear method by using gauss-newton algorithm and experimental kinetics for the sorption of methylene blue onto three different biosorbents. the order of adsorption reaction (n) was found to be between 3.0 and 3.8. from the aforementioned we can conclude that the adsorption of methylene blue by the biomass of the fungi is governed by a reaction order higher than 2. but the model gives nova biotechnologica 9-2 (2009) 203 statistically insignificant coefficients (in the case it is kn) for all three biosorbents (statistics data are not shown). this excludes the use of this model for the description of our experimental data. we can’t forget that this model, contrary to the two others, is a three parameter model. on the one hand the higher number of parameters gives us a better fit, but on the other hand the statistical significance of the regression coefficients is lower. one possible way to increase the statistical significance of the regression coefficients is to increase the number of experimental measurements. because of this, in this work, the pseudo-second order kinetic model seems to be the best fitting for all three biosorbents (fig. 1). 3.3 comparison of linear and non-linear regression analysis to compare model fits between the different linearizations and the nonlinear pseudo-second order kinetic model equation, the best-fit lines for each linearization were transformed back to sorbed concentrations (qt) and error analysis as well as aic were using for comparison goodness-of-fit measure between the different linearizations and the non-linear equation (table 5). table 5. statistic parameters for the nonlinear and linearized pseudo-second order kinetic equations for three different biosorbents. kinetic model biosorbent statistic* parameter model 2 model 3 model 4 nonlinear model sse 32.4 31.8 31.8 30.9 sae 13.6 13.4 13.9 13.8 red χ2 0.086 0.084 0.089 0.084 rhizopus sp. r-18 aic 20.9 20.7 20.7 20.4 sse 5.71 5.49 5.71 5.72 sae 6.1 5.90 6.10 6.23 red χ2 0.016 0.015 0.016 0.016 penicillium candidum aic 0.088 -0.38 0.088 0.115 sse 42.5 42.4 28.4 28.1 sae 18.2 17.6 14.2 14.2 red χ2 0.160 0.165 0.126 0.124 penicillium chrysogenum aic 24.3 24.3 19.9 19.8 * ssethe sum of the squares of the errors; saethe sum of the absolute errors; red χ2reduced chi-square analysis; aicakaike’s information criterion . from the statistical analysis we can conclude, that the linearized models 2, 3 and 4 give us very similar fits to the non-linear models. the best example for this is biosorbent p. candidum with an aic value of -0.38 (model 3). this fact can be described in the following way: the linear regression analysis was done using the leastsquares methods. in order to use this method some conditions known from statistics must be met. if we want to use the linear forms of the kinetic equations of the pseudo204 vrtoch, ľ. and augustín, j. second order the data from the measurement must be transformed. the transformation probably changed the conditions in a positive way, and that caused that the linear models (except model 1) give very similar or better fits than the non-linear models (look at p. candidum in the table 5). as mentioned in the literature this doesn’t always have to be the case the data transformation can also negatively affect the estimate of the regressive model. therefore, the primary drawback to linearization is not the inability to provide similar parameter estimates as the nonlinear equation but rather the inability to provide poor (or good) model fits when the data don’t (or do) conform to the kinetic model. 4. conclusions the sorption of methylene blue by three fungal biosorbents was found to be rapid whereby the equilibrium was reached during one hour. the results confirmed the applicability several kinetic models, namely pseudo-second and pseudo nth order kinetic models. present investigation further showed that the search for best-fit kinetic model using linearization technique is not an appropriate technique to predict biosorption kinetics. the non-linear methods would be more appropriate techniques in predicting the biosorption kinetics. references aksu, z.: application of biosorption for the removal of organic pollutants: a review. process. biochem., 40, 2005, 997-1026. al-degs, y. s., el-barghouthi, m. i., issa, a. a., khraisheh, m. a., walker, g. m.: sorption of zn(ii), pb(ii) and co(ii) using natural sorbents: equilibrium and kinetic studies. water res., 40, 2006, 2645-2658. bolster, h. c., hornberger, m. g.: on the use of linearized langmuir equations. soil sci. soc. am. j., 71, 2007, 796-1806. ho, y. s., mckay, g.: kinetic models for the sorption of dye from aqueous solution by wood. trans. inst. chem. eng., 76, 1998, 183-191. ho, y. s., ng, j. c. y., mckay, g.: kinetics of pollutant sorption by biosorbent: review. sep. pur. methods, 29, 2000, 189-232. kumar, k. v.: linear and non-linear regression analysis for the sorption kinetics of methylene blue onto activated carbon. j. hazard. mater., 137, 2006, 1538-1544. lagergren, s.: about the theory of so-called adsorption of soluble substances. k. sven. vetenskapsakad. handl., 24, 1898, 1-39. özer, a.: removal of pb(ii) ions from aqueous solutions by sulphuric acid-treated wheat bran. j. hazard. mater., 141, 2007, 753-761. svilović, s., stipišić, d. r.: modeling batch kinetics of copper ions sorption using synthetic zeolite nax. j. hazard. mater., 2009 (in press). microsoft word maresova nb 9-3.doc nova biotechnologica 9-3 (2009) 333 influence of anionic surfactants on zn2+ and sr2+ uptake by ivy (hedera helix l.) leaves jana marešová, miroslav horník, martin pipíška, jozef augustín department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (jana.maresova@ucm.sk) abstract: surfactants are frequently used as adjuvants for improving the efficiency of foliar applied fertilizers, pesticides and other biologically active substances. in our paper we used detached leaves of ivy (hedera helix l.) for the study of the influence of anionic surfactants sodium dodecylsulfate (sds) and sodium dicyclohexyl sulfosuccinate (dcss) on zinc and strontium uptake by leaf surface and transport by radiotracer technique with 65zncl2 and 85srcl2. accumulated amounts of zn2+ and sr2+ ions by the surface of detached intact ivy leaves were 5.0 and 1.1 μg/g, respectively. ivy leaves pretreated for 24 h in 1 mm sds or dcss solutions accumulated approx. twice more zn and five time more sr than non treated leaves. pretreatment with surfactants increased mobility of zinc and strontium in leaf tissues. separate experiments showed that both sds and dcss were sorbed onto the leaf tissue reaching equilibrium within several hours of immersing leaf blades to surfactant solutions. the process can be described in terms of partition equilibria p = [c]leaf/[c]solution with log p = 1.396 within surfactant concentration studied co ≤ 100 μmol/l. the mechanism of action of surfactants on metal ion uptake is discussed. key words: ivy, h. helix l., foliar uptake, surfactants, zinc, 65zncl2, strontium, 85srcl2 1. introduction the cuticle is the main interface between plants and their environment. it covers the epidermis of all aerial primary parts of plant organs as a continuous extracellular matrix. this hydrophobic natural composite consists mainly of the biopolymers, cutin, and cuticular lipids called waxes. water-repellent cuticle or waxes on a plant surface is the major barrier to the spreading, retention and penetration of solutes (bargel et al., 2006). surfactants are frequently used as surface-acting adjuvants that improve the absorbing, emulsifying, dispersing, spreading, sticking, wetting or penetrating properties of foliar applied fertilizers. on the other hands, surfactants alone are able to accumulate in plants and to change physico-chemical properties of the leaf surface. it has been proposed that there is a requirement for surfactants to be absorbed into plant leaves at rates similar to those for the active ingredient for the best uptake results (stock et al., 1993). however, there have been only a few studies of surfactant uptake (zabkiewicz et al., 1995) compared to the multitude of studies quantifying active ingredient uptake, and all of these are reported on a percentage basis. the finding that active ingredient mass uptake can be related to initial dose applied (forster et al., 2004; nielsen et al., 2005) raises the question of whether the surfactant component of a typical spray formulation will behave in a similar fashion. our paper is a step towards addressing the question of the influence of anionic surfactants sodium dodecylsulfate (sds) and sodium dicyclohexyl sulfosuccinate (dcss) on the uptake of zn and sr as bivalent metals by the leaf surface of ivy (hedera helix l.). 334 marešová, j. et al. ivy (h. helix) is a common, easily available species, which possesses a number of advantages. the ultra-structure of leaves is well described (canet et al., 1996; gilly et al., 1997). ivy leaf cuticle was used as a model to investigate cuticle permeability (chamel, 1986). fine structure and permeability of ivy leaf cuticles in relation to foliar development and after selective chemical treatments and relationship between structure and permeability are well described (gilly et al., 1997). in our previous papers are described some properties and behavior of sulfosuccinate esters in biological systems (vrbanová et al. 1997; cserháti et al. 1997) and leaf uptake and distribution of zn ions by ivy (marešová et al., 2009). 2. materials and methods 2.1 chemicals standardized 65zncl2 solution (0.877 mbq/cm 3, 50 mg/dm3 zncl2 in 3 g/dm 3 hcl) and 85srcl2 (2.665 mbq/cm 3, 20 mg/dm3 zncl2 in 3 g/dm 3 hcl) were obtained from the czech institute for metrology, prague. sodium dodecylsulfate (sds) was obtained from sigma, sodium dicyclohexyl sulfosuccinate (dcss) from cytec corp., u.s.a. solutions were prepared in deionized water, conductivity 0.05 μs/cm, ph was adjusted with naoh. 2.2 plant material ivy branches (h. helix l.) were picked during spring months from freely grown garden vegetation. the upper part of branches were cut from a wild ivy plant, washed repeatedly in deionized water and used for experiments. leaves about 0.2 – 0.3 g of fresh weight and leaf area 11.5 12.5 cm2 were used in experiments. fig. 1. macro photo of ivy leaves in experiments. leaf blades immersed by both sides in nutrient media in petri dishes. characteristic signs: short petioles; shallow sinus; well developed veins; terminal, lateral and basal lobes. 2.3 bioaccumulation experiments pretreatment of leaves by surfactants was made by immersing of detached leaf blades into sds or dcss solution in deionized water. for metal uptake experiments leaf blades were immersed in 10 ml 25% hm medium supplemented with 5 µmol/l 65zncl2 or 85srcl2 in dishes covered with plastic lids (fig. 1.) in cultivation room at 22±2°c illuminated with artificial light (2 000 lx) in 12h/12h light/dark cycle. the following molar concentrations of salts were present in full-strength hm (mm): nova biotechnologica 9-3 (2009) 335 mgso4.7h2o – 1.5; kno3 – 4.0; cacl2 – 4.0; nah2po4.2h2o – 1.87; na2hpo4.12h2o – 0.13; feso4.7h2o – 0.06; nano3 – 4.0; nh4cl – 3.17; nh4no3 – 2.0; h3bo3 – 0.14; na2moo4.2h2o – 0.0025; mnso4.5h2o – 0.21; znso4.7h2o – 0.023; cuso4.5h2o – 0.033. 2.4 radiometric analysis a gamma spectrometric assembly using the well type scintillation detector 54bp54/2-x, nai(tl) (scionix, the netherlands) and data processing software scintivision 32 (ortec, usa) were used for 65zn and 85sr determination in leaf biomass and solutions. counting time 600 s allowed obtaining data with measurement error <2 %, which do not reflect other source of errors. 2.5 surfactant analysis anionic surfactants were determined by the mbas method (arand et. al., 1992). shortly, anionic detergents react with methylene blue to form a blue colored complex that is extracted into chloroform and blue coloration is measured at 651 nm. 3. results and discussions 3.1 uptake of surfactants by ivy (h. helix l.) leaves leaf surface of h. helix is able to accumulate anionic surfactants sds and dcss from water solutions (fig. 2.). concentration equilibrium csolid/cwater is reached at 20 °c within several hours. such slow processes are typical for partition equilibria between solution and solid materials at which the rate limiting step is the diffusion process in existing membrane systems. 0 1 2 3 4 23 24 0,0 0,5 1,0 1,5 2,0 2,5 q s d s [μ m ol /g ] time [h] c sds = 25 μ mol/dm3 c sds = 50 μ mol/dm3 c sds = 100 μ mol/dm3 fig. 2. kinetics of sds uptake (µmol/dm3, w.w.) by detached ivy leaves (h. helix l.) at 20 °c. the initial sds concentrations: 25.0 (■-■-■), 50.0 (●-●-●) and 100.0 (▲-▲-▲) µmol/dm3. leaf biomass: 40.3 ± 0.15 g/dm3 w.w. (±sd), leaf blades area 1 654 ± 58.0 cm2/dm3 (±sd). 336 marešová, j. et al. qsds and qdcss (μmol/g, d.w.) values are proportional with the initial concentration of surfactants in solution within the concentration range studied c0 ≤ 100 μmol/l (fig. 3.). the process of sds and dcss accumulation by h. helix leaf tissues can be described in terms of partition equilibria with partition coefficient log p = 1.349 for both substances. both sds and dcss contain c12 hydrocarbon part and one ionisable anionic group, what explain similar behaviour in the contact with the leaf structures. 0 25 50 75 100 0,0 0,5 1,0 1,5 2,0 2,5 q 24 [μ m ol /g ] c 0 [μmol/l] sds dcss fig. 3. uptake of sds (■) and dcss (●) by the surface of detached ivy leaves (h. helix l.), expressed as q24 (µmol/g) in dependence on the initial concentration c0 in solution. log p = 1.394 3.2 influence of pretreatment of leaves by surfactants on sr and zn uptake and distribution ivy leaves treated with sds or dcss solution accumulated higher amounts of zn2+ and sr2+ ions, comparing with non-treated control leaves. enhancement ratio er for zn and sr is shown in tab. 1. non-treated ivy leaves accumulated 4.55 times more zinc than strontium and sds-treated leaves 3.2 times more zinc than strontium. similar behavior can be expected also in the case of other bivalent metals. the effect of surfactants on metal sorption of inorganic sorbents was studied by ahn et al. (2009). sds and dcss -impregnated activated carbon sorbed cd2+ up to 0.198 mmol/g, which was more than one order of magnitude better than cd2+ sorption by activated carbon without surfactants. treating of ivy leaves with sds or dcss solution caused the increase of zinc and strontium mobility in plant tissues. as can be seen from data presented in fig. 4 both metals are transported with higher efficiency from immersed part of leaf blades to petiols and to other parts of plants. strontium and zinc foliar uptake and transfer in tomato plants (lycopersicum esculentum l.) was studied by brambilla et al. (2002). leaf to fruit transfer coefficient for 65zn was one order magnitude higher than for 85sr. nova biotechnologica 9-3 (2009) 337 tab.1. zinc and strontium uptake (μg/g, d.w.) by non treated and pretreated leaf surface of ivy (h. helix l.) after 24 h in 1.0 mmol.dm-3 sds or dcss. zn and sr uptake from 25% hm spiked with 65zncl2 (5 µmol.dm-3) or 85srcl2 (5 µmol.dm-3). uptake [μg/g] uptake [μg/g] metal non-treated sdspretreated er* non-treated dcsspretreated er* zn2+ 5.0 17.0 3.4 3.9 11.1 2.8 sr2+ 1.1 5.4 4.9 * enhancement ratio er is the ratio of the metal concentration in leaves treated with surfactants to the metal concentration in non-treated leaves. 0 2 0 4 0 6 0 8 0 q z n [n m ol /g ] n o n t r e a t e d s d s p r e t r e a t e d a 0 2 0 4 0 6 0 8 0 q s r [ nm ol /g ] n o n t r e a t e d s d s p r e t r e a t e d b fig. 4. influence of sds pretreatment on zn (a) and sr (b) uptake and distribution in ivy leaves (h. helix l.). leaves were pretreated for 24h with 1.0 mmol/dm3 sds, then immersed in 5.0 µmol/dm3 zncl2 or srcl2 in 25 % hm, spiked with 65zncl2 (114 kbq/dm3) or 85srcl2 (311 kbq/dm3) without sds. uptake via the surface of fully immersed leaves. cultivation at illumination 12h/12h light/darkness (2 000 lx), ph 5.5 and 22±2°c. data as the mean of two replicates. error bars represent standard deviation (sd) of the mean. wet weight of leaves: a. 0.29 ± 0.02 (±sd) g /10 ml, b. 0.24 ± 0.01 (±sd) g/10 ml. leaf area [cm2] – a. 10.82 ± 0.67 (±sd), b. 11.7 ± 0.36 (±sd). data of leaf blades (■■) and non immersed leaf stalks (■■) expressed as zn or sr concentration (nmol/g), d.w. 338 marešová, j. et al. to explain the effect of anionic surfactants on metal uptake by leaf surface and distribution in leaf structures will require a more detailed study. stimulating effect can be caused by the following factors: increase of capacity of polar or water path for ion transport or the decrease of viscosity of cuticular and wax structures on the leaf surface, or metal cation surfactant anion interactions improving metal mobility in the lipophilic leaf structures. riederer and schönherr (1999) showed that treating the outer surfaces of isolated cuticles of seville orange (citrus aurantium l.) and pear (pyrus communis l. cv. bartlett) leaves with a number of nonionic (polyoxyethylene) surfactants increased their permeability to water by factors ranging from 4.1 to 14.7 and from 7.2 to 152.4, respectively. none of the surfactant treatments altered the amounts or composition of waxes in the cuticles used for transport measurements. anionic surfactants can react with cations of bivalent ions. talens-alesson (2007) found that sds micelles are able to bind zn2+ ions and ions of other metals. however in all sorption experiments we used sds and dcss solutions in concentrations ≤ 1 mmol/l, what is approximately 7 times lower concentration than critical concentration of micelle formation cmc = 6.9 mm (nakagarajan, 2003). according to kirkwood (1999) the physico-chemical properties of the cuticle may affect the rate and efficiency of cuticle permeation. the permeation of organic solutes is influenced by their solubility characteristics as indicated by octanol/water partition coefficients (log kow) and cuticle/water partition coefficient (log kcw). penetration of hydrophillic organic compounds (low log kow) may be enhanced by hydration of the cuticle, while transcuticular transport of non-polar organic solutes (high log kow) is increased by factors which reduce the wax viscosity. 4. conclusions anionic surfactants sodium dodecylsulfate c12h25oso3na and sodium dicyclohexyl sulfosuccinate c16h25o4so3na are sorbed by ivy leaves (hedera helix l.) immersed in surfactant water solution. pretreatment of leaves by surfactants increases their capacity to sorb zn2+ and sr2+ ions. obtained data are discussed from the point of view of the effect of surfactants on the leaf structures and metal uptake. references ahn, ch.k., park, d., woo, s.h., park, j.m.: removal of cationic heavy metals from aqueous solution by activated carbon impregnated with anionic surfactants. j. hazard. mater., 164, 2009, 1130-1136. arand, m., friedberg, t., oesch, f.: colorimetric quantitation of trace amounts of sodium lauryl sulfate in the presence of nucleic acids and proteins. anal. biochem., 207, 1992, 73-75. bargel, h., koch, k., cerman, z., neihuis, ch.: structure-function relationships of the plant cuticle and cuticular waxes: a smart material? funct. plant biol., 33, 2006, 893-910. nova biotechnologica 9-3 (2009) 339 brambilla, m., fortunati, p., carini, f.: foliar and root uptake of 134cs, 85sr and 65zn in processing tomato plats (lycopersicon esculentum mill.). j. environ. radioact., 60, 2002, 351-363. canet, d., rohr, r., chamel, a., guillain, f.: atomic force microscopy study of isolated ivy leaf cuticles observed directly and after embeding in epon®. new phytol., 134, 1996, 571-577. cserháti, t., csiktusnádi kiss, g., augustín, j.: the use of principal component analysis for the study of the interaction of anionic surfactants with hydroxypropyl-β-cyclodextrin. j. incl. macro. phenom., 33, 1997, 123-133. chamel, a.: foliar absorption of herbicides: study of the cuticular penetration using isolated cuticles. physiol. veg., 24, 1986, 491–508. forster, w.a., zabkiewicz, j.a., reiderer, m.: mechanisms of cuticular uptake of xenobiotics into living plants: 1. influence of xenobiotic dose on the uptake of three model compounds, applied in the absence and presence of surfactants into chenopodium album, hedera helix and stephanotis floribunda leaves. pest manag. sci., 60, 2004, 1105-1113. gilly, c., rohr, r., chamel, a.: ultrastructure and radiolabelling of leaf cuticles from ivy (hedera helix l.) plants in vitro and during ex vitro acclimatization. ann. bot., 80, 1997, 139-145. kirkwood, r.c.: recent developments in our understanding of the plant cuticle as barrier to the foliar uptake of pesticides. pestic. sci., 55, 1999, 69-77. marešová, j., horník, m., pipíška, m., augustín, j.: zinc uptake and distribution in ivy (hedera helix l.) leaves. nova biotechnol., 9, 2009, 73-82. nakagarajan, r.: theory of micelle formation. quantitative approach to predict micellar properties from surfactant molecular structure. in: esumi, k., minoru, v. (eds.): surfactant science series: structure-performance relationships in surfactants. marcel dekker, ag., basel, 112, 2003, 1-110. nielsen, c.m., stelle, k.d., forster, w.a., zabkiewicz, j.a.: influence of dose and molecular weight on foliar mass uptake of surfactant. new zealand plant protect., 58, 2005, 174-178. riederer, m., schönherr, j.: effects of surfactants on water permeability of isolated plant cuticles and on the composition of their cuticular waxes. pestic. sci., 29, 1999, 85–94. stock, d., holloway, p.j., grayson, b.t., whitehouse, p.: development of a predictive uptake model to rationalize selection of polyoxythylene surfactant adjuvants for foliage-applied agrochemicals. pestic. sci., 37, 1993, 233-245. talens-alesson, f.i.: behaviour of sds micelles bound to mixtures of divalent and trivalent cations during ultrafiltration. colloid. surface. a, 299, 2007, 169-179. vrbanová, a., prokšová, m., augustín, j., ziegler, w.: function and parameters of sorption and primary biodegradation of a series of alkyl sulphosuccinates by comamonas errigena n3h. biologia, 52, 1997, 747-751. zabkiewicz, j.a., forster, w.a., steele, k.d., liu, z.q.: comparison of uptake into field bean (vicia faba) and wheat (triticum aestivum) of organosilicone and non-silicone surfactants. in: gaskin, r.e. (eds.) adjuvants for agrochemicals. rotorua, new zealand, 1995, 219–224. microsoft word guldanova et al_nb 2010.doc nova biotechnologica 10-2 (2010) 95 bioaccumulation and distribution of 137cs in tobacco cultivated under hydroponic conditions jana guldanová, miroslav horník, jana marešová, martin pipíška, jozef augustín department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (hornikm@ucm.sk) abstract: potential of plants to uptake metals from soil solution can be successfully applied for removal of long-lived radionuclides such as radiostrontium 90sr or radiocaesium 137cs. this work deals with bioaccumulation of cs in tobacco plants (nicotiana tabacum l.) hydroponically grown in diluted hoagland media (hm) spiked with 137cscl. speciation analysis using a program visual minteq showed, that more than 97% of caesium in hm occurred in the form of cs+ ions. we found that bioaccumulation of cs significantly decreased from the value 100% to the value 20% removing of cs from media after 8 days cultivation of plants with increasing hm concentration. however, the concentration ratio (cr) [cs]shoot : [cs]root increased with increasing hm concentration from the value 0.10 to the value 0.85. bioaccumulation of cs by tobacco plants significantly decreased with increasing cscl concentration in media from the value 95% found at concentration of cscl 10 µmol/dm3 to the value 44% at concentration of cscl 1 000 µmol/dm3. we did not found visual symptoms of cs toxicity on plants after 8 days cultivation or significant differences in growth rate or transpiration activity at cscl concentration up to 0.2 mm. however, at > 0.2 mm cscl concentration the decrease of growth rate and necrosis of young leaves or die-back of leaves (> 2 mm cscl) were observed. the cr ([cs]shoot : [cs]root) increased with increasing concentration of cscl (10 – 1 000 µmol/dm3) in media from the value 0.10 to the value 0.40. the obtained data suggest that fast growing plant species with high biomass production like tobacco might be a suitable in phytoextraction or rhizofiltration technologies used for 137cs removing from environment. key words: 137cs, radiocaesium, bioaccumulation, tobacco, nicotiana tabacum, phytoremediation 1. introduction the presence of radionuclides in soil and water exposes the stability of ecosystem and poses serious risk to human health through the food chain. radionuclides can enter to the environment from natural or artificial sources. the artificial sources of radionuclides in the environment represent: testing of nuclear weapons, nuclear waste disposal, accidents resulting from nuclear power generation as well as manipulation with nuclear fuel (see e.g. hu et al., 2010). contamination caused by long-lived radionuclides, particularly 137cs and 90sr, poses a long-term environmental problem. caesium can exist in at least 39 isotopes forms, mostly within the range of atomic masses 112cs to 151cs. of these, the two cs isotopes are of environmental concern owing to their rapid incorporation into biological systems as physico-chemical analogue of potassium, their relatively long half-lives and emissions of β and γ-radiation during decay, are 134cs (τ = 2.06 y) and 137cs (τ = 30.2 y) (white and broadley, 2000). 96 guldanová, j. et al. from the monograph kabata-pendias and pendias (2001) results that cs contents in world soils are in the range from several tenths to tens of mg/kg. in some canadian soils range from 0.3 to 5 mg/kg, in bulgarian soils from 2 to 17 mg/kg, in ukrainian soils the mean value is 3.2 mg/kg. the background values of cs contents in slovakian soils are 5 mg/kg, similarly to the other world soils (čurlík and šefčík, 1999). as the result of chernobyl accident, in ukraine alone, over 260 000 km2 have received more than 40 gbq/km2 of contamination with 137cs (dushenkov, 2003). median values for the inventory of 137cs in topsoil of belarus, about a decade after the chernobyl accident, ranged from 103 to 1 500 kbq/m2, and the highest concentration was as much as 600-times higher than the pre-accident value (kagan and kadatsky, 1996). these authors estimated that about 90% of the inventory of 137cs is contained in the top 3 to 7 cm soil layer. nowadays, it can be expected that the radionuclide contamination is lower and significantly decrease with time. kashparov et al. (2003) found that in the year 2000 radionuclide contamination within the 30-km chernobyl zone was 2 – 3 times lower than the previous estimates. however, the release of new contamination still persists. also, climate/environmental changes e.g. global warming (dowdall et al., 2008) can play a possible role in distribution of radionuclides in soils as well as in soil-to-plant transfer processes. remediation of areas contaminated with toxic metals or radionuclides represent one of the objects of recent science research. remediation of contaminated areas by traditional physico-chemical methods e.g. precipitation, coagulation, soil leaching, flocculation, ion-exchange, reverse osmosis, adsorption, ultrafiltration, using of disperse and chelating agents, is financially unbearable and in many cases also ecologically unacceptable. bioremediation as a using of biological systems (microorganisms, algae and plants) in remediation processes represent new potential approach to solve the problems with toxic metals and radionuclides contaminations. phytoremediation technologies involve the processes, which use of plants for remediation of contaminated environment. several comprehensive reviews (see over the past five years e.g. gardea-torresdey et al., 2005; ghosh and singh, 2005; prasad et al., 2005; pilon-smits, 2005; leduc and terry, 2005; audet and charest, 2006; leštan et al., 2008; vangronsveld et al., 2009 and wenzel, 2009) and monographs (raskin and ensley, 2000; terry and bañuelos, 2000; mackova et al., 2006; morel et al., 2006, singh and tripathi, 2007; willey, 2007) have been written, summarizing many important aspects of this technology. these reviews also give general guidance and recommendations for applying phytoremediation in practice. plants ideal for phytoremediation of contaminated soils should fulfill some requirements: fast growing and high biomass (more than 8 tons/ha for year), extensive root system, easy to harvest, ability to tolerate and accumulate of high amount of metals in the aboveground parts (more than 1 g/kg). however, plants are able to metal uptake only from soil solution. tobacco was selected as a model of plant mainly for the reason its fast-growth rate and high biomass production over 12 tons/ha (keller et al., 2003). also, tobacco is nova biotechnologica 10-2 (2010) 97 important agricultural plant, which represent important source mainly of cd exposure from smoking cigarettes (who, 1992). in our previous papers we study cobalt 60co (horník et al., 2006; vrtoch et al., 2007) and caesium 137cs (vrtoch et al., 2006) bioaccumulation by tobacco as well as effect of complexing agents on cd, co and zn bioaccumulation and distribution in tissues of tobacco plants (horník et al., 2009a; 2009b; 2009c). in this work we present results from studying of effect of macroand microelements as well as cscl concentration on bioaccumulation and translocation of caesium in tissues of tobacco (nicotiana tabacum l.) cultivated in nutrient media spiked with 137cscl. 2. materials and methods 2.1 plant material seeds of tobacco (nicotiana tabacum l.) were germinated and grown at photoperiod 12h light/12 h dark (illumination 2 000 lx) and 22°c in pots filled with granulated perlite as an inert carrier. plants were watered with diluted solution of hoagland medium (hoagland, 1920). the composition of the full strength (100%) nutrient solution was (mmol/dm3): mgso4.7h2o – 1.5; kno3 – 4.0; cacl2 – 4.0; nah2po4.2h2o – 2.1; na2hpo4.12h2o – 0.13; feso4.7h2o – 6.4.10 -2; nano3 – 4.0; nh4cl – 4.0; nh4no3 – 2.0; h3bo3 – 0.14; na2moo4.2h2o – 2.5.10 -4; mnso4.5h2o – 2.1.10 -2; znso4.7h2o – 2.3.10 -3; cuso4.5h2o – 3.2.10 -3 (ph 6). after 4 weeks pre-cultivation seedlings were gently removed from perlite, roots were washed by deionized water for removing of perlite granules and used in bioaccumulation experiments. 2.2 bioaccumulation experiments uniformed plants (approx. 15 cm height and 7 g wet weight) from pre-cultivation phase were transferred into series of 250 ml erlenmeyer flask with 120 ml of 25% hoagland medium with the known 133cscl concentration and added radioactivity of 137cscl as a tracer. the ph of nutrient solutions was adjusted to 6.0 using 1 m naoh solution. all flasks were covered with black foil to protect plant roots against the lights. plants were hydroponically cultivated during 8 days in triplicate series under the same conditions as was mentioned in pre-cultivation phase without the addition of radionuclides. in time intervals aliquot samples of nutrient solution were taken, 137cs radioactivity was measured by gammaspectrometry and subsequently samples were returned into the cultivation medium. at the same time reduction of nutrient medium volume caused by transpiration of water was recorded and the difference was compensated by glass balls. at the end of the experiments plants were removed from nutrient solutions, roots were carefully washed in deionized water and incorporated 137cs radioactivity in roots, stems and leaves was measured. plant parts were dried at 60°c for 48 hours and dry weights were determined. 98 guldanová, j. et al. growth value (gv) was calculated using the following equation (1): 0 0 m mm gv t − = (1) where mt or m0 are fresh weight of plants at the start or end of experiments, respectively. 2.3 radiometric analysis for determination of 137cs radioactivity in plant parts and nutrient solution gammaspectrometric scintillation detectors 54bp54/2-x and 76bp76/3 with well type crystal nai(tl) (scionix, nl) and data processing software scintivision32 (ortec, usa) were used. a library of radionuclides was built by selecting characteristic γ-ray peaks (88.04 kev for 109cd, 661.66 kev for 137cs, 834.81 kev for 54mn and 1115.52 kev for 65zn) for energy and efficiency calibration. standardized solution of 137cs in the form of 137cscl (5.723 mbq/cm3, 20 mg/dm3 cscl in 3 g/dm 3 hcl) was provided from czech metrological institute (prague, cr). 2.4 speciation modelling the percentage occurrence of individual cs ionic forms in nutrient solution was determine by equilibrium speciation modelling software visual minteq (ver. 2.53) obtained from gustafsson (2010). this speciation modelling program allows the calculation of metals speciation in solutions as a function of total salt concentration, solution ph, ionic strength and temperature. 2.5 statistical analysis all analytical determinations were performed in triplicate. statistical significance of differences in calculated values of cs bioaccumulation in plant tissues were evaluated by kruskal-wallis non-parametric test and analysis of variance, respectively. this was followed by multiple range test to ascertain differences between individual groups. the level of significance was 0.05 in all cases. origin 7.0 (originlab corp., usa) and statgraphics centurion ver. 15 (statpoint, inc., usa) were used for graphing and statistical analyses, respectively. 3. results and discussion necessary assumption for successful remediation of contaminated soils with radiocaesium is ability of plants to accumulate significant amount of radioactivity from soil to aboveground part of plants. however, irreversible incorporation of 137cs onto clay minerals is a limiting factor of these processes. sorption reactions in soil-water boundary line are important phenomena, which determine the behaviour, bioavailability and transport of cs in environment. caesium has very low hydratation nova biotechnologica 10-2 (2010) 99 energy, thus electrostatic attraction between cs+ ions and clay particles is high and therefore cs ions are preferentially bound onto clay particles. however, sesquioxides mainly organically bound aluminium and iron oxides are responsible also for cs sorption in soils (chiang et al., 2010). despite these facts, phytoremediation of soil could be effective method of soil decontamination during the first years after radiocaesium penetration into the soil (willey, 2007; willey and collins, 2007). the results of laboratory experiments with plants can not be direct applied for phytoextraction of cs in real conditions. however, they can be useful for study of three processes take place in phytoextraction independently i.e. solubilization of metals from soil and their transport to root system of plants, uptake of metals by root system of plants and translocation of metals into aboveground biomass. experiments with hydroponics can be useful for study of two last mentioned processes (see e.g. tandy et al., 2006). also, this configuration of experiments allows quantitative study of individual speciation forms of metals under strictly defined conditions. in the case of plant cultivation in soil a complex equilibrium system is formed when the concentration of all substances in soil solution is in equilibrium with concentration of substances bound onto soil particles. also, inorganic nutrients occurred in soil solution or in synthetic solutions formed a very complex equilibrium system defined by dissociation constants, concentration, temperature and ph value. experimental determination of all speciation forms of metals is very difficult. therefore, for description of this system is very useful to use the speciation models. in our work we used the speciation modelling program visual minteq ver. 2.53 for calculation of individual ionic forms of cs in hoagland media (hm). we found that caesium at ph 6.0 and 22°c was occurred practically only in the form of cs+ ions (≥ 97%) in the case of all nutrient media containing different concentration of macro and microelements (8.3% – 100% hm) as well as concentrations of cscl (10 – 50 000 µmol/dm3). 0 1 2 3 4 5 6 7 8 0 20 40 60 80 100 100% hm 50% hm 25% hm 8.3% hm time [day] b io ac cu m ul at io n of c s [% ] 0,00 0,05 0,10 0,15 0,20 0,25 0,30 b io ac cu m ul at io n of c s [μ m ol /g ]; w .w . 100 50 25 8.3 hoagland medium [%] fig. 1. influence of nutrient salts concentration in hm on cs bioaccumulation by roots of tobacco plants (n. tabacum l.) during 8 days hydroponic cultivation in 100%, 50%, 25% or 8.3% hm containing 10 μmol/dm3 cscl (34.0 kbq/dm3 137cscl), ph 6.0, at photoperiod 12h light/12h dark (2 000 lx) and 22°c. bioaccumulation of cs expressed in per cent of the total amount of cscl in medium (a; kinetic) and in µmol/g (wet weight) (b; after 8 days cultivation). error bars represent standard deviation of the mean (n = 3). all values of means of cs bioaccumulation were significantly different at the p < 0.05 level based on multiple range test (fig. 1b). a b 100 guldanová, j. et al. fig. 1 a depicts cs bioaccumulation by tobacco hydroponics during 8 days at different concentration of hm containing 10 μmol/dm3 cscl. we found that at the lowest concentration of 8.3% hm (0.83 mm k+ and nh4 + ions) the bioaccumulation reached 100% already after 5 days of cultivation. in the case of 25% hm (2.5 mm k+ and nh4 + ions) the total removing of cs from medium was reached after 8 days of plants cultivation and in the case of 50% (5 mm k+ and nh4 + ions) or 100% hm (10 mm k+ and nh4 + ions) at this time was observed only 40% or 20% bioaccumulation of cs, respectively. similar effect can be seen on the fig. 1 b, where bioaccumulation of cs in μmol/g (wet weight) decreased with increasing hm concentration after 8 days plants cultivation. a kruskal-wallis test confirmed significant effects (at p < 0.05) of the cultivation conditions on cs bioaccumulation. on the basis of results of authors soudek et al. (2004; 2006) it can be expect that differences in cs bioaccumulation at different concentration of hm will be caused mainly in change of k+ and nh4 + concentration in media as competitive ions for cs uptake by plants. sandeep and manjaiah (2008) in the case of mustard, spinach and wheat plants cultivated in contaminated soil with 134cs observed that cs bioaccumulation decreased with increasing concentration of k in soil. similar result found also singh et al. (2008). 0 1 2 3 4 5 6 7 8 9 0 20 40 60 80 100 b io ac cu m ul at io n of c s [% ] time [day] 10 μmol/dm3 20 μmol/dm3 50 μmol/dm3 100 μmol/dm3 200 μmol/dm3 500 μmol/dm3 1000 μmol/dm3 0 200 400 600 800 1000 0 2 4 6 8 10 12 linear regression: y = a + b * x parameter value error a 0.200 0.009 b 0.012 0.0001 -------------------------------------------- r2 sd n p 0.985 5.97 7 <0.0001 b io ac cu m ul at io n of c s [μ m ol /g ]; w .w . c 0 cscl [μmol/dm3] fig. 2. influence of cscl concentration on cs bioaccumulation by roots of tobacco plants (n. tabacum l.) hydroponically cultivated in 8.3% hm containing 10, 20, 50, 100, 200, 500 or 1 000 μmol/dm3 cscl (33.5 kbq/dm3 137cscl), ph 6.0, at photoperiod 12h light/12h dark (2 000 lx) and 22°c. bioaccumulation of cs expressed in per cent of the total amount of cscl in medium (a; during 8 days cultivation) and in µmol/g (wet weight) (b; after 8 days cultivation). error bars represent standard deviation of the mean (n = 3). it is known that the uptake of cs is operated mainly via two transport pathways on plant root cell membranes, namely k+ transporters or k+ channels (zhu and smolders, 2000). however the role of cs+ in plant nutrition is no known (marschner, 1995; white and broadley, 2000). cs+ is nontoxic to plants at external cs+ concentrations below approx. 200 µmol/dm3, although this limit depends critically on the concentrations of other ions (k+, nh4 +) in the substrate. the toxicity symptoms induced by unnaturally high cs concentrations include necrosis of shoot and root tissues (kordan, 1987; white and broadley, 2000). a b nova biotechnologica 10-2 (2010) 101 in the next experiments we observed influence of cscl concentration in the range 10 – 50 000 μmol/dm3 on plant growth as well as on cs bioaccumulation by tobacco roots. for evaluation of phytotoxic effects on tobacco plants the calculation of growth value (the ratio of fresh weight of plants at the start or end of experiments difference to fresh weight of plants at the start of experiments) and macroscopic observation of phytotoxic symptoms on leaves were used. we did not found visual symptoms of cs toxicity on plants after 8 days cultivation or significant differences in growth rate at cscl concentration up to 0.2 mm. however, at cscl concentration above 0.2 mm cscl the decrease of growth rate and necrosis of young leaves or die-back of leaves (> 2 mm cscl) were observed (data not shown). the results in fig. 2 a show that cs bioaccumulation by tobacco plants significantly decreased with increasing cscl concentration in media from the value 95% found at concentration of cscl 10 µmol/dm3 to the value 44% at concentration of cscl 1 000 µmol/dm3. after conversion to µmol cs per gram (wet weight), we found linear correlation (r2 = 0.985) between cs bioaccumulation by tobacco roots and cscl concentration in nutrient media containing 0.33 mm k+ ions (fig. 2 b). it can be assumed, that this correlation corresponds with correlation between k uptake in plant and k concentration in soil solution. it means, that at higher cs concentration than concentration of k in cells of root system will be this correlation affected not only with specific uptake of k or cs ions by k+ transporters (often ≤ 0.3 mm), but also with low specific uptake via ion channels (for k+ ions). the k+ transporters operating at low k concentrations can transport cs+ efficiently whereas cs+ permeates only slowly in k+ channels operating at k concentrations above 0.5 – 1 mm (zhu and smolders, 2000). however, we did not found saturation of the first mentioned system and beginning the second system, what should be reflect to form a characteristic double phase isotherm relationship between cs+ influx to roots and external cs+ concentrations. 0 20 40 60 80 100 c b aa b b a a c b a 8.3 25 50 100 d is tr ib ut io n of c s [% ] root stem leaves hoagland medium [%] a 0,0 0,2 0,4 0,6 0,8 1,0 c b a 1005025 [c s] sh oo t / [c s] ro ot 8.3 hoagland medium [%] a fig. 3. influence of nutrient salts concentration in hm on cs translocation from roots to shoots of tobacco plants (n. tabacum l.) after 8 days hydroponic cultivation in 100%, 50%, 25% or 8.3% hm containing 10 μmol/dm3 cscl (34.0 kbq/dm3 137cscl), ph 6.0, at photoperiod 12h light/12h dark (2 000 lx) and 22°c. distribution expressed in per cent of the total cs uptake by plant (a) and as concentration ratio [cs]shoot : [cs]root (bq/g; dry weight) (b). error bars represent standard deviation of the mean (n = 3). means with the same letter at columns are not significantly different at the p < 0.05 level based on multiple range test. a b 102 guldanová, j. et al. the reviews of authors zhu and smolders (2000) or white and broadley (2000) describe the studies demonstrated that k+ and cs+ competed for influx to excised roots, suggesting that the influx of these cations to root cells is mediated by the same molecular mechanism. the molecular identity and electrophysiological signature of many k+ transporters expressed in the plasma membrane of root cells have been also described. the inward-rectifying k+ (kir), outward-rectifying k+ (kor) and voltage-insensitive cation (vic) channels are all permeable also to cs+. by modelling cation fluxes through these transporters into a stereotypical root cell, it can be predicted that vic channels mediate most (30 – 90%) of the cs+ influx under physiological conditions and that the kup transporters mediate the bulk of the remainder. cation influx through kir channels is likely to be blocked by extracellular cs+ under typical ionic conditions in the soil. from point of view of phytoremediation technologies it is important that toxic metals and radionuclides have to be accumulated mainly in aboveground parts of plants. for evaluation of caesium mobility in conductive tissues of plants in the term of caesium translocation efficiency we established non-dimensional concentration ratio (cr), which represent the ratio of caesium concentration in aboveground part of plants [cs]shoot to caesium concentration in root system of plants [cs]root. 0 20 40 60 80 100 ee dcdbc a ab e d d c bc a ab e e d cdbc a 1000500200100502010 d is tr ib ut io n of c s [% ] c 0 cscl [μmol/dm3] root stem leaves ab 0 200 400 600 800 1000 0,0 0,1 0,2 0,3 0,4 0,5 [c s] sh oo t / [c s] ro ot c 0 cscl [μmol/dm3] fig. 4. influence of cscl concentration on cs translocation from roots to shoots of tobacco plants (n. tabacum l.) after 8 days hydroponic cultivation in 8.3% hm containing 10, 20, 50, 100, 200, 500 or 1 000 μmol/dm3 cscl (33.5 kbq/dm3 137cscl), ph 6.0, at photoperiod 12h light/12h dark (2 000 lx) and 22°c. distribution expressed: a. in per cent of the total cs uptake by plant; b. as concentration ratio [cs]shoot : [cs]root (bq/g; dry weight). error bars represent standard deviation of the mean (n = 3). means with the same letter at columns are not significantly different at the p < 0.05 level based on multiple range test. our previous results obtained by direct analyzing of radioactivity in individual plant tissues as well as from autoradiography of whole plants showed that caesium was localized mainly in root system and young leaves (vrtoch et al., 2006). in this work we found that percentual content of cs in root, stem and leaves are affected with hm concentration i.e. macroand microelements concentrations (significant differences are shown in fig. 3a at the p < 0.05 level based on multiple range test). the concentration a b nova biotechnologica 10-2 (2010) 103 ratio (cr) [cs]shoot : [cs]root increased with increasing hm concentration from the value 0.10 to the value 0.85, particularly at 50% and 100% hm, when concentration of monovalent cations (k+ and nh4 +; [k+] : [nh4 +] = 2 : 3) were 5 mm and 10 mm, respectively (significant differences are shown in fig. 3b at the p < 0.05 level based on multiple range test). similar effect was observed in our previous work with sunflower hydroponics, when shoot-to-root specific 137cs radioactivity ratio (bq/g : bq/g; wet weight) increased with increasing concentration of hm from the value 0.10 to the value 0.69 (horník et al., 2005). buysse et al. (1996) and smolders et al. (1996) found that shoot-to-root cs concentration ratio decreased with decreasing k supply. it can therefore be assumed that the main influence in this respect will be concentration of k+ ions in media. fig. 4 depicts influence of cscl concentration in nutrient media on cs distribution in tissues of tobacco plants cultivated in 8.3% hm. there is a significant change in cs distribution in root, stem and leaves of tobacco. the percentual content of cs in aboveground part of tobacco increased with increasing cscl concentration in media (significant differences are shown in fig. 4a at the p < 0.05 level based on multiple range test). also, the cr ([cs]shoot : [cs]root) increased with increasing concentration of cscl (10 – 1 000 µmol/dm3) from the value 0.1 to the value 0.4. however, at concentration of cscl in media > 500 µmol/dm3 the cr was not changed (fig. 4b). 4. conclusions the results from hydroponic cultivation of tobacco plants (n. tabacum) indicate that bioaccumulation of cs was significantly affected by concentration of hoagland medium particularly monovalent cations as well as concentration of cscl in media. on the other side, higher concentration of hoagland medium and concentration of cscl in media have a positively effect on translocation of cs into aboveground parts of tobacco plants. it may be concluded that fast growing plant species with high biomass production like tobacco might be a suitable in phytoextraction or rhizofiltration technologies used for 137cs removing from environment. however, it should be proven by field experiments, because hydroponic experiments are only a principal model to study metals uptake but do not give information on soil-plant relations and metals uptake under the real conditions. acknowledgement: this work was supported by the ministry of education, science, research and sport of the slovak republic within the bounds of project conrelmat, decree no. cd-2009-36909/39460-1:11. references audet, p., charest, c.: heavy metal phytoremediation from a meta-analytical perspective. environ. pollut., 147, 2006, 231-237. buysse, j., van de brande, k., merckx r.: genotypic differences in the uptake and distribution of radiocaesium in plants. plant soil, 178, 1996, 265-271. 104 guldanová, j. et al. čurlík, j., šefčík, p.: geochemický atlas slovenskej republiky. časť v: pôdy. mžp sr, bratislava, 1999, 99 pp. dowdall, m., standring, w., shaw, g., strand, p.: will global warming affect soil-to-plant transfer of radionuclides? j. environ. radioact., 99, 2008, 17361745. dushenkov, s.: trends in phytoremediation of radionuclides. plant soil, 249, 2003, 167-175. gardea-torresdey, j.l., peralta-videa, j.r., de la rosa, g., pardone, j.g.: phytoremediation of heavy metals and study of the metal coordination by x-ray absorption spectroscopy. coord. chem. rev., 249, 2005, 1797-1810. ghosh, m., singh, s.p.: a review on phytoremediation of heavy metals and utilization of its byproducts. appl. ecol. environ. res., 3, 2005, 1-18. gustafsson, j.p.: visual minteq. web (april 2010): http://www.lwr.kth.se/english/our software/vminteq/index.htm. horník, m., pipíška, m., vrtoch, ľ., augustín, j., lesný, j.: bioaccumulation of 137cs and 60co by helianthus annuus. nukleonika, 50, 2005, 49-52. horník, m., pipíška, m., vrtoch, ľ., augustín, j., lesný, j.: bioaccumulation and translocation of cobalt in nicotiana tabacum l.. in: szilágyi, m., szentmihályi, k. 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j. exp. bot., 51, 2000, 1635-1645. microsoft word kraic.revision.doc nova biotechnologica 8-1 (2008) 71 influence of nutrients in soil and vine leaves and meteorological factors upon vine crop and must quality filip kraic1, jan mocak1,2, miroslav argay 3 1department of biotechnology, university of ss. cyril and methodius, j. herdu 2, sk-917 01, trnava, (filip.kraic@ucm.sk) 2 institute of analytical chemistry, slovak university of technology, radlinskeho 9, sk-812 37 bratislava 3 research institute of oenology and viticulture, matuskova 25, sk-831 01 bratislava abstract: the aim of this work was to analyze the supply of vine by nutrients by determining the content of nitrogen, phosphorus, potassium, calcium and magnesium in the vineyard soil and the vine leaves. its further goal was to discover mutual relationships among the content of determined nutrients, the vine crop, and the quality of the produced must. must quality was here defined by the contents of sugar and acids. the final goal was to investigate the relations among the nutrients, vine crop and its quality and meteorological factors measured during the whole year cycle, and especially at the time of important vegetation changes. the necessary evaluations were performed using multidimensional data analysis. key words: vine, soil analysis, nutrients, electroultrafiltration, principal component analysis, discriminant analysis 1. introduction the relations among the nutrients content in vine leaves and in the soil extracts are complicated and established by the concentration levels as well as antagonistic/ synergic interrelations of the individual influences. the applicable agrochemical methods of the soil analysis utilize different extraction agents of different concentration and ionic strength (németh, 1979; németh, 1985). the nutrients were extracted by different solvents: water (electroultrafiltration method), calcium lactate solution adjusted to ph 3.7 (phosphorus determination by egner), strong polar solvents like the mixture of acetic acid, ammonium fluoride and ammonium chloride in hydrochloric acid (melich method), and the mixture of ammonium acetate and ammonium oxalate (potassium by schachtschabel). different analytical methods were used also for the nutrients analysis in leaves, where the concentration of the most important elements was determined. all corresponding results together with meteorological factors (expressed mainly by temperature, quantity of rainfall and extent of the sunshine in individual vegetation cycles) represent a large number of factors related to the vine crop, must quality, and consequently, to the quality of the produced wine. the primary goal of this work was to determine the supply of vine by nutrients, which was obtained by analyses of nitrogen, phosphorus, potassium, calcium and magnesium in the vineyard soil and the vine leaves. the second goal was to discover 72 kraic, f. et al. the mutual relationships among the determined content of nutrients and three target variables: vine crop, sugar content and content of acids. the last goal was to uncover the hidden relations among the nutrients, vine crop and its quality (sugar, acids) and the meteorological factors measured during the whole year cycle, mainly in the time of important vegetation changes. 2. material and methods 2.1 analytical determinations the extractions of nutrients present in soil were made in two ways: (a) by water (when using electroultrafiltration method), (b) by 0.21 mol/l ammonium chloride in 0.012 mol/l hydrochloric acid (when melich method was applied). determinations of nutrients in leaves were performed after the leaves had been dried and mineralized. for determining the nutrient elements p, k, ca, and mg in soil two quantitative methods were used: electroultrafiltration (németh, 1982; mengel and kirkby, 2001) and melich method (melich, 1984; jones, 2001). 2.2 exploited data the studied data originated from the research institute of oenology and viticulture in bratislava, slovakia. the number of sampling sites, all localized in southern slovakia, was 24. the total number of the considered variables (factors) was 41, among them 4 target variables (vine crop, sugar content in must, content of acids in must, and year of the vine production), 10 variables concerning vine leaves given by the content of 5 elements (n, p, k, ca, mg) at the beginning (april may, denoted by number 1, e.g. k1) and the end (september october, denoted by 2) of the vegetation cycle, 8 variables concerning the vineyard soil given by the content of 4 elements (p, k, ca, mg) found by two analytical methods (electroultrafiltration and melich), and 19 meteorological factors given by 7 temperature indicators t4, t5, t9, t10, t1t12, t4t9, t4t10 (with the number related to the month or to the time interval between two months), 6 rainfall indicators rain4, rain5, rain9, rain10, rain1t12, rain4t10, and finally 6 sunshine indicators sun4, sun5, sun9, sun10, sun1t12, and sun4t10. 2.3 multidimensional (multivariate) data analysis assessment of the interrelations among the selected target variable and individual soil, leaves and meteorological factors was made using several techniques of multidimensional data analysis, namely principal component analysis, cluster analysis, and discriminant analysis (sharma, 1996; everitt and dunn, 2001). the statistical technique of correlation analysis was also implemented. the calculations were performed using commercial software packages statgraphics (ver. 5.1), spss (ver. 15), and sas jmp (ver. 5.1). nova biotechnologica 8-1 (2008) 73 3. results and discussion 3.1 correlation analysis table 1 brings a summary of the most important pair correlations, which represents an extract from a much larger correlation table. each row of the table shows the pair correlation coefficients between the selected target variable (specified in the left column of the table) and the given variable. some observations follow from the results given in table 1. target variable crop is mostly influenced by the amount of rainfall and sunshine in september as well as by the nitrogen content in the first phenophase (with a negative correlation!). target variable sugar is mostly influenced by the amount of rainfall and sunshine in april, the temperature level from april to september, the temperature during the whole year and the rainfall in september. sugar is also highly correlated with the potassium content in the second phenophase. target variable acids mostly correlates with the content of calcium and phosphorus in the second and magnesium and potassium content in the first phenophase. the highest and negative correlation is between acids and sugar. all mentioned important correlations concern the content of nutrients in vine leaves. the nutrients content in soil (not included into table 1) seems to be less important for the selected three target variables. tab. 1. most important correlations extracted from the correlation table at the significance level α=0.05 %. target variable highest positive correlation coefficient (in parentheses) highest negative correlation coefficient (in parentheses) crop rain9 (0.510) n1 (0.579), sun9 (0.500) sugar rain4 (0.640), t4 (0.625), t4t9 (0.620), k2 (0.545), sumt1t12 (0.545) acids (0.666), rain9 (0.538) acids ca2 (0.563), mg1 (0.534) sugar (0.666), k1(0.534), p2 (0.501) target variable highest absolute values of the pair correlation coefficient (in parentheses) crop n1 (0.579), rain9 (0.510), sun9 (0.500) sugar acids (0.666), rain4 (0.640), t4 (0.625), sumt4t9 (0.620), k2 (0.545), sumt1t12 (0.545), rain9 (0.538) acids sugar (0.666), ca2 (0.563), k1(0.534), mg1 (0.534), p2 (0.501) 3.2 cluster analysis ward method and squared euclidean distance were applied since they were found as the most effective way of cluster analysis in preliminary calculations. obtained 74 kraic, f. et al. results of cluster analysis are in good accordance with the performed correlation analysis (figure 1). target variable crop is clustered first of all with the phosphorus content in vine leaves (p1, p2) and the magnesium content in soil (mgme, mgeuf). target variable sugar is clustered mainly with meteorological variables – temperature in april (t4) and in the period april – september (t4t9) and rainfall in april (rain4). target variable acids is clustered mainly with the magnesium content in vine leaves (mg1, mg2) and the calcium content in soil (came). 3.3 principal component analysis the sampling sites represented the objects and analytical results and meteorological factors in different year period represented the variables in principal component analysis. they are depicted together in the form of a pca biplot in figure 2. it is clearly seen that the objects create three natural clusters, which (according to object numbers in a more detailed analysis) belong to three years of the vine crop: 2000, 2001 and 1999 (from the left to the right). fig. 1. dendrogram of cluster analysis of all variables involved in the vine crop and must quality (defined in the text). ward’s clustering technique. software statgraphics plus 5.1. it was also important to find, which variables are closely related to the three selected target variables, namely crop (in kg per vine shrub), sugar (sugar content in kg/hl in must), and acids (content of acids in g/l in must), which reflect the crop quantity as well as the must quality. the smallest angle between crop and rain9 indicates the importance of the september rainfall. almost 180° angle between crop and n1 as well as crop and sun9 indicates a large negative dependence of crop upon nitrogen content in leaves during the first phenophase as well as amount of sunshine in september. these q d is ta nc e 0 200 400 600 800 1000 n 1 n 2 p 1 p 2 k 1 k 2 c a1 c a2 m g1 m g2 p m e p e u f k m e k e u f c am e c ae u f m gm e m ge u f c ro p s ug ar a ci ds t 4 r ai n4 s un 4 t 5 r ai n5 s un 5 t 9 r ai n9 s un 9 t 10 r ai n1 0 s un 10 t 1t 12 r ai n1 t1 2 s un 1t 12 t 4t 9 t 4t 10 r ai n4 t1 0 s un 4t 10 nova biotechnologica 8-1 (2008) 75 findings are in accordance with the results of correlation analysis, as may be expected. moreover, the crop ray points to the 2001 cluster, which means that this year was most successful with regard to the crop of vine. a close position of sugar and rain4 rays indicates the importance of the april rainfall for the sugar content in must. the importance of the april temperature and the temperature level from april to september is indicated in a similar way. almost opposite position of the sugar and acids rays demonstrates a high content of sugar simultaneously with a low acids content in must and vice versa. finally, from the interposition of the samples corresponding to the years 2000 and 1999, respectively, it is visible a large impact of sunshine on the year 2000 samples as well as rainfall on the year 1999 samples. fig. 2. biplot pca showing 40 variables and 24 sampling sites. three clusters of the sampling sites represent the year of sampling: 2000, 2001 and 1999 – from the left to the right. software statgraphics plus 5.1. 3.4 linear discriminant analysis classification of the objects, which in our case mean the sampling sites, is generally made according to the selected target variable, which has to be categorical. a categorical variable should reflect a known categorization of an object into the chosen category. if it happens, the classification algorithm categorizes the given object into a different category than was the pre-determined category, which is then accompanied by decrease of the classification performance. after calculating the discrimination model, it is possible to use further objects either to validate the performance of the discrimination model by an independent way, or to predict the category where an unknown object should be classified. 76 kraic, f. et al. observed classification performance depended, as expected, on the selected categorical target variable. figure 3 shows the case when year was used as the target variable. here 100.0 % of the originally grouped objects were correctly classified when calculating the discrimination model and 87.5 % of the cross-validated objects were correctly classified using leave-one-out method; altogether 3 objects out of 24 were categorized into a different category then supposed. a 100.0 % performance was observed also in case when crop was the target variable and all 40 independent variables were used. the same performance was achieved with 20 independent variables without using the meteorological factors. performance characteristics were obtained also for other combination of variables: soil and meteorological, leaves and meteorological, etc. fig. 3. plot of discriminant functions (df’s) showing 24 sampling sites clustered by year. a 100 % performance was achieved for the sampling sites classification in discrimination model; 87.5 % performance was achieved for the sampling sites classification using leave-one-out validation. all 40 variables were used in calculations. software spss 15. 4. conclusions crop of vine and quality of the produced must are interrelated to the results of elemental analysis of the vineyard soil and the vine leaves. they are also influenced by the meteorological factors like sunshine, rainfall in different months and various years. nova biotechnologica 8-1 (2008) 77 most important factors influencing the vine crop, the sugar content and the content of acids in must were determined, as well. sampling sites in south-western slovakia were classified according to the target variables crop, sugar and acids by linear discriminant analysis and a very high classification performance was achieved. acknowledgment: the support of this work by the grants vega 1/3584/06 and av-4/2025/08 is highly acknowledged. references everitt, b.s., dunn, g.: applied multivariate data analysis, arnold, london, 2001. jones, j. b.: 1. laboratory guide for conducting soil tests and plant analysis, crc press, boca ranton, 2001. melich m.: melich-3 soil test extractant: a modification of the melich-2 extractant. comm. soil sci plant anal., 15, 1984, 1409-1416. mengel, k., kirkby, e.a.: principles of plant nutrition, kluwer academic publishers, bern 2001. németh, k.: recent advances in euf research (1980-1983). plant soil, 83, 1985, 1-19. németh, k.: application of electro-ultrafiltration in agricultural production, kluwer academic publishers, bern 1982. németh, k.: the availability of nutrients in the soil as determined by electroultrafiltration (euf). adv. agronomy, 31, 1979, 155-188. sharma, s.: applied multivariate techniques, j. wiley, new york, 1996. microsoft word ukropcova nb 9-3.doc nova biotechnologica 9-3 (2009) 255 biotechnology commercialisation in europe dana ukropcova1, ernest sturdik2 1bioscience slovakia limited, kostliveho 10, bratislava, sk-821 03, slovak republic (dana.ukropcova@orangemail.sk) 2institute of biochemistry, nutrition and health protection, faculty of chemical and food technology, slovak university of technology, radlinskeho 9, bratislava, sk-812 37, slovak republic (ernest.sturdik@stuba.sk) abstract: european biotechnology was mapped for research and commercialisation from various information sources. social and economical benefits from technology transfer in biotechnology are clearly visible in those european countries where proper attention is given to bioscience entrepreneurship education alongside with scientific base creation and spin-off development. successful bioscience commercialisation is a result of a coordinated support of research and specific commercialisation programmes leading to spinoff establishment and industry partnership. key words: commercialisation, biotechnology, technology transfer, spin-off 1. introduction europe is considered a continent where education and research are undoubtedly strongly supported. biotechnology has deep roots in extensive european life science research and globally competitive results. however, the phase of commercialisation of biotechnology research and development is a different matter. the usa has accumulated and maintained a large absolute advantage in innovative biotechnology activities compare to the rest of the developed world (allansdottir et al., 2002). europe began investing into life sciences commercialisation later than us and made a great effort to become the principal world competitor in biotechnology business (hodgson, 2006; duchene et al., 2007; beuzekom and arundel, 2009). there are many studies being published whether universities and research institutions should accept the role of active players for commercialisation activities or stay in the traditional role of an educator and fundamental research base (muldur et al., 2003). universities were traditionally funded by public money, ensuring a certain amount of neutrality and independence (downie and herder, 2007). for those universities and research institutions that decided to become entrepreneurial more than a decade ago, the results are evident in financial resources from licensing, royalties and other economic provisions flowing back to research and development (siegel et al., 2007). these make prestigious universities modern institutions and, consequently, have an advantage at the competitive global market. spin-off companies are increasing seen as a favoured route for commercialisation of university intellectual property. thus research spin-offs are companies what have been created in order to commercialise intellectual property arising out of a research where staff are transferred from the research institution to a new firm. intellectual property from the institution is licensed to the founding intellectual property of the firm (thornburn, 2000; 256 ukropcova, d. and sturdik, e. jarrett, 2007). the success of the biotechnology commercialisation depends on an array of interwoven factors, not the least of which is the protection, preservation and promotion of public trust in the science, the scientists and the regulators (chalmers and nicol, 2004; savage, 2006). the idea of entrepreneurial research institutions and new firms creation disseminates and yet further research and development is necessary (rothaermel et al., 2007; mroczkowski, 2009). the european commission can play a major role in fostering innovations in the life sciences sector, through its framework programmes for research and technological development, as well as through other policy initiatives implementing a coherent eu strategy for biotechnology and life sciences (kütt et al., 2003). the european council and the european parliament have recognised the importance of life sciences and biotechnology, and the european commission has put forward an action plan to address the challenges and opportunities involved. this ‘strategy on life sciences and biotechnology’, adopted by the european commission in 2002, proposed a 30 point action plan involving the commission, the other european institutions and other stakeholders and it runs until 2010 (european commission, 2007). in this article, we map biotechnology commercialisation in europe and summarize the papers and reports on this topic in european countries. in the concluding section, we discuss some organizational and societal issues that arise from past experience and gained knowledge in biotechnology commercialisation. 2. history and present status biotechnology commercialisation began in europe at the end of 1980s, more than a decade later than in usa. the first to stir up the market appeared united kingdom followed by germany, france and switzerland. some of the smaller european countries, particularly ireland, denmark, the netherlands and the scandinavians countries, are also focused on biotechnology (allansdottir et al., 2002). united kingdom united kingdom (uk) leads europe in biotechnology, although it is still some way behind the united states (sainsbury, 1999; dectera et al., 2007). almost a quarter of all european biotechnology companies are located in the uk. despite the maturity of uk biotechnology, r&d institutions seeking to license intellectual property or use it as the basis for a new life science venture still face several challenges in finding the relevant expertise and creating a fertile environment to facilitate start-up activity (searle et al., 2003). in 2003, there were 455 firms involved in biotechnology in uk. these spent in public-private partnership $ 2008.4 million on r&d and employed 22405 fulltime equivalent employees, 59% of which were working in the healthcare sector (van beuzekom and arundel, 2006; smith and bagchi-sen, 2006). germany although the european biotechnology industry is large and growing larger, individual countries represent very different stages of biotechnology growth and nova biotechnologica 9-3 (2009) 257 venture capital progress. germany provides a good example of how directed changes in governmental policy and commitment can lead to flourishing biotechnology and venture capital industries. before the 1990s, germany had an extensive research base but little interest in commercialising its results. in 1996, the german minister for science and technology launched the bioregio contest to promote the commercialisation of biotechnology. bioregio was a government initiative that promoted the development of biotechnology 'clusters', with the winning 'model' regions receiving $25 million over five years. at the same time, germany set up a program called "risk capital for small technology companies" investing into new biotechnology enterprises (howell et al., 2003). in 2004, there were 607 firms involved in biotechnology or biotechnology-related businesses and 24134 biotechactive employees, of which 11958 (50%) were employed by core biotech firms and 10995 (46%) by large life science firms (van beuzekom and arundel, 2006). france the financial, fiscal, and research landscape for innovation and biotechnology has dramatically improved in france between 2001 and 2006. france became an attractive country for research, innovation, and the rise up of innovative small and middle enterprise. the "young innovative company" status was conceived in france and already benefits more than 300 of the country's biotechnology companies and their shareholders. proposal ‘excellence valo’ aims at creating a favourable environment for technology transfer and the emergence of start-up projects in research organisation (notably universities) thanks to a france-wide competition designed to spread good technology transfer practices and better funding to technology transfer teams throughout france (carmagnol, 2007). according to the french biotechnology association and the industry representative france biotech, the sector has been buoyed by venture capital investments throughout the first half of 2008, particularly pumppriming rounds, with investment of eur 87mil. (costa and carmagol, 2008). in 2003, 755 firms undertook biotechnology r&d in france, which represented 11% of all firms undertaking r&d, and spent $ 1342.0 million on biotechnology r&d in private-public partnership, which represented 5.6% of total business enterprise r&d spending (van beuzekom and arundel, 2006). switzerland out of a total of 693 european biotechnology products in the development pipelines of listed companies, 97 come from switzerland (alexakis, 2007). this second-place ranking in europe shows the country’s innovative capabilities. with 137 enterprises and 81 suppliers, switzerland boasts the world’s highest per capita biotechnology density. in 2006, the swiss biotechnology industry generated a turnover of more than 6 milliard chf and had a workforce of over 14 300 employees. the basis for this ongoing success is the intensified collaboration and exchange of knowledge within a network of institutes, universities and private businesses. switzerland, with over 40 venture capital firms and private equity funds, as well as various science parks and incubators, is a very inviting environment for innovative start-up companies. 258 ukropcova, d. and sturdik, e. denmark danish biotechnology not only outperforms its european counterparts but is on the rise. in 2006, companies raised unprecedented amounts of venture capital, pried open the window for initial public offerings and tempted investors with follow-up offerings (giovannetti and jaggi, 2007). the danish biotech industry has developed at a rapid pace over the past ten years. in the late 1990s, there were around 30 businesses in denmark, the number rose to 117 in 2004, whereas in 2007 it was 82 active biotech companies. geographically, 77 percent of the companies are located in the danish part of the cluster medicon valley (moran, 2006). a new report from ernst & young shows that denmark ranks third in europe in terms of number of biotech products under development (sorensen et al., 2008). scandinavian states the research council of norway supported biotechnology, biology, biomedicine and functional genomics research in total funding in 2003 close to eur 70 million (johne, 2004). in the business sector, had the highest r&d expenditures on biotechnology (public-private partnership $11.2 million), followed by the chemical sector (public-private partnership $10.2 million). in 2003, 1440 biotechnology researchers worked in the higher education and institute sector; about half of these were women. scandinavia has a strong life science and healthcare industry (brazil, 2008). medcoast scandinavia is a norwegian/swedish network organization with the aim to strength and develops the biomedical sector in the göteborg-oslo region. it includes 400 biomedical companies with 10 000 employees, every year about 20 start-ups are and the biotech venture capital investment about eur 50 million annually (oxford research, 2004). belgium in 2006, there were over 140 biotechnology companies operating in belgium, 7% of all such companies in europe and two are elite companies (tab. 2). belgian biotechnology companies accounted for 16% of european turnover and almost 10% of r&d expenditure (belgian federal government, 2008). ireland irish biotechnology sector is strongly supported by state funding. because commercialising technology is vital to achieve sustainable growth this aim, state agency enterprise ireland has established expert commercialisation teams working across the academic-industry interface in three key technology areas: life sciences and food, informatics and industrial technologies. proof of concept funding is available to individuals or small groups who wish to demonstrate that their ideas have originality and true commercial potential. commercialisation of research and development funding aims to bring a new product idea or business venture from third level educational institutions to market. these funding encourages knowledge based campus companies and academic entrepreneurs and to the year 2004 created 49 spin-off nova biotechnologica 9-3 (2009) 259 companies. the bio-incubators at trinity college dublin and national university of ireland in galway, funded by enterprise ireland in 2004, already house a number of spin-off companies (enterprise ireland, 2008). 3. biotechnology and life sciences clusters there are sixteen biotechnology and life sciences clusters in europe. for a number of years now the area where denmark and sweden meet, copenhagen capacity and region skane, has been branded ‘medicon valley’. this area is a life science cluster of research and development, as well as being home to a number of other institutions important for r&d, including several universities and contract research organisations (brazil, 2008). the trinational biovalley is a unique, global, cross-border life sciences cluster, has one of the world's highest densities of life science activities, is one of the biggest life science clusters in europe, has a strong scientific basis: more than 600 companies including big pharma, medtech and start-up companies, four renowned universities basel, freiburg, mulhouse and strasbourg (daniel et al., 2006). central and eastern europe the two main features of the restructuring are increased autonomy for scientists and the beginnings of competitive research funding. in some countries in central and eastern europe, high proportion of research funding is still allocated as block grants to institutes and/or universities. however, these funds may be allocated to institutes dedicated to a specific area of research, e.g. molecular biology. none of the countries reach the eu-25 average for gross domestic expenditure on r&d as a percentage of gdp, which was 1.86% in 2004; most are significantly below this figure (enzing, 2007). the agencies that fund research are normally separated from those that fund its commercialisation through support to applied research, technology development, industrial research grants, university-industry research collaboration and measures to encourage the creation of small firms. applied research and development was carried out in industrial research institutes under specific ministries and was completely separate from the enterprises and there was little in-house industrial r&d. the czech and slovak republic differ from this general pattern and over half of r&d was performed in the business sector. only four countries have attempted to implement this priority by allocating funds to biotechspecific research programmes – bulgaria, hungary, lithuania and slovak republic. there is limited data on commercialisation in nms and ams and this probably reflects the early stage of development of biotechnology in these countries. for instance, there is no data on venture capital investment in biotechnology firms or on initial public offerings (firms floated on stock markets). growth in publications output over time, particularly the capacity to sustain and increase growth of publications, provides a basis to cluster countries with similar performance into three groups: group 1: the czech republic, estonia, hungary and slovenia are closing the gap with the eu-25. group 2: cyprus, croatia, poland and slovak republic are making progress. 260 ukropcova, d. and sturdik, e. group 3: bulgaria, latvia, lithuania, malta, romania and turkey have weak performance. 4. measuring biotechnology commercialisation the socio-economic impact of biotechnology has become very important for policy makers, industry, and consumers. a broad range of indicators measures commercial orientation of the various countries in biotechnology: patents and scientific publications, number of spin-off companies, distribution of employees and venture capital investment. patents and scientific publications commercialisation begins with science. evidence suggests that public expenditure on biotechnology research has been increasing steeply in recent years. several european countries have declared the fostering of biotechnology to be a key priority in their science and technology policies. for example, ireland dedicated a substantial sum of 635 million euro to promoting excellence in scientific research in strategic areas such as biotechnology and information and communication technologies (muldur et al., 2003). in order to obtain a general impression of the commercial orientation of european countries in biotechnology, authors of the report biopolis related the patent output as a measure for technology generation and commercial interest to the scientific publications output, which could be considered as a measure for scientific activities (enzing, 2007). in 2003 almost similar numbers of patent applications are observed from europe and the united states while in the preceding years a clear lead of the united states could be detected. biotechnology seems to gain more importance in smaller high-performing countries, such as sweden, finland, denmark, iceland or switzerland (muldur, 2003). these countries also perform best according to measure of relative scientific output in biotechnology and show the highest relative growth rates. performance in terms of technology generation as measured by patent applications on a per-capita basis also reveals a broad variety among european countries. top performing countries in the most recent years are iceland, denmark and switzerland (muldur, 2003). biotechnological industry – companies and employees as number of biotechnology companies indicates a quality level of biotechnology commercialisation, report critical i in the year 2006 compares the biotechnology sectors across some eighteen european nations and the united states (hodgson, 2006). the survey for 2006 report critical i gathered data from 4 154 companies, of which 2 163 were in europe and 1 991 in the usa. european companies are younger and tend to be smaller. while the healthcare sector in europe accounts for just over a third of companies, it employs 50 000 people, approximately 52% of the european biotechnology workforce (fig. 1). nova biotechnologica 9-3 (2009) 261 agbio and environmental 11% services 34% human healthcare 37% biodiagnostics 18% fig. 1. distribution of biotechnology companies in europe by sector (hodgson, 2006). venture capital in biotechnology measuring commercialisation of biotechnology in terms of venture capital investment reveals an increasing flow of venture capital into almost all countries considered since the mid 1990s (tab. 1). tab. 1. a snapshot of the eu biotech sector data collected from dedicated biotech companies (hodgson, 2006). interestingly, for most countries where data is available (denmark, austria, united kingdom, sweden and finland), we observe a continuous growth over years 19952004. the past few years marked a recovery period for the european biotechnology sector. looking at 2006, it appears that it is now on the right track of sustained progress (tab. 2). a record year of financing, with 4.7 milliard euros raised with 45 percent increase demonstrates the robustness and growing strength of the european biotechnology sector (duchene, 2007). although venture capital funding did not increase at similar rates, the amount of private equity raised by european companies set an all-time record, passing 1.5 milliard euro for the first time ever. with 722 million euros in proceeds from 32 initial public offerings a stable trend in european public company financing appears to be emerging (ernst & young report, 2008). indicator europe us number of companies 2 330 1 991 number of new companies formed 131 78 number of employees 98 500 190 500 r & d expenditure (in mld €) 7.6 21 revenue (in mld €) 21.5 41.5 venture capital raised (in mld €) 1.02 3.2 equity raised (in mld €) 3.65 11.3 debt raised (in mld €) 0.81 7.4 262 ukropcova, d. and sturdik, e. tab. 2. top venture funding in europe (hodgson, 2006). 5. conclusions there is no doubt that biotechnology is the key technology of modern economies. biotechnology industry is built upon excellent science and market demand. if adequate attention is given to creation of scientific base and consequently commercialisation, economic rewards are beneficial for the academic and business community. the results are clearly visible in old member states of european union where combination of biotechnology specific and generic policy instruments have been adopted since end of 1990s (sainsbury, 1999). the analysis confirmed the trend towards commercialisation even in those countries that fell behind the european overage. the new member states and accession countries of european union suffer from lack of public resources to invest into research in general. fragmented and uncoordinated support of biotechnology research makes it difficult for any commercialisation at all. government science and technology policy, significant factors that can often explain the biotechnology performance, are restructuring to make the support system more effective (enzing, 2007). european biotechnology companies are part of a smaller industry due to lower amounts of capital available, and therefore take longer to raise capital (hodgson 2006). they face slow growth beyond the start-up stage than those in us and remain sub-critical size. a recent study showed that there are close to 1 400 technology transfer offices in europe (european commission, 2009). many started as industry liaison offices and also developed services to encourage commercialisation of research results. over time, many of these have developed specialised staff and services for assessing disclosed inventions, patenting, licensing, and developing and funding spinoffs, but also for actively approaching firms for contract based arrangements. name country round amount raised (in mil eur) movetis belgium first round 49 chroma therapeutics uk third round 44 cerenis therapeutics france second round 43 nabriva therapeutics austria first round 42 ablynx belgium fourth round 40 palau pharma spain first round 40 santaris pharma denmark fourth round 40 nowimmune switzerland second round 37 chiasma israel second round 35 esba tech switzerland third round 32 neuropharma spain second round 32 genextra italy second round 30 nova biotechnologica 9-3 (2009) 263 aknowledgement: the project was supported by european social fund, ministry of labour, social affairs and family of the slovak republic, project code dp 161/05i/32-2. references alexakis, a.: swiss biotech report 2007, druckerei feldegg ag, zollikerberg, 2007. allansdottir, a., bonaccorsi, a., gambardella, a., mariani, m., orsenigo, l., pammolli, f., riccaboni, m.: innovation and competitiveness in european biotechnology, office for official publications of the european communities, brussels, 2002. belgian federal government: biotech in belgium, belgian public services, brussels, 2008. brazil, m.: special report: scandinavian biotech, nature publishing group, 2008. carmagnol, a.f.c.: 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h.l., bagchi-sen, s.: university-industry interactions: the case of the uk biotech industry, industry & innovation, 2006, 371 – 392. sorensen, b.l., almen, l., carlsen, s.: biotech in denmark growing stronger. ernst & young denmark, copenhagen, 2008. thornburn, l.: government policies to encourage creation of spin-off firms from academic institutions. sapporo: apec symposium on intellectual property rights, 2000. van beuzekom, b., arundel, a.: oecd biotechnology statistics 2006, oecd publishing, paris, 2006. van beuzekom, b., arundel, a.: oecd biotechnology statistics 2009, oecd, paris, 2009. microsoft word fecko et al 2010-1.doc nova biotechnologica 10-1 (2010) 71 bio-treatment of soil from international airport in ostrava peter fečko, radmila kučerová, eva pertile, lucie nezvalová, nikolas mucha, iva janáková, vladimír čablík vsb – technical university of ostrava, faculty of mining and geology, 17. listopadu 15, 708 33 ostrava poruba, czech republic (peter.fecko@vsb.cz) abstract: the paper deals with an examination of possible application of biodegradation in the decontamination of soil samples from international airport in ostrava. the laboratory biodegradation tests were carried out with a pure bacterial culture of pseudomonas putida, a pure laboratory culture of rhodococcus sp, their mixture and a mixture prepared combining their media free of bacteria. the results of the paper imply that for biodegradation of airport pollutants is most suitable to apply a mixed bacterial culture of pseudomonas putida and rhodococcus sp. the results show that the biodegradation method is applicable for the pollution. keywords: biodegradation, pseudomonas putida, rhodococcus sp., mixed culture 1. introduction biodegradation (biological decontamination) is grounded in the capacity of certain bacterial strains to use hydrocarbons as a source of carbon and energy for their growth and in this way, decomposition of contaminants occur all the way to harmless products carbon dioxide and water. in short, biodegradation is a special case of degradation during which decomposition of polymers takes place due to the action of biological factors. it makes part of natural processes taking place in water and soil. for example, there is spontaneous degradation of biologically degradable oil substances at contamination of soil by oil substances. however, the process is slow and meanwhile contamination may spread into the surroundings. in the locality some resistant substances remain. in order to speed up the rate of degradation, it was necessary to make the process more intense and to remove resistant substances bacterial mixtures may be utilized. the ability of microorganisms to degrade hydrocarbons has been known since 1895, when miyoshi described growth of yeast on paraffin and shortly after the capacity of bacteria to make use of methane as a source of carbon was discovered. gradually, it was demonstrated that they are able to decompose practically all components of crude oil and many other hydrocarbons. at present, over 200 types of microorganisms have been described that are able to degrade hydrocarbons. some are able to make use of one hydrocarbon only (e.g. methane), but no microbial strain is known to degrade a whole range of hydrocarbons present in crude oil, for example. therefore, these are rather microbial associations that participate in degradation (novotný, 2005). the objective of the paper was to examine the application of bacterial leaching to the decontamination of soil samples from the leos janacek airport in ostrava – these samples were taken from two sampling points. 72 fečko, p. et al. 2. materials and methods 2.1 leos janacek airport in ostrava the first mention of air traffic at the territory of the present airport is from 1939 when german luftwaffe built a field aerodrome to attack poland. the modern history began in 1956 when the current airport began to be constructed. before 1989, the airport was used mainly for the needs of the air force. civil aviation was ensured by csa, namely for domestic flights, rarely for international ones. a significant turning point was the year of 1993 when the military traffic was terminated at the airport. on 13 december 2006 the airport was ceremonially christened after the composer of leos janacek and a new departure hall was put into operation. with regard to its excellent technical parameters, a pronounced development of this traffic junction is expected in the future (see references). several oil leaks have occurred at the airport and figure 1 and figure 2 show an oil interceptor lapol d and lapol 2, which intercepts leaks of hazardous substances. table 1 summarizes leaks of hazardous substances since 2005. at the same time, it gives substances that penetrated into the environment and the materials by used for their elimination. table 1. leaks of oil substances at the airport. date leak locality substance qty of leaked subst. [l ] material used for disposal leak into sewer system 23.6.2005 central passenger terminal jet a – 1 200 vapex, cansorb no 21.6.2006 central passenger terminal jet a – 1 not determ. cansorb no 28.7.2006 central passenger terminal hydraulic oil not determ. cansorb no 12.9.2006 central passenger terminal jet a – 1 not determ. cansorb no 18.9.2006 taxiway jet a – 1 50 vapex, cansorb, wa ter, surfaceactive agent no 27.9.2006 northern stand jet a – 1 30 cansorb no 29.3.2007 central passenger terminal jet a – 1 30 cansorb no 28.3.2008 bunkers of airport propellants oil products – closely unspecified not determ. vapex, sorption layer, absorption heaps, sewer seal yes nova biotechnologica 10-1 (2010) 73 it is apparent from the table that accidents with environmental impact prevail, accompanied by leaks of aviation turbine fuel jet a-1. fig. 1. lapol d – leaks of oil products. fig. 2. lapol 2. 2.2 characteristics of drawn samples the soil samples were taken directly from the airfield of the leos janacek airport in ostrava – mosnov see figure 3. fig. 3. view of the sampling point. the mineralogical analyses were implemented in the laboratories of the institute of geological engineering at mining college technical university of ostrava by means of an x-ray diffraction. the results of the mineralogical analyses of sample 1 (figure 4) imply that the samples contain about 18 % of amorphous phase, majority of quartz about 57 % and followed by calcite, chlorite, muscovite, orthoclase and albite. fig. 4. mineralogical analysis of the sample no.1. 74 fečko, p. et al. the results of the mineralogical analyses of sample 2 (figure 5) imply that the samples contain about 30 % of amorphous phase, majority of quartz about 42 %. . fig. 5. mineralogical analysis of the sample no.2. 2.3 characteristics of bacterial cultures and the method of laboratory tests for biodegradation of the samples, pure bacterial cultures of pseudomonas putida and rhodococcus sp were used. the bacterial cultures are shown in figures 6 a, b (fečko et al., 2004). fig. 6. pseudomonas putida (a) and rhodococcus sp. rha1 (b) (fečko et al., 2004). the culture media were the liquid medium of m1 for pseudomonas putida and medium of m96 for rhodococcus sp. the laboratory experiments were carried out with pure bacterial cultures of psedomonas putida and rhodococcus sp., mixed culture and bacterial medium made of 50 % mi medium and 50 % of m96 medium. the experiments were carried out in the laboratories of the institute of environmental engineering at vsb-tu ostrava, where 28day bacterial degradation took place. each sample was placed into a 2 l glass beaker. aeration was secured by means of aquarium pumps placed into the beakers. the necks of the beakers were sealed with a foil and then the beakers were moved into the chemical hood. in the course of 4-week degradation the volume in the beakers was regularly filled with distilled water as gradual evaporation occurred. having finished the experiment, the samples were filtered, dried and sent to further chemical analyses into the brown coal research institute in most. nova biotechnologica 10-1 (2010) 75 3. results and discussion the results of laboratory biodegradation tests after one-month biodegradation with applied pure bacterial cultures and mixed culture are stated in tables 2, 3. the tables imply that in the course of biodegradation tests, gradual degradation of harmful substances content from the sample occurred. for biodegradation the following pure bacterial cultures were used: pseudomonas putida pp, rhodococcus sp. r, mixed culture rhodococcus sp. and pseudomonas putida r+pp, and a check sample from media mixtures k. table 2. course of degradation the selected contaminants by means of rhodococcus r, pseudomonas putida pp and mixed culture pp+r, check test – k. evaluation of the biodegradation test of sample 1 input r removal degree pp removal degree pp+r removal degree check test k removal degree parameter mg/kg mg/kg % mg/kg % mg/kg % mg/kg % nel*) 196 127 35.2 120 38.78 72 63.27 89 54.59 anthracene 11.4 1.3 88.6 1.03 90.96 2.22 80.53 4.28 62.46 benzo(a) anthracene 65.8 8.2 87.54 5.83 91.14 12.88 80.43 29.09 55.79 benzo(b) fluoranthene 67.2 11.51 82.87 7.74 88.48 14.61 78.26 40.22 40.15 benzo(k) fluoranthene 61.2 54.16 11.5 5.97 90.25 11.81 80.7 27.36 55.29 benzo(a) pyrene 105 102.29 2.58 3.71 96.47 5.78 94.5 17.66 83.18 benzo(ghi) perylene 56.5 49.76 11.93 3.09 94.53 6.13 89.15 14.31 74.67 fenantrene 208.8 32.03 84.66 22.69 89.13 46.36 77.8 114.38 45.22 fluoranthene 264 24.05 90.89 18.88 92.85 36 86.36 96.96 63.27 chrysene 86.9 0.32 99.63 0.72 99.17 16.41 81.12 42.57 51.01 indeno (1,2,3-d)pyrene 18.7 0.1 99.47 11.98 35.94 4.44 76.26 18.67 0.16 naftalene 12.3 1.35 89.02 0.95 92.28 2.14 82.6 3.71 69.84 pyrene 230.9 4.08 98.23 12.45 94.61 25.16 89.1 64.17 72.21 σ pah 1188.7 289.15 75.68 95.04 92 183.94 84.53 473.38 60.18 pcb 28 0.01 0.01 0 0.01 0 0.01 0 0.01 0 pcb 52 0.01 0.01 0 0.01 0 0.01 0 0.01 0 pcb 101 0.01 0.01 0 0.01 0 0.01 0 0.01 0 pcb 118 0.02 0.01 50 0.01 50 0.01 50 0.02 0 pcb 138 0.02 0.02 25 0.02 25 0.01 45 0.02 0 pcb 153 0.06 0.02 57.89 0.04 36.84 0.02 59.65 0.04 24.56 pcb 180 0.02 0.01 50 0.02 15 0.01 50 0.01 50 σ pcb 0.15 0.09 39.46 0.11 26.53 0,08 42.86 0.12 16.33 *) nel – hydrocarbons c10 – c40 76 fečko, p. et al. it is apparent from the results of four-week biodegradation test that the most suitable application for the sample 1 is that of the pure bacterial cultures of pseudomonas putida, where the degradation of contaminants of pah was 92 %. in terms of degradation of pcb the best was the application of mixed culture, i.e. 42.9 %. in this case, the efficiency of the mixed bacterial culture was very positive as is visible from the following removed quantities: 63.3 % of nel, 84.50 % of pah and 42.9 % of pcb. table 3. course of degradation the selected contaminants by means of rhodococcus r, pseudomonas putida pp and mixed culture pp+r, check test – k. evaluation of the biodegradation test of sample 2 input r removal degree pp removal degree pp+r removal degree check test k removal degree parameter mg/kg mg/kg % mg/kg % mg/kg % mg/kg % nel 150 100 33.3 60 60 35 76.6 120 20 antracene 0.7 0.49 30 0.07 90 0.1 85.71 0.12 82.86 benzo(a) antracene 2.36 1.89 19.92 0.33 86.02 0.44 81.36 0.48 79.66 benzo(b) fluoranthene 5.2 4.95 4.81 0.6 88.46 0.69 86.73 0.71 86.35 benzo(k) fluoranthene 3.1 2.99 3.55 0.43 86.13 0.54 82.58 0.59 80.97 benzo(a) pyrene 5.31 4,67 12.05 0.53 90.02 0.59 88.89 0.74 86.06 benzo(ghi) perylene 3.5 1.16 66.86 0.21 94 0.27 92.29 0.28 92 fenantrene 2.65 2.45 7.55 0.86 67.55 0.14 94.72 1.45 45.28 fluoranthene 5.7 0.27 95.26 0.19 96.67 0.41 92.81 0.58 89.82 chrysene 2.7 0.13 95,19 0.25 90.74 0.02 99.26 0.02 99.26 indeno(1,2,3-cd) pyrene 6.99 6.66 4.72 2.39 65.81 0.11 98.43 0.56 91.99 naftalene 0.09 0.07 22.22 0.07 22.22 0.07 22.22 0.07 22.22 pyrene 4.75 2.69 43.37 0.6 87.37 0.17 96.42 1.3 72.63 σ pah 43.05 28.42 33.98 6.53 84.83 3.55 91.75 6.9 83.97 pcb 28 <0.01 <0.01 <0.01 <0.01 <0.01 pcb 52 <0.01 <0.01 <0.01 <0.01 <0.01 pcb 101 <0.01 <0.01 <0.01 <0.01 <0.01 pcb 118 <0.01 <0.01 <0.01 <0.01 <0.01 pcb 138 <0.01 <0.01 <0.01 <0.01 <0.01 pcb 153 <0.01 <0.01 <0.01 <0.01 <0.01 pcb 180 <0.01 <0.01 <0.01 <0.01 <0.01 σ 7 pcb <0.07 <0.07 <0.07 <0.07 <0.07 *) nel – hydrocarbons c10 – c40 nova biotechnologica 10-1 (2010) 77 it is apparent from the results of four-week biodegradation test that the most suitable application for the sample 2 is that of mixed bacterial cultures of pseudomonas putida and rhodococcus, where the degradation of contaminants of pah was 91.75 %. the amount of pcb was lower as detection limit. in this case, the efficiency of the mixed bacterial culture was very positive as is visible from the following removed quantities: 76.6 % of nel, 84.83 % of pah and 0 % of pcb. 4. conclusions the objective of the paper was to examine the application of biodegradation in the decontamination of soil sample from the leos janacek airport in ostrava. for the laboratory biodegradation tests soil samples from the airport of the leos janacek in ostrava – mosnov was used. the laboratory biodegradation tests were implemented with pure bacterial culture of pseudomonas putida, pure bacterial culture of rhodococcus sp, their mixture and mixture made combining their media free of bacteria. the efficiency of the biodegradation of sample 1 after one-month leaching with pure bacterial culture of pseudomonas putida (pp) was 38.8 % for nel, 92% for pah, 26.5 % for pcb, by means of pure bacterial culture of rhodococcus sp. (r) was 35.2 % for nel, 75.7 % for pah, 39.5 % for pcb, by means of mixed bacterial culture it was 63.3 % for nel, 84.5 % for pah, and 42.9 % for pcb. the efficiency of the biodegradation of sample 2 after one-month leaching with pure bacterial culture of pseudomonas putida (pp) was 60 % for nel, 84.5% for pah, 0 % for pcb, by means of pure bacterial culture of rhodococcus sp. (r) was 33.3 % for nel, 28.5 % for pah, <0.07 % for pcb, by means of mixed bacterial culture it was 76.6 % for nel, 91.8 % for pah and 0 % for pcb. the paper results imply that the laboratory sample 1 biodegradation efficiency with the selected contaminants ranged from 35.2 – 63.3 % for nel, 60.2 92 % for pah and 16.3 – 42.9 % for pcb. the paper results imply that the biodegradation efficiency of laboratory sample 2 with the selected contaminants ranged from 33.3 – 76.6 % for nel, 33.98 – 91.75 % for pah. the amount of pcb was lower as detection limit. the best efficiency was obtained in the laboratory biodegradation of pah. intermediate efficiency was reached with biodegradation of nel and pcb. the highest amount of nel and pcb were removed by mixed bacterial culture (pp+r). for soil biodegradation, it is thus the most suitable to apply the mixed bacterial culture of pseudomonas putida and rhodococcus sp. the results demonstrate that for the given type of contamination the method of biodegradation is suitable. acknowledgement: the work was carried out within the vav sp/2f2/98/07 project solution “research in the utilization of waste as substitutes for primary raw material sources” and supported by the ministry of environment (moe) of the cr. 78 fečko, p. et al. references novotný, c.: biodegradace a biotechnologie, ostravska univerzita, ostrava, 2005. 96 pp. basic information on the airport [online]. 2009 [cit. 2009-03-02]. available from www: <http://www.airport-ostrava.cz/cz/page-zakladni informace>. history of the airport [online]. 2008 [cit. 2009-03-02]. available from www: <www.airport-ostrava.cz/cz/page-historie-vznik-vyvoj>. technical data on the airport [online]. 2008 [cit. 2009-03-02]. available from www: <http://www.airport-ostrava.cz/cz/page-technicke-udaje>. mosnov municipality [online]. 2009 [cit. 2009-03-02]. available from www: <http://www.mosnov.cz/index.php?lang=1&str=002>. fecko, p. et al.: environmentalní biotechnologie, ostrava, vsbtu ostrava, 2004. 180 pp. nova biotechnol chim (2022) 21(1): e1246 doi: 10.36547/nbc.1246 1 nova biotechnologica et chimica low abundant bovine colostrum proteins in combination with amaranth oil reveal topical analgesic activity nail j. sagdiev1, jamolitdin f. ziyavitdinov1, nodir sh. berdiev1, soyib s. bozorov1, tohir a. khudoyberdiev1, shukhratjon s. olimjonov1, natalya l. vypova1, akmal m. asrorov1, 2,  1institute of bioorganic chemistry of uzbekistan academy of sciences, 100125, 83, m. ulughbek str., tashkent, uzbekistan 2ajou university in tashkent, 100204, 113a, asalobod str., tashkent, uzbekistan  corresponding author: akmal84a@gmail.com article info article history: received: 21st october 2021 accepted: 1st december 2021 keywords: amaranth oil analgesic activity bovine colostrum low-abundant proteins water-alcohol-soluble fraction abstract colostrum is an arsenal of proteins and peptides required at the earliest stage of a newborn development. proteins involved in all biological processes of an infant development have been found in its composition. it is expected as cost-effective source of biologically active proteins and peptides. in this work, we separated wateralcohol-soluble low abundant proteins from bovine colostrum in preparative amounts that were further utilized in combination with amaranth oil for topical cream composition. the ratio of the obtained proteins’ fraction made a thousandth of the colostrum dry mass. the partial sequences of 37 identified proteins were established by mass-spectrometer and using blast search in ncbi database. in our previous work, we established the chemical composition of amaranth seed oil with ~6 % squalene by mass. the physical mixtures of these natural resources were fabricated into cream using hyaluronic acid as moisturizing agent and their analgesic activities were established. the optimal ratio of proteins and oil was determined in terms of their effects as analgesic means by experiments carried out on mice. several proteins could possibly be responsible for the revealed biological efficacy. among them, g-protein coupled receptor and synaptotagmin were previously linked with analgesic activity. establishing an optimum ratio of ingredients proved also the contribution of higher quantity of amaranth oil, a rich source of squalene and unsaturated fatty acids. introduction colostrum is a rich source of proteins, fats, carbohydrates, and other ingredients. as a product secreted after birth, it has a higher content of proteins than milk. immunoglobulins, lactoferrin, lysozyme, and different growth factors compile the main content of colostrum (silva et al. 2019). albumin, heavy chains of immunoglobulins m and g, antitestosterone antibody, lactotransferrin, and polymeric immunoglobulin receptors were demonstrated as the main content of the colostrum whey (zhang et al. 2011). by two-dimensional analysis, ighg1 (immunoglobulin heavy constant gamma 1), c1 with uncharacterized function and vi1a protein were found as low abundant proteins in bovine colostrum whey (golinelli et al. 2011). recent research analysis revealed that exosomes of colostrum are enriched with proteins regulating the immune response and growth. the biological roles mailto:akmal84a@gmail.com nova biotechnol chim (2022) 21(1): e1246 2 of the proteins from exosomes were found to belong to immune regulation, growth and infant development (samuel et al. 2017). a number of colostrum-based products have reached the market as cosmetics, bioadditives, and other means. in combination with other ingredients, colostrum has been patented as a pharmaceutical and cosmetic composition (gobbi 2007). in another document, different ratios of horse colostrum and milk were earlier patented with other ingredients as painrelieving agents against sun burns and fire burns (gobbi 1993). in-depth analysis of bovine colostrum with 2d-lcms/ms analysis identified 403 proteins in the nonfractionated colostrum (nissen et al. 2012). in the fluid phase, 69 additional proteins were found. among all identified proteins, 38 % of them belonged to cellular processes, biological regulations, response to stimulus, and regulations of biological processes. studying the proteomic changes in bovine colostrum and milk revealed that the highest number of proteins belonged to immunity, transport and cell processes both in colostrum and milk (zhang et al. 2015). number of proteins in colostrum involved in cellular process, response to stimulus, metabolic processes, and regulation of biological processes were found as the highest both in bovine colostrum and milk; but all of them made lower number in the composition of milk (nissen et al. 2017). a study on the effects of heat treatment on bovine colostrum protein profile demonstrated that low abundant proteins, involved in cellular processes and immune response, are affected by heat (tacoma et al. 2017). besides, higher number of low abundant proteins was found to belong to metabolic processes, biological regulation, multicellular organismal processes and response to stimulus. recent advances in research allowing find low and very low abundant proteins from colostrum and milk. using so-called combinatorial peptide library technology (cpll) treatments made it possible to detect the signal of proteins masked by major proteins that constitute >95 % of total bovine colostrum protein mass (altomarea et al. 2016). another tandem with lc-ms/ms in this approach used isobaric tag for relative and absolute quantification (itraq) (yang et al. 2017). thus, the method allowed to compare differentially expressed proteins in whey of human and bovine colostrum and milk. in this work, we identified the low-abundant proteins in bovine colostrum after enrichment and by eluting with water-alcohol solution. the enrichment process was carried by trapping the proteins in polytetrafluoroethylene sorbent that was further eluted with water-alcohol solution. the idea came from the isolation of the ones of more hydrophobic nature. the proteins of hydrophobic interaction could have higher membrane positioning and thus cross the membrane more easily (lomize et al. 2017). sequences of the isolated proteins and peptides were established by mass-spectrometric analysis. further analgesic activity of the fraction was studied in combination with amaranth oil and hyaluronic acid; optimum mass ratio of the ingredients were established. in our previous work, we demonstrated high hyperlipidemic efficiency of amaranth oil that could be resulted from high concentration of unsaturated fatty acids and squalene (bozorov et al. 2018). oil samples obtained from local amaranth varieties (olimjonov et al. 2020) can contribute to economic competitiveness. skin surface lipids contain up to 13 % squalene (passi et al. 2002) that can contribute to higher interaction with squalene that ease the penetration of active compounds. experimental separation of proteins from bovine colostrum bovine colostrum samples obtained from cattle (bos taurus) on the 1st, 2nd, and 3rd days of the birth were gathered and the mixture was further used for analysis. to precipitate high-molecular-weight proteins, 60 ml of 54 % (w/v) perchloric acid (hclo4) solution was added to 945 ml of colostrum and kept in the refrigerator for 24 h capped. the precipitate was centrifuged and supernatant was collected, the volume was 600 ml. the excess amount of hclo4 in the obtained supernatant was neutralized with potassium alkali until ph reached 7.5 and kept in refrigerator for 10 h. the resulted mixture was centrifuged to remove excess of kclo4. supernatant volume compiled was 550 ml. it was left in the refrigerator overnight and used for column chromatography. nova biotechnol chim (2022) 21(1): e1246 3 column chromatography hydrophobic chromatography with a sorbent polytetrafluoroethylene (polychrome-1) filled in a 400 ml column connected to lkb bromma pump chromatography system was used to separate the fractions in a 550 ml mixture and desalinate the solution. for that, the obtained solution was first passed through a column. the column was washed out with water; the elution of the trapped proteins from the sorbent was carried out using an aqueous solution of 60 % ethyl alcohol (120 ml solution was used once with a flow rate 60 ml.h-1). the eluents were collected and the excess alcohol was removed using a rotory evaporator (re 2000a). further, the solutions were gathered and lyophilized (the virtis automatic freeze-dryer, 10-010). mass spectroscopy analysis of low-abundant fraction of proteins for mass-spectral analysis, 2 – 5 µl of digested protein samples were subjected to chip (150 µm × 43 mm) zorbax sb c18 column in agilent technologies 1260 cap pump with a flow rate of 4 µl per min, elution rate made 0.6 µl per min. b solution was managed as following: 5 %, 3 min; 80 %, 25 – 30 min. the solution was degassed in agilent technologies 1260 µ-degasser. mass detection was performed in chip-q-tof lc-ms agilent technologies 6520в series mass spectrometer. mass data was recorded in positive ionization mode (ionization source – chip interface, gas flow rate – 4 l.min-1 at 350 oc, skimmer current power – 65 v, ms/ms – 50 – 2400 m/z, mobile phase: a – 0.1 % formic acid, b – acetonitrile + 0.1 % formic acid) and partial sequences were identified with spectrum mill database. for functional annotations, obtained fragments were further searched by blast analysis in the ncbi database (https://blast.ncbi.nlm.nih.gov/blast.cgi): taxid: 9903 (belonging to bos taurus) identity was chosen as organism. for functional annotations and reliability, ncbi search results of the identified peptides with percentage identity higher than 85 % were checked with higher identities in the proteome of other mammals. establishment of amaranth oil composition the chemical composition of amaranth oil was established as described in our previous work (bozorov et al. 2019). the seeds of helios variety were ground in a porcelain mortar using quartz sand. the oil was extracted from the resulting powder with acetone by stirring for 30 min. the squalene content in the oil sample was 5.7 %. fabrication of the cream the basic components of the cream included 5 g of t-1 emulsifier and 0.2 g of sorbic acid as antibacterial means in a total mass of 100 g cream. in order to establish optimum ratio of putative biologically active ingredients, we suggested 10 compositions using various ratios of bovine colostrum proteins, amaranth oil (lacatusu et al. 2018) and hyaluronic acid (juncan et al. 2021) (table 1). the composition variants were added to the cream basis respectively, and filled up with water to a final mass of 100 g and further emulsified using a mixer. the obtained cream was of a dairy colour that is stable for more than a year when stored at 4 oc. analgesic activity the efficiencies of the compositions were determined by the analgesic activity during contactthermal irritation in the hot plate test following a commonly used method as described by deuis et al. (2017). the thermal irritation model reveals a central component in the mechanism of the analgesic action of a substance. the analgesic activity was established by the change in the threshold of pain sensitivity in animals during contact-thermal stimulation under the influence of the cream. the initial reaction of the white outbred mice (the time to start licking the hind limbs) was determined when they were placed on a standard plate with a temperature of 55 °c. ethical norms of evaluating pain behaviors, described by deuis et al. (2017), were followed when analgesic activity of cream samples was determined. then the rate of the same reaction was taken into account after certain time of applying the cream compositions to the animals on the hind and fore paws (15, 30 and https://blast.ncbi.nlm.nih.gov/blast.cgi nova biotechnol chim (2022) 21(1): e1246 2 45 min before the study), and its measurements were expressed as a percentage relative to the initial value. animal experiments were carried out according to requirements used for scientific purposes (european directive 2010/63/eu). table 1. establishment of optimum ratio of ingredients to fabricate analgesic means. compositions amaranth oil [g] bovine colostrum proteins [mg] hyaluronic acid [g] 1 5 0 0 2 0 10 0 3 0 0 2 4 6 8 1 5 5 10 2 6 4 7 3 7 3 10 1 8 2 10 2 9 5 8 3 10 1 9 4 samples that revealed highest analgesic activity are indicated in bold. general and acute toxicity the study of the acute toxicity and general action of the investigated cream was carried out on white outbred mice weighing 20 ± 1.5 g. five mice were included in each group. the tested substance was orally administered at doses up to 5,000 mg.kg-1; cream samples were dermally used at doses up to 2500 mg.kg-1. the observation was carried out 3 times a day for 2 – 3 days in a laboratory condition and further for 10 days in a vivarium. results proteins were isolated from bovine colostrum as above described. obtained fraction of the wateralcohol-soluble fraction of low-abundant proteins made by dry mass approximately a thousandth of the colostrum dry mass. proteins, highly abundant in colostrum such as casein, immunoglobulins, and lactoglobulins, are mainly transport proteins and/or ones involved in immune processes (nissen et al. 2017). our hypothesis originated from isolating the ones that interact with hydrophobic sorbent polytetrafluoroethylene – polychrome (despite fluorine is electronegative, the polymerized sorbent is of hydrophobic nature). in total, ~1.4 g of protein fraction of low abundant ones were obtained and subjected to sequence analysis. in table 2, were provided the sequence of the identified proteins, ncbi annotation as well as accession number, query cover, and percentage identity. query cover only higher than 90 % were selected. our results by analgesic activity showed the initial reaction of mice in the control group occurred after 9.3 ± 0.7 seconds. some of the cream samples that did not contain either amaranth oil alone or amaranth oil and bovine colostrum fractions (samples 2 and 3) revealed lowest effect that could be attributed to their presence. they enhanced analgesic activity in 45 min by 175.3 and 207.5 %, respectively. the effect of hyaluronic acid in pain relief can be noticed in samples 6 and 10 that contain 3 and 4 g of hyaluronic acid, respectively. samples 7, 8, and 10 turned out to be moderately active despite the highest quantity of protein fraction included. when these samples were applied, analgesic activity in 45 min increased by 296.8 % (36.9 ± 3.6 s), 267.7 % (34.2 ± 3.2 s) and 284.9 % (35.8 ± 4.3 s), respectively, compared to the initial latent period. the most active ones were samples 5 and 9, the analgesic activity of which enhanced almost 3.84 % (45.0 ± 3.8 s) and 3.6 times (42.6 ± 4.1 s), respectively (table 3). the observed time lag in pain relief can be concluded with highest quantity of amaranth oil, bovine colostrum protein fraction, and hyaluronic acid. based on the obtained results of the studied samples, the most optimal choice in terms of the ratio of components was sample 5 that contained 5 g of amaranth oil, 10 mg of bovine colostrum protein fraction and 2 g of hyaluronic acid (table 1). in relation to the initial indicators, this composition increased the analgesic activity by 131.2 %, 230.1 % and 383.9 % after 15, 30, 45 min of topical application, respectively (table 3). based on the data obtained, we developed cosmetic cream that was named "zumara". the cream consists of t1 emulsifier, hyaluronic acid, bovine colostrum proteins, amaranth oil, ethyl alcohol, sorbic acid and purified water. the highest biological activity among samples belonged to samples 5 and 9 that could be attributed to higher content of bovine colostrum protein fraction, amaranth oil that contains 4 nova biotechnol chim (2022) 21(1): e1246 3 table 2. identified sequences of low-abundant proteins in bovine colostrum and their accession numbers, query covers and percentage identities in ncbi database. sequences identified by mass analysis accession number query cover per. identity proteins name/ncbi annotation 1. rlvrfypradrvmsvclrvelygclwkaglxstqtg xp_019841431.1 100 % 100 % discoidin domain receptor family, member 1 2. vllgwgsllimlknegfyssmcpaenstnstqdeqrr xp_027418094.1 91 % 85.29 % neutral amino acids transporter small subunit 3 3. nisyggcmaqmfflswsvaaevllftamaydryva xp_027385372.1 100 % 77.14 % olfactory receptor 13a1-like 4. aiyhcreqpasdwpeavclligrqdlskqa xp_005891645.1 100 % 100 % anaphase-promoting complex subunit 1 isoform x1 5. vlsvelpgllaqlarsfalllpvyalgylglsfswvllalallaw xp_027394252.1 97 % 88.64 % extended synaptotagmin-2 6. siggtpirgalqi xp_027405056.1 100 % 100 % tyrosine phosphatase, receptor type, δ isoform 1 precursor 7. siphlllpla np_001192519.1 90 % 87.50 % integrin alpha-10 8. qllsqwsahamevyswklvhptd elr61599.1 100 % 95.95 % cytoplasmic fmr-1 interacting protein 9. vlvvasfdelqrraseargkiivynqp elr52717.1 100 % 77.78 % plasma glutamate carboxypeptidase 10. qlekswsqylacpedisgllhs xp_005892933.1 100 % 95.45 % huntingtin interacting protein 1 11. ssgpyltsvmgkaageref abg67017.1 94 % 77.78 % transcobalamin ii precursor 12. gllcvcwspdgkyivtggeddlvtv xp_024837551.1 100 % 96 % wd repeat containing protein 20 13. pketpflvsfiiaqivttcsgilsypf aic38345.1 100 % 92.59 % adp/atp translocase 4 14. gepgktgppglpgaggsgaistatyttvprvafyaglknph elr50847.1 100 % 100 % crf, c1q-related factor 15. npvacelkafv daa15478.1 90 % 80 % dna-directed rna polymerase iii 16. sfqtgswlldhcqesyceptvcq np_001074209.1 100 % 86.96 % keratin associated protein 11-1 17. lyrwqgifvpfedlsie xp_005907599.1 100 % 100 % very large g protein-coupled receptor 18. slpppplqp xp_027384342.1 100 % 100 % the peroxisome proliferator-activated receptor γ coactivator-1 5 https://www.ncbi.nlm.nih.gov/protein/xp_019841431.1?report=genbank&log$=prottop&blast_rank=1&rid=nbnvkgtz016 https://www.ncbi.nlm.nih.gov/protein/xp_027418094.1?report=genbank&log$=prottop&blast_rank=1&rid=nes9vpm3014 https://www.ncbi.nlm.nih.gov/protein/xp_027385372.1?report=genbank&log$=prottop&blast_rank=1&rid=nm3gv1bn014 https://www.ncbi.nlm.nih.gov/protein/xp_005891645.1?report=genbank&log$=prottop&blast_rank=1&rid=net27gzv014 https://www.ncbi.nlm.nih.gov/protein/xp_027394252.1?report=genbank&log$=prottop&blast_rank=1&rid=net63xyt014 https://www.ncbi.nlm.nih.gov/protein/xp_027405056.1?report=genbank&log$=prottop&blast_rank=2&rid=netatm43014 https://www.ncbi.nlm.nih.gov/protein/np_001192519.1?report=genbank&log$=prottop&blast_rank=3&rid=netsud3a014 https://www.ncbi.nlm.nih.gov/protein/elr61599.1?report=genbank&log$=prottop&blast_rank=4&rid=nkyjywhf016 https://www.ncbi.nlm.nih.gov/protein/elr52717.1?report=genbank&log$=prottop&blast_rank=1&rid=neuftm8f01r https://www.ncbi.nlm.nih.gov/protein/xp_005892933.1?report=genbank&log$=prottop&blast_rank=2&rid=neummzs101r https://www.ncbi.nlm.nih.gov/protein/abg67017.1?report=genbank&log$=prottop&blast_rank=2&rid=nhstf1sc016 https://www.ncbi.nlm.nih.gov/protein/xp_024837551.1?report=genbank&log$=prottop&blast_rank=2&rid=nevarxdd014 https://www.ncbi.nlm.nih.gov/protein/aic38345.1?report=genbank&log$=prottop&blast_rank=3&rid=nevfvbbj016 https://www.ncbi.nlm.nih.gov/protein/elr50847.1?report=genbank&log$=prottop&blast_rank=1&rid=new1hfgb016 https://www.ncbi.nlm.nih.gov/protein/daa15478.1?report=genbank&log$=prottop&blast_rank=1&rid=nex9ncnc016 https://www.ncbi.nlm.nih.gov/protein/np_001074209.1?report=genbank&log$=prottop&blast_rank=3&rid=ney2yc5z016 https://www.ncbi.nlm.nih.gov/protein/xp_005907599.1?report=genbank&log$=prottop&blast_rank=3&rid=neycvsnd016 https://www.ncbi.nlm.nih.gov/protein/xp_027384342.1?report=genbank&log$=prottop&blast_rank=1&rid=neytt5v9014 nova biotechnol chim (2022) 21(1): e1246 2 table 2. identified sequences of low-abundant proteins in bovine colostrum and their accession numbers, query covers and percentage identities in ncbi database – continued. sequences identified by mass analysis accession number query cover per. identity proteins name/ncbi annotation 19. mlenysnlvflglavskpqlvtfleqr elr60893.1 96 % 80.77% zink finger protein znf720 20. iqvtdddineiiqidgtgdnssaeeg xp_027411025.1 100 % 72 % transcription factor iia α/β 21. tssihskeifhslt xp_027422939.1 100 % 92.7% dna polymerase epsilon (ε) catalytic subunit a 22. iqgayyprrgsseiafhtipliqr xp_027411356.1 96 % 86.4 % retinol saturase 23. efrrrdqfpltrgraiqecrspvpppa xp_027369964.1 96 % 95.8 % striated muscle preferentially expressed protein kinase (speg) 24. qgveyakavplgtpiqs np_991380.2 100 % 94.45 % γ-protocadherin-c3 isoform 25. tillgpmstlvhtdqistpetp np_991380.2 100 % 95.45 % hepatocyte nuclear factor 4 γ 26. misqlvleqfllighckd xp_024835263.1 94 % 64.7 % zinc finger and scan domain containing protein 4d 27. qkleqqlkvvprfqpisehqt xp_024846738.1 100 % 95.24 % a kinase anchor protein 9 28. dstfaniskddsdliystygedpdlpsdfsih xp_005218672.1 100 % 100 % bromodomain containing protein 7 29. hiviyssieekttlkdknalhlfsingkylgsqi xp_027420629.1 100 % 85.3 % neurobeachin-like protein 1 30. prgpkghmgdsvigqkger np_001160001.2 100 % 84.21 % collagen 4 alpha 3 31. vhgeplgygv xp_027413406.1 100 % 80 % serine/threonine-protein kinase lats2 32. nlldlllnhqvlvpgcldpfpllsayv daa13671.1 100 % 100 % glutathione s-transferase p 33. siqsqssltitvsstp np_776434.1 93 % 73.33 % hematopoietic progenitor cell antigen cd34 34. apnvqsrelnyd np_001179782.1 100 % 83.33 % parathyroid hormone b1 35. ghrntvlltwkppd xp_027370093.1 100 % 100 % obscurin-like 1 36. sdkihfps e1bpq3.1 100 % 87.5 % g-protein coupled receptor family c gr. 6 member a 37. svnsevlgatrvkrhknllaerweahiya xp_005209316.1 96 % 75 % mitotic interactor and substrate of plk1 6 https://www.ncbi.nlm.nih.gov/protein/elr60893.1?report=genbank&log$=prottop&blast_rank=1&rid=neyueea2014 https://www.ncbi.nlm.nih.gov/protein/xp_027411025.1?report=genbank&log$=prottop&blast_rank=1&rid=nm2u02vz014 https://www.ncbi.nlm.nih.gov/protein/xp_027422939.1?report=genbank&log$=prottop&blast_rank=2&rid=nez4s03n016 https://www.ncbi.nlm.nih.gov/protein/xp_027411356.1?report=genbank&log$=prottop&blast_rank=3&rid=nezabedj016 https://www.ncbi.nlm.nih.gov/protein/xp_027369964.1?report=genbank&log$=prottop&blast_rank=1&rid=nezhayer014 https://www.ncbi.nlm.nih.gov/protein/np_991380.2?report=genbank&log$=prottop&blast_rank=1&rid=nezs3bh2016 https://www.researchgate.net/publication/306270257_the_g-protocadherin-c3_isoform_inhibits_canonical_wnt_signalling_by_binding_to_and_stabilizing_axin1_at_the_membrane https://www.ncbi.nlm.nih.gov/protein/np_991380.2?report=genbank&log$=prottop&blast_rank=1&rid=nm3ve0tz014 https://www.ncbi.nlm.nih.gov/protein/xp_024835263.1?report=genbank&log$=prottop&blast_rank=3&rid=nf012vj5014 https://www.ncbi.nlm.nih.gov/protein/xp_024846738.1?report=genbank&log$=prottop&blast_rank=3&rid=nf01wswa016 https://www.ncbi.nlm.nih.gov/protein/xp_005218672.1?report=genbank&log$=prottop&blast_rank=3&rid=nf063a2k016 https://www.ncbi.nlm.nih.gov/protein/xp_027420629.1?report=genbank&log$=prottop&blast_rank=2&rid=nf0bmu5y016 https://www.ncbi.nlm.nih.gov/protein/np_001160001.2?report=genbank&log$=prottop&blast_rank=2&rid=nf0f024b016 https://www.ncbi.nlm.nih.gov/protein/xp_027413406.1?report=genbank&log$=prottop&blast_rank=1&rid=nf0jtnu3016 https://www.ncbi.nlm.nih.gov/protein/daa13671.1?report=genbank&log$=prottop&blast_rank=3&rid=nf0kac5c016 https://www.ncbi.nlm.nih.gov/protein/np_776434.1?report=genbank&log$=prottop&blast_rank=2&rid=nf0rhtp3014 https://www.ncbi.nlm.nih.gov/protein/np_001179782.1?report=genbank&log$=prottop&blast_rank=2&rid=nhrzzrwg014 https://www.ncbi.nlm.nih.gov/protein/xp_027370093.1?report=genbank&log$=prottop&blast_rank=1&rid=nhs6d0fr016 https://www.ncbi.nlm.nih.gov/protein/e1bpq3.1?report=genbank&log$=prottop&blast_rank=1&rid=nhs5tw7u016 https://www.ncbi.nlm.nih.gov/protein/xp_005209316.1?report=genbank&log$=prottop&blast_rank=1&rid=nhsd5y33016 nova biotechnol chim (2022) 21(1): e1246 3 table 3. effects of the fabricated creams on the latency period and analgesic activity in the thermal nociceptive hot plate test (m  m; n = 6). fabricated cream compositions time with the moment of cream application [min] 15 30 45 latent period (seconds) analgesic activity (percentage increased by) latent period (seconds) analgesic activity (percentage increased by) latent period (seconds) analgesic activity (percentage increased by) control (initial) 9.3 ± 0.7 9.3 ± 0.7 9.3 ± 0.7 1 18.2 ± 1.8 95.7 20.0 ± 2.0 115.1 22.5 ± 3.4 141.9 2 10.5 ± 2 12.9 12.2 ± 0.7 31.2 25.6 ± 2.8 175.3 3 15.3 ± 1.5 64.5 17.5 ± 1.8 88.2 28.6 ± 3.2 207.5 4 20.3 ± 1.5 118.3 25.6 ± 1.5 175.3 40.2 ± 3.5 332.3 5 21.5 ± 1.8 131.2 30.7 ± 2.2 230.1 45.0 ± 3.8 383.9 6 20.3 ± 2.0 118.3 25.7 ± 0.7 176.3 38.7 ± 2.8 316.1 7 19.6 ± 1.8 110.8 24.5 ± 2.0 163.4 36.9 ± 3.6 296.8 8 18.4 ± 1.5 97.8 23.6 ± 1.5 153.8 34.2 ± 3.2 267.7 9 22.5 ± 1.8 141.9 31.2 ± 2.2 235.5 42.6 ± 4.1 358.1 10 19.6 ± 2.0 110.8 24.6 ± 0.7 164.5 35.8 ± 4.3 284.9 р < 0.01 – reliability in relation to the mean value of initial control. samples revealing highest activity and their effects, respectively, are shown in bold letters. ± bars indicate standard deviation value of a data set. squalene up to 5.7 % in the composition and hyaluronic acid. further analysis will prove our hypothesis. discussion previous researchers demonstrated different mechanisms of analgesic activities of peptides. dooley et al. (1994) reported rfwink peptide that do not resemble to any known opioid peptide. reported six amino acid-long peptide was demonstrated to induce long-lasting analgesia in mice. the peptide was suggested to cross blood brain barrier. in another work, gycaekgircddihcctglkckcnasgyncv crkk peptide, isolated from alopecosa marikovskyi spider venom, was found to possess analgesic activity by influencing purinergic p2x3 receptors. the maximum bioactivity of the peptide, studied by thermal hypersensitivity test, was 0.5 mg.kg-1when administered intravenously (palikova et al. 2019). zrccnggcssrwcrdhsrcc peptide (siiia) and similar structures are expected to be potent blocking agents of ttx-r and ttx-s sodium channels (bulaj et al. 2005; green et al. 2007). μ-conotoxins were established to block voltagegated sodium channels (vgsc) and more than twenty sequences belonging to conotoxins were already identified that interact with vgsc subtypes (green et al. 2014). another mechanism of analgesic effect of peptides was shown to be connected to a linkage with clathrin heavy chain. synthetic peptide, obtained by modifying salmon calcitonin sct16-21 fragment retained the activity of a full molecule (kotin et al. 2019). some peptides, identified in this work, were reported to be associated with analgesic mechanisms. very large g-protein coupled receptor 1 (vlgr1) is one of these peptides included in these analgesic processes. the large ectodomain of vlgrl contains calcium exchanger repeats that possess resemblance to sodium-calcium exchanger proteins. very large g protein-coupled receptor 1 (vlgr1), having a large ectodomain, contains multiple calcium exchanger repeats resembling regulatory domains of sodium-calcium exchanger proteins. (mcmillan et al. 2002). g proteincoupled receptors mediate (gpcr) human sense of vision, smell, taste and pain, and are involved in 7 nova biotechnol chim (2022) 21(1): e1246 2 cell recognition processes. they structurally are classified as gpcr possessing short n-terminal ectodomain (50 – 80 residues) and long ectodomain (80 – 600 residues). the long n-terminus can be involved in ligand recognition; the bound ligand can possibly move to the transmembrane region to activate g protein (vaidehi et al. 2002). vlgr1 is a core component for the development of ear inner cells. mutations in vlgr1 genes were defined to cause usher syndrome with symptoms congenital hearing loss and progressive retinitis pigmentosa (hu et al. 2014). we suggested synaptotagmin-2 (syt-2) protein as the next one involved in analgesic processes. syt-2 was reported to be involved in neuronal processes (bouhours et al. 2017). extended synaptotagminlike proteins, are a family of proteins that are involved in ca2+-regulated secretion and characterized by an n-terminal transmembrane sequence and two c-terminal c2 domains (c2a and c2b) (rizo and rosenmund 2008). e-syts consist of a short, nonconserved n-terminal transmembrane region and c2 domains. their c2 domains generally act as ca2+and phospholipidbinding domains and/or as protein–protein interaction domain (min et al. 2007). the e-syt2 c2a domain binds up to four ca2+ ions, whereas the c2b domain does not bind ca2+ (xu et al. 2014). other proteins that could be involved in analgesic activity among the ones, identified in this work, are receptor protein tyrosine phosphatase (rptp) delta isoform, corticotropin releasing factor, and γprotocadherin c3. since analgesic effects of these proteins were not conducted separately, our suggestions are speculative only. however, obtained fraction of water-alcohol soluble proteins, possibly of more hydrophobic nature, has been demonstrated as proper natural resources that can be obtained in preparative amounts. further researches will clarify deeper fundamental basis of the work. amaranth oil is another factor contributing to the efficiency of the cream. beneficial effects of squalene in the composition of creams and other means of cosmetic dermatology provide advantages to amaranth oil over other sources (huang et al. 2009). up to 13 % concentration among the components of the skin surface lipids (passi et al. 2002) causes higher interaction of the cream composition with a high ratio of squalene that contributes to the penetration of active compounds. the study of the general and acute toxicity of the substances and the cosmetic cream showed that they belong to practically harmless substances. ld50 level of the product reached more than 25,000 mg.kg-1 with cutaneous and oral administration in mice and rats. the study of the specific toxicology of the substance and the cream showed that they do not have a local irritating effect on the skin of rats and the conjunctiva of the eyes of rabbits and do not have an allergenic effect. the study of the chronic toxicity of the substance and the developed cream showed that the cream revealed prolonged action and did not lead to any changes in the biochemical parameters of the peripheral blood and internal organs of the experimental animals (results are not shown). the study of specific activity showed that the cream smoothes the skin by retaining moisture resulted from hyaluronic acid (sundaram et al. 2018; lubart et al. 2019). hyaluronic acid, another significant component in the composition of the product, is found in many connective tissues (gupta et al. 2019; chang et al. 2021). it is mostly used moisturizing agent used in cosmetology. hyaluronic acid is responsible for the regeneration process of the skin: it helps the synthesis of collagen (silva et al. 2017) and elastin (joddar and ramamurthi 2006), thus contributing to their correct positioning. thanks to this, the turgor and elasticity of the skin are improved. in addition, the unique ability of hyaluronic acid to retain water provides stability to the product. in the upper layers of the skin, it works as a conductor of nutrients and facilitates their diffusion (dovedytis et al. 2020). conclusion in this work, we identified 37 low abundant proteins in bovine colostrum that are soluble in 60 % ethanol. the identified peptides contribute to the extension of proteomic data of bovine colostrum. the used method can serve to identify lowabundant proteins from the colostrum and milk of other mammals. the sum of these peptides was found likely to possess analgesic effects when used as topical cream. we hypothesized the observed 8 nova biotechnol chim (2022) 21(1): e1246 3 analgesic activity might be attributed to at least two proteins: vlgr1 and synaptotagmin-2. amaranth oil containing high amount of squalene was concluded as efficient additive that likely contributes to biological efficiency. acknowledgement the work was supported with 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itraq-coupled lc-ms/ms. int. j. food sci. nutr. 6: 971-681. zhang l, boeren s, hageman ja, van hooijdonk t, vervoort j, hettinga k (2015) bovine milk proteome in the first 9 days: protein interactions in maturation of the immune and digestive system of the newborn. plos one 10: e0116710. zhang l, wang j, yang y, bu d, li s, zhou l (2011) comparative proteomic analysis of changes in the bovine whey proteome during the transition from colostrum to milk. asian-aust. j. anim. sci. 2: 272-278. 10 microsoft word ivanova et al 2010-1.doc nova biotechnologica 10-1 (2010) 45 stability of immobilized biosorbents and its influence on biosorption of copper dana ivánová1, hedviga horváthová2, jana kaduková2, jana kavuličová1 1technical university of košice, faculty of metallurgy, department of chemistry, letná 9, košice, sk-040 13, slovak republic (dana.ivanova@tuke.sk) 2technical university of košice, faculty of metallurgy, department of non-ferrous metals and waste treatment, letná 9, košice, sk-040 13, slovak republic (hedviga.horvathova@tuke.sk) abstract: biomass immobilization in a polymeric matrix may improve biosorption capacity and facilitate the separation of biomass from metal-bearing solutions. many polymers are studied as immobilizing agents for biosorption including biopolymers such as sodium alginate. in the article swelling behaviour of gel and dry beads has been studied in aqueous solutions with different acid-base character. swelling of gel or dry calcium alginate beads was found in all acidic and basic solutions except of the gel beads in strong acidic solutions, which exhibited the tendency to shrink. dry beads in diluted acidic solutions had the greatest stability because they exhibited minimum swelling. dry and gel beads were completely dissolved in concentrated solutions of sodium and potassium hydroxides. the potential use of immobilized algal biomass in ca-alginate beads for removal of copper ions from aqueous solution was also investigated. the results of the kinetic studies showed that the sorption of copper ions on gel immobilized beads are the most suitable. key words: calcium alginate beads, ph, stability, dissolution, swelling, biosorption, chlorella kessleri 1. introduction heavy metal pollution is one of the most important environmental problems today. one of the processes used for heavy metal removal from waste water and even their recovery can be biosorption that utilizes various natural materials of biologic origin (volesky, 2003; kaduková et al., 2008; wang and chen, 2009). biosorption can be defined as the passive sequestering of metal ions by metabolically inactive biomass via various physicochemical mechanisms. metal uptake may take place by different metal-binding mechanisms such as physical adsorption, chemisorption, ion exchange, microprecipitation, complexation, coordination, chelation (volesky, 2003; kaduková and štofko, 2006; wang and chen, 2009). for industrial application of biosorption, it is important to utilize an appropriate immobilization technique to prepare commercial biosorbents. disadvantage of the free microbial cells used in laboratory conditions is that they are basically small particles, with low density, poor mechanical strength and little rigidity. in real application they may come up with the solid-liquid separation problems, possible biomass swelling, inability to regenerate/reuse and development of high pressure drop in the column mode. high pressures can cause disintegration of free biomass. the immobilization of the biomass in solid structures would create a biosorbent material with the right size, mechanical strength, rigidity and porosity 46 ivánová, d . et al. necessary for use in practical processes. various methods are available for biomass immobilization. these include entrapment in polymers, adsorption, cross-linking and covalent linkage (volesky, 1990). the polymeric matrix determines the mechanical strength and chemical resistance of the final biosorbent particle to be utilized for successive sorption-desorption cycles (wang and chen, 2009). important immobilization matrices used in biosorbent immobilization include sodium or calcium alginate. alginate is a linear polysaccharide isolated from many strains of marine brown algae. alginates constitute a family of unbranched binary copolymers of 1-4 linked β-d-mannuronic acid (m) and α-l-guluronic acid (g) residues (vijaya et al., 2008; vreeker et al., 2008). the aim of this work was to study the stability of the different calcium alginate beads in acidobasic conditions with respect to application in biosorption and desorption experiments. the technical feasibility of using immobilized algal biomass for the removal of copper ions from aqueous solutions and the effect of beads preparation on kinetics of biosorption were investigated. 2. materials and methods 2.1 preparation of calcium alginate beads sodium alginate was dissolved in distilled water at a concentration of 3wt. %. after sodium alginate was completely dissolved, the solution was left undisturbly for 30 minutes to eliminate the air bubbles. the solution was then dropped from a height of 20 cm into gelling medium of 0.2m calcium chloride solution using a syringe with a needle. on the principle of simple ion exchange water soluble sodium alginate was converted to water insoluble and stable calcium alginate salt. the formed beads were cured in the gelling medium for 30 min. calcium alginate beads were divided to three groups: gel beads: fresh prepared calcium alginate beads were washed with distilled water and used for experiments. the average size of the bead was found to be 3.03 mm. dry beads a: prepared calcium alginate beads were rinsed with distilled water, dried and dipped in distilled water for 5 days. the average size of the bead was found to be 1.02 mm. dry beads b: prepared calcium alginate beads were rinsed with distilled water and dried, resulting in particles with a diameter of 0.89 mm. 2.2 study of the prepared beads stability the concentrated and diluted solutions of hydrochloric, nitric and sulphuric acids and sodium and potassium hydroxides were prepared. ph values were measured for each solution. approximately 8-16 beads from each fraction were immersed in the tubs with 10 ml of acidic and basic solutions and stored at laboratory temperature and changes in the form of beads were observed. nova biotechnologica 10-1 (2010) 47 during the whole experiment average diameter of beads were measured by using the micrometer screw gauge and these data were used for % volume change calculation according to the formula: 100. i if c v vv v − = (1) where cv volume change [%] fv final volume [mm 3] iv initial volume [mm 3] 2.3 preparation of immobilized algae in calcium alginate beads powder prepared from dried alga chlorella kessleri was mixed with 3% sodium alginate solution in the ration 1:9. immobilized beads were prepared with the same technique like calcium alginate beads (in section 2.1). prepared beads were consequently divided into three groups. a gel immobilized beads (stored in distilled water) b dry immobilized beads before using dipped in distilled water for 12 hours c dry immobilized beads 2.4 copper biosorption experiments immobilized algae were used for biosorption of cu2+ ions from solution with initial concentration 140 mg l-1. ph value of used solutions was 5.00. each fraction (a, b, c) of beads at concentration 2 g l-1 of beads were added into the solutions. these solutions were mixed during 24 hours. at selected time intervals, solution samples were withdrawn for metal analysis. the concentration of cu2+ ions was measured by atomic absorption spectroscopy (varian aa20+). the metal uptake q was calculated from the mass balance equation as follows: m ccv q e )( 0 −= (2) where q the quantity of metal uptake by biomass [mg g-1] c0 the initial metal concentration [mg l -1] ce final (after sorption at equilibrium) metal concentration [mg l -1] v the volume of solution [l] m dry weight of the biomass added [g] 3. results and discussion 3.1 calcium alginate beads stability obtained characteristics of the behaviour in acidic conditions are shown for gel beads (table 1) and dry beads (table 2-3). the changes of the gel beads and dry beads in the basic solutions are listed in table 4 6. 48 ivánová, d . et al. table 1. ph values of the acidic solutions, average diameter of the beads, their volume with % volume change before and at the end of experiment with gel beads. initial diameter and initial volume of beads were 3.03 mm and 14.57 mm3, respectively. ph of the solution hcl hno3 h2so4 hcl hno3 h2so4 initial 1.13 1.14 1.29 2.64 2.78 2.79 with gel beads 1.13 1.14 1.3 3.23 3.34 3.32 diameter of beads [mm] 2.82 2.38 2.25 3.6 3.5 3.9 volume [mm3] 11.74 7.06 5.96 24.43 22.45 31.06 volume change [%] -19.4 -51.5 -59.1 67.7 54.1 113.2 table 2. ph values of the acidic solutions, average diameter of the beads, their volume with % volume change before and at the end of experiment with dry beads a. initial diameter and initial volume of beads were 1.02 mm and 0.56 mm3, respectively. ph of the solution hcl hno3 h2so4 hcl hno3 h2so4 initial 1.13 1.14 1.29 2.64 2.78 2.79 with dry beads a 1.15 1.15 1.31 3.77 3.68 3.99 diameter of beads [mm] 1.30 1.23 1.30 1.07 1.07 1.03 volume [mm3] 1.15 0.97 1.15 0.64 0.64 0.57 volume change [%] 107 75.4 107 15.4 15.4 3 table 3. ph values of the acidic solutions, average diameter of the beads, their volume with % volume change before and at the end of experiment with dry beads b. initial diameter and initial volume of beads were 0.89 mm and 0.37 mm3, respectively. ph of the solution hcl hno3 h2so4 hcl hno3 h2so4 initial 1.13 1.14 1.29 2.64 2.78 2.79 with dry beads b 1.14 1.14 1.3 3.66 3.54 3.62 diameter of beads [mm] 1.23 1.3 1.34 1.17 1.05 1.04 volume [mm3] 0.97 1.15 1.26 0.84 0.61 0.59 deviation [%] 164.0 211.6 241.3 127.2 64.2 59. 6 table 4. ph values of the basic solutions, average diameter of the beads, their volume with % volume change before and at the end of experiment with gel beads. initial diameter and initial volume of beads were 3.03 mm and 14.57 mm3, respectively. ph of the solution koh naoh naoh koh initial 10.73 11.12 12.73 12.98 with gel beads 8.07 8.33 12.04 12.36 diameter of beads [mm] 3.6 4.1 dissolved by 3 days volume [mm3] 24.43 36.09 volume change [%] 67.7 147.8 from the presented results it is obvious that diameter and volume of the gel and dry beads of all groups in diluted acidic solutions of hcl, hno3, h2so4 and in concentrated and diluted acidic solutions of naoh, koh have increased in comparison with initial state and exhibited the tendency to swell. the dry beads b in nova biotechnologica 10-1 (2010) 49 concentrated solutions of the acids reached the highest swelling. from table 2 it is visible that the dry beads a in diluted acidic solutions with ph values 2.64-2.79 have showed out minimal volume changes from 3-15.4%. the dry beads for both groups were more swelled in concentrated acidic solutions than in diluted acidic solutions. in according to hoffman (2002) swelling of hydrogels is mainly attributed to the hydration of the hydrophilic groups of alginate. also in this study is assumed that diluted acidic solution penetrates inside the beads in order to fill the inert pores among the polymer chains. after obtaining of maximum swelling degree dry and gel beads began indicate dissolution or degradation. table 5. ph values of the basic solutions, average diameter of the beads, their volume with % volume change before and at the end of experiment with dry beads a. initial diameter and initial volume of beads were 1.02 mm and 0.56 mm3, respectively. ph of the solution koh naoh naoh koh initial 10.73 11.12 12.73 12.98 with dry beads a 8.02 8.43 12.24 11.5 diameter of beads [mm] 1.13 1.25 dissolved by 3 days volume [mm3] 0.76 1.02 volume change [%] 36 84.1 table 6. ph values of the basic solutions, average diameter of the beads, their volume with % volume change before and at the end of experiment with dry beads b. initial diameter and initial volume of beads were 0.89 mm and 0.37 mm3, respectively. ph of the solution koh naoh naoh koh initial 10.73 11.12 12.73 12.98 with dry beads b 7.96 8 12 11.53 diameter of beads [mm] 1.1 1.1 dissolved by 4 days volume [mm3] 0.70 0.70 deviation [%] 88.8 88.8 the changes of ph values were observed in the all solutions, excepting the concentrated acidic solutions. ph values in acidic solutions were higher and in alkaline solutions were lower after the swelling of calcium alginate beads. whether with gel or with dry beads, ph values in concentrated acidic solutions remained without changes. the gel and dry beads were dissolved by 3 days in concentrated solutions of sodium and potassium hydroxides. observed degradability of alginate beads is caused by the presence of high concentrations of non-gelling ions, such as na+ and k+ (bajpai and sharma, 2004). the swelling mechanism and subsequent degradation of the beads in presence of the solution of naoh/koh is related with the ca2+ and na+/ k+ exchange. the gel beads tended to shrink when exposed to the concentrated acidic solutions of hcl, hno3 and h2so4 (table 1). at low ph values the carboxylic groups of alginate are protonized and hence the electrostatic repulsion among these groups lessens and shrinkage is favoured (hoffman, 2002; pasparakis and bouropoulos, 2006). 50 ivánová, d . et al. 3.2 application of immobilized algae for the removal of cu2+ heavy metal ions from aqueous solutions the results obtained from kinetic studies on cu2+ sorption by immobilized algal beads of group a, b and c are shown in fig. 1. 0 5 10 15 20 25 30 35 40 45 0 120 240 360 480 600 720 840 960 1080 1200 1320 1440 time [min] s pe ci fic a ds or pt io n [m g g -1 ] group a group b group c fig. 1. specific adsorption of copper solutions by immobilized algal beads of groups a, b, c. it can be seen that biosorption capacity expressed as specific adsorption (q) has reached 33.8 mg g-1 of cu2+ ions (50%) in first hour during the experiment when gel beads a were used. when beads from group b and c were used specific adsorption capacity 35 mg g-1 of cu2+ ions from model solutions took 2 and 4 hours for beads from group b and c, respectively. group a and b reached equilibrium after 6 hours of biosorption. group c reached equilibrium within 8 hours. after 24 hours all group a, b and c removed 63% cu2+ ions from solutions and it corresponds with specific adsorption capacity 43 mg g-1 of cu2+ ions. from these results seems to be the fastest biosorption by gel beads from group a. on the other site dry beads from group b and c had higher mechanical stability. kinetics of biosorption of cu2+ ions by immobilized algae was compared with free powdered algae chlorella kessleri (horváthová et al., 2008). in the case of using free powdered algae equilibrium was reached within 10 minutes and the biosorption capacity was 46.6 mg g-1 of cu2+ ions. 4. conclusions this study shows that the stability of calcium alginate beads depends on ph values of the aqueous solutions and the initial physical state of the beads. behaviour of gel or dry beads is interesting because of the influence of individual metal ions in solutions. calcium alginate beads are sensitive to the solutions nova biotechnologica 10-1 (2010) 51 containing sodium and/or potassium ions. in these solutions they are swelled and subsequently dissolved by ion-exchange process taking place between na+/k+ ions in solution and ca2+ ions in the beads. their presence is the limiting factor for utilization of calcium alginate in metal biosorption. dry beads a treated with diluted acidic solutions exhibited minimum swelling and they are the most preferable in bioreactors because of their high mechanical stability. in contrast to the dry beads with the minimal swelling, the beads with tendency to increase are suitable when swelling and dissolution of the gels is required – an important factor in all applications where recovery of the cells is essential. this study proved the practical feasibility of using immobilized algal beads for the removal of copper from aqueous solution. from studied groups of immobilized algal beads are gel immobilized beads the most suitable because of the fastest copper removal from model solutions. acknowledgement: this work was supported by vega 1/0134/09. references bajpai, s.k., sharma, s.: investigation of swelling/degradation behaviour of alginate beads crosslinked with ca2+ and ba2+ ions. react. funct. polym., 59, 2004, 129-140. hoffman, a.s.: hydrogels for biomedical applications. adv. drug deliv. rev., 43, 2002, 3-12. horváthová h., kaduková j., mražíková a., slafkovská, g., štofko m.: effect of nickel on copper and zinc biosorption. in: acta metallurgica slovaca, 14, special issue, 1/2008, 5th. international conference (kammel’s quo vadis hydrometallurgy 5) košice, 19.-22.may, 2008, 78-82. kaduková, j., štofko, m.: environmentálne biotechnológie pre hutníkov, knkaso hf tu v kosiciach, košice, 2006, 141 pp. kaduková, j., horváthová, h., mražíková, a., štofko, m.: biosorption of cu, ni and zn from single metal solutions and their mixture by alga chlorella kessleri, e-proceedings from 4th european bioremediation conference, chania, greece, september 3-6, 2008, id188. pasparakis, g., bouropoulos, n.: swelling studies and in vitro reklease of verapamil from calcium alginate and calcium alginate-chitosan beads. int. j. pharm., 323, 2006, 34-42. vijaya, y., popuri, s.r., boddu, v.m., krishnaiah, a.: modified chitosan and calcium alginate biopolymer sorbents for removal of nickel (ii) through adsorption. carbohydr. polymer., 72, 2008, 261-271. volesky, b.: biosorption of heavy metals i. crc press, inc., boston, 1990, 381 pp. volesky, b.: sorption and biosorption, bv sorbex, inc., montreal, 2003, 316 pp. vreeker r., li l., fang y., appelqvist i., mendes e.: drying and rehydration of calcium alginate gels. food biophys., 3, 2008, 361-369. wang, j., chen, c.: biosorbents for heavy metals removal and their future. biotechnol. adv., 27, 2009, 195-226. microsoft word urickova et al nbc 12-2.doc nova biotechnologica et chimica 12-2 (2013) 83 doi 10.2478/nbec-2013-0010 © university of ss. cyril and methodius in trnava right-angle fluorescence spectroscopy for differentiation of distilled alcoholic beverages veronika uríčková, jana sádecká*, pavel májek institute of analytical chemistry, faculty of chemical and food technology, slovak university of technology, radlinského 9, sk-812 37 bratislava, slovak republic (jana.sadecka@stuba.sk) abstract: total luminescence and synchronous scanning fluorescence spectroscopic techniques were investigated for differentiating brandies from mixed wine spirits. the studies were performed on 16 brandies from 3 different producers and 30 mixed wine spirits from 5 different producers. differentiation between samples was accomplished by multivariate data analysis methods (principal component analysis, hierarchical cluster analysis, and linear discriminant analysis). correct classification was obtained using emission spectra (400–650 nm) recorded at excitation wavelength 390 nm, excitation spectra (225–460 nm) obtained at emission wavelength 470 nm and synchronous fluorescence spectra (200–700 nm) collected at wavelength interval 80 nm. these results indicate that right-angle fluorescence spectroscopy offers a promising approach for the authentication of brandies as neither sample preparation nor special qualification of the personnel are required, and data acquisition and analysis are relatively simple when compared to front-face technique. keywords: brandy, mixed wine spirit, fluorescence, chemometric, authentication 1. introduction brandy is a general term for distilled wine, usually 40–60% ethanol by volume. types of brandies, originally at least, tended to be location-specific. brandy has to be aged for a certain period in oak casks. toasting wood to be used in oak casks for aging brandy produces a great number of volatile and odoriferous substances. in addition, aging involves various processes producing significant changes in the composition of ageing spirit and being very important for the quality of the final products (taste, flavor and color) (mosedale and puech, 1998). mixed wine spirits are legally produced using wine distillates diluted with ethanol from other sources. they are frequently blended with sugar, brandy aroma and caramel. some mixed wine spirits contain honey or colorants. owing to the low price of this mixed wine spirit, it is sometimes used for a counterfeiting brandy. this usually occurs in restaurants and therefore affects the consumer and places honest traders at a financial disadvantage. for this reason, there is a need for a rapid method for drink authentication to reassure consumers, protect regional designations and ensure fair competition. several analytical methods have been published for classification of brandies. gas chromatography-mass spectrometry is a powerful tool in the analysis of volatile components of brandies (caldeira et al., 2004; watts et al., 2003). highperformance liquid chromatography has been widely used for the analysis of phenolic compounds and furanic derivatives of brandies. the direct injection allows classification of the brandies according to the botanical species of the wood barrel (canas et al., 2003). capillary zone electrophoresis has been applied successfully in the analysis of phenolic compounds. counterfeit brandy is easy to recognize by the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:05 utc 84 uríčková, v. et al. absence of sinapaldehyde, syringaldehyde, and coniferaldehyde (panossian et al., 2001). analytical method used to verify the quality and authenticity of alcoholic beverages should to perform an analysis without sample pre-treatment. in addition, it should to accomplish a fast data acquisition and carry out the data treatment accurately with relatively low costs. the combination of chemometric and spectroscopic methods is a good way to reach these premises. the aim of this paper is to show that right-angle fluorescence spectroscopy and multivariate statistical methods (pca, hca, linear discriminant analysis (lda)) can be used for distinguishing between commercial samples of brandies and mixed wine spirits as an alternative method to front-face fluorescence technique (sádecká et al., 2009), exigent of special front surface accessory. 2. materials and methods 2.1. samples and chemicals the studies were performed on 16 brandies (b) from 3 different producers (b1, n=8; b2, n=4; b3, n=4) and 30 mixed wine spirits (d) from 5 different producers (d1, n=12; d2, n=10; d4, n=4; d5, n=2; d6, n=2). samples were purchased from the local supermarkets besides four brandies which were obtained directly from the manufacturers. brandy b1, a traditional slovak product from the small carpathian viticulture region, is made of the grape using a classic method of aging wine spirit in small 300 l oak barrels for a minimum five years. the wine spirit then goes to 20,000 l barrels for next 3 years. brandy b2 is made of the pure high quality foreign wine spirit matured by natural way in oak barrels. b3 is made of the wine spirit from the east slovak viticulture region matured by natural way in oak barrels. mixed wine spirits are produced using wine spirits diluted with ethanol from other sources. they are frequently blended with sugar, brandy aroma and caramel (e 150a). mixed wine spirits d1 contain honey and colorants (e 102, e 110, e 120, e 122, e 132 and e 151). samples were stored in the dark at room temperature until the day of analysis. the plain caramel (e 150a, color no. 525) was a commercial caramel produced by d.d.williamson (uk). the physicochemical characteristics of this product are: color intensity (absorbance of 0.1% w/v solution at 610 nm through a 1 cm square cell) 0.030−0.035, specific gravity (15.56°c) 1.340−1.360 kg l-1, baume (15.56°c) 36.8−38.4, ph, as is 3.0−4.0. a stock solution, 1.000 g l-1, of caramel product was prepared in ethanol:water (40:60, v/v). the stock solution of caramel was diluted with ethanol:water (40:60, v/v) in order to have system with absorbance values similar to those of mixed wine spirit. ethanol, acs spectrophotometric grade, 95.0%, from sigma-aldrich and water (milli-q system) were used. 2.2. fluorescence spectroscopy fluorescence spectra were recorded using a perkin-elmer ls 50 luminescence spectrometer equipped with a xenon lamp. excitation and emission slits were both set at 5 nm. scan speed was 200 nm/min. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:05 utc nova biotechnologica et chimica 12-2 (2013) 85 fluorescence excitation spectra were recorded between 200 and 500 nm (increment 0.5 nm), repeatedly, at emission wavelengths from 300 to 600 nm, spaced by 10 nm interval in the emission domain. fluorescence emission spectra were recorded from 250 to 700 nm (increment 0.5 nm), repeatedly, at excitation wavelengths from 200 to 500 nm, spaced by 10 nm interval in the excitation domain. synchronous fluorescence spectra were collected by simultaneously scanning the excitation and emission monochromator in the excitation wavelength range 200– 700 nm, with constant wavelength differences δλ between them. spectra were recorded for δλ interval from 10 to 100 nm, in steps of 5 nm. fluorescence intensities were plotted as a function of the excitation wavelength. the right-angle geometry was used for samples in 10 mm × 10 mm × 45 mm quartz cell. fluorescence measurements were done in triplicate for each sample. the spectrometer was connected to a computer supplied with fl data manager software (perkin-elmer) for spectral acquisition and data processing. contour maps of total luminescence and synchronous scan fluorescence spectra were plotted using windows-based software originpro 7.5 (originlab, usa, 2002). 2.3. uv-visible absorption spectroscopy absorption spectra were obtained with a spectronic 20 genesys spectrophotometer (thermo fisher scientific) using 10 mm square cuvette and 8 nm slit width. the measurements were made between 325 nm and 700 nm. 2.4. mathematical analysis of data to investigate the differences between fluorescence spectra of the samples pca and hca were applied. pca is an unsupervised (we have no prior knowledge of the groups to be expected) pattern recognition method that reduces the dimensionality of the original data matrix while retaining the maximum amount of variability as well as recognizing the data structure (geladi, 2003). hca is an unsupervised pattern recognition method detecting natural grouping in data. objects are grouped in clusters in terms of their similarity. we used hierarchical (agglomerative) cluster analysis, where similarity extent was measured by manhattan (city-block) distances and cluster aggregation was based on ward’s method. finally, a supervised pattern recognition method, lda, was used to classify samples according to their origin. lda method is an excellent tool to obtain vectors showing the maximal resolution among categories, maximal separation and compactness of the categories. microsoft excel 2000 and statistica software version 6.0 (statsoft, usa, 2001) were used for statistical analysis. 3. results and discussion 3.1. total luminescence spectra figure 1 shows total luminescence spectra of the brandy and mixed wine spirit as contour maps, constructed in such a way that x-axis represents the emission and y-axis bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:05 utc 86 uríčková, v. et al. the excitation wavelengths, while the contours are plotted by linking the points of equal fluorescence intensity. brandy total luminescence contour map spreads in the excitation wavelength range 380–500 nm, in contrast to mixed wine spirit that spreads in the excitation wavelength range 200–240 nm and 300–500 nm, respectively. the contours for brandy are concentrated in the emission wavelength region 510–570 nm and excitation wavelength region 430–480 nm, while the contours for mixed wine spirit are concentrated in the emission wavelength region 460–530 nm and the excitation wavelength region 380–420 nm. in general, spectral features and fluorescence intensity values of all brandies are typical of brandies of similar origin and nature. the fluorescence spectra of brandies b1 are characterized by the main fluorophores centered at an excitation/emission wavelength pair of 460/540 nm. the other brandies have the following excitation/emission maxima: b2 450/523 nm, b3 450/524 nm. however, the excitation/emission wavelength values of the major peaks of the brandies are generally longer than those usually measured for mixed wine spirits, e.g., d1 396/494 nm, d2 390/476 nm. as mentioned, the mixed wine spirits are frequently colored with caramel (e 150a) to imitate the effect of long aging in wooden casks. therefore we assumed that the major peak originates from this colorant. to support this assumption, the stock solution of caramel was diluted with ethanol:water (40:60, v/v) in order to have system with ethanol volume and absorbance values similar to those of mixed wine spirit, and total luminescence spectra of caramel were recorded. emission spectra recorded after excitation at 395 nm showed a maximum located around 494 nm (fig. 1c). the light observed at 450–550 nm under excitation at 200–240 nm is probably scatter originating from caramel colloidal aggregates. a comparison of fluorescence spectra, particularly of those recorded at excitation/emission maxima of caramel supports an earlier assumption. we should note, however, that exact position of maxima of caramel fluorescence vary from one mixed wine spirit to another, which may results from difference in the respective caramel composition and/or from the technological process used. as it is clearly shown in fig. 1 the shape and the fluorescence intensities are different between the two classes of beverages. it appears that total luminescence spectroscopy can be used to characterize brandy and mixed wine spirit samples using suitable wavelength regions. 300 400 500 600 700 200 250 300 350 400 450 500 e xc ita tio n w av el en gt h (n m ) emission wavelength (nm) a 300 400 500 600 700 200 250 300 350 400 450 500 e xc ita tio n w av el en gt h (n m ) emission wavelength (nm) b 300 400 500 600 700 200 250 300 350 400 450 500 e xc ita tio n w av el en gt h (n m ) emission wavelength (nm) c fig. 1. contour plots of total luminescence spectra of brandy (a), mixed wine spirit (b), and caramel (0.750 g l-1 in ethanol:water, 40:60, v/v) (c). contours join the points of equal fluorescence intensity. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:05 utc nova biotechnologica et chimica 12-2 (2013) 87 besides the fluorescence band, fig. 1 shows the presence of the first and the second order rayleigh scatter bands. the first order rayleigh scatter line is centered at the emission wavelength equals excitation wavelength, the second order rayleigh scatter at the emission equals twice the excitation. scattering is much more intense and/or heterogenous for mixed wine spirits than for brandies. this phenomenon may result from the higher heterogeneity of mixed wine spirit, e.g., presence of colloids, with respect to brandy. the light observed at 450–550 nm under excitation at 200– 240 nm is probably scatter, partially originated from caramel colloids in mixed wine spirit. hence, the differences between brandy and mixed wine spirit are also due to scatter bands. because these bands have no information about the fluorophores in the samples the wavelength range where they are located will be discarded from the subsequent data analysis. 3.2. total synchronous fluorescence spectra the contour plots of total synchronous fluorescence (tsf) spectra were obtained by plotting the fluorescence intensity (z-axis) as a function of excitation wavelength (x-axis) and wavelength interval δλ (y-axis). the tsf spectra of a brandy sample are given in fig. 2a. it shows that the tsf contour map spreads in the excitation wavelength 380–550 nm and δλ 10–100 nm. the contours are concentrated in the excitation wavelength region 440–480 nm and δλ 60–100 nm. the plot shows only one fluorescence maximum blue-shifted from 500 to 450 nm with increasing δλ. the maximum fluorescence intensity was recorded at excitation wavelength 460 nm (δλ = 80 nm), 450 nm (δλ = 75 nm) and 450 nm (δλ = 75 nm) for brandy b1, b2 and b3, respectively. the tsf spectra of a mixed wine spirit sample are given in fig. 2b. the contour map spreads in the excitation wavelength 320–550 nm and δλ 10–100 nm. two overlapping bands with maxima at 420 and 479 nm are apparent for δλ = 10 nm. for δλ = 30 nm, the fluorescence intensity of bands increased, changes in their relative intensities were noted, and maxima were blue-shifted to 407 and 460 nm, respectively. at increased δλ, the first band was shifted to 405 and 399 nm and the second one to 452 and 435 nm for δλ = 40 and 60 nm, respectively. the further increase of intensity and band broadening was apparent for δλ = 70 nm, resulting in only one band with maxima at 400 nm. the banding intensity increases at higher δλ values. the shape and intensity of the fluorescence maxima varied from one mixed wine spirit to another. for example, the maximum fluorescence intensity for sample d1 and d2 was observed at excitation wavelength 396 nm (δλ = 95 nm) and 391 nm (δλ = 85 nm), respectively. generally, the fluorescence maxima shift to shorter wavelengths with increasing δλ for both brandy and mixed wine spirit. brandies give a longer wavelength less intensive fluorescence band, while mixed wine spirits give a shorter high intensive band. the shape and intensity of the synchronous fluorescence spectra of caramel depend on the difference between excitation and emission wavelengths δλ (fig. 2c). for δλ = 10 nm, two bands with maxima at 420 and 478 nm are observed. at increased δλ, the first band was shifted to 407, 404 and 400 nm and the second one to 460, 453 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:05 utc 88 uríčková, v. et al. and 435 nm for δλ 30, 40 and 60 nm, respectively. for δλ = 70 nm, only one band with maxima at 399 nm was recorded. comparison shows that the maxima observed for caramel at different δλ-values are consistent with the respective maxima in mixed wine spirits. 200 300 400 500 600 700 20 40 60 80 100 w av el en gt h in te rv al ( nm ) excitation wavelength (nm) a 200 300 400 500 600 700 20 40 60 80 100 w av el en gt h in te rv al ( nm ) excitation wavelength (nm) b 200 300 400 500 600 700 20 40 60 80 100 w av el en gt h in te rv al ( nm ) excitation wavelength (nm) c fig. 2. contour plots of total synchronous fluorescence spectra of brandy (a), mixed wine spirit (b), and caramel (0.750 g l-1 in ethanol:water, 40:60, v/v) (c). contours join the points of equal fluorescence intensity. 3.3. multivariate analysis of excitation and emission spectra the pca was carried out separately on the excitation and emission spectra to investigate differences between samples. the best classification was achieved using excitation spectra (225–460 nm) recorded at emission wavelength 470 nm or emission spectra (400-650 nm) recorded at excitation wavelength 390 nm. the fluorescence spectra showed different shapes. the width of excitation spectra was larger for mixed wine spirits than those for brandies. mixed wine spirits had higher fluorescence intensity regardless of wavelength but they were also more heterogeneous in this respect. excitation and emission spectra obtained for the mixed wine spirit at the excitation/emission wavelength pair 390/470 nm are similar to those of caramel. moreover, the absorption spectrum is in good agreement with that of caramel in the wavelength range 325−600 nm. the pca output indicates that the first pc (pc1) has the largest eigenvalue at 38.0 and accounts for 82.6% of the total variance in the collected data. the second pc (pc2) has next largest eigenvalue at 7.8 and accounts for 17.0% of the data variation. the remaining pcs account for 0.4% of the variation. according to the eigenvalue results displayed in the scree plot (eigenvalue vs number of eigenvalues), only the first two pcs were used for the purpose of pca illustration. the variances of the subsequent components are similar and correspond to experimental noise, scatter band residuals and baseline fluctuations. the general guideline in pca applications is to select those pcs which account, cumulatively, for at least 80% to 90% of the data variation. cumulatively, the first two pcs from the collected data account for 99.6% of the total variance indicating that two pcs appear sufficient to explain the structures in the data. the similarity map defined by the pc1 and pc2 of the pca performed on excitation spectra allowed a good discrimination of beverages (fig. 3a). spectral pattern associated with the pcs provide the characteristic wavelengths that may be used to discriminate between spectra. the spectral pattern associated with the pc1 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:05 utc nova biotechnologica et chimica 12-2 (2013) 89 exhibited a positive peak around 280 nm and a broad positive peak between 350– 450 nm corresponding to caramel. the spectral pattern associated with the pc2 exhibited positive peaks at 250 and 300 nm (results are not presented). while a discrimination of the brandy and mixed wine spirit samples was achieved with excitation data collection, a different trend was observed with emission spectra. the pc1 has an eigenvalue at 40.2 and accounts for 87.4% of the total variance in the collected data. the pc2 has an eigenvalue at 5.7 and accounts for 12.4% of the data variation. cumulatively, the first two pcs from the collected data account for 99.8% of the total variance indicating that two pcs appear sufficient to explain the structures in the data. although the pca similarity map defined by the pc1 and pc2 did not lead to a clear discrimination of beverages, a general trend pointing out the brandies and mixed wine spirits was observed on the map (fig. 3b). brandies b1 constitute one subgroup, while brandies b2 and b3 constitute another subgroup. finally, there is a group of mixed wine spirits. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 p c 2 pc1 a 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.4 0.5 0.6 0.7 0.8 0.9 1.0 p c 2 pc1 b b1 b2,b3 fig. 3. principal component analysis similarity map (score plot) determined by principal component 1 (pc1) and principal component 2 (pc2) for excitation fluorescence spectra recorded at emission wavelength 470 nm (a) and emission spectra recorded after excitation at 390 nm (b) on brandy (□) and mixed wine spirit (o) samples. in a second step, hca was carried out separately on the excitation and emission spectra in order to evaluate the number of subsets of similar samples appearing in the complete data set. for the sake of simplicity, a reduced number of samples were included in the figures, but from them we can clearly recognize the clustering common to all the samples. the evaluation of the above mentioned excitation spectral region provided two main clusters (fig. 4a). all mixed wine spirits are linked together in the first main cluster. this main cluster is heterogeneous enough but consists of various small groups of very similar products. for instance most of the mixed wine spirits from producer 1 (d1) is found as such in one subgroup with 97% of similarity among them. all brandies are linked together in the second main cluster. one subcluster is constituted of b1 (brandy from producer 2) with 98% of similarity. another subcluster is constituted of b2 and b3 with 95% of similarity among them and 90% in relation to b1. fig. 4b shows the results from hca concerning the emission spectra. the dendrogram shows that the brandies are well separated from the mixed wine spirits. the first main cluster contains mixed wine spirit samples, while the second one contains brandy samples. mixed wine spirits form three smaller groups. one small group is constituted of samples d2 with 98% of similarity among them and by sample bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:05 utc 90 uríčková, v. et al. d5 with 92% of similarity with the samples d2 of this group. another group is constituted of d2, d4 and d6 with 96% of similarity among them and 88% in relation to the group of d1. samples d1 show a similarity of 94% to each other. brandy samples form three small subclusters of the second main cluster corresponding to three different producers. one subcluster is constituted of b1 with 95% of similarity. another subcluster is constituted of b2 and b3 with 96 % of similarity among them and 73% in relation to b1. d23 d22 d24 d51 d15 d25 d41 d42 d21 d61 d13 d12 d16 d14 d11 b14 b13 b12 b11 b22 b21 b32 b31 0 20 40 60 80 100 120 (d lin k/ d m ax )* 10 0 d24d23d22d51d25d42d61d21d41d15d13d12d16d14d11b14b13b12b11b22b21b32b31 0 20 40 60 80 100 120 (d lin k/ d m ax )* 10 0 fig. 4. hierarchical cluster analysis dendrogram using manhattan distance for excitation fluorescence spectra recorded at emission wavelength 470 nm (a) and emission spectra recorded after excitation at 390 nm (b) on brandies (b) and mixed wine spirits (d). 3.4. multivariate analysis of synchronous fluorescence spectra pca was applied separately on synchronous spectra measured at δλ 10–100 nm. the best classification was achieved using fluorescence spectra recorded at δλ = 80 nm. the synchronous fluorescence spectra showed different shapes. the width of synchronous spectra was larger for mixed wine spirits than those for brandies. mixed wine spirits had higher fluorescence intensity regardless of wavelength but they were also more heterogeneous in this respect. synchronous fluorescence spectra of brandy, mixed wine spirit and caramel (e 150a) obtained at δλ = 80 nm confirmed an earlier assignment of the band. figure 5a shows that the plot of the first two pcs lead to a good discrimination of beverages according to origin. the pc1 has an eigenvalue at 38.6 and accounts for 84.0% of the total variance. the pc2 has an eigenvalue at 7.2 and accounts for 15.6% of the data variation. cumulatively, the first two pcs from the collected data account for 99.6% of the total variance. the spectral pattern (fig. 5b) associated with the pc1 presented a positive peak at 280 nm, a broad positive peak between 320−460 nm and a negative peak at 550 nm. the spectral pattern associated with the pc2 exhibited a negative peak at 250 nm and a positive peak around 500 nm. applying hca to fluorescence spectra recorded at δλ = 80 nm, the dendrogram shows that the mixed wine spirits are well separated from brandies (fig. 6). the first main cluster contains mixed wine spirit samples only, while the second one contains brandy samples. mixed wine spirits cluster consists of various small groups of very similar products. one small group is constituted of samples d2 with 99% of similarity among them and by sample d5 with 95% of similarity with the samples d2 of this a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:05 utc nova biotechnologica et chimica 12-2 (2013) 91 group. another group consists of d1 with 96% of similarity among them and 80% in relation to the previous group (d2, d4, d5, and d6). brandy samples form three small subclusters of the second main cluster corresponding to three different producers. one subcluster is constituted of b1 with 97% of similarity. another subcluster is constituted of b2 and b3 with 95 % of similarity among them and 86% in relation to b1. 0,0 0,2 0,4 0,6 0,8 1,0 0,0 0,2 0,4 0,6 0,8 1,0 p c 2 pc1 a 200 300 400 500 600 700 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 wavelength (nm) p c b pc1 pc2 fig. 5. principal component analysis similarity map (score plot) determined by principal components 1 (pc1) and principal component 2 (pc2) (a) and spectral pattern (loading) corresponding to pc1 and pc2 (b) for synchronous fluorescence spectra recorded at δλ = 80 nm on brandy (□) and mixed wine spirit (o) samples. d23 d24 d22 d51 d42 d25 d61 d21 d41 d15 d13 d12 d16 d14 d11 b14 b12 b13 b11 b22 b21 b32 b31 0 20 40 60 80 100 120 (d lin k/ d m ax )* 10 0 fig. 6. hierarchical cluster analysis dendrogram using manhattan distance for synchronous fluorescence spectra recorded at δλ = 80 nm on brandy (b) and mixed wine spirit (d) samples. finally, a supervised pattern recognition method, lda, was applied to fluorescence spectra recorded at δλ = 80 nm to classify samples according to their origin. lda starts with number of objects whose group membership is known. a basic problem in lda is deciding which variables (excitation wavelengths) should be included in the analysis. in stepwise discriminant function analysis, a model of discrimination is built step-by-step (forward or backward). specifically, at each step all variables are reviewed and evaluated (fischer’s statistics—f to enter and f to remove values) to determine which one will contribute most to the discrimination between groups. this variable will then be included in the model, and the process bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:05 utc 92 uríčková, v. et al. starts again. after performing backward lda, a classification function was obtained for individual analyzed beverages containing five variables (excitation wavelengths): 280, 388, 396, 402, and 498 nm, which provide 99.6% correct predictions for brandies and wine distillates samples these results show that complete synchronous spectra are not required to discriminate between beverages. instead of them, fluorescence intensity could be measured at selected wavelengths. 4. conclusions this study shows that brandies and mixed wine spirits can be discriminated using differences in their fluorescence spectra. differentiation between samples was accomplished by multivariate data analysis methods (pca, hca, and lda). comparison of the results obtained from multivariate data analysis indicated that better classification was obtained from synchronous fluorescence spectra than from the excitation/emission fluorescence spectra. right-angle fluorescence spectroscopy offers a promising approach for the authentication of brandies as neither sample preparation nor special qualification of the personnel are required, and data acquisition and analysis are more simple when compared to front-face technique. acknowledgments: this work was supported by the slovak research and development agency under the contract no. apvv-0797-11. references caldeira, i., pereira, r., climaco, m. c., belchior, a. p., bruno de sousa, v.: improved method for extraction of aroma compounds in aged brandies and aqueous alcoholic wood extracts using ultrasound. anal. chim. acta, 513, 2004, 125–134. canas, s., belchior, a. p., spranger, m. i., bruno-de-sousa, r.: highperformance liquid chromatography method for analysis of phenolic acids, phenolic aldehydes, and furanic derivatives in brandies. development and validation. j. sep. sci., 26, 2003, 496–50. geladi, p.: chemometrics in spectroscopy. part i. classical chemometrics. spectrochim. acta b, 58, 2003, 767–782. mosedale, j. r., puech, j.-l.: wood maturation of distilled beverages. trends food sci. tech., 9, 1998, 95–101. panossian, a., mamikonyan, g., torosyan, m., gabrielyan, e., mkhitaryan, s.: analysis of aromatic aldehydes in brandy and wine by highperformance capillary electrophoresis. anal. chem., 73, 2001, 4379–4383. sádecká, j., tóthová, j., májek, p.: classification of brandies and wine distillates using front face fluorescence spectroscopy. food chem. 117, 2009, 491– 498. watts, v. a., butzke, c. e., boulton, r. b.: study of aged cognac using solid-phase microextraction and partial least-squares regression. j. agr. food chem., 51, 2003, 7738–7742. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:05 utc microsoft word durcekova 9-1 2009.doc nova biotechnologica 9-1 (2009) 83 relationship between administration of statins and blood serum levels of selected biochemical parameters tatiana ďurčeková1, ján mocák1, ján balla2, gabriela gromanová2, katarína boronová1 1department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (tatiana.durcekova@ucm.sk) 2analytical-diagnostic laboratory, kováčska 15, prešov, sk-080 01, slovak republic (jan.balla@mail.t-com.sk) abstract: results of biochemical tests of 172 patient data (among them 84 men data and 88 women data, resp.) before and after administration of statins were studied. all monitored patients are characterized by lipoprotein metabolism failure or other kind of lipidaemia. the calculations were performed using several chemometrical methods pursuing visualization of most important biochemical parameters and classification of the patient blood samples by means of up-to-date software packages. the dependences of the content of most common biochemical parameters upon the treatment of patients by statins were elucidated in detail. key words: coronary artery diseases, statins, biochemical parameters, multivariate data analysis. 1. introduction coronary heart disease remains the leading cause of mortality in the civilized countries. in medicine it is now an accepted principle that cholesterol lowering is of major importance in diminishing the risk of a developing coronary artery disease. most patients with coronary artery disease require intensive drug therapy to achieve target lipid levels (kastelein, 1999; shimokata et al., 2004). the introduction of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme a reductase (statins) in 1987 was a major advance in prevention and treatment of cardiovascular diseases. several clinical trials have demonstrated the benefit of lipid lowering caused by statins for the primary and secondary prevention of coronary heart disease (liao, 2002; istvan and deisenhofer, 2001). the clinical practise of cardiovascular medicine has been innovated by the advent of the statins. the statins have a wide range of beneficial biologic effects in addition to lipid lowering, a phenomenon commonly termed a pleiotropic effect (fujita et al., 2008; gounari et al., 2009). statins act by inhibiting 3-hydroxy-3-methyl-glutaryl coenzyme a (hmg-coa) reductase and thereby reducing cholesterol synthesis (garcia et al., 2008). 2. material and methods 2.1 description of data data from the out-patient department in prešov were obtained in cooperation with the specialist internal (gromanova, 2008). in this study, biochemical data steming 84 ďurčeková, t. et al. from 172 probands were analyzed by several chemometrical methods in order to reveal relationships between administration of statins and the level of biochemical parameters. the investigated patients were characterized by lipoprotein metabolism failure and other kind of lipidaemia. two data tables (for men 84 samples, and for women – 88 samples) containing the sample origin (patients) in rows and investigated variables in columns (10 biochemical parameters plus 1 combined parameter and age of the patient) were assembled. the following biochemical variables (often called as parameters) were selected: total cholesterol (tchol), high density lipoprotein cholesterol (hdlc), low density lipoprotein cholesterol (ldlc), triacylglycerols (tg), aterogenic index (ai) the combined parameter, creatinine (crea), aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), creatinine kinase (ck), and gamma-glutamyl transferase (ggt). the parameter values were measured before administration of statins and one year after the statin treatment. the patient samples objects were therefore divided into two groups: class 1 – parameter values before the treatment by statins and class 2 – parameter values one year after the treatment. 2.2 multidimensional data analysis the results of biochemical tests were subjected to multidimensional data analysis using four software packages. statgraphics plus 5.1 was utilized to perform cluster analysis (vandeginste et al., 1997) as well as principal component analysis (massart et al., 1997); spss 15 and sas packages enabled to accomplish discriminant analysis and logistic regression (otto, 1999; sharma, 1996). correlation analysis (meloun and militky, 2002) was also made in addition to the mentioned more sophisticated statistical techniques using software jmp 6.0. 3. results and discussion 3.1 correlation analysis correlation analysis was performed in common for the patients’ results obtained before and after the statins administration. it is represented by the correlation table containing the calculated pair (pearson) correlation coefficients, which indicate the strength of correlation between all pairs of variables biochemical parameters for men and women, distinctively. in order to see the measure of their importance, the correlation coefficient values were compared to the critical value of the sample correlation coefficient, r = 0.215 for men dta set and 0.210 for women data set, which depends on the number of degrees of freedom (the sample number minus two) and the selected probability (usually 0.05). consequently, all values in the correlation tables (table 1, table 2) indicating significant correlations are marked in bold. it can be seen from the observed values that many of the observed significant correlations (marked bold) are very well understandable, as e.g. between tchol a ldlc (strongest correlation for men and women), tchol and its component parts (ldlc and hdlc), nova biotechnologica 9-1 (2009) 85 tchol and tg (for men only), as well as the negative correlation between index of aterogenity ai and so called “good” cholesterol hdlc (high values of hdlc correspond to low values of ai). similarly, significant correlations between the pairs ast – alt and gmt – alt are expectable. what is also interesting are the negative correlations for men between age and any of the variables tg, and ai, which is pronounced after the statins intake. strong positive correlations of tg with tchol and with ai as well as strong negative correlation of tg with hdlc, all observed only after the statins, may also be remarkable. table 1. summary of correlation coefficients expressing mutual correlation between the pairs of investigated biochemical parameters for men data set. variable age crea ast alt gmt alp age 1.000 crea 0.170 1.000 ast -0.081 -0.128 1.000 alt -0.287 -0.090 0.889 1.000 gmt -0.170 0.137 0.168 0.358 1.000 alp 0.290 -0.042 -0.039 -0.048 0.021 1.000 ck 0.021 0.048 0.110 0.039 0.036 -0.298 tchol -0.111 0.040 -0.033 0.054 -0.035 0.001 hdlc 0.171 -0.128 0.094 0.135 0.022 -0.130 ldlc -0.020 0.002 -0.034 0.025 -0.102 0.098 tg -0.352 0.181 -0.073 0.006 0.122 -0.143 ai -0.281 0.156 -0.092 -0.053 -0.072 0.129 continuation ck tchol hdlc ldlc tg ai ck 1.000 tchol -0.084 1.000 hdlc 0.123 0.346 1.000 ldlc -0.094 0.927 0.240 1.000 tg -0.099 0.403 -0.205 0.099 1.000 ai -0.176 0.588 -0.526 0.608 0.528 1.000 3.2 cluster analysis cluster analysis was performed distinctively for the men data set and the women data set. the cluster analysis results are most frequently represented by a dendrogram. figure 1 depicts the dendrogram, which was calculated using squared euclidean distance, commonly used in practice. it shows three significant clusters. the first significant cluster is created by tchol, ldlc, tg and ai, all representing high cardiovascular risk. the second significant cluster is formed by ast, alt (characterizing liver enzymes) and third cluster is created by hdlc with ck, crea with gmt, to which alp and age variables are added at larger distance value (figure 1), which means that these variables are less similar compared to the four ones mentioned. the results for women data set (figure 2) are similar except the observed clustering of age, this time with crea instead of alp. however, with regard to the most important variables indicating lipidaemia, the situation among men and women is similar. 86 ďurčeková, t. et al. table 2. summary of correlation coefficients expressing mutual correlation between the pairs of investigated biochemical parameters for women data set. variable age crea ast alt gmt alp age 1.000 crea 0.389 1.000 ast 0.079 0.105 1.000 alt -0.257 -0.113 0.680 1.000 gmt -0.103 0.163 0.188 0.279 1.000 alp 0.207 -0.017 0.055 -0.003 0.028 1.000 ck 0.085 0.036 0.455 0.253 -0.039 0.105 tchol 0.120 0.082 0.130 0.095 0.172 0.156 hdlc 0.329 0.124 0.418 0.133 0.004 -0.186 ldlc 0.037 -0.016 -0.031 -0.003 0.100 0.164 tg -0.090 0.065 -0.210 0.040 0.253 0.341 ai -0.197 0.023 -0.375 -0.119 0.101 0.328 continuation ck tchol hdlc ldlc tg ai ck 1.000 tchol -0.110 1.000 hdlc 0.007 0.368 1.000 ldlc -0.143 0.859 0.122 1.000 tg -0.068 0.168 -0.477 0.074 1.000 ai -0.084 0.320 -0.670 0.458 0.642 1.000 d is ta nc e 0 200 400 600 800 1000 a ge c r e a a s t a lt g m t a lp c k tc h o l h d lc ld lc t g a i fig. 1. dendrogram of cluster analysis of 12 variables for 84 objects (men). ward`s clustering method, squared euclidean distance. software statgraphics plus 5.1. 3.3 principal component analysis the obtained principal component analysis results are visualized in the biplot form (figure 3). biplot simultaneously represents the samples together with twelve selected nova biotechnologica 9-1 (2009) 87 variables, depicted by the rays, which start from the origin and end at the point determining the variable position in the plane of principal components. 0 200 400 600 800 1000 d is ta nc e ag e c r e a a s t a lt g m t a lpc k tc h o l h d lc ld lct g a i fig. 2. dendrogram of cluster analysis of 12 variables for 88 objects (women). ward`s clustering method, squared euclidean distance. software statgraphics plus 5.1. a close position of tchol, ai, ldlc and tgt at the shown pca biplot confirms their mutual dependence found also by correlation analysis and it helps to understanding the pc1 axis as that expressing the cardiovascular risk. almost opposite position of hdlc is in correspondence with the previous statement. the variables ast, alt and gmt as well as their opponents alp and age are in the perpendicular position (located along the pc2 axis) to the cholesterol variables, which mean their independence with respect to them. this is also in accordance with the results of correlation analysis. 3.4 discriminant analysis discriminant analysis makes a linear combination of the originally selected variables in the way that the discrimination between the classes is mostly pronounced. table 3 shows a summary of the results of linear discriminant analysis (lda), quadratic discriminant analysis (qda) and logistic regression (lr) for the men data set. all these methods are used for classification of the patient samples into two classes – before (class 1) and after (class 2) treatment by statins. the results for training set (used for calculating the classification model) and leave-one-out method validation (one object is left out from the training set in a stepwise mode until all objects are interchanged) are represented in per cents. success in validation step, where the samples independent of the calculations performed in the training process, is more important for overall assessment of the classification method. it is obvious that the predictive ability is better for lda than the qda. however, the most promising are the results achieved by logistic regression. 88 ďurčeková, t. et al. fig. 3. biplot pc1 vs. pc2 (men), 12 variables (with the symbols specified in part 2), 84 patient samples. software statgraphics plus 5.1. table 3. results of discriminant analysis (lda, qda) and lr for two software packages (spss, sas) – classification of the men samples into two categories: before and after statin treatment. spss sas method results training set leave-one-out training set leave-one-out true/all 73/84 67/84 73/84 67/84 lda % true 86.9 79.8 86.9 79.8 true/all 73/84 n.a. 70/84 55/84 qda % true 86.9 n.a. 83.3 65.5 true/all 75/84 n.a. 75/84 72/84 lr % true 89.3 n.a. 89.3 85.7 n.a. – not accessed. tabable 4.: results of discriminant analysis (lda, qda) and lr for two software packages (spss, sas) – classification of the women samples into two categories: before and after statin treatment. spss sas method results training set leave-one-out training set leave-one-out true/all 81/88 74/88 81/88 74/88 lda % true 92.0 84.1 92.0 84.1 true/all 81/88 n.a. 76/88 61/88 qda % true 92.0 n.a. 86.4 69.3 true/all 79/88 n.a. 79/88 77/88 lr % true 89.8 n.a. 89.8 87.5 n.a. – not accessed. nova biotechnologica 9-1 (2009) 89 df1 n o class 1 2 -2,7 -1,7 -0,7 0,3 1,3 2,3 3,3 4,3 -10 10 30 50 70 90 fig. 4. lda scatterplot of 84 patient samples (men) marked by number (no) calculated from 12 variables. df1 is the only discriminant function. class 1before, class 2 – after treatment by statins. software statgraphics plus 5.1. a good separation between the investigated two categories of the patient samples is confirmed also in figure 4, calculated also for the men data set. the results for women data set are collected in table 4. all results referring to the training set are close to or over 90% of the correct results, the best outcome in validation by leave-one-out method was obtained for lr. 4. conclusions treatment of patients with the risk of a developing coronary artery disease can be successfully monitored using the visualization and classification methods of multidimensional statistical analysis. the interrelation of eleven biochemical laboratory parameters as well as the patient’s age and gender was investigated in detail. achieved results enable to evaluate the treatment by statins in each individual case. they may also help in estimation of the pertaining cardiovascular risk after the statin drug administration and in monitoring the dynamic progression of the disease. acknowledgement: the support of this work by the grants vega 1/1005/09 and vvce-000407 is acknowledged. references fujita, m., yamazakib, t., hayashic, d., kohroc, t., okadac, y., nagai, r.: pleiotropic effects on cardiovascular events in the japanese coronary artery disease study. int. j. cardiol., 129, 2008, 294-296. garcia, i., munteanu, c.r., fall, y., gómez, g., uriarte, e., gonzález-díaz, h.: qsar and complex network study of the chiral hmgr inhibitor structural diversity. bioorg. med. chem., 17, 2009, 165-175. 90 ďurčeková, t. et al. gounari, p., tousoulis, d., antoniades, c., kampoli, a.m., stougiannos, p., papageorgiou, n., roulia, g., stefanadi, e., siasos, g., tsioufis, c., stefanadis, c.: rosuvastatin but not ezetimibe improves endothelial function in patients with heart failure, by mechanisms independent of lipid lowering. int. j. cardiol., 2009, (in press) . gromanová, g.: influence of hypolipidaemic treatment by statins on levels of selected biochemical parameters (in slovak). diploma thesis, tu trnava, 2008. istvan, e., deisenhofer, j.: statin inhibition of hmg-coa reductase: a 3dimensional view. atherosclerosis suppl., 4, 2003, 3-8. kastelein, j.j.p.: the future of best practise. atherosclerosis 143, 1999, s17-s21. liao, j.k.: beyond lipid lowering: the role of statins in vascular protection. int. j. cardiol., 86, 2002, 5-18. massart, d.l., vandeginste, b., buydens, l., de jong, s., lewi, p., smeyers-verbeke, j.: handbook of chemometrics and qualimetrics, part a, elsevier, amsterdam, 1997, 886 pp. meloun, m., militký j.: compendium of statistical data processing (in czech), academia, praha, 2002, 764 pp. otto, m.: chemometrics: statistics and computer application in analytical chemistry, wiley, weinheim, 1999, 314 pp. sharma, s.: applied multivariate techniques, j. wiley, new york, 1996, 588 pp. shimokata, k., yamadac, y., kondo, t., ichihara, s., izawa, h., nagata, k., murohara, t., ohnod, m., yokota, m.: association of gene polymorphisms with coronary artery disease in individuals with or without nonfamilial hypercholesterolemia. atherosclerosis, 172, 2004, 167-173. vandeginste, b., massart, d.l., buydens, l., de jong, s., lewi p., smeyers-verbeke, j.: handbook of chemometrics and qualimetrics, part a, amsterdam, elsevier, 1997, 876 pp. microsoft word moravcik nbc 12-2.doc nova biotechnologica et chimica 12-2 (2013) 108 doi 10.2478/nbec-2013-0013 © university of ss. cyril and methodius in trnava high – performance liquid chromatographic method for enantioseparation of underivatized α-amino acids using cyclofructan – based chiral stationary phases jakub moravčík, katarína hroboňová institute of analytical chemistry, faculty of chemical and food technology, slovak university of technology, radlinského 9, bratislava, sk-812 37, slovak republic (jakub.moravcik@stuba.sk) abstract: chromatographic columns with chiral stationary phases based on chemically – bonded derivatized cyclofructans were evaluated for hplc enantioresolution of underivatized α-amino acids. the analytical study of chiral separation of these analytes was focused on the selection of suitable chiral stationary phase and mobile phase. using isopropyl carbamate cyclofructan 6 as a chiral stationary phase, αamino acid optical isomers were separated. the retention and enantioseparation of chiral amino acids were also influenced by a mobile phase composition. the mixture methanol/acetonitrile/acetic acid/triethylamine (75/25/0.3/0.2 v/v/v/v) was found to be the most effective mobile phase for hplc separation of studied compounds. hplc enantioresolution of chiral amino acids was thermodynamically studied. based on the enthalpy and entropy contribution values calculated from the van´t hoff equation, hplc enantioseparation under chosen chromatographic conditions was found to be an enthalpically driven. key words: amino acids, cyclofructan – based chiral selectors, hplc, thermodynamic study 1. introduction chirality was discovered in 1848 by louis pasteur (flack, 2009). since most biologically important compounds exist in two enantiomeric forms that can possess differences in their physiological activity, the impetus for the separation of optical isomers and the preparation of enantiomerically pure compounds in the field of biotechnology, chemistry and pharmacology is still increasing (ilisz et al., 2012). chromatographic methods, i.e. capillary electrophoresis (ce), gas chromatography (gc), high – performance liquid chromatography (hplc), supercritical fluid chromatography (sfc), thin layer chromatography (tlc), have been frequently used for the enantioresolution of chiral analytes. among them, enantioselective hplc method is one of the most powerful and widely used separation techniques at both analytical and preparative scales (ding et al., 2004). amino acids play an important role in living organisms. they serve as the basic structural units of peptides and proteins. most proteinogenic α-amino acids contain an asymmetric carbon atom. their stereoisomers can differ markedly in physiological effects. chiral separation of these substances is a great analytical challenge in several research areas, such as asymmetric syntheses in organic chemistry, biochemistry of amino acids, pharmaceutics, the dating of archaeological materials and the study of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:14 utc nova biotechnologica et chimica 12-2 (2013) 109 food processes in human body (shpigun et al., 2002). the recent direct hplc separations of amino acid stereoisomers and related compounds are summarized in table 1. table 1. summary of chiral stationary phases for direct hplc separations of amino acid stereoisomers. separated stereoisomers chiral selectors mobile phases references cyclodextrins 20 unusual amino acids α-cd, β-cd, acetylated β-cd, 3,5dimethylphenylcarbamoylated β-cd 0.1% aqueous tfaa (ph 4-5) remsburg et al., 2008 crown-ether β-amino acids, proteinogenic d,l-amino acids, aliphatic and aromatic β3-amino acids (+)-(18-crown-6)2,3,11,12-tetracarboxylic acid h2o/meoh + 10 mmo.l -1 acoh, h2o/meoh (80/20-20/80 v/v) + 5-10 mmol.l-1 acoh, h2o/meoh (50/50 v/v) + 0.02 mol.l-1 h2so4 chen et al., 2006; wang et al., 2010; berkecz et al., 2006 β-amino acids (3,3´-diphenyl-1,1´binaphtyl)-20-crown-6 h2o/mecn (80/20 v/v) + 0.01 mol.l-1 h2so4 + 1.0 mmol.l-1 ch3co2nh4 choi et al., 2008 proteinogenic amino acids pseudo-18-crown-6 having 1-phenyl-1,2cyclohexanediol unit hexane/etoh/tfaa/h2o (75/25/0.5/0.2 v/v/v/v) hirose et al., 2005 macrocyclic glycopeptide n-methyloxycarbonyl-α-amino acids ristocetin a, teicoplanin, vancomycin 15 mmol.l-1 nh4oac (ph 4.1)/meoh (80/20 v/v), meoh/mecn/acoh/tea (25/75/0.25/ 0.25 v/v/v/v) boesten et al., 2006 tryptophan, phenylalanin, leucin teicoplanin, teicoplanin aglycone, methylated teicoplanin aglycone 1% tfaa/meoh (60/40 v/v) meoh/mecn/acoh/tea (55/45/0.3/0.2 v/v/v/v) xiao et al., 2006 acoh – acetic acid, nh4oac – ammonium acetate, cd – cyclodextrin, csp – chiral stationary phase, etoh – ethanol, mecn – acetonitrile, meoh – methanol, tea – triethylamine, tfaa – trifluoroacetic acid cyclofructans (cfs) belong to a relatively small group of macrocyclic oligosaccharides. they consist of six or more β-(2→1) linked d-fructofuranose units. native cyclofructans have limited capabilities as chiral selectors (sun et al., 2009). recently, it was found that aliphatic and aromatic functionalized cfs could be highly selective for chiral molecules containing a primary amine functional group (jiang et al., 2009). in the present work derivatized cyclofructans were tested as chiral selectors for the direct hplc enantioseparation of underivatized α-amino acids. isopropyl carbamate bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:14 utc 110 moravčík, j. and hroboňová, k. cyclofructan 6 (ip-cf6), (r)-naphtylethyl carbamate cyclofructan 6 (rn-cf6) and dimethylphenyl carbamate cyclofructan 7 (dmp-cf7) (fig. 1) chiral stationary phases (csp) were selected. the influence of the mobile phase composition on the values of chromatographic parameters (retention factor, resolution) was investigated. the enantioseparation process was thermodynamically characterized by the values of enthalpy and entropy contributions calculated from the van´t hoff equation. or o ro o or n r = derivatization group n = 6 (cyclofructan 6, cf6), 7 (cyclofructan 7, cf7) derivatization groups o nh2 o h3c h n h c3 ch 3 h n o isopropyl carbamate (ip) (r)-naphtylethyl carbamate (rn) dimethylphenyl carbamate (dmp) fig. 1. scheme of stationary phases based on chemically bonded derivatized cyclofructan and chemical structures of used derivatizing groups. coohh2 n cooh nh 2 cooh nh 2 alanine (ala) valine (val) leucine (leu) cooh nh 2 coohnh 2 cooh nh 2 s norleucine (nle) γ-aminobutyric acid (gaba) methionine (met) cooh nh 2 cooh nh 2 n h phenylalanine (phe) tryptophan (trp) fig. 2. chemical structures of separated proteinogenic α-amino acids and γ-aminobutyric acid. covalent linker silica gel bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:14 utc nova biotechnologica et chimica 12-2 (2013) 111 2. materials and methods 2.1 chemicals and samples racemic mixtures of certain proteinogenic amino acids (alanine, leucine, methionine, norleucine, phenylalanine, tryptophan, valine) and γ-aminobutyric acid (fig. 2) were purchased from sigma aldrich. acetonitrile, methanol, acetic acid and triethylamine were purchased from merck. the solutions of racemic mixtures of each amino acid were prepared by dissolving in methanol (concentration 10 mg.ml-1). the homogenization of prepared solutions was performed by an ultrasonic bath (bandelin sonorex rk 100h). 2.2 hplc analysis the hplc separation of chiral α-amino acids were carried out on a agilent (series 1100) hplc system equipped with a binary pump, an injection valve (rheodyne) with a 20 μl injection loop, column thermostat, spectrophotometric detector and a chromatographic datasystem. direct separation of α-amino acid enantiomers were performed on chromatographic columns with chiral stationary phases based on chemically – bonded derivatized cyclofructans ip-cf6, rn-cf6 and dmp-cf7 (4×250 mm i.d, 5 μm). the temperature range of 0 – 35 °c was chosen for the thermodynamic study of the hplc enantioseparation of selected analytes. the mobile phases that were tested consisted of methanol and acetonitrile as organic solvents and acetic acid and triethylamine as ionic modifiers. the different concentration of methanol (50, 65, 75 and 85 vol. %) was used to investigate the influence of the mobile phase composition on the retention and enantioresolution of αamino acid racemic mixtures. the ratio of ionic modifiers was constant (0.3/0.2 v/v). the flow rate was 0.8 ml.min-1. the chromatograms of eluted optical isomers were sxcanned at the wavelength of 210 nm. 3. results and discussion 3.1 the influence of the chiral stationary phase composition on the retention and enantioseparation of chiral amino acids chiral columns based on chemically – bonded cyclofructans with aliphatic (ip-cf6) and aromatic moieties (rn-cf6, dmp-cf7) were tested in order to select the appropriate csp for hplc separation of studied chiral amino acids. the mobile phase methanol/acetonitrile/acetic acid/triethylamine (75/25/0.3/0.2 v/v/v/v) was used for all chromatographic separations. results given in table 2 show that the rn-cf6 chiral selector exhibits no enantioselectivity for the amino acid enantiomers used in this study. all chiral amino acids eluted with retention factors from 0.09 to 0.25. the highest retention factor values were obtained for aromatic amino acids (phenylalanine and tryptophan). the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:14 utc 112 moravčík, j. and hroboňová, k. lower retention factor value was obtained for aliphatic amino acids. the enantioseparation of amino acid enantiomers was not achieved. the dmp-cf7 chiral column was also evaluated. this chromatographic column is based on a chemically – bonded cf7 with an aromatic substituent which allows different types of interactions between separated enantiomers and the chiral selector. among them, π-π interactions, dipole-dipole interactions and hydrogen bonds generally occur. using this csp, the hplc enantioresolution of amino acid optical isomers was not achieved. compared to the rn-cf6 chiral column, the retention factor values were higher (table 2). based on the relationship between a structure of analytes and their retention behavior the amino acids with more polar functional groups were retained in the dmp-cf7 csp for a longer period of time. table 2. chromatographic results for separation of studied chiral amino acids by cyclofructan – based chiral stationary phases. csp rn-cf6 dmpcf7 ipcf6 amino acid k k k1 k2 rs leu 0.22 ± 0.01 0.25 ± 0.01 0.31 ± 0.01 val 0.28 ± 0.01 0.26 ± 0.01 trp 0.23 ± 0.01 0.30 ± 0.01 0.27 ± 0.01 0.32 ± 0.01 0.33 ± 0.01 phe 0.25 ± 0.00 0.30 ± 0.01 0.29 ± 0.01 0.35 ± 0.01 0.36 ± 0.02 nle 0.37 ± 0.02 0.40 ± 0.01 0.40 ± 0.02 met 0.09 ± 0.00 0.36 ± 0.02 0.46 ± 0.02 0.50 ± 0.02 0.25 ± 0.01 gaba 0.10 ± 0.00 0.37 ± 0.02 0.47 ± 0.02 0.52 ± 0.02 0.44 ± 0.02 ala 0.19 ± 0.01 0.39 ± 0.02 0.69 ± 0.03 0.76 ± 0.03 0.51 ± 0.02 t m [min] 5.04 4.14 4.88 the hplc enantioseparation of proteinogenic amino acids was achieved by the ipcf6 chiral column. resolution values given in table 2 show that enantiomers of some analytes were partially separated (rs = 0.25 – 0.51). the highest enantioresolution was obtained for amino acids with aliphatic substituents. amino acids with aromatic moieties were separated with lower resolution values. based on these results, steric effects can be predicted to have a dominant role during the enantioseparation process under the mentioned conditions (ip-cf6 csp, polar – organic separation mode). 3.2 the influence of the mobile phase composition on the retention and enantioseparation of chiral amino acids the influence of mobile phase composition on the retention and enantioresolution of selected amino acids was observed. all chromatographic separations were performed in the polar – organic separation mode (methanol/acetonitrile/acetic acid/triethylamine). the values of methanol concentration in tested mobile phases were 50, 65, 75 and 85 vol. %. small amounts of ionic modifiers (0.3 vol. % of acetic acid, 0.2 vol. % of triethylamine) were added to prepared mobile phases. the resolution values of chiral amino acids (table 3) were calculated for tested mobile phases. results show that the hplc enantioseparation of studied analytes is markedly bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:14 utc nova biotechnologica et chimica 12-2 (2013) 113 influenced by the mobile phase composition. with an increasing value of methanol volume percent concentration the resolution values were decreasing. based on these results, the mixture methanol/acetonitrile/acetic acid/triethylamine (75/25/0.3/0.2, v/v/v/v) was found to be the most effective mobile phase for the hplc separation of all set of chiral α-amino acids. the resolution values obtained for this mobile phase composition were from the interval 0.25-0.51. table 3. the influence of methanol concentration in used mobile phases on the resolution values of chiral amino acids. methanol/acetonitrile/acetic acid/triethylamine (v/v/v/v) 50/50/0.3/0.2 65/35/0.3/0.2 75/25/0.3/0.2 85/15/0.3/0.2 amino acid rs gaba 0.76 ± 0.03 0.67 ± 0.03 0.44 ± 0.02 0.35 ± 0.01 ala 0.65 ± 0.03 0.59 ± 0.02 0.51 ± 0.02 0.40 ± 0.01 leu 0.81 ± 0.03 0.44 ± 0.02 0.31 ± 0.01 0.26 ± 0.01 met 0.58 ± 0.02 0.34 ± 0.01 0.25 ± 0.01 0.21 ± 0.01 nle 0.55 ± 0.02 0.46 ± 0.02 0.40 ± 0.01 0.35 ± 0.01 phe 0.54 ± 0.02 0.42 ± 0.02 0.36 ± 0.01 0.31 ± 0.01 trp 0.56 ± 0.02 0.42 ± 0.02 0.33 ± 0.01 0.28 ± 0.01 t m [min] 4.09 4.06 4.27 4.50 fig. 3. the dependence of retention factors (k1, k2) of methionine (■,●) and phenylalanine (♦,▲) on the concentration of methanol in a mobile phase. the retention factor values of eluted amino acid enantiomers exhibited an observable dependence on the change of methanol volume percent concentration. this dependence for selected analytes (methionine, phenylalanine) is graphically k1, k2 concentration of methanol in a mobile phase (vol. %) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:14 utc 114 moravčík, j. and hroboňová, k. represented in fig. 3. the increase in methanol content in a mobile phase has the same effect on the retention of studied compounds as on the resolution. 3.3 the influence of the column temperature on the retention and enantioseparation of chiral amino acids the column temperature is one of the factors that can affect elution parameters. moreover, increasing the temperature usually causes the enantioresolution values to get lower. thermodynamic study of chiral separation provides useful information about enantioseparation mechanism and the nature of the interactions between optically active analytes and chiral stationary phases. based on the values of enthalpy and entropy contribution calculated from the van´t hoff equation, separation of optical isomers can be characterized as an enthalpically or an entropically driven process. thermodynamics is the effective tool for evaluating both enantioselective separation and chiral selectors (weng et al., 2004). [min.] time 2 4 6 8 [mv] v ol ta ge 15 20 25 30 t = 273 k t = 283 k t = 296 k fig. 4. hplc chromatograms of methionine racemic mixture in the temperature interval 273 – 296 k. chromatographic conditions: stationary phase: isopropyl carbamate cyclofructan 6, mobile phase: methanol/acetonitrile/acetic acid/ triethylamine (75/25/0.3/0.2 v/v/v/v), flow rate: 0.8 ml.min-1, detection: spectrophotometric (λ = 210 nm), injected volume: 20 μl. the effect of the column temperature on the retention and resolution of chiral amino acids was investigated. the hplc enantioseparation of selected analytes was carried out at eight different temperature values (0 °c, 5 °c, 10 °c, 15 °c, 20 °c, 23 °c, 30 °c and 35 °c). when the column temperature was increasing, the retention factor values of eluted amino acid racemic mixtures were decreasing. the hplc chromatograms of methionine enantioseparation in the temperature interval 273 – 296 are shown in fig. 4. the resolution values (table 4) were also markedly influenced by the temperature at which enantiomeric separations were realized. the influence of the column temperature on the enantioseparation of selected amino acids exhibited the same trend as the temperature dependence of the retention of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:14 utc nova biotechnologica et chimica 12-2 (2013) 115 separated amino acid enantiomers. the resolution of chosen chiral amino acids reached its maximum value at 0 ºc. with the increasing column temperature, the hplc enantioseparation of studied compounds was getting worse (lower values of rs). for the thermodynamic characterization of the chromatographic enantioseparation of studied compounds, the van´t hoff equation was used. this equation represents the dependence of the natural logarithm of the retention factor value (ln k) on reciprocal value of the column temperature (1/t). the van´t hoff analysis is suitable for the determination of the fundamental thermodynamic parameters, i.e. the standard partial molar enthalpy of transfer (δhi) and the standard partial molar entropy of transfer (δsi). the values of these parameters were calculated according to the equation: ln ki = -δhi/rt + δsi/r + ln φ where ki is the retention factor, δhi [jmol -1] is the standard partial molar enthalpy of transfer, δsi [jk -1mol-1] is the standard partial molar entropy of transfer, r is the gas constant (8.314 jk-1mol-1), t [k] is the column temperature and φ is the phase ratio defined as the volume of the stationary phase (vs) divided by the volume of the mobile phase (vm). the value of the standard partial molar enthalpy of transfer (δhi) was calculated from a slope of -δhi/r and the value of the standard partial molar entropy of transfer (δsi) was calculated from an intercept of δsi/r. the determination of these thermodynamic characteristics requires the knowledge of the phase ratio. it is usually difficult to calculate the phase ratio because the value of the stationary phase volume (vs) is approximate in many cases. its value is relatively easy to calculate only for liquid – liquid chromatography (chester et al., 2003). the values of the standard partial molar enthalpy δ(δhi) and entropy change δ(δsi*) of chosen amino acid racemic mixtures are given in table 5. the values of the entropic contribution δ(δsi*) also included the value of the phase ratio. the values of the standard partial molar gibbs energy (δgi) of both amino acid enantiomers (table 5) were calculated for two different temperatures (273 k and 308 k) according to the gibbs – hemholtz equation: δgi = δhi tδsi* the standard partial molar gibbs energy change δ(δgi) of chosen amino acid racemic mixtures (table 5) was also determined for temperatures of 273 k and 308 k. the standard partial molar enthalpy of the enantiomer with a higher value of retention time (δh 2 ) reached higher values than the standard partial molar enthalpy of the first eluted enantiomer (δh 1 ). this fact was observed for all separated amino acid racemic mixtures. enantioselective interactions between a chiral selector and the second eluted enantiomer are more preferred than enantioselective interactions between a chiral selector and the first eluted optical isomer. higher values of the standard partial molar enthalpy change δ(δhi) were obtained for racemic mixtures of α-amino acids with an aliphatic substitution (leucine, methionine, norleucine). the standard partial molar enthalpy change δ(δhi) exhibited lower values for alanine, γ-aminobutyric acid and phenylalanine. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:14 utc 116 moravčík, j. and hroboňová, k. table 4. the resolution values of the chosen chiral amino acids in the temperature interval 273 – 308 k. t [k] 273 278 283 288 293 296 303 308 amino acid rs trp 0.47 ± 0.02 0.44 ± 0.02 0.41 ± 0.01 0.38 ± 0.01 0.35 ± 0.01 0.33 ± 0.01 0.30 ± 0.01 0.28 ± 0.01 leu 0.66 ± 0.02 0.60 ± 0.02 0.48 ± 0.02 0.44 ± 0.02 0.36 ± 0.01 0.31 ± 0.01 0.29 ± 0.01 0 phe 0.53 ± 0.02 0.50 ± 0.02 0.47 ± 0.02 0.42 ± 0.01 0.38 ± 0.01 0.35 ± 0.01 0.32 ± 0.01 0.29 ± 0.01 nle 0.72 ± 0.03 0.62 ± 0.02 0.56 ± 0.02 0.49 ± 0.02 0.43 ± 0.02 0.40 ± 0.01 0.37 ± 0.01 0 met 0.71 ± 0.03 0.68 ± 0.03 0.62 ± 0.02 0.54 ± 0.02 0.37 ± 0.01 0.25 ± 0.01 0.21 ± 0.01 0.19 ± 0.00 gaba 0.63 ± 0.02 0.61 ± 0.02 0.57 ± 0.02 0.52 ± 0.02 0.48 ± 0.02 0.44 ± 0.02 0.38 ± 0.01 0.35 ± 0.01 ala 0.70 ± 0.03 0.67 ± 0.03 0.61 ± 0.02 0.55 ± 0.02 0.53 ± 0.02 0.51 ± 0.02 0.49 ± 0.02 0.46 ± 0.02 table 5. the values of thermodynamic parameters of chosen chiral amino acids calculated from the van't hoff equation. amino acid δ (δh°) [jmol-1] δ (δs°*) [jk-1mol-1] (δg1°)273 k [jmol-1] (δg2°)273 k [jmol-1] δ (δg°)273 k [jmol -1] (δg1°) 308 k [jmol-1] (δg2°) 308 k [jmol-1] δ (δg°)308 k [jmol -1] phe -286 ± 15 0.35 ± 0.05 1953 ± 179 1528 ± 140 -425 ± 22 3544 ± 324 3151 ± 288 -393 ± 20 met -993 ± 91 -2.69 ± 0.32 871 ± 80 613 ± 57 -258 ± 14 2407 ± 220 2243 ± 205 -164 ± 15 trp -985 ± 90 -2.06 ± 0.25 2281 ± 209 1858 ± 170 -423 ± 22 3688 ± 337 3337 ± 305 -351 ± 18 gaba -616 ± 57 -1.37 ± 0.17 838 ± 77 597 ± 56 -241 ± 13 2360 ± 216 2167 ± 198 -193 ± 10 ala -290 ± 15 -0.31 ± 0.04 -108 ± 10 -313 ± 16 -205 ± 11 1400 ± 128 1206 ± 110 -194 ± 10 leu -2130 ± 195 -6.10 ± 0.80 2069 ± 190 1605 ± 147 -464 ± 24 4663 ± 426 4413 ± 403 -250 ± 13 nle -1007 ± 92 -2.63 ± 0.31 1487 ± 136 1198 ± 110 -289 ± 15 2997 ± 274 2800 ± 256 -197 ± 10 b ereitgestellt von s lovenská poľnohospodárska knižnica | h eruntergeladen 16.01.20 15:14 u tc nova biotechnologica et chimica 12-2 (2013) 117 the chromatographic enantioseparation is also influenced by steric effects. the diffusion of separated optical isomers into interaction parts of a chiral selector is dependent on the size of analyte molecules. the standard partial molar entropy of enantiomers (δs 1 *, δs 2 *) is connected with steric interactions and can be also used for the enantioselectivity evaluation. the entropic contribution of the second eluted enantiomer (δs 2 *) exhibited higher values than the entropic contribution of the enantiomer with a lower value of retention time (δs 1 *). this means that molecules of the second eluted enantiomer were retained in more interaction parts of a chiral selector than molecules of the first eluted enantiomer. based on the values of the enthalpic and entropic contribution to the change of gibbs energy, the hplc enantioseparation of chosen amino acids under selected chromatographic conditions (ip-cf6 column, polar – organic separation mode) was found to be an enthapically driven process. it means that the second eluted enantiomer and a chiral selector form a diastereomeric complex with a higher stability constant value than the enantiomer with a shorter elution time. the van't hoff plots of chosen chiral amino acids are graphically represented in fig. 5. the coefficient of determination values (r2~0.99) indicate the linear dependence of retention of studied compounds on the column temperature. the retention mechanism did not change with the temperature change. fig. 5. the van't hoff plots of chosen chiral amino acids. (■,▲) phenylalanine, (●,♦) methionine 4. conclusions different types of cyclofructan – based chiral selectors were tested for hplc separation of chiral amino acids. among them, isopropyl carbamate cyclofructan 6 showed the ability to separate optical isomers in the polar – organic separation mode ln k1, ln k2 1/t [k-1] bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:14 utc 118 moravčík, j. and hroboňová, k. (mobile phase: methanol/acetonitrile/acetic acid/triethylamine, 75/25/0.3/0.2 v/v/v/v). based on the results of thermodynamic study in the temperature interval 0 ºc – 35 ºc, the hplc enantioseparation of studied racemic mixtures can be characterized as an enthapically driven process. acknowledgements: this study was financially supported by the scientific grant agency of the ministry of education, science, research and sport of the slovak republic and of the slovak academy of sciences (grant vega no. 1/0164/11). the authors thanks to d. w. armstrong for the donation of chiral column. references berkecz, r., sztojkov-ivanov, a., ilisz, i., forró, e., fülöp, f., hyun, m. h., péter, a.: high-performance liquid chromatographic enantioseparation of β-amino acid stereoisomers on a (+)-(18-crown-6)-2,3,11,12tetracarboxylic acid-based chiral stationary phase. j. chromatogr. a, 1125, 2006, 138-143. boesten, j.m.m., berkheij, m., schoemaker, h.e., hiemstra, h., duchateau, a.l.l.: enantioselective high-performance liquid hromatographic separation of n-methyloxycarbonyl unsaturated amino acids on macrocyclic glycopeptide stationary phases. j. chromatogr. a, 1108, 2008, 26-30. chen, j.j., zhang, d.t., shen, b.c., zhang, x.j., xu, b.j., xu, x.z.: enantioseparation of d,l-α-amino acids on crown ether chiral stationary phases. j. anal. chem., 34, 2006, 1535-1540. chester, t.l., coym, j.w.: effect of phase ratio on van´t hoff analysis in reversed-phase liquid chromatography, and phase-ratio-independent estimation of transfer enthalpy. j. chromatogr. a, 1003, 2003, 101-111. choi, h. j., ha, h. j., han, s. c., hyun, m. h.: liquid chromatographic resolution of β-amino acids on csps based on optically active (3,3´-diphenyl-1,1´binaphtyl)-20-crown-6. anal. chim. acta, 619, 2008, 122-128. ding, g.-s., ying, l., cong, r.-z., wang, j.-d.: chiral separation of enantiomers of amino acid derivatives by high-performance liquid chromatography on a norvancomycin-bonded chiral stationary phase. talanta, 62, 2004, 997-1003. flack, a.: louis pasteur´s discovery of molecular chirality and spontaneous resolution in 1848, together with a complete review of his crystallographic and chemical work. acta cryst., 65, 2009, 371-389. hirose, k., jin, i.z., nakamura, t., nishioka, r., ueshige, t., tobe, y.: preparation and evaluation of a chiral stationary phase covalently bound with chiral pseudo-18-crown-6-ether having 1-phenyl-1,2-cyclohexanediol as a chiral unit. j. chromatogr. a, 1078, 2005, 35-41. ilisz, i., aranyi, a., pataj, z., péter, a.: recent advances in the direct and indirect liquid chromatographic enantioseparation of amino acids and related compounds: a review. j. pharm. biomed. anal., 69, 2012, 28-41. jiang, ch., tong, m.-y., breitbach, z.s., armstrong, d.w.: synthesis and examination of sulphated cyclofructans as a novel class of chiral selectors for ce. electrophoresis, 30, 2009, 3897-3909. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:14 utc nova biotechnologica et chimica 12-2 (2013) 119 remsburg, j.w., armstrong, d.w., péter, a., tóth, g.: lc enantiomeric separation of unusual amino acids using cyclodextrin-based stationary phases. j. liq. chromatogr. rel. technol., 31, 2008, 219-230. shpigun, o.a., shapovalova, e.n., ananieva, i.a., pirogov, a.v.: separation and enantioseparation of derivatized amino acids and biogenic amines by high-performance liquid chromatography with reversed and chiral stationary phases. j. chromatogr. a, 979, 2002, 191-199. sun, p., wang, ch., breitbach, z.s., zhang, y., armstrong, d.w.: development of new hplc chiral stationary phases based on native and derivatized cyclofructans. anal. chem., 81, 2009, 10215-10226. wang, y., young, d.j., tan, t.t. y., ng, s.c.: click preparation of hindered cyclodextrin chiral stationary phases and their efficient resolution in high performance liquid chromatography. j. chromatogr. a, 1217, 2010, 7878-7883. weng, w., yao, b., chen, x., lin, w., zeng, q.: thermodynamic studies on mechanism of chiral resolution by liquid chromatography. progress in chemistry, 18, 2006, 1056-1064. xiao, t.l., tesařová, e., anderson, j.l., egger, m., armstrong, d.w.: evaluation and comparison of a methylated teicoplanin aglycone to teicoplanin aglycon and natural teicoplanin chiral stationary phases. j. sep. sci., 29, 2006, 429-445. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:14 utc nova biotechnol chim (2022) 21(2): e1155 doi: 10.36547/nbc.1155 1 nova biotechnologica et chimica characterization of functional effects of different lab isolated from sourdoughs in turkey zuhal alkay, ezgi metin sagir, muhammed ozgolet, muhammed zeki durak department of food engineering, faculty of chemical and metallurgical engineering, yildiz technical university, istanbul, turkey  corresponding author: zuhalakay21@hotmail.com article info article history: received: 7th september 2021 accepted: 22nd november 2021 keywords: antifungal antimicrobial lactic acid bacteria phytase activity proteolytic sourdough abstract in this study, the technological properties of five different lactic acid bacteria (lab) isolated from sourdough collected from three cities of turkey (gümüşhane, manisa, ankara) and cyprus were investigated. for this purpose, antimicrobial, antifungal, phytase, and proteolytic activities of these bacteria and their effect on ph were examined. the ph of the prepared solutions decreased to 3.8 and 4.4 from 6.3 by lab addition following 24 h incubation. lactiplantibacillus plantarum o6f-25 strain showed the best inhibitory effect against four gram-positive and four gramnegative bacteria. in terms of proteolytic activity, lactiplantibacillus plantarum 45mk-32 was the most effective strain. the antifungal effects of lab were tested against aspergillus flavus, aspergillus niger, and penicillium carneum. levilactobacillus brevis kco-48 was the most effective lab strain. phytase activities (710.40 – 840.37 u.ml-1) of lab studied except limosilactobacillus fermentum (29gt-19), which has the lowest phytase activity, were not significantly different (p < 0.05). this study revealed that sourdough labs have the potential to be used as biopreservative and produce functional food products. introduction sourdough technology is one of the earliest fields of biotechnological applications, mainly originated from the fermentation of grain matrices by natural lactic acid bacteria (lab) (gobbetti et al. 2014). however, the trend bringing about the consumption of health-promoting foods has increased the production of functional and nutritional products (teleky et al. 2020). sourdough bread meets this demand of conscious consumers because it offers several important health benefits such as lowering glycemic index, increasing mineral bioavailability, antimicrobial agent, and bioactive peptide formation, thus promoting clean label bread production (rizzello et al. 2017; tsafrakidou et al. 2020). internal and external factors influence sourdough production. flour type and quality, fermentation temperature and time, water activity, relative humidity, dough yield, ph and acidification, oxygen tension, and backslopping dynamics determine sourdough characteristics (de vuyst et al. 2017). since traditional spontaneous sourdough is time-consuming and trying, it needs skilled labour, and causes differences in quality of final bread, liquid or dried starter cultures are chosen for industrial processes (siepmann et al. 2018). the demand for the stable composition of starter cultures in the modern sourdough-making industry encouraged various studies on the selection of starter cultures with superior functionality (gänzle and ripari 2016). the sourdough microbiota is the primary influencer of the technological and mailto:zuhalakay21@hotmail.com nova biotechnol chim (2022) 21(2): e1155 2 nutritional value of the sourdough bread (sakandar et al. 2019). sourdough microbiota consists mainly of lab and yeast of various genera and species. most lab species isolated from sourdoughs belong to the genus lactobacillus with more than 60 species (de vuyst et al. 2017). lb. brevis, lb. fermentum, lb. paralimentarius, and lb. plantarum are among the most prevalent lab present in sourdough (minervini et al. 2014). many of the studies have focused on specific technological and nourishing effects of sourdough lab such as proteolytic activity (ozturkoglubudak et al. 2016; melini and melini 2018), phytase activity (cizeikiene et al. 2015; karaman et al. 2018), antibacterial activity (cizeikiene et al. 2013; petkova et al. 2021), antifungal activity (demirbaş et al. 2017; quattrini et al. 2019). in addition, low ph resulting from lab’s acid production increases the activities of amylases and proteases present in the flour, thus contributing to bread rheology and flavor via influencing gluten structure (yildirim-mavis et al. 2019). furthermore, antimicrobial and antifungal activities sourced from lab metabolites improve the shelf life and safety of the final sourdough bread (demirbaş et al. 2017). therefore, antifungal starter cultures have been successfully applied in bread (axel et al. 2016). another essential feature of sourdough fermentation is the stimulating impact on phytic acid degrading enzymes. phytic acid degradation leads to increased mineral, free amino acid, and protein bioavailability (gobbetti et al. 2014). many studies revealed strain dependency of lab characteristics (cizeikiene et al. 2013; melini and melini 2018; cizeikiene et al. 2015; ozturkoglubudak et al. 2016; demirbaş et al. 2017). in this study, five lab species among 17 isolates of sourdoughs were selected via 16s rdna gene sequencing and ft-ir spectroscopy. the study aims were to assess the technological and nutritional characteristics of selected lab. for this purpose, antibacterial, antifungal, proteolytic, phytase activities and effects of these labs were analysed. experimental bacterial strains and growth conditions lab strains used within the scope of this study were isolated from sourdoughs collected from four different regions of turkey. the type of flour, dough yield and depository used in the collected sourdoughs were given in table 1. each strain was grown in de man rogosa sharpe (mrs, merck kgaa, darmstadt, germany) medium for 24 h at 37 °c and under anaerobic conditions. five lab were chosen among 17 isolated bacteria based on 16s rdna gene sequencing and ft-ir spectroscopy for further analysis. table 1. the type of flour, dough yield and depository used in the collected sourdoughs. sourdough code flour type depository efficiency of sourdough 29gt-19 wheat and rye bakery 250 06b-2 wheat bakery 250 o6f-25 wheat bakery 250 kco-48 wheat home made 150 45mk-32 wheat bakery 250 ft-ir measurements the isolated sourdough lab strains were anaerobically cultivated on all purpose tween agar (apt) at 34 °c for 24 h. 25 μl of cell suspension of grown cells suspended and homogenized in 100 μl distilled water was placed on znse table and then dried at 40 °c. the spectral measurements were practiced by a tensor 27 spectrophotometer (bruker optic gmbh & co. kg, ettlingen, germany) equipped with an htsxt unit in the range of 600 – 4,000 cm-1 wavenumbers. the spectra of the isolates were processed using opus software (karaman et al. 2018). proteolytic activity of lactic acid bacteria milk agar plates, which include 28 g.l-1 skim milk powder, 5 g.l-1 casein peptone, 2.5 g.l-1 yeast extract, 1 g.l-1 glucose, and 15 g.l-1 agar were prepared to evaluate the proteolytic activity of lab strains. then, four 20 μl cell spots of lab strains were placed on the prepared agar, and plates were incubated anaerobically for 16 h at 37 °c. following 48 h incubation, proteolytic activity was indicated as clear zone surrounding lab cell spots (axel et al. 2016). nova biotechnol chim (2022) 21(2): e1155 2 quantitative determination of extracellular phytase activity of lab strains the phytase activities of lab isolated from sourdough samples were determined by adding 0.1 ml of the broth, including lab strains previously grown in fresh mrs medium into 4.9 ml modified mrs medium with 0.1 % naphytate and 0.2 % glucose. following incubation at 30 °c for 24 h, the lab cells were separated from the medium by centrifugation at 9,000 rpm at 4 °c. then, 250 μl cell-free supernatant and 250 μl substrate prepared by solving 2 mmol.l-1 naphytate in 0.1 mol.l-1 sodium acetate-acetic acid were mixed. the mixture was allowed for the reaction at 50 °c for 15 min. in order to cease the reaction, 500 μl of a 10 % (w/v) trichloroacetic acid (tca) solution was used. a mixture of 2.5 % (w/v) ammonium molybdate solved in a 5.5 % (v/v) sulfuric acid solution and 2.5 % (w/v) ferrous sulfate solution in the ratio of 4 : 1 was used to obtain the color reagent. kh2po4 at diverse concentrations were used as phosphorous source to form calibration curve. the absorbance values of samples were measured at 700 nm (yildirim and arici 2019). determination of the antibacterial activity of sourdough lab strains the antimicrobial activities of lab sourdough strains were determined by the method adapted from demirbaş et al. (2017). the inhibitory effects of lab were tested against four gram-negative pathogens; salmonella typhimurium atcc 0402, escherichia coli atcc 25922, pseudomonas aeruginosa atcc 27853, klebsiella pneumoniae atcc 06023 and four gram-positive pathogens; staphylococcus aureus atcc 25923, listeria monocytogenes atcc 13932, bacillus cereus atcc 11778, and bacillus subtilis atcc 6633. the isolated sourdough strains were grown overnight with 1 % inoculation in 10 ml of mrs broth to determine antibacterial activity. the grown cells in the culture were separated at 14,000 × g for 5 min, 4 °c by centrifugation. after the centrifuge, with a sterile 0.22 μm syringe filter, the supernatant was filtered to remove all bacteria cells that had remained in the supernatant. cell-free supernatant (cfs) was adjusted to ph 6.0 with naoh then treated with catalase (merck kgaa, darmstadt, germany) at 25 °c for 30 min. thus, possible inhibition effects of organic acids with h2o2 were eliminated. 20 μl supernatant was poured into the wells punched in tryptic soy agar plates where pathogen strains previously spread and allowed to incubate at 37 °c for 24 h. clear zones were measured as mm around the wells. determination of antifungal activities of sourdough isolates the lactic acid bacteria isolated from sourdough were grown at 37 °c for 24 h with 1 % inoculation in 10 ml of mrs broth. the suspensions were arranged at concentration 107 cfu.ml-1, and following the overnight growth, two 5 μl cell spots of lab strains were placed on the mrs agar plates, and plates were incubated anaerobically for 48 h at 37 °c. afterward, 10 ml of soft malt extract agar (2 % glucose, 0.8 % agar) at suitable temperature comprising 104 cfu.ml-1 penicillium carneum, aspergillus flavus, aspergillus niger were inoculated on lactic acid bacteria in plates and incubated for 2 – 3 d at 25 °c. after that, the antifungal activity was classified as no inhibitory (-), delayed spore formation with no clear zone of inhibition (+), delayed spore formation with the small clear zone of inhibition (++), delayed spore formation with the good clear zone of inhibition (+++), and broad inhibition on spore formation and mycelial growth with remarkable zones around a colony (++++) (demirbaş et al. 2017). ph decrease by lactic acid bacteria metabolites to determine the effect of metabolites produced by lab strains and ph decrease, cultures were grown with 1 % inoculation in 10 ml of mrs broth. the ph measurement was fulfilled after 3, 6, 9, and 24 h by ph meter (hanna instruments deutschland gmbh, vöhringen, germany). statistical analysis one-way analysis of variance (anova) followed by tukey’s test (p < 0.05) was used to determine the difference between strains depending on the measured characteristics. minitab version 17.3.1 (minitab, llc, state college, usa) and jmp 9 version was used for this purpose. each calculation 3 nova biotechnol chim (2022) 21(2): e1155 2 was given as mean ± standard deviation of three replicates. results ft-ir spectra of lab isolates ft-ir microspectroscopy was exploited to identify the microorganisms. this technique is particularly utilized to characterize lab in the microbiological field. this study provided a quick description of lab biodiversity in sourdough samples. lab identification was accomplished thanks to both 16s rdna gene sequencing and ft-ir spectroscopy at hts-xt module. as demonstrated in fig. 1, 16 lab isolates were clustered and obtained dendrogram gave five different groups. the groups have included 6 lactiplantibacillus plantarum, 5 levilactobacillus. brevis, 2 limosilactobacillus fermentum, 2 bacillus haynesii, 1 lactiplantibacillus pentosus, respectively. 16s-rdna based phylogenetic tree was formed as shown in fig. 2 attributed to the dendrogram. therefore, 5 different lactobacillus species, which are levilactobacillus brevis kco-48, lactiplantibacillus plantarum 06f-25, companilactobacillus paralimentarus 06b-2 and limosilactobacillus fermentum 29gt-19 and lactiplantibacillus plantarum 45mk-32 were selected. then, these species were tested for their technological characteristics. fig 1. dendrogram obtained based on ftir spectra of the isolated lab. spectral ranges: 600 – 3,996 and 3,032 – 2,829 cm-1. ward’s algorithm and correlation with standard (euclidean distance). 4 nova biotechnol chim (2022) 21(2): e1155 2 fig 2. phylogenetic tree for 17 isolates based on 16s rdna gene sequencing. phylogenetic reconstruction was generated via neighbor-joining method with a length of 406 nucleotides, 16s rdna fragments. the count of bootstrap replicates is 2,000. bootstrap values higher than 70 % are displayed. the bar shows the length corresponding to 0.1 nucleotide substitution. determination of proteolytic activity five lab were screened for their protease activity on casein-containing plates. the proteolytic activities of lab isolates were determined by measuring clear zone diameter derived from protease enzymes produced by lab on skim milk agar. lab isolates studied except levl. brevis kco-48 demonstrated proteolytic activity. clear zone diameters ranged from 9.97 to 12.60 mm as stated in table 2. table 2. proteolytic activity diameters of clear zones. microorganisms clear zone diameter [mm] levl. brevis kco-48 liml. fermentum 29gt-19 10.0 ± 0.00b lpb. plantarum 06f-25 10.0 ± 0.00b coml. paralimenterius 06b-2 10.0 ± 0.00b lpb. plantarum 45mk-32 12.6 ± 0.29a a-bdifferent letters indicates the significant differences between the selected strains (p < 0.05). the highest proteolytic activity was produced by lpb. plantarum 45mk-32. differences in proteolytic activities of liml. fermentum 29gt-19, lpb. plantarum 06f-25, coml. paralimenterius 06b-2 were negligible (p ˃0.05). extracellular phytase activity extracellular phytase activity of lab isolated from sourdough were determined exploiting the addition of sodium phytate as substrate to mrs broth and release of phosphorous compound by phytase activity produced by lab. as shown in fig. 3, the phytase activities were found between 710 u.ml-1 and 841 u.ml-1. the lab strain with the highest phytase activity is levl. brevis kco-48 with the value of 840.6 u.ml-1 whereas the lowest phytase activity occurred by liml. fermentum 29gt-19 with the value of 710.4 u.ml-1. the phytase activities of lpb. plantarum 45mk-32 and lpb. plantarum 06f-25 were found 825.2 and 821.67 u.ml-1, respectively. the phytase activity of coml. paralimentarius was detected similarly to the lpb. plantarum species. no significant difference was found among phytase activities of lab studied in the study, excluding liml. fermentum 29gt-19. 5 nova biotechnol chim (2022) 21(2): e1155 2 fig. 3. extracellular phytase activities of lab isolates (u.ml-1). a-nwithin the column, the letters indicate that there is significant difference between the strains (p < 0.05). antimicrobial activity of lab against pathogens in this study, the antimicrobial capacity of neutralized and catalase-treated cell-free supernatants (cfs) of lab growth media was tested against 8 pathogenic bacteria to determine the inhibitory action of bacteriocin-like inhibitory substances (blis). in agar well diffusion method, clear zone diameter has been studied to determine the antimicrobial activity. the zone diameters of the inhibition area were given in table 3 and the figure of the lpb. plantarum strain that creates the best antimicrobial zone is given in fig. 4. table 3. antimicrobial activity of cfs of lab strains against pathogenic bacteria. pathogens zone of inhibition [mm] liml. fermentum 29gt-19 lpb. plantarum 06f-25 coml. paralimenterius 06b-2 levl. brevis kco-48 lpb. plantarum 45mk-32 k. pneumoniae atcc 06023 10.0 ± 0.00 a 10.0 ± 0.00 a 10.0 ± 0.00 a 10.0±0.00 a 10.0±0.00 a p. aeruginosa atcc 27853 nd 10.0 ± 0.00 a nd nd nd b. subtilis atcc 6633 10.2 ± 0.03 a 10.0 ± 0.00 a 10.0 ± 0.00 a 10.0 ± 0.00 a 10.0 ± 0.00 a l. monocytogenes atcc 13932 12.0 ± 0.00 a 11.6 ± 0.03 b 10.0 ± 0.00 e 10.2 ± 0.03 d 11.0 ± 0.00 c b. cereus atcc 11788 12.0 ± 0.00 d 13.2 ± 0.03 a 12.8 ± 0.03 b 10.8 ± 0.03 e 12.6 ± 0.03 c s. typhimurium atcc 0402 12.0 ± 0.00 a 12.0 ± 0.00 a 11.8 ± 0.03 b 11.0 ± 0.00 d 11.6 ± 0.03 c e. coli atcc 25922 10.0 ± 0.00 a 10.0 ± 0.00 a 10.0 ± 0.00 a 10.0 ± 0.00 a 10.0 ± 0.00 a s. aureus atcc 25923 12.2 ± 0.03 a 12.2 ± 0.03 a 11.8 ± 0.03 b 11.8 ± 0.03 b 10.0 ± 0.00 c a-b different letters indicates the significant differences between the selected (p < 0.05). nd – no inhibition zone was detected. 6 nova biotechnol chim (2022) 21(2): e1155 2 all bacteria displayed mild or moderate inhibition of pathogens. the highest antimicrobial activity of the supernatants was detected against b. cereus atcc 11778. lpb. plantarum 06f-25 was only strain that inhibits all pathogens. p. aeruginosa inhibited only by lpb. plantarum 06f-25 metabolites has become the most resistant bacteria. the highest zone diameters were observed against b. cereus atcc 11778 by lpb. plantarum 06f-25, coml. paralimentarius 06b-2, lpb. plantarum 45mk-32. even though lpb. plantarum 06f-25 and lpb. plantarum 45mk-32 are the same species, they have differed in antibacterial effect against 6 out of 8 pathogens. lab were found more efficient on gram-positive pathogens than gramnegative pathogens. s. typhimurium atcc 0402 was the least resistant gram-negative pathogen with an average 11.7 mm zone diameter. however, the zone diameters resulted from cfs on other gramnegative bacteria ≤ 10 mm. the lowest inhibition on gram-positive pathogens was observed on b. subtilis atcc 6633 with an average 10.0 mm zone diameter. fig. 4. figure of best zone measurement. antifungal activities of lactic acid bacteria the influence of lab grown in mrs broth on several mould species such as penicillium carneum, a. flavus, and a. niger was analyzed to test antifungal activity. their effects on the moulds were given in table 4. p. carneum was not inhibited by lab. even though any lab did not have any antifungal effect on p. carneum, all tested lab inhibited a. flavus. all lab excluding coml. paralimentarius 06b-2 also showed inhibitory impact on aspergillus niger. the highest inhibitory impact was demonstrated against a. flavus and a. niger by coml. paralimentarius 06b-2 and liml. fermentum 29gt-19. lpb. plantarum species 45mk-32 and 06f-25 demonstrated lower inhibitory action with a small clear zone with delayed spore formation than liml. fermentum and levl. brevis against a. niger. 7 nova biotechnol chim (2022) 21(2): e1155 2 table 4. inhibition of several moulds by lab isolates. microorganisms inhibitory spectrum of lab supernatants penicillium carneum aspergillus flavus aspergillus niger liml. fermentum 29gt-19 ++ ++++ lpb. plantarum 06f-25 ++ + coml. paralimenterius 06b-2 ++++ levl. brevis kco-48 +++ +++ lpb. plantarum 45mk-32 ++ + the mould inhibition of lab was classified as; (-) no inhibition, (+) spore formation delayed but no clear zone, (++), spore formation delayed with a small clear zone around the colony, (+++) spore formation delayed with good clear zone around the colony, (++++) extensive inhibition of spore formation and mycelial growth with definite clear zones around colonies. ph decrease due to the metabolites produced by lab the ph values of mrs broths including lab isolates were measured every 3 h in the first 9 h and 24th h. the measurement results were given in table 5. all lab have a strong impact on the ph value of the mrs broth medium. whereas initial ph values of broths ranged between 6.51 and 6.56, the last ph of broths varied from 3.83 to 4.41. the highest decrease in ph value by 3.83 was observed in lpb. plantarum 45mk-32 containing broth. liml. fermentum 29-gt-19 was found the least effective strain on ph. table 5. ph decreasing effect of lab. microorganisms time [h] 3 h 6 h 9 h 24 h lpb. plantarum 06-f25 6.38 ± 0.02aba 6.18 ± 0.02bb 5.14 ± 0.02dc 4.04 ± 0.01cd levl. brevis k-co48 6.40 ± 0.03aba 6.36 ± 0.02aa 6.09 ± 0.02ab 3.92 ± 0.03dc coml. paralimenterius 06-b2 6.27 ± 0.03ca 6.00 ± 0.03cb 5.31 ± 0.02cc 4.33 ± 0.03bd liml. fermentum 29-gt19 6.29 ± 0.03bca 6.04 ± 0.02cb 5.39 ± 0.03cc 4.41 ± 0.02ad lpb. plantarum 45-mk32 6.41 ± 0.03aa 6.37 ± 0.02aa 5.70 ± 0.02bb 3.83 ± 0.01ec * capital letters – differences in uppercase letters indicate significant difference (p < 0.05) in a row. * small letters – differences in lowercase letters indicate significant difference (p < 0.05) in a column. discussion ft-ir analysis is a thought reliable method to identify bacteria and yeast with chemometric techniques (wenning and scherer 2013). ft-ir analysis was conducted on 17 sourdough lab isolates brought from 7 different regions of turkey. first derivatives of the obtained spectra included in 1,500 – 1,200 cm-1, 1,200 – 900 cm-1, 900 – 700 cm-1 spectral regions were utilized to differentiate spectra of lactobacillus species that had been studied before (akman et al. 2021). in this study, lab isolated from sourdough samples was chosen exploiting ft-ir cluster analysis, which gives faster results than rapd-pcr. according to the cluster analysis, 16 lab isolates were genotypically identified. based on 16s rdna sequencing, among 17 lab, 9 lpb. plantarum, 2 levl. brevis, 1 liml. fermentum, 1 coml. paralimentarius, and 4 bacillus haynesii were determined. in addition, 16s rdna-based phylogenetic tree of lab isolates using the neighbor-joining method presented the conformable phylogenetic cluster. whereas 45mk32 and 06f-25 isolates were clustered on the same branch, 29gt-19, kco-48, and 06b-2 isolates were clustered on distinct branches. the similarity of lpb. plantarum isolates were specified by phylogenetic clustering. it was concluded that cluster analysis of 17 lab isolates was consistent with grouping attributed to 16s rdna and separated these isolates into different groups phylogenetically. proteolytic activity has an important role in the quality of bread. proteolysis derived from lab increases amino acid amount in sourdough; hence, it affects flavor development by reaction between amino acids and reducing sugar during baking (gilcardoso et al. 2021), and atanasova et al. (2014) found that the antimicrobial-active peptides were 8 nova biotechnol chim (2022) 21(2): e1155 2 formed by lab grown in goat milk. lab isolated from yogurt samples and described by biochemical and morphological analysis for their proteolytic activities were studied, and it was found that all lab isolates have proteolytic activities (phyu et al. 2015). moreover, axel et al. (2016) investigated lab isolated from sourdough samples and the lowest proteolytic activity were detected in levl. brevis incompatible with the finding in our research. two different lpb. plantarum strains 45mk-32 and 06f-25 have different clear zone diameters displaying that proteolytic activity of lab is strain-specific. microbial acidification provides favorable environment (ph 3.5 – 4.0) for aspartic proteinases (gänzle et al. 2008). hence, extracellular proteinases of lab are influenced by medium ph. lpb. plantarum 45mk-32, which has the highest proteolytic activity in our study, was the most efficient to alter the ph of mrs medium leading to the formation of favourable conditions for proteinase activity. whole meal bread consumption has remarkably increased since awareness of its nutritionally valuable components, such as dietary fibers, vitamins, minerals, complex carbohydrates, and proteins (mohammadi‐kouchesfahani et al. 2019). however, phytic acid, the major phosphorus form in cereal grains including wheat, impedes the absorption of fe+2, zn+2, ca+2, mg+2, cu+2, and mn+2; thus, mineral deficiencies are promoted (ficco et al. 2009; liu et al. 2019). lab does not degrade phytate directly; however, it presents favourable conditions by decreasing ph for extracellular phytase activity (reale et al. 2007). nevertheless, some studies have been conducted which indicate intracellular phytase activity of lab (nuobariene et al. 2015; yildirim and arici 2019). in the study conducted by karaman et al. (2018) the extracellular phytase activities of lab strains obtained from sourdough were found to range from 703.1 u-ml-1 to 1153.8 u-ml-1. yildirim and arici (2019) also examined 64 lab isolates from sourdough in terms of extracellular phytase activity. the results varied between 725.58 u-ml-1 and 803.95 u-ml-1 in accordance with the results in our study. sourdough bread consumption offers health-promoting effect since lab can degrade phytate in whole wheat flour which contains high amount of phytate. therefore, the decrease in mineral uptake can be prevented (lopez et al. 2003; rizzello et al. 2017). furthermore, lab's phytase activity benefits the small intestine via decomposing the remaining phytate (haros et al. 2009). moreover, the usage of phytase-active lab enables to minimize antinutritional characteristics of animal feeding (humer et al. 2015). it is a fact that lab's antimicrobial characteristics can be exploited to prevent spoilage of foods and kill pathogenic bacteria and the benefits in the large intestine as probiotic. lab produces organic acids, hydrogen peroxide, diacetyl, and bacteriocin-like inhibitory substances which inhibit bacterial growth. bacteriocins can promote invasion of beneficial strains in the gastrointestinal tract via inhibiting competing or pathogen strains; hence, the microbiota and host immune system are regulated (arqués et al. 2015). in our study, almost all lab showed an inhibitory effect on common pathogens following neutralization and catalase treatment. bacteriocin-producing ability of 437 lab isolated from 70 sourdough samples were tested (petkova et al. 2021). whereas 85 of these strains are effective on indicator organisms without removing acids and other antimicrobial agents in cell-free supernatant (cfs), only five lab strains inhibited indicator organisms after neutralization, catalase treatment and sterilization by filtration. venkadesan et al. (2015) investigated the antimicrobial effect of 9 lab isolated from milk products on different pathogens. liml. fermentum strains led to moderate inhibition on e. coli, s. aureus and s. typhi and l. monocytogenes atcc 13932 while lpb. plantarum showed weak inhibition. cizeikiene et al. (cizeikiene et al. 2013) analyzed five lab isolated from rye sourdough to specify their antimicrobial effect against 23 pathogenic bacteria, including b. cereus, b. subtilis, various pseudomonas spp., staphylococcus aureus, listeria monocytogenes, e. coli, and salmonella spp. strains, the results showed that the blis of all lab studied inhibited b. subtilis strains whereas no inhibitory effect was detected on other studied pathogens moreover, our study is consistent with the studies maintaining mild, low, or no inhibitory impact of lab metabolites on well-recognized pathogens. consequently, the antimicrobial capacity of lab supernatants without acid and h2o2 removal is considerable; however, catalase treated and neutralized cfs influence on pathogenic bacteria is limited. 9 nova biotechnol chim (2022) 21(2): e1155 2 mould growth is a major spoilage reason for bakery products. the most prevalent mould species are aspergillus, fusarium, and penicillium. biopreservation techniques have been considered one of the main strategies to prevent spoilage owing to the chemical-free food demand of society (melini and melini 2018). lab produces various antifungal compounds such as organic and carboxylic acids, reuterin, fatty acids, and cyclic dipeptides (crowley et al. 2013). sevgi and tsveteslava (2015) reported that six different lpb. plantarum strains retarded a. niger growth. in our study, lpb. plantarum strains showed mild inhibitory effect by delaying spore formation on a. niger growth and their antifungal impacts were expressed as clear zone diameter around the lab colonies. moreover, demirbaş et al. (2017) found that lpb. plantarum has a moderate impact on a. niger with a small clear zone. kivanc et al. (2011) analyzed 45 different lab against 7 mould species by means of dual agar overlay and well method, levl. brevis strains studied have clear inhibition zone around the colonies against a. flavus in accordance with levl. brevis kco-48 inhibitory impact on a. flavus. antifungal action of lab depends on mould species. although coml. paralimenterius showed the highest antifungal effect on a. flavus, it did not demonstrate any inhibiting impact on penicillium carneum and aspergillus niger. it was suggested that the metabolites produced by lab inhibited mould growth and extended shelf life, thus demonstrating that lab could be exploited as biopreservative (muhialdin et al. 2011). similarly, liml. fermentum 29gt-19 showed extensive inhibition on a. niger with a good clear zone and delayed spore formation and moderate inhibition on a. flavus. the results showed that lab inhibits a. niger and a. flavus differently depending on mould and lab species. penicillium carneum is the most resistant mould against all studied lab. antifungal metabolites producing lab could be utilized as biopreservative in bakery products. conclusion in summary, lab isolates isolated from sourdough samples were tested for some technological and nutritional properties. these strains had antagonistic effects against pathogenic bacteria. they tend to degrade and multiply phytic acid that binds minerals. therefore, they have the potential to improve human health and product quality. it can be said that bioactive peptides and aroma compounds inhibit mould growth and lower the ph as much as preventing the survival of pathogens and spoilage microorganisms. the superior properties of lab differentiate from each other. levl. brevis kco-48 demonstrated the highest phytase activity, lpb. plantarum 06f-25 was only lab that inhibits all tested pathogens. lpb. plantarum 45mk-32 have the highest proteolytic activity and highest ph decreasing effect. liml. fermentum 29gt-19 and levl. brevis kco-48 were most effective to hinder mould growth and spore formation considering total effects on studied moulds. the studied lab can be exploited to promote human health and develop/preserve food quality when taking all their characteristics into account. moreover, the utilization of these lab as combination have promised functional products since they may meet different requirements in terms of product quality. acknowledgments this study was partly funded by the scientific research projects of yildiz technical university with the grant number fdk-2020-3885. we thank hakan dogan and danedolu team for their assistance in providing the sourdough samples. 12 conflict of interest the authors declare that they have no conflict of interest. references akman pk, ozulku g, tornuk f, yetim h (2021) potential probiotic lactic acid bacteria isolated from fermented gilaburu and shalgam beverages. lwt. 149: 111705. arqués jl, rodríguez e, langa s, landete jm, medina m (2015) antimicrobial activity of lactic acid 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m, scherer s (2013) identification of microorganisms by ftir spectroscopy: perspectives and limitations of the method. applied microbiol. biotechnol. 97: 7111-7120 yildirim rm, arici m (2019) effect of the fermentation temperature on the degradation of phytic acid in wholewheat sourdough bread. lwt 112: 108224. yildirim-mavis c, yilmaz mt, dertli e, arici m, ozmen d (2019) non-linear rheological (laos) behavior of sourdough-based dough. food hydrocoll. 96: 481-492. 12 1 microsoft word luptakova nb.doc nova biotechnologica vii-i (2007) 17 importance of sulphate-reducing bacteria in environment alena luptakova department of mineral biotechnologies, institute of geotechnics of slovak academy of sciences, watsonova 45, sk-043 53 kosice, slovak republic (luptakal@saske.sk) abstract: the sulphate-reducing bacteria represent the part of the biosphere, the active component in the cycle of elements in the biosphere and as shown by the existing knowledge they are becoming also the part of the environmental industrial technologies. the objective of this work was to give principal information concerning characteristics, the occurrence and the importance of sulphate-reducing bacteria in environment. key words: sulphate-reducing bacteria, sulphuretum, heavy metals 1. introduction sulphate-reducing bacteria (srb) are a ubiquitous group of anaerobic prokaryotes. they are unified by a shared ability to carry out sulphate reduction as a principal component of their bioenergetic processes. they are found in diverse environments and are of great utilitarian and academic interest. their practical importance arises from both economic and environmental concerns. srb are of significant economic interest in many industrial sectors. their academic importance arises from the unique ecological and evolutionary roles they play. sulphate reducers are important organisms in many anaerobic environments in nature, and their physiological activities are of profound importance for many ecological communities. furthermore, there is increasing biochemical and genetic evidence suggesting that these organisms began evolutionary divergence at an early time. this long evolutionary history has led to development in these organisms of a variety of unique proteins and biochemical processes with profound academic importance and fundamental insights. 1.1 characteristics of sulphate-reducing bacteria the srb represents a group of chemoorganotrophic and strictly anaerobic bacteria that may be divided into four groups based on rrna sequence analysis (castro et al., 1999): gram-negative mesophilic srb, gram-positive spore forming srb, thermophilic bacterial srband thermophilic archaeal srb. they include genera such as desulfovibrio, desulfomicrobium, desulfobacter, desulfosarcina, desulfotomacullum, thermodesulfobacterium, archaeoglobus, etc. the basic metabolic process of srb is the anaerobic reduction of sulphates in which organic substrate (lactate, malate, etc.) or gaseous hydrogen is the electron donor and sulphate is the electron acceptor. considering the inorganic or organic 18 luptakova, a. character of energy source of srb there are two types of anaerobic respiration of sulphates (odom and rivers singleton, 1993): 1. authotrophic reduction of sulphates – the energy source is a gaseous hydrogen, the reaction proceeds in several stages and the whole process can be summarily expressed by equation (1): srb 4 h2 + so4 2 ⎯⎯⎯→ s2+ 4 h2o (1) 2. heterotrophic reduction of sulphates – the energy sources are simple organic substances (lactate, fumarate, pyruvate, some alcohols and the like). depending on the final product of organic substrate oxidation we know: incomplete heterotrophic oxidation of organic substrate in which the final product is acetate (equation 2): srb 2 ch3chohcoo + so4 2 ⎯⎯⎯→ 2 ch3coo+ 2 hco3+ h2s (2) complete heterotrophic oxidation of organic substrate in which the final products are co2 and h2o (equation 3): srb 4 ch3cocoona + 5 mgso4 → 5 mgco3 + 2 na2co3 + 5 h2s + 5 co2 + h2o (3) in the process of anaerobic respiration of sulphates srb produce a considerable quantity of gaseous hydrogen sulphide (h2s) which reacts easily in the water medium with heavy metal cations forming not easily dissoluble sulphides of the given metals (equation 4): me2+ + h2s ⎯⎯⎯→ mes + 2h+ (me2+ metal cation) (4) the typical species of srb is desulfovibrio desulfuricans (fig. 1). fig. 1. scanning electron micrograph of desulfovibrio desulfuricans (luptáková, 2002) 1.2 occurrence of sulphate-reducing bacteria srb are a unique and ubiquitous group of prokaryotic mo, found in a variety of anaerobic environmental niches such as: soil (ronald, 1995), nova biotechnologica vii-i (2007) 19 mud and sediments of freshwaters (rivers, lakes, …), thermal or no-thermal sulphur springs (karavajko et al., 1988, ho-yong j. et al., 1996), mining waters from sulphide deposits (groudeva and groudev, 1997), waters from deposits of mineral oil and natural gas (armstrong et al., 1995), industrial waste waters (metallurgical industry (luptáková, 2002), sea and ocean (postgate, 1982), gastrointestinal tract of man and animals (sneath et al., 1996). 1.3 importance of sulphate-reducing bacteria the metabolic actions of srb described by equations 1-4 are the fundamental of srb importance in environment. they are generator of many processes both positive and negative. despite the odoriferous physiology of these organisms, they are important to study for both practical and academic reasons. their practical importance stems from the significant economic impact they have in many industrial and ecological areas. their academic importance is related to their unique environmental and evolutionary roles in nature. because srb are ubiquitous to oil-bearing shale and strata they play an important economic role in many aspects of oil technology (dockins et al., 1980). they are responsible for extensive corrosion of drilling and pumping machinery and storage tanks. furthermore, because they contaminate resulting crude oil the organisms cause release of hydrogen sulphide into petroleum products, thereby increasing the sulphur level of fuels. they are also important in secondary oil recovery processes, where bacterial growth in injection waters can plug machinery used in these processes. it has also been suggested that these organisms may play a role in biogenesis of oil hydrocarbons. for all of these reasons srb are of vital importance in petroleumproducing and processing-industries (postgate, 1982). a major economic consequence resulting from the metabolic action of srb is anaerobic corrosion of underground buried ferrous metals such as tanks and pipelines. srb play a vital role in a variety of other important economic areas. in many industrial processes they produced iron sulphides, thereby causing blackening and discoloration of products. for example, in the paper industries srb can contaminate processing water, leading to sulphide production, which causes blackening of paper (odom and rivers singleton, 1993). 1.4 environmental importance of srb these organisms are also of great ecological importance, which is in part related to their economic importance. pivotal ecological roles of srb result from their participation in the sulphur cycle, which is a major biogeological cycle in nature. sulphur is widely distributed on earth and occurs predominantly bound as sulphides in the form of sulphates or even as elemental sulphur. in nature sulphur circulates permanently because it is continuously oxidized or reduced by chemical or biological processes. in such a biogeochemical sulphur cycle (fig. 2) the biological transformations may have either assimilatory or dissimilatory metabolic functions. 20 luptakova, a. with the exception of animals and humans, most plants, fungi and bacteria are capable of performing an assimilatory reduction of sulphate to sulphide which is necessary for the biosynthesis of sulphur containing cell compounds. on the other hand the energy producing dissimilatory sulphur metabolism is restricted to a few groups of bacteria. these groups include mo such as: anaerobic dissimilatory sulphate reducers (desulfovibrio, desulfotomaculum, desulfomonas,….), anaerobic dissimilatory sulphur reducers (desulfuromonas, beggiatoa,….), anaerobic phototrophic sulphur oxidisers (some cyanobacteria and most anoxygenic phototrophic bacteria), aerobic chemotrophic sulphur oxidisers (thiobacillus, sulfolobus, thiospira, thiobacterium,….), anaerobic chemotrophic sulphur oxidisers (thibacillus denitrificans, thiomicrospira denitrificans,..) (rehm and reed, 1981). fig. 2. the biological sulphur cycle (rehm and reed, 1981). 1 assimilatory sulphate reduction by plants, fungi and bacteria; 2 death and decomposition by fungi and bacteria; 3 sulphide assimilation by bacteria and some plants; 4 excretion of sulphate by animals; 5 dissimilatory sulphate-reducing bacteria; 6 dissimilatory sulphur-reducing bacteria; 7 phototrophic and chemotrophic sulphide-oxdizing bacteria; 8 phototrophic and chemotrophic sulphur-oxidizing bacteria. fig. 3. schematic diagram illustrating the chemical processes of a sulphuretum (odom and rivers singleton, 1993) as can be seen from fig. 2 the srb miss the assimilatory part of the cycle and produce sulphides. the microbial population of this dissimilatory part is called 8 4 so4 2 biologically bound sulphur s0 s2 1 5 2 3 7 6 assimilatory dissimilatory part accumulates 0 aerobic sulphur-oxidisers (at top) so42 feeds in hs srb hs anaerobic sulphate-reducers (at bottom) diffusion so42so42nova biotechnologica vii-i (2007) 21 “sulphuretum” and its activity from the chemical processes point of view is shown in fig. 3. sulphate ion from the surrounding land mass is leached into and diffuses to the bottom of these small aquatic systems, where it is reduced to s2by anaerobic sulphate-reducing bacteria. sulphite ion then diffuses upward through the water column to the surface layers, where it is oxidized by aerobic organisms to either elemental sulphur (s0) or so4 2-. the latter diffuses to the bottom, anaerobic region where the cycle continues. because of its low solubility in water, however, s0 precipitates from solution and forms a yellow crust on the water surface and near the edges of these systems. the activity of sulphuretum is a basis of many processes in nature and it finds its application in industry, for example: the removal of heavy metals from industrial waste waters (e.g. acid mine drainage) (kontopoulos, 1998; boonstra et al., 1999), the removal of so42from mine waters (kontopoulos, 1998; glombitza, 2001) , the production of sulphur from waste waters (postgate, 1982), the removal of sulphur from crude oil, petrol and coal (desulphurization) (armstrong, 1997). 1.5 evolutionary importance of srb there appears to be clear geochemical evidence that process of biological sulphate reduction is evolutionarily ancient. this conclusion grows from the observation that bacterial reduction of sulphate shows a kinetic isotope effect in which 32so4 2is used more rapidly than the heavier isotope 34so4 2-. consequently, geological sulphur deposits that are enriched in the lighter isotope, relative to the heavier isotope are believed to be biological in origin. evidence of this nature suggests that many of the world´s sulphur reserves were biologically formed and that this action occurred prior to significant accumulation of atmospheric oxygen. this line of reasoning clearly suggests that the process of sulphate reduction was of bioenergetic significance before the process of oxygen reduction. present arguments suggest that development of dissimilatory sulphate reduction occurred between 2.8 and 2.5 x 109 years ago and was simultaneous with oxygenic photosynthesis by ancestral cyanobacteria but prior to aerobic respiration (widdel, 1988). 2. conclusion this paper attempts to give the notion that srb were unified by shared ability to utilize sulphate reduction as an important component of their bioenergetics processes. srb represent the part of the biosphere, the active component in the cycle of elements in the biosphere and as shown by the existing knowledge they are becoming also the part of the environmental industrial technologies. these bacteria are extremely diverse from metabolic, morphological, antigenic as well as ecological perspectives. this diversity will have profound implications for our understanding of the environmental and economic role that these bacteria play in the natural world. 22 luptakova, a. acknowledgment: this work was supported by the slovak research and development agency under the contract no. apvv-51-027705 and grant agency vega for project no. 2/5148/27. references armstrong, s.m., sankey, b.m., voordouw, g.: converzion of dibenzothiophene to biphenyl by sulfate-reducing bacteria isolated from oil field production facilities. biotechnol. lett., 17, 1995, 1133-1136. armstrong, s.m., sankey, b.m., voordouw, g.: evaluation of sulfatereducing bacteria for desulfurizing bitumen or its fractions. fuel, 76,1997, 223227. boonstra, j., van lier, r., janssen, g., dijkman, h., buisman, c. j. n.: biological treatment of acid mine drainage. in: r. amils, a. ballester (eds.) biohydrometallurgy and the environment toward the mining of the 21st century, (eds.). elsevier, amsterdam, 1999, 559-566. castro, h. f., williams, n. h., ogram, a.: phylogeny of sulphate-reducing bacteria. fems microbiol. ecol., 31, 1999, 1-9. dockins, w. s., olson, g.j., mcfeters, g. a., turbak, s. c.: dissimilatory bascterial sulphate reduction in montana ground water. geomicrobiol. j., 2, 1980, 83-98. glombitza, f.: treatment of acid lignite mine flooding water by means of microbial sulfate reduction. waste manag., 21, 2001, 197-203. groudeva, v.i., groudev, s.n.: bioremediation of acide drainage waters. proceedings of international biohydrometallurgy symposium. sydney, australia, 1997, 4-7. jin, h. y., lee, d. h., zo, y. g., kang, c. s., kim, s. j.: distribution and activity of sulfate-reducing bacteria in lake soyang sediments. j. microbiol., 34, 1996, 131-136. karavajko, g. i., rossi, g., agate, a. d., groudev, s. n., avakyan, z. n.: biotechnology of metals, centre of projects gknt, moscow, 1988, 59 pp. kontopoulos, a.: acid mine drainage control. in: effluent treatment in the mining industry, univesity of concepcion, chile, 1998, 57 pp. luptáková, a., kušnierová, m., fečko, p.: minerálne biotechnológie ii., sulfuretum v prírode a v priemysle. es všb – technická univerzita ostrava, 2002. odom, j. m., rivers singleton, j. r.: the sulfate-reducing bacteria: contemporary perspectives, springer-verlag, new york, 1993. postgate, j.: mikróby a my, praha, pyramída, 1982. rehm, h.j., reed, g.: biotechnology, vol. 6b, verlag chemie gmbh, weuheim, 1981, 473-475. ronald, m. a.: principles of microbiology, mosbi -year book, 1995. sneath, p.h., mair, n.s., sharpe, m.e., holt, j.g.: bergey´s manual of systematic bacteriology, vol. 2, vol. 8, sydney, 1200-1202, 1996, 663-679. widdel, f.: microbiology and ecology of sulphateand sulphurreducing bacteria. in: a.j.b zehner (ed.) biology of anaerobic organisms, john wile, new york, 1988, 469-585. nova biotechnol chim (2022) 21(2): e1226 doi: 10.36547/nbc.1226 1 nova biotechnologica et chimica optimization of procedural factors for advanced xylanase synthesis by lysinibacillus fusiformis using kolanut husk as substrate suliat olatidayo omisore, daniel juwon arotupin, michael tosin bayode department of microbiology, federal university of technology, p.m.b. 704, akure, nigeria  corresponding author: omisore34@gmail.com article info article history: received: 13th october 2021 accepted: 1st december 2022 keywords: kolanut husk lysinibacillus fusiformis optimization substrate xylan xylanase abstract xylan is a complex hetero-polysaccharide consisting of different monosaccharides held together by glycosidic and ester bonds. extracellular xylanase fashioned by numerous microbes principally from bacterial species such as bacillus species are responsible for cleaving the glycosidic linkages. microbial xylanases exhibit different substrate specificities and biochemical peculiarities. this study was carried out for optimization of cultivation conditions for xylanase production using the bacterium lysinibacillus fusiformis and kolanut husk as a component of cultivation medium. the bacterium was isolated from kolanut plantation waste soil and screened for the production of xylanase qualitatively on xylan nutrient agar and quantitatively under submerged fermentation. the different conditions optimized included substrate concentration, additional sugars, incubation period, temperature, initial ph, nitrogen supplementation and inoculum mass through one factor at a time approach. maximum xylanase production was obtained at substrate concentration of (1 % xylan and 1.5 % kolanut husk), nitrogen source (yeast extract plus peptone), carbon source (sucrose), incubation period (24 h), ph (5.0), temperature (35 oc) and inoculum size (1 %). lysinibacillus fusiformis has been proven to be a promising bacterium for xylanase production using kolanut husk as substrate. the use of kolanut husk as foremost carbon source is predominantly precious as being an agricultural waste, affordable, and locally available compared to expensive commercially sold xylan. introduction hemicellulose serves as the second most ample source of polysaccharides in nature, exhibiting 25 % 30 % of the dry weight of lignocellulose biomass (coman et al. 2011; walia et al. 2015). it is the major amount of lignocellulose, comprising of varied 5and 6-carbon sugars such as arabinose, galactose, glucose, mannose, and xylose (girisha 2018). xylan is a polymer of xylose molecules which plays the pivotal role in holding the plant cell walls together (irfan et al. 2016; girisha 2018). considerable xylanolytic enzymes comprising α-arabino-furanosidase, act collectively for total disintegration of xylan. the most important endo β1, 4-xylanase plays an imperative part to catalyze the breakdown of xylan into diminutive oligosaccharides (irfan et al. 2012; khusro et al. 2016). udey et al. (2016) reported the latent applications of xylanase in numerous fields such as saccharification of lignocellulosic biomass for ethanol and xylo-oligosaccharides generation in the biofuel industry. its use as a fading agent and mailto:omisore34@gmail.com nova biotechnol chim (2022) 21(2): e1226 2 lighting up of pulp to reduce chlorine in paper and pulp production operations was also reported (raghukumar et al. 2004; sridevi et al. 2016; patel and dudhagara 2020a). it helps in the smooth relocation of the water core in bread dough ensuring a softer dough formation (beg et al. 2001; garg et al. 2010). furthermore, xylanolytic enzymes have been reported by garg et al. (2010) for its application in the animal industry for the conversion of lignocellulose into feedstocks and fuels. xylanases are produced by a diverse number of microbes such as bacteria, fungi and actinomycetes (battan et al. 2006; chakdar et al. 2016; ramanjaneyulu et al. 2017). however, xylanases from bacteria are the most-preferred for industrial applications due to thermo-stability and ability to withstand alkali-substrates (nagar et al. 2013; aarti et al. 2015; mandal et al. 2015). xylanolytic bacteria are the generally striking producers of high-level extracellular xylanase as compared to fungi and actinomycetes, which have timeconsuming generation time alongside the production of extremely glutinous polymers and deprived oxygen transport. there is limited exploitation of xylanase commercially due to its high cost of production. in this view, the search for cost-effective means of production, such as the use of low-cost agro agricultural residues has captivated the attention of scientists (patel and dudhagara 2020b). gautério et al. (2020) and ratnadewi et al. (2020) also buttressed the utility of agriculture waste derivatives like wheat bran, corn cobs, rice straw for exploitation as a lowgrade substrate for xylanase production. kolanut husk alongside its testa has been proven as a budding substrate to produce the microbial enzyme particularly xylanase (fabunmi et al. 2018). therefore, the study was carried out to determine the optimal process parameters needed to improve the yield of xylanase produced from simple inexpensive kolanut husk using lysinibacillus fusiformis. in biotechnological process, the best results are achieved when the individual and combined effects of parameters involved are considered (tandon et al. 2016). the current study aims at optimizing the cultural conditions such as carbon source, nitrogen supplementation, temperature, incubation time and inoculum sizes for xylanase production by lysinibacillus fusiformis, an isolate of kolanut plantation waste soil using a medium with kolanut husk as substrate in other to know the best conditions for highest xylanase. experimental kolanut plantation soil and kolanut husk collection kolanut husks and kolanut plantation soil from the kolanut husk dumpsite were obtained from a kolanut farm at ile ife city, osun state, nigeria (7.4905° n, 4.5521° e). sample transported to the laboratory of microbiology department, federal university of technology akure, ondo state in a ziploc bag. the soil samples were preserved at -20 ºc before use. isolation and identification of bacteria isolates one gram of the soil sample was suspended in 100 ml of distilled water then incubated in a control incubator shaker (gallenkamp, cambridge, uk) at 200 rpm for 30 min. mixtures were allowed to settle, and serial dilutions of up to 104 diluents were prepared using sterile distilled water. bacterial isolation was conducted by aseptically plating 100 µl from the diluent on xylan nutrient agar (nutrient agar supplemented with 1 % xylan) and incubated aerobically for 37 ºc for 24 h. purification of bacteria was done by repeated streaking and sub-culturing for purity and preserved at 4 ºc until use (kartik et al. 2016). authentication of the xylanolytic ability of isolates by well plate diffusion assay the organism was however authenticated qualitatively for positive xylanase production by growing isolates on nutrient agar medium containing 1 % birchwood xylan. the media was sterilized, poured, and allowed to solidify. upon solidification, wells were made with the aid of sterile cork-borer of 10 mm diameter. to each well bored in the solidified media, 100 µl of the overnight pure bacterial culture was poured and incubated for 24 – 36 h. the plate was flooded with nova biotechnol chim (2022) 21(2): e1226 3 0.25 % congo red solution for 15 min after which it was decolorized with 0.5 % acetic acid for 15 min and finally washed twice by 1 m nacl for de-staining as described (chan et al. 2016). clear zones around the well further authenticated xylanolytic activity. submerged fermentation for xylanase production submerged fermentation was conducted using kolanut husk as the substrate in addition to minimal salt medium containing (g.l-1): xylan 10.0, k2hpo4 0.1, cacl2 0.5, mgso4.7 h2o 0.5, peptone 2.0, feso4·7 h2o in trace amount, ph 6.8, sterilized at 121 ºc for 15 min. 50 ml of the sterile minimal salt medium in 250 ml flasks were inoculated with 1 ml of bacterial culture in broth and incubated at 37 oc for 24 h in a rotary shaker (hunterlab digital colorimeter, reston, usa) with agitation speed of 150 rpm. after the incubation period, the enzyme production medium was subjected to cold centrifugation at 10,000 rpm for 10 min to eliminate the bacterial cells and superfluous elements. the supernatant considered as source of the crude enzyme was gently decanted into sterile vials (sari et al. 2016). xylanase assay xylanase activity in the supernatant was assayed by determining the concentration of reducing sugar released by the activity of the crude enzyme on its substrate xylan by 3,5-dinitrosalicylic acid (dnsa) reagent (miller 1959). reaction mixture containing 0.5 ml of culture enzyme with 0.5 ml 1 % of birchwood xylan solution primed in phosphate buffer ph 6.8 was incubated for 20 min at 45 oc. the control experiment was set up to authenticate the results from the actual testing; with the control tube comprising a similar volume of a substrate with enzyme solution being replaced with sterile distilled water. after incubation, the reaction was terminated by adding 1 ml of dnsa reagent as described by miller (1959). the tubes enclosing the resultant blend were excited in the boiling water bath for 10 min, observed for brick red colour development and cooled swiftly in a water bath at room temperature. the optical density of the cooled mixture was measured with a spectrophotometer (lab-tech digital colorimeter) at 540 nm against a blank and articulated as xylose equivalent. one unit of endo-1,4-β-xylanase was defined as the amount of enzyme requisite to discharge 1 µmol of xylose per minute in model assay conditions as depicted by sharma and bhajaj (2005); bhalla et al. (2015). protein estimation the amount of protein released in the supernatant was estimated as described bradford (1976) using bovine serum albumin (bsa) (sigma-aldrich, darmstadt, germany) as standard. 1ml of the enzyme extract was taken in a glass tube and added to it 3 ml of bradford’s reagent, the mixture was incubated at room temperature for 7 – 10 min. the absorbance was measured at 595 nm with a spectrophotometer (hunterlab digital colorimeter, virginia, usa) and compared with the bovine serum albumin standard curve. optimization of cultural and nutritional conditions the optimization of xylanase production by lysinibacillus fusiformis was carried out using “one factor at a time technique” (varying one factor while keeping the rest constant) in order to estimate xylanase activity for each varied parameter. parameters such as the effect of kolanut husk as carbon source at concentration (0.5 – 2.5 %), kolanut husk induction with xylan (0.5 – 1.5 %), additional carbon source at 0.5 % (glucose, fructose, sucrose, galactose and lactose), nitrogen supplementation at 0.25 % (yeast extract, urea, gelatin, tryptone, casein, ammonium chloride, ammonium nitrate, ammonium sulfate, potassium nitrate), incubation period (0 – 42 h), ph (2 – 10), temperature (25 – 45 oc) and inoculum size (0.5 – 2.5 %) were optimized for enhanced xylanase production by lysinibacillus fusiformis. the fermentation broth was separated from the bacterial cells by centrifugation, the supernatant served as a source of a crude enzyme tested for xylanase activity. the entire experimentations were done in triplicates with each mean shown in the result section. each experiment had its control with the control tube not having the tested process parameters. nova biotechnol chim (2022) 21(2): e1226 2 statistical analysis the data obtained after the optimization studies were statistically evaluated using the one-way analysis of variance (anova) at significance level of p < 0.05 using the statistical package ibm spss statistics v. 23.0 (chicago, usa). results screening and authentication of obtained xylanolytic bacteria isolate appeared cream-coloured, regularly shaped, round edges with 1 – 2 mm diameter on nutrient agar plate. it was gram-positive and rod-shaped and conforms to its earlier identification by fabunmi et al. (2018) as l. fusiformis. organisms showed zones of hydrolysis on xylan agar plates after incubation, indicating its ability to degrade xylan in the media alongside its growth during the incubation period with an average of 29 mm diameter zone of hydrolysis on xylan agar medium. optimization of culture and nutritional conditions the effect of different substrate (kolanut husk) concentration (0.5 – 2.5 %) as sole source of xylan in submerged fermentation by l. fusiformis is in table 1. result indicated that the highest enzyme activity was at kolanut husk concentration of 2.0 % with value of 419.28 µmol.ml-1.min-1 while the least at 0.5 % with a value of 201.52 µmol.ml-1.min-1. table 1. upshot of substrate concentration (kolanut husk) on xylanase production by l. fusiformis under smf. values are obtainable as mean ± s.e (n = 3). values with different superscripts are statistically significantly different (p < 0.05). smf – submerged fermentation. effect of xylan supplementation with kolanut husk on xylan production by l. fusiformis the effect of different concentrations of a husk (0.5, 1.0, 1.5, 2.0, and 2.5 %) in combination with a commercial birchwood xylan was supplemented to the fermentation medium for enhanced production of xylanase as shown in table 2. xylan supplementation as inducer showed maximum activity at 1.5 % kolanut husk supplemented with 1 % commercial xylan with xylanase activity of 422.63 µmol.ml-1.min-1 and lowest at 1 % kolanut husk with 0.5 % xylan with activity of 303.06 µmol.ml-1.min-1. effect of carbon source on xylanase production by lysinibacillus fusiformis effect of sugar optimization revealed sucrose to having the highest activity effect amongst others with enzyme activity of 549.75 µmol.ml-1.min-1 exceeding other sugars as shown in table 3. the monosaccharide galactose gave the least activity of 494.5 µmol.ml-1.min-1, the remaining sugars gave moderate improvement on enzyme production but not comparable to sucrose. kolanut husk [%] substrate optimization absorbance enzyme activity [µmol.min-1.ml-1] protein concentration specific enzyme activity [µmol.min-1.mg-1] 0.5 0.54 ± 0.00a 206.52 ± 2.02a 1.96 ± 0.06a 100.18 ± 0.16b 1.0 0.70 ± 0.00b 266.83 ± 2.04b 2.17 ± 0.01b 124.28 ± 0.11e 1.5 0.92 ± 0.00c 341.43 ± 0.29c 3.89 ± 0.02d 88.34 ± 0.08a 2.0 1.11 ± 0.00e 419.28 ± 0.36e 3.91 ± 0.01d 106.83 ± 0.12c 2.5 1.01 ± 0.00d 381.36 ± 0.31d 3.11 ± 0.06c 121.65 ± 0.03d 4 nova biotechnol chim (2022) 21(2): e1226 3 table 2. effect of xylan (x) supplementation as inducer with varied kolanut husk concentration on xylanase production by l. fusiformis under smf. values are obtainable as mean ± s.e (n = 3). values with different superscript are statistically significantly different (p < 0.05). smf – submerged fermentation. table 3. effect of sugar supplementation (carbon source) on xylanase production by lysinibacillus fusiformis under smf. carbon sources [%] carbon sources optimization absorbance enzyme activity [µmol.min-1.ml-1] protein concentration specific enzyme activity [µmol.min-1.mg-1] control (peptone water) 0.86± 0.02a 320.66 ± 0.88a 1.79 ± 0.05d 177.61 ± 0.87a glucose 1.39 ± 0.01d 521.74 ± 0.80e 2.06 ± 0.02e 250.47 ± 0.86b fructose 1.38 ± 0.01d 518.02 ± 1.13d 1.21 ± 0.04c 452.10 ± 0.58d sucrose 1.45 ± 0.02e 550.58 ± 0.71f 1.08 ± 0.02b 500.57 ± 0.86e galactose 1.32 ± 0.01c 495.16 ± 0.44c 0.88 ± 0.01a 566.38 ± 2.34f lactose 1.25 ± 0.02b 476.16 ± 1.48b 1.76 ± 0.02d 264.52 ± 0.86c values are obtainable as mean ± s.e (n = 3). values with different superscript are statistically significantly different (p < 0.05). smf – submerged fermentation. effect of organic nitrogen on xylanase production by lysinibacillus fusiformis nitrogen sources revealed yeast extract giving the highest xylanase titre (437.75 µmol.ml-1.min-1) and gelatin (339.38 µmol.ml-1.min-1) displayed the lowest enzyme titre (table 4). table 4. effect of organic nitrogen source on xylanase production by lysinibacillus fusiformis under smf. inoculum [%] organic sources optimization absorbance enzyme activity [µmol.min-1.ml-1] protein concentration specific enzyme [µmol.min-1.mg-1] control (peptone water) 1.05 ± 0.00b 401.06 ± 0.23b 2.76 ± 0.07b 149.06 ± 0.38b gelatin 0.86 ± 0.01a 340.12 ± 0.47a 2.79 ± 0.06b 116.61 ± 0.45a casein 1.12 ± 0.00c 426.90 ± 0.31c 2.72 ± 0.01b 155.44 ± 0.29c urea 1.13 ± 0.00c 430.72 ± 0.14d 2.39 ± 0.01a 181.06 ± 0.52e tryptone 1.15 ± 0.00d 437.98 ± 0.89e 2.36 ± 0.01a 184.73 ± 0.63f yeast extract 1.15 ± 0.00d 438.55 ± 0.47e 2.70 ± 0.01b 162.89 ± 0.58d values are obtainable as mean ± s.e (n = 3). values with different superscript are statistically significantly different (p < 0.05). smf – submerged fermentation. effect of inorganic nitrogen on xylanase production by lysinibacillus fusiformis table 5 showed the effect of inorganic nitrogen with kno3 resulting in the highest xylanase activity (464.25 µmol.ml-1.min-1) and nh4co3 with the least (325.70 µmol.ml-1.min-1). kolanut husk + xylan [%] substrate optimization absorbance enzyme activity [µmol.min-1.ml-1] protein concentration specific enzyme activity [µmol.min-1.mg-1] k1.0 + x0.5 0.80 ± 0.00a 303.06 ± 0.54a 1.87 ± 0.01a 162.84 ± 0.07d ki.0+ x1.0 1.01 ± 0.00d 384.13 ± 0.59e 2.09 ± 0.05b 190.69 ± 0.85f k0.5+x1.0 0.97 ± 0.01c 367.12 ± 1.16c 2.38 ± 0.01c 153.37 ± 0.49b k1.5+x0.5 0.79 ± 0.00a 374.65 ± 0.87d 2.38 ± 0.07c 166.72 ± 0.43d k1.5+x1.0 1.12 ± 0.00e 422.54 ± 1.44f 2.39 ± 0.01c 178.55 ± 0.23e 2.0k.h 0.92 ± 0.00b 348.36 ± 0.31b 2.36 ± 0.02c 147.21 ± 1.17a 5 nova biotechnol chim (2022) 21(2): e1226 2 table 5. outcome of inorganic nitrogen source on xylanase production by lysinibacillus fusiformis under smf. inoculum [%] inorganic sources optimization absorbance enzyme activity [µmol.min-1.ml-1] protein concentration specific enzyme [µmol.min-1.mg-1] control (peptone water) 1.05 ± 0.00b 400.56 ± 0.86b 2.68 ± 0.02a 148.96 ± 1.15e kno3 1.22 ± 0.01d 463.74 ± 0.90d 3.83 ± 0.01c 121.27 ± 0.36d (nh4)2so4 1.05 ± 0.00c 398.01 ± 0.57b 3.52 ± 0.02b 113.49 ± 0.86c nano3 1.07 ± 0.01c 405.86 ± 0.59c 4.30 ± 0.05d 96.44 ± 0.28b nh4co3 0.85 ± 0.00 325.70 ± 1.19a 4.50 ± 0.01e 92.45 ± 0.27a values are obtainable as mean ± s.e (n = 3). values with different superscript are statistically significantly different (p < 0.05). smf – submerged fermentation. effect of ph on xylanase synthesis by lysinibacillus fusiformis media ph is an extra significant dynamic considered in table 6, enzyme activity was highest value of 537.79 µmol.ml-1.min-1 at ph of 5.0 and the lowest enzyme activity at ph of 2.0 (368.20 µmol.ml-1.min-1). a supplementary boost in ph above 5.0 resulted in a significant drop-off in the activity recorded. table 6. upshot of ph on xylanase synthesis by lysinibacillus fusiformis under smf. ph ph optimization absorbance enzyme activity [µmol.min-1.ml-1] protein concentration specific enzyme [µmol.min-1.mg-1] 2 0.88 ± 0.08a 368.20 ± 0.90a 3.55 ± 0.02d 102.69 ± 0.11a 3 1.11 ± 0.01bc 424.08 ± 0.46b 3.52 ± 0.03cd 121.53 ± 0.03b 4 1.20 ± 0.00cd 456.88 ± 1.55c 3.05 ± 0.02b 148.83 ± 0.06d 5 1.41 ± 0.00g 537.79 ± 1.10h 3.73 ± 0.12e 141.29 ± 0.12c 6 1.39 ± 0.00fg 528.31 ± 0.84g 3.35 ± 0.07c 161.22 ± 0.14g 7 1.31 ± 0.00ef 497.30 ± 0.84f 3.14 ± 0.03b 159.14 ± 0.13f 8 1.28 ± 0.00de 486.26 ± 1.86d 3.10 ± 0.05b 161.25 ± 0.14g 9 1.20 ± 0.00cd 458.18 ± 0.92cc 2.99 ± 0.05b 152.71 ± 0.11e 10 1.09 ± 0.00b 490.56 ± 0.72e 2.75 ± 0.02a 141.44 ± 0.02c values are obtainable as mean ± s.e (n = 3). values with different superscript are statistically significantly different (p < 0.05). smf – submerged fermentation. effect of temperature on xylanase production by lysinibacillus fusiformis xylanase production was at its peak at 35 oc (572.83 µmol.ml-1.min-1) and least at 45 oc (347.33 µmol.ml-1.min-1) (table 7). table 7. effect of temperature on xylanase synthesis by lysinibacillus fusiformis under smf. temp [ºc] temperature optimization absorbance enzyme activity [µmol.min-1.ml-1] protein concentration specific enzyme [µmol.min-1.mg-1] 25 1.20 ± 0.00c 457.64 ± 1.30c 3.07 ± 0.06a 151.82 ± 0.03c 30 1.39 ± 0.00d 531.41 ± 0.33d 3.11 ± 0.11a 169.65 ± 0.17d 35 1.51 ± 0.00e 572.77 ± 1.29e 3.50 ± 0.05b 167.62 ± 1.44d 40 1.10 ± 0.00b 419.86 ± 0.32b 3.39 ± 0.05b 124.51 ± 0.86b 45 0.91 ± 0.00a 346.11 ± 0.67a 2.94 ± 0.02a 119.35 ± 0.87a data are presented as mean ± s.e (n = 3). values with different superscript are statistically significantly different (p < 0.05). smf – submerged fermentation. 6 nova biotechnol chim (2022) 21(2): e1226 3 effect of incubation time on xylanase production by lysinibacillus fusiformis incubation period optimization results showed that 24-h cultivation (624.29 µmol.ml-1.min-1) gave the best production as seen in fig. 1 and further increase revealed a corresponding reduction in enzyme activity. fig.1. effect of time on xylanase production by lysinibacillus fusiformis under submerged fermentation. effect of inoculum size on xylanase production by lysinibacillus fusiformis inoculum size is a vital dynamic in enzyme synthesis and maximum enzyme activity was recorded at 1.5 % with a value of 647.63 µmol.ml1.min-1 and the least observed at 2.5 % with 616.07 µmol.ml-1.min-1 (table 8). table 8. effect of inoculum size on xylanase production by lysinibacillus fusiformis under smf. inoculum [%] inoculum optimization absorbance enzyme activity [µmol.min-1.ml-1] protein concentration specific enzyme [µmol.min-1.mg-1] 0.5 1.62 ± 0.00a 615.27 ± 0.36a 0.89 ± 0.01b 697.68 ± 0.71d 1.0 1.67 ± 0.00b 624.07 ± 0.51b 0.96 ± 0.01c 653.53 ± 0.74b 1.5 1.70 ± 0.00d 647.63 ± 0.32d 1.25 ± 0.01d 518.78 ± 0.32a 2.0 1.69 ± 0.00c 643.35 ± 0.86c 0.97 ± 0.01c 671.07 ± 0.52c 2.5 1.62 ± 0.00a 616.07 ± 0.51a 0.84 ± 0.00a 732.31 ± 0.56e data are presented as mean ± s.e (n = 3). values with different superscript are statistically significantly different (p < 0.05). smf – submerged fermentation. 7 nova biotechnol chim (2022) 21(2): e1226 2 discussion kolanut husk in nature is composed of hemicellulose (40.41 %), lignin (21.29 %), cellulose (38.72 %) as reported by adeyi (2010) and patel and dudhagara (2020a), making it an appropriate substrate for xylanase production. they are mostly disposed of as agricultural waste at production site of kolanut forming a landfill and environmental problems. nevertheless, as a substrate for enzyme production could help to solve environmental pollution problems as well as reduction in production cost of xylanase. kolanut husk as an enzyme source was optimized as a component of the microbial fermentation medium. among the concentration of kolanut husk tested 2.0 % gave the maximum xylanase production. concentration above 2.0 % demonstrated a declined xylanase production but not as low as when lower concentrations of 0.5 % and 1.0 % was used (table 1). this indicated that adding further substrate does not show any noteworthy change in xylanase synthesis. a general boost in substrate absorption could lead to swell in enzyme output velocity at certain point till it reaches substrate saturation where no enzyme activity could be recorded (heskia et al. 2018). furthermore, the fermentation medium was induced with commercial birchwood xylan alongside kolanut husk. this was intended to boost xylanase production in the medium. optimum xylanase production was attained at a lesser substrate concentration in contrast to medium with solely kolanut husk. different xylan concentrations have also been reported to give maximum xylanase activity of 0.5 % (annamalai et al. 2009; heskia et al. 2018). thus, indicating that xylanase stimulation is an intricate trend in which the intensity of reaction varies with altered concentration combinations. decreases in enzyme activity at higher substrate concentration might be owed to the formation of thick suspension in the presence of higher substrate concentration which could have resulted in improper mixing of the substrate or non-uniform circulation of a nutrient under agitation condition (karim et al. 2015). karim et al. (2015) has revealed that substrate concentration used is a decisive element for utmost xylanase optimization and that there are different concentrations for various ligno-cellulose substrates. supplementation of medium with additional carbon sources involving both monosaccharide and disaccharide improved the enzyme activity produced. sucrose had the highest enzyme activity and closely followed by other sugars (table 2 and table 3). the results obtained agree with singh et al. (2011) who stated that amylase synthesis is favored by galactose, linking to the fact growth and enzyme productions of any organism are greatly influenced by both environmental conditions as well as the nutrient present in the growth medium. however, these findings contradict reports that wholesome sugars only have good growth but poor xylanase synthesis (shah and madamwar 2005) and that of seyis and aksoz (2005) that sucrose and glucose gave inhibitory effect on enzyme activity. their reports were attributed to catabolite repression of xylanase genetic material which is guarded at two levels as reported by de graaff et al. (1994), repressed gene transcription and ultimately by repressed transcriptional activator. in addition to carbon sources, nitrogen sources have a significant end product on the synthesis of xylanolytic enzymes produced by bacteria. overall, crude sources gave a higher xylanase production than inorganic sources (table 4 and table 5). this agrees with the report of bajaj et al. (2010), who stated that peptone combined with yeast extract as the largely reasonable and accustomed nitrogen sources in terms of elevated output of xylanase from diverse bacteria. nagar et al. (2010) reported maximum xylanase production by b. pumilus sv85s using peptone followed by yeast extract as nitrogen source. however, it is contrary with the report of sepahy et al. (2011) where tryptone in combination with yeast extract gave the maximum xylanase titre. peptone and yeast extract are multifaceted nitrogen sources rich in diverse growth factors such as minerals and vitamins and hence improve the growth of bacteria and enzyme synthesis (bibi et al. 2014) as few among the inorganic kno3 resulted in higher titre among others. sepahy et al. (2011) stated the ph of the medium has a strong influence on enzymatic processes as it affects the growth and metabolic activities of 8 nova biotechnol chim (2022) 21(2): e1226 3 individual microorganisms. influences of ph ranging from acidic to alkaline were tested for xylanase production as b. megaterium had the highest production at ph 5.0, indicating that acidic favours this particular organism when acting on kolanut husk as substrate. the alkalinity or acidity of the medium affect bacteria growth coupled with other enzymatic reactions through transport of ions, metabolites, and enzyme structure (liang et al. 2010). our results revealed that as the ph of the medium increases from extremely acidic (ph 2.0) to slightly acidic (ph 5.0), so does the enzyme activity increased but tending towards neutral ph, there was a drop in the value and the least value recorded at alkaline ph (table 6). this report is in contrary with the observations of sepahy et al. (2011); irfan et al. (2016) who reported the highest xylanase synthesis at ph 8.0 using b. mojanvensis and b. subtilis under submerged fermentation. this could possibly be attributed to the alkaline medium possessing an inhibitory influence on lysinibacillus fusiformis and moreover each microorganism has its ph for optimum growth and enzyme production. temperature is another key dynamic that influences the success of optimization system. the best medium temperature was 35 oc which is close to the optimal temperature for utmost xylanase titre in submerged fermentation (smf) (table 7) from other bacillus species reported as 37 °c (qureshi et al. 2002; battan et al. 2007; geetha and gunasekaran 2010; nagar et al. 2012) but contradicts the report of 50 °c temperature (sapereira et al. 2002; azeri et al. 2010; sepahy et al. 2011), 50 – 55 °c (anuradha et al. 2007; sharma et al. 2011) and 55 °c (annamalai et al. 2009). this indicates that productions of xylanase are favored at ambient temperature as compared to extremophilic temperature. this could be due to the fact that mesophilic temperature favors the growth of organisms and hence their ability to produce the desired end product. though, thermophilic temperature is best preferred as it diminishes the risk of mesophilic microbial defects (yeoman et al. 2010). generally, bacillus sp. shows ability to grow and produce enzymes at 37 oc under suitable fermentation conditions (sanghi et al. 2008; nagar et al. 2010). the finest temperature for microbial enzyme synthesis may fluctuate as it is linked to their proliferation. the organism used in this study showed relatively high xylanase production within a short period of incubation which could be attractive for large-scale xylanase production. xylanase activity was found to decrease with prolonged incubation which could be linked to depletion in the available nutrient as time progresses and accumulation of end-products (fig. 1). production of toxic metabolites inhibits enzyme synthesis also (irfan et al. 2016). apparently, the increased incubation period affected any xylanase yield but reduced enzyme activity. this appears to be similar to simphiwe et al. (2011) who reported 24-h as an utmost incubation period using digested bran as substrate. in another report (anuradha et al. 2007), xylanase synthesis by bacillus species was growth-related with highest production at 24-h incubation period. however, prakash et al. 2011 reported contrarily with maximum production after 48-h of fermentation with b. habdurans. the decline in xylanase production during the later stage of fermentation could be attributed to the liberation of a limited volume of protease from the mature cells penetrating into autolysis or shortage of impenetrable xylan particles in the medium. limited fermentation time presented better and gainful production which is a favourable condition in large-scale industrial production. optimal inoculum size is crucial for preserving balance between growth sizes and obtainable nutrients in order to obtain the utmost yield. the outcome of this study as illustrated in table 8 is parallel to irfan et al. (2012) where 1.5 % inoculum size as peak of yield using b. megaterium on corn cob but in variance with that of nagar et al. (2010) who tested higher inoculum size (1.0 – 5.0 %) for xylanase production. the reduction might be related to the depletion of available nutrients from the fermentation medium as inoculum level increases which could have resulted to decline in enzyme synthesis. conclusion the search for agricultural waste such as kolanut husk as potential source of enzyme production rather than environmental nuisance is desirable as it reduces cost of production and increases bioconversion of waste to value-added compounds 9 nova biotechnol chim (2022) 21(2): e1226 2 by economically feasible techniques. the results obtained therein showed that l. fusiformis can produce xylanase via kolanut husk as a substrate under submerged fermentation. results indicated that cultural and nutritional parameters are paramount to enzyme production. the optimization process is an important pre-requisite to be considered for a cost-effective production and since kolanut husk is an agro-waste, it could be an alternative to the expensive commercial xylan as substrate especially at large scale. acknowledgements authors cherish the efforts of the technical staff of the department of microbiology, federal university of technology, akure, (futa) nigeria for their technical support in this study. conflict of interest the authors declare that there is no conflict of interest. references aarti c, arasu mv, agastian p (2015) lignin degradation: a microbial approach. south indian j. biol. sci. 1: 119-127. adeyi o (2010) proximate composition of some agricultural wastes in nigeria and their potential use in activated carbon. j. appl.sci. environ. manag. 14: 1. annamalai n, thavasi, r, jayalakshmi s, balasubramanian t (2009) thermostable and alkaline tolerant xylanase production by bacillus subtilis isolated from marine environment. indian j. biotechnol. 8: 291-297. anuradha p, vijayalaxmi k, prasanna nd, sridevi k (2007) production and properties of alkaline xylanases from bacillus sp. isolated from sugarcane field. curr. sci. 90: 1283-1286. azeri c, tamer au, oskay m. 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invitski et al., 1999). the recent well published million-dollar food recalls due to food poisoning bacteria such as listeria monocytogenes (lin et al., 2005; donnelly, 2001; schlech, 2000) has increased the need for more rapid, sensitive and specific methods of detecting these microbial contaminants. the aim of this overview is to summarize the rapid and sensitive detection methods of pathogens in foods. here reviewed methods are focused particularly on detection of antigens (immunological methods), and on detection of species-specific dna sequences (molecular genetic methods). the highlights of these methods lie on their high specificity, rapidity and sensitivity purposes. 2. conventional detection methods traditionally, the detection and identification of bacteria mainly rely on specific microbiological and biochemical identification. although these methods can be sensitive, inexpensive and give both qualitative and quantitative information on the number and the nature of the microorganisms tested, they are greatly restricted by 6 vidová, b. et al. assay time, with initial enrichment needed to detect pathogens, which typically occur in low numbers in food and water (leonard et al., 2003). culture is the term used to describe the biological amplification of cultivatable bacteria using artificial growth media. these media may be liquid (broth), solid (due to the addition of agar, and used for their growth-supporting surface), enriched (with growth-promoting substrates) and selective (with inhibitors to reduce growth of unwanted species) or contain specific indicators to reveal growth of species of particular interest. bacterial growth also requires the right temperature and other physiochemical and chemical conditions. the bacteria have first to be recovered in pure culture. this requires growth of each, individual species in single colonies. the first identification tests performed include observation of bacterial colony morphology, alteration of the surrounding agar medium, the gram stain, termed benchtop tests such as coagulase, catalase and oxidase reactions and specific antibody agglutination reactions (chow et al., 2006). 3. immunological methods detection with antibodies is perhaps the only technique that has been successfully employed for the detection of cells, spores, viruses and toxins alike (iqbal et al., 2000). polyclonal antibodies can be raised quickly and cheaply and do not require the time or expertise associated with the production of monoclonal antibodies (cahill et al., 1995). however, polyclonal antibodies are limited both in terms of their specificity and abundance. since the development of hybridoma techniques (köhler and milstein, 1975) and the emergence of recombinant antibody phage display technology, developed during the past decade (petty et al., 2007), immunological detection of microbial contamination has become more sensitive, specific, reproducible and reliable with many commercial immunoassays available for the detection of a wide variety of microbes and their products (leonard et al., 2003). some of these methods are briefly listed below. 3.1 serotyping serotyping is most widely applied to gram-negative enteric bacterial pathogens such as salmonella and escherichia. among gram-positives, serotyping is important for the genus listeria. the gist of a typical serotyping scheme is the use of specific antibodies (antiserum) to identify homologous antigens (fig. 1). in many foodborne pathogens, the antigens are particulate, and agglutination methods are employed (jo et al., 2004; lukinmaa et al., 2004; bailey et al., 2002; gorman and adley, 2004). 3.2 fluorescent antibody (fa) this technique has had extensive use in both clinical and food microbiology since its development in 1942. an antibody to a given antigen is made fluorescent by coupling it to a fluorescent compound and when the antibody reacts with its antigen, nova biotechnologica 8-1 (2008) 7 the antigen-antibody complex emits fluorescence and can be detected by the use of fluorescent microscope (fig. 2). a b c fig. 1. group b streptococcus [gbs] serotyping test fy essum ab. (a) placing of reagent onto the agglutination site of the test plate. (b) picking some fresh colonies from the plate where the bacteria have been isolated and their transfer adjacent to the test plate. (c) gentle agitating the test plate and reading the agglutination pattern while agitating (processed according to persson et al., 2004) the fluorescent markers used are rhodamine b, fluorescein isocyanate, and fluorescein isothiocyanate, with the last being the most widely used. the fa technique obviates the necessity of pure culture isolations of salmonellae oh globulin labeled with fluorescein isothiocyanate with somatic groups a to z represented (hilker and solberg, 1973; thomason, 1971; insalata et al., 1973). because of the cross-reactivity of salmonellae antisera with other closely related organisms (e.g., arizona, citrobacter, e. coli), false positive results are to be expected when naturally 8 vidová, b. et al. contaminated foods are examined (cherry and moody, 1965). an indirect fluorescent antibody technique based on highly specific monoclonal antibody for streptococcus inae was developed and was found to be suitable for the detection and identification of s. inae from experimentally and naturally infected tilapia (klesius et al. 2006). fig. 2. fluorescent antibody identification of yersinia pestis. (photo: abdul ghaffar) 3.3 radioimmunoassay (ria) this technique consist of adding a radioactive label to an antigen, allowing the labeled antigen to react with its specific antibody, and measuring the amount of antigen that combined with the antibody by a counter to measure radioactivity. the label used by many workers is 125i. the ria technique lends itself to the examination of foods for other biological hazards such as endotoxins, paralytic shellfish toxins, etc. the detection and identification of bacterial cells within 8-10 minutes have been achieved (strange et al. 1971) by the use of 125i-labeled homologous antibody filtered and washed on a millipore membrane. because of its requirement for an isotope and its lack of portability, the ria method is rarely used now for food microbiology. 3.4 enzyme-linked immunosorbent assay (elisa) the elisa (enzyme immunoassay or eia) is an immunological method similar to ria but employing an enzyme coupled to antigen or an antibody rather than a nova biotechnologica 8-1 (2008) 9 radioactive isotope. a typical elisa is performed with a solid-phase (polystyrene) coated with antigen and incubated with antiserum. following incubation and washing, an enzyme-labeled reparation of anti-immunoglobulin is added (fig. 3a). after gentle washing, the enzyme remaining in the tube or microtiter well is assayed to determine the amount of specific antibodies in the initial serum. the amount of enzyme present is ascertained by the colorimetric determination of enzyme substrate. variation of this basic elisa consists of a “sandwich” elisa that allows detection and quantification of pathogen related antigen (fig. 3b) (valdivieso-garcia et al., 2001; gilroya and smith, 2003). in this case, the antibody specific for the antigen is coated to the surface of microtiter wells to which the antigen containing test sample is added. after unbound antigens are washed away, the antibody antigen complexes are than detected by an enzyme-linked antibody, which is specific for a different epitope on the antigen. next, unbound reporter antibodies are washed away, the substrate is added and the coloured reaction product is measured. the “double sandwich” elisa is a variation of the latter method, and it employs a third antibody. the elisa technique is used widely to detect and quantitate organisms and/or their products in foods (bennett, 2005). a b fig. 3 elisa assay (a) typical elisa assay (b) sandwich elisa. 3.5 immunomagnetic separation this method employs paramagnetic beads (about 2-3 μm in size, about 106108/ml) (fig. 4) that are surface activated and can be coated with antibody by incubating in the refrigerator for varying periods of time up to 24 hours. the unabsorbed antibody is removed by washing. when properly treated, the coated beads are added to food slurry that contains the homologous antigen (toxin or whole cells in gram-negative bacteria), thoroughly mixed, and allowed to incubate from a few minutes to several hours to allow for reaction of antigen with antibody-coated beads. the concentrated antigen is assayed by other methods. in one study, immunomagnetic 10 vidová, b. et al. separation was combined with flow cytometry for the detection of e. coli o157:h7. the antigens were labeled with fluorescent antibody, which was measured by flow cytometry, and the combined method could detect <103 cfu/g of pure culture or 103104 cfu/g in ground beef (fu et al., 2005; drysdale et al., 2004; tsai et al., 2006). this method was also used for measuring the number of l. monocytogenes cells (hibi et al., 2006). fig. 4 the aureon biosystems salmonella a-beads™: polydisperse 1.5 um cluster type paramagnetic particles with affinity-purified antibodies against salmonella spp. covalently bound to the surface. the salmonella a-beads™ will bind specifically to salmonella in a mixed flora sample and is designed for the rapid and specific isolation of salmonella from food and environmental samples (edited according photo by charles j. dicomo). 4. molecular genetic methods the progress made in nucleic acids techniques for diagnostics is enormous in relation to the period during which it has been applied to the development of assay systems as compared to that during which phenotypic in vitro analysis has developed. indeed, nucleic acid technology has now assumed an essential role in various areas of in vitro diagnosis. basically, this approach employs genetic materials (dna and rna) as diagnostic targets for identifying suspected pathogens. the rapid advancement of nucleic acid diagnostic is a consequent of two essential advantages: (1) nucleic acids can be rapidly and sensitively measured, and (2) the sequence of nucleotides in a given gene is highly specific and it can be used to distinguish closely related serotypes nova biotechnologica 8-1 (2008) 11 (kwang, 2006). the 16s rrna species-specific sequences are reliable tool for bacterial diagnosis. the importance of 16s rrna was shown at proposing the establishment of three kingdoms of life forms, namely eukaryotes, archaebacteria, and prokaryotes. the 70s bacterial ribosome contains three definable rrna fractions, 5s, 16s and 23s. the 5s contains ca. 120 nucleotides while 16s and 23s contain ca. 1.500 and ca. 3.000, respectively. the 16s rrna has been shown to be an excellent chronometer of life forms, especially bacteria. it was by the use of 16s rrna sequence and hybridization data that the class proteobacteria was established. by use of these sequences, new bacterial genera can be defined on a genotypic rather than a phenotypic basis. from this and other molecular genetic information, the genus pseudomonas has been reduced by the transfer of over 50 species to 10 new genera. the existence of many of gene banks for many other bacterial taxa of importance in foods suggests that generic and species realignments will continue (jay et al. 2005). besides, nucleic acids detection offers many advantages over immunological assays. nucleic acids are more stable than proteins to high temperatures, high ph, organic solvents, and other chemicals; hence samples can be treated in a relatively harsh manner without destroying the nucleic acid for detection. tools used for application of nucleic acid detection like dna primers and probes are more defined entities than antibodies and antigens, and their composition can be accurately checked by sequence analysis, and produced in dna synthesizers whenever necessary. the more commonly applied nucleic acid assay technologies in diagnostic include nucleic acid probes and polymerase chain reaction (kwang, 2006). 4.1 nucleic acid probes the fundament of the specific association of nucleic acid sequences by complementary base pairing through hydrogen bonding, which centralizes based on adenine complementarities to thymine and uridine, while guanine complementary to cytosine, has allowed the development of several nucleic acid diagnostic methods for detection. nucleic acid probes are short fragments of nucleic acid sequences that detect specific sequences associated with pathogen by nucleic acid hybridization with highly conserved sequences. if pathogen contains sequences complementary to the probe, the two sequences can hybridize to form a double-stranded molecule. the probe can be chemically synthesized according to the target gene sequence, which can be derived if a short amino acid sequence of the protein encoded by the target gene is known. during synthesis of the probe, labeled nucleotides are incorporated, to enable detection of the probe after hybridization. the types of labeled probes include radioactive (which gives a radioactive signal), biotin (which generate a color) and chemiluminescent (which is enzyme-linked). nucleic acid hybridization is often performed in the form of southern blotting that detects for dna and northern blotting that detects for rna. in both southern and western blotting, denatured dna and rna samples are separated by electrophoresis and immobilized onto a solid support system, usually nitrocellulose or nylon membrane, respectively, before detection with radioisotope-labeled or enzyme-labeled sequence-specific probe(s). hybridization is allowed to occur at an optimized 12 vidová, b. et al. temperature in which considerable sequence homology between the target sequence and the probe is necessary to form a stable duplex. following washing to remove unhybridized probe, the hybridization is detect according to how the probe was labeled (dooley, 1994). nucleic acid probes can be designed for identifying all serotypes or some interesting serotypes of a pathogen. however, when used alone probes may not be very sensitive. although, nucleic acid hybridization procedures can be combined with pcr due to provide a powerful tool, which exhibits remarkable specificity and sensitivity. it is especially useful when the target pathogen cannot be cultivated, or is difficult to cultivate. on the other hand, nucleic acid probes in diagnostics is designed for use in the food industry, such as for the detection of salmonella and staphylococcus employ probe dipsticks, which remove hybridized dna from liquid solution. for this method, a two-component probe is used, in which one serves to bind to the specific sequence of the pathogen, and the other serves to bind to the dipstick. following hybridization of the two-component probe to target sequence, the dipstick is inserted into the hybridization solution to remove the hybridized dna for measurement (jay et al., 2005). 4.2 polymerase chain reaction (pcr) this method is fast becoming the most widely used of all molecular genetic methods for detecting and identifying bacteria in foods. its increasing use is due to its high sensitivity, specificity, its availability in many formats, and the commercial availability of pcr-based methods in kit-like formats. the polymerase chain reaction diagnostic methods detect for specific nucleic acid sequence found in the genome of pathogens (chotár et al., 2006, fig. 5). fig. 5. determinations of specificity of primers in pcr assay; amplification products of the different primer combinations were analyzed by electrophoresis in 0.9 % agarose gel. lanes: 1–3 – dna s. agalactiae with primers saga1 and saga2 and sip3(f) and sip4(r) (1), ecoli1 and ecoli2 (2), sau1 and sau2 (3); 4–6 – dna e. coli with primers saga1 and saga2 (4), ecoli1 and ecoli2 (5), sau1 and sau2 (6); 7–9 – dna s. aureus with primers saga1 and saga2 (7), ecoli1 and ecoli2 (8), sau1 and sau2 (9); 10 – multiplex pcr with all 8 primers; m – 100-bp dna ladder (biolabs) (chotár et al., 2006, photo: barbora vidová). nova biotechnologica 8-1 (2008) 13 fig. 6. polymerase chain reaction procedure (drawn by michael gaines). it is based on dna replication in vitro to produce large quantities of a target sequence of pathogen isolated from a complex mixture of heterogeneous dna 14 vidová, b. et al. molecules (e.g. host genome, nucleic acid from other microbes present). the reaction is set up using the following reagents reaction buffer, dna template, deoxynucleotides (dntps), primers and thermo stable dna polymerase. a pair of primers is used for the reaction. the primers are single-stranded dna molecules of about 20-30 nucleotides long (oligonucleotides) designed from the sequence flanking the two ends of the target genome. these primers are specific to the pathogen to maximize the specificity of the assay. the amplification of dna by pcr is accomplished through cycling a succession of incubation steps of different temperatures optimized for dna replication (fig. 6). briefly, a sample containing mixture of dna molecules is heat-denatured to separate complementary strands of dna into single-stranded molecules for reaction. next, the reaction is brought down to a lower temperature to allow pathogen-specific primers to search and anneal to the complementary sequence on opposite strands of the target dna. this step is followed by dna extension at a higher temperature, with dna polymerase adding on dntps using the annealed dna sequence as a template to produce copies of the targeted sequence. the three steps make up one cycling reaction, with each cycle producing duplicates of the dna targets. the cycle of steps is then repeated, in which the newly synthesized dna strands can also serve as templates for primer extension. these steps are repeated 20-40 times, yielding exponential amplification of the target dna sequences. the amplified products can then be visualized on agarose gel, or further assayed by nucleic acid probing for increased sensitivity and specificity (snustad and simmons, 2000). pcr offers a major advantage of detecting pathogens in a single cell particle. this saves the hassle of isolating the pathogens in culture. pcr has distinct advantages over culture and other standard methods for the detection of microbial pathogens and offers the advantages of specificity, sensitivity, rapidity, accuracy and capacity to detect small amounts of target nucleic acid in a sample (toze, 1999). pcr have been used extensively for several years for identification and characterization of bacteria in food samples, including meat and dairy products (hill, 1996; wang et al., 1997; aslam et al., 2003; ercolini et al., 2004; alarcón et al., 2006; jensen et al., 1993; fach and popoff, 1997; tsen et al., 1998), viruses (schwab et al., 1996; dubois et al., 1997; traore et al., 1998), protozoa (rochelle et al., 1997; stinear et al., 1996) and helminths (geary, 1996; mathis et al., 1996) and multiple primers can be used to detect different pathogens in one multiplex reaction. alternatively, diagnostic methods like nucleic acid probes, restriction fragment length polymorphism, or sequence analysis can also be used to confirm the identity of the pcr product and to further characterize the genome (vogel et al., 2004). 4.3 real-time polymerase chain reaction real-time pcr, also known as kinetic pcr or quantitative real time pcr, has been increasingly employed in diagnostic microbiology, virology and parasitology. in the biotechnology, real-time pcr has been used to genotype identification of genetically modified organisms, monitor gene expression in bioprocesses and for evaluating the nova biotechnologica 8-1 (2008) 15 safety of biologicals. the importance and wide use of real-time pcr as a quantitative tool in the life sciences is due to the inherent wide dynamic range of quantification (107-109 fold), high assay sensitivity (detection of less than 10 copies), and high precision with no post-pcr manipulations, thus minimizing the risk of crosscontaminations. thus, in view of the high accuracy of measurement and reliability of real-time pcr, this technology will continue to be an essential tool for the quantification of gene expressions and genotyping. taqman real-time pcr pathogen detection assays amplify target nucleic acid sequences from select microbes present in samples collected from complex biological environments. specific amplification of target sequences is directed by custom designed primers and probes. the first degree of specificity is achieved by the combination of amplification primer sequences. an additional degree of specificity results from a probe that hybridizes to a region of nucleic acid sequence that identifies the microbe of interest. as pcr amplification proceeds, fluorescence is excited by laser or halogen light and is detected by a charge-coupled device (ccd) camera. because the probe does not inhibit the pcr reaction, these systems give a linear response to template concentration over at least five orders of magnitude. target sequences that confer specificity to pathogenic microorganisms are found within the portion of the microbe’s genome that encodes their virulent agents. the genes or a portion of the genes that contribute to the disease phenotype of the pathogen distinguish a target microbe from its nearest neighbors (johnson et al., 2005; oberst et al., 1998). fig. 7. ribotyping (drawn by michael gaines) 16 vidová, b. et al. 4.4 ribotyping dna is extracted from cells and digested with a restriction endonuclease such as ecori, and the fragments are separated by agarose gel electrophoresis. separated fragments are transferred to a nylon membrane and hybridized with an appropriately labeled copy dna (cdna) probe derived from ribosomal rna (rrna) by reverse transcriptase. the pattern radioactive / chemiluminescent) that is created is recorded. the automated device creates riboprints that are matched or compared to those of known strains stored on computer software (jay et al., 2005; grif et al., 2006) (fig. 7). in a study of salmonella serotype enteritidis, ribotyping was the most discriminating and accurate of the genetic methods used to distinguish among food, water, and pathogenic strains (landers et al., 1998). fig. 8. microarray assay. microarray is produced by selecting (1) and printing (2) probes in an array on s glass plate (microscope slide). the sample (target) is prepared, labelled (3) and then let to react with the microarray in a process called hybridization (4). after hybridization, the slides are washe with a weak salt solution (4) that removes unspecific binding of the target molecules (stringent washing). the target molecules that have bound to the probes on the array are detected (5) using fluorescent scanning or other scanning techniques. finally, quantified values of the “spots” are analysed to give a genotype. 4.5 microarrays a very simple microarray may consist of a solid surface (such as a nylon membrane, glass slide, or silicon chips) onto that are attached small quantities of a nova biotechnologica 8-1 (2008) 17 single-stranded dna (ssdna) from different known bacterial species (fig. 8). when ssdna from as many unknown species is exposed to these array (dna chip), complementary strains will bind to their respective sites on the chip. if a reporter molecule is used, the identity of the unknown species can be confirmed. a dna microarray is, in essence, a dot blot set-up with the capacity to obtain and process large amounts of data. a dna microarray for microorganisms begins with the construction of oligonucleotides (primers), probes, hybridization, and data analysis. the overall process has been presented and reviewed by ye et al. (2001). as currently used, several hundred to several thousand specimens or probes may be applied to a solid surface. in a study of xanthomonas pathovars, a 47-probe microarray was employed to fingerprint 14 closely related strains, and the fingerprints showed clear differences between the test strains (kingsley et al., 2002). a dna microarray has been developed for the detection of bacteria in the microbial consortium of ready-to-eat vegetable salads, with as few as 100 cfu being detectable in < 1 hour (rudi et al., 2002). 5. conclusion in the field of food protection, it is extremely important to be able to identify and to type pathogenic and spoilage microorganisms at an early stage. here reviewed methods based on immunochemical and nucleic acid technologies provide a very useful alternative against conventional microbiological laboratory methods, because conventional methods involve enriching the food sample and performing various media-based metabolic tests (agar plates or slants), which typically require 3–7 days to obtain a result. on the other hand rapid-screening tests based on immunological or nucleic acid technologies developed for food testing can provide results within hours. an important area for further investigation will lie in the expansion of whole genome sequence comparisons to related pathogens to identify those genes that confer detrimental properties, and to other microorganisms including closely related nonpathogenic species to ascertain evolutionary relationships and to identify novel virulence factors. the use of dna microarrays will facilitate whole genome comparisons among diverse strains and the identification of strainand lineagespecific sequences. specific (serotype) markers could be used to develop accurate identification and subtyping methods for use in both health institutions and the food industry. the combination of such pathogen specific markers with detection on dna microarrays might provide a more sensitive, rapid and informative detection of pathogens in food and food production sites than do classical culture methods. more rigorous and systematic assessment and development procedures are needed to realize the full potential of microarrays for use in industrial and clinical settings, however, because currently this technique is prohibitively expensive and requires highly specialized knowledge. efficient cooperation between academia and industry is required so that dna microarray technology and other novel detection methods can be tailored to the practical needs of the food industry and health institutions. there is also a need to understand better the ways in which pathogens can protect themselves against the 18 vidová, b. et al. conditions that are used in food preservation, because this might lead to the optimization of current foodprocessing strategies. complete genome sequences have already provided us with an unprecedented insight into the evolution and lifestyles of food-borne pathogens, including species that previously have not been studied so well. the availability of several microbial genome sequences has catalyzed a burst of genomics-driven fundamental research in the ecology, physiology and virulence of foodborne pathogens. the coming years will bring the first practical benefits to the field of microbial food safety, including strategies and tools for the detection, identification and control of these pathogens. acknowledgements: the work was supported by projects of the ministry of agriculture of slovak republic (2003 sp 27/028, oe 02/028, oe 02-01-03). we are very grateful to dr. abdul ghaffar (department of microbiology and immunology, university of south carolina, school of medicine, columbia 29208, usa) for permission to publish his photo in this paper as figure 2. also we are very grateful to dr. charles j. dicomo (aureon laboratories, inc., 28 wells ave. yonkers, ny 10701, usa) for permission to edit and publish his photo of salmonella a-beads™ assay as the figure 4. our gratitude belongs also to professor michael s. gaines (department of biology, university of miami, florida 33124-042, usa) for giving us permission to show his two pictures of pcr procedure (figure 6) and ribotyping (figure 7) in this paper. references alarcón, b., vicedo, b., aznar, r.:. pcr-based procedures for detection and quantification of staphylococcus aureus and their application in food. j. appl. microbiol., 100, 2006, 352-364. aslam, m., hogan, j., smith, k.l.: development of a pcr-based assay to detect shiga toxin-producing escherichia coli, listeria monocytogenes, and salmonella in milk. food microbiol., 20, 2003, 345-350. bailey, j.s., fedorka-cray, p.j., stern, n.j., craven, s.e., cox, n.a., cosby, d.e.: serotyping and ribotyping of salmonella using restriction enzyme pvuii. j food prot., 65, 2002, 1005-1007. bennett, r.w.: staphylococcal enterotoxin and its rapid identification in foods by enzyme-linked immunosorbent assay-based methodology. j food 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fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic dna analysis, ribotyping, and pcr-restriction fragment length polymorphism analysis. j. food prot., 67, 2004, 1656-1665. wang, r.f., cao, w.w., cerniglia, c.e.: a universal protocol for pcr detection of 13 species of foodborne pathogens in foods. j. appl. microbiol., 83, 1997, 727-736. ye, r.w., wang, t., bedzyk, l., croker, k.m.: applications of dna microarrays in microbial systems. j. microbiol. meth., 47, 2001, 257-272. nova biotechnol chim (2022) 21(2): e1264 doi: 10.36547/nbc.1264 1 nova biotechnologica et chimica antibacterial activity of medicinal plants against streptococcus agalactiae marina gisel novosak1,, daniana lilian winnik1, margarita ester laczeski1,2, marina inés quiroga1 1department of microbiology, faculty of exact chemical and natural sciences, national university of misiones, conicet, misiones, argentina 2molecular biotechnology laboratory, misiones institute of biotechnology, faculty of exact chemical and natural sciences, national university of misiones, conicet, "dra. maría ebe reca", misiones, argentina  corresponding author: marinanovosak2008@gmail.com article info article history: received: 15th november 2021 accepted: 1st april 2022 keywords: aqueous extract bacterial drug resistance ethanolic extract plants, medicinal streptococcus agalactiae abstract streptococcus agalactiae, group b streptococcus (gbs), infects and causes severe diseases in humans and numerous animal species, including fish, given its ability to cross the host-specific barrier. the emergence of antibiotic resistant gbs strains makes it necessary to look for alternatives to treat and prevent infections that it produces. the aim of the present study was to determine the antibacterial activity of ethanolic and aqueous extracts of medicinal plants from misiones province, northeast argentina, against gbs from humans and fish. we used human streptococcus agalactiae atcc® baa-611™ and tilapia streptococcus agalactiae atcc® 51487™ strains. minimum inhibitory dose (mid) was determined by the disc diffusion method. minimum inhibitory concentration (mic), minimum bactericidal concentration (mbc), mbc/mic ratio, drug synergism with commercial antibiotics, and resistance assays were determined with extracts that showed antibacterial activity. medium lethal dose 50 (ld50) was determined by the artemia salina assay. for ethanolic and aqueous eugenia uniflora l. extracts, we obtained a mid = 0.5 mg.disc-1. for both extracts of eugenia uniflora l., the mic and mbc values were 1 mg.ml-1 and 5 mg.ml-1, respectively. the mici (mbc/mic ratio) = 5 qualified the action of these extracts as bacteriostatic. the drug synergism assay with ampicillin, erythromycin, and clindamycin combination and extracts showed indifference. the ld50 of the aqueous extract was 0.82 mg.ml-1 indicating moderate toxicity. this work is a first step to identify chemical compounds in native medicinal plants of misiones, argentina, that could mean an alternative for the treatment of streptococcus agalactiae infections. introduction streptococcus agalactiae, group b streptococcus (gbs), is a commensal bacterium of human gastrointestinal and genitourinary tracts (tan et al. 2017). it is the primary cause of life-threatening infections in newborns and infants, such as sepsis, meningitis, and pneumonia. its transmission by gbs-colonized pregnant women is the main route of neonatal infestation (raabe 2019). infections by gbs in newborns decreased with the detection of this microorganism in pregnant women between 35 – 37 gestation weeks and the implementation of intrapartum antimicrobial prophylaxis. however, the rate of invasive disease in older adults and people with underlying chronic disease is increasing (sendi et al. 2016). mailto:marinanovosak2008@gmail.com nova biotechnol chim (2022) 21(2): e1264 2 streptococcus agalactiae infects a wide variety of animal species due to its ability to cross the hostspecific barrier. authors have shown that some human gbs strains come from fish and other aquatic animals (skov sorensen et al. 2019). since gbs is the leading cause of septicemia and meningoencephalitis outbreaks in freshwater fish, it is problematic in aquaculture with a significant economic impact on the fish industry (mian et al. 2009; tavares et al. 2019). streptococcus agalactiae is the most important pathogen of nile tilapia (niloticus oreochromis). the tilapia farming industry has recently been hampered by streptococcosis outbreaks associated with high fish densities. in brazil, it causes up to 90 % of deaths in tilapia farms (banrie 2012). similar problems are expected to manifest themselves in misiones province, argentina, where there are more than 4,000 aquaculture producers recorded through provincial development programs (ministry of agriculture, livestock, and fisheries 2020). several authors have identified virulence genes from human gbs strains in fish gbs strains (delannoy et al. 2013; liu et al. 2013) and suggested that colonization with gbs serotypes ia and ib in humans is associated with the consumption of infected fish (foxman et al. 2007). this shows that human and fish gbs strains are closely related. penicillin, a beta-lactam family antibiotic, is recommended as first-line therapy against gbs infections. macrolides (erythromycin) and lincosamides (clindamycin) are second-line antibiotics. these are usually prescribed for betalactams allergy patients (le doare et al. 2017). also, penicillin is commonly used in animal farming and aquaculture for prophylactic or treatment purposes (ibrahim et al. 2020). the number of human clinical gbs isolates with reduced penicillin susceptibility, and a multidrug resistance tendency increased between 2005 – 2006 and 2012 – 2013 in japan (seki et al. 2015). furthermore, a study in china described the appearance of gbs strains resistant to penicillin in human lesions and tilapia (nagano et al. 2019). the appearance of multidrug resistance and gbs not susceptible to penicillin inside and outside health centers is a concern (nagano et al. 2019). also, resistances to erythromycin (14.5 to 70 %) and clindamycin (8.2 to 70 %) have been reported worldwide (nagano et al. 2012). in misiones, 6 % resistance to erythromycin and 5 % resistance to clindamycin were reported in human gbs strains, respectively (novosak et al. 2020). to date, in argentina and brazil, isolates show susceptibility to ceftriaxone, penicillin, and vancomycin. however, researchers reported strains with reduced susceptibility to these antimicrobial agents in japan, the usa, the uk, and canada (bonofiglio et al. 2018; li et al. 2020). the streptococcosis treatment is performed with fish food administration supplemented with antibiotics. however, the intensive antibiotic use in fish farms could lead to resistant strains emergence (amal et al. 2011). in this context, it is necessary to search for alternatives for streptococcosis treatment. medicinal plants have been used to treat several human diseases worldwide for thousands of years. the world health organization (who) has recorded more than 20,000 species of medicinal plants with a variety of potential uses (silva et al. 2021). also, their products are used as alternatives to antibiotics and chemotherapy to prevent and control diseases in aquaculture (cheesman et al. 2017). medicinal plants are sources of nutrients and can be used in whole, in part or as an extract, alone or in combination, by aquatic route or food supplement, or even mixed with other bioactive compounds (doan et al. 2019). moreover, medicinal plants usually have stimulating and immunostimulating properties of fish growth (awad et al. 2017). these have a wide range of bioactive compounds that are generally cheaper, safer, and more accessible than their synthetic equivalents (cheesman et al. 2017), such as phenolic, polyphenolic, alkaloid, quinone, terpenoid, lectin, and polypeptide compounds (abuajah et al. 2015). at least 93 species of native medicinal plants belonging to 21 families have been described in misiones, including apocynaceae, asteraceae, lythraceae, and myrtaceae. previous studies (guida et al. 2003; jerke et al. 2008; bargardi et al. 2021) suggest that these possess metabolites able to inhibit the growth of human and animal pathogenic bacteria. however, the effect of many of these plants against gbs isolates has not been described so far. the aim of the present study was to determine the antibacterial activity of the ethanolic nova biotechnol chim (2022) 21(2): e1264 3 and aqueous extracts of medicinal plants from misiones province, northeast argentina, against gbs from humans and fish. experimental plants collection and identification the leaves of psidium guajava l., eugenia uniflora l., tabernaemontana catharinensis a.dc., baccharis crispa spreng., cecropia pachystachya trécul., and acanthospermum australe (loefl.) kuntze were collected in different misiones province locations. the taxonomic identification was carried out in the chair of pharmacobotany of the faculty of exact, chemical and natural sciences (fceqyn). extraction leaves were dried at room temperature for ten days, stirring periodically. then, dry leaves were crushed in a numak f 100 460 w blade mill (instrumentación científica s. a., buenos aires, argentina). the powder was sieved through a nominal mesh aperture of 1.4 mm with w.s. tyler™ o-tap sieve shaker rx-29 (wstyler, ohio, usa). extracts were obtained by digestion (argentine pharmacopoeia 2013) with water and 96 % hydroalcoholic solution (commercial alcohol) and concentrated with a rotary evaporator laborota 4000-efficient (heidolph instruments gmbh & co. kg, schwabach, germany). powder was stored in a dark and dry place. bacterial strains the bacterial strains used in the assays were streptococcus agalactiae lehmann and neumann (atcc® baa-611™, aatc, manassas, usa) (clinical specimen–human) and streptococcus agalactiae (atcc® 51487™, aatc, manassas, usa) (tilapia sp. brain, israel). minimum inhibitory dose (mid) assay minimum inhibitory dose (mid) was determined by the agar diffusion assay according to seyyednejad et al. (2014) with modifications. extracts effective doses were 0.5, 1, 5, 10, and 15 mg. extract solutions were prepared using dimethyl sulfoxide (dmso) as a solvent for ethanolic extracts and sterile distilled water for aqueous extracts. thirty microliters of these solutions were impregnated on filter paper discs. ampicillin discs (10 μg) (laboratorios britania s.a., buenos aires, argentina) were used as positive controls. discs impregnated with dmso were used as negative control for the ethanolic extracts, whereas discs impregnated with sterile distilled water were used as negative control for aqueous extracts. the bacterial suspension equivalent to mcfarland 0.5 was inoculated on müeller-hinton agar (mha) plates (laboratorios britania s.a., buenos aires, argentina) supplemented with 5 % sheep blood using a sterile swab. discs were placed on the surface of inoculated plates. plates were incubated at 35 – 37 °c for 24 h for the subsequent measurement of inhibition diameters (id). mid was considered the minimum quantity of the extract included in a paper disc able to show a visual inhibition of microbial growth. only extracts with antibacterial activity were used in assays that follow. the percentage of inhibitory effect was obtained by the following expression (eq. 1; martinez et al. 1996): minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) assays, and mbc / mic ratio mic and mbc assays were carried out following clinical and laboratory standards institute (clsi) recommendations (clsi 2015). the concentration range tested for each selected extract was from 20 to 0.0625 mg.ml-1. mic was defined as the lowest concentration without visible development. to determine mbc, mic cultures that did not show visible growth were seeded in mha with 5 % sheep blood and incubated for 24 h at 35 – 37 °c in a 10 % co2 atmosphere. mbc was defined as the lowest concentration of the extract capable of totally inhibiting microbial growth (procedures in clinical microbiology 2000). mic and mbc assays were performed in triplicate. when (1) nova biotechnol chim (2022) 21(2): e1264 2 mbc/mic ratio was ≤2 the effect of the active fraction was considered bactericidal, >2 and <16 bacteriostatic, and ≥16 ineffective (shanmughapriya et al. 2008). induction of resistance assay gbs strains were sub-cultured in sub-mic concentration for ten consecutive days to investigate their ability to develop drug resistance (su et al. 2015). tubes with mh broth and 0.5 mg.ml-1 of extract were prepared and stored in tubes at -20 ºc. a tube was used each day. on the first day of an assay, a tube was inoculated with 0.5 mcfarland of streptococcus agalactiae atcc® baa-611™ and incubated in an oven at 35 – 37 °c for 24 h. the next day, 50 µl from the first tube was inoculated into a second tube with the extract. in addition, each day the broth was plated on a nutritive agar plate supplemented with 5 % of sheep blood and incubated at 35 – 37 ºc for 24 h to evaluate possible contamination. the assay was repeated until the tenth day. mic was determined on test day 11. the same assay as in the streptococcus agalactiae atcc® baa-611™ was carried out with streptococcus agalactiae atcc® 51487™. assays were carried out in triplicate. drug synergism assay between extracts and commercial antibiotics it was performed on streptococcus agalactiae atcc® baa-611™ and streptococcus agalactiae atcc® 51487™ by the double-disc assay described by sachdeva et al. (2017) with modifications. commercial antibiotics used for the treatment of gbs were used: ampicillin (10 μg), erythromycin (15 μg), and clindamycin (2 μg) discs (laboratorios britania s.a., argentina). bacterial suspensions equivalent to mcfarland 0.5 were inoculated with a sterile swab on mha supplemented with 5 % sheep blood. a disc with 1 mg of eugenia uniflora l. extract was placed in the center of the plate. commercial antibiotic discs were placed on the sides 2 cm from center to center. the plates were incubated at 35 – 37 ºc for 24 h. the increased inhibition halo in the proximity of discs was interpreted as drug synergism. assays were carried out in triplicate. toxicity assay the artemia salina larvae assay was used to determine the extract toxicity (meyer et al. 1982). approximately 0.1 g of artemia salina cysts (aquagreen®, argentina) was added to one liter of a saline solution (containing 10 g of nacl per liter of distilled water). the container was kept at room temperature (28 – 30 °c), with air supply through a pump submersible pump bl-200 (baojie, zhejiang, china) and constant illumination. after 24 – 48 h the cysts hatched, and the larvae were taken in groups of ten to submit them to different extract concentrations in a 96-well plastic microplate. each well was filled with 200 μl of saline solution (containing 10 artemia salina larvae), 12.5 µl of eugenia uniflora l. aqueous extract dissolved in dmso and the final volume of 250 µl was completed with the same saline solution. concentrations tested were 0.0625, 0.125, 0.25, 0.5, 0.75, 1, 2 mg.ml-1. the microplate was incubated under illumination in a previously saturated glass container (humid atmosphere) at 28 – 30 °c and for 24 h. then, the number of surviving larvae in each well is counted with a stereoscopic magnifying glass nikon smz 445 (nikon corp. tokyo, japan). two controls were carried out: a growth control containing only the larvae in saline solution and a dmso control, which contained larvae, saline solution, and 12.5 µl of dmso without extract. the larvae death was established by the total lack of movement during 10 seconds of observation (vanhaecke et al. 1984). the lethality percentage in each well was calculated by the following equation (eq. 2): the lethal concentration 50 (lc50) was determined by graphic estimation, representing the lethality percentage of the larvae depending on the extract concentration. the lc50 value was obtained by the linear regression method using the software statgraphics centurion xvii (statgraphics technologies, inc., the plains, usa). (2) 4 nova biotechnol chim (2022) 21(2): e1264 3 results minimum inhibitory dose (mid) assay ethanolic and aqueous extracts of eugenia uniflora l. presented antibacterial activity against streptococcus agalactiae atcc® baa-611™ and streptococcus agalactiae atcc® 51487™ at the concentrations tested the mid was 0.5 mg.disc-1 for both extracts (table 1 and fig. 1). while psidium guajava l., tabernaemontana catharinensis a.dc., baccharis crispa spreng., cecropia pachystachya trécul. and acanthospermum australe (loefl.) kuntze did not show antibacterial activity. thus, following assays were conducted only with aqueous and ethanolic extracts of eugenia uniflora l. table 1. inhibition zone diameters and relative inhibitory effect percentages for ethanolic and aqueous eugenia uniflora l. extracts by agar diffusion assay against streptococcus agalactiae atcc® baa-611™ and streptococcus agalactiae atcc® 51487™. ee – ethanolic extract; ae – aqueous extract. fig. 1. minimal inhibitory dose (mid) – agar diffusion assay of aqueous and ethanolic extract of eugenia uniflora l. ae – aqueous extract; ee – ethanolic extract; c (+)– positive control; c (-) ae – negative control for the aqueous extract; c (-) ee – negative control for the ethanolic extract; mm – millimeter. eugenia uniflora l. extract concentra tions [mg] streptococcus agalactiae atcc® baa-611™ streptococcus agalactiae atcc® 514871™ ee ae ee ae inhibition zone diameters [mm] relative inhibitory effect [%] inhibition zone diameters [mm] relative inhibitor y effect [%] inhibition zone diameters [mm] relative inhibitor y effect [%] inhibition zone diameters [mm] relative inhibitory effect [%] 0.5 9.0 ± 0.0 28.12 9.0 ± 0.0 28.12 9.0 ± 0.0 28.12 9.0 ± 0.0 28.90 1 10.66 ± 0.57 33.33 11.0 ± 0.0 34.37 10.66 ± 0.57 33.33 11.33 ± 0.57 35.41 5 12.0 ± 0.0 37.5 11.66 ± 0.57 36.45 12.33 ± 0.57 38.54 12.33 ± 0.57 38.54 10 13.33 ± 0.57 41.66 13.33 ± 0.57 41.66 14.0 ± 0.0 43.75 13.66 ± 0.57 42.70 15 14.66 ± 0.57 45.83 14.0 ± 0.0 43.75 15.33 ± 0.57 47.91 15.0 ± 0.0 46.87 5 nova biotechnol chim (2022) 21(2): e1264 3 mic and mbc assays, and mbc / mic ratio mic and mbc obtained for ethanolic and aqueous eugenia uniflora l. extracts were 1 mg.ml-1 and 5 mg.ml-1, respectively. the mbc/mic ratio was 5 for both extracts. the action of extracts was bacteriostatic according to shanmughapriya et al. (2008) criteria. induction of resistance assay the mic value was 1 mg.ml-1 after gbs exposure to sub mic extract concentrations, suggesting that the microorganism would not develop resistance to the active principles of eugenia uniflora l. drug synergism assay between extracts and commercial antibiotics drug synergism trial showed indifference by the double-disc method. toxicity assay ld50 obtained for the aqueous extract was 0.82 mg.ml1, indicating moderate toxicity. the larval control group maintained 100 % viability and showed no behavioral changes during the test. discussion the results of current research revealed the antibacterial activity of eugenia uniflora l. against streptococcus agalactiae atcc® baa-611™ and streptococcus agalactiae atcc® 51487™. psidium guajava l., tabernaemontana catharinensis a.dc., baccharis crispa spreng., cecropia pachystachya trécul., and acanthospermum australe (loefl.) kuntze did not show any antibacterial activity at the different concentrations assayed. psidium guajava l. is used in the treatment of diarrhea, dysentery, menstrual disorders, vertigo, anorexia, digestive problems, gastric insufficiency, inflamed mucous membrane, laryngitis, skin problems, ulcers, vaginal discharge, and cough (díaz de cerio et al. 2017). silva et al. (2016) informed a low activity of aqueous and ethanolic extracts of psidium guajava l. against gbs but high on other bacterial species. sivananthan et al. (2013) mentioned that psidium guajava l. leaves extract showed antibacterial activity against staphylococcus aureus and streptococcus agalactiae. but the solvent used for the extraction procedure was chloroform. other authors informed the activity of ethanolic and aqueous psidium guajava l. extract against escherichia coli, pseudomonas aeruginosa, saphylococcus aureus, klebsiella pneumoniae y streptococcus pneumoniae (ifeanyichukwu et al. 2015; kenneth et al. 2017). the genus tabernaemontana has an important biological activity for the treatment and prevention of diseases, such as sore throat, hypertension, abdominal pain, and pulmonary disease (naidoo et al. 2021). richard et al. (2021) suggested that the crude latex of tabernaemontana catharinensis a. dc. displays an antimicrobial effect against alicyclobacillus, with potential for application in the food industry. goncalves et al. (2011) informed the in vitro antimicrobial activity of the tabernaemontana catharinensis a. dc. extract against staphyloccocus aureus and pseudomonas aeruginosa. however, no similar studies were found with gbs. baccharis crispa spreng. is used in infusion or decoction, as hepatic, diuretic, as a drying agent of ulcers and antiseptic in external use (rodriguez et al. 2008). palacios et al. (1983) informed antibacterial activity of bacharis crispa spreng. against bacillus subtilis and micrococcus luteus but no activity against staphylococcus aureus. no similar studies against gbs were reported. cecropia pachystachya trécul. is used in traditional medicine to treat respiratory disorders, renal diseases, and for its anti-inflammatory, diuretic, anti-hypertensive, and anti-diabetic properties, antidepressant-like, cardiotonic, sedative, and antimalarial effects (machado et al. 2021). de andrade et al. (2021) informed antibacterial activity of ethanolic extract of cecropia pachystachya trécul. against staphylococcus aureus atcc 29213, staphylococcus aureus atcc 25925, streptococcus pyogenes 1033b with halos that ranged from 12 mm to 18 mm. acanthospermum australe (loefl.) kuntze is used in folk medicine for the treatment of various 6 nova biotechnol chim (2022) 21(2): e1264 2 conditions such as diarrhea, skin diseases, blennorrhagia, dyspepsia, parasitic worms, and malaria. mallman et al. (2018) studied the efficacy of aqueous and hydroalcoholic extracts of acanthospermum australe (loefl.) kuntze against diarrhea-inducing bacteria. the hydroalcoholic root extract was unique in presenting a bactericidal effect against shigella dysenteriae. none of the extracts showed bacteriostatic or bactericidal activities against yersinia enterocolitica and enterococcus faecalis. similar works with extracts of cecropia pachystachya trécul. and acanthospermum australe (loefl.) kuntze against gbs strains were not reported. eugenia uniflora l. is often used in folk medicine as antidiarrheal, antihypertensive, antirheumatic, anti-inflammatory, respiratory disorders, digestive disorders, and numerous infections (de souza et al. 2018). several authors reported the activity of this plant against gram-positive and gram-negative bacteria. falcão et al. (2018) demonstrated activity against staphylococcus aureus atcc 25923, staphylococcus epidermidis incqs 00016, enterococcus faecalis atcc 29212, salmonella enteritidis incqs 00258, and pseudomonas aeruginosa atcc. however, the extract did not show activity against methicillin-resistant staphylococcus aureus and escherichia coli atcc 25922. also, victoria et al. (2012) obtained good activity of the essential oil of the leaves of eugenia uniflora l. against staphylococcus aureus and listeria monocytogenes. oliveira et al. (2008) worked with the lectin from eugenia uniflora l., obtaining antibacterial activity against staphylococcus aureus, pseudomonas aeruginosa, and klebsiella sp. although antibacterial activity has been reported for this plant, there are no published data against gbs so far. the antibacterial action of a plant extract is considered high when its percentage of relative inhibition is > 70 %, intermediate between 50 and 70 %, and low when it is 50 % (cruz-carrillo et al. 2010). for eugenia uniflora l. extracts, it was observed a relative inhibition percentage of less than 50 %, indicating low antibacterial activity. however, secondary metabolites with antibacterial activity are often found at low concentrations proportionally in the weight of dry extracts (compean et al. 2005). so, the extract may contain significant antimicrobial activity but in concentrations that are not high enough to inhibit bacterial growth considerably. according to the criteria of avellaneda et al. (2005) a bacterial strain has a high susceptibility when the tested substance has a mic less than 12.5 mg.ml-1, moderate susceptibility, between 12.5 and 50 mg.ml-1, and low susceptibility when the mic is between 50 and 100 mg.ml-1. during mic evaluation, the growth inhibition was obtained with both eugenia uniflora l. extracts to a lower concentration than 12.5 mg.ml-1. these results indicate high activity of eugenia uniflora l. extracts against the gbs strains studied. mic is considered the fundamental parameter for comparing the bacterium susceptibility against an antibacterial (struthers et al. 2005). it is the most reliable technique to determine substance antimicrobial properties. in brazil, lazzarottofigueiró et al. (2021) reported a mic value of 0.87 mg.ml-1 for the ethanolic extract of eugenia uniflora l. leaves when testing against staphylococcus aureus strains. they obtained higher mic values when testing with gramnegative bacilli (5 mg.ml-1 for escherichia coli, 20 mg.ml-1 for pseudomonas aeruginosa, and 10 mg.ml-1 for salmonella typhymurium). borges monteiro et al. (2019) obtained a mic value of 0.128 mg.ml-1, but they worked with methanolic extracts of eugenia uniflora l. leaves and h. pylori strains. in this study, mbc values were lower than 20 mg.ml-1 with both extracts, which indicates a high activity against gbs, according to the criteria of avellaneda et al. (2005). the mbc/mic ratio was 5 for both extracts, indicating the bacteriostatic extract capacity according to the criteria of shanmughapriya et al. (2008). medicinal plant extracts with intrinsic antimicrobial properties effectively prevent or reduce antimicrobial resistance. however, a combined approach that allows for a drug synergism between plant extracts and conventional antibiotics is possibly the most effective method of combating antibacterial resistance (inui et al. 2007; cheesman et al. 2017). in addition, the drug synergy between the bioactive plant product and antibiotics can prevent problems of toxicity or overdose, as two lower concentrations of agents that are combined in the treatment are required. in 7 nova biotechnol chim (2022) 21(2): e1264 3 this study, we did not observe drug synergy or inhibition effects between eugenia uniflora l. ethanolic and aqueous extracts and commercial antibiotics tested against gbs strains. coutinho et al. (2010) demonstrated a pharmacological synergism between the eugenia uniflora l. ethanolic extract and amikacin, gentamicin, kanamycin, neomycin, and tobramycin when testing against clinical isolates of staphylococcus aureus. however, the clinical isolates of escherichia coli did not show pharmacological synergism with this extract and antibiotics. the exposure of a bacterial population to the antimicrobial action usually produces a deleterious effect, either by inhibiting its growth or producing its death. this effect is not always observed due to the emergence of resistance mechanisms or the selection of resistant mutants (meyer et al. 1982). when determining mic after exposing gbs to submic concentrations of aqueous and ethanolic eugenia uniflora l. extracts for ten consecutive days, results revealed no change in value. therefore, gbs would not be expected to develop resistance to the active substance in the extract during treatment. it is also necessary to evaluate the toxicity of a product which can limit its benefit (santos et al. 2013). the toxicity criteria used were established by leos-rivas et al. (2016), where ld50> 1 mg.ml-1 represents low toxicity, ≥ 0.5 and ≤ 1 mg.ml-1 moderately toxic, and < 0.2 mg.ml-1 high toxicity. ld50 is defined as the substance concentration that causes 50 % of individuals to die in a study population. for ld50 determination, there are different methods using cell lines, laboratory animals, or artemia salina larvae. the latter is fast, economical, and simple. it does not require any special equipment or training and uses a relatively small test sample (leos-rivas et al. 2016). our research group have had previously determined that the toxicity of the ethanolic extract of eugenia uniflora l. is moderate [ld50 0.61 (0.51 – 0.74) mg.ml-1] (bobadilla et al. 2018). in this work, the ld50 value of the aqueous extract of eugenia uniflora l. was 0.82 mg.ml-1 (0.76 – 0.90 mg.ml-1), indicating also moderate toxicity. arcanjo et al. (2012) obtained an ld50 value of 288.46 µg.ml-1 (194.24 – 433.67 µg.ml-1) for the ethanolic extract of eugenia uniflora l. in their tests with artemia salina. however, toxicity studies carried out with this plant present wide methodological variations, making it difficult to compare the observed biological effects. the emergence of a gbs clone with zoonotic potential produced a sepsis outbreak in humans through raw fish consumption in singapore (kalimuddin et al. 2017). the application of antibiotics and chemotherapeutics to control infectious diseases in aquaculture eradicates microflora and emerges the resistant bacteria and accumulates residues in the human body (who 2006). challenges in treating infectious diseases in humans and fish have increased due to the resistance emergence. so, it is necessary to search for alternatives for antibacterial therapy development. natural products are a good option as they are less toxic and have fewer adverse effects than synthetic products. so, the present study expands the knowledge of natural antibacterial options existing in the region. conclusion aqueous and ethanolic extracts of eugenia uniflora l. have antibacterial activity against gbs of human and fish origin. these do not exhibit drug synergism with commercial antibiotics, the intratreatment resistance would not develop, and have moderate toxicity. aqueous and ethanolic extracts from psidium guajava l., tabernaemontana catharinensis a.dc., baccharis crispa spreng., cecropia pachystachya trécul. and acanthospermum australe (loefl.) kuntze did not show antibacterial activity against gbs. more studies are required with other solvents for secondary metabolite extraction from these plant species. this work is a first step to identify chemical compounds in native medicinal plants of misiones, argentina, that could mean an alternative for the treatment of streptococcus agalactiae infections. acknowledgments we thank marta yajía for identifying the plant species studied. this research was funded by pio conicet–unam nº. 237-201601-00007. 8 nova biotechnol chim (2022) 21(2): e1264 2 conflict of interest the authors declare that they have no conflict of interest. references abuajah ci, ogbonna ac, osuji cm (2015) functional components and medicinal properties of food: a review. j. food sci. technol. 52: 2522-2529. amal mna, zamri-saad m (2011) streptococcosis in tilapia (oreochromis niloticus): a review. pertanika j. trop. agric. sci., 34: 195-206. arcanjo ddr, albuquerque acm, melo-neto b, santana lclr, medeiros mgf, citó amgl (2012) bioactivity evaluation against artemia salina leach of medicinal plants used in brazilian northeastern folk medicine. braz. j. biol. 72: 505-509. argentine pharmacopoeia (2013) ministry of health of the nation. 7th eds., vol. iv, buenos aires, argentina. avellaneda ss, rojas hn, cuéllar ca, fonseca juárez m (2005) antibacterial activity of diphysa minutifolia rose. rev. cubana plant med. 10: 1-10. awad e, awaad a (2012) role of medicinal plants on growth performance and immune status in fish. fish shellfish immunol. 67: 40-54. banrie (2012) impact of streptococcosis on tilapia in brazil, aquavac strep sa for management. https://thefishsite.com/articles/impact-of-streptococcosison-tilapia-in-brazil-aquavac-strep-sa-for-management. bargardi s, kramer l, medvedeff m, jordá g, guida a (2001) antibacterial activity of peschiera australis (müell) miers against staphylococcus aureus and bacillus subtilis. rev. cienc. tecnol. 4. bobadilla fj, novosak mg, winnik dl, kachuk av, laczeski me, quiroga mi (2018) antibacterial activity, and toxicity of the ethanolic extract of eugenia uniflora l. leaves on pseudomonas aeruginosa. j. microbiol. biotechnol. food sci. 8: 842-846. bonofiglio l, gagetti p, garcía gabarrot g, kaufman s, mollerach m (2018) susceptibility to β-lactams in βhemolytic streptococci. rev. argent. microbiol. 50: 431435. cheesman mj., ilanko a, blonk b, cock ie (2017) developing new antimicrobial therapies: are synergistic combinations of plant extracts/compounds with conventional antibiotics the solution? 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jacob rg, alves d (2012) essential oil of the leaves of eugenia uniflora l.: antioxidant and antimicrobial properties. food chem. toxicol. 50: 2668-2674. who (2006) report of a joint fao/oie/who expert consultation on antimicrobial use in aquaculture and antimicrobial resistance, seoul, republic of korea. https://apps.who.int/iris/handle/10665/133869. 11 https://apps.who.int/iris/handle/10665/133869 microsoft word janos, lesny nb.doc nova biotechnologica vii-i (2007) 85 czech and slovak young brown coals in environmental applications pavel janoš1, juraj lesný2, lucia závodská3, sylvie kříženecká1, lucie herzogová1 1faculty of the environment, university of jan evangelista purkyně, králova výšina 3132/7, ústí nad labem, cz-400 60, czech republic (pavel.janos@ujep.cz) 2faculty of natural sciences, university of ss. cyril and methodius, nám. j. herdu 2, trnava, sk-917 01, slovak republic (lesnyj@ucm.sk) 2department of projecting&nuclear safety, ekosur, jaslovské bohumice, sk-919 31 slovak republic (zavodska@ekosur.sk) abstract: in recent time, various kinds of young brown (low-rank) coals are utilized increasingly not only as fuels, but also as valuable materials in advanced environmental applications. it should be noted in this context that significant deposits of the young brown coals can be found both in the czech republic as well as in slovakia. for their effective applications e.g. in wastewater treatment or in soil remediation, the properties of the coals should be studied in more details and numerous physico-chemical characteristics should be measured. as a part of a common czech-slovak project, a series of various kinds of coals was collected, including north-bohemian oxihumolites, lignite from south moravia and several lignites from slovakia (baňa zahorie). basic properties were measured, such as acid-base titration curves, contents of main functional groups and sorption capability towards heavy metal cations (cu2+, zn2+, co2+) and some organic pollutants. the contents of humic substances – main active constituents of the young coals – were also determined. selected environmental applications of the brown coals will be mentioned further, e.g. for the removal of metal cations from waters or in phytoremediation of contaminated soils. key words: lignite, oxihumolite, humic acids, metal, sorption 1. introduction young brown coals have been frequently used to improve the soil quality for a long time. large deposits of various kinds of young brown coals can be found in the czech republic (north bohemia, south moravia) as well as in slovakia. young brown coals contain large amounts of humic substances and may serve as an exogenous source of organic soil-constituents. due to their complex-forming ability, humic acids play a crucial role in the mobility of nutrients, essential elements and heavy metals in the environment and in the bioavailability of these elements to plants. a special kind of weathered and oxidatively altered lignite from the north-bohemian coal basin is called “oxihumolite” (veselá et al., 2005). it occurs at the surfaces of lignite deposits and is characterized by a high content of humic substances, unique sorption properties and high biological activity. similar kinds of low-rank coals from other locations are called leonardites. because of their sorption ability towards various kinds of chemical species, they are increasingly used also in other environmental applications, such as in wastewater treatment for the removal of metal cations (lao et al., 2005; janoš et al., 2007b; mizera et al., 2007), hexavalent chromium (lakatoš et al., 2002), ionogenic organic substances, such as synthetic dyes 86 janoš, p. et al. (janoš et al., 2005; janoš et al., 2007a), or low-polarity organic compound, e.g. phenanthrene (kalaitzidis et al., 2006). to exploit successfully the unique properties of various kinds of young brown coals, numerous physico-chemical characteristics should be measured and examined in more details. the following properties of the young brown coals are measured as a part of the common czech-slovak project: • contents and elemental composition of the organic part of coals, which is important for the plant nutrition as well as for the interaction of coals with some organic pollutants (kalaitzidis et al., 2006). • contents, elemental and phase composition of the inorganic part of coals – identification of the clay minerals, determination of the contents of heavy and toxic metals often present in coals, which may limit some environmental applications. • contents of total and extractable humic substances, as these compounds play a crucial role in soil fertility, biological activity as well as in the mobility of pollutants. • acid-base properties and contents of acidic functional groups, as these groups govern the mobility and bioavailability of various species in soils, metal cations in the first place. • radiochemical properties of coals also important for some environmental applications. • surface properties (surface area, pore-size distribution, etc.) that are significant for those applications where the sorption ability of the coals is exploited. some preliminary results of the mentioned project are presented in this contribution. 2. materials and methods various kinds of brown coals were collected from the selected deposits in the czech republic and slovakia. for the sake of comparison, also leonardite from gascoyne (usa) was involved in the study; it is recommended as a standard/reference material by the international humic substances society. the samples were air-dried in laboratory and then used for the measurements without an additional pre-treatment except of grinding and a size classification by sieving, if not specified otherwise in the respective measurement procedure. typically, the fraction with grain sizes less than 2 mm was used for laboratory experiments. the coals used in this study are listed in table 1. the total contents of humic acids and the contents of extractable (free) humic acids in coals were determined according to the standard procedure stn iso 5073. for the investigations of the acid-base properties of the coals and measurements of titration curves, the classical batch method of topp and pepper (topp and pepper, 1949) was adopted. the original method was modified as follows (janoš et al., in press): constants amounts (typically 0.25 g) of the coal were weighted into the series of 100 nova biotechnologica vii-i (2007) 87 ml pe bottles. different amounts the naoh solution were added to each of the bottles and mixed with different amounts of the nacl solution to keep a desired constant ionic strength after adjusting to the constant volume of 50 ml. the solutions were purged with nitrogen for several minutes, the pe bottles containing the samples under a nitrogen atmosphere were closed and equilibrated for 72 hours with intermittent shaking. then the equilibrium ph values were measured using a combined glass electrode. table 1. young-brown coals used in the present study name of the sample kind of coal deposit/sampling site ox-vršany oxihumolite – naturally weathered young brown coal mining site vršany, north-bohemian coal basin, czech republic ox-jiří oxihumolite – naturally weathered young brown coal mining site jiří near sokolov, northbohemian coal basin, czech republic ox-václav oxihumolite – naturally weathered young brown coal mining site václav near duchcov, north-bohemian coal basin, czech republic li-mikulčice lignite mining site mikulčice, south moravia, czech republic leonardit leonardite naturally weathered young brown coal gascoyne, usa li-zahorie i lignite mining site zahorie, slovakia li-zahorie ii lignite mining site zahorie, slovakia li-zahorie iii lignite mining site zahorie, slovakia li-zahorie iv lignite mining site zahorie, slovakia equilibrium sorption experiments (sorption of metal cations) were carried out by shaking a known amount of the coal (typically 0.4 g) with 50 ml of solution of the respective metal with desired concentration (typically ranging from 0.1 to 4 mmol dm3) in pe bottles. the metal solution together with the sorbent was agitated in the closed pe bottle for 72 hours using a horizontal shaker with an intensity of agitation 2 rps. then the solid phase was separated by sedimentation/centrifugation, the concentration of the metal in the solution was determined immediately by ion chromatography or icp-oes, and the sorbed amounts were calculated. 3. results and discussion as mentioned above, humic acids (ha) represent the main active constituents of young brown coals and the content of ha is a decisive factor in potential environmental applications of the coals. various kinds of brown coals may differ 88 janoš, p. et al. significantly in the contents of ha, as can be seen from fig. 1, where the total contents of ha as well as the contents of free ha are compared. as can be seen, all the examined coals contain significant amounts of ha and extremely high contents of ha was found in oxihumolite václav. functional groups in ha, mainly carboxylic and phenolic groups, govern the acid-base properties of coals. acid-base titration curves of selected coals were measured using a discontinuous batch method. according to ritchie and perdue (ritchie and perdue, 2003), the content of carboxylic groups was calculated from the base consumption for titration up to ph 8, whereas the phenolic group content was calculated from the two-fold consumption for the titration between ph 8 and 10. examples of the determinations are listed in table 2. it seems that the contents of functional groups may be related to the contents of ha (see a high content of carboxylic groups in oxihumolite václav), but preliminary results do not allow to make definite conclusions. table 2. contents of acidic functional groups in chosen samples of young brown coals (mmol g-1) name of the sample carboxylic groups phenolic groups ox-jiří 1.20 1.20 ox-václav 3.90 2.20 li-mikulčice 1.45 2.00 li-zahorie i 1.50 3.00 li-zahorie ii 1.55 2.30 0 20 40 60 80 100 ox -v rša ny ox -ji ří ox -v ác lav limi ku lči ce le on ar dit liza ho rie i liza ho rie ii liza ho rie iii liza ho rie iv c on te nt o f h a (% ) total ha free ha fig. 1. contents of humic acids in coals nova biotechnologica vii-i (2007) 89 the sorption capabilities of the czech and slovak brown coals were studied extensively, focusing on their ability to retain heavy metal cations, in the first place. the sorption isotherms were measured for several metal cations and evaluated using various models (langmuir, langmuir-freundlich) – details and model parameters can be found elsewhere (janoš et al., 2007 b). for direct comparison of an ability of different coals to retain metal cations from aqueous solutions, a parameter called “nominal removal efficiency” was introduced in this work. it is defined as an efficiency of the metal removal (in %) under given conditions. these conditions are specified as follows: weight of the sorbent (coal) 1g, volume of the metal solution 50 ml, initial metal concentration 1 mmol dm-3, contact time 72 hours. the removal efficiency was measured for all the above listed coals and selected metal cations cu2+, zn2+, co2+. as can be seen from fig. 2, most of the examined coals exhibited a very high ability to retain metal cations and could be potentially used for the removal of heavy metals from wastewaters or other industrial effluents. 0 50 100 ox -v rša ny ox -ji ří (s ok olo v) ox -v ác lav (d uc hc ov ) limi ku lči ce le on ar dit g as co yn e liza ho rie i liza ho rie ii liza ho rie iii liza ho rie iv r em ov al e ff ic ie nc y (% ) cu zn co fig. 2. nominal removal efficiency for cu2+, zn2+, co2+ ions. (conditions: weight of coal 1g, volume of metal solution 50 ml, initial metal concentration 1 mmol dm-3, contact time 72 hours) however, the sorption ability also affects the mobility of metals and the metal bioavailability, when the coals are used in soil amendments. further experiments consisted in an addition of coals and other ha-containing materials to soils contaminated with heavy metals. consequently, the mobility of heavy metal in the soils was examined using a standardized extraction test (a sequential bcr test) 90 janoš, p. et al. (janoš et al., 2004). preliminary results showed that an addition of ha affected significantly a fractionation of heavy metals in soils. 4. conclusions the total – end free humic acid contents, the content of carboxylic – and phenolic groups as well as by the authors newly introduced parameter, the “nominal removal efficiency” towards cu2+, zn2+ and co2+ have been determined for selected oxihumolite and lignite samples from sampling sites/deposits in czech republic (north bohemia, south moravia) and slovakia (mining site zahorie). a comparison of relevant characteristics for the mentioned samples and for a well known leonardit sample (mining site gascoyne, usa) has been done. the results confirm the correlation between the functional group concentration and the relevant concentration of humic acids. results of the “nominal removal efficiency” towards cu2+, zn2+ and co2+ show a generally high ability to retain metal cations for all the examined samples with exception of the naturally weathered young brown coal from mining site jiří (near sokolov). references janoš, p., herzogová, l., rejnek, j., hodslavská, j.: assessment of heavy metals leachability from metallo-organic sorbent-iron humate-with the aid of sequential extraction test. talanta, 62, 2004, 497-501. janoš, p., kříženecká, s, madronová, l.: acid-base titration curves of solid humic acids. react. funct. polymers, (in press). janoš, p., michálek, p., turek, l.: sorption of ionic dyes onto untreated lowrank coal – oxihumolite: a kinetic study. dyes pigm., 74, 2007a, 363-370. janoš, p., sypecká, j., mlčkovská, p., kuráň, p., pilařová, v.: removal of metal ions from aqueous solutions by sorption onto untreated low-rank coal (oxihumolite). separ. purif. technol., 53, 2007b, 322-329. janoš, p., šedivý, p., rýznarová, m., grötschelová, s. : sorption of basic and acid dyes from aqueous solutions onto oxihumolite. chemosphere, 59, 2005, 881-886. kalaitzidis, s., karapanagioti, h.k., christianis, k., bouzinos, a., iliopoulou, e.: evaluation of peat and lignite phenanthrene sorption properties in relation to coal petrography: the impact of inertinite. inter. j. coal geol., 68, 2006, 30-38. lakatos, j., brown, s.d., snape, c.e.: coals as sorbents for the removal and reduction of hexavalent chromium from aqueous waste streams. fuel, 81, 2002, 691-698. lao, c., zoldán, z., gamisans, x., solé, m. : sorption of cd(ii) and pb(ii) from aqueous solutions by a low-rank coal (leonardite). separ. purif. technol., 45, 2005, 79-85. mizera, j., mizerová, g., machovič, v., borecká, l.: sorption of cesium, cobalt and europium on low-rank coal and chitosan. water res., 41, 2007, 620626. nova biotechnologica vii-i (2007) 91 ritchie, j.d., perdue, e.m.: proton-binding study of standard and reference fulvic acids, humic acids, and natural organic matter. geochim, cosmochim. acta, 67, 2003, 85-96. topp, n.e., pepper, k.w.: properties of ion exchange resins in relation to their structure. i. titration curves. j. chem. soc., 1949, 3299-3303. veselá, l., kubal, m., kozler, j., innemanová, p.: struktura a vlastnosti přírodních huminových látek typu oxihumolitu. chem. listy, 99, 2005, 711-717. nova biotechnol chim (2022) 21(1): e1175 doi: 10.36547/nbc.1175 1 nova biotechnologica et chimica effects of ultrasonic-assisted alkaline pretreatment on xylose production from pineapple peel waste choosit hongkulsup, panchalee pathanibul department of food science and technology, faculty of science and technology, suan sunandha rajabhat university, bangkok, thailand  corresponding author: choosit.ho@ssru.ac.th article info article history: received: 16th september 2021 accepted: 1st december 2021 keywords: aeromonas acid hydrolysis pineapple peel ultrasonic-assisted alkaline pretreatment xylose abstract the pineapple industry generates large amounts of unusable waste (peel and core) with adverse environmental impacts. this experimental study aims to systemize the potential of ultrasonic-assisted alkaline pretreatment for xylose production from pineapple peel waste. the best condition for single alkaline pretreatment (1 % naoh w/v, 100 c, 60 min) has obtained hemicellulose, cellulose, and lignin composition at 34.80 %, 32.16 %, and 8.66 %, respectively, retained in the biomass. meanwhile, a combination of alkaline (1 % naoh, w/v) and ultrasonic (frequency 40 khz, 45 min) pretreatment has obtained the percentage yield of hemicellulose and lignin at 51.15 % and 7.15 %, respectively. both single alkaline and ultrasonicassisted alkaline pretreated samples were subsequently hydrolyzed with 2 % h2so4 (w/v). after acid hydrolysis for 30 min, the maximum xylose concentration of 48.85 g.l-1 was achieved by using ultrasonic-assisted alkaline pretreatment, while single alkaline pretreatment contributed to the lowest yield of xylose (37.11 g.l-1). it is shown that the ultrasonic-assisted alkaline treatment is more favorable than single alkaline pretreatment as it can produce high xylose concentration after the subsequent hydrolysis. these results indicated that ultrasonic-assisted alkaline pretreatment and its subsequent acid hydrolysis were appropriate for producing xylose from pineapple peel waste. introduction smooth cayenne pineapple (ananas comosus l. merr.) is one of the most valuable and popular tropical fruits in the world. as stated by the ministry of agriculture and cooperatives of thailand, thailand is ranked the first in the production and export of pineapple, with approximately 50 % of the international market share (wattanakul et al. 2020). in thailand, different pineapple products are exported to the global market, including fresh, frozen, canned, and juice products. however, according to the agricultural informatics center (2018), the demand and supply of pineapple products are always fluctuated. as a result, production of pineapples is higher than domestic consumption and export demand, resulting in an oversupply of pineapple in thailand. this consequently leads to a decline in the price of pineapple products. the manufacturing of canned pineapple or pineapple juice usually generates large amounts of pineapple waste including 29 – 40 % (w/w) of peel, 9 – 10 % (w/w) of core, and 2 – 5 % (w/w) of stem, which is mainly used as a supplement in animal feed or discarded in landfills at a very low price (ketnawa mailto:choosit.ho@ssru.ac.thsk nova biotechnol chim (2022) 21(1): e1175 2 et al. 2012). in recent years, thailand’s manufacturing process of pineapple products generated approximately 1.62 million tons of pineapple residues (ritthisorn 2016; banerjee et al. 2019). the greater expansion of pineapple production and export will subsequently generate more pineapple waste. however, pineapple peel wastes contain a major source of lignocellulosic materials. the chemical composition of pineapple residues contains approximately 38% to 50 % of cellulose, 23 % to 32 % of hemicellulose, and 13% to 30 % of lignin (sierra et al. 2008). therefore, hemi-cellulosic resources, including agricultural and industrial residues, offer a suitable alternative for the extraction of high-value renewable products (kunaver et al. 2016). lignocellulosic waste has become an alternative natural resource for the extraction of a valuable sugar, xylose. the interesting challenges for sustainability in industrial chemistry and biotechnology have encouraged the development of renewable agricultural residues. apparently, the conversion of agricultural wastes to alternative raw materials requires additional techniques to develop economic and effective procedures (binder and raines 2010). the typical conversion of lignocellulosic residue to reducing sugars consists of two main steps: (1) pretreatment and (2) hydrolysis. the removal and depolymerization of unwanted components and other impurities found in pineapple peel waste by various pretreatments have been widely studied. the valuable sugar can be extracted by different hydrolysis methods using chemicals and enzymes (chongkhong and tongurai 2017). the application of ultrasonication in solid-liquid extraction processes is considered as mechanical energy by particles vibrating or moving within a medium. theoretically, the formation of microbubbles during sonication treatments allows pretreated substrates to be more readily hydrolyzed by increasing accessible surface area, decreasing particle size and crystallinity (yunus et al. 2010). some studies reported that pretreatment of lignocellulosic materials with ultrasonic irradiation at working frequency higher than 20 khz could be advised for the intensification of bioconversion with different physical and chemical effects (asakura et al. 2008). the utilization of sonication for the improvement of agricultural by-product seems to be a promising technology that can be readily applied for the extraction of value-added sugar from pineapple waste (ong et al. 2019). however, the excessively long reaction time and the complicated procedure for enzymatic hydrolysis as well as the higher costs compared to chemical method have limited process applicability on an industrial scale (niwaswong et al. 2014). in addition, the direct usage of lignocellulosic residue from the pineapple industry as a hemicellulose extract for xylose production has been limited. agriculturalists and local enterprises play an important role in the utilization of agricultural residues. nevertheless, they have limited professional knowledge and lack the ability to use novel technology for converting agricultural waste into value-added products. consequently, the government should provide a policy environment and adequate financial support that encourages the sustainable development of agricultural waste utilization (xia and ruan 2020). the improvement of pretreatment processes to produce xylose and its derivatives is therefore necessary. the effect of ultrasonication on the pineapple peel prior to acid hydrolysis was evaluated. this experimental study aims to demonstrate the potential of ultrasoundassisted alkaline pretreatment and acid hydrolysis for xylose extraction from pineapple peel waste in a systematic way. experimental fresh pineapple peels were collected from the fruit market in prachuap khiri khan province, thailand. firstly, the pineapple peels were washed with tap water to remove foreign materials and left under the sun to dry. washed and dried peels were cut into small pieces and further dried at 60 °c in a hot-air oven until the moisture content is less than 10 % of wet basis. after that, the dried peels were ground and sieved to obtain particle sizes of less than 2 mm. the ground dried peels were then packed in sealed plastic bags and stored at room temperature before use. the smaller particle size is expected to increase the accessible surface area, improving the efficiency of ultrasonic treatment (zheng et al. 2009). proximate analysis of pineapple peel wastes was evaluated in accordance with the standard aoac (2012). nova biotechnol chim (2022) 21(1): e1175 3 chemical pretreatment dried pineapple peel waste (20 g) was treated under different solvent conditions: water; 1 %, 2 %, and 4 % h2so4 (w/v); 1 %, 2 %, and 4 % naoh (w/v); a solid-to-liquid ratio of 1 : 10 (w/v) and stirred at ambient temperature for 15 min. the chemical pretreated sample was heated at 100 °c and atmospheric pressure for 60 min in a sealed 1 l glass beaker placed in a water bath with temperature controller (sukruansuwan and napathorn 2018). it was then neutralized and filtered by water washing to remove a solvent on the surface of the sample. the neutralized pineapple peel was dried overnight at 60 °c in a hot air oven. the dried sample was then ground to a mesh size of 0.75 – 1 mm by a laboratory mill machine (ct 293 cyclotectm, foss, hillerød, denmark). the chemical composition of pretreated samples was analyzed for hemicellulose, cellulose, and lignin following the detergent fiber analysis according to van soest et al. (1991). the best conditions for chemical pretreatment of the pineapple peel obtained were used for the next experiment. ultrasonic pretreatment the ultrasonic pretreatment procedure of pineapple peels was performed with 1 % w/v naoh (the pretreatment condition obtained from the previous section) in a solid-to-liquid ratio of 1 : 10 (w/v). the mixture samples then underwent sonication using 4 different ultrasonication times (15, 30, 45, and 60 min) under the following conditions: temperature 50 °c; working frequency 40 khz; and ultrasonic power 200 w (lt-200-pro, tierratech®, guarnizo, spain). the control sample was unsonicated (0 min) but was added to 1 % naoh (w/v), incubated at 50 °c and stirred for 60 min. after ultrasonication, the pretreated samples were washed using distilled water and filtered using a filter paper. the solid residue was then dried at temperature of 60 °c overnight in a hot air oven until a constant weight was reached. the ultrasonic pretreated samples were kept in vacuum packaging before subsequent chemical hydrolysis (koutsianitis et al. 2015). acid hydrolysis pretreated pineapple peel (10 g) was completely mixed with 2 % h2so4 (w/v) in a solid-to-liquid ratio of 1 : 10 (w/v). the acid hydrolysis treatments were conducted at 100 °c and atmospheric pressure for 30 min in a sealed 1 l glass beaker placed in a water bath with temperature controller. after hydrolysis, the hydrolysates were neutralized by 6 m naoh and filtered using a 0.22 µm membrane filter. the filtrates (hemicellulosic fraction) were then kept in vials prior to xylose analysis by hplc (yunus et al. 2010). characterization of pineapple peel the detergent fiber method (van soest et al. 1991) for the analysis of fiber-rich feeds, and currently most frequently applied for fiber analysis in forage, provides a more suitable alternative to better characterize the carbohydrates in the plant cell walls. in this technique, the fractions of fiber that are insoluble either in acid detergent or in neutral detergents were measured, and the residue after treatment of acid detergent fiber (adf) fraction with 12 mol.l-1 sulfuric acid was considered to be acid detergent lignin (adl) and calculated by the following equations (eq. 1; eq. 2): % cellulose = adf – adl (1) % hemicellulose = ndf adf (2) where ndf is the neutral detergent fiber (%, w/w) and adf is the acid detergent fiber (%, w/w), while adl represents the acid detergent lignin (%, w/w). hplc analysis the oligomer composition of sugar supernatant was determined by high performance liquid chromatography (hplc) (shimadzu, kyoto, japan) equipped with two lc-20ad pumps, a dgu-20a5 degasser, an sil-20ac auto sampler and a refractive index detector (rid-10a). the column was an inertsil nh2 column (5 µm particle size, 250 mm x 4.6 mm, gl sciences inc., nova biotechnol chim (2022) 21(1): e1175 2 tokyo, japan) and its temperature was maintained at 40 c. the mobile phase was 85 % acetonitrile and 15 % ultra-pure water at a flow rate of 0.50 ml/min and 10 μl of sample was injected through a 0.22 µm filter into the hplc (veena et al. 2018). d-(+)-xylose standard purchased from merck life science uk limited (gillingham, uk) was used as standard. the peak areas were used to plot the calibration curve for the determination of the amount of oligomer in the sample, which were determined from the calibration curves of the corresponding standards. statistical analysis all data were statistically considered by analysis of variance (anova) followed by post hoc test using duncan‘s multiple range test (dmrt) and mean plots treatment. statistical processing was analysed by spss 19.0 software (ibm, somer ny, usa) for the window statistical package. the differences between the mean values were carried out at 95 % confidence interval (p < 0.05). all data described in figures and tables were the mean values of triplicate determinations. results and discussion proximate composition of pineapple peel the chemical compositions of the pineapple peels were determined following the methods described above. the results obtained are given in table 1. it is evident that the values given compare favorably with the normal values for fiber and carbohydrate content (37.30 % and 52.33 %, respectively) reported by zakaria et al. (2021). the fiber content of pineapple peels was about 31.31 %, while the total carbohydrate content is approximately 56.94 %. in general, pineapple waste was rich in lignocellulosic material, especially the peels and the leaves of the fruit (sukruansuwan and napathorn 2018). table 1. proximate composition on % (w/w) dry basis of pineapple peel. sample chemical compositions [%, dry basis] moisture ash protein fat fiber carbohydrate pineapple peel 12.51 ± 0.62 4.82 ± 0.35 6.29 ± 0.55 0.64 ± 0.10 31.31 ± 0.91 56.94 ± 2.03 effects of different chemical pretreatments on pineapple peels the major lignocellulosic components, such as cellulose, hemicellulose and lignin, were determined by the detergent fiber analysis based on the gravimetric method. as shown in table 2, the percentage yields of cellulose, hemicelluloses, and lignin in the raw pineapple peel powder were 31.56 %, 26.35 %, and 10.70 %, respectively. in the current study, the effect of pretreatment using different solvents, such as water, acid, and alkaline, was systematically investigated. it is clear from table 2 that pineapple peels pretreated with water and acid solution (h2so4) yielded the lower amount of hemicellulose, whereas pretreated with alkaline solution (naoh) still retained a high hemicellulose content of 31.57 % to 34.80 %. this observation suggests that hemicellulose degraded more easily under acid treatment than under alkaline treatment (ying et al. 2014). owing to its amorphous structure, hemicellulose is more prone to hydrolysis by mild acid solution compared to crystallized cellulose which likely requires more severe treatment conditions (zhang et al. 2012). considering the utilization of hemicellulose, its component is usually composed of xylan which can further be converted into xylose monosaccharides. for lignin analysis, the lignin contents from water and acid pretreatment were similar or slightly lower than that of the untreated sample, which demonstrated that a small part of the lignin was removed during pretreatment (ying et al. 2014). noticeably, the removal of hemicellulose and cellulose may slightly increase the percentage of lignin in fiber. in contrast, alkaline pretreatment was more capable in removing lignin; where up to 33.36 % of lignin content was removed resulting in 4 nova biotechnol chim (2022) 21(1): e1175 2 the availability of cellulose and hemicellulose for chemical hydrolysis (pandey et al. 2000). considering, both hemicellulose yield and lignin removal, alkaline pretreatment using 1 % naoh was chosen for obtaining the highest hemicellulose content and deemed to be suitable for further experiments. table 2. effects of different chemical pretreatments on characterization of pineapple peel. pretreatment condition composition of pineapple peel [%, dry basis] hemicellulose cellulose lignin untreated 31.56 ± 1.90b 26.35 ± 1.45b 10.70 ± 1.04a water 28.72 ± 1.79c 23.48 ± 2.33c 11.16 ± 1.02a 1 % naoh 34.80 ± 1.10a 32.16 ± 2.16a 8.66 ± 0.98b 2 % naoh 33.36 ± 1.94a 31.36 ± 1.45a 7.56 ± 0.81c 4 % naoh 31.57 ± 1.35b 28.83 ± 2.11b 7.13 ± 0.86c 1 % h2so4 26.97 ± 1.90 c 23.44 ± 1.45c 10.30 ± 0.98a 2 % h2so4 24.43 ± 1.78 d 21.53 ± 1.28cd 10.95 ± 1.32a 4 % h2so4 20.28 ± 1.54 e 20.51 ± 1.22d 9.46 ± 1.07ab * untreated sample is raw pineapple peel. ** percent composition is percentage of retained lignocellulosic materials in a sample. *** values with different superscripts (a-e) in the same column are statistically different (p ≤ 0.05). effect of ultrasonic-assisted alkaline pretreatment on pineapple peel the ultrasonic-assisted alkaline pretreatment of pineapple peel powder was undertaken under five conditions depending on varying ultrasonication time from 0 to 60 min. based on the results of the previous experiment; an alkali pretreatment (1 % naoh) was chosen to determine the effect of ultrasonic-assisted alkaline pretreatment on the composition of pineapple peel. table 3 demonstrates that the combination of ultrasonic and alkaline pretreatments could significantly decrease the total lignin content with time up to 60 min to reach a minimum value of 6.94 %. a significant percentage of hemicellulose increases gradually with irradiation time up to 45 min, and then it decreases slightly to 50.63 %. the application of excessive ultrasonication time did not further increase hemicellulose yield. during pretreatment, ultrasonication helped in delignification due to cleavage of interand intramolecular linkages and the cavitation of microbubbles which increased the available surface area of the cellulose and hemicellulose (koutsianitis et al. 2015). table 3. effects of ultrasonication time and 1% naoh pretreatment on characterization of pineapple peel. ultrasonication time [min] after pretreatment with 1 % naoh [%, dry basis] hemicellulose cellulose lignin 0 33.22 ± 2.13d 28.31 ± 1.32c 9.96 ± 0.94a 15 40.88 ± 2.45c 26.80 ± 1.90bc 8.67 ± 0.86b 30 47.19 ± 2.16b 27.62 ± 1.10bc 7.46 ± 0.98bc 45 51.15 ± 1.90a 29.82 ± 1.83ab 7.15 ± 0.97c 60 50.63 ± 2.77a 30.01 ± 1.68a 6.94 ± 1.18c * percent composition is percentage of retained lignocellulosic materials in a sample. ** values with different superscripts (a-d) in the same column are statistically different (p ≤ 0.05). a similar research was demonstrated by zhang et al. (2018) who stated that there was a reduction in lignin content after ultrasonication on rice straw. this is also in agreement with the report of loow et al. (2016), who stated that the ultrasonic method helped to decrease quantities of lignin in biomass 5 nova biotechnol chim (2022) 21(1): e1175 2 for reducing sugar recovery. in addition, ultrasonic application has been shown to increase the delignification of lignocellulosic materials, as the degradation of lignin-carbohydrate bonds would increase the accessibility for solvent penetration which may improve the extraction of reducing sugar (ong et al. 2019). therefore, ultrasonication time of 45 min is suitable for obtaining the highest hemicellulose yield from pineapple peel. effect of ultrasonic-assisted alkaline pretreatment and acid hydrolysis on xylose production basically, xylose is a reducing sugar obtained by hydrolysis of hemicelluloses, as it can be converted to valuable by-products such as xylitol, ethanol, and furfural. as shown in table 4, the ultrasonication time of 45 min followed by 2 % h2so4 with hydrolysis time of 30 min could yield a maximum xylose concentration around 48.85 g.l-1 which is a considerable improvement in comparison with the highest xylose concentration (24.1 ± 0.4 g.l-1) obtained in previous studies (sukruansuwan and napathorn 2018; banerjee et al. 2019; ariffin et al. 2020). an upward trend in sugar yield by using ultrasonic-assisted alkaline pretreatment was observed with a reduction in lignin content. these results were obtained due to the breakdown of the lignin matrix leading to an increase in the accessible surface of hemicelluloses within the pineapple peel. however, an excessive ultrasonic irradiation time of more than 45 min caused no further increment in xylose yield. this finding suggested that prolonged ultrasound duration would not be necessary for producing higher sugar yield but unfavorably it may cause adverse effects due to collision and aggregation between particles (baruah et al. 2018; ong et al. 2019). similar findings were revealed by ong et al. (2019) and yunus et al. (2010) who stated that ultrasonic irradiation helps to increase xylose contents extracted from oil palm fronds and empty fruit bunches, respectively. under the conditions obtained from this study (naoh concentration of 1 %, ultrasonication time of 45 min, and hydrolysis time of 30 min), the maximum xylose yield was achieved. thus, the data obtained from the best conditions of xylose production from pineapple peel waste using combination pretreatment would be potentially useful information not only for producing value-added product but also helps solve the environmental pollution. table 4. effect of ultrasonic-assisted alkaline pretreatment and acid hydrolysis on xylose production. pretreatment with 1 % naoh after 2 % h2so4 hydrolysis 30 min ultrasonication time [min] xylose [g.l-1] 0 37.11 ± 2.10d 15 40.13 ± 2.94c 30 44.05 ± 2.76b 45 48.85 ± 2.45a 60 47.20 ± 3.12ab * values with different superscripts (a-d) in the same column are statistically different (p ≤ 0.05). conclusions the feasibility of using pineapple peel waste as a lignocellulosic material for xylose production was evaluated. the present work represents a systematic attempt to investigate the effects of ultrasonic-assisted alkaline pretreatment on the best conditions of xylose production. the results have shown that alkaline pretreatment using simple ultrasonic technology is a practical alternative for production of valuable xylose sugar compared to costly catalysts or expensive enzymes. in this study, the best ultrasonic condition at 40 khz for 45 min with 1 % naoh pretreatment and hydrolysis time of 30 min successfully achieved 33.18 % delignification while producing high xylose concentration up to 48.85 g.l-1. the ultrasonicassisted alkaline pretreatment with acid hydrolysis exhibited better xylose yields and lower costs of extraction in comparison with an enzymatic method. thus, the data obtained from the effect of ultrasonication on alkaline pretreatment and acid 6 nova biotechnol chim (2022) 21(1): e1175 2 hydrolysis would be potentially useful for the application of pineapple peel waste as raw material for large scale xylose production. acknowledgements the authors are grateful to thailand science research and innovation fund for providing financial support (grant no. 0002406). the authors would also like to thank science center, faculty of science and technology, suan sunandha rajabhat university for facilitating hplc analysis. conflict of interest the authors declare that they have no conflict of interest. references agricultural informatics center (2018) agricultural statistics of thailand, 2017. office of agricultural economics. ministry of agriculture and cooperatives, bangkok, 232 p. aoac (2012) official methods of analysis of aoac international. 19th ed. aoac international, gaithersburg, md, usa. methods 930.15, 930.05, 990.03, 945.16, 942.05, and 985.01. ariffin kk, masngut n, seman mna, saufi sm, jamek s, sueb msm (2020) dilute acid hydrolysis pretreatment for 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(2021) microwave-assisted extraction of pectin from pineapple peel. mal. j. fund. appl. sci.17: 33-38. 7 nova biotechnol chim (2022) 21(1): e1175 3 zhang d, ong yl, li z, wu jc (2012) optimization of dilute acid-catalyzed hydrolysis of oil palm empty fruit bunch for high yield production of xylose. chem. eng. j. 181182: 636-642. zhang h, zhang p, ye j, wu y, liu j, fang w, xu d,wang b, yan l, zeng g (2018) comparison of various pretreatments for ethanol production enhancement from solid residue after rumen fluid digestion of rice straw. bioresour. technol. 247: 147-156. zheng y, pan z, zhang r (2009) overview of biomass pretreatment for cellulosic ethanol production. int. j. agric. biol. eng. 2: 51-68. 8 microsoft word att00007.doc nova biotechnologica 8-1 (2008) 45 dna damage by oxidized fatty acids detected by dna/spe biosensor ľudmila sirotová, marcela matulová food research institute, department biocentre, kostolná 7, sk900 01 modra, slovak republic (sirotova@vup.sk) abstract: electrochemical dna/screen-printed electrode biosensor (dna/spe biosensor) was tested for the detection of alterations in dna formed as a consequence of the reaction between dna and oxidative products of fatty acids. interaction of dna with a mixture of products generated during the oxidation of linoleic and oleic acids manifested dna damage depending on a tested fatty acid and the presence of hydroperoxides and thiobarbituric acid reactive substances (tbars) determined after the oxidation of fatty acids. a bigger extent of the dna damage was registered in the case of the interaction with oxidized linoleic acid with the high content of tbars. the results achieved suggest the possible application of dna/spe biosensor in the detection of an interaction between dna and products of fatty acid oxidation. key words: dna/spe biosensor, fatty acid oxidation, dna damage 1. introduction the damage of dna and its consequences have become the closely observed topics in the last years (marnett, 2000; marnett et al., 2003). mutagenicity and genotoxicity of various endogenous and exogenous reactive compounds acting on dna have been connected with ageing, the formation of such illnesses as cancer, cardiovascular and neurodegenerative diseases and immune system decline (hwang and bowen, 2007). some of the studies suggest that the oxidation of fatty acids and the consequent dna damage play an important role in such processes (marnett, 2002; kanner, 2007). the polyunsaturated fatty acids are extremely sensitive to attack of reactive oxygen species. the first products of polyunsaturated fatty acid oxidation are relatively short-lived lipid hydroperoxides. they are reduced to unreactive fatty acid alcohols or react with metals to aldehydes, ketones, alcohols, short fatty acids, esters, hydrocarbons, furans, and lactones, named as secondary products (burcham, 1998). some products are rather long-lived and can drift far from membranes and damage nucleic acids. dna damages may occur as single-strand breaks, double-strand breaks, abasic site formation, sister chromatide exchange, dnadna and dna-protein cross-links, damages to deoxyribose and base adduct formation. the phosphodiester backbone can be also damaged, leaving abnormal ends. such alterations to dna have been shown to disrupt transcription, translation, dna replication and lead to mutations, cell senescence or death (termini, 2000; blair, 2001). methods used for evaluation of dna damage assess the overall state of dna (cleavage of dna strand, changes in structure, presence of bound compounds affecting the properties of dna) or they are based on detecting the formed adducts. gel electrophoresis (bozko et al., 2005; philips et al., 2005) and comet assay (detection of cell dna damage) (gontijo et al., 2001) are the methods currently 46 sirotová, ľ. and matulová, m. employed for assessing the changes in dna. methods for detection of formed adducts allow qualitative and quantitative assessment of damage of individual bases. the detection of the adducts is possible using 32p-postlabelling hplc method (nath et al., 1996), by the chromatographic techniques as gas chromatography/electron capture/negative chemical ionization/mass spectrometry (gc/ec nci/ms) (rouzer et al., 1997), liquid chromatography with electrospray tandem mass spectrometry (lc/esi-ms/ms) (chaudhary et al., 1995; jeong et al., 2005; singh and farmer, 2006) or by immunochemical techniques (kawai et al., 2002). biosensors bring the new possibilities in the area of monitoring of the dna damage (drummond et al., 2003). particularly electrochemical dna/screen printed electrode biosensor (dna/spe biosensor) constitutes a device for a rapid, cheap but sensitive detection of the changes on dna (tudorache and bala, 2007). the detection schemes are based on a redox signal of the dna base (adenine or often guanine) (xie et al., 2007) and/or the special electrochemical indicators (tansil et al., 2005; niu et al., 2006). biosensors have been applied for observing of the alterations of dna due to multiple chemical nucleases, antibiotics (labuda et al., 2000), samples from the environment (mascini, 2001) and have also been used for observing the impacts of the antioxidatively active substances on dna (heilerová et al., 2003; labuda et al., 2003; liu et al., 2006). in our study we have tested dna/spe biosensor as a detection system for the assessment of the influence of oxidized fatty acids on dna. 2. material and methods 2.1 materials oleic acid, linoleic acid and tween 20 were purchased from sigma (steinheim, germany). stock solution of calf thymus dna (calbiochem, darmstadt, germany) 1 mg.ml-1 was prepared in te solution (10 mm tris-hcl with 1 mm edta, ph 8). electrochemical marker [co(phen)3]clo4 was supplied by fchpt stu (bratislava, slovakia). all other chemicals were of analytical grade. 2.2 fatty acid oxidation both linoleic and oleic acid (3 g) were oxidized in rancimat 743 (metrohm, herisau, switzerland) test tube at 150°c for 4 h. the amount of 0.01 g of oxidized fatty acid was homogenized with 100 μl of tween 20 solution (0.5 g/10 ml deionized water). 2.3 peroxide value determination peroxide value was determined by the idf method (shantha and decker, 1994). briefly, oxidized fatty acid (0.01-0.3 g) was mixed in test tube with a mixture of chloroform:methanol 7:3 (v/v). then nh4scn and subsequently fe(ii) solutions were added to the fatty acid solution. the absorbance was measured nova biotechnologica 8-1 (2008) 47 spectrophotometrically at 500 nm against blank solution after 5 min incubation of the mixture in dark. peroxide value was expressed in milliequivalents of peroxides per kg of fatty acid. 2.4 tbars value determination oxidized fatty acid and 2 ml of each 1% tba and 20% iced acetic acid and 1 ml of deionized water were added to the mixture and then heated in a water bath to 100°c for 1 h. after cooling, 5 ml of chloroform were added to the mixture, then the entire mixture was centrifuged at 2 000xg for 10 min. the absorbance of supernatant was measured spectrophotometrically at 532 nm. the value of tbars was expressed in nmol of mda equivalent per g of fatty acid. 2.5 dna/spe biosensor a computerized voltammentric analyzer ecapol 110 (istran, bratislava, slovakia) was equipped by a screen-printed three electrode assembly (vup biocemtrum, modra, slovakia) including a carbon working electrode and the silver/silver chloride reference and counter electrodes. the working electrode, used without any chemical preconditioning, was modified by covering with 5 μl of the dna stock solution and leaving the electrode to dry overnight. dna/spe biosensor was pretreated by immersion in 5 mm phosphate buffer ph 7.0 under stirring for 2 min, then rinsed with water. the [co(phen)3] 3+ indicator was accumulated under stirring for 120 s at an open circuit from 5 ml of its 5.10-5 m solution in 5 mm phosphate buffer. the differential pulse voltammogram (dpv) was recorded immediately from 400 to -400 mv at a pulze amplitude of 100 mv and 2 mv scan step at the scan rate of 25 mv/s. the indicator peak current (i0) was evaluated against the base-line using analyzer software and corrected by a subtraction of the mean indicator peak current measured at the unmodified spe under the same conditions. then, the dna/spe biosensor was regenerated by a removal of the accumulated [co(phen)3] 3+ ions from the dna layer by treating the sensor in 100 mm phosphate buffer ph 7.0 under stirring for 120 s. the peak current i0 was obtained in triplicate. the dna layer was covered with 5 µl of the oxidized fatty acid sample. after 20 min the dna/spe biosensor was treated for 120 s in surfactant solution (0.5 g/100 ml deionized water) (procter and gamble, rakona, czech republic) under stirring at open circuit conditions. the sensor was rinsed with deionized water and then immersed into 5 mm phosphate buffer with [co(phen)3] 3+ indicator for 120 s for its immobilization. the dpv was recorded immediately at the same conditions as above. the peak current i was obtained in triplicate. the degree of dna damage was expressed as the ratio i/i0, the indicator peak current after interaction of dna with oxidized fatty acid (i) against the indicator peak current of unmodified dna on spe (i0). both i and i0 were corrected by a subtraction of the mean indicator peak current measured at the unmodified spe under the same conditions. 48 sirotová, ľ. and matulová, m. 2.6 statistical analysis all data are expressed as the mean ± standard deviation. all raw data were processed using standard ms-excel statistical package. 3. results and discussion dna immobilized on a screen-printed electrode interacted with the solution of oxidized linoleic and oleic acid. the interaction was evaluated using the current response signal of the electrochemical intercalating indicator [co(phen)3] 3+ measured by the dpv technique. a drop in the current response of the indicator indicates the damage of a dna strand or the presence of a competing compound, creating the steric obstacles to the intercalation of the indicator. the lower value of i/i0 ratio the bigger dna damage occurs. the dna damage by oxidized linoleic and oleic acids is showed in fig. 1. the extent of the dna damage was dependent on the conditions of the fatty acid oxidation. the influence of unoxidized fatty acids under the selected conditions showed a moderate decrease in the current response signal of the indicator (by 13% for linoleic acid and by 3% for oleic acid). this could be due to the oxidative products already present in the samples, what can be confirmed by pv (peroxide value) and tbars (thiobarbituric acid reactive substances) values determined for the respective fatty acids (fig. 2 and 3). the dna damage was bigger with the increasing time of oxidation of the tested fatty acid. the course of this dependence had an exponential character. after a 4-hour oxidation the current response signal of the indicator was lowered by 57% in the case of linoleic acid and by 48% for oleic acid. the achieved degree of the dna damage is a result of the interaction between the products formed in the respective stage of fatty acid oxidation and their reactivity towards dna. the dna/spe biosensor detected the bigger extent of the dna damage in the case of the reaction mixture of oxidized linoleic acid compared to oxidized oleic acid. 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 0 1 2 3 4 t (h) i/i 0 linoleic acid oleic acid fig. 1: the dependence of the dna damage on the time of fatty acid oxidation assessed by dna/spe biosensor. nova biotechnologica 8-1 (2008) 49 during oxidation of fatty acids a mixture of products is formed depending on the type of fatty acid and the conditions of oxidation. oxidation of fatty acids may be initiated by various ways, such as the presence of reactive oxygen species or other oxidatively active compounds, the activity of the specific enzymes or the increased temperature. as a consequence of oxidation of fatty acid, primary carbon centered radicals l• are produced to give rise to peroxyl radicals loo• which are transformed to hydroperoxides looh. these compounds are further transformed to alkoxyl radicals lo• or decompose rapidly into a multitude of volatile and non-volatile products. alcohols, saturated aldehydes, α,β-unsaturated aldehydes and epoxy compounds have been reported as the major secondary oxidation products (frankel et al., 1992; burcham, 1998). the presence of the respective compounds determines the degree of fatty acid oxidation and this can be detected by laboratory tests such as determination of the peroxide value (determines the amount of hydroperoxides produced – primary products) or tbars value (determines the amount of substances able to react with thiobarbituric acid – secondary products). the comparison of linoleic and oleic acid shows that with the increasing time of oxidation the peroxide value fell but the tbars value rose (fig. 2 and 3). at the same time, the peroxide value was higher in the case of oleic acid, while the tbars value was higher for linoleic acid. a drop in the peroxide value and a rise in the tbars value indicate the successive transformation of hydroperoxides of fatty acids to the secondary products. this transformation is more noticeable in the case of linoleic acid compared with oleic acid. this can be explained by the fact that linoleic acid is more prone to oxidation when compared to oleic acid because of the higher number of multiple bonds. 0 5 10 15 20 25 30 0 1 2 3 4 t (h) p v (m ili ek vi va le nt o f pe ro xi de .k g1 ) linoleic acid oleic acid fig. 2: peroxide value of oxidized fatty acids. based on the comparison of a mixture of oxidized fatty acids and the dna damage it is possible to suggest that under the selected conditions the presence of the secondary products has a more significant influence on the dna damage than the presence of hydroperoxides. the extent of the dna damage is increased with the increasing tbars value. studies on interactions between the dna and hydroperoxides indicate that these compounds react with the dna and cause cleavage 50 sirotová, ľ. and matulová, m. of the double-stranded dna or formation of the hydroperoxide-induced dna adduct (termini, 2000; blair, 2001; kawai et al., 2002; williams et al., 2006). 0 100 200 300 400 0 1 2 3 4 t (h) tb a r s (n m ol .g -1 ) linoleic acid oleic acid fig. 3: tbars amount in oxidized fatty acids. base adducts are preferentially formed by α,β-unsaturated aldehydes, ketones and their epoxides and alkenes (eide et al., 1995). especially the adducts with acrolein, crotonaldehyde (marnett, 1994), malondialdehyde (mda) (marnett, 1999) and 4-hydroxy-2-nonenal (yang et al., 2003), which serve as biomarkers of certain diseases have been thoroughly studied. therefore, the lowered current response signal of the indicator could be the result of the structural changes in the dna strand or due to the base adducts formed, which hinder the indicator from intercalating into the minor and major grooves of dna. 4. conclusions under the selected conditions dna/spe biosensor sensitively responded to the dna damage caused by oxidized linoleic and oleic acids. though the biosensor and the given indicator system does not enable to define the concrete type of the dna damage, it can be used for proving the oxidative alterations in fatty acids or other lipidic samples. to effectively use the biosensor for evaluation of interactions between dna and the oxidative products of fatty acids it is important to take into consideration reactivity of individual generated products towards dna, a mechanism of their action, a type of the damage they cause and the selection of the proper technique and indicator for detection of the dna damage. the mentioned parameters are a subject of further studies. acknowledgement: this work was supported by science and technology assistance agency under the 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(2008) 53 williams, m.v., lee, s.h., pollack, m., blair, i.a.: endogenous lipid hydroperoxide-mediated dna-adduct formation in min mice. j. biol. chem., 281, 2006, 10127-10133. xie, h., yang, d., heller, a., gao, z.: electrocatalytic oxidation of guanine, guanosine and guanosine monophosphate. biophys. j., 2007, l01-l02. yang, y., sharma, r., sharma, a., awasthi, s., awasthi, y.c.: lipid peroxidation and cell cycle signaling: 4-hydroxynonenal, a key molecule in stress mediated signaling. acta bioch. pol., 50, 2003, 319-336. microsoft word farago nb 9-3.doc nova biotechnologica 9-3 (2009) 279 the use of biotechnology in hop (humulus lupulus l.) improvement juraj faragó1,2, ivana pšenáková2, natália faragová1 1plant production research centre, research institute of plant production (ripp), bratislavská cesta 122, piešťany, sk-921 68, slovak republic (farago@vurv.sk) 2department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic abstract: hop (humulus lupulus l.) is a clonally propagated, dioecious, perennial, climbing plant used commercially for their secondary metabolites. the resins containing αand β-acids, and essential oils produced by the lupulin glands, present on the female flowers are used to add bitterness, aroma and flavour to beer. recently, flavonoids, including chalcones and flavanones, of hops have been shown to exert a variety of biological activities, including oestrogenic and anticancerogenic characteristics. in this review, we provide a overview of the techniques and opportunities presented by the integration of plant biotechnology into hop improvement. the use of tissue culture techniques such as micropropagation, meristem culture, in vitro storage, adventitious shoot induction, callus culture and cell suspension culture in hops are briefly reviewed. the usefullness of genetic transformation technology to introduce novel traits into hop is also discussed. keywords: hops, biotechnology, tissue culture, genetic transformation, secondary metabolites 1. introduction the hop plant (humulus lupulus l.) is widely cultivated throughout the temperate zones of the world for its female inflorescences (commonly referred to as ‘cones’), which are used in the brewing industry to add bitterness, aroma and flavour to beer. before using in brewing industry, hops were traditionally used as herbal medicines, mainly for the treatment of sleeping disorders, as a mild sedative, and for the activation of gastric functions as bitter stomachic (zanoli and zavatti, 2008). currently, the beer brewing industry accounts for 98% of the world use of hops (moir, 2000). hop oil fractions and hop extracts are also used as flavoring products in nonalcoholic beverages and foods. in addition to improving the agronomic traits, hop breeding also aims to tailor the components of the plant important for brewing to the demands of the market. thus, there is an ever increasing need for cultivars exhibiting superior performance in multiple traits, such as high levels of α-acids, excellent flavour, good storage stability and high yield. until very recently, plant breeding relied solely on the accumulated experience of generations of farmers and breeders, that is, on sexual transfer of genes between related plant species. in the last 150 years, however, plant breeding has developed into a complex discipline that now incorporates information from many branches of science and mathematics. the most recent development is the utilization of biotechnology for plant improvement (pauls, 1995). for the purposes of this review, 280 faragó, j. et al. plant biotechnology can be defined as the application of tissue culture and molecular genetics to develop or produce a commodity from plants. plant tissue culture refers to the maintenance and propagation of plant parts (organs, tissues, and single cells) in biologically pure (axenic) and controlled environments, while molecular genetics includes the techniques for isolating, characterizing, recombining, and transferring discrete fragments of dna containing genes coding for specific traits into other, nonrelated, genetic backgrounds. the purpose of this review is to provide a brief overview of the techniques and opportunities presented by the integration of plant biotechnology into improvement of hop (h. lupulus l.) species. 2. biological characteristics of hops 2.1 taxonomy the genus humulus belongs to the family of cannabinaceae, previously placed in the order urticales, but in 2003 incorporated in the order rosales (bremer et al., 2003). the genus consists of three species: humulus lupulus linneus, humulus japonicus siebold & zucc. and humulus yuannensis hu (small, 1978; neve, 1991). the centre of origin of the genus is considered to be china, because all humulus species have been found in this area (neve, 1991; murakami et al., 2006). h. japonicuc is native to japan, taiwan and china, while h. yuannensis is native to high altitudes of the yunan province in china. h. lupulus, which is almost exclusively cultivated for brewing purposes, was first domesticated in central europe in the middle of the ninth century, and is currently naturalized throughout the north temperate regions of the world as well as some temperate regions in australia, south africa, and south america (chadwick et al., 2006). based on their morphological characteristics and geographical locations small (1978) classified the species humulus lupulus into five taxonomic varieties: var. lupulus small (european hops), var. cordifolius small (japanese hops), var. neomexicanus nelson & cockerell, var. pubescens small and var. lupuloides small (north american hops). 2.2 botany common hop is a dioecious perennial plant which produces new shoots each year in early spring from rhizomes of an underground rootstock. the outgrowing climbing vine grows up to 6-9 m in length and senesces to the perennial rootstock in autumn (krištín, 1987; rybáček, 1980). the stems twist around their support in a clockwise direction. the dioecious nature of the plant means that the male and female flowers are on separate plants (fig. 1). however, individual monoecious plants have been found in some wild north american hop populations (haunold et al., 1993). the male flowers are 7-14 cm long racemes (fig. 1a), while the female inforescences are 2.5-5.5 cm long cone-like catkins (strobiles, fig. 1b), made up of overlapping flattened membranaceous bracts. at the base of cone bracts a number of yellow glandular trichomes can be found, which secrete a resinous substance, known as lupulin (rybáček, 1980). only female plants are cultivated in hop gardens, while nova biotechnologica 9-3 (2009) 281 male plants are essential in hop breeding programs aimed at developing new varieties through controlled hybridization (rybáček, 1980; neve, 1991). the cultivated hop is a short-day plant that initiates flowering when the plant is at a critical size (about 6 m, 20-24 nodes) and critical daylength is about 16 h, beyond which flowering cannot be induced (villacorta et al., 2008). the minimum daylength, below which the plant ceases vegetative growth and forms dormant terminal buds, is considered to be 8-10 h. from the cytological point of view, cultivated hop is a diploid (2n = 20) species with heteromorphic sex chromosomes (xx in female plants, xy in males) (danilova and karlov, 2006). fig. 1. male (a) and female (b) inflorescences of hop. (foto: j. farago) 2.3 breeding at the earliest hop plants were collected in the wild. according to wilson (1975) the cultivation of hops started in germany the middle of the ninth century, between a.d. 859 and 875. the interest in selection of new and improved hop variants grew significantly during the nineteenth century when selections were made by the growers from variants observed in their own gardens (moir, 2000). hop breeding started with these clonal selections from adapted wild hops, which has lead to cultivation of lanraces like fuggle and goldings in england, tettnanger and hallertauer mittlefruer in germany and saazer in czech republic. these varieties and their derivatives are still in production today and are typical representatives of european aroma hops with relatively low yield and resin content (stajner et al., 2008). modern cultivars of hops are derived by sexual hybridization which allows the tailoring of hop aroma and bitterness, as well as the introduction of new traits. current breeding practices are aimed primarily at improving the disease resistance (verticillium wilt, downy mildew, and powdery mildew), increasing the resin content, and combining traits by utilization of cultivars and breeding lines or wild hops with favourable properties. today’s basic sources of plant breeding material are maintained in hop germplasm collections, which have been established at main hop breeding centres (stajner et al., 2008). hop is sensitive to different types of pathogens, such as viruses, viroids, bacteria and fungi, which adversely affects its yield and product quality. there is still a shortage of effective chemicals for pest and disease control, and crop yields are consequently subject to dramatic seasonal variation. in most hop-growing countries 282 faragó, j. et al. one of the main objectives of recent breeding programs have been to raise the α-acid content. there is also an increased demand for improved varieties of hops which differ in their resin content, produce different bitterness levels in beers and have increased ability to preserve beer. in some countries, for example in australia and england, tetraploid parents play an important role in hop breeding, as they can be crossed with diploids to produce triploid progeny. the large number of grown hop varieties fall generally into two groups: alpha (or bitter hops) and aroma hops. bitter hops are high alpha acid content, whereas aroma hops have much lower content (<5%). 2.4 phytochemistry more than 1000 chemicals have been identified from hops (chadwick et al., 2006). the main structural classes of chemical compounds identified in hops include terpenes, bitter acids, and chalcones. hundreds of terpenoid compounds identified in the essential oil component of hops account for 0.3-1.0% of strobile weight. the study of the composition of hop essential oils revealed that hydrocarbons usually constitute 40–80% of the oil and, apart from a few simple alkanes, they all are of terpenoid origin. a complex group of at least 40 acyclic, monocyclic, bicyclic, and tricyclic sesquiterpenes is usually dominated by α-humulene, β-caryophyllene, and farnesene. of the monoterpenes, myrcene is the primary component of hop essential oils (moir, 2000; roberts and lewis, 2002). the analysis of hop essential oils helps in determining both hop quality and varietal discrimination (roberts and lewis, 2002). the bitter acids comprise 5-20% of hop strobile weight. they are classified as either α-acids or β-acids which are, respectively, dior tri-prenylated phloroglucinol derivatives. in addition, they each contain a 3-, 4-, 5-, or 6-carbon oxo-alkyl side chain (verzele and keukeleire, 1991). the bitter acids are present in hop cones as a complex mixture of variable composition and concentrations, depending on the variety and growing conditions. the α-acids, particularly humulone (35–70% of total α-acids), cohumulone (20–65%), and adhumulone (10–15%) are regarded as the most important constituents in determining the quality of hops (verzele and keukeleire, 1991; chadwick et al., 2006). the biochemistry (biosynthesis, isomerization, oxidation and degradation) of hop bitter acids have been extensively studied (verzele and keukeleire, 1991; goese et al., 1999). the dried hop cones contain 4–14% polyphenols, mainly phenolic acids, prenylated chalcones, flavonoids, catechins and proanthocyanidins (gerhäuser, 2005). the flavonoids of hops include flavonol glycosides, condensed tannins, and prenylflavonoids (stevens et al., 1998). the majority of known flavonoids from hops can be considered as derivatives of the compound 2',4,4',6'-tetrahydroxy-3'prenylchalcone, commonly known as desmethylxanthohumol (dmx) (chadwick et al., 2006). the most abundant and most important prenylated flavonoid in fresh, and properly preserved hops, is the chalcone xanthohumol, present at concentrations of 0.01–0.5% (stevens et al., 1999). recently, xanthohumol (xn) was identified as a broad-spectrum cancer chemopreventive agent with inhibitory mechanisms in the initiation, promotion and progression phase of carcinogenesis (miranda et al., 1999). in addition, another prenylated flavonoid of hop, 8-prenylnaringenin (8-pn) was identified as a very potent phytoestrogen (milligan et al., 1999). nova biotechnologica 9-3 (2009) 283 3. biotechnology of hop improvement 3.1 micropropagation traditionally, hop cultivars are vegetatively propagated by layering young vines under the soil or by rooting soft-wood nodal cuttings during the growing season (neve, 1991). tissue culture techniques can be used as alternative means of plant multiplication. the clonal propagation of plants in vitro is called micropropagation. thus micropropagation can be characterized as the multiplication of genetically identical individuals by asexual reproduction. a significant advantage offered by the aseptic methods of clonal propagation over the conventional methods is that in a relatively short span of time and space a much larger number of plants can be produced from an individual (chawla, 2002). in vitro micropropagation of hop is feasible starting from node cuttings, apical tip explants (roy et al., 2001; smýkalová et al., 2001), meristems (adams, 1975), and parts of the shoot or leaves (batista et al., 1996; peredo et al., 2006). roy et al. (2001) used nodal explants of hop variety h138 to test the effect of plant growth regulators and different media formulations on shoot multiplication efficiency. they achieved efficient culture establishment (96.6% of explants) on murashige and skoog (ms) media supported with plant growth regulators indoleacetic acid (iaa, 0.57 μm) and 6-benzylaminopurine (ba, 2.22 μm). however, in subsequent multiplication media, addition of the cytokinin n-phenyl-n'-1,2,3-thidiazol-5-ylurea (tdz) resulted in significantly higher number of shoots and nodes generated per explant compared to media containing ba, 6-(γ,γ-dimethylallylamino)purine (2ip) or kinetin (kin). smýkalová et al. (2001) developed an efficient in vitro micropropagation system for czech and several foreign hop mericlones using low sugar, starch-gelrite media. the use of this medium resulted in lower cost of micropropagation of healthy hop cultures without exhibition of vitrification. in clonal propagation of plants, such as commercial cultivars of hops, it is very important to keep the genetic stability of recovered plants. however, due to genetic and epigenetic changes induced by the in vitro culture environment, the phenotype of the regenerated plants may be different from the original mother plant in some cases (kaeppler and phillips, 1993). the use of molecular techniques as a method for evaluating genetic stability of tissue culture derived plants is frequently described in the literature since molecular markers can characterize the fenomenon of somaclonal variation with much greater precision and lesser effort than karyological and phenotypic analyses. in hops, peredo et al. (2006) used the amplified fragmentlength polymorphism (aflp) and methylation-sensitive amplified polymorphism (msap) techniques to characterize the genetic and epigenetic variation in hop plants regenerated from sequential subcultures of organogenic calli. they detected no major genetic rearrangements in the callus-derived plants since none of the aflp loci were polymorphic, however, epigenetic changes due to a demethylation process were detected by the msap technique. the epigenetic changes were found to increase when the calli were maintained over a longer period in tissue culture. micropropagation is often used to mass propagate virus-free stock plants obtained by the use of meristem culture technique. 284 faragó, j. et al. 3.2 meristem culture among the prevalent tissue culture techniques used in agricultural biotechnology, the meristem and shoot tip culture have been exploited at a much wider scale primarily due to their potential applicability in diverse areas, such as virus elimination and production of virus-free stock plants, rapid clonal multiplication of vegetatively propagated plants, and germplasm preservation of both vegetatively and seed propagated crop species (nehra and kartha, 1994). the meristem-tip propagation technique used for production of virus-free plants was the first direct application of tissue culture technology to hop improvement by vine and jones (1969) at east malling research station in united kingdom. this approach proved to be very successful and was extended by gippert et al. (1974) in germany, by adams (1975) in the uk, by kubo et al. (1975) in japan, by samyn and welvaert (1983) in belgium, by popov et al. (1985) in the previous ussr, and by probasco and winslow (1986) in the usa. more recently the meristem culture technique was applied to produce virus-free stock plants also in czech republic (svoboda, 1992) and slovakia (faragó and nešťáková, 1998), respectively. in our laboratory at ripp piešťany, meristem culture was successfully applied to all ten cultivars tested. in average around 70% of meristems developed 1 to 4 shoots during the 28 day-long culture period on msc induction medium. in total 299 mericlones were obtained and subjected to thermotherapy. the viability of cultures after thermotherapy was in average 65% (faragó and nešťáková, 1998). starting with year 1996, the hop yards in slovak republic were replanted with meristem culture-derived virus-free plants, which caused a substantial increase in yield parameters in subsequent years (for example almost doubled α-acid content) (košútová, 2002, pers. comm.). now, the cultivation area of meristem culturederived hops in slovakia is cca. 350 ha, representing about 0,8% of the total production in the european union. meristem-tip culture has been widely used for the production of virus-free plant material in many species propagated mainly or exclusively by vegetative means. this technique enables the mother plant’s genetic features to be conserved maximally in the regenerated plants. virus elimination through tissue culture techniques, however, requires efficient plant regeneration systems. 3.3 in vitro storage hop germplasm is traditionally preserved in the form of field collections in areas of cultivation (revilla and martínez, 2002). however, conservation of plants in natural conditions is costly in terms of land utilization, labour and risks of losses through environmental hazards and diseases (watt et al., 2000). in addition, virus diseases can accumulate in a field collection and be transferred to additional sites by vegetative propagation (reed et al., 2003). in vitro techniques have been found to be useful in ex situ conservation of a number of plant species (villalobos and engelmann, 1995), including meristem culture-derived virus-free germplasms (fletcher et al., 1998). the storage longevity of in vitro collections depends on many important factors such as explant type, culture medium composition and culture conditions (aynalem et al., 2006; faragó et al., 2008) nova biotechnologica 9-3 (2009) 285 according to reed et al. (2003) the u.s. department of agriculture, ars, national clonal germplasm repository at corvallis (oregon, usa) maintains about 700 accessions of humulus spp. germplasm in field collections, in pots in the screen house, and as tissue cultures in cold storage or as cryopreserved shoot tips in liquid nitrogen. at the plant production research centre – research institute of plant production (piešťany, slovak republic) we maintain more than 100 accessions of 10 cultivars (osvalds clones 31, 72 and 114, bor, sládek, siřem, aromat, lučan, zlatan and premiant) of predominantly meristem culture-derived virus-free hop genotypes (94 clones) using an optimized in vitro storage system (tab. 1). eight clones were introduced into in vitro culture in 1994, 94 clones in 1996 and 11 clones in the year 2000. accessions are represented by 4 culture vessels (baby food jars, fig. x) with 67 nodal explants in each. the subculture interval of cultures maintained in slow growth conditions ranges from 12 to 18 weeks. the cultures are regularly inspected for the potential occurrence of latent endocontaminating bacteria and the genetic stability of in vitro stored plant material is studied by molecular analysis of microsatellite markers (faragó et al., 2008). table 1. optimization of tissue culture system for in vitro storage of meristem culture-derived hop germplasm. variants marked with bold tetters were chosen to slow down the growth of in vitro shoot cultures. parameter variant mineral content of medium full strength, ½ strength, ¼ strength sugar concentration 2, 3, 4 and 6% glucose; 2, 3, 4 and 6% sucrose solidifying agent 2,5 % phytagel, 7 % agar plant growth regulators 0, 0.05, 0.1, 0.15, 0.2, 0.25 mg.l -1 bap; 0, 0.5, 1.0, 1.5, 2.0 mg.l-1 ga3 culture vessel type baby food jars, industrial glass, magenta vessels, test tubes vitrovent vessels explant type growth tip, defoliated nodal segment, non-defoliated nodal segment cold treatment 25/20°c, 15/10°c and 5±2°c plant growth retardants ancymidol and paclobutrazol at 0, 1, 2.5, 5 and 10 μm; mannitol and sorbitol at 0, 10, 20, 30 and 40 mg.l-1 fig. 2. in vitro storage of hop germplasm using the optimized slow growth method (photo: j. farago). 286 faragó, j. et al. 3.4 adventitious shoot regeneration beside the importance in different tissue culture techniques, regeneration of plants in in vitro culture is also an essential prerequisite for generation of transgenic plants. however, only a few reports are available to date on the regeneration of hop tissues other than meristems (motegi, 1976; rakouský and matoušek, 1994; batista et al., 1996; gurriarán et al., 1999). motegi (1976) reported a successfulr regeneration of plants from callus cultures derived from leaf explants. rakouský and matoušek (1994) published a protocol for direct organogenesis of two commercial czech hops on media containing bap or zeatin. they achieved the highest regeneration of shoots from either petioles or internodes at frequencies 2l % and 52 %, respectively. batista et al. (1996) tested the organogenic ability of petiole and stem segments of two hop varieties on three different basal media supplemented with 0.025 mg.l-1 indole-3-acetic acid (iaa) and 2.0 mg.l-1 6benzylaminopurine (bap). these conditions induced rather heterogeneous responses, which depended mainly on the explant source and the genotype. the regeneration rate of cultures was also highly correlated with the production of green, nodular areas in the compact calli. gurriarán et al. (1999) established a very efficient protocol for plant regeneration from two commercial hop cultivars, brewers gold and nugget, and studied the morphogenetic potential of explants cultured on adams modified medium supplemented with several concentrations of cytokinins and auxins. regeneration rates of 60 and 29% were achieved for nugget and brewers gold, respectively. we developed a highly efficient in vitro system for adventitious shoot regeneration from leaf and internode explants of 12 hop genotypes (fig. 3; faragó and kollárová, results not published). the highest percentage of plant regeneration (52.7 %) was achieved in genotype zlatan/1/2t from internode explants cultured on media supplemented with 2.0 mg.l-1 bap and 2.0 mg.l-1 naa and when maltose was used as carbon source. all the tested genotypes produced adventitious shoots with average frequencies of 1.4-17.4%. the regeneration frequency depended on the explant type (internode segment vs. leaf segment), auxin type (2,4-d vs. naa), and csource (glucose vs. maltose). fig. 3. adventitious shoot regeneration from internode and leaf segment explants of the hop genotype k72/6/13 (photo: j. farago). nova biotechnologica 9-3 (2009) 287 3.5 callus culture secondary metabolites of hops important for the brewing of beer include α-acids and ß-acids, however, another group of compounds present in hops, such as prenylated chalcones, xanthohumol, and desmethylxanthohumol, were recently found to exhibit interesting bioactive properties. the increased demand for medicinally important secondary metabolites increases the pressure to produce these compounds via alternative ways, especially using cell/tissue cultures and transgenic plants, respectively. griffin and coley-smith (1968) first initiated hop callus cultures with the aim of study in aseptic conditions the infection process of fungus pseudoperonospora humuli. callus cultures in hops have been used experimentally to provide a source of regenerationg green nodular callus, for attempted selection of novel disease resistance to verticillium albo-atrum in somaclonal variants, as well as providing a sterile source of leaf mesophyll protoplasts (connell and heale, 1986b; heale et al., 1989). there were also attempts to establish regenerating, organogenic callus caltures from different types of explants (motegi, 1976; batista et al., 1996). undifferentiated, white calli was produced from stem internodal segments, petioles, and leaf segments (connell and heale, 1986b; heale et al., 1989; pšenáková et al., 2009) currently, in line with a growing interest in the health benefits of medicinal plants, hop has received particular attention by the researchers due to the identification of pharmacologically relevant compounds, such as flavanones, chalcones, and phloroglucinol derivatives in hop cones (zanoli and zavatti, 2008). therefore, we study the possibility to establish a convenient in vitro system, based on the induction of callogenesis and establishment of cell suspension culture in hops for chemical analyses of constituents of in vitro cultures and for potential production of interesting flavonoids in in vitro culture systems (pšenáková et al., 2009). our system is based on induction of callus formation from two types of explants (internodal segments and leaf segments isolated from in vitro grown shoot cultures) on media containing the combination of auxin and cytokinin growth regulators bap, naa, and 2,4-d. content of total polyphenols depended on culture conditions and ranged from 76.6 to 158.5 mg.l-1 of gallic acid equivalent (gae) in our callus cultures (pšenáková et al., 2009). 3.6 cell suspension culture the culture of plant cells on a large scale has long been regarded as a potential source of secondary products. large-scale tissue cultures were and are seen as a more convenient and reliable source of secondary products than intact plants (collin, 2001). plant cell suspension cultures offer the manufacturer independence from fluctuations in supply of the raw plant material that might have been created by vagaries in the climate, agriculture and political activities of the source country. growing plant cells in vitro also permit a stricter control of the quality of the products. problems connected with gathering, storing, processing and disposal of huge amounts of biomass, connected with extraction of active substances from in vivo plants are also 288 faragó, j. et al. solved (ionkova, 2007). suspension cultures are of special interest due to their high growth rate and short cycle of reproduction. the first cell suspension cultures of hops have been established by robbins and ratcliffe (1984) to study the intracellular distribution of phosphate in hop cells growing in media with elevated phosphate concentrations. later, cell suspension cultures were established with the aim of attempted production of α-acid compounds (robbins et al., 1985; furze et al., 1987). connell et al. (1986a) studied the interaction of verticillium wilt toxins with suspension cells. various research groups performed some biological and phytochemical studies on hop callus and cell cultures. for example langezaal and scheffer (1992) initiated cell suspension cultures of various hop cultivars and screened them for the presence of bitter acids by hplc and of volatile compounds by capillary gc. however, they could not detect neither bitter acids nor volatile compounds. trevisan et al. (1997) studied the effect of elicitation on peroxidase activity in cell suspension cultures of one variety and four cultivars of hop. we are interested in the potential of plant cell cultures to produce secondary metabolites, such as polyphenols, flavonoids and prenylated chalcons. as a model system we use hop callus and cell suspension cultures. we established cell suspension cultures from stabilized callus cultures of hop cultivar osvald's clone 72 in liquid ms media containing different combinations of plant growth regulators. cell suspension cultures were derived from two types of explants, stem segments and leaf segments and were cultured in two types of culture conditions. they showed high biomass accumulation (fw and dw), depending on the explant type, medium composition and culture conditions. the viability of cells (assessed as % of ttc-positive cells) depended on the concentration of pectinase added to liquid media to liberate cells from cell clumps and ranged from 60.9-90.6 % in media without pectinase to 36.2-65.4 % in media with 1000 μl pectinase.g-1 tissue fw. content of total polyphenols depended on the type of in vitro culture and ranged 60.5-137.1 mg.l-1 of gae in comparison to 121.4 mg.l-1 gae in the source shoot cultures of hops. using hplc analysis, we were able to detect also the production of xanthohumol in cell suspension cultures of hops. the highest production of xanthohumol was observed in cell suspension cultures established from leaf segment-derived calli in medium containing 1.0 mg.l-1 bap in combination with 1.0 mg.l-1 2,4-d without pectinase and cultured in dark conditions (pšenáková et al., 2009). 3.7 genetic transformation genetic engineering, or the ability to transfer genetic information from one species to another, has been possible for about three decades. the advantages of genetic engineering in hop breeding are obvious. in contrast to conventional breeding programs involving cross-pollination, the agronomic and qualitative traits of hops can be improved by inserting a single (or a few) gene(s) of interest with no other changes in the genome. a renewed interest in genetic transformation of hops has arisen from the finding that some hop compounds have remarkable bioactive properties showing strong estrogenic and anticancer activities, and therefore hop is regarded as strong candidate to molecular farming. nova biotechnologica 9-3 (2009) 289 genetic transformation of hop using agrobacterium tumefaciens is regarded as the most promising approach since hop in one of the natural hosts of this soil bacterium (de cleene and de ley, 1976). however, only a few reports have been published on genetic transformation of hop using this technique, moreover, these experiments were not successful (becker, 2000), or irreproducible (oriniaková and matoušek, 1996). the first report on successful transformation of hop tissues by a. tumefaciens, and regeneration of transgenic plants of cv. tettnanger was reported by horlemann et al. (2003). only two other papers have been reported on the successful production of transgenic hop plants containing gus reporter and/or nptii selection genes with the use of a. tumefaciens-mediated system [okada et al., 2003 (cv. osvald’s clone 72); škof and luthar, 2005 (cv. aurora)]. recently schwekendiek et al. (2007) reported on hop (cv. tettnanger) transformation with a grapevine stilbene synthase gene using the system developed by horlemann et al. (2003). biolistic transformation has proven to be a powerful and versatile technique and an alternative to agrobacterium-mediated transformation. recently batista et al. (2008) developed a successful transformation system for hops using particle bombardment. they were able to establish a reproducible and efficient method for gene transfer and transgenic plant regeneration in hop var. eroica. 4. conclusions biotechnology is now applicable to many aspects of hop improvement. to be effective, plant biotechnology must be well integrated into established plant breeding programs and crop production practices. plant cell cultures of hops can produce many valuable secondary metabolites including novel medicinal agents and in combination with synthetic chemistry, the tissue culture techniques can afford an attractive route to synthesis of complex natural products and related compounds of industrial importance. application of biotechnology to hop improvement will also make diverse germplasm sources more valuable than ever before, because genetic material from different species, genera and even kingdoms can now be stably incorporated and expressed in hops. very few reports on genetic transformation of hop have been published so far, and, in part, this can be explained by the difficulty to establish efficient plant regeneration systems that are still highly genotype dependent. nevertheless, genetic transformation may be a powerful tool for improving agronomic and qualitative traits of hops as well as enhancing the productivity of secondary metabolites in transgenic cell cultures. references adams, a.n.: elimination of viruses from the hop (humulus lupulus) by heat and meristem culture. j. hort. sci., 50, 1975, 151-160. aynalem, h.m.; righetti, t.l.; reed, b.m.: iron formulation affects in vitro storage of hops an image analysis. in vitro cell. dev. biol. – plant, 42, 2006, 405-410. 290 faragó, j. et al. batista, d.; sousa, m.j.; pais, m.s.: plant regeneration from stem and petiolederived callus of humulus lupulus l. 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zavatti, m.: pharmacognostic and pharmacological profile of humulus lupulus l. j. ethnopharmacol., 116, 2008, 383-396. microsoft word farago nb.doc nova biotechnologica vii-i (2007) 63 biotechnological tools to improve ethanol production from plant biomass juraj farago1,2 1slovak agricultural research center, research institute of plant production, bratislavská cesta 122, piešťany, sk-921 68, slovak republic (farago@vurv.sk) 2department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic abstract: increasing concerns for security of the fossil fuel supply emphasizes the need to complement fossil fuel-based energy sources with renewable energy sources. plant biomass represents an abundant renewable resource for the production of bioenergy and biomaterials. this review summarizes the last advancements in the use of biotechnological tools to improve bioethanol production from plant biomass through genetic engineering the starch content and composition and lignocellulosic matter characteristics, and increasing the capacity of plants to produce harvestable yield and ameliorating the negative abiotic stresses on plants so as to increase yield. key words: biotechnology, genetic engineering, starch, lignocellulose, crop yield 1. introduction the production of ethanol for use as a transportation fuel is not a new technology. it was first introduced in the u.s.a. in the 1930s, when the ford model t was able to run on either gasoline or ethanol (kovarik, 1998). post world war ii, however, the interest in the use of agricultural crops for ethanol production dropped, mainly because of the availability of cheap and abundant fuel from fossil sources. renewed interest in ethanol as fuel developed in the 1970s (bothast and schlicher, 2005). brazil was the world’s first country to run large-scale program for using ethanol as fuel (de oliveira et al., 2005), and in the united states ethanol has been utilized as a fuel source since the turn of the century (bothast and schlicher, 2005). in slovak republic, the production of corn-based bioethanol started in this year (p. kostík, enviral, personal communication). it is now understood, that it is important to use plant biomass energy, that would complement solar, wind, and other intermittent energy sources. biomass is considered an interesting energy source for several reasons (see lin and tanaka, 2006). most agricultural biomass containing starch or sucrose can be used as a substrate for ethanol fermentation by microbial processes. these “energy crops” include maize (corn), wheat, rice, potato, cassava, sugarcane and sugarbeet. currently used crops, however, are non-optimized for biofuel production. thousands of years of traditional breeding through phenotypic selection have resulted in modifications that are better fit for harvesting for feed or food uses (mclaren, 2005). genetic engineering can offer viable opportunities to increase bioethanol production from crop plants principally in two ways: modifying the biomass properties 64 farago, j. and/or increasing biomass yield. this article will review the latest advancements in the research and development focused on these two topics. 2. biomass properties 2.1 starch content and structure starch is by far the most important storage carbohydrate found in plants. annually, 20-30.106 tons of starch are produced to serve a wide range of industrial applications such as coating of textiles and paper, or as a thickening or gelling agent in the food industry (heyer, 2000). most of the starch is isolated from corn, but also potato, wheat and cassava are sources of starch. starch is deposited in the plastidic compartment of plants as granular material, for example in the amyloplasts of plant storage organs, such as seed andosperms or tubers. it is composed of two glucose polymers, amylose and amylopectin. in the linear polymer amylose, the glucose monomers are linked by α-1,4-glycosidic linkages. in contrast, the molecule of amylopectin is branched and various amount (about 5% in maize) of its glucose units are linked by α-1,6-glycosidic linkages. the molecular weight of amylopectin is higher (107-108 da) than that of amylose (105-106 da). normally, starches contain 70-80% amylopectin in contrast to 20-30% amylose (heyer et al., 1999). the biosynthesis of starch is sufficiently understood to allow genetic engineering of plants for higher accumulation or modified composition. alteration of starch structure and content can be achieved by modifying genes coding for enzymes responsible for starch synthesis (heyer, 2000; jobling, 2004). transgenic plants with modified expression of different starch synthases, starch branching enzymes or starch debranching enzymes have been produced to increase the accumulation of starch (tjaden et al., 1998; mckibbin et al., 2006) or to alter the crystallinity of starches and thus increase the accessibility to enzymatic digestion (baga et al., 1999; otani et al., 2007). stark et al. (1992) demonstrated an increase of up to 135% of control starch content was obtained following the constitutive expression of a regulatory variant of bacterial adpglucose pyrophosphorylase (agpase). another possibility is to decrease the gelatinization temperature of modified starches to reduce the energy requirement for the conversion of starch to ethanol. recently, for example, chiang et al. (2005) showed that overexpression of a thermostable and bifunctional starch hydrolase, amylopullulanase (apu) from thermoanaerobacter ethanolicus 39e in rice seeds resulted in starch, that could be hydrolyzed with optimal temperatures between 85 and 95 °c to complete conversion into soluble sugars. 2.2 lignocellulosic biomass lignocellulusic biomass is renewable, cheap and readily available with over over 10-50.109 tons produced per year at the global level (sticklen, 2006). lignocellulose is a more complex substrate than starch. it is composed of a mixture of different carbohydrate polymers (cellulose and hemicelluluse) and lignin. nova biotechnologica vii-i (2007) 65 the biological process for converting the lignocellulose requires several steps: (1) delignification to liberate cellulose and hemicellulose from their complex with lignin, (2) depolymerization of the carbohydrate polymers to produce free sugars, and (3) fermentation of mixed hexose and pentose sugar to produce ethanol (lin and tanaka, 2006). the major costs of biomass refineries include the pre-treatment processing of the lignocellulosic matter at the delignification step, and the cost of production of the microbial cellulases needed to convert the cellulose biomass into fermentable sugars (kabel et al., 2005; sticklen, 2006). genetic engineering provides tools to decrease both of these costs. several approaches have been use to accomplish these goals. firstly, genetic transformation of plants can decrease the lignin content and/or change the composition of lignin. lignins are complex three-dimensional polymers embedded in the cell walls of plant cells. in order for cellulases to access the cellulose for degradation, costly and harsh heat or acid pretreatment of biomass is required to remove lignin and hemicellulose from the lignocellulosic matter. transgenic manipulation of different lignin biosynthetic pathway genes have been attempted to decrease lignin content (piquemal et al., 2002; chen and dixon, 2007) or alter lignin composition (ralph et al., 2006) and thus reduce the pre-treatment costs. secondly, transgenic plants can be designed to overexpress enzymes for cellulases, such as endoglucanases, exoglucanases and βglucosidases, or produce recombinant (microbial) cellulases within the biomass crop. recently, montalvo-rodriguez et al. (2000) have used this approach to generate transgenic plants constitutively producing either a hyperthermofilic αglucosidase or β-glycosidase using genes derived from the archeon sulfolobus solfataricus. the authors showed that transgenic plant protein extracts released glucose from purified polysaccharide substrates at appreciable rates during incubation in high-temperature reactions. in another study, oraby et al. (2007) constitutively expressed the catalytic domain of acidothermus cellulolyticus thermostable endoglucanase gene (e1) in rice and observed that approximately 30% of the cellulose of ammonia fiber explosion-pretreated maize biomass was converted into glucose using rice e1 heterologous enzyme. lastly, genetic engineering can be employed to produce recombinant microbial cellulases and/or ligninases in bioreactors (e.g. galliano et al., 1988, jin et al., 2004). 3. biomass yield biomass yield is a very complex trait. unlike the genes for starch, cellulose and lignin biosynthesis, the majority of genes involved in the yield traits remain elusive. therefore, it was believed, that yield as a trait can not be easily manipulated through genetic engineering approaches. however, current achievements in genetic transformation technology suggest, that biomass yield increase and stabilization can be achieved through understanding and enhancing such mechanisms as stress tolerance (vinocur and altman, 2005, umezawa et al., 2006), nitrogen assimilation (harrison et al., 2000), and carbohydrate metabolism (sakamoto and matsuoka, 2004). crop yields can be increased by increasing leaf photosynthetic rates. considerable effort has been focused, for example, on molecular modification of 66 farago, j. photosynthesis by introducing the precursor pathway for organic acid fixation of co2 (c4 pathway) into c3 species (matsuoka et al., 2000; evans and von caemmerer, 2000). another way to improve photosynthesis may be modifying plant architecture to adjust to the high planting density currently used in agriculture (sakamoto and matsuoka, 2004). in cereals, grain yield is the main target for genetic improvement. adpglucose pyrophosphorylase is frequently referred as the rate limiting enzyme in starch biosynthesis and as such the key enzyme regulating sink strength. recent reports on transgenic deregulation of agp suggest a possibility to increase plant sink strength and subsequently the seed and biomass yield (smidansky et al., 2003, 2007). enhanced stress tolerance in plants through genetic engineering has been achieved through manipulation of effector genes (e.g. biosynthetic enzymes, ion transporters) or regulatory genes (e.g. transcription factors or signal transduction components) (fao, 2001). 4. conclusion in the future, fuel ethanol will remain an attractive option, at least because it is a renewable energy source, creating a substantial new market for different high energy crop supplies and new jobs. genetic engineering can effectively contribute to and provide great potential for future agriculture and biofuel production. however, to fully exploit the potential of modern biotechnology tools, it is necessary to increase the public acceptance of biotech-derived crops. also, in many countries the legislation concerning plant biotechnology and the regulatory system lags behind the advancement of a technology. however, genetic engineering to improve biomass characterization will be economically feasible if the social benefits from the adoption of the technology will be greatest than its social costs. references båga, m, repellin, a., demeke, t., caswell, k., leung, n., chibbar, r.n., abdel-aal, l.-s., hucl, p.: wheat starch modification through biotechnology. starch-stärke, 51, 1999, 111-116. bothast, r.j., schlicher, m.a.: biotechnological processes for conversion of corn into ethanol. appl. microbiol. biotechnol., 67, 2005, 19-25. chen, f., dixon, r.a.: lignin modification improves fermentable sugar yields for biofuel production. nature biotechnol., 25, 2007, 759-761. chiang, c.-m.; yeh, f.-s.; huang, l.-f.; tseng, t.-h.; chung, m.-c.; wang, c.-s.; lur, h.-s.; shaw, j.-f.; yu, s.m.: expression of a bi-functional and thermostable amylopullulanase in transgenic rice seeds leads to autohydrolysis and altered composition of starch. mol. breed., 15, 2005, 125-143. de oliveira, m.e.d., vaughan, b.e., rykiel, e.j.: ethanol as fuel: energy, carbon dioxide balances, and ecological footprint. biosci., 55, 2005, 593-602. evans, j.r., von caemmerer, s.: would c4 rice produce more mass than c3 rice?. in: j.e. sheehy et al. 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biotechnol., 17, 2006, 113-122. vinocur, b., altman, a.: recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. curr. opin. biotechnol., 16, 2006, 123-132. nova biotechnol chim (2022) 21(1): e1083 doi: 10.36547/nbc.1083 1 nova biotechnologica et chimica isolation and statistical optimization of rhodanese (a thiosulphate sulphur transferase) production potential of klebsiella oxytoca jcm 1665 using response surface methodology babamotemi oluwasola itakorode1,2,, raphael emuebie okonji2 1department of chemical sciences, oduduwa university ipetumodu, ile-ife, osun state, nigeria 2department of biochemistry and molecular biology, faculty of science, obafemi awolowo university ile-ife, osun state, nigeria  corresponding author: itakorgsoli29@gmail.com article info article history: received: 2nd august 2021 accepted: 20th december 2021 keywords: klebsiella oxytoca optimization polymerase chain reaction response surface methodology rhodanese 4 abstract microorganisms are increasingly being used in cyanide bioremediation. several organisms have been reported to thrive in cyanide contaminated wastewater due to their ability to produce cyanide detoxifying enzymes. however, to improve the production efficiency of these enzymes combinations of process variables need to be optimized. in this study, klebsiella oxytoca jcm 1665 was isolated from industrial wastewater, identified by sequencing its 16s rrna gene and subjected to rhodanese production using submerged fermentation. the conditions for production were optimized using response surface methodology (rsm). central composite design was employed to evaluate the effects of three production parameters – peptone (1 – 5 %), kcn (0.1 – 0.5 %), and time of incubation (1 – 24 h). second-order polynomial model was used to predict the response. rhodanese activity in the experiments varied from 0.05 to 7.5 ru.mg-1. under the optimum conditions of 4.35 % peptone, 0.4 % kcn and incubation time of 13 hr., the value for rhodanese yield was 7.810 u.ml-1. the r2 value for the model was 0.9925 (r2 = 0.9925). also, the experimental values are in accordance with those predicted, indicating the suitability of the employed model and the success of rsm in optimizing the production conditions. introduction cyanides are toxic chemicals that are widely present in both the abiotic and biotic components of an ecosystem. it acts as a defense mechanism in various organisms such as fungi, bacteria, algae and plants. however, the amount of cyanide generated by these organisms is insignificant when compared to the ones generated through anthropogenic activities (luque-almagro et al. 2016). in industries, cyanide is used for gold extraction and in the synthesis of various agrochemicals such as fertilizers, liming and pesticides. cyanide toxicity is due to its high binding affinity to metal ions; it inhibits metalloenzymes especially the cytochrome c oxidase in the electron transport chain (luquealmagro et al. 2016). the amount of cyanide in the environment has been observed to have increased greatly due to rapid industrialization and cyanide leaching methods have been the major techniques in gold extraction. the demand for gold jewellery in countries like the united states, russia and most african countries has led to the increase mailto:itakorgsoli29@gmail.com nova biotechnol chim (2022) 21(1): e1083 2 in mining activities (mudder et al. 2000). industrial processes such as mining and electroplating have contributed significantly to the cyanide contamination in water bodies. to minimize environmental disasters, cyanide-containing liquid must be properly treated before releasing into the water bodies. the removal of cyanide from the effluent is one of the challenges in the cyanidebased industries to fulfil their standards and recycling of the water (kuyucak and akcil 2013). despite the toxicity of cyanide, cyanotrophic microorganisms such as pseudomonas sp. (akcil and mudder 2003; oyedeji et al. 2013), bacillus pumilus (kandasamy et al. 2015), and bacillus cereus (itakorode et al. 2019) has been reported to survive in the presence of cyanide due to their ability to synthesize cyanide metabolizing enzyme such as rhodanese. rhodanese catalyzes cyanide detoxification by transferring sulfur from a suitable substrate such as thiosulfate to cyanide. this leads to the formation of a less toxic compound (thiocyanate) that can be excreted in the urine (eq. 1): s2o3 2+ cn scn+ so3 2 (1) where s2o3 2 – thiosulfate, cn– cyanide ion, scn– thiocyanate ion, so3 2– sulfite. its role as a detoxification enzyme is supported by its mitochondria and cytosolic localization (cipollone et al. 2007; steiner et al. 2018). although the use of microorganisms has been proven to be viable in cyanide bioremediation, enzymatic proteins may be of good alternative (gianfreda et al. 2004; 2010). enzymes can retain its activity at extreme conditions that limit microbial activities. also, they are not inhibited by inhibitors of microbial metabolism and the technique is eco-friendly. therefore, the importance of producing cyanide metabolizing enzymes for industrial application cannot be overemphasized. many factors including incubation time, the temperature, the ph, carbon and nitrogen sources contribute to the efficacy of metabolite production by organisms (adekunle et al. 2017; itakorode et al. 2019). optimization of metabolites production may be achieved by either one factor at a time or statistical means (rodrigues et al. 2008; annegowda et al. 2012). response surface methodology (rsm) is a statistical experimental protocol used in mathematical modelling (triveni et al. 2001; gong et al. 2012). rsm technique reduces the experimental assays and improves the statistical interpretation and interaction between variables (tsapatsaris et al. 2004; yim et al. 2012). the rsm can give a mathematical equation. it is also helpful to calculate the response value when different levels of variables are set. central composite design is a widely used protocol in response surface methodology (yang et al. 2008; rao 2010). the impact of industrialization on the quality of the environment is evident and there is a need to have eco-friendly strategies to treat effluent for the sustainable development of industries. hence, this study aims to investigate optimal production conditions of rhodanese by klebsiella oxytoca. experimental wastewater was collected from iron and steel smelting company in sterile plastic bottles and stored at 4 oc. it was centrifuged at 5,000 × g for 10 min. the clarified supernatant was collected and used for further analysis. physicochemical parameters such as ph, turbidity, chemical oxygen demand, total dissolved solids, dissolved oxygen, cadmium and lead were determined using the methods described by ademoroti (1996). isolation and screening for rhodanese production a loopful of diluted sample was spread on a modified bushnell hass agar plate and incubated at 37 °c for 96 h to select for cyanide degrading bacteria. rhodanese producing potential of the isolates was done by employed the method described by zlosnik and williams (2004). production medium consists of nacl (0.5 g/100 ml), bacteriological peptone (1 g/100 ml) and kcn (0.3 g/100 ml) at ph of 6.0. the growth media prepared were inoculated with standardized cells suspension. the culture media were checked for rhodanese activity for 24 h at an interval of 2 h. rhodanese nova biotechnol chim (2022) 21(1): e1083 3 isolates identification and characterization the most productive strain was identified by sequencing its 16s rrna gene. this was carried out at the bioscience centre of the international institute of tropical agriculture (ibadan, oyo state, nigeria). dna extraction was carried out using modified method of trindade et al. (2007). the assay mixture for polymerase chain reaction (pcr) amplification consists of 4 µl of the dna solution, 0.4 µl of 10 mm dntps, 2 µl of 25 mm mgcl2, 1 µl of 10 pmol each of primer (forward 5’-ccagcagccgcggtaatacg-3’ and reverse 5’-tcggctaccttgttacgacttc-3’) (yamamoto and harayama 1995), 0.24 µl of taq polymerase (1 u.µl-1) (promega usa) and the 5 µl of 5× pcr buffer. sterile dnase free water was added to make a volume of 25 µl. pcr was conducted in an automated thermal cycler. the thermal conditions were as follows: denaturation at 94 oc for 1 min, annealing at 56 oc for 1 min and 1 min extension at 72 oc. the pcr amplicons were visualized using 1.5 % agarose gel electrophoresis. enzyme assay and experimental design sodium thiosulfate (na2s2o3) and potassium cyanide (kcn) were used as substrates for rhodanese activity. 1 ml of the assay mixture consists of 50 mm borate buffer (ph 9.4), 0.25 m kcn, and na2s2o3 and 0.1 ml of the enzyme. the enzyme reaction was terminated with 0.5 ml 38 % formaldehyde after 1 min of incubation at 25 oc (lee et al. 1995). concentration of thiocyanate produced was quantified by the addition of 1.5 ml sorbo reagent (which is made up of 10 g fe (no3)2·9h2o, 20 ml hno3 and 80 ml distilled water) (sorbo 1953). the absorbance of the reaction medium was taken at 460 nm. the unit of rhodanese activity (ru) is defined as the micromoles of the product (thiocyanate) formed in one minute. the protein concentration was determined by the method of bradford (1976), bovine serum albumin (bsa) was used as standard. the extracellular production of rhodanese was established by rsm which was employed to determine the best combination of variables for optimum rhodanese production by k. oxytoca. the independent variables used in this study were peptone concentration (a – % (w/v)), potassium cyanide (b – % (w/v)), and time (c – h) while response was rhodanese activity (ru.mg-1). the coded and uncoded levels of the independent variables are presented in table 1. table 1. coded and decoded levels of independent variables used in the rsm design. run a: peptone b: kcn c: time 1 -1 (0.36) 0 (0.30) 0 (12.50) 2 -1 (1.00) +1 (0.50) -1 (1.00) 3 +1 (6.30) 0 (0.30) 0 (12.50) 4 0 (3.00) 0 (0.30) 0 (12.50) 5 -1 (1.00) -1 (0.10) +1 (24.00) 6 +1 (5.00) +1 (0.50) -1 (1.00) 7 0 (3.00) 0 (0.30) 0 (12.50) 8 -1 (1.00) +1 (0.50) +1 (24.00) 9 +1 (5.00) -1 (0.10) +1 (24.00) 10 0 (3.00) -1 (0.03) 0 (12.50) 11 0 (3.00) 0 (0.30) +1 (31.84) 12 0 (3.00) 0 (0.30) 0 (12.50) 13 0 (3.00) +1 (0.60) 0 (12.50) 14 0 (3.00) 0 (0.30) 0 (12.50) 15 0 (3.00) 0 (0.30) 0 (12.50) 16 +1 (5.00) +1 (0.50) +1 (24.00) 17 0 (3.00) 0 (0.30) -1 (6.84) 18 +1 (5.00) -1 (0.10) -1 (1.00) 19 0 (3.00) 0 (0.30) 0 (12.50) 20 -1 (1.00) -1 (0.10) -1 (1.00) statistical analysis the experimental data were analyzed using jmp 11’s response surface regression algorithm (statistical analysis system inc., sas). p-values under 0.05 (p < 0.05) were considered significant. anova was used to assess the quality of the mathematical models fitted by rsm, based on the f-test and the percentage of total explained variance (r), as well as the adjusted determination coefficient (r2adj), which provide a measurement of how much of the variability in the observed response values could be explained by the experimental factors and their linear and quadratic interactions (granato et al. 2012). to fit the data, a second-order polynomial quadratic equation (eq. 2) was used. (2) nova biotechnol chim (2022) 21(1): e1083 2 where y is the predicted response, β0, βi, βii, βij, are the correlation coefficients for intercept, linear, quadratic and interaction terms, respectively and xi and xj are the levels of the independent variables. experimental data were fitted to the chosen regression model to have a better understanding of the relationship between each variable and response. the predictive equation of rsm was used to find the optimal conditions for the production of rhodanese. the validity of the model was determined by comparing the experimental and predicted response values. results isolation and identification of the bacteria isolate table 2 shows the physicochemical properties of the wastewater such as the chemical oxygen demand, ph, turbidity, total dissolved solids, dissolved oxygen, lead, and cadmium. the selection of microorganisms from the wastewater led to the isolation of nine gram-positive and twogram negative bacteria. one strain was chosen for further study due to its appreciable rhodanese production. molecular analysis based on 16s rrna gene sequencing revealed that isolates jcm 1665 shared 99.65 % homology with k. oxytoca ay873801, 99.72 % homology with k. oxytoca mf144436 and 99.79 % homology with k. oxytoca mt568561 strain (table 3). table 2. physiochemical analysis of the wastewater. parameter ph 6.0 ± 1 concentration of parameter [mg.l-1] turbidity 8.23 ± 2.74 chemical oxygen demand 74.38 ± 5.19 total dissolved solids 536 ± 10.6 lead 0.02 ± 0.002 cyanide 67.49 ± 9.2 cadmium 0.024 ± 0.006 dissolved oxygen 3.94 ± 0.88 table 3. sequence identity of k. oxytoca (mn590525) with other klebsiella oxytoca. klebsiella oxytoca % identity accession in the genbank database mt568561 99.79 mt568561 mf144436 99.72 mf144436 ay873801 99.65 ay873801 the sequence was deposited in the genbank database of the ncbi as accession mn590525 (https://www.ncbi.nlm.nih.gov/nucleotide/). analysis of the model the result for the analysis of the model for the production optimization is shown in table 4. in this model, two linear (b and c) and five quadratic models (ab, ac, a2, b2, and c2) were found to be significant at the level of p < 0.05. table 4. experimental and predicted result for rhodanese activity. run response (activity u.mg-1) actual value predicted value 1 5.00 5.00 5.30 2 2.50 2.50 2.13 3 6.00 6.00 5.69 4 7.10 7.10 7.25 5 5.70 5.70 5.50 6 5.50 5.50 5.71 7 7.20 7.20 7.25 8 5.70 5.70 5.71 9 2.00 2.00 2.38 10 3.50 3.50 3.35 11 3.50 3.50 3.43 12 7.10 7.10 7.25 13 6.50 6.50 6.64 14 7.50 7.50 7.25 15 7.20 7.20 7.25 16 6.00 6.00 5.94 17 0.05 0.05 0.10 18 2.00 2.00 2.00 19 7.40 7.40 7.25 20 1.70 1.70 1.77 the result for the fitting quadratic model is listed in table 5. the result of the analysis of variance (anova) indicates that the model was significant (p < 0.05) for the response of the dependent variables (rhodanese activity). the result also indicates a good model performance with correlation coefficient (r2) values of 0.9925. the fitted quadratic model for rhodanese production is shown in eq. 3. 4 https://www.ncbi.nlm.nih.gov/nucleotide/ nova biotechnol chim (2022) 21(1): e1083 3 table 5. analysis of variance (anova) for response surface quadratic model for the production of rhodanese. source ss df ms f-value p-value model 99.35 9 11.04 147.52 < 0.0001* a-peptone 0.1832 1 0.1832 2.45 0.1487 b-kcn 13.04 1 13.04 174.28 < 0.0001* c-time 13.35 1 13.35 178.40 < 0.0001* ab 5.61 1 5.61 74.99 < 0.0001* ac 5.61 1 5.61 74.99 < 0.0001* bc 0.0112 1 0.0112 0.1503 0.7063 a² 5.56 1 5.56 74.36 < 0.0001* b² 9.18 1 9.18 122.69 < 0.0001* c² 54.15 1 54.15 723.62 < 0.0001* ss – sum of squares; df – degrees of freedom; ms – mean square. r2 = 0.9925; r2 adj = 0.9858; cv = 5.52 %. (*values statistically significant at p < 0.05). analysis of response surface the best way of expressing the relationship between the independent variables and dependent variables is to graphically plot the response surface plots generated by the model (fig. 1, 2, 3). fig. 1 showed the interaction between potassium cyanide (kcn) and incubation time while holding peptone at the center point (0). fig. 2 showed the interaction between potassium cyanide (kcn) and peptone while incubation time is held constant. fig. 3 showed the interaction between incubation time and peptone while kcn is held constant. fig. 1. response surface plots showing the effects of kcn (% w/v) and peptone (% w/v) on rhodanese production. fig. 2. response surface plots showing the effects of incubation time (hr) and peptone (% w/v) on rhodanese production. 5 nova biotechnol chim (2022) 21(1): e1083 2 fig. 3. response surface plots showing the effects of incubation time (hr) and kcn (% w/v) on rhodanese production. discussion klebsiella oxytoca was isolated from an industrial effluent, identified and subjected to extracellular rhodanese production. rhodanese production ability has been identified in a variety of bacteria species such as e. coli (ray et al. 2000), p. aeruginosa (cipollone et al. 2008), azotobacter vinelandii (kaewkannetra et al. 2009), bacillus brevis (oyedeji et al. 2013) and bacillus cereus (itakorode et al. 2019). enzyme production by an organism is often dependent on the growth of the bacterium in the appropriate media composition. the optimization of culture media and environmental conditions is essential for effective production as it tends to reduce cost of production by increasing yield (george-okafor et al. 2012). the optimum nutritional requirement for rhodanese production was determined to obtain maximum enzyme production by k. oxytoca. a central composite design (20 runs) was chosen with start and center points. the design was rotatable; this means that the designs have points that are equidistant from the center. experiments at the center were carried out to obtain an estimation of the experimental error. the designed experiment matrix and the experimental results are presented in tables 1 and 4. the rhodanese yield varied (from 0.05 to 7.5 ru.mg-1). the rhodanese production yield reached its maximum value (7.5 ru.mg-1) at 3 % of peptone, 0.3 % of kcn and incubation time of 12.5 h (run 17). as noted, rhodanese producing ability of k. oxytoca was affected by the time of incubation. the analysis of the overall data set indicated that incubation time, kcn and interaction ac had the most pronounced effects on the response (table 5). also, from eq. 3, it is evidence that kcn (b) concentration and incubation time (c) had the highest positive effect on the rhodanese production while interaction (ac) and (c2) had the highest negative effect on production. analysis of variance (anova) was important in determining the adequacy and significance of the quadratic model. anova summary is shown in table 5. the fitness of the model was expressed by the r2 value, which is 0.9925, indicating that 99.25 % of the variability in the response can be explained by the model. the adjusted r2 value of 0.9858 suggested that the model was significant. a very low value of coefficient of the variation (cv) 5.52 % indicated a very high degree of precision and a good deal of reliability of the experimental values. the interaction between kcn and peptone concentration at a constant time of incubation is shown in fig. 1. the result showed that rhodanese production increases as the concentration of both variables increases. at high concentrations, a gradual fall in production was observed. research had shown kcn and peptone to influence the production of cyanide detoxifying enzymes (adekunle et al. 2017). itakorode et al. (2019) reported kcn as the best carbon source for b. cereus. the ability of p. putida and b. pumilus to make use of cyanide as carbon source was also reported by kandasamy et al. (2015). okonji et al. (2018) also reported the ability of pseudomonas straminea to make use of peptone as nitrogen source. the high cyanide tolerance of k. oxytoca observed was probably due to its ability to synthesize rhodanese as a means of surviving in cyanide contaminated environment. fig. 2 showed the interaction effect of peptone concentration and incubation time while keeping kcn concentration constant. in this interaction, minimal rhodanese production was observed at a low concentration of peptone. however, as concentration and incubation 6 nova biotechnol chim (2022) 21(1): e1083 3 time increases, an increase in rhodanese activity was recorded. the same trend was also observed in the interaction between incubation time and kcn. however, a decline in production was noted at high time of incubation. the decline in rhodanese production may be due to exhaustion of the nutrients or accumulation of other products or metabolites which are both inhibitory to the growth of the bacterium and rhodanese production. this result agrees with the findings of okonji et al. (2018) who observed a decrease in metabolite production after 15 hours of incubation. linawati et al. (2002) also reported the influence of environmental conditions on the production of pigment by serratia marcescens. the optimal value of the independent variables for rhodanese production was examined using the maximum desirability. the result of optimal conditions used to obtain the highest rhodanese production by k. oxytoca were 4.35 % peptone, 0.4 % kcn and incubation time of 13 hours, at which rhodanese yield was 7.810 ru.mg-1. conclusion it can be concluded that the regression equations obtained in this study can be used to maximize the rhodanese production by k. oxytoca. also, further study is required to improve the production of the enzyme through genetic engineering and to examine the cyanide bioremediation potential of the rhodanese synthesized by the organism. conflict of interest the authors declare that they have no conflict of interest. references adekunle ap, torimiro n, okonji re (2017) a study on 3mercaptopyruvate sulphurtransferase (3-mst) produced under submerged 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(2004) method of assaying cyanide in bacterial culture supernatant. lett. appl. microbiol. 38: 360-365. 8 microsoft word jeric nb 9-2.doc nova biotechnologica 9-2 (2009) 155 chemometric characterization of textile waste waters from different processes tina jerič1, alenka majcen le marechal1, darja kavšek2, darinka brodnjak vončina3 1faculty of mechanical engineering, university of maribor, smetanova 17, 2000 maribor, slovenia 2regional technological centre zasavje, chemical-technological laboratory, nasipi 48, 1420 trbovlje, slovenia 3faculty of chemistry and chemical engineering, university of maribor, smetanova 17, 2000 maribor, slovenia abstract: the aim of this work is focused on water quality classification of the textile waste water streams and evaluation of pollution. data from the chemical characterization of the effluents were elaborated to identify a useful separation in potentially treatment for reuse. this was done with the aim of realizing a full scale characterization of effluents. in the two textile companies analyzed, machineries are used to carry out different production processes such as sizing and desizing, weaving, scouring, bleaching, mercerizing, carbonizing, fulling, dying and finishing. different process effluents from the same machinery were found to be very diverse in pollution level. 25 and 49 samples of textile waste waters from two different textile companies were analysed and physical chemical measurements were performed. the following physicochemical and chemical water quality parameters were controlled: absorbance measured at three different wavelengths, ph, conductivity, turbidity, total suspended solids, volatile suspended solids, chemical oxygen demand, metals content (ba, ca, cu, mn, k, sr, fe, al, na) and total nitrogen content. for handling the results, basic statistical methods for the determination of mean and median values, standard deviations, minimal and maximal values of measured parameters and their mutual correlation coefficients, were performed. different chemometric methods, namely, principal component analysis (pca), cluster analysis (ca), and linear discriminant analysis (lda) were used to find hidden information about textile waste water quality. key words: textile waste waters, water quality, chemometrics, principal component analysis, classification 1. introduction the chemical and physicochemical studies on textile waste water streams from two textile companies were performed in the time period 2008-2009. the quality of the textile waste water streams was followed and the classification has been made according to different textile process outlets. the classification is based on 19 chemical and physicochemical parameters. the aim of this work is to find the correlation between different textile process outlets and the variables obtained by physicochemical and chemical measurements, which can be used to construct a fast decision model for separating different textile waste water quality outlets. chemometric methods have been often used for the classification and comparison of different water samples (massart et al., 1997). some examples of chemometric characterizations of waste water and pollution of water are described, for instance, in the case of chemi-thermo-mechanical pulp mill (tønning et al., 2005), micro156 jerič, t. et al. electronics industry (velinova and koumanova, 1995), and in the case of waste waters in the lagoon of venice (carrer and leardi, 2006). chemometric methods have been used also for surface water quality and pollution estimation (astel et al., 2008; kowalkowski et al., 2006; astel et al., 2007), for characterization of water springs (ragno et al., 2007), for natural mineral waters (sarbu and zwanziger, 2001; silva et al., 2002), for classification of ground waters (panero and da silva, 2008), and for the oceanographic characterization of water (ferreira et al., 1999). the quality of the textile waste water streams was studied through the years 2008 and 2009. the monitoring programme was performed on different textile process waste water effluents (outlets) from two textile companies. textile waste water effluents were gathered from different textile processes (printing, finishing, dyeing, bleaching, washing, rinsing, mercerizing, softening and desizing) and from different machinery. altogether 19 characteristic features were measured for 25 and 49 samples of textile waste waters from two textile companies, respectively. water samples were collected and analysed during two years. several chemometric methods were applied in order to visualize multivariate data and to enable a quick classification of samples, regarding to textile process waste water effluents (outlets). 2. materials and methods a standard method was used for sampling (iso 5667-01: 1996 (e)). textile waste water samples were collected in polyethylene bottles at the outlet. standard analytical methods were used for the determination of 19 chemical and physicochemical variables. all reagents were analytical grade. the milli-q system was used for purifying the water. 25 and 49 samples of textile waste water from two textile companies respectively, are characterized by 19 chemical and physicochemical variables: (1) ba, (2) cu, (3) mn, (4) k, (5) sr, (6) fe, (7) al, (8) na, (9) ca, (10) total nitrogen, (11) ph, (12) conductivity, (13) turbidity, (14) chemical oxygen demand, (15) total suspended solids, (16) volatile suspended solids, (17) absorbance at 436 nm, (18) absorbance at 525 nm, and (19) absorbance at 620 nm. the results of all measurements have been investigated by different chemometric methods (massart et al., 1997): the basic statistical methods for the determination of mean and median values, standard deviations, minimal and maximal values of measured variables and their mutual coefficients. the principal component analysis (pca) (massart et al., 1997; teach/me datalab 2.002, 1999) was applied for grouping of textile waste water samples due to measured variables. all the calculations and plots in the following (pca) section were done with the teach/me software (teach/me datalab 2.002, 1999) using teach/me data analysis option. 3. results and discussion 3.1 statistical screening of data the mutual correlation was sought for all measured variables. the maximal correlation coefficient for the data of 25 waste water samples from the first textile nova biotechnologica 9-2 (2009) 157 company was found between measurements of absorbance at 620 nm and copper (r = 0.99), between absorbance at 620 nm and absorbance at 525 nm (r = 0.99), between absorbance at 525 nm and copper (r = 0.98), between total suspended solids and volatile suspended solids (r = 0.98), between absorbance at 620 nm and potassium (r = 0.97), and between absorbance at 525 nm and potassium (r = 0.96). the maximal correlation coefficient for the data of 49 waste water samples from second textile company was found between measurements of absorbance at 525 nm and absorbance at 436 nm (r = 0.99), between measurements of sodium content and electrical conductivity (r = 0.98), between total suspended solids and volatile suspended solids (r = 0.98), between potassium and manganese (r = 0.90), between absorbance at 620 nm and calcium (r = 0.86), and between absorbance at 436 nm and sodium (r = 0.86). fig. 1. dendrogram of 49 textile waste water samples from the second textile company. different shades correspond to 9 different textile process waste water outlets (classes). cluster analysis resulted in dendrogram shown in fig. 1, where 49 textile waste water samples are divided into number of clusters, depending on the level of similarity based on ward distance. first group of textile waste water samples (the right-most cluster) is quite well distinguished from others. this was also the most polluted water stream, as it was first outlet after dyeing process. 3.2 principal component analysis (pca) pca was performed to find the correlation of 25 or 49 textile waste water samples from two different textile companies, respectively, described with physicochemical 158 jerič, t. et al. and chemical variables (fig. 2). quality of textile waste waters depends on different textile processes and different waste water outlets. first outlet is always the most polluted one, as it belongs to the dyeing process. all other outlets are less polluted as they belong to rinsing processes. pca was applied on the matrix composed of 25x19 and 49x19 elements. 25 or 49 rows represent textile waste water samples from two textile companies, respectively, composed of 19 variables. "column centering" of the data was used, which means that the mean value of each column was subtracted from individual (25 or 49) elements. fig. 2. pca for 25 textile waste water samples from five different textile process waste water outlets denoted by class numbers from 1 to 5 and for 49 textile waste water samples from nine different textile process waste water outlets denoted by class numbers from 1 to 9. fig. 3. pca for textile waste water samples from nine and five different textile process waste water outlets; the loadings in 19 pc axes are shown. the pca of the textile waste water samples represented with 19 variables is shown in fig. 3. it can be seen that the first component, pc1, is associated with a group of variables such as conductivity and concentration of sodium, describing inorganic nova biotechnologica 9-2 (2009) 159 pollution, while the second component pc2 represents mainly the dependence on chemical oxygen demand, which is responsible for organic pollution. 3.3 linear discriminant analysis (lda) lda is a supervised learning method, which can determine the classification into predetermined classes. from figures 4a and 4b it can be seen that first class (first outlet from dyeing) for samples from both textile companies is well distinguished from all other outlets, belonging to rinsing processes. plot of discriminant functions -13 -3 7 17 27 37 47 function 1 -5,4 -3,4 -1,4 0,6 2,6 4,6 fu nc tio n 2 class 1 2 3 4 5 centroids fig. 4a. linear discriminant analysis for 25 samples. plot of discriminant functions -4,6 -2,6 -0,6 1,4 3,4 5,4 function 1 -7 -4 -1 2 5 8 fu nc tio n 2 class 1 2 3 4 5 6 7 8 9 centroids fig. 4b. linear discriminant analysis for 49 samples. 4. conclusions the aim of this work is to find the best solution for both textile companies for reuse of waste waters. for water recycling it is necessary to find correlation between different textile waste water streams. chemometric methods can help in characterization of textile waste water effluents and thus can be used for separation of concentrated textile waste water streams from non concentrated ones. after collecting concentrated textile waste waters, evapoconcentration treatment process will be used before additional cleaning at waste water treatment plant. for treatment of non concentrated textile waste water streams aop processes, like h2o2/uv process, and 160 jerič, t. et al. membrane filtration processes will be used. the cleaned waste water with the best quality can then be reused in different textile production processes. references astel, a., tsakovski, s., barbieri, p., simeonov, v.: comparison of selforganizing maps classification approach with cluster and principal components analysis for large environmental data sets. water res., 41, 2007, 4566-4578. astel, a., tsakovski, s., simeonov, v., reisenhofer, e., piselli, s., barbieri, p.: multivariate classification and modeling in surface water pollution estimation. anal. bioanal. chem., 390, 2008, 1283-1292. carrer, s., leardi, r.: characterizing the pollution produced by an industrial area: chemometric methods applied to the lagoon of venice. sci. total environ., 370, 2006, 99-116. ferreira, m.m.c., faria, c.g., paes, e.t.: oceanographic characterization of northern sao paulo coast: a chemometric study. chemometr. intell. lab. syst., 47, 1999, 289-297. kowalkowski, t., zbytniewski, r., szpejna, j., buszewski, b.: application of chemometrics in river water classification. water res., 40, 2006, 744-752. massart, d.l., vandeginste, b.g.m., buydens, l.m.c., de jong, s., lewi, p.j., verbeke, j.s.: handbook of chemometrics and qualimetrics: part a, elsevier, amsterdam, 1997. panero, f.s., da silva, h.e.b.: application of exploratory data analysis for the characterization of tubular wells of the north of brazil. microchem. j., 88, 2008, 194-200. ragno, g., de luca, m., ioele, g.: an application of cluster analysis and multivariate classification methods to spring water monitoring data. microchem. j., 87, 2007, 119-127. sarbu, c., zwanziger, h.w.: fuzzy classification and comparison of some romanian and german mineral waters. anal. lett., 34, 2001, 1541-1552. silva, f.v., kamogawa, m.y., ferreira, m.m.c., nobrega, j.a., nogueira, a.r.a.: geographical discrimination of mineral waters from são paulo state through exploratory analysis. ecletica quim, 27, 2002, 91–102. teach/me, sdl software development lohninger; teach/me datalab 2.002, 1999, springer, berlin, developed by h. lohninger and the teach/me people. tønning, e., sapelnikova, s., christensen, j., carlsson, c., winther-nielsen, m., dock, e., solna, r., skladel, p., nørgaard, l., ruzgas, t., emnéus, j.: chemometric exploration of an amperometric biosensor array for fast determination of wastewater quality. biosens. bioelectron., 21, 2005, 608-617. velinova, r.r., koumanova, b.k.: statistical modelling of wastewater quality: the case of micro-electronics industry. water res., 29, 1995, 2541-2547. water quality-samplingpart 10: guidance on sampling of waste water iso 5667-01: 1996 (e). microsoft word vrtoch nb.doc nova biotechnologica vii-i (2007) 41 bioaccumulation of 60co2+ ions in tobacco plants ľuboš vrtoch, martin pipíška, miroslav horník, jozef augustín department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (vrtochl@ucm.sk) abstract: tobacco has previously been used in investigations of metals and radionuclide uptake. this study presents determination of bioaccumulation and translocation of 60co2+ ions in tobacco plants (nicotiana tabacum l.) grown in hoagland’s nutrient solution. cobalt concentration in tobacco plants increased with increasing concentration in nutrient solution. bioaccumulation from the initial concentration c0 = 0.96 μm co reached 90% after 7 day cultivation. only small amounts of co accumulated in roots, up to 2 4 % were removable from roots by washing with 0.1 m cocl2, indicating that this portion of co is bound to the root surface in ion-exchangeable form. tobacco roots retained approximately 2/3 of accumulated cobalt and 1/3 was transported to shoots. autoradiography revealed that 60co was preferentially localized in younger leaves. prolongation of cultivation time did not change the [co]roots : [co]shoots ratio significantly. relationships between growth rate, transpiration rate, uptake and distribution of cobalt in plant tissue are discussed. key words: cobalt, 60co2+, nicotiana tabacum l., accumulation, hydroponics, autoradiography 1. introduction many papers explaining mechanism of uptake, translocation and detoxication of cobalt have been published (bakkaus et al., 2005; perez-espinoza et al., 2005; woodard et al., 2003; moreno-caselles et al., 1997). a number of mechanisms have been proposed on how cobalt is taken up and transported through the plant (woodard et al., 2003). early literature suggested that cobalt was present in xylem tissues in tomato as an inorganic cation (tiffin, 1967). others have suggested an organic anion of molecular weight from 1000 to 5000 da as found in ricinus communis (wiersma and van goor, 1979). it has been reported previously that cobalt is one of those metals that do not activate phytochelatin synthase (zenk, 1996). it was therefore suggested that cobalt cannot be detoxified via the phytochelatin system in plant cell (oven et al., 2002). this is in contrast with results obtained for other metals such as cd and zn. on the other hand, oven et al. (2002) identified cysteine as a compound involved in co complexation in cobalt hyperaccumulator crotalaria cobalticola suspension cells. cobalt also induced cysteine increase in non-hyperaccumulator species, suggesting that there are also other cellular mechanisms that enable cobalt tolerance and hyperaccumulation. tobacco has previously been used in other investigations of metals and radionuclides uptake (fuhrmann and lanzirotti, 2005; mench and martin, 1991). an excellent review on utilization of genetically modified tobacco 42 vrtoch, ľ. et al. and other plants for phytoremediation of toxic metal contaminated soil was published by eapen and d´souza (2005). in our paper, the accumulation of 60co by tobacco plants nicotiana tabacum l. has been studied with the aim to characterize cobalt bioaccumulation and translocation in roots, stems and leaves at hydroponic cultivation. the tobacco was chosen as a model system due to its high biomass production, easy hydroponic cultivation, and as a model of widely cultivated agricultural plant. 2. material and methods 2.1 plant material seeds of tobacco were germinated and grown in pots filled with granulated moist perlite as an inert carrier in day/night photoperiod 12/12 h (illumination with two fluorescent tubes fluora 550 lm, l18w/77, osram; one tube cool white 1150 lm, f18w/33, tungsram), temperature 22 ± 1°c, and relative humidity 50%. plants were irrigated with 25% hoagland nutrient medium (hoagland, 1920). after 2 months, seedlings were removed from perlite, roots were washed free of any adhering perlite granules by distilled water and pre-cultured for 7 days in 25 % hoagland nutrient medium in erlenmeyer flasks and used for experiments. plants used were about 15 cm tall and about 4.0 – 6.0 g of fresh weight. 2.2 bioaccumulation experiments plants were cultivated in 25 or 50% hoagland´s medium supplemented with 0.96; 9.6; 25 or 50 µmol.l-1 cocl2 spiked with 60cocl2 (125 kbq.l -1 ) for 7 or 14 days under the conditions as described in the previous section. bioaccumulation experiments were replicated three times. in 24 h intervals aliquot samples of nutrient solution were taken and 60co radioactivity measured by gamma spectrometry. at the end of experiments plants were harvested and roots carefully rinsed in 0.1 m cocl2 and distilled water. growth value (gv) was calculated following the equation gv = (m – m0) / m0, where m and m0 is plant fresh weight at the end and at the beginning of experiments respectively (soudek et al., 2006). radioactivity incorporated in roots, stems and leaves was measured by gamma spectrometry. material was dried in oven at 60 °c to a constant weight. after cultivation in the presence and in the absence of co2+ ions, tobacco leaves were observed with jenatech light microscope (carl zeiss). micrographs were taken with digital camera olympus camedia 4000. 2.3 radiometric analysis a gamma spectrometric assembly using the well type scintillation detector 54bp54/2-x, nai(tl) (scionix, the netherlands) and data processing software scintivision32 (ortec, usa) were used for 60co determination in plant and solution. counting time 600s allowed obtaining data with relative measurement error <2%, nova biotechnologica vii-i (2007) 43 which do not reflect other source of errors. standardized 60cocl2 solution (20 mg/l cocl2 in 3 g/l hcl) with specific radioactivity 5.181 mbq/ml was obtained from the czech institute of metrology, prague, czech republic). before autoradiography, roots were rinsed with distilled water, drops were wiped off; plants were pressed between two layers of filter papers and dried at laboratory temperature. dried plants were mounted on the x-ray film (hr-gb 100 nif, fujifilm, japan), and developed after 40 days. developed films were scanned and black and white film was converted to color scale gradient by software photoshop cs2 ver. 9.0 (adobe, usa). 2.4 transpiration rate transpiration rates (tr) were calculated from the daily weight losses of cultivation vessels containing plants, corrected for water evaporation of vessels media without plants. transpired water was daily replenished with distilled water to the initial volume. leaf area (m2) was calculated from weights of leaf copies (g) made from the paper sheet of known specific weight (g/m2). 3. results and discussion 3.1 cobalt uptake tobacco plants (nicotiana tabacum l.) cultivated in 25 % hoagland’s medium were tested to the ability accumulate co2+ ions from nutrient solutions. bioaccumulation at the initial cobalt concentration c0 = 0.96 μm reached 90 % within 7 days (fig. 1). the same kinetics (data not shown) and comparable uptake values 89.5±1.9 and 86.8±3.3% were observed for c0 = 9.6 and 25 μm respectively. at c0 = 50 μm cocl2, toxic effects appeared and total uptake decreased to 54.2±5.5% (table 1). transpiration rate (ml/day) at 22 ± 1°c and 50 % humidity was linear within 7 days (fig. 1). only small amount of cobalt, up to 2-4 % was removable from roots at the end of the experiment by washing with 0.1 m cocl2, indicating that this portion of cobalt was bound to the surface of the roots in ion-exchangeable form. at concentrations c0 ≥ 50μm cocl2, significant decrease in biomass production expressed by growth values (table 1), decrease in co uptake and transpiration rates (table 3) and chlorosis localized in interveinal areas of younger and new leaves were observed. the cobalt treatments within concentration range c0 = 0.96 25 μm had no significant effect on transpiration rates and therefore induced no toxic effect. the average transpiration rates 11.5, 11.6 and 11.9 ml.m-2.h-1 at 0.96, 9.6 and 25 μm respectively were obtained. the transpiration rate of tobacco plants was reduced to 8.3 ml.m-2.h-1 under higher co exposure (50 μm) (table 3). on the other hand, increasing amounts of co in the shoots was observed (table 1), indicating that under higher co exposure, the co transport mainly through the path of phloem and driven by the concentration gradient of co in the roots. similar cu transport in rice seedlings under extreme cu exposure observed chen et al., (2004). 44 vrtoch, ľ. et al. uptake of cations by root system is influenced by other ions present in solution. the increase of hoagland´s strength from 25 % to 50 % caused decrease of cobalt uptake from 90 % to 54 % at co = 9.6 μm cocl2 (table 2). this fact can be explained by competitive effects of bivalent cations. in more concentrated hoagland’s solution such as 50 %, also suppression of transpiration rate and lower gv values were also observed (table 2 and 3). inhibition of cobalt uptake by sunflower from 50 % hoagland´s medium was described in our previous paper (hornik et al., 2005). inhibition of transpiration by higher nutrient concentrations was described by kang and van iersel (2004). 0 30 60 90 120 150 180 0,0 0,2 0,4 0,6 0,8 1,0 c o2 + [μ m ol .l -1 ] t [h] 0 10 20 30 40 50 t ranspiration [m l] fig. 1. kinetic of co2+ bioaccumulation (□-□) and transpiration ( ) by tobacco plants grown in 25% hoagland medium at 22±1°c; c0 = 0.96 μm co2+. each point is the mean of three replicates. error bars represent standard deviation (sd) of the mean. tab. 1. uptake and distribution of co2+ ions in roots and aboveground parts of tobacco plants cultivated 7 days in 25 % hoagland’s hydroponic medium. co2+ [μg.g-1 dw ± sd] co2+ [μmol.l-1] gv co uptake [%] [co]root / [co]shoot] root stem leaves 0.96 0.26±0.06 90.5±0.2 9.3 27.2±12.0 1.8±1.6 1.1±0.6 9.6 0.25±0.07 89.5±1.9 11.8 582±97 27±11 22±3 25 0.23±0.06 86.8±3.3 10.7 1077±113 51±10 50±14 50 0.12±0.01 54.2±5.5 3.5 711±119 124±9 80±20 3.2 translocation of cobalt tobacco roots retained approx. 2/3 of bioaccummulated cobalt and only 1/3 was translocated into shoots. these data expressed in term of the cobalt ratio in root to shoot (65:35) changed only slightly with the increase of the initial cobalt concentration within the range c0 = 0.96 to 25 μm. more pronounced translocation of cobalt to shoots was observed at 50 μm cobalt in medium where cobalt ratio in root to shoot has changed to 44:56 (fig. 2). nova biotechnologica vii-i (2007) 45 autoradiography also demonstrated that 60co is preferentially localized in the roots and in young leaves. in leaves 60co is distributed around the veins and significant amount is also localized in leafstalks (fig. 4). gamma spectrometry showed the following cobalt 60co distribution: root 68 %; young leaves (leaf no. 4, 5, 6) approx. 20 %. stem and older leaves (leaf no 1, 2, 3) contained 8.7 % and 3.2 % 60co respectively. however, higher concentrations of nutrient salts in cultivation medium did not influence cobalt translocation in tobacco plants significantly (fig. 3a). 0 20 40 60 80 100 r el at iv e c o2 + di st rib ut io n in p la nt [% ] cocl2 [μmol.l -1] root stem leaves 0,96 9,6 25 50 fig. 2. relative distribution of co2+ ions in tobacco roots, stems and leaves after 7 days cultivation in 25% hoagland medium with initial concentration c0 = 0.96, 9.6, 25 and 50 μmol/dm3 cocl2 at 20 ± 2°c. each column is the mean of three replicates. error bars represent standard deviation (sd) of the mean. tab. 2. influence of strength of hoagland medium and cultivation time on uptake and translocation of co2+ ions by tobacco plants. c0 = 9.6 umol/l cocl2. time [day] gv hoagland´s strength [%] co uptake [%] co (root/shoot) ratio [μg.g-1 dw] /[μg.g-1 dw] 7 0.25±0.07 25 89.5±1.9 11.8 7 0.15±0.04 50 54.8±4.1 4.7 14 0.46±0.04 25 85.8±0.8 8.8 tab. 3. relation between co2+ uptake and transpiration rate (tr) of tobacco at different initial co2+ concentration in cultivation medium after 7 days cultivation. co2+ [μmol.l-1] co uptake [%] tr [ml.m-2.h-1] hoagland´s strength [%] 0.96 90.5±0.2 11.5±1.5 25 9.6 89.5±1.9 11.8±0.6 25 9.6 54.8±4.1 8.8±0.9 50 25 86.8±3.3 11.9±0.8 25 50 54.2±5.5 8.3±0.9 25 in our experiments cobalt was added to the nutrient solution as a single dose in such amounts that more than 90 % uptake was reached within 7 days of cultivation. we expected that during prolonged cultivation in the same cultivation medium, where 46 vrtoch, ľ. et al. cobalt was depleted, would result in additional cobalt translocation from roots to shoots by transpiration stream. this hypothesis was not confirmed. prolongation of cultivation from 7 to 14 days did not change root to shoot cobalt ratio significantly. as can be seen in fig. 3b, additional translocation of cobalt primarily localized in roots into shoots was not observed. the only cobalt movement was slight translocation from stems to leaves. on the contrary, page and feller (2005) observed negligible mobility of cobalt in wheat. cobalt 57co was trapped mainly in roots and bound so tightly, that was not significantly translocated to wheat shoots during the next 50 days of cultivation. differences in cobalt mobility in plant organs described in literature are explained by differences in biochemical composition of plant organs and differences in transport systems. 0,0 0,2 0,4 0,6 0,8 1,0 r el at iv e c o2 + di st rib ut io n in p la nt 9.6 μm co2+ 25% hoag 9.6 μm co2+ 50% hoag root leaves stem a 0,0 0,2 0,4 0,6 0,8 1,0 r el at iv e c o2 + di st rib ut io n in p la nt 9.6 μm co2+ 7 days 9.6 μm co2+ 14 days root leaves stem b fig. 3. relative distribution of co2+ ions in tobacco roots, stems and leaves in dependence on the concentration of hoagland nutrient medium (a), and on the cultivation time (b). each column is the mean of three replicates. error bars represent standard deviation (sd) of the mean. 3.3 phytotoxicity of cobalt it has been established that co like other pollutant elements are relatively toxic to plants when given in supranormal doses (chatterjee and chatterjee 2000; pandey et al., 2002; gopal et al., 2003). after 50 μm co treatment, tobacco showed interveinal chlorosis on young leaves and significant suppression of growth (fig. 5, table 1). similar toxic effect of cobalt on tomato plants was described by gopal et al., (2003). chlorosis is considered as indirect effect caused by: alterations nova biotechnologica vii-i (2007) 47 fig. 4. autoradiography of 60co2+ distribution in root, stem and leaves of tobacco after 7 days cultivation in 25% hoagland medium with initial concentration c0 = 1,9 μmol/l cocl2 (263 kbq/l 60cocl2) at 20 ± 2°c. autoradiogram was converted to color scale gradient by software photoshop cs2. single leaves are numbered as: 1 – the oldest (lower), 7 – the youngest (upper). relative distribution of 60co in plant parts calculated from gama-spectrometric data: 68.0% root; 8.7% stem; 0.8% leaf 1; 1.2% leaf 2; 1.4% leaf 3; 2.8% leaf 4; 5.4% leaf 5; 11.7% leaf 6+7. fig. 5. visible symptoms of tobacco plants to excess supply of co2+ (50 μm) after 7 day cultivation in nutrient solution: a-plant cultivated in the absence of co2+ (magnification 0,4 ×), b – plant cultivated in the presence of 50 μm co2+ (magnification 0,5 ×), c – leaf of co2+ non-treated plant (magnification 30 ×), d – leaf of 50 μm co2+ treated plant with developed interveinal chlorosis (magnification 10 ×). 5 6 1 2 3 7 4 48 vrtoch, ľ. et al. in the concentrations of essential mineral nutrients, a decrease in net photosynthesis as a consequence of stomatal closure, reduced intercellular spaces and by alterations within chloroplasts (chatterjee and chatterjee 2000; vazquez et al., 1987). on molecular level it was found, that co in supranormal concentrations caused inhibition of catalase activity, decrease of chlorophyll content connected with chlorosis (chatterjee and chatterjee, 2000; pandey and sharma, 2002). toxicity of cobalt at c0 50 μm resulted in both decrease of tobacco transpiration rates and cobalt uptake. 4. conclusion uptake of co2+ ions from nutrient hydroponic solution by tobacco plants is a process dependent on transpiration rate. cobalt is translocated from root to shot biomass in the ratio 70: 30. at concentrations c0 ≥ 50 μm co 2+, phytotoxic effects such as intervenial chlorosis was observed. uptake of co2+ ions by tobacco roots and translocation to shoots is influenced by the presence of other bivalent metal ions. cobalt can be considered as a next toxic agents accumulating in tobacco grown in contaminated soils. on the other hand, roots contain cobalt in concentrations 27 times higher than in the aboveground part calculated on dry weight biomass, what have to be taken in consideration in calculations of cobalt and radiocobalt persistence in soil/plant system. references bakkaus, e., gouget, b., gallien, j.p., khojda, h., carrot, f., morel, j.l., collins, r.: concentration and distribution of cobalt in higher plants: the use of micro-pixe spectroscopy. nucl. instrum. methods phys. res. section b, 231, 2005, 350-356. chatterjee, j., chatterjee, c.: phytotoxicity of cobalt, chromium and copper in cauliflower. environ. pollut., 109, 2000, 69-74. chen, ch-t., chen, t-h., lo, k-f., chiu, ch-y.: effects of proline on copper transport in rice seedlings under excess copper stress. plant sci., 166, 2004, 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23 metals recovery from acid mine drainage alena luptakova1, magdalena balintova2, jana jencarova1, eva macingova1, maria prascakova1 1department of mineral biotechnologies, institute of geotechnics of the slovak academy of sciences, watsonova 45, košice, sk-043 53, slovak republic (luptakal@saske.sk, jencarova@saske.sk, macingova@saske.sk, prascak@saske.sk) 2institute of building and environmental engineering, civil engineering faculty, technical university of košice, vysokoškolská 4, košice, sk-042 00, slovak republic (magdalena.balintova@tuke.sk) abstract: the objectives of the present work give the results view of some physicochemical, chemical and biological-chemical methods for the heavy metals removal from acid mine drainage (amd). the background of the studied physicochemical methods was the adsorption by turf, chemical methods the heavy metals precipitation by the neutralization with naoh. the principles of the biological-chemical methods were the bioprecipitation by the applications of sulphate-reducing bacteria (srb), the sorption by the bacterially produced iron sulphides and sorption by brown coal bio-modified by micromycetes. key words: acid mine drainage, neutralization, bioprecipitation, sorption, biosorption 1. introduction acid mine drainage (amd) is unique among industrial contaminants on the subject of mainly the mining industry of the sulphide minerals. amd generation proceeds increased considerably after the closure of mines. acid mine waters have low ph-values, and typically high concentrations of sulphates, iron and non-ferrous metals (kontopoulos, 1988), which serve as buffering systems. development of cost-effective and sustainable remediation solutions for the mine water problem has been the subject of extensive review. in addition to monitored natural attenuation, the two broad philosophies which have been pursued in the treatment and abatement of mine water pollution include measures directed towards prevention at source, usually involving physical intervention of one form or another, and measures directed at the resulting effluent, including active or passive remedial systems. both active and passive systems may be implemented using physicochemical, chemical or biological-chemical treatment technologies (skousen et al., 1998; kadukova and stofko, 2006a). nowadays is pay attention to physical, chemical and biological methods for the selective recovery of metals from amd. these methods constitute the possibility of the recovery metals in the suitable forms for commercial value. current research of heavy metals removal in water environment is aimed to application of natural materials as well as industrial wastes that are regarded as cost effective sorbents 24 luptakova, a . et al. (garcia sanchez et al. 1999). among the most often tested sorbents of heavy metals belong: zeolite, carbonate, clays, turf, oxide and hydroxide of iron. deorkar and tavlarides (1998) development of amd physicochemical treatment by application of an adsorption process comprised of inorganic chemically active adsorbents to selectively recover of fe, cu, zn, cd and pb from amd without neutralization. jacke and diebold (1983) evaluated the chemical treatment of amd on the ground of metal recovery from amd by addition of sulphides followed by oxidation and selective titration. cu and zn precipitated as sulphides and fe, al, mn and mg were recovered as hydroxides. tabak et al. (2003) conducted the biotreatment of amd by selective sequential precipitation to recover metals as hydroxides and sulphides. for the metals removal from amd can be used different biosorbents on the base of sawdust, algae, microscopic fungi, biogenous ferrous sulphides biomass and etc. (kadukova and stofko, 2006b; kadukova and vircikova, 2003). in slovak republic there are some localities with existing amd generation conditions. our previous research demonstrates the amd critical values in the abandoned cu – fe ore deposit smolnik (luptakova et al., 2006). it is necessary to develop methods for their treatment. that was the reason for starting a systematic monitoring of geochemical development in acid mine drainage in 2004 in order to prepare a prognosis in terms of environmental risk and use of these waters as an atypical source of a wide range of elements. on this account was our research oriented on the development suitable methods or their combination for the amd effluent treatment from deposit smolnik. we studied some physicochemical, chemical and biological-chemical methods. heavy metals model solutions and amd from smolnik were used for experiments. the background of the used physicochemical methods was the adsorption by turf brush peatsorb. experiments of the chemical methods were oriented on to study of the metals selective precipitation in the hydroxides form by the neutralization with naoh. the principle was the endpoint titration. the backgrounds of the biological-chemical methods were the bioprecipitation by the applications of sulphate-reducing bacteria (srb), the sorption by the bacterially produced iron sulphides and sorption by brown coal modified by different micromycetes strains. the metabolic process of srb is the anaerobic reduction of sulphates by the formation of hydrogen sulphide reacting in the water with cations of metals forming little soluble sulphides. investigated was the process of the heavy metals precipitation by bacterially produced hydrogen sulphide with the combination of the metals precipitation by sodium hydroxide at the various amd ph values. in the literature this methods is named as the selective sequential precipitation (ssp) (tabak et al., 2003). besides srb application gives rise to fe sulphides (fexsy) with the magnetic properties, which are suitable bio-sorbents of heavy metals and are utilizable for amd treatment in combination with the magnetic separation. for the heavy metals removal from solutions also unconventional sorbents prepared from brown coal by micromycetes were used. it was studied bio-modification of brown coal sorption materials with the aim to enhance its sorption properties. the micromycetes (aspergillus niger, aspergillus clavatus, nova biotechnologica 10-1 (2010) 25 penicillium glabrum and trichoderma viride) have been selected for biological activation of coal samples. 2. material and methods 2.1 model solutions stock solutions of each metal: cu(ii) containing 30-300 mg/l, zn(ii) containing 30-300 mg/l and cd(ii) containing 50-100 mg/l were prepared by dissolving cuso4.5h2o, znso4.h2o and 3cdso4.8h2o of analytical grade in distilled water. 2.2 acid mine drainage the experiments were conducted with amd coming from the abandoned and flooded deposit of smolnik (slovak republic). the average values of ph and the major metals composition of acid mine drainage was following: ph 3.9, so4 2 2 938 mg/l, fe 307 mg/l, mn 26 mg/l, cu 5 mg/l, zn 11 mg/l, al 77 mg/l. 2.3 adsorption metals by turf based on our previous results, where various adsorbents were tested (balintova and kovalikova, 2008) for our study of cu, fe, al, zn ions removal from acid mine drainage by adsorption, turf brush peatsorb (reo amos slovakia) was used. the dependence of cu, fe, al, zn concentration decreasing on time (1, 3, 5 and 10 min), was investigated under dynamic conditions using turf brush peatsorb. to intensify the adsorption process, sample of amd was continuously stirred with 5 g of turf brush peatsorb. in filtrate was determined ph (mettler toledo) and cu, fe, al, zn by colorimeter dr 890 (hach lange). 2.4 metals precipitation by naoh the precipitation by 1m naoh solutions was used for the removal of metals as hydroxides from amd. experiments were carried out by raw amd samples of 100 ml and each were titrated to ph end points ranging from 5 to 9 using 1m naoh. when the preset ph end point was reached, the titrated solution was filtered to precipitated metals removing. during titration the amd solution was continuously stirred, the ph was monitored and the concentration of metals was determined too. 2.5 metals precipitation by biogenic h2s and naoh for the production of the bacterially h2s the cultures of srb (genera desulfovibrio) were used. these bacteria were isolated from a mixed culture obtained from the potable mineral water (gajdovka spring, the locality kosice-north, slovakia). 26 luptakova, a . et al. this biological-chemical method contains several process steps and can be divided in to these main steps, as well as: the bacterially h2s production by srb; the heavy metals precipitation by the bacterially produced h2s; the heavy metal sulphides separation by the filtration; the setting ph of the filtrate from previous steps by 1m naoh (the precipitation of metals as hydroxides); the heavy metal hydroxides separation by the filtration; the subsequent precipitation of the heavy metals by bacterially produced h2s. values of ph for the heavy metals precipitation were assigned on the ground of the study of the orientation conditions for the selected metal removal from amd by precipitation using naoh and our previous works and enumerations (luptakova et al., 2003). the concentration of metals was determined by atomic absorption spectrometry using spectrometer aa-30 varian instrument. a glass ph electrode combined with the reference ag/agcl electrode was used to measure ph. digital phmeter gprt 144 agl was used. 2.6 sorption of metals by bacterially produced iron sulphides the preparation of biogenic sulphides was realized in the bioreactor filled with 400 ml of modified nutrient medium dsm-63 and inoculated with 100 ml of a culture of srb during 21 days at 30°c under anaerobic conditions. these conditions were generated by introducing an inert gas (n2) and chemically with sodium thioglycolate. the ph of the medium was adjusted to the value 6.8 with sodium hydroxide. preparation was realized under 2 different modes. during discontinuous mode the bioreactor worked without addition of fresh nutrient medium. during semicontinuous process of preparation the bioreactor worked 4 days in batch mode and then 3 days in continuous mode (i.e. fresh medium was supplied into reactor). batch sorption experiments were performed in 100 ml erlenmeyer flasks with the sorbent dose 1 g/l. sampling was conducted during 90 minutes. the concentration of zinc and cadmium was determined by atomic absorption spectrometry. 2.7 sorption by bio-modified brown coal the cultures of selected micromycetes were grown in sabouraud agar medium. suspension was injected (5 ml) of every 14-day-old culture spores in sab to the 10 g of brown coal mixed with 10 ml of sab medium. the cultivation was executed in the dark at ambient temperature. the leaching process took 7 weeks. after leaching, the suspensions were filtered, washed with distilled water, dried and prepared for adsorption experiments. all the adsorption experiments were conducted at ambient temperature in a laboratory shaker. metal solutions of known concentrations were introduced into the glass erlenmeyer bottles containing defined amounts (10 g/l) of the adsorbent. the bottles were shaken horizontally and the adsorbent was removed by filtration after 1 hour adsorption. the equilibrium concentrations of heavy metals were determined by atomic adsorption spectroscopy and the metal uptake was calculated from the difference. the langmuir adsorption isotherms have been constructed and the maximum adsorption capacity of the adsorbents has been nova biotechnologica 10-1 (2010) 27 determined. for cu(ii) sorption the ph reached 5, as cu(ii) ions undergo hydrolysis reactions in water and form insoluble aqueous complexes with increasing ph. zn(ii) sorption experiments were performed at ph 7 for the same reason as in case of cu(ii) adsorption. 3. results and discussion 3.1 adsorption metals by turf table 1 documents the physical-chemical method studies and there are also given the results of ph measuring in amd turf brush mixture depending on time and influence of adsorption processes on cu, fe, al, zn concentrations in individual samples. from the results follows, that it is possible to decrease the cu, fe, al and zn concentrations in polluted surface water by physical adsorption. the highest turf brush efficiency was observed for zinc removal, where the decrease of concentration in solution was 95.71%. then follows copper (decreasing in amd about 55.88%), iron (21.19%) and aluminium (15.6%). based on experimental results we can also state that chosen adsorbent have not influenced the ph increasing above 4 that is connected with precipitation of metals. table 1. dependence of cu, fe, al and zn removal from amd versus adsorption time. cu fe al zn adsorption time ph [mg/l] amd 3.95 1.36 358.2 54.5 17.5 1 min 3.10 0.86 347.8 52.7 1.5 3 min 2.95 0.61 353.3 46.4 1.2 5 min 2.97 0.61 333.5 46.2 1.0 10 min 2.93 0.60 282.3 46.0 0.75 3.2 metals precipitation by naoh the results of the titration test amd by 0.2m naoh documents fig. 1. it is a documentation of the titration curve and elemental amd analysis. the titration curve of amd can be divided into a number of by its different slopes. it shows that the optimum ph for metals precipitation is different for each metal: for al – ph 5; for cu – ph 6; for zn – ph 7; for fe – ph 8; for mn – ph 9. table 3 demonstrates results of metals precipitation by naoh that is titration of amd to ph end points for the individual metals ranging from 5 to 9 using 1m naoh. as it is seen from table 2 was removed 97% of al at ph 5; 99.9% of cu at ph 6; 99.9% of zn at ph 7; 99.9% of fe at ph 8 and 99.9% of mn at ph 9. initial assumption about precipitation of followed metals up to ph 9 has been confirmed. 28 luptakova, a . et al. 3.3 metals precipitation by biogenic h2s and naoh the selective sequential precipitation of heavy metals form amd sample was performed in two interconnected bioreactors with a capacity 1000 ml (the first bioreactor) and 250 ml (the second bioreactor), which operated at the semi-continual conditions. using the operating conditions and obtained results of the selective sequential precipitation of heavy metals form amd sample illustrated in table 3. the bacterially produced hydrogen sulphide by srb at ph 3.9 (initial ph of amd), 4.5 and 6.0 realized the selective sequential precipitation of cu, zn and fe, respectively (i.e. steps 1, 3 and 5). al and mn were precipitated as aluminium and manganese hydroxide at ph 6.0 and 9.0, respectively (i.e. steps 4 and 6). fe was precipitated predominantly as hydroxide (steps 2, 4 and 6). fig. 1. titration curve and metal amd analysis of amd from the deposit of smolnik. table 2. precipitation of al, cu, zn, fe and mn in ph dependence (initial ph of amd – 3.9). ph al (mg/l) cu (mg/l) zn (mg/l) fe (mg/l) mn (mg/l) 3.9 58.8 1.38 6.88 338.6 22.88 5 0.4 0.65 6.75 268.8 22.88 6 0.4 <0.02 5.38 9.8 22.88 7 0.4 <0.02 <0.03 2.5 20.38 8 0.4 <0.02 <0.03 <0.03 12.25 9 0.4 <0.02 <0.03 <0.03 0.03 table 3. metals precipitation by bacterially produced h2s and naoh (initial amd ph – 3.9). step 1. step 2. step 3. step 4. step 5. step 6. ph 3.9 4.5 4.5 6.0 6.0 9.0 precipitating agent h2s naoh h2s naoh h2s naoh removed metals cu fe, al zn al, fe fe mn, fe form of removed metals (solid phase) cus fe(oh)3 al(oh)3 zns al(oh)3 fe(oh)3 fes mn(oh)2 fe(oh)2 filtrate composition concerning of metals presence (liquid phase) fe, al, zn, mn fe, al, zn, mn fe, al, mn fe, mn fe, mn ----- nova biotechnologica 10-1 (2010) 29 3.4 sorption of metals by bacterially produced iron sulphides fig. 2 shows sorption of cadmium and zinc ions from model solutions by iron sulphides during 90 minutes by semi-continuous and discontinuous sorbents, when initial concentration of metal ions in model solutions was 50 mg/l. 0 5 10 15 20 25 0 10 20 30 40 50 60 70 80 90 time [min] so rp ti on [ m g/ g ] cd-ss (50) cd-ds (50) zn-ds (50) zn-ss (50) fig. 2. sorption of cadmium and zinc ions from model solutions. the quantities of metal ions that iron sulphides captured from 100 ml of solution are in calculation on 1 g weights of dry the sorbent. we can see that the sorption value for cadmium after 90 minutes for semi-continuous sorbent (cd-ss) is 21.96 mg/g and 21.83 mg/g for discontinuous sorbent (cd-ds). values of zinc sorption are 9.49 mg/g for semicontinuous sorbent (zn-ss) and 5.22 mg/g for discontinuous sorbent (zn-ds). 0 5 10 15 20 25 30 35 40 45 50 0 10 20 30 40 50 60 70 80 90 time [min] so rp ti o n [ m g /g ] cd-ss (100) cd-ds (100) zn-ds (100) zn-ss (100) fig. 3. sorption of cadmium and zinc ions from model solutions. 30 luptakova, a . et al. fig. 3 compares sorption of zinc and cadmium ions during 90 minutes by semicontinuous and discontinuous sorbents, when initial concentration of metal ions in model solutions was 100mg/l. in this case the highest value was obtained for zinc sorption by semi-continuous sorbent (zn-ss) 43.13 mg/g and the lower value belong to cadmium sorption by semicontinuous sorbent (cd-ss) 22.75 mg/g. 3.5 sorption by bio-modified brown coal fig. 4 presents adsorption isotherms of copper and zinc adsorption by biomodified coal adsorbents. experimental data were fitted by langmuir equation. the correlation coefficient ranged from 0.96 to 0.99. experimental results showed that maximum increase of metals uptake for biologically activated brown coal was achieved by the activation by penicillium glabrum (sample s3), i.e. 8.8 mg cu(ii)/g of sorbent and 14.3 mg zn(ii)/g of sorbent. 0 50 100 150 200 0 2 4 6 8 10 cu adsorption q [m g/ g] c e [mg/l] coal + a. niger coal + a. clavatus coal + p. glabrum coal + t. viride coal 0 50 100 150 200 0 2 4 6 8 10 coal + a. niger coal + a. clavatus coal + p. glabrum coal + t. viride coal zn adsorption q [m g/ g] c e [mg/l] fig. 4. langmuir adsorption isotherms of cu (a) and zn (b) adsorption on brown coal treated by different microorganisms. the results of adsorption experiments showed that selected type of sorbent preparation had positive influence on sorption properties of activated material and prepared sorbents had good affinity to selected metals. obtained results confirmed that there is a possibility to prepare the sorbents by new non-conventional methods and the obtained results appear as promising for the research and development in utilisation of non-energetic coal. 4. conclusions the major metal ions in the amd from the abandoned and flooded deposit of smolnik (slovak republic) were fe, al, ca and mg, among which fe and al were potentially valuable, while others such as cu, zn and mn were present as minor metals at significantly low concentrations. the value of ph, contend of fe, al, zn, cu, mn nova biotechnologica 10-1 (2010) 31 and mg of amd did not meet the effluent limitation of nv sr no. 296/2005 z.z. up to now amd was not treated at the site. results of the amd titration test document that the titration curve can be divided into four ranges (i to iv). its show the different optimum ph for metals precipitation of each metal: for al – ph 5; for cu – ph 6; for zn – ph 7; for fe – ph 8; for mn – ph 9. the metals precipitation using 1m naoh confirms that was removed 97% of al at ph 5; 99.9% of cu at ph 6; 99.9% of zn at ph 7; 99.9% of fe at ph 8 and 99.9% of mn at ph 9. metals removal by precipitation is possible up to ph 9. the study of the selective sequential precipitation and bio recovery of metals from aforementioned amd was realized by the combination of the metals precipitation by the bacterially produced hydrogen sulphide and the precipitation of metals by sodium hydroxide at the various values of ph amd. for the removal of cu and zn in the form of sulphides were received excellent results. was not come to good results point of view of the fe, al and mn the selective precipitation, because was determined the co-precipitation of fe and al or fe and mn. obtained results can be used for suggestion of technology for selective metal recovery from acid mine drainage from smolnik. the base this technology will be the combination of chemical and biological agent application. acknowledgements: this work was supported by the slovak research and development agency under the contract no. apvv-51-027705 and bilateral project apvv sk-cz-0105-07. references balintova, m., kovalikova, n.: removal of heavy metals from acid mine drainage using adsorption methods. proceedings of the 8th international scientific conference modern management of mine producing, geology and environmental protection sgem 2008, albena, 2008, 155-160. boonstra, j., van lier, r., janssen, g., dikman, h., buisman, c.j.n.: biological treatment of acid mine drainage. proceedings of the 13th international biohydrometallurgy symposium, biohydrometallurgy and the environment toward the mining of the 21st century ii., ibs, madrid, 1999, 559-566. deorkar, n.v., tavlarides, l.l.: adsorption process for metal recovery from acid mine waste: the berkeley pit problem. environ. prog., 17, 1998, 120-125. foucher, s., battaglia-brunet, f., ignatiadis, i., morin, d.: treatment by sulfate-reducing bacteria of chessy acid-mine drainage and metals recovery. chem. eng. sci., 56, 2001, 1639-1645. garcia sanchez, a., alavarez ayuso, e., jimenez de blas, o.: sorption of heavy metals from industrial waste water by low-cost mineral silicates. clay miner., 34, 1999, 469-477. janke, d.r., diebold, f.e.: recovery of valuable metals from acid mine drainage by selective titration. water res., 17, 1983, 1639-1645. kadukova, j., vircikova, e.: minerálne biotechnológie iii., biosorpcia kovov z roztokov, všb tu ostrava, 2003, isbn 80-248-0244-9. 32 luptakova, a . et al. kadukova, j., stofko, m.: environmentálne biotechnológie pre hutníkov, equilibria, košice, 2006a, isbn 80-8073-496-8. kadukova, j., stofko, m.: biosorption of heavy metals ions from aqueous solutions. environmental research trends, nova publishers, 2006b, isbn 160021-556-4. kalin, m., fyson, a., wheeler, n.w.: the chemistry of conventional and alternative treatment systems for the neutralization of acid mine drainage. sci. tot. environ., 366, 2006, 395-408. kontopouls, a.: acid mine drainage control. in: castro, s.h., vergara, f., sánches, m.a. (eds.) effluent treatment in the mining industry. university of concepcion, chile, 1988, 57-118. luptakova, a., kusnierova, m., bezovska, m., fecko, p.: the selective precipitation of heavy metals by sulphate-reducing bacteria. proceedings of the 15th international biohydrometallurgy symposium, nereus congress and conferences, athens, greece, 2003, 665-672. luptakova, a., kusnierova, m., slesarova, a.: environmental risks and impact of old mine loads in the slovak republic. proceedings of 2nd international conference on environmental research and assessment, university of bucharest, centre for environmental research and impact studies, bucharest, 2006, 254-257. skousen, j., rose, a., geidel, g., foreman, j., evans, r., hellier, w.: a handbook of technologies for avoidance and reclamation of acid mine drainage, morgantown, wv: national mine land reclamation center, west virginia university, 1998. tabak, h.h., scharp, r., burckle, j., kawaharai, f.k., govind, r.: advances in biotreatment of acid mine drainage and biorecovery of metals: 1. metal precipitation for recovery and recycle. biodegradation, 14, 2003, 423436. veeken, a.h.m., rulkens, w.h.: innovative developments in the selective removal and reuse of heavy metals from waste-waters. water sci. technol., 47, 2003, 9-16. microsoft word sunovska et al 2013_nbec_final.doc nova biotechnologica et chimica 12-2 (2013) 141 doi 10.2478/nbec-2013-0016 © university of ss. cyril and methodius in trnava characterization of soil additive derived from sewage sludge anna šuňovská, miroslav horník, martin pipíška, juraj lesný, jozef augustín, stanislav hostin department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, sk-917 01 trnava, slovak republic (anna.sunovska@ucm.sk) abstract: the aim of the present work is to characterize the soil additive derived from sewage sludge as potentially economically acceptable material for agricultural production as well as for soil and environment protection. the soil additive consisting of sewage sludge obtained from the wastewater treatment plant pannon-víz zrt. (győr, hungary) and agricultural byproducts represented by wastes from grain mill industry and crushed corn cobs was prepared using the low-capacity granulator equipment constructed by energy agency public nonprofit ltd. (hungary). the characterization of sewage sludge as primary composite and prepared soil additive includes the determination of physico-chemical parameters such as ph determined in suspension with distilled water, 0.01 mol/dm3 cacl2 or 1 mol/dm3 kcl solutions, phzpc predicted by potentiometric titration and protofit software, water holding capacity (whc), cationexchange capacity (cec) and total organic carbon (toc). the elemental analysis by x-ray fluorescence spectrometry revealed that sewage sludge as well as prepared soil additive contain significant amount of zn and cu as important microelements in plant nutrition. also, it was found that prepared soil additive represents the considerable source of a significant proportion, strong bound and in this way gradually released microelements. obtained results suggest on the application potential of prepared soil additive in agricultural production as well as in remediation and reclamation of contaminated or degraded soil. key words: sewage sludge, waste, soil additive, agriculture, remediation, lysimeter 1. introduction in the 20th century, industrialization and ore mining led to the increasing of the number of sites contaminated with metals or organic xenobiotics. nowadays, environmental issues also have been broadened in connection with concepts involving sustainable development, which implies not only ecological, but economical and social responsibilities as well. however, globally the intensification of production processes and relative human activities raise the production of gaseous, liquid and solid wastes. the european community generates approximately two billion tons of waste per year (düring and gäth, 2002). the handling of sewage sludge (ssl) represents one of the most significant challenges in wastewater management (fytili and zabaniotou, 2008). ssl is defined by the u.s. environmental protection agency (epa) as the “solid, semi-solid, or liquid residue generated during the treatment of domestic sewage in a treatment works” (nrc, 2002). within wastewater treatment plant the ssl represents the residue generated during the primary (physical and/or chemical), the secondary (biological) and the tertiary (additional to secondary, often nutrient removal) treatment (fytili and zabaniotou, 2008). the composition of ssl is bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:46 utc 142 šuňovská, a. et al. dependent on its origin in terms of the type of wastewater treatment, and therefore is variable and unpredictable as well. in this way, the public concern and definition of regulations or directives are primarily associated with toxic metals, organic xenobiotics or pathogen occurrence in this type of wastes. ssl treatment by a variety of processes including aerobic digestion, anaerobic digestion, composting, heat drying, air drying, lime stabilization, and chemical fixation is the possible key to minimizing the risks in terms of the quantitative occurrence or negative impacts of mentioned components (mathney, 2011). also, the proportion of macroor microelements and ph levels, alkalinity or organic acid content are important parameters from the point of view of the ssl ultimate disposal as well as the process of anaerobic digestion (tchobanoglous et al., 2003), respectively. all these factors play an important role in decision with regard to the ultimate ssl disposal, which can involve: composting, landfilling, agricultural reuse as well as sea or surface waters disposal and incineration of ssl (donatello and cheeseman, 2013). during the last decades there has been a major change in the ways ssl is disposed (fytili and zabaniotou, 2008). prior to 1998, municipal ssl was primarily disposed at seawaters or was used in agriculture (odegaard et al., 2002). additional solutions include incineration of ssl or landfilling. since 1998 onwards, european legislation prohibits the sea disposal of ssl (fytili and zabaniotou, 2008). large differences between individual eu countries exist in current ssl disposal concerning on the character of disposal operations or their proportion on ssl disposal. some papers have been reported that ssl, except the conventional methods of disposal, can be disposed by pyrolysis or incineration for biochar (méndez et al., 2013) or ash production as building construction materials (chen and lin, 2009) as well as can be used in remediation of soils contaminated with heavy metals (garrido et al., 2012). approximately 5.6 million tons of dry ssl are used or disposed of annually in the united states, of which 60 % is used for land application or public distribution (nrc, 2002). in europe, dry weight per capita production of ssl resulting from primary, secondary and even tertiary treatment represents more than 90 g per person per day (davis, 1996). in conditions of slovak republic the production of ssl within the last decade is relative constant, e.g. in 2002 represent 51,270 tons per year and in 2012 58,706 tons per year. in mentioned year 2012, the ssl was predominantly used for compost production (62.7 %), reclamation (16.4 %), landfilling (13.5 %), incineration (5.5 %) and direct application into the soil (1.9 %) (according to data of mžp sr, 2012). previous works deal with the possibility of application of the sludge originated from sewage treatment plants as potential sorbent for co removing from contaminated solutions (frišták et al., 2013) or application of simultaneous and sequential extraction protocols as tools for determination of zinc bioavailability in dried anaerobic sludge (frišták et al., 2012). the aim of the present work is to characterize the soil additive derived from ssl as potentially economically acceptable material for agricultural production as well as for remediation and reclamation of contaminated or degraded soil. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:46 utc nova biotechnologica et chimica 12-2 (2013) 143 2. materials and methods 2.1 sewage sludge and soil samples dried samples of sewage sludge (ssl) as primary composite for soil additive preparation were obtained from the wastewater treatment plant pannon-víz zrt. (győr, hungary). the sample represented concentrated, anaerobically digested, dewatered and dried ssl. soil samples as a model of agricultural used soil were obtained from experimental fields of plant production research center piešťany (pprc piešťany) in borovce (spring period, 2013; altitude 160 m, north latitude 48°34´, east longitude 17°44´). all obtained samples were kept under laboratory conditions without their special treatment. dry weight of studied matrices was determined at 105 °c in drying chamber and the distribution of particles size of individual samples was evaluated by sieve analysis with standard sieves using. 2.2 soil additive derived from sewage sludge dried samples of soil additive consisting of mentioned ssl and agricultural byproducts represented by wastes from grain mill industry and crushed corn cobs (top feed & cargo hungary holding zrt., hungary) were prepared in volume ratio 1 : 1.5 using the low-capacity granulator equipment (fig. 1) designed by energy agency public nonprofit ltd. (budapest, hungary). the low-capacity granulator provided the mixing of both primary composites and thermal treatment (~ 75 °c) for inhibition of present microorganisms. fig. 1. low-capacity granulator equipment designed by energy agency public nonprofit ltd. (hungary) (a) for production of designed soil additive (b) – primary pellets (left) and crushed material applied in experiments (right). 2.3 determination of ph and cation-exchange capacity the ph determination or cation-exchange capacity (cec) of ssl, prepared soil additive and agricultural used soil were realized according to iso standard method no. 10390 or no. 11260, respectively. a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:46 utc 144 šuňovská, a. et al. according to mentioned standard method soil suspensions with deionized water, 1 mol/dm3 kcl or 0.01 mol/dm3 cacl2 at volume ratio v(matrix) : v(solution) = 1 : 5 were stirred on orbital incubator shaker (150 min-1) within 1 h at 25 ºc and subsequently the ph value of these suspensions was measured using glass electrode. in the case of cec determination the studied matrix was suspended in bacl2 solution for the reason ba binding in matrix and removing of others present cations. subsequently, the matrix saturated with ba was suspended with mgso4 solution and remaining, non-binding amount of mg2+ cations was analyzed by chelatometric titration using edta-na2 on eriochrome black t indicator. 2.4 elemental and total organic carbon analysis the elemental analysis of ssl and prepared soil additive samples was performed by x-ray fluorescence spectrometry using the high performance x-ray fluorescence spectrometer x-lab 2000, spectro (germany). the content of as, ca, cd, cr, cu, fe, mg, mn, ni, pb, sb, se and zn was analyzed in samples treated to granularity < 0.063 mm. total organic carbon (toc) was determined using toc analyzer toc-vcpn shimadzu (japan). 2.5 potentiometric titration with the aim to prediction of protonation constants pka and binding sites concentration of functional group can on the surface of studied matrices the potentiometric titration according to modified procedure described by zhang et al. (2010) was carried out. the sample was transferred into erlenmeyer flask containing a mixture of 0.1 mol/dm3 hcl and 0.1 mol/dm3 nacl, stirred on orbital shaker and centrifuged. the studied matrix was suspended in 0.1 mol/dm3 nacl solution and in this suspension the titration solution 0.l mol/dm3 naoh was gradually added with the parallel measuring ph. the obtained data describing the relationship between the volume of added titration solution and the measured ph values were analyzed by modelling program protofit ver. 2.1.(turner and fein, 2006). 2.6 determination of water holding capacity the determination of water holding capacity (whc) value was realized in 30 cm length glass column with internal diameter 2.5 cm. the defined amount (in grams) of individual samples of ssl, soil additive and soil or the mixture soil additive : soil in weight ratios 1 : 9; 1 : 4; 1 : 1.5 or 1 : 1.25 was transferred into the column with the bottom cap. subsequently, the defined amount of distilled water (in grams) equivalent with the amount of matrix sample was added into the mentioned system. after 30 min the bottom of the column was opened and the water eluate was collected into erlenmeyer flask during the next 30 min. the whc value was calculated bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:46 utc nova biotechnologica et chimica 12-2 (2013) 145 according to gravimetrically evaluation of water balance between added water into system and obtained amount of eluate. 2.7 single extraction of zn and cu from studied matrices for the evaluation of available microelements zn and cu amount in studied matrices the mehlich method was used. the suspension of matrix and mehlich-iii solution in weight ratio 1 : 10 was stirred on orbital shaker during 10 min. the mehlich-iii extraction solution consisted of 0.2 mol/dm3 ch3cooh, 0.25 mol/dm3 nh4no3, 0.015 mol/dm 3 nh4f, 0.013 mol/dm 3 hno3 and 0.001 mol/dm3 edta (mehlich, 1984). subsequently, the suspension was filtered and in solution the concentrations of zn and cu were analyzed by atomic absorption spectrometer (aa-7000 shimadzu, japan) with electrothermal atomization (gfa-7000 shimadzu, japan). from the obtained concentration of zn and cu the available amount of zn and cu in milligrams per kilogram of matrix was calculated. 2.8 laboratory lysimetric experiment a plastic laboratory lysimeter (ecotech, germany) with diameter 30 cm and height 50 cm was filled with 45 kg of dried agricultural used soil (content of water max. 2.1 %). mentioned laboratory lysimeter was equipped with: sprinkling head for soil watering, temperature sensors and tensiometers located in soil column within 10 cm, 20 cm or 30 cm from the surface, suction bottom with controlled vacuum pump for sampling of soil eluates, and datalogger for data acquisition with communication and evaluation software. the whole lysimeter system containing soil column with 0.28 m2 of surface and 40 cm height was maintained under defined conditions of plant growth chamber (kbwf 720 binder, germany) and in the given time intervals weighted for water balancing in the system. 3. results and discussion 3.1 physico-chemical characterization of studied matrices in the first step of sewage sludge (ssl), prepared soil additive and model of agricultural used soil characterization the analyses aimed to the determination of ph (phh2o, phcacl2 and phkcl), cation-exchange capacity (cec) and total organic carbon (toc) values were carried out. obtained results showed that studied matrices were significantly differed in all evaluated values (table 1). the lowest ph value was determined for designed soil additive consisting of ssl as primary studied composite and agricultural byproducts represented by wastes from grain mill industry and crushed corn cobs. on the other side, the sample of soil additive showed higher values of cation-exchange capacity (cec) and total organic carbon (toc) in comparison with ssl and agricultural used soil. this fact suggests on the potential bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:46 utc 146 šuňovská, a. et al. positive effect of such soil additive, particularly in the case of basic soils, in terms of the increasing metals availability for plant production purposes as well as in remediation of soils contaminated with toxic metals using selected plant species applied in phytoremediation methods (chiu et al., 2006). table 1. determined values of ph (phh2o, phcacl2 and phkcl), cation-exchange capacity (cec) and total organic carbon (toc) for studied matrices. matrix phh2o phcacl2 phkcl cec (meq/100 g) toc sewage sludge* 6.73 6.66 6.57 28.2 28.6 % soil additive ** 5.75 5.61 5.38 33.2 37.4 % soil *** 7.26 6.78 6.30 29.0 1.10 % (cox) * sewage sludge obtained from the company pannon-víz zrt. (hungary) as primary composite of prepared soil additive; ** soil additive consisting of sewage sludge and agricultural byproducts represented by wastes from grain mill industry and crushed corn cobs was prepared using the low-capacity granulator equipment constructed by energy agency public nonprofit ltd. (hungary); *** soil obtained from the experimental fields of pprc piešťany as a model of agricultural used soil.   2 3 4 5 6 7 8 9 10 11 12 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ph soil additive sewage sludge a dd iti on o f 0 .1 m ol /d m 3 n ao h [c m 3 ] 0 20 40 60 80 100 soil soil additive p ro po rt io n of fr ac tio ns [% ] sewage sludge < 0.15 mm 0.31 mm 0.15 mm 0.63 mm 0.31 mm 1.20 mm 0.63 mm > 1.2mm fig. 2. a. potentiometric titration curves for sewage sludge and designed soil additive (3.0 g/dm3). the titration was performed with addition of 0.1 mol/dm3 naoh and 0.1 mol/dm3 nacl as background electrolyte and at 25 °c. b. sieve analysis of studied matrices for determination of fraction proportion of individual particles on the basis of their size. in the context with the cec value, the potentiometric titration analysis of ssl and prepared soil additive was realized (fig. 2a). this analysis confirmed that prepared soil additive containing except the evaluated ssl also agricultural byproducts represented by wastes from grain mill industry and crushed corn cobs showed higher proportion of important functional groups from the point of view of metal cations binding such as carboxyl (–cooh), hydroxyl (–oh), phosphate (–po3h2) or amino (–nh2) groups. protofit software predicted that in prepared soil additive the concentrations of mentioned functional groups were minimally 4-times higher in comparison with the primary composite – sewage sludge, particularly a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:46 utc nova biotechnologica et chimica 12-2 (2013) 147 in the case of basic functional groups (–nh2 and –oh). from the potentiometric titration data fitted by the program protofit the values of phzpc were also predicted, whereby the phzpc value (ph of zero point charge) represents the ph at which the matrix has a net zero surface charge. the matrix surface has a net positive charge at ph < phzpc, while at ph > phzpc, the surface has a net negative charge (sun et al., 2011). ssl showed practically neutral value of phzpc = 7.8 in contrast to the prepared soil additive, when the phzpc reached the value 9.4. in cation binding the specific surface of matrix particles plays also a decisive role. fig. 2b depicts the fraction proportion of individual particles on the basis of their size for studied matrices determined by sieve analysis. 0 20 40 60 80 100 120 mixture soil + soil additive 80 %40 %20 %10 %soil additive sewage sludge soil w h c [g h 2o /1 00 g ] weight proportion of soil additive in mixture fig. 3. water holding capacity (whc) determined for individual studied matrices or for mixture of model agricultural used soil and prepared soil additive with different weight proportion of soil additive. in the last decades, the climatic changes in terms of floods and dry season rotation play an important role in agricultural production. therefore, the soil additive of this type and its characteristics should take into account the mentioned negative effects of climatic changes on the soil quality. in experiments evaluating the values of water holding capacity (whc), we found that soil additive prepared from ssl and agricultural by-products represented by wastes from grain mill industry and crushed corn cobs showed the highest values of whc in comparison with ssl and model of agricultural used soil. also, the addition of prepared soil additive had the positive effect on the increasing of whc values of mixture soil additive : soil (fig. 3). from the point of view of ssl application into the soil is very important to know the presence and concentration of toxic substances as well as microbial contamination. these factors are reflected in the relevant eu legislative directions. as we mentioned, the ssl added as composite into the designed soil additive was thermally treated with the aim of microorganisms inhibition. in the context of toxic substances presence in ssl, we focused on toxic metals as well as important macroand microelements content (table 2). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:46 utc 148 šuňovská, a. et al. united states environmental pollution agency (us epa) developed parameters the effects range low (erl) and the effects range median (erm) as predictive tools for contamination characterizing in sediments. the erl represents the tenth percentile of the effects database, below which harmful effects on aquatic biota are rarely observed. the erm represents the fiftieth percentile of the effects data and is indicative of concentrations above which harmful effects are often observed (us epa, 2002). also, these parameters can be applied for ssl as water sediments. from the elemental analysis by x-ray fluorescence spectrometry, we found that studied ssl showed overload of the erl parameter in the case of cr and ni and overload of the erm parameter for cu and zn. despite this fact, it can be concluded that the concentrations of cr and ni do not represent a serious risk of soil contamination with heavy metals after designed soil additive application into the soil in the case of observance of rules defined by legislation of the slovak republic (act no. 188/2003) determining the maximal amount of applied ssl into the soil 15 tons (dry weight; d.w.) per ha and per 5 years. also, this act no. 188/2003 determines the limits of maximal concentrations for mentioned metals cr and ni in ssl applied into soil, which represent the values 1,000 and 300 mg/kg (d.w.), respectively. on the other hand, in the case of soils deficient in zn or cu as important microelements in plant nutrition, the prepared soil additive containing zn and cu in this level can represents a valuable source of these nutrients. table 2. elemental analysis of studied matrices by x-ray fluorescence spectrometry. element sewage sludge* soil additive** as 8.0 ppm 6.5 ppm ca 4.74 % 3.21 % cd < 2 ppm < 2 ppm cr 84.7 ppm a 67.5 ppm cu 654 ppm b 583 ppm b fe 3.92 % 3.13 % mg 0.61 % 0.21 % mn 0.03 % 0.03 % ni 42 ppm a 44 ppm a pb 36 ppm 26 ppm sb 4.67 ppm < 2 ppm se 2.67 ppm < 1 ppm zn 1,940 ppm b 1,510 ppm b a overload of the effects range low (erl); b overload of the effects range median (erm). 3.2 single extraction of zn and cu from studied matrices in the context with previous results indicating the fact that ssl as well as prepared soil additive showed high content of zn and cu as important microelements in plant bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:46 utc nova biotechnologica et chimica 12-2 (2013) 149 nutrition, the experiment evaluating availability of these metals for plants were carried out. for these purposes, the method according to mehlich (mehlich, 1984) was applied. from the fig. 4a, it is evident that more than 2-times higher amount of zn was extracted by mehlich-iii solution from prepared soil additive in comparison with ssl (130 mg/kg; d.w.) as primary composite. in the case of cu up to 6-times higher extractable amount for soil additive (10 mg/kg; d.w.) was found. for comparison, the extractable amount of zn and cu for sample of agricultural used soil is also mentioned in fig. 4a. within these results, the fact of higher total content of zn and cu in the case of ssl as well as 3-times higher total amount of zn in comparison with cu in studied matrices should be taken into account (table 2). in this way, the proportion of available zn and cu for soil additive represents more than 19 % and 11 %, respectively (fig. 4b). it can be concluded that the addition of agricultural by-products represented by wastes from grain mill industry and crushed corn cobs and the treatment of ssl at production of designed soil additive using the low-capacity granulator equipment caused the changes responsible for increasing of available zn and cu proportion in this matrix. also, it can be supposed that the proportion of zn and cu will be lower in the case of agricultural by-products represented by wastes from grain mill industry and crushed corn cobs in comparison with ssl. therefore the mentioned changes probably take place significantly within the ssl. 0 50 100 150 200 250 300 e xt ra ct ab le a m ou nt [m g/ kg ]; d. w . zn cu soil sewage sludge soil additive soil soil additive sewage sludge 0 5 10 15 20 25 p ro po rt io n of a va ila bl e m et al [% ] zn cu soil additive sewage sludge sewage sludge soil additive fig. 4. the single step extraction of zn and cu from agricultural used soil, sewage sludge and designed soil additive by mehlich-iii solution expressed as the total extractable amount of metal (in mg/kg) (a) or the ratio of extracted amount of metal to the total amount of metal in matrix (b). yu et al. (2004) found that between the total amount of cu in soil and extractable amount of cu by mehlich-iii solution the linear dependence exists. also, these authors observed the proportion of extractable amount of cu in comparison with the total amount of cu in degraded soil more than 50 %. it is generally known that the availability of heavy metal such as zn and cu is also strongly determined by factors e.g. ph, organic matter, clay minerals, oxyhydroxides, and others (kabata-pendias, 2011). obtained results showed that the availability of zn and cu in soil additive is relatively low. this fact suggests that prepared soil additive represents the source of a significant proportion, strong bound and in this way gradually released a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:46 utc 150 šuňovská, a. et al. microelements. these characteristics correspond with suitable parameters for agricultural production. strong binding of metals or relative high cec values can be also utilized in remediation and reclamation of contaminated or degraded soil. 3.3 laboratory lysimetric experiment in this part the potential of laboratory lysimetric system application in further study of positive or negative characteristics of soil additive derived from ssl for agricultural production or remediation and reclamation of contaminated or degraded soil is demonstrated. the following preliminary experiment under laboratory conditions was focused on evaluating the possibility to imitate the behaviour of mentioned soil additive in conditions of agricultural used, contaminated or degraded soils. in this term, the possibility to control the soil column watering, the obtaining of soil eluate and vertical distribution of soil moisture, as well as the effect of temperature, relative humidity or illumination on these processes were studied. 0 100 200 300 400 500 600 -800 -700 -600 -500 -400 -300 -200 -100 0 100 2b -10 0 10 20 30 40 5 0 6 0 70 80 90 100 110 120 13 0 -800 -700 -600 -500 -400 -300 -200 -100 0 100 p re ss u re p o te n tia l [ h p a ] t im e [h] 1 2 a 3 a 3b p re ss ur e po te nt ia l [ hp a] time [h] 10 cm from the soil surface 20 cm from the soil surface 30 cm from the soil surface 1 2a a 3c 3d 3e a 4 fig. 5. the changes in the soil moisture within the soil column (diameter 30 cm; height 40 cm) located in the laboratory lysimeter caused by watering, suction of soil eluate, evapotranspiration of water under given conditions in plant growth chamber (temperature, relative humidity or illumination). 1. start of the experiment with column of dried soil (content of water max. 2.1 %); 2. watering of soil with distilled water using lysimeter sprinkling head: a – during 6 h with the watering rate 0.66 dm3/h, b – during 3 h with the watering rate 0.66 dm3/h; 3. suction of soil eluate by controlled vacuum pump: a – during 4 h under pressure 20 – 40 hpa, b – during 6 h under pressure 20 – 40 hpa, c – during 1 h under pressure 50 – 100 hpa, d – during 24 h under pressure 20 – 40 hpa, e – during 72 h under pressure 20 – 40 hpa; 4. activation of illumination 16 h day / 8 h night with 11 450 lx intensity up to end of experiment. the fig. 5 depicts the tensiometric data of water pressure potential defining the changes in the soil moisture within the soil column (diameter 30 cm; height 40 cm) installed in the laboratory lysimeter caused by watering, suction of soil eluate or evapotranspiration of water under given conditions in plant growth chamber (temperature, relative humidity or illumination). at the start of experiment the sample bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:46 utc nova biotechnologica et chimica 12-2 (2013) 151 of agricultural used soil in individual 10 cm layers from the soil surface showed the values of water pressure potential up to 700 hpa, which correspond with the water content approx. 2 %. the values of water pressure potential near to 0 hpa represent the water saturated soil. it can be seen that the application of 4 dm3 distilled water in the amount and rate typical for natural rain conditions caused significant increasing of water content in soil column (45 kg; d.w.) near to soil saturation with water, but without soil eluate obtaining. within the next 6 days the gradually decreasing of soil moisture in all analyzed vertical soil layers supported by 2 suction under pressure was observed. subsequently, the repeated soil watering with 2 dm3 distilled water was carried out. the next decreasing of soil moisture was sequentially supported by suction of soil eluate in different time duration as well as illumination (16 h day / 8 h night with 11 450 lx intensity) provided by plant growth chamber. it was found that under mentioned conditions, it is possible to significantly change the soil moisture within short time correlating with the real natural conditions. moreover, the different quantitative distribution of soil moisture within the vertical location of analyzed soil layers was obtained. in the fig. 6, the conditions related with temperature and relative humidity within lysimetric experiment are described. 0 100 200 300 400 500 600 0 5 10 15 20 25 30 temperature relative humidity time [h] t em pe ra tu re [° c ] day night 0 20 40 60 80 100 r elative hum idity [% ] fig. 6. the changes in the values of temperature and relative humidity within the lysimetric experiment described in the fig. 5. 4. conclusions the soil additive consisting of sewage sludge and agricultural by-products represented by wastes from grain mill industry and crushed corn cobs, and prepared by designed low-capacity granulator equipment was characterized on the basis of physico-chemical parameters such as ph, phzpc, cation-exchange capacity (cec), water holding capacity (whc), total organic carbon (toc) and the content of toxic metals or microelements. the elemental analysis revealed that applied sewage sludge as well as prepared soil additive contain significant amount of zn and cu as important microelements in plant nutrition. in this context, it was found that prepared soil bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:46 utc 152 šuňovská, a. et al. additive represents the considerable source of a significant proportion, strong bound and in this way gradually released microelements. these characteristics correspond with suitable parameters for agricultural production as well as for remediation and reclamation of contaminated or degraded soil. in a separate part of the work the potential of laboratory lysimetric system application in further study of positive or negative characteristics of soil additive derived from sewage sludge for mentioned purposes was evaluated. acknowledgement: this work was supported by the project of the cross-border co-operation programme and co-financed with european regional development fund (erdf), the grant number husk/1101/1.2.1/0148 as well as the project of the operational program research and development and co-financed with european regional development fund (erdf), the grant number itms 26220220106. also, authors want to thank dr. daniela mackových for x-ray fluorescence spectrometric and toc analyses. references act no. 188/2003 (zákon č. 188/2003 z. z.): zákon o aplikácii čistiarenského kalu a dnových sedimentov do pôdy a o doplnení zákona č. 223/2001 z. z. o odpadoch a o zmene a doplnení niektorých zákonov v znení neskorších predpisov, národná rada slovenskej republiky, 2003. davis, r.d.: the impact of eu and uk environmental pressures on the future of sludge treatment and disposal. water environ. j., 10, 1996, 65-69. donatello, s., cheeseman, c.r.: recycling and recovery routes for incinerated sewage sludge ash (issa): a review. waste manag., 33, 2013, 2328-2340. düring, r.a., gäth, s.: utilization of municipal organic wastes in agriculture: where do we stand, where will we go? j. plant nutr. soil sci., 165, 2002, 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mohamed m. el-zahed, m. i. abou-dobara, ahmed k.a. el-sayed, zakaria a. m. baka department of botany and microbiology, faculty of science, damietta university, new damietta, egypt  corresponding author: mohamed.marzouq91@du.edu.eg article info article history: received: 20th june 2021 accepted: 8th november 2021 keywords: antimicrobial biosynthesis escherichia coli nanocomposite silica silver abstract silica (sio2) has a fundamental role in the recuperation of plants in response to environmental stresses, besides the induction of resistance against plant diseases. silver nanoparticles (agnps) have a superior antimicrobial activity. the combination between sio2 and agnps is a promising approach due to their antimicrobial activity, biological activity, low toxicity, and high stability of the produced nanocomposite. the current study postulated a green method for silver/silica nanocomposite (ag/sio2nc) synthesis at room temperature using the crude metabolites of escherichia coli d8 (mf062579) strain in the presence of sunlight. uv-vis spectrophotometry, x-ray diffraction (xrd), fourier transforminfrared spectroscopy (ftir), and transmission electron microscopy (tem) analyses have characterized the biosynthesized nanocomposite. tem study of ag/sio2nc showed an average particle size of ~32 – 48 nm whereas agnps showed a mean size of 18 – 24 nm. the negative charged ag/sio2nc (-31.0 mv) showed potent antimicrobial activity against bacillus cereus atcc6633, klebsiella pneumoniae atcc33495, staphylococcus aureus (atcc25923), e. coli (atcc25922), candida albicans (atcc10231), and botrytis cinerea (pers: fr.). the minimum inhibitory concentration (mic) test showed a dose-dependent manner of ag/sio2nc antimicrobial action. mic values of ag/sio2nc against the tested pathogens exhibited 125 and 6.25 μg.ml-1 as antibacterial and antifungal agents, respectively. tem micrographs showed changes in the pathogens treated with ag/sio2nc including wrinkling, damage, and rupture of the bacterial cell membrane. in addition, the formation of a mucilage matrix connecting the hyphal cells, the appearance of big vacuoles and lipid droplets with severe leakage of cytoplasmic contents of the treated b. cinerea were also recorded. introduction nowadays, there is a tendency to use materials after converting them into their nano-form, because of the new and promising advantages and unique properties gained in the new nano-form such as antimicrobial activity, chemical, magnetic, electronic, or mechanical properties because of the change of quantum and surface boundary effects compared with their bulk materials (chhipa and joshi 2016; musere et al. 2021). nanomaterials can be described as materials with a size of 1 – 100 nm, known as the nano-scale range. synthesis of nanometals can be done using different methods such as chemical, physical or biological techniques. conventional hypothesis of chemical synthesis of nanoparticles (nps) might possess several serious problems due to using expensive toxic chemicals (zhang et al. 2021). mailto:mohamed.marzouq91@du.edu.eg nova biotechnol chim (2022) 21(1): e1023 2 physical methods include high radiation and stabilizing agents that might be dangerous for human health and to the environment (awwad et al. 2020). accordingly, the green synthesis of nps by plant extracts or microbial metabolites plays an important role in the reduction of metal ions into nps and capping them in supporting their stability (parveen et al. 2016). biological nps have different characters compared with chemical or physical nps, with superior stability and suitable dimensions due to the one-step technique (narayanan and sakthivel 2011). silver nanoparticles (agnps) were used as a potent antimicrobial agent during the last decades (hamad et al. 2020). it showed antimicrobial activity against pathogens such as escherichia coli (yang et al. 2021), staphylococcus aureus (enan et al. 2021), klebsiella pneumoniae (pareek et al. 2021), candida albicans (takamiya et al. 2021), and botrytis cinerea (ouda 2014). the aggregation of agnps is a common problem that decreases their biological activity (el-dein et al. 2021). one way to enhance the metal nps stability is by stabilizing them by embedding them inside a polymer, which prevents their aggregation, even at high-volume fractions (baheiraei et al. 2012). several previous studies have focused on the synthesis of stable monodisperse silica-coated with nanometals, mainly ag and gold (au) (chen et al. 2017; si et al. 2019; li et al. 2021). silica (sio2) can act as the platform for developing nps moreover having antimicrobial properties owing to their large surface area, positive surface charge, and monodispersity. gankhuyag et al. (2021) reported that sio2 might increase the stability of the nanometals and prevent their aggregation. in addition, the net positive charge of sio2 facilitates a greater number of agnps to interact with the negatively charged surface of bacteria, resulting in highly efficient antimicrobial activity (jayasuriya 2017). egger et al. (2009) and sohrabnezhad et al. (2020) demonstrated the antibacterial activity of silver/silica nanocomposite (ag/sio2nc) against both e. coli and s. aureus. ag/sio2nc revealed marked changes in the bacterial cell contents, including the cell wall integrity, metabolism, and genetic stability of pseudomonas aeruginosa (anas et al. 2013). also, xu et al. (2009) reported the antibacterial effects of ag/sio2 core-shell particles against e. coli and s. aureus. ag/sio2nc had antifungal potential against b. cinerea as reported by oh et al. (2006). youssef and roberto (2021) demonstrated the antifungal activity of chitosan/silica nanocomposite against b. cinerea. ag/sio2nc showed fungicidal activity against the pathogenic fungi in the soybean plants (fusarium oxysporum and rhizoctonia solani) as reported by nguyen et al. (2016) and aspergillus flavus as reported by diagne et al. (2020). hence, new distinctive structures of ag/sio2nc could present a new prospect for its antimicrobial activity. the present study aimed to evaluate the ability of the crude metabolite of e. coli d8 (mf062579) for reducing the silver nitrate (agno3) into agnps extracellularly and also their binding with sio2 in a new one-step green approach. the antimicrobial potential of ag/sio2nc was studied against some pathogenic strains, comparing their activity to the standard commercial antibiotics. experimental microbial cultures e. coli d8 (ac: mf062579) and the pathogenic bacterial and fungal strains were obtained from the culture collection of botany and microbiology department, faculty of science, damietta university, egypt. chemicals the chemicals included silver nitrate (panreac quimica s.l.u, barcelona, spain), silica (silicon dioxide nanoparticles, particle size 190 – 250 nm, mesoporous, pore size 4 nm, sigma-aldrich), culture media, and other chemicals (sigma aldrich chemical pvt. ltd., india). penicillin g potassium (buffered pfizerpen) and fluconazole (diflucan) were purchased from pfizer inc., new york, ny. biosynthesis of silver nanoparticles and silver/silica nanocomposite silver nanoparticles were prepared according to elzahed et al. (2021). in brief, e. coli d8 agar slants were sub-cultured on nutrient agar plates (37 °c, 24 h). then the grown colonies were inoculated into a nutrient broth medium with 0.5 mcfarland standard (1 – 2 × 108 cfu.ml-1) and incubated at nova biotechnol chim (2022) 21(1): e1023 3 37 °c/150 rpm for 48 h. later, the cell-free metabolites of e. coli d8 were collected by centrifugation (3h24ri intelligent high-speed refrigerated centrifuge, herexi instrument, and equipment co., ltd) at 8,000 rpm for 20 min and filtration through a sterile 0.22 µm syringe filter (millex gv, millipore). for the synthesis of agnps, 1.5 mm of agno3 solution was mixed with cell-free metabolites (1 % v/v) at room temperature and sunlight. for the synthesis of ag/sio2nc, 0.5g of agno3 was dissolved into 50 ml of distilled water and then added to another beaker that included 100 g of sio2. at room temperature, the previous solution was mixed well with 20 ml of e. coli d8 cell-free metabolites in the presence of sunlight. the first indicator for the agnps and nanocomposite (nc) formation was the color change from colorless (agno3) or white (sio2) into brown. after 20 min, the agnps and ag/sio2nc were collected separately by centrifugation at 10,000 rpm for 15 min several times and then dried in an oven at 50 °c for 24 h. then, the nps and nc were dried at 185 °c for 5 h (sadeghi et al. 2013). characterization of silver/silica nanocomposite silver/silica nanocomposite spectra were scanned by uv/vis/nir spectrophotometer (v-630, jasco corporation, japan). the x-ray diffraction (xrd) pattern of the ag/sio2nc was performed at 2θ values (l = 1.54 °a in the range 10 – 80 °) using a cu x-ray tube at 40 kv and 30 ma with the xray diffractometer (model labx xrd-6000, shimadzu, japan). fourier transform infrared spectroscopy (ftir) spectrum of the ag/sio2nc was recorded by jasco ft/ir-4100typea in the 400 – 4,000 cm-1 range. the size and morphology of agnps and ag/sio2nc were investigated by tem (jeol, jem-2100, japan) at an accelerating voltage of 200 kv and using a carbon-coated copper grid (type g 200, 3.05 μm diameter, taap, usa). charge of agnps and size distribution by volume were recorded by zeta potential analyzer (malvern zetasizer nano-zs90, malvern, uk). antimicrobial potential the antimicrobial potential of ag/sio2nc was tested against gram-positive bacteria (s. aureus atcc25923 and bacillus cereus atcc6633), gram-negative bacteria (e. coli atcc25922 and k. pneumoniae atcc33495), yeast (c. albicans atcc10231), and the phytopathogenic fungus (b. cinerea pers: fr.) by agar well diffusion and broth dilution methods. the bacterial, yeast and fungal strains were grown and tested using mueller hinton agar (mha), bacto-casitone agar, and potato dextrose agar (pda) plates, respectively. 200 μl of 0.5 mcfarland standard (1 – 2 × 108 cfu.ml-1) of microbial suspension was used as an initial inoculum for each test. agar well diffusion method agar well diffusion assay was performed in vitro against the microbial strains according to the guidelines of the clinical and laboratory standards institute (clinical and laboratory standards 2006). about 200 μl of 150 μg.ml-1 of sio2, ag/sio2nc, agno3, penicillin g potassium (antibacterial), and fluconazole (antifungal) were prepared and added separately into small wells (5 mm diameter of size) that were made into the solidified agar plates. plates were incubated at 37 °c for 48 h, 30 °c for 48 h, or 28 °c for 5 days, for bacteria, yeast, and fungi, respectively. after the incubation period, inhibition zones were measured in millimeters (mm). broth dilution method mueller hinton, bacto-casitone and potato dextrose broth media test tubes were prepared, autoclaved, and inoculated by 100 μl of microbial suspensions (0.5 mcfarland standard (1 – 2 × 108 cfu.ml-1)) in three sets of test tubes containing different dosages of ag/sio2nc and penicillin g potassium (antibacterial) or fluconazole (antifungal) concentrations (6.25 – 125 μg.ml-1). then, the tubes were incubated at 37 °c/120 rpm for 24 h, 30 °c/120 rpm for 24 h, or 28 °c/120 rpm for 5 days, for bacteria, yeast, and fungi, respectively. the minimal inhibition concentration (mic) for the tested pathogenic strains was determined by measuring their growth spectrophotometrically at nova biotechnol chim (2022) 21(1): e1023 2 600 nm against negative controls (exclusive of ag/sio2nc). the growth inhibition percentage was calculated using the following formula (eq. 1): % growth inhibition = (1) where the negative control (broth media exclusive of ag/sio2nc) optical density; odc and the ag/sio2nc-treated tested sample optical density; odt (clinical and laboratory standards 2008; 2017). ultrastructural study the ultrastructure of ag/sio2nc treated e. coli and b. cinerea was studied with tem (jeol, jem2100, japan, 200kv) according to bozzola (2007). the tested strains were subjected to ag/sio2nc (mic, 6.25 μg.ml-1) for 2 h and compared with untreated bacteria and fungi as controls. the samples were fixed in 2.5 % glutaraldehyde in 0.1m cacodylate buffer at ph 7.0 and then postfixed in 1 % osmium tetroxide, dehydrated with a graded series of ethanol, embedded in a plastic resin, and sectioned on an ultramicrotome. ultrathin sections were double‐stained with uranyl acetate and lead citrate and then loaded on carboncoated copper grids (type g 200, 3.05 μm diameter, taap, u.s.a.). statistical analysis spss software version 18 was used for all the statistical analysis. all values in the experiments were expressed as the mean ± standard deviation (sd) and were analyzed with a one-way analysis of variance (anova) with a significant level set at p < 0.05. results and discussion synthesis and characterization of ag/sio2nc synthesis and characterization of ag/sio2nc have attracted the attention of the materials community because of their promising properties (zaferani 2018). the green synthesis approach of those ncs with controllable size and properties has applications in miniaturized catalysts, photonics, optical devices, medical applications moreover it could be used as a potential nanomicrobicide and nanoscale growth regulator in agriculture (das et al. 2019). endless progression of microbial antibiotic-resistant mechanisms claims continuous searching for alternative approaches to deal with their risk to humans and plants (rai et al. 2012). the present study provided a green approach for the synthesis of antimicrobial ag/sio2nc mediated by the cell-free metabolites of e. coli d8. ftir spectrum confirmed the presence of proteins during the bio-reduction process. these proteins, in the e. coli d8 metabolite, might be including the reducing enzymes and/or some redox agents such as sulfurcontaining proteins resulting in the bio-reduction of silver ions (ag+) into agnps (krishnaraj et al. 2012). also, quinones (menaquinone, demethylmenaquinone, and ubiquinone) found in the e. coli d8 metabolite act as an electron shuttle compound and reduced ag+ into agnps in the presence of sunlight as reported by duan et al. (2015) and sharma et al. (2012). the biosynthesis of agnps was confirmed through visual observation of the color change of the mixture into brown color producing an obvious absorption peak at 430 nm (fig. 1). the brown color is because of the excitation of the agnps surface plasmon resonance (el-dein et al. 2021). granbohm et al. (2018) found the uv-vis spectra of ag/sio2nc powders showed the silver spr peak at 410 nm. fig. 1. the uv-vis spectra of sio2 and ag/sio2nc. the xrd patterns of sio2 were examined and showed an amorphous sio2 characteristic diffraction peak at 22.4 °. ag/sio2nc xrd pattern revealed peaks at 2θ angles of 32.25 °, 38.25 ° and 44.4 ° corresponding to the reflections of (110), (111) and (200) crystalline planes of the face4 nova biotechnol chim (2022) 21(1): e1023 3 centered cubic (fcc) structure of agnps (fig. 2). also, we had found no other diffraction peaks for silver oxide in the ag/sio2nc xrd pattern which showed the coverage of the nc with pure agnps (xu et al. 2015). fig. 2. the xrd patterns of sio2 and ag/sio2nc. the ftir spectra of ag/sio2nc were analyzed in the region of 400 – 4,000cm-1 (fig. 3). the vibration bands around 3,431 and 1,613 cm-1 are attributed to the oh and carbonyl group (c=o), respectively. these signals clearly confirm the presence of bacterial compounds bounded on the surface of ag/sio2nc that affect protection and stability of the nc. the intense peaks around 3,431 and 2,914 cm-1 are attributed to the primary and secondary amines vibrations bands, respectively. the stretch c-n vibration of aliphatic amines existed at 1,078 cm-1 bands. these signals confirmed the presence of proteins in the ag/sio2nc synthesis. water bands were appeared at around 1,613 cm-1 corresponding to bending vibrations indicating the hygroscopic character of the powdered samples (singh and ahmed 2012). si–o–si and si–oh absorptions bands have been observed at 1,078; 783; and 462 cm-1. the si–o–ag linkages stretching were also seen at around 691 cm-1. the band appears in the ag/sio2nc suggesting bonding between the agnps and the oxygen bonded to sio2. the peaks 450 – 800 cm-1 are probably related to the pseudolattice vibrations (mathur et al. 2006). fig. 3. the ftir spectra of sio2 and ag/sio2nc. the ag/sio2nc was examined by the tem (fig. 4) to investigate the morphology and size of the agnps (fig. 4b). agnps are embedded within the matrix and on the surface of the sio2. tem image showed small spherical shaped agnps having a diameter between 18 – 24 nm. gu et al. (2011) reported that the agnps average particle size on the surfaces of sio2 had a little increase from 10 to 25 nm as reaction temperature increased. this should be attributed to the higher reduction rate of ag+ at the elevated reaction temperature. sio2 has a net positive charge, while ag/sio2nc may have a positive or negative surface charge depending on the surface functional group and solution ph (jana et al. 2007; jayasuriya 2017). the synthesis of ag/sio2nc included binding primary amines as confirmed by the ftir analysis. the primary amines were deprotonated during the bio-reduction process, were leading to a gradual decrease in the surface positive charge of sio2 and might approach zero (jana et al. 2007). on the other hand, the binding between the agnps which are capped with highly negative proteins (el-dein et al. 2021), and sio2 to give negatively charged ag/sio2nc. the biosynthesized ag/sio2nc had a negative charge, -31.0 mv (fig. 5), which matched with shanthil et al. (2012) results, -33 ± 2 mv and was better than zhao et al. (2016) and elsheshtawy et al. (2020) results (-16.10 mv and -15 mv, respectively). different studies (verma and stellacci 2010; anas et al. 2013) have studied the interaction of charged nanomaterials with cells showing that positively charged nanomaterials have 5 nova biotechnol chim (2022) 21(1): e1023 2 the greatest efficacy in penetrating the cell membrane. other studies (fuller et al. 2008; martin et al. 2008) have examined the cellular uptake of negatively charged nanomaterials and proposed that negatively charged nanomaterials generate reactive oxygen species (ros) contributes towards potent bacterial toxicity (ivask et al. 2010; agnihotri et al. 2013). a further study of the synthesized ag/sio2nc should be taken into account to improve the antimicrobial efficacy of ag/sio2nc to have a positive surface charge. the positive charge of the nanomaterials increases the efficient electrostatic interaction with the negative charges of the microbial cell wall (li et al. 2011). fig. 4. (a) tem micrograph of sio2. (b) tem micrograph of ag/sio2nc. fig. 5. zeta potential measurement analysis of ag/sio2nc (-31.0 mv). antimicrobial potential of ag/sio2nc the pathogenic bacteria, yeast, and fungi appeared to be more tolerant to sio2 than ag/sio2nc. in this study, ag/sio2nc was investigated to determine its antimicrobial action (fig. 6 and table 1). the nc revealed very good antimicrobial potential against a wide range of microorganisms such as k. pneumoniae, s. aureus, and b. cinerea. the inhibition of microbial growth due to surface contact with the sio2 nanocomposite containing agnps demonstrated that nc functionalized with 6 nova biotechnol chim (2022) 21(1): e1023 2 the agnps has better antimicrobial action than bulk sio2. the antibacterial potential results of ag/sio2nc in he et al. (2012) study revealed that ag/sio2nc were sensitive to s. aureus and e. coli and with the inhibition zone diameter 15.3 mm and 10.4 mm, respectively. lu et al. (2017) studied the combination between chlorhexidine and ag/sio2nc and recorded that combination might produce synergistic bactericidal and candidacidal effects and improve the microbicidal efficiency. in addition, ag/sio2nc showed antifungal potential against b. cinerea besides the antibacterial and anticandidal actions. rodríguez-cutiño et al. (2018) confirmed the antimicrobial properties of ag/sio2nc against bacteria such as e. coli, b. cereus, s. typhimurium, and s. aureus in addition to the green squash fungi: b. cinerea and r. solani. fig. 6. antimicrobial activity of sio2, agnps, and ag/sio2nc; (a) b. cereus, (b) e. coli, (c) k. pneumoniae, (d) s. aureus, (e) c. albicans, and (f) b. cinerea. table 1. antimicrobial activity of sio2, agnps and ag/sio2nc against the pathogenic microbial strains (highly significant = *p < 0.05; n = 3). antibacterial activity (inhibition zone, mm ± sd) substance b. cereus e. coli k. pneumoniae s. aureus agno3 24 ± 0.06* 26 ± 0* 34 ± 0* 29 ± 0* sio2 -ve -ve -ve -ve agnps 30 ± 0.14* 30 ± 0.14* 38 ± 0* 37 ± 0* ag/sio2nc 20 ± 0.06* 22 ± 0.14* 36 ± 0* 34 ± 0* penicillin g potassium 29 ± 0* 30 ± 0* -ve 26 ± 0.06* antifungal activity (inhibition zone, mm ± sd) substance c. albicans b. cinerea agno3 13 ± 0.06* 13 ± 0.14* sio2 -ve -ve agnps 16 ± 0.06* 21 ± 0.14* ag/sio2nc 14 ± 0.06* 24 ± 0.14* fluconazole 15 ± 0* 16 ± 0.14* agnps and penicillin g potassium showed a similar manner of mic values (6.25 μg.ml-1) against s. aureus, b. cereus, and e. coli compared to ag/sio2nc (mic value, 125 μg. ml -1). the 7 nova biotechnol chim (2022) 21(1): e1023 2 mic values against b. cinerea were 6.25 and 25 μg.ml-1 for ag/sio2nc and fluconazole, respectively. the better growth inhibition percentage of ag/sio2nc was against b. cinerea (50.2 %) followed by b. cereus (31.1 %), s. aureus (30.7 %), e. coli (26.6 %), and c. albicans (6.6 %) showing a dose-dependent manner of ag/sio2nc antimicrobial action (fig. 7). the minimum antibacterial concentration of the ag/sio2nc is 0.2 and 0.3 μg.ml-1 for bacillus sp. and e. coli, respectively (huang 2008). qasim et al. (2015) suggested ag/sio2nc to be a potential antifungal agent for c. albicans 077 showing that this tested human pathogenic fungus was sensitive to ag/sio2nc with mic∼6 μg.ml -1 of ag/sio2nc. vladkova et al. (2020) presented that tio2/sio2/ag nanocomposite totally inhibited the e. coli growth within 30 min to 2 h. the growth of b. cinerea was almost completely inhibited (98.4 %) by ag/sio2nc (6.4 µg.ml -1) treatment compared with agnps alone (72.43 %, 6.4 µg.ml-1 as reported by kim (2011). fig. 7. growth inhibition percentage of ag/sio2nc, agnps, and antibiotics at mic values against s. aureus, b. cereus, e. coli, c. albicans, and b. cinerea. it is known that both, ag+ and agnps are effective antimicrobial agents even though their antimicrobial mechanism is not fully understood (kędziora et al. 2018). several studies (feng et al. 2000; lara et al. 2011) reported the different mechanism of the antimicrobial action of nanomaterials such as penetrating the cell wall and plasma membrane, ending with damaging dna molecules. others suggested that nanomaterials might interact with thiol groups in proteins, which induces the inactivation of microbial proteins. in the presented study, agnps bonded on the surface of sio2 have an opposite charge with grampositive bacteria, in that way killing them more easily than gram-negative bacteria due to the electrostatic attraction. the antimicrobial activities of ag/sio2nc are investigated using e. coli and b. cinerea as two model microorganisms. as shown in fig. 8, untreated e. coli was typically rod-shaped with smooth and intact cell walls. after being treated with ag/sio2nc, cell walls became wrinkled and damaged. the separation between the bacterial cell wall and cell membrane was also noted. fig. 8. the antibacterial action of ag/sio2nc on the ultrastructure of e. coli. (a) a negative control (without ag/sio2nc). (b) a treated sample (150 μg.ml -1), there are irregular rods (arrows) with lysed cell walls (ly) and complete cell lysis (cl). also, note the separation that occurs between the bacterial cell wall and cell membrane. ag/sio2nc 8 nova biotechnol chim (2022) 21(1): e1023 3 with treated b. cinerea (fig. 9), tem micrographs showed many changes, including the reduced size of treated cells, the formation of a mucilage matrix connecting the hyphal cells together, the appearance of big vacuole and lipid droplets with severe leakage of cytoplasmic contents in comparing to the control. the separation between the fungal cell wall and plasma membrane was also detected in the treated cells. the observed damages of the e. coli and b. cinerea cells after the treatment by ag/sio2nc could be because of cellular interactions with the agnps. the combined action of adhesion and penetration of agnps might illustrate the biocidal action of the nc, plasma membrane being the target of rapid antimicrobial action of agnps in e. coli and b. cinerea (rai et al. 2012). eckhardt et al. (2013) reported that the binding of agnps with microbial proteins might inactivate the electron transport chain, in that way suppressing the respiration and growth of the microbial cells. to establish that the advantages of silver nanocomposites (agncs) and the possible mechanisms of their antimicrobial action outweigh the possible risks, the toxicity of agnps and agncs must be investigated. w cy v pm v l (a) (b) fig. 9. the antifungal activity of ag/sio2nc on the ultrastructure of b. cinerea. (a) negative control (without ag/sio2nc). note normal cell wall (w), plasma membrane (pm), vacuole (v), and compact cytoplasm (cy). (b) the treated sample, note, the big vacuole (v) and lipid droplets (l). also, note the separation that occurs between the fungal cell wall and plasma membrane (arrow). conclusion the embedded agnps in ag/sio2nc mediated by e. coli d8 were characterized as negativecharged (-31.0 mv) and spherical with an average size ranging between 18 and 24 nm. ag/sio2nc showed a good antimicrobial potential against gram-negative and gram-positive bacteria and pathogenic yeast and fungi. the ag/sio2nc has brought many biomedical and agriculture applications (non-toxic to humans in minute concentrations). further study will be designed to elucidate the mode of action of ag/sio2nc as an antimicrobial agent. conflict of interest the authors declare that they have no conflict of interest. references agnihotri s, mukherji s, mukherji s (2013) immobilized silver nanoparticles enhance contact killing and show highest efficacy: elucidation of the mechanism of bactericidal action of silver. nanoscale 5: 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hollow nanoparticles. sci. rep. 6: 1-12. 11 microsoft word juristová nb.doc nova biotechnologica vii-i (2007) 107 synthesis and reactions of [1]benzofuro[3,2-c]pyridine nadežda juristová1, eleonóra štefanovová1, tatiana ďurčeková1, naďa prónayová2, anton gatial3, alžbeta krutošíková1 1department of chemistry, faculty of natural sciences, university of ss. cyril and methodius, sk-917 01 trnava, slovak republic e-mail: alzbeta.krutosikova@ucm.sk 2institute of analytical chemistry, department of nmr and ms spectroscopy, slovak university of technology, radlinského 9, sk-812 37 bratislava, slovak republic 3institute of physical chemistry and chemical physics, slovak university of technology, radlinského 9, sk-812 37 bratislava, slovak republic abstract: (e)-3-(1-benzofuran-2-yl)propenoic acid (i) was prepared from 1-benzofuran-2-carbaldehyde under the doebner’s conditions. the obtained acid was converted to the corresponding azide ii, which was cyclized by heating in diphenyl ether to [1]benzofuro[3,2-c]pyridin-1(2h)-one (iii). this compound was aromatized with phosphorus oxychloride to chloroderivative iv which was reduced with zinc and acetic acid to the title compound v. [1]benzofuro[3,2-c]pyridin-2-oxide (vi) was synthesized by reaction of v with 3chloroperoxybenzoic acid in dichloromethane. treatment vi with benzoyl chloride and potassium cyanide (reissert-henze reaction) was shown to produce the corresponding [1]benzofuro[3,2-c]pyridin-1carbonitrile (vii). the title compound was used for preparation of complex compounds viii, ix key words: [1]benzofuro[3,2-c]pyridine, n-oxide, 1-carbonitrile, 1h, 13c nmr and ir spectra, metalorganic compounds 1. introduction furopyridines are of chemical interest due to their similarity to quinoline, isoquinoline, and benzofuran, which are important ring systems present in many biologically active compounds. the antihypertensitive drug cicletanine is a furo[3,2c]pyridine derivative. the furopyridine skeleton from which this compound is derived is not associated with any known pharmacological classes (sherman, 1996). in the past one of us has been interested in studying the synthesis and reactivity of various furo[3,2-c]pyridines (bobošík et al., 1995; krutošíková et al., 1992, 1994, 1995; krutošíková and sleziak, 1996). the authors (bencková and krutošíková, 1995; 1999) were concentrated on transformations on the pyridine ring of some of this type of compounds. later on some furo[3,2-c]pyridines have been used for preparation cu(ii) and ni(ii) complexes (krutošíková et al., 2001). furo[3,2-c]pyridine and its 2-methyl-, 2,3-dimethyl and benzo[4,5] derivative were used for the first time as ligands to synthesize potentially new werner clathrates and their structural characterization, spectral and magnetic properties of isothiocyanate nickel(ii) complexes (miklovič et al., 2004, mojumdar et al., 2005). magneto structural correlations in heteroleptic furo[3,2-c]pyridineni(ii) complexes were 108 juristová, n. et al. published by the research group (baran et al., 2005). recently two papers dealing about the syntheses and crystal structures of tetra-μ-acetato-bis(1-benzofuro[3,2c]pyridinecopper(ii) and bis(1-benzofuro[3,2-c]pyridine-κn)dichlorocobalt(ii) were published (vrabel et al., 2007). in this paper, which continues our previous papers (bencková and krutošíková, 1999; krutošíková et al., 1992, 1994, 1995) we report the synthesis of [1]benzofuro[3,2-c]pyridine and its 2-oxide (scheme 1). o chα hβ c co2h o nh o o n o n cl ch2(co2h)2 et3n, clco2et o cho i ii iiiivv o n o nan3 2 2 2 vi o chα hβ c con3 2 pocl3 bu3n ph2o zn acoh scheme 1 3 4 5 6 7 3 4 5 6 73 4 5 6 3 4 56 7 6 7 3 4 5 6 7 8 9 8 99 8 9 111 1 8 7 4 5 3-clpba 2 2 3 2. experimental melting points were determined using kofler hot plate. all solvents were distilled and dried before use. all reagents were commercially available and were used without purification. elemental analyses were determined using an eager 300 at institute of inorganic chemistry, technology and materials stu in bratislava. ir spectra were taken on a ftir nicolet nexus 470 spectrophotometer using kbr technique (0.5 mg in 300 mg kbr) in region 4000 – 400 cm-1 at institute of physical chemistry and chemical physics, stu in bratislava. for interpretation of ir spectra following abbreviations are used: m = medium band (a value of transmittance: 36-50%) w = weak (a value of transmittance: over 50%), without marking are strong bands (a value of transmittance: 0-35%). 1h nmr spectra were measured in dmso-d6 using varian inova 600 (for 1h 599,782 mhz and for 13c 150.830 mhz) spectrometer at institute of analytical chemistry, department of nmr and ms spectroscopy, stu in bratislava. chemical shifts (δ-scale) are quoted in parts per million and following abbreviations are used: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet, coupling constants (j) are given in hz. 2.1 (2e)-3-(1-benzofuran-2-yl)propenoic acid (i) a mixture of 1-benzofuran-2-carbaldehyde (5.1 g; 35 mmol), malonic acid (3.7 g; 35 mmol), pyridine (39.3 cm3) and piperidine (17 drops) was heated on steam bath for 8 h. the reaction mixture was poured on ice, acidified with 0.5 m hydrochloric acid. the separate precipitate was filtered off and crystallized. yield: 11.74 g (88.9%); white crystals, m.p. 222-223 °c (ethanol). for c11h7n3o2 (mr = 188.18) wi (calc.): 70.21% c, 4.29% h; wi (found): 70.41% c, 4.26% h. nova biotechnologica vii-i (2007) 109 1h nmr spectrum: 12.6 ( bs co2h); 7.69 (d, 3j(4,5) = 7.8 hz, 4j(4,6) = 1.2 hz, h4); 7.59 (dd, 3j(7,6) = 8.1 hz, 4j(7,5) = 1.2 hz, h7); 7.57 (d, 3j(b,a) = 15.6 hz, hb); 7.39 (t, 3j(6,7) = 7.8 hz, 4j(6,4) = 1.2 hz, h6); 7.34 (s, h3); 7.28 (t, 3j(5,6) = 7.8 hz, 4j(5,7) = 1.2 hz, h5); 6.41 (d, 3j(a,b) = 15.6 hz, ha). 13c nmr spectrum: 167.1 (c=o); 154.9 (c7a); 152.1 (c2); 131.2 (cb); 128.1 (c3a); 126.7 (c6); 123.6 (c5); 122.2 (c4); 119.4 (ca); 111.7 (c3); 111.4(c7). ir spectrum: 1671; 1630; 1451; 1419; 1304; 1268; 1200; 1127; 1007w; 978w; 951; 882w; 834m; 753; 738; 664w; 568m; 456w. 2.2 (2e)-3-(1-benzofuran-2-yl)propenoyl azide (ii) propenoic acid i (3.76 g; 20 mmol) suspended in dry acetone (40 cm3) was cooled to -10 °c and triethylamine (3.2 cm3) in dry acetone (5 cm3) was added. then solution of ethyl chloroformate (2.4 cm3) in dry acetone (5 cm3) was added dropwise to the stirred reaction mixture at temperature lower than 0 °c. the reaction mixture was stirred for another 30 min at the same temperature. solution of sodium azide (2.0 g; 30.8 mmol) in water (10 cm3) was added at temperature below 0 °c. the mixture was stirred for an additional hour and allowed to warm to room temperature, poured onto crushed ice and precipitate was filtered off, washed with water and crystallized. yield: 4.07 g; (95.6%), m.p. 114-116 °c white crystals. for c11h7n3o2 (mr = 213.19) wi (calc.): 61.97% c, 3.31% h, 19.71% n; wi (found): 61.82% c, 3.27% h, 19.78% n. 1h nmr spectrum: 7.74 (d, 3j(b,a) = 15.5 hz, hβ); 7.73 (dd, 3j(4,5) = 8.2 hz, 4j(4,6) = 1.2 hz, h4); 7.61 (dd, 3j(7,6) = 8.2 hz, 4j(7,5) = 0.88 hz, h7); 7.50 (s, h3); 7.44 (t, 3j(6,5) = 7.2 hz, 4j(6,4) = 1.2 hz, h6); 7.30 (t, 3j(5,6) = 7.38 hz, 4j(5,7) = 0.88 hz, h5); 6.46 (d, 3j(a,b) = 15.5 hz, ha). 13c nmr spectrum: 170.9 (c=o); 155.2 (c7a); 151.5 (c2); 133.1 (cb); 127.9 (c3a); 127.4 (c6); 123.7 (c5); 122.5 (c4); 118.6 (ca); 114.2 (c3); 111.4 (c7). ir spectrum: 2150; 1677; 1619; 1544; 1471; 1448; 1348; 1330; 1272; 1169; 1127; 1096; 999; 951; 824; 788; 687; 630; 610m; 544m; 520m; 441m; 402m; 2.3 [1]benzofuro[3,2-c]pyridin-1(2h)-one (iii) azide ii (8.67 g; 41 mmol) dissolved in toluene (67 cm3) was added dropwise into diphenyl ether (35 cm3), and tributylamine (10.7 cm3) were at 180-200 °c for 45 min, while toluene was distilled out. then the reaction mixture was heated up 215 °c for 15 min. after cooling, diethyl ether was added the precipitate was filtered off and washed with diethyl ether. yield: 6.22 g (82.6%), m.p. 207-209 °c (ethanol) white crystals. for c11h7no2 (mr = 185.18) wi (calc.): 71.35% c, 3.81% h, 7.56% n; wi (found): 71.22% c, 3.78% h, 7.66% n. 1h nmr spectrum: 11.83 (bs, nh); 8.01 (dd, 3j(9,8) = 7.33 hz, 4j(9,7) = 1.76 hz, h9); 7.678 (dd, 3j(6,7) = 7.04 hz, 4j(6,8) = 1.76 hz, h6); 7.41 (t, 3j(7,8) = 7.33 hz, 4j(7,9) = 1.47 hz, h7); 7.407 (t, 3j(8,7) = 7.33 hz, 4j(8,6) = 1.47 hz, h8); 7.56 (d, 3j(3,4) = 7.34 hz, h3); 6.76 (d, 3j(4,3) = 7.34 hz, h4). 13c nmr spectrum: 162.5 (c4a); 159.5 (c1); 154.1 (c5a); 135.2 (c3); 125.7 (c7); 124.2 (c8); 123.6 (c9a); 120.8 (c9); 111.3 (c6); 110.0 (c9b); 94.4 (c4). 110 juristová, n. et al. ir spectrum: 1647; 1565; 1486; 1453; 1428; 1389; 1388m; 1258m; 1233; 1183; 1068; 1000m; 947m; 906m; 846m; 788; 760; 677m; 653m; 587; 543; 43. 2.4 1-chloro[1]benzofuro[3,2-c]pyridine (iv) a pyridone iii (4.5 g; 24 mmol) was refluxed in phosphorus oxychloride (8.6 cm3) for 4 h. pocl3 was removed at reduced pressure and the ice was added to the residue. the mixture was then made basic with diluted aqueous ammonia. the precipitate was filtered out, washed with water, dried and crystallized. yield: 4.71 g (91.2%), m.p. 65 67 °c (hexane) white crystals. for c11h6clno (mr = 203.62) wi (calc.): 64.88% c, 2.97% h, 6.88% n; wi (found): 64.73% c, 2.95% h, 6.79% n. 1h nmr spectrum: 8.44 (d, 3j(3,4) = 5.57 hz, h3); 8.16 (dd, 3j(9,8) = 7.8 hz, 4j(9,7) = 1.8 hz, h9); 7.798 (d, 3j(4,3) = 5.57 hz, h4); 7.782 (dd, 3j(6,7) = 8.3 hz, 4j(6,8) = 0.88 hz, h6); 7.64 (t, 3j(7,8) = 7.6 hz, 4j(7,9) = 1.8 hz, h7); 7.50 (t, 3j(8,7) = 7.6 hz, 4j(8,6) = 0.88 hz, h8). 13c nmr spectrum: 161.5 (c4a); 155.1 (c5a); 147.1 (c3); 143.6 (c1); 129.3 (c7); 124.5 (c8); 122.1 (c9); 119.7 (c9b); 118.8 (c9b); 112.0 (c6); 107.6 (c4). ir spectrum: 1589; 1561; 1457m; 1428; 1331m; 1294m; 1261m; 1233m; 1186; 1152m; 1016w; 995w; 947; 842m; 829; 755; 614m; 520w; 434w. 2.5 [1]benzofuro[3,2-c]pyridine (v) zinc powder (12.07 g; 185 mmol), was added to iv (9.0 g; 44 mmol) and acetic acid (15 cm3) and mixture was refluxed for 8 h, then filtered and the solvent was distilled off under reduced pressure. the residue was alkalized with diluted sodium hydroxide solution and extracted with chloroform. the solution was dried with sodium sulfate and the solvent was evaporated. yield: 4.65 g (62.5%), m.p. 74-76 °c (hexane) white crystals. for c11h7no (mr = 169.18) wi (calc.): 78.09% c, 4.17% h, 8.28% n; wi (found): 78.12% c, 4.01% h, 8.16% n. 1h nmr spectrum: 9.42 (d, 5j(1,4) = 0.88 hz, h1); 8.65 (d, 3j(3,4) = 5.8 hz, h3); 8.25 (dd, 3j(9,8) = 7.6 hz, 4j(9,7) = 1.2 hz, h9); 7.785 (dd, 3 j(6,7) = 8.2 hz, 5 j(6,9) = 0.88 hz, h6); 7.78 (dd, 3 j(4,3) = 5.8 hz, 5 j(4,1) = 0.88 hz, h4); 7.60 (t, 3 j(7,6) = 8.2 hz, 4 j(7,9) = 1.2 hz, h7); 7.49 (t, 3 j(8,7) = 7.6 hz, 4 j(8,6) = 0.88 hz, h8). 13c nmr spectrum: 160.4 (c4a); 155.7 (c5a); 147.6 (c3); 143.9 (c1); 128.6 (c7); 124.1 (c8); 121.5 (c9); 121.1 (c9b); 120.7 (c9a); 111.9 (c6); 107.5 (c4). ir spectrum: 3063-3037; 1590; 1577; 1481m; 1463; 1446; 1331m; 1293m; 1266m; 1243w; 1207; 1186; 1162; 1106m; 1009m; 995m; 862; 838; 823; 776; 751; 734; 595. 2.6 [1]benzofuro[3,2-c]pyridin-2-oxide (vi) a mixture of v (1.42 g; 8.4 mmol) and 3-chloroperoxybenzoic acid (70%, 3.69 g; 14.6 mmol) in dichloromethane (50 cm3) was stirred at room temperature for 4 h. the nova biotechnologica vii-i (2007) 111 reaction mixture was filtered and extracted with sodium carbonate (10%), and water. the organic layer was dried over sodium sulfate, filtered and solvent was evaporated and residue was crystallized. yield: 1.01 g, 65%., m.p. 205-206 °c (toluene). for c11h7no2 (mr = 185.18) wi (calc.): 71.35% c, 3.81% h, 7.56% n; wi (found): 71.42% c, 3.79% h, 7.38% n. 13c nmr spectrum: 9.21 (d, 4j(1,3) = 1.8 hz, h1); 8.32 (dd, 3j(3,4) = 7.1 hz, 4j(3,1) = 1.8 hz, h3); 8.20 (dd, 3j(9,8) = 7.6 hz, 4j(9,7) = 1.4 hz, h9); 7.82 (d, 3j(4,3) = 7.1 hz, h4); 7.76 (dd, 3j(6,7) = 8.2 hz, 4j(6,8) = 1.7 hz, h6); 7.64 (t, 3j(7,6) = 8.2 hz, 4j(7,9) = 1.4 hz, h7); 7.48 (t, 3j(8,9) = 7.6 hz, 4j(8,6) = 1.7 hz, h8). 13c nmr spectrum: 156.8 (c5a); 151.3 (c4a); 138.3 (c3); 132.6 (c1); 129.7 (c7); 124.2 (c6); 123.0 (c9b); 122.5 (c9); 120.2 (c9a); 112.2 (c8); 109.9 (c4). ir spectrum: 1661; 1470; 1440; 1308m; 1288; 1205; 1158; 1098; 1010m; 929m; 828; 769; 742; 596; 526; 425. 2.7 [1]benzofuro[3,2-c]pyridine-1-carbonitrile (vii) to solution of potassium cyanide (1.25 g, 19 mmol) in water (2 cm3) a solution of [1]benzofuro[3,2-c]pyridin-2-oxide (vi) (3.42 g, 1.85 mmol) in dichloromethane (10 cm3) was added and then dropwise a solution of benzoyl chloride (0.3 cm3, 2.15 mmol) in dichloromethane (10 cm3). after vigorous stirring at room temperature for 2 days, the organic layer of the reaction mixture was separated and aqueous layer was extracted with chloroform. after drying over magnesium sulfate, the combined organic layers were evaporated and residue was purified by column chromatography on silica gel (hexane-ethyl acetate 3:1). the obtained vii was crystallized. yield: 3.05g, (85%), m.p. 135-137 °c (hexane) 139-141 °c (bencková and krutošíková, 1999). for c12h6n2o (mr = 194.19) wi (calc.): 74.22% c, 3.11% h, 14.43% n; wi (found): 74.32% c, 3.18% h, 14.36% n. 13c nmr spectrum: 8.81 (d, 3j(3,4) = 5.57 hz, h3); 8.21 (d, 3j(9,8) = 7.63 hz, h9); 8.14 (d, 3j(4,3) = 5.57 hz, h4); 7.89 (d, 3j(6,7) = 8.5 hz, h6); 7.76 (t, 3j(7,6) = 7.92 hz, 3j(7,8) = 7.63 hz, h7); 7.62 ((t, 3j(8,7) = 7.63 hz, 3j(8,9) = 7.63 hz, h8). 13c nmr spectrum: 160.4 (c4a); 155.8 (c5a); 148.8 (c3); 130.9 (c7); 125.0 (c8); 124.9 (c9b); 124.2 (c1); 121.4 (c9); 118.4 (c9a); 116.2 (cn); 112.6 (c6); 111.4 (c4). ir spectrum: 3093; 2227m; 1627w; 1591; 1567; 1483w; 1458; 1422; 1336; 1295w; 1263; 1231; 1182; 1109; 1072w; 1015m; 1005; 944w; 841; 806; 784w; 748; 641w; 568w; 543m. 2.8 tetra-μ-acetato-bis[([1]benzofuro[3,2-c]pyridine)copper(ii)] (viii) to a cu(ch3co2)2.h2o (1.5 mmol) in ethanol (5 cm 3) was added the solution of the title compound (3.2 mmol) in ethanol (2 cm3). small blue-green crystals were 112 juristová, n. et al. collected after 2 d. these were filtered off, washed with ethanol and crystallized from tetrahydrofuran. for c30h26n2cu2o10 (mr = 701.63) wi (calc.): 51.35% c, 3.73% h, 3.99% n, 18.11% cu; wi (found): 51.43% c, 3.65% h, 3.99% n, 18.15% cu. ir spectrum: 3852; 3820; 3674; 3628; 3441; 3085; 3053; 2569; 2527; 1633; 1605; 1592; 1485; 1466; 1448; 1333; 1292; 1251; 1212; 1177; 1154; 1146; 1041; 1024; 878; 838; 821; 774; 758; 596; 574w; 562w; 523w. 2.9 bis([1]benzofuro[3,2-c]pyridine-κn)dichloridocobalt(ii) (ix) to a cocl2.6h2o (1 mmol) solution in ethanol (4 cm 3) was added the solution of v (3 mmol) in ethanol (4 cm3) at room temperature. small blue crystals were formed after 3 d, which were filtered off, washed with ethanol and crystallized from tetrahydrofuran. for c22h14cl2con2o2 (mr = 468.21) wi (calc.): 56.43% c, 3.01% h, 5.98% n, 12.58 co; wi (found): 56.67% c, 2.96% h, 5.96% n, 11.96 co. ir spectrum: 1626; 1596; 1484m; 1484; 1467; 1450; 1433; 1210m; 1187; 1165; 1018w; 872; 840m; 777; 757m; 681m; 629; 598w; 567w; 523w. 3. results and discussion (e)-3-(1-benzofuran-2-yl)propenoic acid (i) was prepared from 1-benzofuran-2carbaldehyde under the doebner’s conditions. the obtained acid i was converted to the corresponding azide ii, which was cyclized by heating in diphenyl ether to [1]benzofuro[3,2-c]pyridin-1(2h)-one (iii). this compound was aromatized with phosphorus oxychloride to chloroderivative iv which was reduced with zinc and acetic acid to the title compound v. [1]benzofuro[3,2-c]pyridin-2-oxide (vi) was synthesized by reaction of v with 3-chloroperoxybenzoic acid in dichloromethane. treatment vi with benzoyl chloride and potassium cyanide (reissert-henze reaction) was shown to produce the corresponding [1]benzofuro[3,2-c]pyridine-1-carbonitrile (vii). this transformation was easily verified by means of infrared spectra, loss of n-oxide band at 1205 cm-1 in the spectra of vii and appearance of cyano group absorption at 2227 cm-1. disappearance of the most downfield signal in the 1h nmr spectrum (at 9.21 ppm) of the starting compound vi also supported the structure of the compound vii. for the assignment of carbon signals in the synthesized compounds 2d-spectra,gcosy, ghsqcad and ghmbcad were used. 4. conclusions the title compound [1]benzofuro[3,2-c]pyridine (v) was prepared five step synthesis and was used for preparation of complex compounds. the structures of viii, ix were proved by x-ray analysis (figures 1,2). the structural data are presented in (vrabel, 2007). nova biotechnologica vii-i (2007) 113 fig. 1. the molecular structure of the complex compound viii (vrabel et al. 2007). fig. 2. the molecular structure of the complex compound ix (vrabel et al. 2007). acknowledgements: this work was supported by the grant agency of the ministry of education of the slovak republic vega (grant no. 1/3584/06). nmr experimental part of this work was facilitated by support of slovak national research and development program no. 2003sp200280203. references baran, p., boča, m., boča, r., krutošíková, a., miklovič, j., pelikán, j., titiš, j.: structural characterization, spectral and magnetic properties of isothiocyanate nickel(ii) complexes with furopyridine derivatives. polyhedron, 24, 2005, 1510-1516. 114 juristová, n. et al. bencková, m., krutošíková, a.: synthesis of pyrrolo[2´,3´:4,5]furo[3,2c]pyridines. monatsh. chem., 126, 1995, 753-758. bencková, m., krutošíková, a.: 5-aminofuro[3,2-c]pyridinium tosylates and substituted furo[3,2-c]pyridine-n-oxides: synthesis and reactions. collect. czech. chem. commun., 64, 1999, 539-547. bobošík, v., krutošíková, a., jordis, u.: synthesis and reactions of 2,3dimethyfuro[3,2-c]pyridines. monatsh. chem., 126, 1995, 747-752. krutošíková, a., dandárová, m., chylová, j., végh, d.: condensed on-heterocycles by the transformation of azidoacrylates. monatsh. chem., 123, 1992, 807-815. krutošíková, a., dandárová, m., alföldi, j.: substituted vinyl azides in the synthesis of condensed nitrogen heterocycles. chem. papers, 48, 1994, 268273. krutošíková, a., sleziak, r: synthesis of 2-arylfuro[3,2-c]pyridines and their derivatives. collect. czech. chem. commun., 61, 1996, 1627-1636. krutošíková, a., mitasová, b., jóna, e., bobošíková, m.: synthesis, thermal and spectral properties of cu(ii) and ni(ii) complexes with furopyridines or quinoline. chem. papers, 55, 2001, 290-293. miklovič, j., krutošíková, a., baran, p.: two furopyridine complexes of nickel(ii) isothiocyanate. acta cryst., c60, 2004, m227-m230. mojumdar, s.c., miklovič, j., krutošíková, a., valigura, d., stewart, j. m.: furopyridines and furopyridine-(ni(ii) complexes: synthesis, thermal and spectral characterization. j. therm. anal. cal., 81, 2005, 211-215. sherman, a.r.: bicyclic 5-6 systems: two heteroatoms 1:1. in: c.a. ramsden (ed.) comprehensive heterocyclic chemistry ii. pergamon press, oxford, 1996, 167-227. vrábel, v., švorc, ľ., juristová, n., miklovič, j., kožíšek, j.: tetra-μacetato-bis[(benzofuro[3,2-c]pyridine]copper(ii). acta cryst., e63, 2007, m2112m2113. vrábel, v., švorc, ľ., juristová, n., miklovič, j., kožíšek, j.: bis(1benzofuro[3,2-c]pyridine-κn)dichloridocobalt(ii). acta cryst., e63, 2007, m24272428. nova biotechnologica et chimica 13-1 (2014) 13 doi 10.2478/nbec-2014-0002 © university of ss. cyril and methodius in trnava interplay between bacteriophages and restriction-modification systems in enterococci peter pristas, anna vandzurova, peter javorsky institute of animal physiology, slovak academy of sciences, soltesovej 4-6, 040 01 kosice, slovakia, (pristas@saske.sk) abstract: the complete genomes of enterococcus faecalis bacteriophages were analyzed for tetranucleotide words avoidance. very similar tetranucleotide composition was found in all tested genomes with strong underrepresentation of palindromic gatc and ggcc words. this avoidance could be explained as a protection mechanism against host restriction-modification systems as a clear correlation was found between avoidance of palindromic words and the specificity of e. faecalis restriction and modification systems. no similar avoidance of tetranucleotide words was observed for non-palindromic words. a weak correlation was observed between avoidance of tetranucleotide palindromes in bacteriophage genomes and the possession of phage encoded dna methyltransferases confirming the interrelation between bacteriophage genomes composition and restriction and modification systems in enterococci. key words: enterococcus, bacteriophage, restriction modification systems, palindrome avoidance 1. introduction type ii restriction-modification systems entail a dna methyltransferase and an endonuclease of the same recognition sequence specificity. the endonuclease digests foreign dna that enters the cell, thereby protecting the bacteria from genetic subversion. the methylase modifies the cell's dna, thereby protecting it from similar digestion. it is generally accepted that restriction systems in bacteria primarily act to protect the organism from foreign dna, particularly from infection by bacteriophages (bickle and kruger, 1993). bacteriophages on other hand have evolved antirestriction mechanisms, encode for their own methyltransferases, and are frequently deficient in recognition sites for restriction endonucleases (samson et al., 2013). the aim of our study was to analyze the tetranucleotide composition of enterococcal bacteriophage genomes, to analyze the effect of phage encoded modification methyltransferases on avoidance of tetranucleotides, and to compare the frequency and variability of restriction endonucleases encoded by enterococci. enterococci are ubiquitous bacteria present in the environment, in the gastrointestinal tract of healthy animals and humans, and in foods, especially those of animal origin such as dairy products (giraffa, 2003). enterococci entry into milk and milk products through the water supply, equipment, and unsanitary and unhygienic conditions during production and handling. in milk products, they are used as probiotics resulting in positive effects on human digestibility. thanks to the efficient utilisation of organic acids, enterococci contribute to the development of unique sensory characteristics in fermented dairy products. in contrast to these positive roles, some enterococcal strains were suspected to have pathogenic properties for bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:56 utc 14 pristas, p. et al. humans (hunt, 1998). enterococcal bacteriophages with very effective bactericidal activity are potentially used as novel antibacterial agents to control enterococcal infections (deresinski, 2009), however in dairy industry the bacteriophage infections result in unacceptably low production of lactic acid and flavour compounds along with reduced proteolysis and led to the major losses in fermentation processes (mc grath et al., 2007). better understanding of bacteriophage genetics and biology could improve this dual role of enterococcal bacteriophages. we have used markov chain analysis to evaluate which nucleotide dna words are overor underrepresented in the genomes of e. faecalis bacteriophages. markov analysis seems to be the most widely used to evaluate palindrome distribution (panina et al., 2000). briefly, using this model it is possible to calculate how often a word should appear in a sequence on the basis of knowledge of the distribution of the word’s fragments. under the maximum applicable order of the markov model, the expected count of a particular tetranucleotide (k), e.g. gatc sequence, is defined as: ( ) ( ) ( ) ( )atatcgatgatc nnnk /×= (1) where n(gat), n(atc) and n(at) are the observed counts of oligonucleotides. similar formulas have been widely used (fuglsang, 2003). this number can then be compared with the actual count of the word in the sequence. however, the difference itself does not tell to what extent a word is overor underrepresented. for this purpose, the normalized statistic z is used, as proposed by schbath (1997), and used by others for similar purposes. z (contrast value) is positive for overrepresented words and negative for underrepresented words, and is calculated as: ( ) ( )[ ] ( )gatc gatcgatc v nkn z /− = (2) where n is number of nucleotides in analysed sequence and the variance v(gatc) is calculated as: ( ) ( ) ( ) ( )[ ] ( ) ( )[ ] ( )at gatatatcat gatcgatc n nnnn kv 2) −×− = (3) based on the normalized nature of z, the probability of random observation of |z| > 3.29 is <0.001. the data obtained indicate strong underrepresentation of palindromic tetranucleotides in e. faecalis bacteriophage sequences which could be probably explained as a protection mechanism against host restriction-modification systems. 2. materials and methods complete genome sequences of e. faecalis bacteriophages and e. faecalis genome (refseq accession number nc_004668) were taken from the refseq ncbi reference bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:56 utc nova biotechnologica et chimica 13-1 (2014) 15 sequence database available at http://www.ncbi.nlm.nih.gov/refseq/. the presence (marked as y in table 1) or absence (marked as n in table 1) of modification methyltransferase in the genomes of bacteriophages was taken from annotations available in refseq database. for the list of bacteriophage sequences used through the study see table 1. the frequency of restriction endonucleases in e. faecalis was taken from rebase database (roberts et al., 2010). table 1. the list and corresponding z score of bacteriophage sequences used in the study. tetranucleotide average z-score bacteriophage accession number genome size (bp) mtase a all palindrome the most underreprese nted word (score) reference phifl4a nc_013644 37856 y 0 -3.62 gatc (-9.55) yasmin et al. 2010 phifl3a nc_013648 39576 y 0 -3.39 gatc (-9.34) yasmin et al. 2010 phifl2a nc_013643 36270 y 0 -3.19 gatc (-9.52) yasmin et al. 2010 phifl1a nc_013646 38764 y 0 -3.14 gatc (-8.77) yasmin et al. 2010 phief24c nc_009904 142072 n 0 -5.85 gatc (-27.68) uchiyama et al. 2008 phief11 nc_013696 42822 y 0 -3.21 gatc (-9.09) stevens et al. 2011 efrm31 nc_015270 16945 n 0 -2.95 gatc (-8.85) fard et al. 2010 efap-1 nc_012419 21115 n 0 -2.77 gatc (-12.76) son et al. 2010 ef62phi nc_017732 30505 n 0 -2.82 cgcg (-6.07) brede et al. 2011 bc-611 nc_018086 53996 n 0 -5.55 gatc (-19.79) horiuchi et al. 2012 ay in the mtase column indicate the presence, n the absence of modification methyltransferase in the genome of bacteriophage tetranucleotide counts, z score values, and the pearson’s correlation coefficients were calculated using tetra software (teeling et al., 2004).the matrix of correlation coefficients was converted into distance matrix using dambe software version 5 and similarity dendrogram was constructed using neighbor-joining algorithm implemented in the software (xia, 2013). statistica® software package (statsoft, tulsa, oklahoma) was used to compare the z (contrast value) distribution between datasets using pearson’s correlation coefficient. 3. results and discussion bacteriophages, although simple in organization, are the most diverse life forms in the biosphere (ackermann and kropinski, 2007). bacteriophage life cycle completely relies on bacterial host. during the lytic cycle, bacteriophage infection redirects host metabolism towards the replication of the phage nucleic acid and assembly of new phage particles, which are then released upon cell death and lysis. the bacteriophages thus have an important role in bacterial evolution and have led to a great variety of defense mechanisms. bacteriophages, for their part, have developed bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:56 utc 16 pristas, p. et al. counter defense mechanisms to evade the bacterial defense mechanisms (samson et al., 2013). one of the best studied systems to protect bacteria form invading bacteriophages is the possession of restriction-modification systems. these systems entail a dna methyltransferase and an endonuclease of the same recognition sequence specificity. the endonuclease recognizes short, usually palindromic oligonucleotides and digests bacteriophage dna that enters the cell, thereby protecting the bacteria from genetic subversion. the methylase modifies the cell's dna, thereby protecting it from similar digestion (wilson, 1991). bacteriophages have developed antirestriction systems and are frequently deficient in oligonucleotides recognized by restriction endonucleases. bacteriophages changed their genomes by adopting point mutations which reduce the number of restriction sites (tock and dryden, 2005). using tetra software palindrome deficiency in genomes of all available enterococcal bacteriophages was analyzed. a very similar pattern of palindrome avoidance was observed in genomes of enterococcal bacteriophages as well as in e. faecalis genome. while the frequencies of all tetranucleotide words were found to be normally distributed around z value 0, frequencies of palindromes were found to be strongly underrepresented in both bacteriophage and e. faecalis genomes (fig. 1). for bacteriophages z values of tetranucleotide palindromes were in range from 7.51 to 27.68. average z value of tetranucleotide palindromes for all bacteriophages were in range from -2.77 to -12.09, indicating strong underrepresentation of tetranucleotide palindrome words in genomes of e. faecalis bacteriophages. the most underrepresented word in all but one tested bacteriophage genome was gatc word (average z value -12.09, see table 1). fig. 1. correlation of z values of enterococcus faecalis v583 complete genome (refseq accession number nc_004668) and genome of phief11 bacteriophage (refseq accession number nc_013696). correlation of tetranucleotide palindromes (part a) compared to all palindromes (part b) is shown. several authors showed that the lowest contrast values show palindromes which serve as a recognition sites of most frequently occurring endonucleases. rocha et al. (1998), reported that gatc and ggcc palindromes are between 6 the most underrepresented tetranucleotide palindromes in complete genome of bacillus subtilis. surprisingly, much higher degree of tetranucleotide palindrome avoidance was bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:56 utc nova biotechnologica et chimica 13-1 (2014) 17 observed in the genome of e. faecalis v583 compared to bacteriophage genomes (table 2). while average z score observed for bacteriophage genomes was -3.70 much lower z score (-30.55) was observed for e. faecalis v583 chromosome. however a correlation was observed between z values of bacteriophages and chromosome (pearson correlation coefficient r=0.68). gatc and ggcc tetranucleotide palindromes are among 4 the most underrepresented words in both genomes. clear correlation was observed between the word avoidance and the existence of restrictionmodification systems with given specificity (table 2). from the e. faecalis bacterium restriction endonucleases recognizing gatc, ggcc, ccgg, and cgcg tetranucleotide are described (vanat et al., 1993; radlinska et al., 2005; anonymous at rebase.neb.com, and our unpublished data) and these 4 tetranucleotide words are the most underrepresented in the genome of e. faecalis. these 4 words show the lowest z score compared to other words as well. significant underrepresentation of other tetranucleotide words was observed in the genome of e. faecalis v583 indicating that much higher number of restriction-modification systems than currently known has occurred in the genome of the e. faecalis, each of them leaving a trace of underrepresentation of a short palindrome. table 2. correlation between the frequency of tetranucleotide palindromes in the genomes of e. faecalis bacteriophages and e. faecalis v583 complete genome and frequency of restriction-modification systems in e. faecalis. a denotes lack of rms, + the presence of rms bacteriophages frequently employ additional strategies to overcome host restriction and modification systems e.g. site specific modification of the phage genome by bacteriophage encoded modification methyltransferases (samson et al., 2013). among e. faecalis bacteriophages modification methyltransferase was found in the genomes of 5 from 10 tested bacteriophages (table 1). based on degree of 4 bp palindromes avoidance the matrix of similarity coefficients between all pairs of bacteriophage genomes was constructed and the similarity tree was constructed indicating that bacteriophages encoding bacteriophages chromosome tetranucleotide z-score rank z-score rank known rmsa aatt -6.21 3 -29.54 11 agct -2.16 12 -34.78 7 acgt -3.66 8 -10.78 14 atat 1.03 15 -5.00 15 gatc -12.69 1 -66.48 1 + ggcc -4.81 4 -51.21 2 + gcgc -2.81 11 -3.93 16 gtac 1.09 16 -14.21 13 catg -6.27 2 -30.05 10 cgcg -4.79 5 -43.59 4 + ccgg -4.34 6 -44.99 3 + ctag -3.27 9 -15.07 12 tata -3.98 7 -36.53 6 tgca -1.82 12 -31.68 8 tcga -2.93 10 -31.25 9 + ttaa -1.54 14 -39.77 5 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:56 utc 18 pristas, p. et al. methyltransferase (phifl4a, phifl3a, phifl2a, phifl1a, and phief11, shown in bold in fig. 2) have slightly different 4 bp palindrome composition. while average z score of palindrome avoidance in bacteriophages possessing modification methyltransferase was -3.31, z score in bacteriophages lacking modification methyltransferase was -3.99. this is probably due to protective effect of methyltransferases and decreased pressure on avoidance of tetranucleotide words. fig. 2. neighbor-joining tree showing the relatedness of palindrome avoidance in bacteriophages possessing modification methyltransferase gene (shown in bold) or not. the bar indicates distance level 0.05. 4. conclusions strong underrepresentation of palindromic tetranucleotide words was observed in genomes of enterococcus faecalis bacteriophages and host bacterium. the most underrepresented are gatc and ggcc words. this avoidance could be explained as a protection mechanism against host restriction-modification systems as a clear correlation was found between avoidance of palindromic words and the specificity of e. faecalis restriction-modification systems. no similar avoidance of tetranucleotide words was observed for non-palindromic words. a weak correlation was observed between avoidance of tetranucleotide palindromes in bacteriophage genomes and the possession of phage encoded dna methyltransferases confirming the interrelation between bacteriophage genomes composition and restriction-modification systems in enterococci. acknowledgments: the work was financially supported by the european regional development fund project 26220220065. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:56 utc nova biotechnologica et chimica 13-1 (2014) 19 references ackermann, h.w., kropinski, a.m.: curated list of prokaryote viruses with fully sequenced 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30, 2013, 1720-1728. yasmin, a., kenny, j. g., shankar, j., darby, a.c., hall, n., edwards, c., horsburgh, m. j.: comparative genomics and transduction potential of enterococcus faecalis temperate bacteriophages. j. bacteriol., 192, 2010, 1122-1130. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:56 utc nova biotechnol chim (2022) 21(1): e1320 doi: 10.36547/nbc.1320 1 nova biotechnologica et chimica genetic diversity and phylogenetic analyses of vitex agnus-castus l. populations using issr-pcr and chloroplast dna trnl intron and trnl-f sequences emre sevindik faculty of agriculture, department of agricultural biotechnology, aydın adnan menderes university, cakmar, turkey  corresponding author: ph.d.-emre@hotmail.com article info article history: received: 12th december 2021 accepted: 2nd march 2022 keywords: issr-pcr trnl intron trnl-f phylogenetic vitex agnus-castus abstract in this study, genetic diversity and phylogenetic analysis of some vitex agnus-castus l. populations were conducted based on issr-pcr and chloroplast dna trnl intron, and trnl-f sequence analyses. vitex agnus-castus populations were detected in aydın province, turkey. fresh leaf samples from the populations were collected and brought to the laboratory for genomic dna isolation. 15 issr primers were used to determine the genetic diversity ofe vitex agnus-castus populations. a total of 138 bands were obtained in issr analysis, 85 of which were polymorphic and 53 were monomorphic. polymorphism rate was determined as 61.59 %. trnc, trnd, trne, and trnf primers were used for pcr amplification of the chloroplast trnl intron and trnl-f region. a total of 138 bands were obtained by issr analysis. for trnl intron analyses, nucleotide lengths of 13 populations were between 508 and 516. the average nucleotide composition consisted of 38.5 % t, 18.3 % c, 27.5 % a and 15.7 % g. in trnl-f assays, the nucleotide lengths of the 13 populations ranged from 330 to 353. the average nucleotide composition consisted of 29.4 % t, 18.1 % c, 32.9 % a and 19.6 % g. the results of the phylogenetic trees constructed using some trnl intron and trnl-f sequences of vitex doniana, vitex trifolia, vitex triflora, vitex turczaninowii, vitex queenslandica, vitex axillariflora, vitex rotundifolia and vitex negundo species obtained from ncbi were compared. as a result of the study, polymorphisms were obtained at a rate of 61.59% from the issr analysis. in addition, the phylogenetic relationship between chloroplast trnl intron and trnl-f sequences of vitex agnus-castus populations along with the other species was revealed. introduction medical and aromatic plants have a long history of being used globally for medicinal purposes and have traditionally been an important component of health services throughout human history due to their health benefits (eryigit et al. 2015; chaachouay et al. 2019). the vitex genus includes about 250 species and many of these have important medicinal effects (yao et al. 2016). vitex agnus-castus l., known as chasteberry, belongs to the lamiaceae family (previously included in the verbenaceae family), and it is a 1 – 3 m long highly branched small tree. its trunk is covered with short, dense, soft and grey hairs. it is a deciduous shrub native to europe and central asia (stojković et al. 2011; hürkul and köroğlu 2018; souto et al. 2020; ilhan 2021). this plant is mailto:ph.d.-emre@hotmail.com nova biotechnol chim (2022) 21(1): e1320 2 naturally found in many cities throughout turkey, including amasya, antalya, bursa, muğla, trabzon and çanakkale (yilar et al. 2016). vitex agnuscastus, used in conventional medicine, has a long tradition in folk medicine, and its use dates back to 2,500 years ago (sorensen and katsiotis 1999; lataoui et al. 2014; uçak koç et al. 2017). it is widely used in anatolian folk medicine as diuretics, appetizers, antifungals, antispasmodics, for preterm labor and for pain relief (ilhan 2021). its fruits, flowers and leaves have been reported to contain flavonoids, tannins, iridoids and diterpenoids (sağlam et al. 2007; sarikurkcu et al. 2009). v. agnus-castus fruits have been used as a flavor and spice component in meals, as a hormone-like remedy for menstrual problems and a mild sedative and digestive tool in iranian traditional folk medicine (ghannadi et al. 2012). the aromatic leaves are used as a spice (stojković et al. 2011; mari et al. 2015). genetic diversity is a prerequisite for the short and long-term survival of plant species in their natural environment (verma et al. 2017). morphological, biochemical, and dna markers are used to determine the genetic diversity of plants (sevindik et al. 2020). dna-based molecular markers offer many advantages in determining genetic diversity. among the polymerase chain reaction (pcr) based marker techniques, the issr technique is one of the simplest and most widely used markers. it is also a marker system utilized in the determination of genetic relationships and linkage mapping (alansi et al. 2016; atasagun et. al. 2018; güngör et al. 2020). chloroplast (cp) is an important organelle for green plants as it is the place where photosynthesis, carbon fixation and pigment synthesis take place. the cp is an independent genome (cp genome) with a preserved circular structure and a low molecular weight (zhou et al. 2019; munyao et al. 2020; zhu et al. 2021). chloroplast dna (cpdna), which is inherited by the female in most angiosperm plants, is generally accepted as a single non-recombinant inheritance unit and is widely used to reveal plant phylogenies in interspecies studies (taberlet et al. 1991; gielly and taberlet 1994; dizkirici et al. 2019; sun et.al. 2019). non-coding regions of the chloroplast genome are used in molecular systematic and plant population genetic studies (kalmer and tekpınar 2017). within this study the chloroplast regions with both variable and highly conserved primer sequences were used. the first of these regions is the trnluaa intron; and the second is the trnl(uaa)f(gaa) intergenic spacer (igs). noncoding cpdna trnl-f is located between trnl (uaa) 3' exon and trnf (gaa) gene. in addition, it is more variable towards the coding regions, and some studies have revealed that it shows higher variations and more frequent mutations than the coding regions (taberlet et al. 1991; hartana 2010). the trnl intron is the first group i intron identified in chloroplast dna and is also the first intron identified to cut a trna gene (bakker et al. 2000). in this study, we performed a genetic diversity analysis using issr and cpdna trnl intron and trnl-f region markers for some vitex agnus-castus populations grown in the aydın region of turkey. experimental plant materials, genomic dna isolation and pcr leaf samples belonging to the vitex agnus-castus populations to be used in this study were collected from the center, koçarlı, çine, germencik, i̇ncirliova, and köşk districts of aydın province (fig. 1). fig. 1. location of collecting places in the aydın/turkey (https://www.google.com/maps). leaf samples of the collected vitex agnus-castus plant were brought to the laboratory and prepared for genomic dna isolation. a commercial kit (genemark) was used for the genomic dna isolation from the plant specimens. the obtained gdna samples were stored at -20 °c. the primers, pcr mix and protocol used for the amplification of https://www.google.com/maps nova biotechnol chim (2022) 21(1): e1320 3 issr-pcr, cpdna trnl intron and trnl-f regions are given in table 1 and table 2. table 1. primers used in the issr-pcr reactions, pcr protocols and their tm degrees. issr primers dna sequences (5’-3’) tm [oc] pcr amplification (35 cycle) amplification ubc-831 5’-ctctctctctctctctt-3’ 50 °c 94 °c/1min 94 °c/1min 48-55 °c/1min 72 °c/1 min 72 °c/10min + ubc-830 5’-tgtgtgtgtgtgtgtgg-3’ 52 °c + ubc-807 5'-agagagagagagagagt-3' 50 °c + ubc-808 5’-agagagagagagagagc-3’ 52 °c + ubc-836 5'-agagagagagagagagya-3’ 52 °c + ubc-856 5'–acacacacacacacacya-3’ 52 °c + ubc-853 5'tctctctctctctctcrt-3' 52 °c + ubc-892 5’tagatctgatatctgaat-3’ 52 °c ubc-810 5' -gagagagagagagagat-3' 50 °c + ubc-826 5’-acacacacacacacacc-3’ 52 °c + ubc-811 -gagagagagagagagac-3’ 53 °c + ubc-834 5’-agagagagagagagayt-3’ 52 °c + ubc-873 5’-gacagacagacagaca-3’ 48 °c + ubc-855 5’-acacacacacacacac 52 °c + ubc-880 5’-ggagaggagaggaga-3’ 55 °c + table 2. trnl intron and trnl-f primers used in this study with their designers and pcr protocols. primer name 5’ to 3’ primer sequence pcr amplification (35 cycle) based on (the source publication) forward trnc 5’-cgaaatcggtagacgctacg-3’ 94 °c/4min. 94 °c/1min 50 °c/1min. 72 °c/1 min. 72 °c/10min. taberlet et al. 1991 reverse trnd 5’-ggggatagagggacttgaac-3’ taberlet et al. 1991 forward trnle 5’-ggttcaagtccctctatccc-3’ taberlet et al. 1991 reverse trnff 5’-atttgaactggtgacacgag-3’ taberlet et al. 1991 1 % agarose gel 1x tbe buffer was used for electrophoresis of pcr products. 5 µl of the reaction mixture in pcr tubes was added to 1 µl loading buffer (loading dye solution) and mixed, and 6 µl of this mixture was placed in the wells of the gel. after loading 3 kb dna marker into the first well, the device was subjected to electrophoresis at 100 v for 90 min. then, the dna bands were imaged under uv light and their photos were taken (fig. 2 – 4). fig. 2. gel image of issr-pcr bands amplified with ubc811 primer. fig. 3. gel image of pcr bands amplified with cpdna trnl intron. fig. 4. gel image of pcr bands amplified with cpdna trnlf region. issr-pcr analyses nova biotechnol chim (2022) 21(1): e1320 2 after the pcr analyses, dna bands were scored as follows: “1” was given if there was dna and “0” was given if there was no dna in the bands. a “?” was given for missing data. using bands, the genetic relationships of vitex agnus-castus populations used in the study were analysed using the paup 4.0b10 (swofford 2001) program. cpdna trnl intron and trnl-f sequences service procurement from biotechnology company triogen (istanbul, turkey) was obtained for pcr reactions and purification. to ensure healthy analyses, it was necessary to visually check the accuracy of dna sequences one by one. to this end, professional computer programs bioedit (hall 1999) and finch tv, which are frequently used in molecular systematic studies worldwide, were utilized. a maximum likelihood phylogenetic tree was constructed using the mega 6.0 (tamura et al. 2013) to extract phylogenetic relationships among the sequenced vitex agnus-castus populations. to evaluate the degree of support for given clades, a bootstrap analysis (1,000 replicates) was applied (felsenstein 1985). in addition, the genetic distance matrix between populations was also performed using the same software. results and discussion in recent years, some pcr-based molecular markers have been developed and tested in genetic studies of various organisms (poyraz et al. 2016). in previous studies, genetic diversity, phylogenetic and molecular analyses of vitex species have been conducted using rapd (sevindik et al. 2019), issr (gıachıno and avcı 2017; saedyani et al. 2020), rps14 gene (ayaz et al. 2020; malik et al. 2021), nrits dna, chloroplast ndhf, trnh-psba, trng-trns sequences (bramley et al. 2009; sun et al. 2019), chloroplast rbcl and ndhf sequences (wagstaff et al. 1998), and chloroplast matk and psba-trnh intergenic spacer (phoolcharoen and sukrong 2013). issr analysis in recent years, pcr-based dna marker systems such as aflp, rflp, rapd, ssr and issr have been common for investigating the genetic makeup of populations, genetic characterization, genetic diversity, divergence and phylogenetic studies (li and ge 2001; gajera et al. 2010; chen et al. 2017; hocaoglu-ozyigit et al. 2020). issr markers have been proposed as a new source of genetic markers that are inherited in mendelian fashion and are scored as dominant markers (paul et al. 2020). these markers have the role of analyzing genetic diversity through the classification of varieties (kiani and siahchehreh 2018). in issr analysis 15 primers were used of which 14 yielded positive results while 1 primer did not (table 1). a total of 138 bands were obtained in issr analysis, 85 of which were polymorphic and 53 were monomorphic. polymorphism rate was determined as 61.59 %. the upgma tree generated based on the issr dataset consisted of two clades (fig. 5). fig. 5. the upgma tree generated using issr data. clade 1 was divided into 2 subclades. subclade a consisted of only the çine purple flowered population. çine purple flowered population was found together with koçarlı white flowered, çakmar white flowered, germencik purple flowered and erbeyli white flowered populations in rapd analysis (sevindik et al. 2019). in the subclade b, çine white flower, çine pink flower, çakmar pink flowered, aydın purple flowered and aydın white flowered appeared in the same group. according to sevindik et al. (2019), these populations were found together according to the rapd results. in subclade b, çakmar purple flowered, germencik purple flowered, erbeyli white flowered, çakmar white flowered and i̇ncirliova purple flowered populations emerged together. according to sevindik et al. 4 nova biotechnol chim (2022) 21(1): e1320 3 (2019), germencik purple flowered, çakmar white flowered and erbeyli white flowered were found together, while i̇ncirlova purple flowered and çakmar purple flowered populations detected in separate groups. clade 2 consisted of köşk purple flower and koçarlı white flower populations. according to sevindik et al. (2019), both populations emerged in different groups. according to paup analysis, the closest genetic distances were between germencik purple flowered and erbeyli white flowered populations with a value of 0.10145; and the greatest genetic distance was between çine purple flowered and koçarlı white flowered populations with a value of 0.36957 (table 3). giachino and avci (2017) used 6 of 21 issr primers for vitex agnus-castus populations collected from various parts of the yunt mountain of manisa. the discrimination power of issr primers was evaluated by calculating various marker parameters such as percent polymorphism, polymorphism information content (pic), resolving power (rp), and marker index (mi). in the study, 31 (65.95 %) of a total of 47 useable bands were found to be polymorphic. the polymorphic band ratios of the primers varied from 25 % to 85.7 %. polymorphism information content (pic) values of 6 primers ranged from 0.22 to 0.35, and the mean pic value was calculated as 0.30. the resolution power (rp) values ranged between 0.5 and 3.25, and the marker index (mi) ranged between 0.11 and 1.72. saedyani et al. (2019) determined genetic diversity for 19 vitex agnus-castus genotypes using 13 issr primers. in their study, 74 bands were obtained and the polymorphism rate was 95% on average. they determined the genetic distance of the genotypes between 0.195 and 0.593 using the dice coefficient. cpdna trnl intron and trnl-f sequence analysis phylogenetic analyses of molecular sequences are part of many molecular and phylogenetic biology studies (anisimova et al. 2013). the cpdna trnlf region is located in the large single-copy region of the chloroplast genome and is widely used for phylogenetic analyses (hocaoglu-ozyigit et al. 2020). for the cpdna trnl intron, the base lengths of vitex agnus-castus populations ranged from 508 to 516. the genetic distance between populations was between 0.000 and 0.002 (table 4). the mean nucleotide ratio for the trnl intron was 38.5 % t, 18.3 % c, 27.5 % a, and 15.7 % g. using the mega 6.0 program, tajma's neutrality test (tajima 1989) was calculated based on cpdna trnl intron sequences of vitex agnus-castus populations. numbers of sequences (m) yielded one segregation site (s) revealing very low nucleotide diversity (π) of 0.001422 (table 5). the maximum likelihood phylogenetic tree constructed using 13 populations consisted of two large groups (fig. 6). the first clade is divided into two subgroups. subclade a consisted of i̇ncirliova purple flowered, koçarlı white flowered, germencik purple flowered, çine purple flowered, çakmar white flowered and çakmar purple flowered, and this group was supported with a bootstrap value of 55 % (fig. 6). in the study of sevindik et al. (2019), çakmar purple flowered and i̇ncirliova purple flowered populations were found in a separate group, while koçarlı white flowered, germencik purple flowered, çine purple flowered and çakmar white flowered populations were found in the same group. subclade b consisted of çakmar pink flowered, çine pink flowered, erbeyli white flowered and köşk purple flowered, and this group was supported with a 60 % bootstrap value (fig. 6). sevindik et al. (2019) found erbeyli white flowered and köşk purple flowered populations in separate groups, while çakmar pink flowered and çine pink flowered populations in the same group. clade 2 consisted of aydın white flowered, aydın purple flowered and çine white flowered populations. within this group, aydın white flowered and aydın purple flowered populations were monophyletic with a bootstrap value of 63 % (fig. 6). 5 nova biotechnol chim (2022) 21(1): e1320 3 table 3. pairwise genetic distance matrix obtained from pcr with issr primers. populations 1 2 3 4 5 6 7 8 9 10 11 12 13 çine purple flower 0.19565 0.23915 0.22464 0.20455 0.24638 0.27536 0.30435 0.26087 0.26812 0.26812 0.31159 0.36957 çine white flower 27 0.17391 0.15952 0.13636 0.23913 0.22464 0.21014 0.18116 0.18841 0.20290 0.26087 0.33333 çine pink flower 33 24 0.13043 0.15152 0.15217 0.16667 0.19565 0.19565 0.18841 0.20290 0.27536 0.31884 çakmar pink flower 31 22 18 0.12879 0.13768 0.18116 0.23913 0.21014 0.20290 0.20290 0.27536 0.36232 aydın purple flower 27 18 20 17 0.15152 0.13636 0.20455 0.19697 0.19637 0.18939 0.28788 0.29545 aydın white flower 34 33 21 19 20 0.17391 0.27536 0.24638 0.22464 0.23913 0.32609 0.35507 çakmar purple flower 38 31 23 25 18 24 0.17391 0.17391 0.12319 0.13768 0.25362 0.25362 çakmar white flower 42 29 27 33 27 38 24 0.11594 0.13768 0.13768 0.19565 0.29710 i̇ncirliova purple flower 36 25 27 29 26 34 24 16 0.13768 0.13768 0.19565 0.32609 germencik purple flower 37 26 26 28 26 31 17 19 19 0.10145 0.21739 0.26087 erbeyli white flower 37 28 28 28 25 33 19 19 19 14 0.15942 0.23188 köşk purple flower 43 36 38 38 38 45 35 27 27 30 22 0.20290 koçarlı white flower 51 46 44 50 39 49 35 41 45 36 32 28 6 nova biotechnol chim (2022) 21(1): e1320 2 table 4. pairwise genetic distance matrix obtained from cpdna trnl intron sequences. populations 1 2 3 4 5 6 7 8 9 10 11 12 13 aydın purple flower aydın white flower 0.000 cakmar pink flower 0.002 0.002 cakmar purple flower 0.002 0.002 0.002 cakmar white flower 0.002 0.002 0.002 0.000 cine pink flower 0.002 0.002 0.000 0.002 0.002 cine purple flower 0.002 0.002 0.002 0.000 0.000 0.002 cine white flower 0.002 0.002 0.002 0.002 0.002 0.002 0.002 erbeyli white flower 0.002 0.002 0.000 0.002 0.002 0.000 0.002 0.002 germencik purple flower 0.002 0.002 0.002 0.000 0.000 0.002 0.000 0.002 0.002 incirliova purple flower 0.002 0.002 0.002 0.000 0.000 0.002 0.000 0.002 0.002 0.000 kocarlı white flower 0.002 0.002 0.002 0.000 0.000 0.002 0.000 0.002 0.002 0.000 0.000 kösk purple flower 0.002 0.002 0.000 0.002 0.002 0.000 0.002 0.002 0.000 0.002 0.002 0.002 table 5. tajima’s neutrality test values based on cpdna trnl intron of date vitex agnus-castus populations. no. of sequences “m” no. of segregating sites “s” ps=s/n θ = ps/a1 nucleotide diversity ‘’ π ‘’ tajima test statistic ‘’d’’ 13 1 0.001980 0.000638 0.001422 2.700233 7 nova biotechnol chim (2022) 21(1): e1320 3 fig. 6. the maximum likelihood tree generated using cpdna trnl intron sequences. according to sevindik et al. (2019), these three populations were in the same group. there are both similarities and differences between the upgma dendrogram generated by rapd analysis and the phylogenetic trees constructed with trnl intron sequences. additionally, vitex doniana (mk187249.1), vitex trifolia (aj505539.1), vitex triflora (mk797715.1), vitex turczaninowii (mg836415.1), vitex queenslandica (mg836414.1), vitex axillariflora (mg836413.1), vitex rotundifolia (ab817427.1; ab817638.1; ab817574.1) and vitex negundo (dq304786.1; dq304787.1) species were added to the analyses after obtaining their trnl intron sequences from ncbi. a maximum likelihood phylogenetic tree was generated and the relationship of vitex agnuscastus populations with other species was revealed. the phylogenetic tree consists of 2 clades (fig. 7). the first clade consists of vitex agnus-castus populations, vitex axillariflora, vitex trifolia, vitex rotundifolia and vitex negundo species formed and is supported by a bootstrap value of 95 %. clade 2 is divided into two subclades. subclade a, consists of vitex triflora and vitex doniana species and is supported by a bootstrap value of 97 %, and subclade b, consists of vitex turczaninowii vitex queenslandica species and is supported by a 75 % bootstrap value (fig. 7). fig. 7. the maximum likelihood tree generated using cpdna trnl intron sequences and other species sequences retrieved from ncbi (bootstrap values greater than 50 % are given above branches). for the cpdna trnl-f region, the base lengths of vitex agnus-castus populations ranged from 330 to 353. the genetic distance matrix between populations turned out to be 0.000 (table 6). 8 nova biotechnol chim (2022) 21(1): e1320 3 table 6. pairwise genetic distance matrix obtained from cpdna trnl-f sequences. populations 1 2 3 4 5 6 7 8 9 10 11 12 13 aydın purple flower aydın white flower 0.000 cakmar pink flower 0.000 0.000 cakmar purple flower 0.000 0.000 0.000 cakmar white flower 0.000 0.000 0.000 0.000 cine pink flower 0.000 0.000 0.000 0.000 0.000 cine purple flower 0.000 0.000 0.000 0.000 0.000 0.000 cine white flower 0.000 0.000 0.000 0.000 0.000 0.000 0.000 erbeyli white flower 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 germencik purple flower 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 incirliova purple flower 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 kocarlı white flower 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 kösk purple flower 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 9 nova biotechnol chim (2022) 21(1): e1320 3 the mean nucleotide ratio for the trnl-f region was 29.4 % t, 18.1 % c, 32.9 % a, and 19.6 % g. using the mega 6.0 program, tajma's neutrality test (tajima 1989) was calculated based on cpdna trnl-f sequences of vitex agnus-castus populations. numbers of sequences (m) gave one segregation site (s) revealing very low nucleotide diversity (π) of 0.000000 (table 7). table 7. tajima’s neutrality test values based on cpdna trnl –f of date vitex agnus-castus populations. no. of sequences “m” no. of segregating sites “s” ps=s/n θ = ps/a1 nucleotide diversity ‘’ π ‘’ tajima test statistic ‘’d’’ 13 0 0.000000 0.00000e+000 0.000000 n/c the maximum likelihood tree constructed using trnl-f sequences consists of two clades (fig. 8). clade 1 is divided into 3 subclades in itself. subclade a consists aydın purple flowered, aydın white flowered, çakmar pink flowered, çakmar purple flowered, çine pink flowered and çine purple flowered populations. fig. 8. the maximum likelihood tree generated using cpdna trnl-f sequences. sevindik et al. (2019) detected the çine purple flowered population in a different group, while all other populations were determined in the same group. in subclade b, erbeyli white flowered and köşk purple flowered populations were sister groups, while i̇ncirliova purple flowered populations were close to these two populations. sevindik et al. (2019) identified three populations in different groups. subclade c consists of only the germencik purple flowered population. clade 2 consisted of çine white flowered and koçarlı white flowered populations, which was supported by a bootstrap value of 99 %. according to sevindik et al. (2019), these two populations were included in separate groups. in this study, a maximum likelihood phylogenetic tree was generated using trnl-f sequences of vitex trifolia (aj505539.1), vitex turczaninowii (mg836415.1), vitex queenslandica (mg836414.1) and vitex axillariflora (mg836413.1) from ncbi. the relationship of vitex agnus castus populations with other species was revealed. the phylogenetic tree consists of 2 clades (fig. 9). the first clade consists of vitex agnus castus populations and vitex trifolia species, and this group has a bootstrap value of 94 %. clade 2 consists of vitex turczaninowii, vitex queenslandica and vitex axillariflora species. in trnl intron analysis, vitex agnus-castus populations coexisted with vitex axillariflora and vitex trifolia species. in addition, in trnl-f analysis, vitex turczaninowii, vitex queenslandica and vitex axillariflora were found in one group. vitex axillariflora and vitex trifolia species were found together with vitex agnus-castus populations, and vitex turczaninowii and vitex queenslandica species were found in a separate group in trnl intron analysis. malik et al. (2021) determined the phylogenetic relationship of some species belonging to the lamiaceae family with the chloroplast rps14 gene. vitex agnus-castus and vitex trifolia species were found in the same group in the study. in our trnl intron and trnl-f sequence studies, vitex agnuscastus and vitex trifolia were found in the same group (fig. 7 and fig. 9). ayaz et al. (2020) performed phylogenetic analysis of some species belonging to lamiaceae in pakistan using the rps14 gene. in their study, vitex agnus-castus var pseudonegundo and vitex negundo species were found in the same group. in the ml tree constructed using trnl intron sequences and sequences taken from 10 nova biotechnol chim (2022) 21(1): e1320 2 ncbi, the vitex agnus-castus populations were in the same clade with vitex axillariflora and vitex negundo. wagstaff et al. (1998) used chloroplast rbcl and ndhf sequences to reveal the phylogenetic relationships of some lamiaceae taxa. vitex agnus-castus species formed a sister group with petitia domingensis species in the strict consensus tree of rbcl and ndhf sequences. fig. 9. the maximum likelihood tree generated using cpdna trnl-f sequences and other species sequences retrieved from ncbi (bootstrap values greater than 50 % are given above branches). conclusion as a result of the study, approximately 61.59 % polymorphism were detected among vitex agnuscastus populations based on issr analyzes. in general, the results of this research aiming to analyze the genetic diversity and phylogenetic analysis of vitex agnus-castus populations through issr, cpdna trnl intron and trnl-f sequence comparisons will be used to determine the phylogenetic relationship between vitex agnuscastus species and other species. acknowledgments this research was supported by the aydın adnan menderes university (project 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wang et al. 2020). the core of the vitamin b12 consists of a tetrapyrrolic corrin ring with a central cobalt atom, grouped with four nitrogen’s, one nucleotide base group and one upper ligand. the recommended intake of the vitamin b12 for healthy adults is 2.4 μg/day. (mohammed et al. 2014a). vitamin b12 plays a significant role in the normal functioning of the nervous system, formation of blood, dna synthesis and regulation, fatty acid synthesis and energy production. the deficiency of the vitamin b12 leads to complications such as pernicious anaemia, neurological dysfunctions such as inhibition of the physiological formation of the myelin sheath, altering correct nerve transmission (mohammed et al. 2014b). plants, fungi, prokaryotes, and animals, including humans cannot synthesis vitamin b12 while synthesis is restricted to few classes of bacteria and archaea. four main types of the vitamin b12 are cyanocobalamin (cn-cbl), methylcobalamin (mecbl), hydroxocobalamin (oh-cbl), and deoxyadenosylcobalamine (ado-cbl). the cobalamins have different bioand photochemistries. the predominant forms of vitamin b12 are me-cbl and ado-cbl and they are actively used as cofactors for enzymes methionine synthase and mailto:projectsagnlabs@gmail.com nova biotechnol chim (2023) 22(1): e1339 2 methylmalonyl coa mutase, while oh-cbl and cn-cbl are not used directly instead they are first converted into an active form of b12. all forms of cobalamins are light sensitive, with me-cbl and ado-cbl identified as extremely labile, photodegrading into oh-cbl in a matter of seconds after light exposure. all forms of cobalamin are converted to cn-cbl to use for pharmacological and commercial purposes, because it is the most stable form of the vitamin b12 in the presence of light (heal et al. 2014; mohammed et al. 2014b). when the consumption of animal foods is very low or absent and scarce presence in plant foods, an adherent importance to the molecule is created, making it essential, either through supplements or fortified foods. this deficiency is common among vegetarians. hence, the production of the vitamin b12 has received a great attention due to increasing global requirements. production of cobalamin relies solely on microbes since chemical methods remain economically not feasible due to the technical complexity of the synthetic process. the natural process of the vitamin b12 synthesis consists of approximately 30 enzymes mediated steps proceeding either through aerobically evident in pseudomonas denitrificans, or anaerobically as witnessed in bacillus megaterium, propionibacterium shermanii, salmonella typhimurium, and lactobacillus reuteri (mohammed et al. 2014a). lactobacillus reuteri is a gram-positive, heterofermentative lactic acid bacterium, prevalent throughout the gastrointestinal tract of humans and other animals (santos et al. 2011; walter et al. 2011). lactobacillus reuteri is known to has probiotic properties (taranto et al. 2003) and a functionally active b12 biosynthetic gene cluster (santos et al. 2009). the bacteria were also able to secrete reuterin, a broad-spectrum antimicrobial agent produced during anaerobic sugar-glycerol cofermentation (talarico et al. 1988; talarico and dobrogosz 1989). whey, a by-product of the dairy industry is rich in lactose, proteins, and minerals. lactose and protein content vary from 39 to 45 g.l−1 and 9 – 14 g.l−1 respectively. about 0.7 % (w/v) of the total solid content of whey consists of whey proteins. the notable whey proteins are β-lactoglobulin, α-lactalbumin, bovine serum albumin, immunoglobulins (igg) (kumar et al. 2012; priyanka and rastogi 2018; kaur et al. 2020). the global production of whey is around 160 million tons per year and out of the total production of whey, only 70 % is used in the production of various products, while 30 % is disposed of in rivers and seas. regardless of all, whey is one of the major pollutants in the food processing industry. a million tons of whey are disposed into rivers, lakes, other water bodies which possess serious threat to the ecosystem due to its high biological and biochemical oxygen demand. in india, the production of whey is scattered, unorganized and the production levels vary from small (20 – 50 l/day) to large scale (50,000 – 1,000,000 l/day) hence, may not be processed at one location (kakan et al. 2018). hence, it is crucial to devise strategies for proper utilization of whey. many optimization studies have been carried out using experimental design techniques such as plackett-burman design, box–behnken design and central composite design have been widely used in the literature (şensoy et al. 2020). boxbehnken design, a response surface methodology statistical design was widely used to explore the correlation between process variables and the responses using minimal experiments (jeganathan et al. 2014). the current study aimed to investigate the practicability to use paneer whey as a growth medium for production of the vitamin b12 and to optimize the media components to achieve the maximum vitamin b12 production using boxbehnken design. experimental materials vitamin b12 (cyanocobalamin, 99.9 % pure), hplc water, and methanol was purchased from sigma aldrich chemicals private limited (bangalore, india). all other chemicals and reagents used were of analytical grade. whey procurement and characterization liquid unprocessed paneer whey (lpw) was obtained from a cottage dairy industry in chennai, nova biotechnol chim (2023) 22(1): e1339 3 tamilnadu. lpw was analysed for ph and water activity. the protein and ash content were determined according to indian standard method is 4684-1975 (1976). fat content was estimated by the aoac method 922.06. the carbon and nitrogen content were determined using the chns analyzer at csir-clri, chennai. strain and culture conditions the bacterial strain used in the study was l. reuteri dsm 20016, acquired from metabolic engineering laboratory, anna university, chennai. to obtain a working culture, from the frozen stock, 100 µl of the thawed culture was transferred to 10 ml of the mrs broth (de man et al. 1960) and incubated for 24 h at 37 °c. daily transfer to fresh medium was performed to maintain the cell viability prior to use. cells were cultivated in nonstirred batch cultures under anaerobic conditions (95 % n2 and 5 % co2) unless stated otherwise. preparation of growth medium production of vitamin b12 by l. reuteri dsm 20016 was done in lpw medium consisting of 20 g lpw, 5 g yeast extract, 1 g polysorbate 80,2 g ammonium citrate, 5 g sodium acetate, 0.1 g magnesium sulphate, 0.05 g manganese sulphate and 2 g dipotassium hydrogen phosphate in 1 l of distilled water (ph 6.5). the medium composition was adapted from the mrs medium in which lpw was replaced as a carbon source instead of dextrose. glycerol and cobalt chloride were used as supplements to enhance the vitamin b12 production. the composition of the media was modified according to the experiments. optimization of vitamin b12 production the growth kinetics of l. reuteri dsm 20016 was monitored in mrs and lpw medium at 37 °c. the preliminary screening assessed the effect of varying individual components of lpw yeast extract (2.5 – 15 g.l-1), polysorbate 80 (1 – 6 g.l-1), lpw (20 – 120 g.l-1). the effect of varying levels enhancers glycerol (1 – 8 g.l-1) and cobalt chloride (1 – 8 g.l-1) were studied individually on the production of vitamin b12 was also evaluated. the other components were maintained at a fixed level. the starter culture of od600 = 0.961 (corresponds to cfu =1.14 × 104 cfu.ml-1) was used as inoculum. all experiments were conducted in duplicates and average values were reported. the spss 11.5 software package was used for statistical evaluation of data. the difference between the runs was evaluated using duncan’s test at 5 % significance level. response surface methodology as the subsequent step, the response surface methodology (rsm) was conducted by applying box–behnken design to study the optimal medium formulation and the interactions between the factors for the vitamin b12 production. boxbehnken designs a three-level second-order design developed to fit the second-order response surface model (vellaisamy singaram et al. 2021). the factors of box behnken design along with the levels are as follows – lpw (20 – 80 g.l-1), yeast extract (2.5 – 5 g.l-1), glycerol (1 – 3 g.l-1) and cobalt chloride (1 – 3 g.l-1). the model was developed using a design expert (stat-ease) software version 11.1. the design matrix with actual values and experimental results are shown in table 2. the experimental results were fitted with a second order polynomial function (eq. 1): y= βo + xi + + + (1) where y is the predicted response, k is the number of factors, xi and xj are the coded variables; β0 is the offset term; βi, βj, and βij are the first-order, quadratic, and interaction effects, respectively; i and j are the index numbers for factor; and eij is the residual error. the fit quality of the model was evaluated by r2 and analysis of variance (anova). the lack of fit was used for controlling the statistics adequacy and efficiency of the model. statistical testing of the model was done by fisher’s statistical test (kumar et al. 2012). nova biotechnol chim (2023) 22(1): e1339 2 extraction and analysis of vitamin b12 the vitamin b12 (cyanocobalamin) was quantified using high performance liquid chromatography. hydroxocobalamin is a natural form of cobalamin, formed in the bacterial cell. since it is highly unstable, it is converted to cyanocobalamin using kcn (hajfarajollah et al. 2015). for highperformance liquid chromatography (hplc) analysis, the cell culture broth (40 ml) was centrifuged (9,000 × g for 10 min at 4 °c) to harvest the cells. the supernatant was discarded, and the pellet was washed with 10 ml 0.2 m potassium phosphate buffer (ph 5.5), centrifuged (10,000 × g for 15 min) and re-suspended in 1 ml of 0.2 m potassium phosphate buffer (ph 5.5) containing 0.1 % potassium cyanide. in order to lyse the cells for release of the vitamin b12, the resuspended cells were vortexed and autoclaved (121 °c, 15 min). finally, the samples were vortexed again and centrifuged (10,000 × g, 15 min). the supernatant was filtered (0.22µm syringe filter) and analysed with hplc (hugenschmidt et al. 2010). a shimadzu chromatography system with c18 column (shodex c18-4e, i.d. × length, mm 4.6 × 250) and uv-vis detector was used for the analysis of vitamin b12. the uv detection wavelength, flow rate of a mobile phase, oven temperature, and injection volume were set to 361 nm, 1 ml.min-1, 30 °c, and 20 μl, respectively. the mobile phase was a mixture of hplc water: methanol (50 : 50). an external standard (cyanocobalamin) was used for calibration. each sample was injected twice, and the mean values were reported. results and discussion characterization of liquid paneer whey the compositional characteristics of lpw are shown in table 1. the proximate composition of lpw was 0.44g/100g protein, 4.91 % lactose, 0.16g/100g ash. the lpw has 97.63 % moisture content, and it is devoid of fat. the carbon and nitrogen contents of lpw were determined as 52.941 % and 1.187 %, respectively. the water activity value suggests lpw as a suitable medium for bacterial growth. similar observations on the composition of paneer whey have been reported in the literature (ranvir and adil 2018; singh et al. 2019). table 1. compositional analysis of lpw. s. no. parameters value 1 moisture [%] 97.63 2 ash [g/100g] 0.16 3 protein [g/100g] 0.44 4 fat [g/100g] 0.001 5 lactose [%] 4.91 6 water activity [aw] 0.98 7 ph 5.73 8 density [g.l-1] 1.0178 growth analysis the growth curve of l. reuteri dsm 20016 strain in mrs and lpw medium was studied (fig. 1 and fig. 2). fig. 1. growth curve of l. reuteri dsm 20016 in mrs medium (experiments were conducted in triplicates and the standard deviation was denoted as error bars). fig. 2. growth curve of l. reuteri dsm 20016 in lpw medium (experiments were conducted in triplicates and the standard deviation was denoted as error bars). 4 nova biotechnol chim (2023) 22(1): e1339 3 the exponential phase in the mrs medium reached a peak at 8 h and the stationary phase existed till 18 h. the carbon source in the mrs medium is glucose, which is easily metabolized by lactobacillus sp. after 18 h the amount of nutrients will be depleted, resulting in cell death. lpw was reported to be an excellent liquid carrier on the basis of bacterial shelf-life and presence of lactose and proteins which can serve as carbon and nitrogen sources for microorganisms (tan and li 2018; prakash and arora 2020). in the lpw media the exponential phase reached peak at 18 h and the stationary phase extended till 44 h. the delayed cell growth in the lpw media might be due to the complex nature of lactose since bacterial βgalactosidase activity is necessary to metabolize lactose. l. reuteri possesses low proteolytic activity and therefore supplementation with extra nitrogen sources might improve growth (jantzen et al. 2013). the number of cells obtained in mrs and lpw medium at 18 h is 1.46 × 107 cfu.ml-1 and 2.41 × 109 cfu.ml-1 respectively. since the vitamin b12 is a primary metabolite, the sampling time point in the experiment is chosen as 18 h. effect of media components on vitamin b12 production the effect of media components – yeast extract, polysorbate 80, and lpw on the vitamin b12 production was studied and the results are presented as follows. yeast extract yeast extract is commonly used for the growth of lactic acid bacteria due to their composition in amino acids and vitamins, which is necessary for the cell growth. yeast extract is the most common nitrogen source employed in the published studies concerning l. reuteri (couvreur et al. 2017; ichinose et al. 2020; nguyen et al. 2021). in the present study, the effect of varying the amount of yeast extract (2.5 g – 15 g) on the vitamin b12 production was analysed. the other components of lpw media are as follows – 20 g lpw, 1 g polysorbate 80, 2 g ammonium citrate, 5 g sodium acetate, 0.1 g magnesium sulphate, 0.05 g manganese sulphate, and 2 g dipotassium hydrogen phosphate in 1 l of distilled water (ph 6.5). as shown in fig. 3, the increase in amount of yeast extract from 2.5 g.l-1 – 5 g.l-1 significantly improved (p < 0.05) the vitamin b12 production from 25.578 ± 0.282 µg.l-1 to 32.728 ± 0.215 µg.l1. further increase in the amount of yeast extract reduced the vitamin b12 production. the lowest vitamin b12 (14.861 ± 0.037 µg.l -1) was obtained at 15 g.l-1 of yeast extract. the negative effect of yeast extract might be due to toxicity of yeast extract at higher concentration which in turn decreases the cell concentration. similar results on the negative effect of yeast extract on the growth of lactobacillus have been reported in the literature (ghaly et al. 2003; miloud et al. 2017). the optimal concentration of yeast extract for the production of the vitamin b12 was found to be 5 g.l-1. fig. 3. effect of varying amount of yeast extract on vitamin b12 production. values with different superscripts are significantly different (p < 0.05). polysorbate 80 to further investigate the influence of polysorbate 80 on the vitamin b12 production, polysorbate 80 concentrations from 1 to 6 g.l-1 were used, and other media components are as follows – 20 g lpw, 5 g yeast extract, 2 g ammonium citrate, 5 g sodium acetate, 0.1 g magnesium sulphate, 0.05 g manganese sulphate, and 2 g dipotassium hydrogen phosphate in 1 l of distilled water (ph 6.5). polysorbate 80, a surfactant, is a crucial growth factor in culture media for lactobacilli as it brings unsaturated fatty acids that allow reducing intracellular energy consumption (nguyen et al. 2021). as shown in fig. 4, highest and lowest vitamin b12 production was obtained at 3 g.l -1 (35.796 ± 0.012 µg.l-1) and 6 g.l-1 (30.846 ± 0.058 5 https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/lactobacillus nova biotechnol chim (2023) 22(1): e1339 2 µg.l-1). higher concentrations of polysorbate 80 decreased the vitamin b12 production because polysorbate 80 became toxic at higher concentration due to the destruction of the cell membrane structure and/or loss of the cell membrane function caused by the solubility of lipid bilayer by the surfactant (qi et al. 2009). hence, the optimal concentration of polysorbate 80 to produce the vitamin b12 was found to be 3 g.l -1. fig. 4. effect of varying amount of polysorbate 80 on vitamin b12 production. values with different superscripts are significantly different (p < 0.05). paneer whey for analysis of the effect of lpw on the vitamin b12 production, the yeast extract and polysorbate 80 levels were set at 5 g.l-1 and 3 g.l-1. the level of lpw was varied between 20 to 120 g.l-1 and other components were fixed as follows – lpw medium consisting of, 2 g ammonium citrate, 5 g sodium acetate, 0.1 g magnesium sulphate, 0.05 g manganese sulphate, and 2 g dipotassium hydrogen phosphate in 1 l of distilled water (ph 6.5). as shown in fig. 5, the vitamin b12 production was significantly increased with the increase of lpw percentage from 20 to 80 g.l-1. the highest vitamin b12 was achieved at cultures with 80 g.l -1 lpw. it has been reported that lpw is rich in various components, such as carbohydrate, vitamins and organic acids (patowary et al. 2016). due to the complex nutritional requirements of the lactobacillus, their survival is favoured in the presence of increased carbohydrate, protein, and vitamin sources (gomes et al. 2015). when further increasing the lpw amount into 100 g.l-1 or more, it drastically decreased the production of the vitamin b12. hence, the highest vitamin b12 produced was 65.179 ± 0.093 µg l-1 and the optimal concentration of lpw was 80 g.l-1. after determining the optimal concentration of yeast extract, polysorbate 80 and lpw, the effect of glycerol and cobalt chloride on the vitamin b12 production was analysed. fig. 5. effect of varying amount of lpw on vitamin b12 production. values with different superscripts are significantly different (p < 0.05). effect of glycerol on vitamin b12 production the effect of glycerol was analysed in a medium containing 80 g lpw, 5 g yeast extract, 3 g polysorbate 80, 2 g ammonium citrate, 5 g sodium acetate, 0.1 g magnesium sulphate, 0.05 g manganese sulphate, and 2 g dipotassium hydrogen phosphate in 1 l of distilled water (ph 6.5) with varying amounts of glycerol (1 g.l-1 to 8 g.l-1). l. reuteri was not capable of growing in glycerol as a sole carbon and energy source, even in the presence of complex ingredients of mrs broth (gopal ramakrishnan et al. 2015). during glycerol fermentation of l. reuteri, glycerol serves only as an external hydrogen acceptor (diraviam sriramulu et al. 2008). glycerol plays an important role in the growth of cells and acts as an inducer for vitamin b12 production. the genes responsible for the synthesis of cobalamin are cobt and cbia. the cbia catalyzed amidations to a side chain of cobyrinic acid in the biosynthesis of cobalamin (vitamin b12) from uroporphyrinogen iii. the lower ligand of cobalamin is covalently linked to a phosphoribosyl moiety through an alpha-glycosidic bond formed by the cobt enzyme. however, cobt was reported that it is responsible for the production of analogues. the expression of cbia and cobt was reported to raise significantly with an increase of glycerol supplementation (crofts et al. 6 nova biotechnol chim (2023) 22(1): e1339 3 2014; gu et al. 2015). in the present study, the highest and lowest vitamin b12 production was obtained at 3 g.l-1 (95.704 ± 0.022 µg.l-1) and 8 g.l-1 (29.640 ± 0.001 µg.l-1) (fig. 6). the higher concentrations of glycerol had a negative effect on vitamin b12 production. it was reported that the vitamin b12 production was enhanced with the supplementation of glycerol but high concentrations of glycerol supplementations inhibited the growth of cells which might be due to the activity of glycerol dehydratase inhibited by a quorum sensing of reuterin (bauer et al. 2010; gu et al. 2015). therefore, the optimal concentration of glycerol to produce the vitamin b12 was found to be 3 g.l-1. fig. 6. effect of varying amount of glycerol on vitamin b12 production. values with different superscripts are significantly different (p < 0.05). effect of cobalt chloride on vitamin b12 production lastly, the effect of cobalt chloride was analysed in medium containing 80 g lpw, 5 g yeast extract, 3 g polysorbate 80, 2 g ammonium citrate, 5 g sodium acetate, 0.1 g magnesium sulphate, 0.05 g manganese sulphate, and 2 g dipotassium hydrogen phosphate in 1 l of distilled water (ph 6.5) with varying concentrations of cobalt chloride from 1 g.l-1 to 8 g.l-1. glycerol was not included in the media to study the individual effect of cobalt chloride on the vitamin b12 production. cobalt chloride is a part of the metabolic pathway of the vitamin b12 production. the availability of cobalt chloride ions in the culture medium is essential to the vitamin b12 biosynthesis because it is a central component of the corrinoid ring (burgess et al. 2009). the amount of cocl2.6h2o is significant in affecting the final concentration of the vitamin b12 (hajfarajollah et al. 2015). as shown in fig. 7, maximum vitamin b12 production of 147.633 ± 0.367 µg.l-1 was achieved at the concentration 3 g.l-1. similarly, results of the increasing concentrations of cobalt chloride in enhancing the vitamin b12 production in bacillus megaterium and propionibacterium freudenreichii subsp. shermanii atcc 13673 has been reported in literature (mohammed et al. 2014a; de assis et al. 2020). however, further increase in the concentration to 4 g.l-1 or more depleted the vitamin b12 production. fig. 7. effect of varying amount of cobalt chloride on vitamin b12 production. values with different superscripts are significantly different (p < 0.05). response surface methodology the components in lpw medium, sodium acetate, k2hpo4, ammonium citrate, mgso4·7h2o and mnso4·h2o were considered not to have a significant effect on the biomass production (griet et al. 2018) and based on the single variable experiments, polysorbate 80 doesn’t induce a significant increase in the vitamin b12 production. hence, in the present study, the effect of four factors – paneer whey (a), yeast extract (b), glycerol (c) and cobalt chloride (d) on the vitamin b12 production were optimized using box behnken design. the design matrix along with the experimental values is in table 2. the production of the vitamin b12 varied from 54.863 µg.l -1 to 213.449 µg.l-1 and the maximum production was achieved at the following conditions – paneer whey (80 g.l-1), yeast extract (5 g.l-1), glycerol (2 g.l-1) and cobalt chloride (2 g.l-1). a modified quadratic model was developed to model the experimental data. 7 nova biotechnol chim (2023) 22(1): e1339 2 table 2. box behnken design matrix with actual values and experimental results. std run lpw (a) yeast extract (b) [g.l-1] glycerol (c) [g.l-1] cobalt chloride (d) [g.l-1] vitamin b12 [µg.l-1] 1 2 20 2.5 2 2 54.8634 3 20 20 5 2 2 80.2672 9 17 20 3.75 2 1 72.616 11 25 20 3.75 2 3 81.4221 17 16 20 3.75 1 2 76.8487 19 13 20 3.75 3 2 80.4883 5 28 50 3.75 1 1 108.055 6 7 50 3.75 3 1 121.639 7 24 50 3.75 1 3 124.885 8 18 50 3.75 3 3 129.369 13 26 50 2.5 1 2 85.7037 14 14 50 5 1 2 134.839 15 15 50 2.5 3 2 93.8982 16 21 50 5 3 2 141.774 21 27 50 2.5 2 1 83.4567 22 10 50 5 2 1 131.367 23 23 50 2.5 2 3 91.8839 24 29 50 5 2 3 143.159 25 22 50 3.75 2 2 109.055 26 12 50 3.75 2 2 109.609 27 4 50 3.75 2 2 110.545 28 1 50 3.75 2 2 110.748 29 3 50 3.75 2 2 110.532 30 30 50 3.75 2 2 110.58 2 19 80 2.5 2 2 138.321 4 9 80 5 2 2 213.449 10 6 80 3.75 2 1 175.385 12 8 80 3.75 2 3 189.326 18 5 80 3.75 1 2 176.45 20 11 80 3.75 3 2 190.726 the analysis of variance (anova) results for the model are presented in table 3. table 3. anova for reduced quadratic model. source sum of squares df mean square f-value p-value model 44259.68 12 3688.31 3300.21 < 0.0001 significant a-lpw 33830.15 1 33830.15 30270.42 < 0.0001 b-yeast extract 7337.31 1 7337.31 6565.25 < 0.0001 c-glycerol 217.71 1 217.71 194.80 < 0.0001 d-cobalt chloride 379.98 1 379.98 340.00 < 0.0001 ab 618.12 1 618.12 553.08 < 0.0001 ac 28.28 1 28.28 25.31 0.0001 ad 6.59 1 6.59 5.90 0.0265 cd 20.70 1 20.70 18.52 0.0005 a² 1443.02 1 1443.02 1291.18 < 0.0001 b² 47.83 1 47.83 42.80 < 0.0001 c² 273.95 1 273.95 245.13 < 0.0001 d² 158.43 1 158.43 141.76 < 0.0001 residual 19.00 17 1.12 lack of fit 16.67 12 1.39 2.98 0.1183 not significant pure error 2.33 5 0.4663 cor total 44278.68 29 8 nova biotechnol chim (2023) 22(1): e1339 3 the model was found to be significant (p < 0.05) and the statistical significance of the model was analysed using the fisher test. in the current study, the significant model terms are found to be a, b, c, d, ab, ac, ad, cd a2, b2, c² and d2. the lack of fit f-value of 2.98 implies the lack of fit is not significant.the high value of the coefficient of determination (0.9996) indicates the model is significant. in addition to this, the predicted r2 (0.9983) and adjusted r2 (0.9993) values must be less than 20 % which further suggests the adequacy of the model. the coded equation for the modified quadratic model is given as follows eq. 2: vitamin b12 (µg.l -1) = +110.18 + 53.10 a + 24.73 b + 4.26 c + 5.63 d + 12.43 ab + 2.66 ac + 1.28 ad 2.27 cd + 14.51 a2 2.64 b2 + 6.32 c2 + 4.81 d2 (2) the influence of the process variables on the vitamin b12 production was studied with the help of 3d surface plots (fig. 8). the vitamin b12 production was increased with respect to increasing lpw and yeast extract concentrations. the interactive effect between lpw and yeast extract was positive. the interactive effect of glycerol and cobalt chloride on the vitamin b12 production was negative. the negative effect might be due to the toxic nature of the compounds. fig. 8. 3d surface plots showing the influence of process variables on the vitamin b12 production. conclusion from the current study, it could be concluded that paneer whey is an excellent substrate for the vitamin b12 production. the concentrations of paneer whey, yeast extract, glycerol and cobalt chloride has most distinct effects on the biosynthesis of the vitamin b12 by l. reuteri dsm 20016. response surface methodology was used to optimize the process variables and the optimal condition for the vitamin b12 production was found to be paneer whey (80 g.l-1), yeast extract (5 g.l-1), glycerol (2 g.l-1) and cobalt chloride (2 g.l-1). the maximum of the vitamin b12 production obtained was 213.449 μg.l-1. the current study proposes valuable insights on cost effective carbon source to produce the vitamin b12. acknowledgement this research was funded by the ministry of food processing industries, government of india (research & development project: q-11/38/2018-r&d). conflict of interest the authors declare that they have no conflict of interest. 9 nova biotechnol chim (2023) 22(1): e1339 2 reference aoac 922.06-1922 fat in flour. 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tamilnadu, india abstract: in this study, hypnea valentiae, a red alga is used as a sorbent for the removal of chromium from aqueous solutions. the biosorption potential of hypnea valentiae was investigated in batch experiments. the process parameters were optimized using response surface methodology. based on the central composite design, quadratic model was developed to correlate the variables to the response. the most influential factor on each experimental design response was identified from the analysis of variance (anova). the optimum conditions for the maximum biosorption of chromium are ph – 2.8, temperature – 48.2oc, sorbent dosage – 5.3 g/l, metal concentration – 103 mg/l and contact time – 27 min. at these optimized conditions the maximum removal was found to be 94.5%. key words: hypnea valentiae, optimization, rsm, chromium, algae, biosorption 1. introduction toxic heavy metals are detrimental to human health and ecosystem stability. the removal and recovery of heavy metals from wastewater is important in the protection of the human health and environment. the most toxic metals are aluminium, chromium, iron, cobalt, nickel, copper, zinc, cadmium, mercury, and lead. the major industries that contribute to water pollution by chromium are mining, leather tanning, textile dyeing, electroplating, plants producing industrial inorganic chemicals etc. chromium (cr) bearing wastewater resulted from all these industries must be disposed off after treatment. traditional methods such as chemical precipitation, evaporation, electroplating, adsorption and ion exchange processes have been used to remove chromium from wastewater. however, these technologies are most suitable in situations where the concentrations of the heavy metal ions are relatively high. they are either ineffective or expensive when heavy metals are present in the wastewater at low concentrations, or when very low concentrations of heavy metals in the treated water are required. hence new technologies are required that can reduce heavy metal concentrations to environmentally acceptable levels at affordable costs. biological approaches, especially application of sorbents, have been suggested in the last decade. the advantages offered by sorbents are higher metal loading capacity and greater selectivity for transition and heavy metals. marine algae have been found to be potential suitable sorbents because of their low cost, relatively high surface area and high binding affinity. the use of marine algae for heavy metal removal has been reported by several authors (tien, 2002; jalali et al., 2002; prasannakumar et al., 2007; deng et al., 2007; ozer et al., 2009). 108 rajasimman, m. and murugaiyan, k. rsm is a collection of statistical techniques for designing experiments, building models, evaluating the effects of factors and searching for the optimum conditions. it is widely used for multivariable optimization studies in several processes (ravikumar et al., 2005; korbahti, 2007; aleboyeh et al., 2008; garg et al., 2008; rajasimman et al., 2009; rajeshkannan et al., 2009). so far no work has been carried out for the removal of chromium using h. valentiae. hence the objectives of the present investigation are to quantify the biosorption of chromium to unmodified h. valentiae and to optimize the process parameters for the biosorption of chromium using h. valentiae using response surface methodology. 2. materials and methods 2.1 preparation of adsorbent the red colored marine alga hypnea valentiae was used in the present study. it was collected from the coastal belt of gulf of mannar, tamilnadu, india. the collected algae were washed with deionized water several times to remove impurities. the washing process continued till the wash water contained no dirt. the washed alga was then completely dried in sunlight for 10 days. the dried alga was then cut into small pieces and was powdered using domestic mixer. in the present study the powdered materials in the range of 500–700 μm particle size were then directly used as sorbents without any pre-treatment. 2.2 preparation of solution batch experiments were performed with a magnetic stirrer (remi, india) at 200 rpm using 250 ml beakers containing test solutions. the stock chromium solution (1 g/l) was prepared by dissolving potassium dichromate (sd fine chemicals, india) of analytical grade in deionized water. other concentrations were prepared by dilution of this stock solution and fresh dilutions were used in each experiment. 2.3 experimental design by rsm a full factorial design, which includes all possible factor combinations in each of the factors, is a powerful tool for understanding complex processes for describing factor interactions in multifactor systems. rsm is an empirical statistical technique employed for multiple regression analysis by using quantitative data obtained from properly designed experiments to solve multivariate equations simultaneously. the experiments with different ph, adsorbent dosage, temperature, initial metal concentration and processing time were employed simultaneously covering the spectrum of variables for the removal of chromium in the central composite design. the coded values of the process parameters were determined by the following equation: δx ox ix ix = (1) nova biotechnologica 10-2 (2010) 109 where xi coded value of the i th variable, xi uncoded value of the i th test variable and x0 uncoded value of the i th test variable at center point. the range and levels of individual variables are given in table 1. the experiment design is given in table 2 along with experimental data and predicted responses. the regression analysis was performed to estimate the response function as a second order polynomial ∑ = ∑ = +∑ = +∑ = += k 2j j x ix ijβ 1-k 1i k li 2 i x iiβ i x k 1i iβ 0β y (2) where y is the predicted response, βi, βj, βij are coefficients estimated from regression, they represent the linear, quadratic and cross products of x1,x2,x3 on response. table 1. experimental range and levels of independent process variables. range and levels independent variable 2.38 -1 0 1 +2.38 ph (a) 1 2 3 4 5 temperature, (b) °c 25 35 45 55 65 sorbent dosage, (c) g/l 3 4 5 6 7 chromium concentration, (d) mg/l 50 75 100 125 150 contact time, (e) min 10 20 30 40 50 a statistical program package design expert 7.1.5, was used for regression analysis of the data obtained and to estimate the coefficient of the regression equation. the equations were validated by the statistical tests called the anova analysis. the significance of each term in the equation is to estimate the goodness of fit in each case. response surfaces were drawn to determine the individual and interactive effects of test variable on percentage removal of chromium. after biosorption, the contents of the beakers were centrifuged at 4500 rpm for 3 min and the sorbent was successfully separated from aqueous solution. the supernatants were analyzed for residual chromium concentration using an atomic absorption spectrophotometer (elico, india). 3. results and discussion 3.1 fitting models experiments were performed according to the ccd experimental design given in table 2 in order to search for the optimum combination of parameters for the biosorption of chromium using the red alga. a model f-value of 94.93 implies that the model is significant. the fisher f-test with a very low probability value (pmodel > f = 0.0001) demonstrates a very high significance for the regression model. the fitness of the model is checked by the determination coefficient (r2). the coefficient of determination (r2) was calculated to be 0.9850. this implies that more than 98.5 % of experimental data was compatible with the data predicted by the model (table 2) and only less than 1.5 % of the total variations are not explained by the model. the r2 value is always between 0 and 1, and a value >0.75 indicates aptness of the model. 110 rajasimman, m. and murugaiyan, k. table 2. ccd matrix for the experimental design and responses for chromium removal. % chromium removal run no. a b c d e experimental theoretical 1 1 1 -1 -1 1 65.2 66.50 2 1 1 1 -1 1 76.3 75.51 3 0 0 0 2.38 0 82.5 81.37 4 1 1 -1 -1 -1 69.2 68.84 5 1 -1 1 1 1 79.2 80.92 6 -1 1 1 -1 1 80.3 80.93 7 0 0 0 0 0 93.6 93.41 8 -1 -1 1 1 -1 74.6 73.72 9 -1 1 1 1 1 83.2 82.13 10 -1 -1 -1 -1 -1 79.8 80.67 11 0 0 0 0 0 93.5 93.41 12 -1 -1 1 -1 -1 83.6 82.78 13 -1 1 1 1 -1 86.2 86.73 14 0 2.38 0 0 0 82.2 81.18 15 1 -1 1 -1 -1 83 83.71 16 0 0 0 -2.38 0 79.6 79.17 17 -1 -1 1 1 1 70.8 71.67 18 1 -1 -1 -1 1 78.4 78.82 19 1 -1 -1 1 -1 79.6 79.24 20 0 0 0 0 -2.38 82.3 81.37 21 -1 1 1 -1 -1 87.6 88.72 22 2.38 0 0 0 0 75 73.84 23 1 -1 -1 -1 -1 77.2 78.60 24 -1 -1 1 -1 1 76.2 77.55 25 1 -1 1 1 -1 80.6 80.42 26 1 -1 1 -1 1 81.9 81.02 27 -2.38 0 0 0 0 76.3 75.90 28 1 1 1 -1 -1 80.3 80.76 29 0 0 0 0 0 93.6 93.41 30 1 -1 -1 1 1 83.1 82.66 31 -1 1 -1 1 -1 80.6 81.75 32 -1 -1 -1 1 1 76.2 76.44 33 0 0 0 0 0 93.4 93.41 34 1 1 1 1 -1 83.1 84.55 35 1 1 -1 1 1 76.4 77.40 36 0 0 0 0 0 93.6 93.41 37 1 1 -1 1 -1 76.8 76.55 38 0 0 0 0 0 93.4 93.41 39 -1 -1 -1 -1 1 79.9 78.36 40 0 0 2.38 0 0 76.2 75.44 41 -1 -1 -1 1 -1 74.2 75.56 42 -1 1 -1 -1 1 73.8 74.93 43 0 0 0 0 0 93.5 93.41 44 0 0 0 0 0 93.5 93.41 45 1 1 1 1 1 82.3 82.47 46 0 -2.38 0 0 0 80.9 80.36 47 -1 1 -1 -1 -1 81.2 79.80 48 0 0 0 0 2.38 76.8 76.17 49 0 0 -2.38 0 0 67.7 66.90 50 -1 1 -1 1 1 80.1 80.07 nova biotechnologica 10-2 (2010) 111 for a good statistical model, r2 value should be close to 1.0. the adjusted r2 value corrects the r2 value for the sample size and for the number of terms in the model. the value of the adj r2 (0.9746) is also high to advocate for a high significance of the model. the pred r2 0.9451 is in reasonable agreement with the adj r2. the experimental results are analyzed through rsm to obtain in empirical model for the best response. the results of theoretically predicted response are shown in table 2. the mathematical expression of relationship to the response with variables are shown below % removal of chromium = 93.38-0.43 a + 0.17 b + 1.79c + 0.46 d – 1.09 e – 3.27 a2 – 2.23 b2 – 3.93c2 – 2.32d22.58 e2 – 2.22 ab + 0.75ac + 1.44ad + 0.63 ae + 1.70bc + 1.77bd – 0.64 be – 0.98cd – 0.73ce + 0.80 de (3) the results of multiple linear regressions conducted for the second order response surface model are given in table 3. the significance of each coefficient was determined by student’s t-test and p-values, which are listed in table 3. the larger the magnitude of the t-value and smaller the p-value, the more significant is the corresponding coefficient. values of "prob > f" less than 0.0500 indicate model terms are significant. in this case, a, b, c, d, e, ab, ac, ad, ae, bc, bd, be, cd, ce, de, a2, b2, c2, d2, e2 are significant model terms for the biosorption of chromium. values greater than 0.10 indicate the model terms are not significant. this implies that the linear, square and interactive effects of ph, temperature, sorbent dosage, chromium concentration and contact time are more significant factors. table 3. analysis of variance (anova) for response surface quadratic model source coefficient factor f value p model a b c d e a*a b*b c*c d*d e*e a*b a*c a*d a*e b*c b*d b*e c*d c*e d*e residual lack of fit pure error cor total 93.38 -0.43 0.17 1.79 0.46 -1.09 -3.27 -2.23 -3.93 -2.32 -2.58 -2.22 0.75 1.44 0.63 1.70 1.77 -0.64 -0.98 -0.73 0.80 94.93 6.54 1.01 111.85 7.41 41.58 477.44 221.62 687.34 239.54 297.47 126.72 14.56 53.27 10.33 74.45 80.02 10.53 24.87 13.61 16.30 235.65 < 0.0001a < 0.0160a 0.0328 < 0.0001 0.0109 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0007 <0.0001a 0.0032 <0.0001 <0.0001 0.0030 <0.0001 0.0009 0.0004 <0.0001 std. dev.-1.12 ; r2 0.9850; adj. r2-0.9746; pred. r2 0.9451; c.v.% 1.38; adeq precision 37.158 112 rajasimman, m. and murugaiyan, k. 3.2 response surfaces and contour plots response surface plots as a function of two factors at a time, maintaining all other factors at fixed levels are more helpful in understanding both the main and the interaction effects of these two factors. the response surface curves were plotted to understand the interaction of the variables and to determine the optimum level of each variable for maximum response. the response surface curves for the removal of chromium are shown in figs 1-4. there was a relative significant interaction between every two variables, and there was a maximum predicted yield as indicated by the surface confined in the smallest ellipse in the contour diagrams. figure 1-4 shows the effect of solution ph on chromium removal. the results indicate that the percentage removal of chromium varies from 65% to 93% in the ph range 1 to 5, suggesting that the removal was highly ph-dependent. the biosorption capacity of h. valentiae was high at initial ph 2.8, but decreased considerably from 93% to 71% as the ph of experimental solution increased to 5.0. from fig. 1 it was observed that the biosorption of chromium increased from 65% to 93% with increase in temperature from 25 to 49°c. a maximum biosorption of 93% chromium has been obtained at 49°c. this suggests that biosorption between seaweed and chromium could involve a combination of chemical interaction and physical adsorption. with the increase in temperature, pores in the seaweed enlarge, resulting in increased surface available for the biosorption, diffusion and penetration of chromium ions within the pores of seaweed causing increased biosorption (saleem et al., 2007). amount of sorbent used for the treatment studies is an important parameter, which determines the potential of sorbent to remove chromium at a given initial concentration. as shown in fig. 2, the removal of chromium increased with an increase in sorbent dosage (upto 5 g/l) and then decreased. therefore, the optimum value of sorbent dosage was found to be 5.3 g/l. with increasing the quantity of seaweed, beyond this optimum condition, biosorption capacity decreases for h. valentiae. the decrease in biosorption capacity may be due to splitting effect of concentration gradient between sorbate and sorbent with increasing seaweed concentration causing a decrease in amount of chromium adsorbed onto unit weight of h. valentiae. -2.38 -1.19 0.00 1.19 2.38 -2.38 -1.19 0.00 1.19 2.38 49 60.25 71.5 82.75 94 % r em ov al o f c hr om iu m a: ph b: temperature fig. 1. 3d plot showing interactive effect of ph and temperature on cr removal. nova biotechnologica 10-2 (2010) 113 percentage removal of chromium has been found to be higher at lower concentration of chromium solution (100 mg/l); a maximum chromium removal of 93% has been obtained for h. valentiae (fig. 3). the increase of chromium biosorption capacity of sorbent with an increase in chromium concentration is probably due to higher interaction between metal ions and the sorbent. as seen in fig. 4, the percentage of chromium removal increased by contact time up to 27 min. after that point, there was no considerable change in chromium removal. therefore, optimum contact time was considered as 27 min. -2.38 -1.19 0.00 1.19 2.38 -2.38 -1.19 0.00 1.19 2.38 43 55.75 68.5 81.25 94 % r em ov al o f c hr om iu m a: ph c: sorbent dosage fig. 2. 3d plot showing interactive effect of ph and sorbent dosage on cr removal. -2.38 -1.19 0.00 1.19 2.38 -2.38 -1.19 0.00 1.19 2.38 51 61.75 72.5 83.25 94 % r em ov al o f c hr om iu m a: ph d: chromium concentration fig. 3. 3d plot showing interactive effect of ph and chromium concentration on cr removal. -2.38 -1.19 0.00 1.19 2.38 -2.38 -1.19 0.00 1.19 2.38 55 64.75 74.5 84.25 94 % r em ov al o f c hr om iu m a: ph e: contact time fig. 4. 3d plot showing interactive effect of ph and contact time on cr removal. 114 rajasimman, m. and murugaiyan, k. 3.3 optimum condition for chromium removal optimum conditions for the removal of chromium from aqueous solution using a red alga h. valentiae were found. second order polynomial models obtained in this study were utilized for each response in order to determine the specified optimum conditions. the sequential quadratic programming in matlab 7 is used to solve the second-degree polynomial regression equation 3. the optimum values obtained by substituting the respective coded values of variables are: ph – 2.8, temperature – 48.2oc, sorbent dosage – 5.3 g/l, chromium concentration – 103 mg/l, contact time – 27 min. at this point, the maximum chromium removal was found to be 94.5%. 4. conclusions the biosorption of chromium on h. valentiae, a red alga, was investigated in a batch system. the biosorption conditions of chromium on h. valentiae were optimized by using rsm. the relationship between the response and the independent variables was developed via the quadratic approximating function of chromium biosorption capacity of sorbent. the optimum conditions were determined as initial ph – 2.8, temperature – 48.2°c, biosorbent concentration – 5.3 g/l, initial chromium concentration 103 mg/l and contact time – 27 min. as a result, a chemical waste containing chromium can be removed by using a red alga such as h. valentiae which is abundant and cheaply available. this study also clearly showed that response surface methodology was one of the suitable methods to optimize the operating conditions and maximize the chromium removal. analysis of variance showed a high coefficient of determination value (r2 = 0.9850), thus ensuring a satisfactory adjustment of the second-order regression model with the experimental data. references aleboyeh, a., daneshvar, n, kasiri, m.b.: optimization of c.i. acid red 14 azo dye removal by electrocoagulation batch process with response surface methodology. chem. eng. process., 47, 2008, 827–832. deng, l., su, y., su, h., wang, x., zhu, x.: sorption and desorption of lead (ii) from wastewater by green algae cladophora fascicularis. j. hazard. mater., 143, 2007, 220–225. garg, u.k., kaur, m.p., garg, v.k., sud, d.: removal of nickel(ii) from aqueous solution by adsorption on agricultural waste biomass using a response surface methodological approach. bioresour. technol., 99, 2008, 1325–1331. jalali, r., ghafourian, h., asef, y., davarpanah, s.j., sepehr, s.: removal and recovery of lead using nonliving biomass of marine algae. j. hazard. mater., b92, 2002, 253–262. korbahti, b.k.: response surface optimization of electrochemical treatment of textile dye wastewater. j. hazard. mater., 145, 2007, 277–286. ozer, a., gurbuz, g., alimli, a.c., korbahti, b.k.: biosorption of copper (ii) ions on enteromorpha prolifera: application of response surface methodology. chem. eng. j., 146, 2009, 377–387. nova biotechnologica 10-2 (2010) 115 prasanna kumar, y., king, p., prasad, v.s.r.k.: adsorption of zinc from aqueous solution using marine green algae—ulva fasciata sp. chem. eng. j., 129, 2007, 161–166. rajasimman, m., sangeetha, r., karthic, p.: statistical optimization of process parameters for the extraction of chromium (vi) from pharmaceutical wastewater by emulsion liquid membrane. chem. eng. j.l, 150, 2009, 275-279. rajeshkannan, r., rajamohan, n., rajasimman, m.: removal of malachite green from aqueous solution by sorption on hydrilla verticillata biomass using response surface methodology. front. chem. eng. china, 3, 2009, 146-154. ravikumar, k., pakshirajan, k., swaminathan, t., balu, k.: optimization of batch process parameters using response surface methodology for dye removal by a novel adsorbent. chem. eng. j., 105, 2005, 131–138. saleem, m., pirzada, t., qadeer, r.: sorption of acid violet 17 and direct red 80 dyes on cotton fiber from aqueous solutions. colloids surf. a: physicochem. eng. asp., 292, 2007, 246–250. tien, c.j.: biosorption of metal ions by freshwater algae with different surface characteristics. process biochem., 38, 2002, 605 -/613. microsoft word farago nb 2010-2.doc nova biotechnologica 10-2 (2010) 137 community level physiological profiling of microorganisms in the rhizosphere of transgenic plants – a review juraj faragó1, natália faragová2 1department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic 2plant production research centre, research institute of plant production, plant biology section, bratislavská cesta 122, piešťany, sk-921 68, slovak republic abstract: since 1996, when the first genetically modified seeds were planted in field conditions, the commercial growing of genetically modified crops increased to over 134 millions of hectares in 2009 worldwide. along with the great potential of transgenic plants for future agriculture, considerable concerns on their biosafety have been raised, including their potential impact on soil microbial communities. this review briefly summarizes the important features of soil microorganisms for plant health and ecosystem stability, the numerous methods available for microbial ecologists to study soil microbial diversity, with emphasis on the method of community level physiological profiling (clpp) based on carbon substrate utilization patterning, and finally the use of clpp for assessing the effects of transgenic crops on soil microbial communities. key words: clpp, soil microbial community, transgenic plants, carbon sources 1. introduction the first transgenic plant, tobacco containing a selectable marker gene nptii from a bacterium conferring resistance to kanamycin, was produced at the university of ghent, belgium, in 1983 (zambryski et al., 1983), only 30 years after the discovery of the structure of dna. now, some 27 years later, genetically modified (or transgenic) crops became a common feature of the agricultural landscape in 25 countries of the both americas, europe, asia, africa and australia (james, 2009). the unprecedented high growth rate of adoption of transgenic crops started in the u.s.a. in 1996 with 1.7 million hectares. in 2009, the global area of genetically modified (gm) crops reached 134 million hectares (james 2009), which is more than the total land area of six neighboring countries: slovakia, czech republic, poland, hungary, ukraine and austria together (122 mil. ha). the first commercially grown transgenic crops contained one gene of interest or one introduced novel trait (single trait gm crops). at present, however, combination or stacking different traits or genes in one plant is rapidly gaining popularity and recent growth of gm crops hectarage over the last few years comes largely from deployment of these so called “stacked traits” gm crops (taverniers et al., 2008; james, 2009). several transgenic plants containing two, three or more genes of interest are currently under evaluation by national authorities of different countries for deliberate release of transgenic plants into environment (taverniers et al., 2008). 138 faragó, j. and faragová, n. along with the increasing potential for widespread commercial use and the benefits of transgenic crops, considerable concerns on their safety have been raised, including safety aspects relating to their potential impact on the environment (widmer, 2007). the rapid development of agricultural biotechnology and release of transgenic plants into environment have provided many agronomic and economic benefits, however they also raised a multitude of concerns over the potential ecological effects of transgenic plants. these concerns include unintended consequences of transgene flow to indigenous organisms through pollen transfer, plant invasiveness, the development of resistance in target pests, and direct or non-direct effects on nontarget flora, fauna and microorganisms and ecosystems (seidler and levin, 1994; nielsen et al., 2001; dale et al., 2002; bruinsma et al., 2003; filion, 2008). in addition to the relatively abundantly studied aboveground effects, underground impacts of gm crops have also been recognized as a result of recent methodological advances in soil microbial ecology (bruinsma et al., 2003; zhang et al., 2005). nevertheless, the impact of gm crops on soil-associated microfauna, including soil microbial communities, still represents one of the least studied and understood areas, probably due in part to the inherent technical difficulties involved in the study of soilborne living organisms (filion, 2008). recently several excellent reviews on the effects of transgenic plants on soil microbial communities have been published (e.g. bruinsma et al., 2003; dunfield and germida, 2004; widmer, 2007; filion, 2008). in this review, we focused on the methodological aspects, as well as the use of community level physiological profiling (clpp) based on sole carbon source utilization analysis to study the functional diversity of microbial communities in the rhizosphere of transgenic plants. we provide also a brief overview of the microbial characteristics of soils and methods used for analyses of soil microbial communities. 2. microbial characteristics of soils soil is a complex and dynamic physical, chemical and biological system and a major component in agro-ecosystems (nannipieri et al., 2003). it is considered to be the greatest reservoir of biodiversity on the planet (crawford et al., 2005). prokaryotic organisms comprise more than half of the biodiversity on earth and their diversity in soil has been estimated to be about three orders-of-magnitude greater than in all other environments combined (curtis et al., 2002). microorganisms are fundamentally important components of the soil habitat where they play key roles in ecosystem functioning through controlling nutrient cycling reactions essential for maintaining soil fertility, and they also contribute to the genesis and maintenance of soil structure (kirk et al., 2004). the microbial community of arable soils is a key agent in biologically mediated processes such as degradation of organic residues, transformation of organic matter, mineralization of nutrients, formation of soil aggregates, and soil nutrient balance. soil microorganisms are also a component with multiple functions of importance to soil fertility, such as catalysis of nutrient transformations, storage of nutrients, formation and stabilization of soil structure, and control of plant pathogens (kennedy and papendick, 1995; petersen et al., 2002; gömöryová et al., 2009a). nova biotechnologica 10-2 (2010) 139 soil microbial communities have great potential for temporal and spatial change, and thus represent a powerful tool for understanding community dynamics in both basic and applied ecological research (garland, 1997). the soil microbial community structure is considered as an early and sensitive indicator of anthropogenic effects on soil ecology (nielsen and winding, 2002). soil microorganisms are able to respond more quickly to environmental stress because, compared to higher organisms, they have a high surface-to-volume ratio, which means they are capable of a much more intense exchange of matter and energy with their environment. therefore, changes in size, composition and activity of microbial communities can frequently be observed before detectable changes in soil physical and chemical properties occur (nielsen and winding, 2002; widmer and oberholzer, 2003; gömöryová et al., 2009a). moreover, bacteria interact with plants during their entire life cycles and therefore can act as precise and fast indicators of environmental changes. plants depend on the ability of roots to communicate with microbes. the converse is also true; many bacteria and fungi are dependent on associations with plants that are often regulated by root exudates (bais et al., 2004). crop plants interact with soil microbial communities through a micro-zone at the root-soil interface that is under the influence of the plant root. this complex habitat composed of the fraction of soil adhering to plant roots and altered by root activity is referred to as rhizosphere. the rhizosphere environment hosts a complex microorganism network which interacts very closely with plant roots, the outcome of such interaction being seen in the strong influence plant type has on the microbial ecosystem of the soil (brusetti et al., 2004). it is well known, that rhizosphere exerts a selective influence on the associated microbial communities and these microorganisms in turn have a great impact on root biology, they influence plant growth, nutrition and development, and are important for long-term sustainability of agricultural soils (mantelin and touraine, 2004). a number of studies have shown that the rhizosphere has much higher population densities of bacteria and fungi compared with bulk soil (maloney et al., 1997; semenov et al., 1999). plants modify their rhizosphere environment through release of assimilates in the form of root exudates (uren, 2001). the four types of main ingredients in plant root exudates are carbohydrates, amino acids, fatty acids and nucleotides, other compounds, such as organic acids, steroids, growth hormones, flavones and enzymes are also involved (zhang et al., 2005). this complex mixture of organic compounds provides a source of reduced carbon, nitrogen, and other nutrients for soil inhabiting microorganisms (buyer et al., 2002). the composition of compounds which are released from the roots and interact with bacteria in the rhizosphere vary depending on the plant species, cultivar, and physiological status of the plants (sorensen, 1997; saxena et al., 1999). root exudates are known to mediate both positive and negative interactions in the rhizosphere. the positive interactions include enhancement of growth rates of bacteria (hartwig et al., 1991), acting as chemoattractants to attract bacteria towards the root (dharmatilake and bauer, 1992), and acting as transcriptional signals in the communication between soil bacteria and host plants during nodule formation and biological nitrogen fixation by plants (redmond et al., 1986). on the other side, plant-microbe interactions often involve also plant activities 140 faragó, j. and faragová, n. to suppress growth of bacteria in the rhizosphere, e.g., by secretion of bactericidal substances (garcia-olmedo et al., 1998). bactericidal substances naturally released from the plant roots may include toxic components like benzofurans, terpenoids, butyrolactones, and other phytoalexins (bowen and rovira, 1991). previous studies revealed that qualitative and quantitative differences in root exudation could strongly affect the structure of microbial communities in the rhizosphere (oger et al., 1997; savka and farrand, 1997; maloney et al., 1997). the composition of microbial communities in the rhizosphere is governed mainly by the quality and quantity of carbon substrates that are released as root exudates (maloney et al., 1997). root exudation may create a niche that influences which microorganisms colonize the rhizosphere, thereby altering the composition and diversity of rhizomicrobial communities in a plant-specific manner (grayston et al., 1998). even small modifications, as may exist between different cultivars of the same plant species may cause altered composition of root exudates, which can eventually result in the occurrence of different microbial communities in the rhizosphere (rengel et al., 1998). it was shown previously that microbial population density and diversity can be influenced by introducing genes into plants for the production of novel compounds (oger et al., 1997; savka and farrand, 1997). genetically engineered plants might change the bacterial consortia in the rhizosphere due to the release of transgene products or an altered composition of root exudates (oger et al., 1997). the insertion of transgenes may unintentionally alter plant characteristics of the transgenic crop others then the desired trait. in turn, these changes may influence the growth and species composition of soil microorganisms associated with the roots of these plants, and therefore they may have ecological effects (donegan et al., 1995). an altered composition of root exudates may induce a different community of rhizosphere microorganisms and even small modifications, as may exist between different cultivars of the same plant species, can result in the occurrence of different microbial communities in the rhizospheres (rengel et al., 1998). comparative studies assessing whether there are differences between microbial communities living in the rhizospheres of transgenic and non-transgenic plants represent an important first step in determining if the presence of transgenic material can catalyze changes in the environment. this knowledge of the impact of transgenic crops on soil microbial ecology is essential for understanding the long-term agronomic and environmental effects of genetically modified crops and for developing appropriate management practices for minimizing potential negative impacts (fang et al., 2007). 3. methods of studying soil microbial communities over the past ten years, approaches to studying soil microbial communities changed dramatically. a number of new techniques have been developed and are now available to soil microbiologists to gain deeper insight into community-level responses to changes in soil properties or management. commonly applied bulk soil microbial parameters include measurement of total microbial biomass (vance et al., 1987; liao and xie, 2007; lupwayi et al., nova biotechnologica 10-2 (2010) 141 2007), determination of activities of enzymes involved in c-, nand p-nutrient cycling, such as proteases, cellulases, phosphatases, dehydrogenases, arylsulfatases, and others (rasche et al., 2006; bradley et al., 2007; gorlach-lira and coutinho, 2007), basal soil respiration (visser et al., 1994; lupwayi et al., 2007; raubuch et al., 2010) and specific metabolites (raubuch et al., 2007). these methods can provide information on general microbial activities, but not on specific microbial groups that actually contribute to different types of soil enzymatic activities (liu et al., 2005). the methods for the analysis of microbiological soil characteristics and assessment of the impact of environmental or anthropogenic factors, including the cultivation of genetically modified crops, on soils are generally categorized into two broad groups: (1) culture-dependent methods and (2) culture-independent methods (hill et al., 2000; widmer, 2007). there are several cultivation-dependent methods which are used to retrieve microorganisms from soils. the plating method (syn. plate count method) offers a simple but useful tool to identify and characterize changes of specific microbial strains, or groups of species (brusetti et al., 2004; liu et al., 2005; faragová et al., 2005; mulder et al., 2006). it can be used to detect effects of transgenic plants on specific soil microorganisms such as symbiotic nitrogen-fixing bacteria, degraders of recalcitrant organic matter, nitrifying bacteria, denitrifying bacteria, cellulolytic bacteria, actinomycetes, and others (bruinsma et al., 2003; faragová et al., 2005, faragová and faragó, 2010). one limitation of the method is, however, that it is affected by the cultivation bias precluding analysis of the entire microbial diversity in soils (widmer, 2007). development of the metabolic fingerprinting method referred to as community-level physiological profiling (clpp), syn. community-level substrate utilization (clsu), based on parallel cultivation of soil microorgamisms on a set of different carbon sources have significantly improved the capacity and robustness of cultivation-dependent methods. this method is also a cultural technique, and as such, its use is restricted to culturable microorganisms only (preston-mafham et al., 2002; liu et al., 2005; widmer, 2007). nevertheless, clpp is often used as an efficient method capable of revealing the effects of transgenic plants on soil microbial communities (donegan et al., 1995; di giovanni et al., 1999; dunfield and germida, 2003; griffiths et al., 2000; heuer et al., 2002; tesfaye et al., 2003; brusetti et al., 2004; fang et al., 2005; lupwayi et al., 2007). inherent limitations of culture-based methods have lead soil microbiologists to explore culture independent methods of microbial community analysis. these methods allow to characterize the composition and diversity of soil microbial communities based on the extraction, identification and quantification of molecules from soil that are specific to certain microorganisms or microbial groups (hill et al., 2000). such molecules include fatty acids and nucleic acids. phospholipid fatty acid (plfa) analysis is based on the analysis of the presence and abundance of specific fatty acids in soil which are indicative of specific groups of microorganisms. a biochemical method fatty acid methyl ester (fame) analysis provides information on the microbial community composition based on separation and identification of methylated fatty 142 faragó, j. and faragová, n. acids extracted directly from soil. these methods are capable of providing a quantitative measure of the viable or potentially viable microbial biomass (liu et al., 2005). both, plfa and fame are extensively used to assess the effects of transgenic plants on soil microbial communities (dunfield and germida, 2003; griffiths et al., 2007). advances in molecular biology in last years considerably improved our knowledge of morphological, physiological, biochemical and ecological features of soil microorganisms. using the dna based methods specific marker sequences can be analyzed in soil dna extracts by means of polymerase chain reaction (pcr) amplification (hill et al., 2000; widmer, 2007). of the various marker genes used to estimate microbial community composition and diversity, the most used are those encoding 16s ribosomal rna (rrna) in prokaryotic, and 18s rrna in eukaryotic microorganisms. a number of molecular-based techniques have been developed to assess the microbial community diversity (hill et al., 2000; kirk et al., 2004; widmer, 2007). these include dna guanine plus cytosine content analysis (nusslein and tiedje, 1999), dna reassociation (torsvik et al., 1990), dnadna hybridization (guo et al., 1997), and different pcr-based methods such as terminal restriction fragment length polymorphism (t-rflp) (liu et al, 1997; rasche et al., 2006), amplified ribosomal dna restriction analysis (ardra) (liu et al., 1997; widmer et al., 2001; lottmann et al., 2010), denaturing gradient gel electrophoresis (dgge) (muyzer et al., 1993; castaldini et al., 2005; costa et al., 2006), temperature gradient gel electrophoresis (tgge) (muyzer and smalla, 1998), single-strand conformation polymorphism (sscp) (schweiger and tebbe, 1998; miethling-graff et al., 2010), and ribosomal intergenic spacer analysis (risa) (fisher and triplett, 1999; brusetti et al., 2004). the rflp and ardra techniques distinguish sequences based on different locations of restriction enzyme recognition sites. dgge and tgge rely on differences in the stability of dna duplex, while sscp analysis detects differences in secondary structures of single stranded dna. risa, on the other hand, resolves length differences of the amplified marker gene sequence (widmer, 2007). these methods, used for dna fingerprint analyses, enables cultivation-independent analysis of microbial community structure and diversity through the detection and characterization of microbial nucleic acid sequences within samples (liu et al., 2005), however, they do not provide quantitative analyses of microbial groups in soil. recently, a new dna-dna hybridization technique, taxonomic microarray analysis, has been adopted to assess soil microbial diversity (greene and voordouw, 2003). this technique has been shown to be able to discriminate and characterize bacterial community composition in related biological samples, offering extensive possibilities for systematic exploration of bacterial diversity in different ecosystems (sanguin et al., 2006). 4. community level physiological profiling to understand the role of microbial communities in different environments, it is essential to have knowledge of microbial community function and functional diversity nova biotechnologica 10-2 (2010) 143 (preston-mafham et al., 2002). although molecular techniques are now widely used to characterize soil bacterial communities, these techniques provide little information regarding functional properties of microbial communities, for example, on the ability of microbial communities to metabolize specific chemical compounds. to address these functional features of microbial communities, the method of community level physiological profiling (clpp) using biolog® microplates (biolog, inc., hayward, usa) have been developed. garland and mills (1991) were the first who introduced the use of commercially available biolog® microtitre plate bacterial identification system based on the utilization pattern of 95 different single carbon sources, to assess functional diversity of microorganisms in environmental samples. the biolog® system was originally developed for rapid identification of pure bacterial cultures by sole carbon source utilization, using plastic 96 well microtiter plates containing 95 different carbon substrates in separate wells, and no substrate in one well, which is used as control. among the 95 substrates on biolog gn and gp plates, there are several groups of chemical compounds, such as carbohydrates, amino acids, carboxylic acids, amines, amides and polymers (fig. 1). in addition, each well contains a colourless tetrazolium dye. upon inoculation of plate with a pre-grown isolate, metabolism of the substrate and the production of nadh via cell respiration lead to reduction of colourless tetrazolium to violet formazan (fig. 2). the colour development is measured colorimetrically at specific absorbance and a data set of optical density (o.d.) values is obtained (garland and mills, 1991; prestonmafham et al., 2002). individual species may be identified by a specific pattern of colour change on the plate, providing an identifiable metabolic fingerprint (preston-mafham et al., 2002). differences in sole carbon source utilization have been used successfully to distinguish among different bacterial isolates for more than 60 years. at present, the microbial identification database for biolog systems (biolog, inc.) contains more than 1950 entries for rapid identification of broad spectrum of aerobic and anaerobic bacteria, yeasts and filamentous fungi, mainly of medicinal importance. the commercially available 96 well biolog® microtitre plates were first used to compare metabolic activities of heterotrophic microbial communities from environmental habitats (water, soil and wheat rhizosphere) by garland and mills (1991). currently several different types of biolog plates are available to microbial ecologists, all of them containing different substrates appropriate for specific groups of microorganisms. the biolog gntm plates were developed for identification of gramnegative bacteria and contain substrates appropriate for this group of microorganisms (fig. 1a), whereas biolog gptm plates contain carbon sources appropriate for grampositive bacteria (fig. 1b). even though gn and gp plates were successfully used for ecological microbiology research, biolog ecotm plates have been developed specifically for ecological applications. contrary to gn and gp plates, the eco plates contain only 31 substrates (plus a control well without a substrate) in three replicates on the microtitre plate. these substrates have previously been found to have high relevance to soil bacterial communities, or are known to be included in plant root exudates (preston-mafham et al., 2002). the tetrazolium dye used in gn, gp 144 faragó, j. and faragová, n. and eco plates is not metabolizable by fungi, therefore specific biolog ff plates have been designed for identification of fungal microorganisms. the ff plates contain different carbon sources from gn, gp, and eco plates and a modified tetrazolium dye specifically metabolizable solely by fungi. to prevent interference of growth and colour development by bacteria, an appropriate combination of antibiotics is frequently included into ff plates (buyer et al., 2001). biolog mt plates, containing a redox dye and no substrate, are also available, which allow researchers to produce customized plates (preston-mafham et al., 2002; stefanowitz, 2006). a b fig. 1 carbon sources in gn2 (a) and gp2 (b) microplates (biolog, inc., hayward, usa) nova biotechnologica 10-2 (2010) 145 fig. 2 colour development in biolog gp microplate inoculated with a microbial suspension extracted from the rhizosphere zone of transgenic alfalfa (faragová et al., not published). the use of biolog microtitre plates enables analyzing microbial communities on both, the individualas well as community levels (garland, 1997). at the individual-level, the analysis of microbial communities requires the isolation of microorganisms, isolation and characterization of individual isolates, and description of the community based on the relative abundances and diversities of different isolates. the community-level approach, established by garland and mills (1991), bypasses the isolation step and the environmental samples are directly inoculated into microplates and the resulting response (colour change pattern) is used to describe differences in microbial communities (garland, 1997). community-level biolog analysis is generally accomplished in five consecutive steps: (1) sample processing to obtain suspension of microorganisms, (2) inoculation of aliquots of microbial suspension into wells of biolog® microplates, (3) incubation of plates for 2-8 days in defined culture conditions, (4) regular monitoring the colour development in each well and (5) data processing and statistical analysis of results. for analyses of pure culture isolates, the samples are prepared from freshly grown cells on a standard enriched medium (garland, 1997; konopka et al., 1998). in contrast, analyses of environmental samples have used a number of different sample processing techniques, including direct inoculation of aquatic samples into microtitre plates (garland and mills, 1991), shaking plant roots in a solution to release rhizosphere microorganisms (garland, 1996), and extraction of microbial cells from complex soil matrices using sieving, shaking, blending and sedimentation or centrifugation of samples (haack et al., 1999; konopka et al., 1998; dobler et al., 2001). for comparisons it is important that samples are of equivalent size, in term of volume and/or weight (preston-mafham et al., 2002). in most cases the processed sample is diluted prior to use as inoculum. as bacterial cell density in the inoculum affects colour development in biolog plates (garland and mills, 1991; haack et al., 1995; garland, 1996), it is very important to 146 faragó, j. and faragová, n. determine dilution levels that reduce additional colour development caused by excess utilizable organic matter in the sample (konopka et al., 1998; prestonmafham et al., 2002). it is known, that a minimum number of about 108 metabolically active cells per ml in a well is required for observable colour development (haack et al., 1995). therefore, in dilute inocula, the kinetics of colour development will be a function of time and will follow a sigmoidal curve with an initial lag phase, followed by linear growth phase and a final plateau phase. the lag phase represents the time period during which the inoculum grows to a population density of 108 cells ml-1, while during the linear phase the specific metabolic activity of a microorganism on a specific substrate causes accumulation of formazan and colour development. at the plateau phase the production of formazan upon the oxidation of a specific substrate by the activity of a microorganism reaches its maximum yield due to organic substrate depletion. especially when the goal is identification of pure cultures, it is recommended to adjust all microbial suspensions to a standardized cell density prior to inoculation into biolog® plates. alternatively, with community-level biolog analysis, adoption of appropriate data analysis can account for different inoculum densities (garland and mills, 1991). most studies of soil microbial community composition and diversity employing biolog® plates use fixed incubation temperatures between 15 and 28°c (prestonmafham, 2002). another important variable in biolog analysis is incubation time, as both the colour intensity in individual wells, and the number of positive wells will increase with incubation time. too short incubation times (48 h and less) may cause absence of colour development in some wells (especially with those of slow growing microorganisms), while longer incubations (>48 h) will result in more positive responses and reaching the colour saturation levels in some wells (konopka et al., 1998; preston-mafham et al., 2002). to reduce the effects of differential rates of colour development due to inoculum density and inoculation time, appropriate transformations of original raw data are recommended (garland and mills, 1991; preston-mafham et al., 2002). in principle, the biolog assay is done by colorimetrically measuring tetrazolium dye reduction that is coupled to substrate oxidation. any microtitre plate reader equipped with an appropriate filter (590 nm) can be used to quantify the colour development in individual wells of plates. the degree to which each of the 95 substrates is oxidized is determined after a fixed incubation period. a positive response is identified as an absorbance or optical density value greater than that occurring in the blank well (haack et al. 1995). the number and types of utilized substrates, as well as developed colour intensities, constitute a data set from which the functional diversity of microbial communities involved may be assessed. colour development in each well reflects species activity and density, as well as the ability of the bacterial community to respond to particular substrates (zak et al., 1994). the data obtained by quantifying the colour development in individual wells are usually expressed as individual well optical densities or average well colour development (awcd) (garland and mills, 1991; preston-mafham et al., 2002). the awcd value for each microplate is obtained by calculating the raw difference between the optical density in each well and that in the control well and then summing nova biotechnologica 10-2 (2010) 147 all these values and dividing by 95. on the other hand, sigler (2004) uses two parameters to describe the microbial community structure: the average metabolic response (amr), and community metabolic diversity (cmd). amr describes the average respiration of the c-sources by the microbial community and provides a single metric by which communities can be compared. the amr is calculated as the average of the mean difference between the o.d. of the c-source-containing wells and the control well. the second parameter, cmd, represents the number of substrates utilized by the microbial community and is analogous to community functional richness. cmd is calculated by summing the number of positive responses (violet-coloured wells) observed following incubation (sigler, 2004). the clpp generates a rich data set of 95 values for each sample (if gn or gp plates are used) or 31 values in triplicate (if eco plates are evaluated). data are corrected against the control well (without a carbon substrate) or the initial reading at time zero, before subjecting to multivariate statistical analysis. regardless of the chose of absorbance measurement, i.e. net o.d. of individual substrates, awcd, or parameters received from kinetic analyses, the use of multidimensional statistical analyses is necessary (konopka et al., 1998; stefanowicz, 2006). the most commonly used method is principal component analysis (pca) of the quantitative colour data, which reduces the number of variables to a few significant principal components. the use of cluster analysis based on the presence or absence of colour in wells (zak et al., 1994) is also possible. in kinetic studies, one may process the data by calculating the area under curve for each well o.d. for the entire period of incubation, or by estimation of kinetic parameters by fitting the curve of o.d. versus time to a density dependent logistic growth equation (guckert et al., 1996; lindstrom et al, 1998; insam and goberna, 2004). analysis of microbial community functional diversity using sole carbon source utilization profiles is, despite some limitations (garland, 1997; konopka et al., 1998; preston-mafham et al., 2002), a rapid, reproducible and useful tool to discriminate among bacterial communities from diverse environmental samples. its strength lies in the low manpower it requires, which enables intensive sampling across temporal and spatial scales, and the reliance of the method on metabolic traits that could lead to functionally relevant characterization of changes in microbial communities (garland, 1997). although clpp is a culture based assay, it has been found that non culturable cells also respond to this assay, and therefore this assay seems not to be as biased as the classical culture-based techniques (prestonmafham et al., 2002). 5. assessing the effect of transgenic crops on soil microbial communities using clpp the clpp using biolog® plates has been used to date for characterizing bacterial communities from a range of environments, including comparisons of rhizosphere bacterial communities of transgenic vs. non-transgenic plants. as the method can provide important insights into ecosystem function and stability, it was used to study the metabolic responses of microbial communities from rhizosphere and bulk soils 148 faragó, j. and faragová, n. (söderberg et al., 2004), from the rhizosphere of different plant species (grayston et al., 1998; miethling et al., 2003) or from different parts of the rhizosphere along the root (baudoin et al., 2001). the biolog method is also frequently applied for estimating the influences of different cropping systems (larkin and honeycutt, 2006), agronomic procedures such as tillage (griffiths et al., 2007), soil management (gomez and correa, 2008), or factors involved in ecosystem changes (gömöryová et al., 2009b) on microbial community physiological profiles. currently one of the most frequent applications of biolog® system is for assessing the impacts of different stressing factors, such as heavy metals (dobler et al., 2001; liao and xie, 2007), herbicides (mijangos et al., 2009), explosive elements (anderson et al., 2010) and high salinity and low ph (pankhurst et al., 2001). donegan et al. (1995) first used the biolog gn plates to assess changes in soil microorganisms associated with transgenic cotton plants expressing the bacillus thuringiensis endotoxin (bt). from that time, the clpp method using biolog® gn, gp or eco plates proved to be a quick, reproducible and useful method to discriminate among microbial communities associated with the roots of transgenic vs. nontransgenic plants (di giovanni et al., 1999; schmalenberger and tebbe, 2002; tesfaye et al., 2003; brussetti et al., 2005; fang et al., 2005; mulder et al., 2006; watrud et al., 2006; lupwayi et al., 2007; travis et al., 2007 and many others). some authors (brussetti et al., 2005; fang et al., 2005; griffiths et al., 2007) could not find any statistically significant differences in functional diversities of rhizosphere microorganisms between transgenic and non-transgenic plants using biolog® microplates. in these studies, whether in greenhouse or field conditions, soil texture, crop type and pest management regime, rather than cultivation of transgenic plants affected the rhizosphere microbial communities. other authors reported very few changes in functional diversity of rhizosphere soil bacteria associated with growing gm crops (lupwayi et al., 2007), or significant shifts in the carbon utilization patterns of culturable microbial communities in the rhizospheres of transgenic plants, or soils amended with residues from transgenic plants (di giovanni et al., 1999; tesfaye et al., 2003; fang et al., 2007; travis et al., 2007). at least some of these differences in carbon source utilization patterns may be explained by the possible effects of site of insertion or simply somaclonal variation on the availability of endogenous nutrients in the soil mix on microbial populations (watrud et al., 2006). fang et al. (2007) hypothesized, that amendment of soils with bt corn residues may affect selected activities of soil bacteria carried by specific enzymes. altered composition of root exudates of transgenic plants may also induce a different community of rhizosphere microorganisms. in general, however, if shifts in bacterial communities due to cultivation of transgenic plants were observed, these were similar to those observed when nontransgenic cultivars are compared. in comparison with other factors, the impact of the genetic modification of the plant on soil microbial communities was usually minor and transient, or comparable to variations due to plant genotype, plant physiological and developmental stage, soil type, agricultural practice used and pathogen exposure. nova biotechnologica 10-2 (2010) 149 nevertheless, despite the research performed up to now, it is not yet clear whether gm plants or their products exert any ecological effect on the microbiota and microbial processes in soil. to elucidate this question, more studies on the impact of transgenic plants on soil microbial communities is needed, preferably employing the polyphasic approach using different methods of soil microbial communities analysis, including plate count, biochemical and molecular methods. 6. conclusions community level physiological profiling is a quick and easy method of investigating the functional diversity of microbial communities from a range of environmental habitats. the method, which employs biolog® microplates containing 95 different carbon sources, are gaining increasing importance not only in general soil ecology, but also is increasingly used as a method which is able to provide functional information on the microbial community structure and diversity in the rhizosphere of transgenic plants. the simplicity, speed, and low cost of the method compared with other approaches are very attractive to microbial ecologists and other microbiologists. however, the researcher should keep in mind, that to fully exploit its potential the technique requires very careful data acquisition, appropriate statistical analysis and cautious interpretation of results. acknowledgement: this work was supported partially by the research and development project of the ministry of agriculture of slovak republic no. 2005 uo 27/050 02 06/050 02 06 granted to plant production research centre, piešťany, slovak republic. references anderson, j.a.h., canas, j.e., long, m.k., zak, j.c., cox, s.b.: bacterial community dynamics in high and low bioavailability soils following laboratory exposure to a range of hexahydro-1,3,5-trinitro-1,3,5-triazine concentrations. environ. toxicol. chem., 29, 2010, 38–44. bais, h.p., park, s.-w., weir, t.l., callaway, r.m., vivanco, j.m.: how plants communicate using the underground information superhighway. trends plant sci., 9, 2004, 26-32. baudoin, e., benizri, e., guckert, a.: metabolic fingerprint of microbial communities from distinct maize rhizosphere compartments. eur. j. soil biol., 37, 2001, 85-93. bowen, g.d., rovira, a.d.: the rhizosphere. in: waisel, y., eshel, a., kafkafi, u. 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studies china, 7, 2005, 28–34. 38 nova biotechnologica et chimica 13-1 (2014) doi 10.2478/nbec-2014-0005 © university of ss. cyril and methodius in trnava distribution of zinc and cadmium in tissues of giant reed (arundo donax l.): sequential extraction radiometric study barbora micháleková richveisová, zuzana dürešová, miroslav horník, jozef augustín, martin pipíška department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, j. herdu 2, trnava, sk-917 01, slovak republic (b.richveisova@gmail.sk) abstract: heavy metals are taken up by the vascular plant root system from water solutions in cationic forms. subsequently, during both short and long distance transport to other plant tissues, cation forms are incorporated to many bioorganic compounds differing in stability, ionic character and physico-chemical properties such as solubility in lipid structures and mobility across cell membrane systems. many sequential and single step extraction methods have been elaborated for characterization of the role of individual components of plant cells components in transport and detoxication of heavy metals. in our study, dry biomass of giant reed (arundo donax l.) grown in hydroponic media spiked with 65zncl2 and 109cdcl2 was treated with dithizone solutions as complexing ligand in order to convert free zn2+ and cd2+ ions to corresponding dithizonates. treatment with dithizone showed that up to 67 % of the total plant cd and 56 % of the total plant zn were transformed to dithizonate complexes extracted with chloroform. extraction of biomass with folch reagent showed that up to 48 % of the total root cadmium and up to 18 % of the total shoot cadmium is bound in lipid fraction. zinc was not found in lipid fraction of root and shoot. derivatization of the dried root and shoot lipid fraction by dithizone showed that two third of cd in root and practically all cd in shoot lipid fraction could be transformed to cd-dithizonate. methods of biomass treating with complexing ligands and a method of sequential extraction procedures with non-polar organic solvents and radiotracer methodology seem to be useful methods for the study of metal speciation and distribution in vascular plants. key words: 65zn, 109cd, arundo donax l., sequential extraction, dithizonate, speciation 1. introduction contamination of environment by heavy metals such as cu, cd, pb, hg and zn is a global problem. heavy metals and radionuclides spreading from the source of air pollution are dissolved by rainfall water and sorbed by leaf surface of higher plants or directly sorbed onto soil components. reversibility of the metal sorption at the end of vegetation period determine the next transport of contaminants to the soil and consequently into the food chain in the following vegetation period. plants can bind contaminants from soil and according to the way of binding, these metals are released to the environment in different speed. giant reed (arundo donax) can be considered both as an energy plant and as a tool for toxic metal removal from contaminated land. the plant can survive in wetlands bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:05 utc nova biotechnologica et chimica 13-1 (2014) 39 when the concentration of cu2+, pb2+, cd2+, zn2+, ni2+ and hg2+ is 100 mg/kg and cr6+ 50 mg/kg (han and hu, 2005). the concentration of heavy metals in soil declines with the growth of the plant due to the translocation of heavy metals from peripheral soil to rhizosphere and the phytoextraction and phytovolatilization. the characters of large biomass, exuberant root and good adaptability of a. donax suggested its great potential in remediation of polluted soils. giant reed can be considered also as a sensitive biomonitor of the presence of heavy metals in water and sediments. bonanno (2012) showed that concentrations of metals in plant tissues were significantly dependent on the kind of organ and element. trace element concentrations decreased according to the pattern of root > leaf > stem, implying that roots acted as the main centres of bioaccumulation, and stems as transit organs as a consequence of the general high translocation from roots to leaves. a. donax showed a significant capacity of bioaccumulation in agreement with ecologically similar macrophytes. positive correlations were found between trace concentrations in plant organs and sediment (al, cr, mn, ni, zn), and water (cu, ni, zn). the results of this study suggested that a. donax acts as an ecological indicator of environmental conditions, thus, its application may prove a useful tool during monitoring campaigns of wetlands. in the quantification of the efficiency of energy production processes based on biomass, energy returned on invested energy is an important parameter to be considered. in this context, a. donax showed the best results, thanks to the great yields reached in low input cultivation conditions, with respects to other herbaceous perennial species (pilu et al., 2012; takahashi et al., 2010). the annual stem production can reach from 12 to 23 t dm/ha (mavrogianopoulos et al., 2002). giant reed is therefore considered an important representative of energy plants. for extraction of heavy metals from soils and sediments, a variety of single and sequential extraction techniques can be used (tessier et al., 1979). these techniques were recently reviewed by babel and dacera (2006). however, owing to the wide range of extractants used in these extraction techniques, the results obtained were not comparable. in 1987, the community bureau of reference (bcr) started a programme to harmonize the methodology used in sequential extraction schemes for determination of metals in soils and sediments (ure et al., 1993). the methodology was also successfully applied to different types of biomass. the type of applied sequential extraction scheme can lead to the anomalies in the results, as sequential extraction schemes are greatly influenced by the type of extractants used, the operating conditions (ph, contact time and temperature) and the sequence in which the extraction steps are applied. van hullebusch et al. (2005) compared the sequential extraction procedure by tessier et al. (1979), the procedure according to stover et al. (1976), the bcr protocol and discussed the results of the fractionation of anaerobic metabolism of important metals (co, ni, cu, zn, mn and fe). the phytofiltration of cd and zn by giant reed (a. donax l.) was described and published in previous papers (horník, et al. 2011; dürešová et al., 2013; dürešová et al., 2013). sequential extraction has been successfully used for speciation study of elements in soils and other inorganic matrices (tessier, 1979; mehlich, 1953; mehlich, 1984; lindsay, 1978). however practical applicability for behaviour of toxic metals in plant tissues is rather limited. the use of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:05 utc 40 micháleková richveisová, b. et al. method of sequential extraction of heavy metals in sewage sludge was reported in our previous paper frišták et al. (2012). the aim of this paper was to characterize mode of heavy metals binding in the process of root uptake and translocation to shoots in vascular plants. giant reed (a. donax l.) grown in nutrient media spiked with 65zn and 109cd was used as a model plant. sequential extraction of dried biomass was used in the presence of ligands able to bind heavy metal cations to complexes differing in stability constants log k and estimated in extracts by gamma-spectrometry. 2. material and methods 2.1 chemicals deionized water (conductivity 0.054 μs/cm) was prepared with simplicity 185 (millipore, f). commercially available chemicals of analytical grade, without pretreatment were used: dithizone (c13h12n4s; cas: 60-10-6), chloroform, methanol. 2.2 cultivation media hoagland medium (mg/dm3): nh4no3 – 160.1; h3bo3 – 8.5; na2moo4.2h2o – 0.06; mnso4.5h2o – 5.0; znso4.7h2o – 0.66; cuso4.5h2o – 0.8; mgso4.7h2o – 369.7; kno3 – 404.4; cacl2 – 443.9; nah2po4.2h2o – 291.7; na2hpo4.12h2o – 46.5; feso4.7h2o – 17.9; nano3 – 339.9; nh4cl – 213.9, ph 6.0. basal medium was diluted with deionized water in the ratio 1:3. mineral medium (mm) (mg/dm3): cocl2.6h2o – 0.3; feso4.7h2o – 5.8; kh2po4 – 23.4; mgso4.7h2o – 41.0; co(nh2)2– 91.7; nh4cl – 12.8; ch3coonh4 – 79.4. microelements (μg/dm3): cuso4.5h2o – 80; h3bo3 – 850; mnso4.5h2o – 500; na2moo4.2h2o – 6.0; znso4.2h2o – 66, ph 5.5. 2.3 plants arundo donax l. (var. versicolor) was obtained from the centre for research of plant production (piešťany, slovak republic). plants originated from tissue cultures grown in nutrition media (murashige et skoog, 1962) supplemented with synthetic cytokinine 6-benzylaminopurine and α-naphthylacetic acid for micropropagation of plants. in the next step, plants were cultivated for 2-4 weeks in 25 % hoagland nutrition media (hoagland, 1920) in cultivation box binder (germany), type kbwf 720. photoperiod light/dark 16h/8h at light intensity max 11 450 lx (stepwise 0, 40, 60 and 100 %) and at 28/15 °c, relative humidity 60-80 %. 2.4 zinc and cadmium root uptake pre-cultivated 20 plants (each approx. 20 cm, 40 mg d.w., shoot to root ratio 4:1 d.w.) were transferred to test tubes and cultivated in the first step for 24 h in mineral medium (mm), in the second step for 24 h in the same mineral medium supplemented with 65zncl2 and 109cdcl2 at 20 °c at day/night light cycle. roots were washed in bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:05 utc nova biotechnologica et chimica 13-1 (2014) 41 surplus of deionized water, roots and shoots were air dried, weighted and radioactivity was measured by gamma-spectrometry. 2.5 extraction procedures dried biomass (approx. 200 250 mg) divided to roots and shoots was separately treated according to scheme in fig. 2 and fig. 3. presented data are averages of three independent experiments. 2.5.1 sequential extraction of lipid fraction shoot or root biomass (approx. 110 mg) were subsequently extracted with 2 ml folch solution (chcl3 : meoh = 2 : 1) for 1 h at 25 °c according to the scheme in fig. 3. aliquots of extract solution removed from biomass and in both extract and biomass were 65zn and 109cd radioactivity by gamma-spectrometry measured. after this, all samples of biomass and folch extracts were dried for 1 day at 25 °c. subsequently, the dried samples were extracted with 2 cm3 1 mmol/dm3 chloroform solution of dithizone for 1 h at 25 °c. extracts were removed from biomass and samples of wet biomass were weighted and retained volume of extractants was calculated by subtraction of dry biomass used in experiments. concentration ratio of zn and cd species was calculated from the radioactivity of extracts and remaining biomass by gamma-spectrometry. 2.5.2 sequential extraction combined with derivatization to dithizonates biomass of a. donax (approx. 200-250 mg, d.w.) was separated to roots and shoots, air dried and extracted according to the scheme in fig. 2. shortly, in the first step, biomass was extracted with 2 cm3 0.1 mmol/dm3 dithizone for 1 h at 25 °c, in the next step biomass was extracted with chloroform solution of 1 mm dithizone, and in the last step with 0.1 mol/dm3 hcl. after each extraction the extract was removed, samples of wet biomass were weighted and retained volume of extractants was calculated by subtraction of dry biomass used in experiments. concentration ratio of zn and cd species was calculated from the radioactivity of extracts and remaining biomass by gamma-spectrometry. 2.6 radiometric analysis for determination of 65zn and 109cd gamma-spectrometric scintillation detectors 54bp54/2-x and 76bp76/3 with well type crystal na(tl) (scionix, nl) and data processing software scintivission-32 (ortec, usa) were used. a library of radionuclides was built by selecting characteristic γ-ray peaks (88.04 kev for 109cd and 1115.52 kev for 65zn) for energy and efficiency calibration. standardized solution of 65zncl2 (4.90 mbq/cm 3; 0.05 g/dm3 zncl2 in 3 g/dm 3 hcl) and 109cdcl2 (3.94 mbq/cm3; 0.05 g/dm3 cdcl2 in 3 g/dm 3 hcl) was provided from the czech metrological institute (prague, cr). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:05 utc 42 micháleková richveisová, b. et al. 3. results and discussion giant reed (a. donax) was grown in liquid nutrient media spiked with 65zn and 109cd at subtoxic concentrations 11.0 μmol/dm3 zncl2 and 11.0 μmol/dm 3 cdcl2. it was found that zinc and cadmium were distributed between roots and shoots in the ratio 6.6:1 for cd and 5.4:1 for zn. the ratio of specific radioactivity (bq/g, d.w.) of root biomass to specific radioactivity of shoots biomass calculated from 20 plants of a. donax was 33.7:1 for cd and 11.9:1 for zn. this phenomenon can be explained by higher mobility of zinc as microelement comparing with mobility of phytotoxic cadmium, which is retained in root biomass with higher efficiency. speciation of zinc and cadmium in plants speciation of heavy metals entering the plant tissues plays decisive role in the both long distance and short distance transport of these metals in plant and is responsible for phytotoxic behaviour of heavy metals and participation in metabolic processes. for solving these problems in our experiments we combined sequential extraction procedures and derivatization of plant zinc and cadmium by complexing ligands (fig. 1). 2 + m2+ fig.1. reaction of dithizone (i) with bivalent metals m2+resulting in metal dithizonates formation (ii). dithizone is able to react with free zn2+ and cd2+ ions and also with zinc and cadmium bound in complexes with many organic and inorganic compounds bearing anionic groups such as carboxylic acids, amino acids, inorganic and organic phosphates. concentration equilibrium of this substitution reaction depends on log k values of stability constants of corresponding metal ligands. stability constants of synthetic complexing ligands such as edta, nta and dithizone are higher within orders than stability constants for components of plant tissues (table 1). synthetic complexing ligand could therefore serve as an efficient tool for speciation analysis of microelements and toxic metals entering plant tissues. the main advantage of dithizone comparing with edta is high solubility of zn and cd dithizonate in organic solvents e.g. in chloroform and it can be quantitatively separated from reaction mixture and at utilization of radiotracer methods determined by common radioanalytical equipments. i k ii bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:05 utc nova biotechnologica et chimica 13-1 (2014) 43 . table 1. stability constants log k of naturally occurring and synthetic metal-ligand complexes. log k ligand zn2+ cd2+ naturally occurring acetic acid 1.59 c 1.7 c glutamic acid 5.45 a 4.78 b histidine 6.6 c 5.65 b cysteine 9.8 c 10.30 a adp 4.28 c znh3phytate 7.81 d triphosphates 9.7 c synthetic nta 10.45 c 9.8 b edta 16.5 b 16.5 b other organic ligands 0.8-21.9 e 0.9-23.3 e a bottari and festa (1997); b christensen and izatt (1983); c martell (1964); d crea et al. (2008); e solovjev et al. (2012) fig. 2. sequential extraction of dried a. donax biomass (~ 110 mg, d.w.) combined with zn and cd complexation with dithizone in situ without removal of lipid fraction. plants grown in mineral medium (mm) at c0 = 11 μmol/dm3 zncl2 and 11 μmol/dm3 cdcl2. in our experiments (fig. 2), dried plant biomass was treated with 0.1 and 1.0 mmol/dm3 dithizone in chloroform at 20 °c. this procedure leads to conversion of zn bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:05 utc 44 micháleková richveisová, b. et al. and cd species to corresponding dithizonates and their extraction to chloroform phase. gamma-spectrometry showed, that in root biomass after double extraction was 78 % zn and 76 % cd in non-ion-exchangeable form, which was soluble in 0.1 mol/dm3 hcl. similar ratios were obtained in shoot biomass, where after double extraction was in non-ion-exchangeable form 66 % zn and 57 % cd. residual proportion in biomass after extraction with 0.1 mol/dm3 hcl represented ≤3%, which defines metal binding to metalloproteins or other biomass compartments, which can be for example for steric reasons unavailable in reaction with such effective ligand as hcl. the data are presented in table 2. table 2. ion-exchangeable (dithizonate) and non-ion-exchangeable (non dithizonate) forms of zn and cd in a. donax estimated by in situ conversion of metals to corresponding metal-dithizone complexes with the following extraction into chloroform. a sum of the data of the subsequent reaction with 0.1 mmol/dm3 and 1 mmol/dm3 dithizone in chloroform. fig. 3. sequential extraction of dried a. donax biomass (~ 110 mg d.w.) combined with zn and cd complexation with dithizone in situ after removal of lipid fraction by folch reagents. root (%) shoot (%) fraction zn cd zn cd total biomass 100 100 100 100 dithizonate (a) 22 24 34 43 non dithizonate 78 76 66 57 hcl soluble 75 74.5 66 54 hcl insoluble 3 1.5 0 3 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:05 utc nova biotechnologica et chimica 13-1 (2014) 45 after removal of lipid fraction from biomass (fig. 3) we found, that zn entering the plant via root of a. donax l. from very diluted solutions (11 μmol/dm3 zncl2 and 11 μmol/dm3 cdcl2) was not distributed in root and shoot lipid fraction, or only in small quantities. zn was localized in non-lipid fraction, in forms which are not available for reaction with dithizone in non-polar organic solvent immiscible with water, or is bound to a chemical forms in ion non-exchangeable form (e.g. proteins, nucleic acid…). cadmium is bound in high quantities in root lipid fraction (48 %) and shoot lipid fraction (18 %). derivatization of dried root and shoot lipid fraction by dithizone showed that two third of cd in root and practically all cd in shoot lipid fraction could be transformed to cd-dithizonate. in shoot lipid fraction, substantial part of cadmium is in ion-exchangeable form (16 % total). in root, the proportion of metal in non-ionexchangeable form is higher (15 %). in shoot non lipid fraction, cd in ionexchangeable form is practically in comparable amount as zn (cd 41 % and zn 38 %), but in root non-lipid fraction is higher (zn 8 % and cd 18 %). prevailing part of the zinc in root (up to 92 % of the total) and cadmium (up to 34 % of total) remaining in non-lipid fraction was not convertible to dithizonate. similarly, high proportion of zinc and cadmium remained in non-lipid fraction of shoots (62 % of total zinc and 41 % of total cadmium). all data are presented in table 3. table 3. ion-exchangeable and non-ion-exchangeable forms of zn and cd in lipid and non-lipid fraction of root and shoot tissues of a. donax. see table 2 for details. many papers confirmed (see e.g. hiroshi et al., 1994) that both zn and cd dithizonate in chloroform extract can be completely converted to water soluble form by re-extraction with edta solution what gives the next opportunity of speciation analysis of microelements and toxic metals entering the plant tissues via root and foliar uptake as well as for speciation analysis of metal ions sorbed on sorbents with many different metal binding active centres. 4. conclusions experiments with giant reed (a. donax) plants grown in the presence of non-phytotoxic concentration of 65zncl2 and 109cdcl2 in nutrient media showed that metals are transported from roots to shoots reaching root-to-shoot ratio 6.6:1 and 5.4: 1 for cadmium and zinc, respectively. treatment of dried plant biomass with dithizone showed that up to 24 % of cd and 22 % of zn incorporated in root and up to 43% of cd and 34% of zn incorporated in shoot can be transformed to dithizonate complexes. root (%) shoot (%) fraction zn cd zn cd total biomass 100 100 100 100 lipid fraction 0 48 0 18 dithizonate 0 33 0 16 non dithizonate 0 15 0 2 non lipid fraction dithizonate 8 18 38 41 non dithizonate 92 34 62 41 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:05 utc 46 micháleková richveisová, b. et al. sequential extraction of plants biomass, treatment of extracts and solids by complexing ligands provide metal-ligand complexes of various stabilities. radiotracer methods can be a powerful tool for the study of long distance transport and speciation analysis of bivalent metals entering the plant tissues via root system. acknowledgement: this work was supported by project of the cross-border co-operation programme and co-financed with european regional development fund (erdf) with the grant number husk/1101/1.2.1/0148. also authors want to thank to msc. m. gubišová, the center for research of plant production, piešťany, slovak republic for plant materials obtained by tissue culture cultivation. references babel, s., dacera, d.d.m.: heavy metal removal from contaminated sludge for land application. j. waste manag., 26, 2006, 988-1004. bonanno, g.: arundo donax as a potential biomonitor of trace element contamination in water and sediment. ecotoxicol. environ. saf., 80, 2012, 20-27. bottari, e., festa, m.r.: on the behavior of cysteine as ligand of cadmium (ii). talanta, 44, 1997, 1705-1718. bracklow, u., drews, a., vocks, m., kraume, m.: comparison of nutrients degradation in small-scale mbr fed with synthetic/domestic wastewater. j. hazard. mater., 144, 2007, 620-626. crea, f.: formation and stability of phytate complexes in solutions. coord. chem. rev., 252, 2008, 1108-1120. dürešová, z., horník, m., gubišová, m., gubiš, j.: phytofiltration potential of arundo donax in cd removing from contaminated wastewater. chem. pap., 2014 (accepted). dürešová, z., šuňovská, a., horník, m., pipíška, m., gubišová, m, gubiš, j., augustín, j.: phytofiltration of cd and zn by root system of energy plants. proceedings of the international conference of applied natural sciences. nový smokovec, slovakia, october 2-4, 2013, 26. folch, j., lees, m., sloane stanley, g.h.: a simple method for the isolation and purification of total lipides from animal tissues. j. biol. chem., 226, 1957, 497-509. frišták, v., valovčiaková, m., pipíška, m., augustín, j.: simultaneous and sequential extraction protocols as tools for determination of zinc bioavailability in dried anaerobic sludge. nova biotechnol. chim., 11, 2012, 167175. han, z., hu z.: tolerance of arundo donax to heavy metals. chin. j. appl. ecol., 16, 2005, 161-165. hiroshi, k., hideyuki, i., tomoyuki, o.: mutual separation of metal ions by dithizone extraction-isotachophoresis on the basis of hsab principle. analyst, 9, 1994, 723-726. hoagland, d.r.: optimum nutrient solutions for plants. science, 52, 1920, 562564. horník, m., šuňovská, a., pipíška, m., gubišová, m., gubiš, j., augustín, j.: phytofiltration of cd and zn by roots of vascular plants: effect of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:05 utc nova biotechnologica et chimica 13-1 (2014) 47 metals speciation. proceedings of international conference of applied natural sciences 2011, faculty of natural sciences ucm, trnava 2011, 210-214. christensen, j.j, izatt m.r.: handbook of metal-ligand heats and related thermodynamic quantities. marcel dekker, new york, 1983, 783 pp. lindsay, w.l., w.a. norvell, w.a.: development of a dtpa soil test for zinc, iron, manganese, and copper. soil sci. soc. amer. j., 42, 1978, 421-428. martell, a.e.: stability constants of metal-ion complexes. the chemical society, london, 1964, 754 pp. mavrogianopoulos, g., vogli v., kyrisis, s.: use of wastewater as a nutrient solution in a closed gravel hydroponic culture of giant reed (arundo donax). bioresour. technol., 82, 2002, 103-107. mehlich, a.: mehlich-3 soil test extractant: a modification of mehlich-2 extractant. commn. soil sci. plant anal., 15, 1984, 1409–1416. mehlich, a.: determination of p, ca, mg, k, na and nh4. north carolina soil test division, raleig. 1953. no 1-53. murashige, t., skoog, f.: a revised media for rapid growth and bio assay with tobacco tissue cultures. physiol. plant., 15, 1962, 473-479. pilu, r., bucci, a., badone, f.c., landoni, m.: giant reed (arundo donax l.): a weed plant or a promising energy crop? afr. j. biotechnol., 11, 2012, 91639174. solov´ev, v., sukhno, i., buzko, v., polushin, a., marcou, g., tsivadze, a., varnek, a.: stability constants of complexes of zn2+, cd2+ and hg2+ with organic ligands: qspr consensus modeling and design of new metal binders. j. incl. phenom. macrocycl. chem., 72, 2012, 309-321. takahashi, w., takamizo, t., kobayahi, m., ebina, m.: plant regeneration from calli in giant reed (arundo donax, l). grassland sci., 4, 2010, 56224–56229. tessier, a., campbell, p.g.c., bisson, m.: sequential extraction procedure for the speciation of particulate trace metals. j. anal. chem., 51, 1979, 844-851. ure, a.m., quevauviller, p., muntau, h., griepink, b.: speciation of heavy metals in soils and sediments. an account of the improvement and harmonization of extraction techniques undertaken under the auspices of the bcr of the commission of the european communities. int. j. environ. anal. chem., 51, 1993, 1-4. van hullebusch, e.d., utomo, s., zandvoort, m.h., lens, p.n.l.: comparison of three sequential extraction procedures to describe metal fractionation in anaerobic granular sludges. talanta, 65, 2005, 549-558. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:05 utc nova biotechnol chim (2023) 22(1): e1384 doi: 10.34135/nbc.1384 1 nova biotechnologica et chimica control of magnetic susceptibility of probiotic strain lactobacillus rhamnosus gg svitlana gorobets1, oksana gorobets2, liubov kuzminykh1 1department of bioenergy, bioinformatics and environmental biotechnology, faculty of biotechnology and biotech engineering, national technical university of ukraine "igor sikorsky kyiv polytechnic institute", pr. peremohy 37, kyiv 03056, ukraine 2faculty of physics and mathematics, national technical university of ukraine "igor sikorsky kyiv polytechnic institute", pr. peremohy 37, kyiv 03056, ukraine  corresponding author: l.kuzminykh-2022@kpi.ua article info article history: received: 14th october 2021 accepted: 20th october 2022 keywords: iron chelate lactobacillus rhamnosus gg magnetic susceptibility magnetically-guided vector permanent magnetic field targeted drug delivery abstract the paper investigates the increase in the natural magnetically controlled properties of probiotic microorganisms lactobacillus rhamnosus gg (lgg) and their ability to form magnetically sensitive inclusions (msi). the magnetic susceptibility of lgg was increased by modifying the nutrient medium composition and by cultivating the probiotic culture in a constant magnetic field. therefore, this study can be useful for their further use as magnetically controlled vectors since it is being extensively researched for their use in targeted drug delivery in cancer treatment, for the prevention of chemotherapy complications, etc. the results of this study indicate that growing microorganisms with natural magnetically-guided properties in a modified medium with iron chelate and in an external magnetic field leads to an increase in the magnetic susceptibility of lgg by 1.8 and 2.6 times, respectively, compared with the control. the best magnetic susceptibility was recorded for lgg suspensions, which were grown in a constant magnetic field and cultured in modified medium with iron chelate. the lgg suspension, grown both in a constant magnetic field and on a modified medium with iron chelate, had the highest magnetic susceptibility, and it was 4.8 times larger than the magnetic susceptibility in the control. © university of ss. cyril and methodius in trnava introduction probiotics are used for treating and preventing diseases of the gastrointestinal tract (parian et al. 2018), strengthening the immune system (mendi et al. 2016), overcoming the effects of antibiotic therapy (saarela et al. 2000), for the prevention and treatment of tumor diseases (bedada et al. 2020; lamichhane et al. 2020), as vectors for targeted drug delivery (duong et al. 2019). in particular, studies show a number of advantages while using probiotic microorganisms as vectors comparing to other microorganisms that can have toxic effects on the body (zhou et al. 2018; laliani et al. 2020). the strain of lactic acid bacteria lactobacillus rhamnosus gg or l. rhamnosus atcc 53103 (lgg) is the most studied strains of probiotics due to its valuable properties. the microorganism colonises the gastrointestinal tract and thus protects against pathogens and increases the body's resistance to infections (capurso 2019). it is known that lgg is used as a vector in targeted drug mailto:l.kuzminykh-2022@kpi.ua nova biotechnol chim (2023) 22(1): e1384 2 delivery in the treatment of pathologies and tumors (tangney 2010; bedada et al. 2020). the use of probiotics as vectors for targeted drug delivery has a number of advantages, in particular, the noninvasiveness of probiotic cultures and the affinity for tumor tissue of some probiotic microorganisms (bedada et al. 2020). probiotic microorganisms can specifically target a tumor, causing intra-tissue regression (tangney 2010). in the last few years, the field of biotechnology and medicine has been intensively developing to create minimally invasive systems for the targeted delivery of drugs and genes using biohybrid microrobots – conjugates of bacteria, magnetic nanoparticles, and the target cargo (liposomes, etc.). such microrobots should possess highthroughput fabrication, efficient motility, tissue penetration, multifunctional operation, and external stimuli-responsive control (including control and trigger-triggered cargo release). magnetic nanoparticles in the composition of microrobots are used to provide the possibility of remote control by means of magnetic field application and, accordingly, targeted delivery to the target organ. many bacterial biohybrid microrobots suffer from low to moderate conjugation yields, with only a small fraction of cells carrying artificial cargo. in particular, since bacteria have a short doubling time (e.g., 20 min for wild-type e. coli under optimal conditions), some of the payload will be lost during the growth phase, which results in a loss of payload efficiency and weakens magnetic controllability over long periods of time. thus, for effective magnetic control in medical applications, a highperformance conjugation process and stability of the properties of microrobots for a long time are required. in addition to throughput, the conjugation approach should not adversely affect either the bacteria or the functional properties of the artificial media. in addition, as a result of conjugation, the large size of the microrobot can seriously hinder its mobility and thus affect the performance of the bacterial microrobot. the problem of increasing the motility of the bacterial microrobot is related to the difficulty of penetration of the bacterial vector through an environment with high density and hierarchical structure in real tumors (akolpoglu et al. 2022). a significant part of the biohybrid designs of microrobots reported in the literature have insufficient characteristics of mobility, stability, and controllability. in this regard, further investigation of the mechanical motility of bacteria in confined spaces is needed, together with the possibilities of artificial taxis, such as magnetophoresis, which may create more opportunities for effective penetration and transport of anticancer agents into solid tumors. microorganisms that synthesize biogenic magnetic nanoparticles (bmns) or magnetosomes can be used as magnetically-guided vectors to transfer a large number of therapeutic substances (kuzajewska et al. 2020). these microorganisms lack the disadvantages of bacterial biohybrids as their large size, insufficient stability, and loss of magnetic controllability over time. magnetotactic bacteria are the best-known producers of biogenic magnetic nanoparticles. the genetic apparatus of bmns biosynthesis is unique in all kingdoms of living organisms, as shown by genetic analysis (gorobets et al. 2014a). however, magnetotactic bacteria (mtb) are used with caution as bacterial vectors for drug delivery because their effect on living organisms is not fully studied and because of the lack of clinical trials and regulatory documentation (mathuriya 2015). also, mtb is difficult to cultivate and maintain viability since their habitat is significantly different from the internal environment of a human or animal organism (müller et al. 2020). bmns of nonmagnetotactic microorganisms have been studied in comparison with bmns of mtb (vainshtein et al. 2002 and 2014; gorobets 2012). bmns of nonmagnetotactic microorganisms are perspective as bacterial magnetically guided vectors because they manifest magnetophoresis in gradient magnetic fields (vainshtein et al. 2002 and 2014; gorobets and koralewski 2017). a number of probiotic and non-magnetotactic microorganisms can produce bmns, as summarized in the review of the experimental data (gorobets et al. 2014b). as a result, the use of probiotics that produce bmns and magnetically sensitive inclusions (msi) is a promising solution for this class of problems (vainshtein et al. 2002 and 2014). bacteria from the genus lactobacillus synthesize msi when grown on special media with the addition of ferric ions (ariskina 2003). also, magnetically-guided vectors based on lgg can have additional nova biotechnol chim (2023) 22(1): e1384 3 advantages, as they can control their velocity to the tumor site and are well concentrated in the desired area (mokriani et al. 2021). it is known that tumors can produce an increased amount of bmns compared to healthy tissues (brem et al. 2006; chekchun et al. 2011; gorobets 2017). magnetically-guided vectors are more efficiently attached to tumor cells and accumulate in the target area due to both aerotaxis to hypoxic regions of the tumor and magnetic dipole-dipole interactions of bacterial bmn and bmn of the tumor (mikeshyna et al. 2018; gorobets et al. 2022). magneticallyguided vectors in the tumor area can serve as a magnetic material for therapeutic magnetic hyperthermia, which together with the therapeutic effect of the delivered drugs, can increase the effectiveness of treatment several times (gorobets et al. 2013; felfoul et al. 2016). based on the above issues, the purpose of this work is as follows: the approach is developed increasing the magnetic susceptibility of bacterial vectors not due to loading (conjugation) with artificial magnetic nanoparticles but due to, firstly, the selection of bioinformatics methods of bacterial strains that have genetic mechanism of biomineralization of biogenic magnetic nanoparticles (bmn) and, secondly, due to the modification of the environment for growing bacteria (adding iron salts to the medium during cultivation) and growing conditions (applying an external magnetic field during cultivation) to activate the biomineralization mechanism of bmn. thus, the study of ways to control the magnetic susceptibility of lgg is relevant for the creation of magnetically-guided vectors based on the natural magnetically-guided properties of lgg. experimental physical methods and software the physical methods of the research were optic microscopy, magnetophoresis, and methods of magnetostatics. the scipy package of python programming language was used for the numeric simulation of magnetophoresis in bacterial agglomerates. cultivation of microorganisms the probiotic lyophilized drug acidolac® (1 sachet of the drug contains 4×109 cfu lactobacillus rhamnosus gg) was used for the study. pure cultures of microorganisms were isolated and cultured on lactobacilli-mrs agar medium. the microorganisms were cultured on the standard agar medium and modified medium with the addition of iron chelate for 48 h at a temperature of 36 ± 1 oc. lgg was grown on mrs medium prepared according to a standard recipe. the modified medium was prepared to enhance the natural magnetically-guided properties with the addition of iron chelate at a concentration of 64 mg.l-1. the media were prepared according to the recipe and sterilized by pressure in a steam sterilizer at 121 oc for 15 –20 min. the control group of microorganisms (lgg contr.) was grown on a standard medium under standard conditions. lgg was grown to enhance the natural magneticallyguided properties with modification of the cultivation conditions: grown on а standard medium in a constant magnetic field (lgg + m), grown on a modified medium with the addition of iron chelate (lgg + fe), grown on a modified medium with the addition of iron chelate in a constant magnetic field (lgg + fe + m). the purity of the lgg culture was checked by microscopic examination. research of microorganisms with a two-magnet system the magnetic susceptibility of the lgg suspension was investigated using a system of two magnets according to the method already described (wosik et al. 2018). a drop of suspension with a culture of microorganisms is applied to the glass located on the contact surface of the system of two permanent magnets. the system of two permanent magnets is created so that the highest gradient of the magnetic field is created in the area of their joint. the magnetic particles in the suspension move to the contact line of the magnets, forming a strip. magnetic susceptibility was studied in two ways: concentrating the bacterial suspension on the contact line to determine the width of the formed band and determining the velocity of cell agglomerates to the contact line of the system of two permanent magnets. nova biotechnol chim (2023) 22(1): e1384 2 bandwidth measurements of an lgg cell suspension formed at the contact surface of a system of 2 permanent magnets an aqueous suspension of probiotic strain lgg was applied to a 0.13 mm thick cover slide, placed on a two-magnet system until the liquid was completely dry to measure the width of the strip formed. after drying the product, the bandwidth of the suspension was evaluated using a digital microscope and a microline. series of digital microscope images were taken, and the average width of the formed strip was determined using the irfanview software. determination of the velocity and the average magnetic susceptibility of particles of the lgg suspension a drop of lgg suspension was applied to a glass placed on the contact surface of the system of two permanent magnets. the movement of conglomerates of lgg suspension was recorded on video using a digital microscope. the video was filmed in several repetitions to ensure the reliability of the results. microorganisms were taken from different parts of the petri dish, a suspension was prepared, and individual videos were made moving to the line of contact of the system of two permanent magnets. the experiment was repeated several times with separate generations of lgg cultures to obtain reliable results. an average of 15 videos were made for each type of microorganism cultivation. particles with similar sizes were selected, the motion of which was clearly visible in the video, for the experiments. the number of particles examined was: lgg contr. 57 particles, lgg + m 78 particles, lgg + fe 83 particles, lgg + fe + m 139 particles. particles were selected that were clearly visible, 40-110 μm in size, the average particle size was approx. 70 μm. the magnetic susceptibility of the lgg suspension was determined using python and the formulas in the section results and discussion. the program irfanview has been used to determine the particle movement (µm) to the contact line of the magnets and the particle diameter (µm). the time (s) of movement of the particle was determined by dividing the video into frames by the free video to jpg converter software. the average velocity (µm.s-1) of the particles was determined by dividing the path (µm) by the time (s) of movement of the particles to the contact line of the magnets. table 1 presents the physical parameters taken to determine the magnetic susceptibility of the lgg suspensions. table 1. physical parameters of the system that were used to calculate the average magnetic susceptibility of suspensions in the study with a system of two magnets. parameter dimension value, units saturation magnetization of magnets in a system of two permanent magnets ms 1600 g a dynamic viscosity of water η 0.01 g/(cm·s) magnet length a 0.65, cm glass thickness h 0.13, mm note: a the unit is part of the gaussian system of units or cgs-emu, 1 g = 10−4 tesla (si system). results and discussion determination of bandwidth and velocity of suspension particles lgg the microscopy of the studied of probiotic strain lgg is shown in fig. 1. the video files are linked to fig. 1: 1) the video file “l.rhamnosus gg contr”: the control group of microorganisms (lgg contr.) was grown on a standard medium under the standard conditions; 2) the video file “l.rhamnosus gg + fe”: lgg was grown to enhance the natural magneticallyguided properties with modification of the cultivation conditions grown on a modified medium with the addition of iron chelate (lgg + fe); 3) the video file “l.rhamnosus gg + m”: lgg was grown to enhance the natural magneticallyguided properties with modification of the cultivation conditions: grown on а standard 4 nova biotechnol chim (2023) 22(1): e1384 3 medium in a constant magnetic field (lgg + m); 4) the video file “l.rhamnosus +fe +m”: lgg was grown to enhance the natural magneticallyguided properties with modification of the cultivation conditions: grown on a modified medium with the addition of iron chelate in a constant magnetic field (lgg + fe + m). the cover glass with the bacterial culture was manually shifted several times during the video capture with the purpose to show magnetophoretic movement of bacterial agglomerates to the contact line of two magnets at several typical microscope fields. the method of video capture was the same as for video capture of the magnetophoretic movement of bacterial agglomerates in the reference (gorobets et al. 2023). a b fig. 1. probiotic bacteria l. rhamnosus gg, cultured from acidolac® on mrs medium (a). digital microscope examination of the magnetophoretic mobility of l. rhamnosus gg: a system of two permanent magnets is presented, where a cover glass with a culture concentrating on the contact line is located on top (b). fig. 2 shows a preparation from a suspension of lgg, placed over a system with two magnets. the images show the conglomerates of bacterial cells located along the contact line of the magnetic system. a b c d fig. 2. stripes from a suspension of probiotic strain l. rhamnosus gg, which formed above the contact line of the system of two magnets: lgg contr. (a), lgg +m (b), lgg + fe (c), lgg + fe + m (d). 5 nova biotechnol chim (2023) 22(1): e1384 3 wider and less branched strips form above the contact line when the natural magnetically-guided properties of the lgg culture are enhanced by iron ions and a constant magnetic field. the strip that is formed after cultivation under standard conditions (lgg contr.) is thin, indistinct, with gaps and branches, many particles are not on the line of contact and do not move towards the line. the strip, formed by cultivation in a constant magnetic field (lgg + m) is distinct along its entire length but rather branched; a certain number of suspension particles do not move towards the contact line. the strip that is formed after cultivation on a modified medium with iron chelate (lgg + fe) is somewhat narrower compared to the strip formed by lgg + m. the strip formed for lgg + fe on the contact line of two magnets is distinct, rather uniform, the particles are concentrated on the line, therefore, they form a denser strip with few branches; there are few particles that remain outside the strip and do not move towards the contact line. the strip for lgg + fe is narrower than the strip for lgg + m due to the fact that the particles are more densely concentrated on the contact line. the strip formed during cultivation on a modified medium with the addition of iron chelate and in a constant magnetic field (lgg + fe + m) is the widest strip compared to the strips formed for other cultivation conditions. a distinct, wide strip is formed for lgg + fe + m rather homogeneous and has few branches; few particles remain outside the strip and do not move towards the contact line. fig. 3 shows the results of the average velocity of particles at the line of contact of the system of two magnets. the average velocity (µm.s-1) of the particles was obtained by dividing the path of the particle by time. fig. 3. dependence of the average velocity of the particles in the suspension of l. rhamnosus gg depending on the cultivation conditions. the statistical significance of the results is 0.95. the results in this paper show that the speed of the suspension particles in the two-magnet system increased as the growing conditions changed, and the culture medium was modified. calculation of the magnetic susceptibility of the suspension lgg the main idea of modifying the magnetic properties of bacteria with natural magnetically guided properties is to grow these bacteria under special conditions, namely with the addition of iron chelates to the environment and under the application of an external constant magnetic field during cultivation. this idea is based on experimental data on the influence of cultivation conditions on the amount of bmns in magnetotactic bacteria. the bmns of magnetotactic bacteria increase up to several times due to an increase in the concentration of iron ions in the cultivation medium and the application of an external magnetic field with an induction of about 0.1 t during cultivation. the magnetic susceptibility of the biomass of magnetotactic 6 nova biotechnol chim (2023) 22(1): e1384 2 bacteria also increases (wang et al. 2009). according to gorobets et al. (2014b) and mokriani et al. (2021), it can be assumed that the same influencing factors that regulate the amount of bmns in magnetotactic bacteria will similarly affect the number of bmns and the magnetic susceptibility of non-magnetotactic bacteria with natural magnetically-guided properties. in addition, the novelty of this work is the choice of nonmagnetotactic bacteria with natural magneticallyguided properties that are used as vectors for drug delivery (zhou et al. 2018; bedada et al. 2020). in recent years, methods for creating strong magnetic fields have been intensively developed using systems of highly anisotropic permanent magnets connected in such a way that the directions of the magnetization vectors of these magnets are different at the boundary between them. in this case, strong magnetostatic fields are created at the "sharp angles" of each of the magnets, having a logarithmic divergence at certain points in space. this divergence is not distorted due to the bending of the magnetization vector, if the material from which the magnet is made has a large magnetic anisotropy. akhiezer et al. (1968) and samofalov et al. (2004; 2013) calculated the divergence of the tangential component of the magnetic field outside a uniformly magnetized rectangular parallelepiped. the magnetostatic field increases logarithmically when approaching the edges of the parallelepiped and can exceed the value that is the limiting value for permanent magnets of traditional designs. fig. 4 presents an option of implementation of this idea. the combination of several highly anisotropic ferromagnets of various shapes with different directions of magnetization in one magnet to create strong magnetic fields near their common "sharp angle". fig. 4 presents a magnet where two ferromagnets are connected in such a way that one of them is magnetized along the axis and the other one in the opposite direction. the expression for the tangential component of the magnetostatic field for the kittel’s domain structure of two magnets has the form (eq. 1) (akhiezer et al. 1968; samofalov et al. 2004 and 2013): (1) where contains a logarithmic divergence at z = 0 and . a b fig. 4. scheme of kittel's open domain structure of two magnets (a). schematic representation of magnetic poles of the kittel's open domain structure of two magnets (b): red – south pole, blue – north pole. therefore, a magnet of this shape, made of highly anisotropic material, can be used to create strong magnetic fields in localized volumes of space. the normal component of the magnetostatic field for the kittel’s domain structure of two magnets has the form (eq. 2) (samofalov et al. 2004 and 2013): (2) analysis of the existing theories of particle deposition in a gradient magnetic field has revealed that the theory of magnetophoresis of paraand diamagnetic particles in high-gradient fields has been quite fundamentally developed. the choice of a model to describe the efficiency of the magnetic separator depends on the size of the particles that must be isolated from the working medium. there are two approaches which depend on particle size (more or less some critical size), the particle approach and the continuous medium approach. the continuous medium approach is used to model 7 nova biotechnol chim (2023) 22(1): e1384 3 the separation of particles, usually less than one micron in size. the statistical method is used when it is impossible to estimate the spontaneous brownian motion of each particle and the suspension is considered as a continuous medium. the particle approach gives good results for particles of sufficient size to be neglected in brownian motion. the model using the particle approach was first developed in (friedlander et al. 1981; plyavin’ and blum 1983; vincent-viry et al. 2000; gorobets and mikhailenko 2014). this model takes into account only the gradient magnetic force and the force of hydrodynamic resistance . the force of inertia is neglected, and the balance of forces is written in the following form (eq. 3 and 4): (3) (4) – the strength of the magnetic in the working volume of the magnetic separator, – effective magnetic susceptibility, which is equal to the difference between the susceptibilities of the liquid and the particle , the volume of the particle. the gradient of the magnetic force is directly proportional to the gradient of the magnetic energy density. thus, if the field is locally homogeneous, it means that no force acts on the particles. a nonzero gradient magnetic force exists in an inhomogeneous magnetic field. if the motion can be characterized by small reynolds numbers, the stokes force acting on a spherical particle has the form (eq. 5): (5) assuming that the shape of a particle trapped in an inhomogeneous magnetic field is a sphere of radius which is moving in a liquid with dynamic viscosity , the velocity difference between liquid and particle in a stationary medium. b is the radius of bacterial cell agglomerates in magnetophoretic experiments, in the case of b about 3 microns, the estimated value of the ratio b/a is about 0.001. however, the numeric simulation of mag/netophoresis of the agglomerates of bacterial cells was carried out not for the estimated value of b/a but for an exact one based on the measurement of the radius of every bacterial cell agglomerate using video recording of optic microscopy of magnetophoretic movement of bacterial agglomerates. the capture process is determined as a result of the competition between the magnetic force and the stokes force (friedlander et al. 1981). the magnetic field strength is described by expressions (eq. 1), eq. 2 in the case when an inhomogeneous magnetic field is created by a system of two magnets shown in fig. 4. we assume that a thin layer of liquid on the surface of the system of magnets is stationary, then the velocity of spherical particles in the liquid on the surface of the system of magnets along the axis, and the characteristic radius of the particle. eq. 3 can be converted to the next dimensionless form (eq. 6): (6) where dimensionless coordinates , , and and notation are introduced (eq. 7): (7) as a result of integrating eq. 7, we find the dimensionless time required for the particle to move in the magnetic field of the magnet system from a point with dimensionless coordinate to the line of contact of two magnets with dimensionless coordinate, as a function of dimensionless particle coordinates : (8) the suspension of particles sedimented in the gravitational field before being introduced into the magnetic field, therefore the dimensionless 8 nova biotechnol chim (2023) 22(1): e1384 2 coordinate of the particle was chosen for the calculation, where h is the thickness of the glass on the surface of the system of two magnets. this means that the center of the spherical particle is at a height above the surface of the magnet system equal to the radius of the particle with the addition of the thickness of the glass. it can be assumed with great accuracy that if . (9) calculation of the average dimensionless velocity of the particle along the axis in the region based on expression (eq. 10): (10) the dependence of average dimensionless velocity on x when z=0 is presented in fig. 5. fig. 5. dependence of the average dimensionless velocity of the particle on x at z=0.001 and at calculated by eq. 10. the average velocity of a particle measured in seconds is determined through the dimensionless average velocity by the formula (eq. 11): (11) the effective magnetic susceptibility of a particle moving in a liquid on glass on the surface of a system of two magnets can be determined from the last expression (eq. 12): (12) the dimensionless coordinate of the particle is chosen for calculation, as already mentioned: . the results of the calculation of the average effective magnetic susceptibility of particles depending on the cultivation conditions by formula (eq. 12) are presented in fig. 6. fig. 6. the average effective magnetic susceptibility of the particles of the l. rhamnosus gg suspension depending on the cultivation conditions according to formula eq. 12. the error was determined using the sdm. the statistical significance of the results is 0.95. the approach of increasing of magnetic susceptibility of bacteria for drug delivery applications proposed in this work has the following advantages compared to methods based on conjugation: • in this approach, the rapid division of bacteria does not lead to the loss of increased magnetic susceptibility by daughter cells, so a homogeneous and stable process of distribution 9 nova biotechnol chim (2023) 22(1): e1384 3 of magnetic susceptibility in the bacterial culture occurs. • the size of the bacteria in this approach does not change, so the mobility of the bacterial vector is not reduced, which will facilitate the penetration of the bacterial vector through the environment with high density and hierarchical structure in real tumors. • the proposed approach is also promising as the first stage of creating a bacterial vector with increased magnetic susceptibility, with the aim of further using the method of conjugation with magnetic nanoparticles or magnetoliposomes. in this case, the magnetodipole interaction between the bmn of the bacterial vector and artificial magnetic nanoparticles will create additional magnetic forces that bind this nanocomposite. conclusion the results show that the speed of the suspension particles in the two-magnet system increased as the growing conditions changed and the culture medium was modified. the effective magnetic susceptibility for the lgg control sample is (0.25 ± 0.03) × 10-7. the magnetic susceptibility of the lgg bacterial culture growing in a constant magnetic field was 1.8 times greater in comparison with the magnetic susceptibility for the control sample. the magnetic susceptibility of the lgg bacterial culture growing at the medium modified with iron chelate was 2.6 times greater in comparison with the magnetic susceptibility for the control sample. the magnetic susceptibility of the lgg bacterial culture growing at the medium modified with iron chelate in a constant magnetic field was 4.8 times greater in comparison with the magnetic susceptibility for the control sample. thus, modification of the growth medium with iron chelate and/or cultivation in a constant magnetic field improves the ferrimagnetic properties of the bacterial culture lgg, which can be useful in creating vectors of magnetically-guided delivery. a significant increase in the magnetic susceptibility of lgg bacterial culture was observed when both methods are used together i.e., modification of the medium with iron chelate and cultivation in a constant magnetic field. conflict of interest the authors declare that they have no conflict of interest. references akhiezer ai, barʹyakhtar vg, peletminskii sv (1968) spin waves. amsterdam, north-holland pub. co. akolpoglu mb, alapan y, dogan no, baltaci sf, yasa o, tural ga, sitti m 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133 comparison of ten biochemical laboratory tests before and after treatment by statins tatiana ďurčeková1, ján mocák1*, ján balla2, gabriela gromanová2, katarína boronová1 1department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (tatiana.durcekova@ucm.sk) 2analytical-diagnostic laboratory, kováčska 15, prešov, sk-080 01, slovak republic (jan.balla@mail.t-com.sk) abstract: results of 10 biochemical tests of 172 patient data (among them 84 men data and 88 women data, resp.) before and after administration of statins were thoroughly studied. all monitored patients are characterized by disorders of lipoprotein metabolism or other kind of dislipidaemia. the calculations were performed using four chemometrical methods facilitating quantification and visualization of the statin effect upon most important biochemical parameters, mainly lipid markers, and allowing classification of the patient blood samples taking into account whether the patient has been or has not been medicated by a statin drug. key words: cardiovascular risk, lipid markers, statins, roc curves, anova, knn classification. 1. introduction at the present state of medicine the patients with coronary heart disease (chd) may require intensive drug therapy usually realized by hypolipidemic drugs like statins. the number of individuals who are candidates for lipid-lowering therapy by statins has continued to increase. statins are chemically and pharmacologically diverse class of drugs that lower cholesterol levels in people with or at risk of cardiovascular disease by inhibiting the enzyme hmg-coa reductase, which is the rate-limiting enzyme of the cholesterol synthesis (wierbzbicki et al., 2003; laws et al., 2004; kinlay et al., 2005; deambrosis et al., 2009). statins, the most widely prescribed medications in patients with hyperlipidaemia and coronary heart disease, have a number of pleiotropic actions beyond cholesterol lowering. they improve endothelial function, they have antioxidant and anti-inflammatory effects, they regulate neovascularization and have immunomodulatory activities (kwak et al., 2003; tousoulis et al., 2007; blum et al., 2009). the first part of the results dealing with most important and frequent biochemical tests of 172 patients (among them 84 men and 88 women, respectively) before and after administration of statins and processed by basic chemometrical procedures were given in our previous paper (durcekova et al., 2009). in this work, further techniques of laboratory data processing were applied to inspect a diverse influence of the statin administration upon blood serum levels of the selected biochemical parameters. several statistical/chemometrical tools have been here newly applied: (a) 134 ďurčeková, t. et al. the box-plot representation of the statin drug effect, (b) the comparison of efficiency of the selected variables (biochemical tests plus patient’s age) by means of receiver operating characteristic curves (roc) analysis and gini indices, (c) one-way analysis of variance (anova) demonstrating whether the selected variable is significantly changed with the statin administration, (d) the k-th nearest neighbour (knn) pattern recognition technique used for classification of the patient samples before and after statin administration. this, rather complex approach allows investigate the statin effects on the patient’s status from a different point of view. 2. material and methods 2.1 description of the analyzed biochemical data serum level of the selected biochemical tests was analytically determined for 172 probands (individuals) who were characterized by failure of their lipoprotein metabolism or another kind of lipidaemia. two data matrices were investigated containing the test results for: (1) 84 men samples, and (2) 88 women samples, which represent the rows of the investigated data matrix. its columns contained 12 monitored variables, namely 5 lipid parameters total cholesterol (tchol), high density lipoprotein cholesterol (hdlc), low density lipoprotein cholesterol (ldlc), triacylglycerols (tg), aterogenity index (ai) – obtained by the following calculation: (tchol – hdlc)/hdlc, 6 standard biochemical parameters creatinine (crea), aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), creatinine kinase (ck), and gamma-glutamyl transferase (ggt), and, finally, the age of the patient as the last variable. all mentioned variables were measured before statin administration and one year after it. 2.2 multidimensional data analysis multidimensional data analysis was performed using three software packages: (1) minitab 15 was utilized to perform box-plots, (2) spss 15.0 was used to perform roc analysis and anova and (3) knn was carried out using software package sas enterprise guide 3.0. 3. results and discussion 3.1 comparison of selected biochemical parameters using box-plots box-plots (or box-and-whisker plots) are useful for the comparison of different groups of one-dimensional data. its construction allows a visual representation of the data (massart et al., 1997). the box itself covers inner 50 % of all data values, starting from the lower quartile (25 % of the ordered data) and ending by the upper quartile (75 % of the ordered data); the whiskers, represented by the abcisses, cover the lowest and highest part of the data representing the given variable. the line across the box represents the median. based on known categorization of probands, divided into two categories before and after administration of statins, a comparison of the blood serum levels of the nova biotechnologica 9-2 (2009) 135 studied variable before and after administration of statins was visualized using boxand-whisker plots and demonstrated for all studied variables. in addition, some multicomponent variables, namely pc1 (the first principal component), df1 (the first discriminate function) and logit (the dependent variable in logistic regression) are also shown and compared to individual biochemical tests. the mentioned multicomponent variables were calculated by linear combinations of all original variables by principal component analysis, discriminant analysis and logistic regression (durcekova et al., 2009). further details of these multivariate techniques can be found in khattree et al., 2000; jolliffe et al., 2002; kleinbaum et al., 2005. it is demonstrated in fig. 1 that the selected two categories of probands are relatively well separated in case of variables ldlc, tchol and ai for the men samples and ldlc and tchol for the women samples. nevertheless, it is evident that the best separation of two categories is achieved when using multicomponent variables df1 and logit for the men samples and df1, logit and pc1 for the women samples. the effective utilization of the multicomponent variables in prediction and/or confirmation of clinical diagnosis was discovered in our previous works (balla et al., 2004; mrazova et al., 2009). a minor difference of hdl cholesterol between treated and untreated patients were expected. the change in box-plots of six standard biochemical parameters with the statin treatment was not significant. 3.2 roc analysis the predictive value of an assay can be displayed in the form of a plot of sensitivity against (1 specificity), where sensitivity and specificity are defined in the way common in clinical chemistry (mocak et al., 2003). the area under the corresponding curve, a, is defined between 0.5 (the diagonal line on the roc diagram represents an assay with a zero predictive value and corresponds to 50 % of the total area) and 1.0 (absolutely successful laboratory test corresponds to the total area) (massart et al., 1997; balla et al., 2004). in the roc analysis, the original laboratory variables as well as the multicomponent variables pc1, df1 and logit were used. the variables with the largest roc curve area are shown in fig. 2, which demonstrates that the areas belonging to logit and df1 are clearly larger compared to the best original variables (tchol, ldlc, ai). among the original variables, the most significant statin effect exhibit tchol, ldlc, ai, tg and hdlc for the men samples and tchol, ldlc, ai and tg for the women samples with the roc area greater that 0.6. the observed independence of six standard biochemical parameters characterizing liver and/or renal function upon statin treatment was anticipated and confirmed. an alternative, new-fashioned comparison of the statin effect upon 11 investigated variables is surveyed in table 1 containing gini coefficients, g, defined by means of the roc curve area, a, in the more convenient interval (0, 1): g = 2 a – 1 (1) it is worth noting that negative values of gini coefficients are insignificant since they were caused by calculation errors and denote in fact zero. 136 ďurčeková, t. et al. fig. 1. comparison of the box-and-whisker plots for the best laboratory tests and multicomponent variables df1, pc1 and logit by box-and-whisker plots constructed using 10%, 25%, 50%, 75% and 90% percentiles of the ranked variable. b − blood serum samples before statin uptake, a − after the uptake. men samples are located in the left frame, women samples are in the right frame. software minitab 15.1.0.0. ab 1 .6 1 .2 0 .8 h d l ab 2 .4 1 .6 0 .8 h d l ab 6 4 2 a i ab 5 3 1 a i ab 6 4 2 l d l ab 6 4 2 l d l ab 4 0 -4 p c 1 ab 4 0 -4 p c 1 ab 1 0 0 -1 0 l o g it ab 1 0 0 -1 0 l o g it ab 2 0 -2 d f 1 ab 4 0 -4 d f 1 nova biotechnologica 9-2 (2009) 137 fig. 2. roc curves for the best biochemical parameters tchol, ldlc, ai and tg, and multicomponent variables df1, logit and pc1 selected by the largest area under the curve. 88 women samples. software spss 15.0. table 1. effect of statin administration on all investigated variables (laboratory as well as multicomponent ones) expressed by gini coefficients. men women variable a g variable a g df1 0.949 0.898 logit 0.973 0.946 logit 0.935 0.870 df1 0.968 0.936 tchol 0.931 0.862 tchol 0.940 0.880 ldlc 0.921 0.842 ldlc 0.880 0.760 pc1 0.892 0.784 ai 0.748 0.496 ai 0.798 0.596 pc1 0.710 0.420 tg 0.713 0.426 tg 0.676 0.352 hdlc 0.629 0.258 hdlc 0.562 0.124 crea 0.529 0.058 alt 0.598 0.196 alt 0.512 0.024 alp 0.533 0.066 ast 0.509 0.018 gmt 0.517 0.034 alp 0.501 0.002 ast 0.512 0.024 gmt 0.490 -0.020 crea 0.509 0.018 ck 0.446 -0.108 ck 0.443 -0.114 3.3 analysis of variance the goal of analysis of variance, anova, is to divide the total variance of the given variable into their appropriate components in order to discover whether the variable is significantly affected by the selected factor, which represents the outcome of the investigated problem (massart et al., 1997). this factor is represented by a categorical variable, named here as statmw and indicating whether the treatment by statins was carried out (after drug administration) or not (before) and if the patient is a 138 ďurčeková, t. et al. man or a woman: 1 – men before drug administration, 2 – men after the drug treatment, 3 – women before the treatment, 4 – women after the treatment. two posthoc anova tests were applied: the least significant difference (lsd) test found out the variables tchol, ldlc, tg and ai capable to separate the categories 1 from 2 and 3 from 4 (which is the main goal), eventually also 1 from 4 and 3 from 2; bonferroni test provided the same results. the selection of the most important anova outputs is summarized in table 2 where only the significant categories combinations are included. table 2. anova outputs showing only those variables and categories i and j, which are significantly influenced by the statin treatment (with the significance p-value less than 0.05). multiple comparison 95% confidence interval dependent variables (i) (j) mean difference (i-j) stand. error sig. lower bound upper bound tchol 1 2 1.567 0.172 0.000 1.228 1.905 1 4 1.746 0.170 0.000 1.411 2.081 3 2 1.489 0.170 0.000 1.154 1.824 3 4 1.668 0.168 0.000 1.337 1.999 ldlc 1 2 1.243 0.156 0.000 0.936 1.550 1 4 1.431 0.154 0.000 1.128 1.735 3 2 1.061 0.154 0.000 0.758 1.365 3 4 1.250 0.152 0.000 0.950 1.550 tg 1 2 0.555 0.169 0.001 0.226 0.888 1 3 0.478 0.167 0.005 0.150 0.807 1 4 0.879 0.167 0.000 0.551 1.208 3 1 -0.478 0.167 0.005 -0.817 -0.150 3 4 0.401 0.165 0.016 0.076 0.726 ai 1 2 0.908 0.179 0.000 0.555 1.260 1 3 0.916 0.177 0.000 0.567 1.265 1 4 1.657 0.177 0.000 1.308 2.006 3 1 -0.916 0.177 0.000 -1.265 -0.567 3 4 0.741 0.175 0.000 0.396 1.086 significance level is expressed by p-values rounded to 3 decimal figures; the mean difference is considered significant when p < 0.050. categories: 1 – men before drug administration, 2 – men after drug administration, 3 – women before drug administration, 4 – women after drug administration. software spss 15.0. 3.4 knn classification k-th nearest neighbour, knn, is a non parametric discrimination technique (khattree and naik, 2000) very useful for classification purposes, which does not need any assumption on the distribution of errors. the main variant of this technique is based on the majority vote rule, which means that k neighbour objects, nearest to the classified object, are searched and then the classification of the given object is made according to which class the neighbour objects are predominantly classified. nova biotechnologica 9-2 (2009) 139 knn results were evaluated by the successful classification in % for (a) the training set, from which the classification model is calculated, and for (b) the samples leaved out from the training set by leave-one-out method, which is most important for prediction purposes. it is shown in table 3 that the best classification performance was achieved when using seven nearest neighbours (k = 7) for the men samples and eleven for the woman samples. table 3. classification results by knn method; k = 5 – 11. k results model leave-1-out true/all 69/84 64/84 5 % true 82.1 76.2 true/all 69/84 66/84 7 % true 82.1 78.6 true/all 70/84 64/84 9 % true 83.3 76.2 true/all 69/84 63/84 m 11 % true 82.1 75 true/all 76/88 70/88 5 % true 86.4 79.5 true/all 75/88 73/88 7 % true 85.2 82.9 true/all 76/88 73/88 9 % true 86.4 82.9 true/all 76/88 75/88 w 11 % true 86.4 85.2 m – men samples, w – women samples 4. conclusions positive changes in lipid metabolism after statin treatment of the patients with cardiovascular risk can be unambiguously discovered and monitored by means of statistical and chemometrical techniques, which provide qualitative as well as quantitative judgment, which laboratory parameters are mostly affected by the administration of statin drugs. in this work, box-and-whisker plots and roc curves allowed visualization of the statin effects. knn classification method allowed a clear discrimination of the patients’ samples into two categories – before and after statin treatment. analysis of variance revealed that four variables are capable to differentiate the statin treatment with regard to the patient gender: aterogenity index, low density lipoprotein cholesterol, total cholesterol and triacylglycerols. high density lipoprotein cholesterol as well as all further investigated biochemical parameters were not efficient in differentiating neither men nor women groups before and after statin treatment. a special feature of the achieved results is a very high diagnostic effectiveness of the multicomponent variables composed by linear combination of individual laboratory assays, which predestinates their further utilization in cardiovascular risk confirmation and prediction. there may be considered two potentially serious side effects of statins of which patients need to be aware. occasionally, statin use cause an increase in liver enzymes. if the increase is severe, the patient may need to stop taking the drug, which usually 140 ďurčeková, t. et al. reverses the problem. if there is no increase or it is only mild, one can continue to take the drug. in general, our study has not proved a significant change in the level of liver enzymes with the statin uptake. such an effect should be individually assessed and perhaps further investigation should be made aimed to evaluating the mentioned side effects. acknowledgement: the support of this work by the grants vega 1/1005/09, vega 1/0066/09 and vvce-0004-07 is acknowledged. references balla b., mocak j., pivovarnikova h., balla j.: comparative study of cardiovascular markers data by various techniques of multivariate analysis. chemom. intell. lab. sys., 72, 2004, 259-267. blum a., shamburek r.: the pleiotropic effect of statins on endothelial function, vascular inflammation, immunomodulation and thrombogenesis. atherosclerosis, 203, 2009, 325-330. deambrosis p., terrazzani g., walley t., bader g., giusti p., debetto p., chinellato a.: benefit of statins in daily practise? a six-year retrospective. pharmacol. res., 2009 (in press). durcekova t., mocak j., balla j., gromanova g., boronova k.: relationship between administration of statins and blood serum levels of selected biochemical parameters. nova biotechnol., 9, 2009, 83-90. laws p.e., spark j.i., cowled p.a., fitridge r.a.: the role of statins in vascular disease. eur. j. vasc. endovasc. surg., 27, 2004, 6-16. jolliffe i.t.: principal component analysis, springer-verlag, new york, 2002, 487 pp. kinlay s.: potential vascular benefits of statins. am. j. med., 118, 2005, 625-675. kleinbaum d.g., klein m., pryor e.r.: logistic regression, springer, heidelberg, 2005, 282 pp. khattree r., naik d.n.: multivariate data reduction and discrimination. sas institute, cary, north carolina, 2000, 558 pp. kwak b., mulhaupt f., mach f.: atherosclerosis: anti-inflammatory and immunomodulatory activities of statins. autoimmun. rev., 2, 2003, 332-338. massart, d.l., vandeginste, b., buydens, l., de jong, s., lewi, p., smeyers-verbeke, j.: handbook of chemometrics and qualimetrics, part a, elsevier, amsterdam, 1997, 886 pp. mocak j., balla b., bobrowski a., blazicek p.: proper ways of comparison of two laboratory methods. chem. pap., 57, 2003, 143-146. mrazova v., mocak j., varmusova e., kavkova d., bednarova a.: use of multidimensional data analysis for prediction of lung malignity. j. pharm. biomed. anal., 50, 2009, 210-215. tousoulis d., charakida m., stefanadi e., siasos g., latsios g., stefanadis c.: statins in heart failure. beyond the lipid lowering effect. int. j. cardiol., 115, 2007, 144-150. wierzbicki a.s., poston r., ferro a.: the lipid and non-lipid effects of statins. pharmacol. therapeut., 99, 2003, 95-112. nova biotechnologica et chimica 11-2 (2012) 147 doi 10.2478/v10296-012-0017-9 © university of ss. cyril and methodius in trnava the possibilities of application of bacterial leaching in retrieval of valuable metals from mining waste peter fečko1, vojtěch zechner1, michal guziurek1, barbora lyčková2, eva pertile2 1všb-tu ostrava, institute of clean technologies for extraction and utilization of energy resources, 17.listopadu street 15, 708 33 ostrava, czech republic, (vojtech.zechner@vsb.cz) 2všb-tu ostrava, institute of environmental engineering, 17.listopadu street 15, 708 33 ostrava, czech republic abstract: the paper deals with an application of bacterial leaching on two selected samples from old ecological loads situated in the karlovy vary region. to be specific, they are heaps in prebuz and kraslice. bacterial leaching was applied making use of acidithiobacillus ferrooxidans bacteria and lasted 28 days. the results imply that the given method is suitable for the retrieval of valuable metals from waste and may help to deal with the issue of old heaps and dumps. keywords: bacterial leaching, acidithiobacillus ferrooxidans, mining waste heaps 1. introduction mining of mineral resources and utilization of lithospheric sources always represent a substantial interference with the geological conditions in any territory. mining endangers both the deposits and the natural environment. it causes decreases in the land resources, destruction of the vegetation cover, damage to the agricultural, forest and water management, destruction of settlements, worse environmental conditions and results in the anthropogenic georelief (bosecker, 1997; závada and bouchal, 2010). the major manifestations of the influence on the geomorphology by mining are anthropogenic movements caused by undermining, changes in the georelief (miming forms), influence of mining and waste on the landscape (removal of overburden, heaps, preparation tailings), pollution by preparation processes, collapse of quarry slopes and induced bursts (brombacher et al., 1997; fečko et al., 2004). the remediated anthropogenic forms of landscape georelief may have various functions: protected natural phenomenon, non-toxic waste disposal site, agriculturally cultivated fields, recreation-sports facilities (playgrounds, swimming) or may be used as car parks, etc (ledin and padersen, 1995). a collected evaluation of the environmental impacts of abandoned mine workings (predominantly for mining of building materials, ores, coal and uranium) in the cr showed that out of 2,000 mine workings in the cr 79 % of them do not influence the environment in any way, more than 20 % only marginally and with no long-term consequences. mere 0.5 % of mine workings are critical, with hazardous wastes, slag, contaminants, etc. it is surprising that only mining causes 1/3 of the unfavorable influence on the natural environment. the prevailing part is of a different bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:12 utc 148 fečko, p. et al. origin (unauthorized waste disposal sites, etc.). the consequences of mining are effaced by the auto-regulative powers of the nature with no human intervention only in a low extent. from this point of view, it is necessary to project the environmental settlement for using lithospheric sources into the gross domestic product calculation system. as mentioned above, any extraction of mineral resources produces waste along with the major component. this waste is usually called mining waste. for example, it is waste rock, spoil, preparation tailings, etc. this waste is usually disposed of on heaps and dumps. however, it is often the case that the waste may be rich some elements, e.g. copper, gold. in the past, the elements remained unused because of economic intensity of their retrieval, but at present various methods tend to be applied to retrieve the valuable elements. recently, one of the most effective methods has been bacterial leaching (torma, 1997; maršálek, 1979; borovec, 1989; crundwell et al., 2000; boom, 2001; kraus, 2006; fečko et al., 2008). the objective of the work is to identify what elements in which quantities are to be found in the samples from the above mentioned heaps related to extraction of copper and tin in the karlovy vary region and how to extract them by means of bacterial leaching. 2. materials and methods 2.1. characteristic of the locality prebuz and kraslice in the work two samples are described, namely from the locality of prebuz and the locality of kraslice. fig. 1 shows the geological characteristic of the locality of prebuz and kraslice. fig. 1. locality of prebuz and kraslice. prebuz is found in the district of sokolov, in the karlovy vary region. the mining locality is 1 km southwestwards from prebuz at the foot of the hartelsberg hill. there is the most important tin district in the region. there are remnants of extensive mining and preparation activities from the 16th – 20th century, e.g. shaft otto, the main shaft with a shaft frame construction in good repair (technical monument), 45 rows of cave-ins – surface depressions, drift entrance, heaps of as-mined ore and waste rock, ore preparation plant frame, sludge ponds, tin placers and a lade. on the heaps there are granites in the karlovy bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:12 utc nova biotechnologica et chimica 11-2 (2012) 149 vary pluton (earlier intruded complex), ore mineralization (sn-greisens and several generation veins with ore mineralization of sn, as, w, fe, mn, u, etc. rare minerals can be found there (topaz, opal, secondary minerals of as, cu, bi, u, etc.). it is a locality of supraregional importance to do research in the genesis of tin deposits of the greisens type. from the regional zoning point of view, it is the bohemian massif, crystalline complex and prevariscan paleozoic – saxothuringicum, ore mountains pluton. as for stratigraphy, they are igneous rocks of a hercynian age. figures 2 shows the character of the drawn samples from both the localities. fig. 2. character of the sample after its drawing from the locality of kraslice (a) and prebuz (b). 2.2. mineralogical characteristics of the samples the mineralogical analyses were implemented using an x-ray analyzer in the laboratories of the institute of geological engineering of vsb-tu ostrava. the results imply that the kraslice sample contains barite, cerussite, galena and quartz. the prebuz sample contains muscovite and chlorite. tables 1 and 2 state the percentages of the individual minerals in the tested samples. table 1. mineralogical composition of the kraslice sample. composition ratio (%) barite 56.4 ± 3.0 cerussite 2.4 ± 1.1 galena 2.9 ± 0.7 quartz 38.3 ± 3.0 table 2. mineralogical composition of the prebuz sample. composition ratio (%) chlorite llb-2 20.2 ± 9.6 muscovite_2m1 79.8 ± 9.6 2.3. methodology of bacterial leaching samples of 100 % grain-size below 0.071 mm were prepared for bacterial leaching. a pure bacterial culture of acidithiobacillus ferrooxidans was applied, coming from the czech collection of microorganisms in brno. a 5-litre bioreactor was used, into which 500 ml of the bacterial culture were poured, followed by the tested samples and then bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:12 utc 150 fečko, p. et al. silverman medium 9k was added. stirring in the bioreactor was ensured by means of aquarium pumps. the ph value of the solution was maintained at the range from 1.8 to 2.0 by means of 1m sulfuric acid during the overall experiment. bacterial leaching was conducted at the laboratory temperature of 20 °c. the time of the experiment was 28 days and having finished the experiment, the samples were analyzed on an x-ray fluorescence analyzer. 3. results and discussion the evaluated test results are summarized in tables 3 and 4. the stated results revealed that the highest recovery of cu and pb, over 40 %, was obtained in the sample from kraslice. from the presented results it is clear that the recovery with bacterial leaching depends on the primary concentration of an element in the waste rock. of toxic elements, as exhibited variable behavior during leaching by acidithiobacillus ferrooxidans; the ability of the element to be released is probably connected with the original concentration of the element in the waste rock (fig. 3). table 3. results of bacterial leaching of the kraslice sample. element prior to leaching (ppm) post leaching (ppm) recovery (%) ca 3686 3572 3.1 ti 116102 112663 2.7 cr 3342 2944 11.9 mn 1653 1085 34.4 fe 7148 5383 24.7 ba 414756 330033 20.4 cu 5332 3040 43.0 zn 1545 1338 13.4 as 2752 2456 10.8 pb 87988 48557 44.8 sr 3099 2834 8.6 sn 738 0.001 99.9 v 18849 17908 5.0 table 4. results of bacterial leaching of the prebuz sample. element prior to leaching (ppm) post leaching (ppm) recovery (%) ca 17801 15164 14.8 ti 9637 1774 81.6 cr 680 0.001 99.9 mn 1680 1300 22.6 fe 234998 148019 37.0 ba 7947 0.001 99.9 cu 2447 0.001 99.9 zn 3606 300 91.7 as 207 74 64.3 pb 1669 0.001 99.9 sr 89 18 79.8 sn 2988 0.001 99.9 the dependence of the concentration of elements in the waste rock on the recovery with bacterial leaching shows a power trend with a high value of the correlation bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:12 utc nova biotechnologica et chimica 11-2 (2012) 151 coefficient, i.e. > 0.98. the activity of the bacteria may be affected by a relatively high concentration of toxic as in the environment of the bioreactor (stolz et al., 2006). in the case of the přebuz sample, better results were obtained; the concentration of metals in the waste rock was very low. the recovery was high, almost 100% in the majority of the elements, except for fe and mn. fig. 3. relationship between recovery of elements (as, cu) during bacterial leaching and concentration of elements in waste rock. 4. conclusion the objective of the work was to test bacterial leaching using waste material samples from the localities of kraslice and prebuz applying the bacteria of acidithiobacillus ferrooxidans. the obtained results imply that the given technology is suitable for the waste material drawn from the locality of prebuz. in case of the slag material from the locality of kraslice, it would be advisable to prolong the time of leaching. it is apparent from the above mentioned that applying an ecological technology it is possible to retrieve valuable metals from old heaps. acknowledgments: article has been done in connection with project institute of clean technologies for mining and utilization of raw materials for energy use, reg. no. cz.1.05/2.1.00/03.0082 supported by research and development for innovations operational programme financed by structural founds of europe union and from the means of state budget of the czech republic. references boom, m.: the mechanism of direct and indirect bacterial oxidation of sulfide minerals. hydrometallurgy, 62, 2001, 67-70. borovec, z.: mikrobiologická oxidace sulfidických rud. rudy, 37, 1989, 261-268. bosecker, k.: bioleaching: metal solubilization by microorganisms. fems microbiol. rev., 20, 1997, 591-604. brombacher, c., bachofen, r., brandl, a.: biohydrometallurgical processing of solids: a patent review. appl. microbiol. biotechnol., 48, 1997, 577-587. crundwell, f. k., holmes, p. r., fowler, t. a.: how do bacteria interact with minerals. j. s. afr. inst. min. metall., 100, 2000, 399-401. fečko, p.; kušnierová, m.; čablík, v.; pečtová, i.: environmentální biotechnologie, všb – tuo, ostrava, 2004, 180 pp. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:12 utc 152 fečko, p. et al. fečko, p.; kušnierová, m.; sochorková, a.; praščáková, m.; ovčaří, p.; mucha, n.; janáková, i.: biotechnólogie v úprave uhlia, všb – tuo, ostrava, 2008, 156 pp. kraus, s.: metale pretioase-vol ii. matrix rom, bucuresti, romania, 2006. ledin, m., pedersen, k.: the environmental impact of mine wastes role of microorganisms and their significance in treatment of mine wastes survey. göteborg university, lundeberg institute, 1995, 104 pp. maršálek, j.: thiobacillus ferrooxidans a jeho kultivace v procesu biologického loužení rud. sntl nakl. technické literatury, praha, 1979, 143 pp. stolz, j. f.; basu p.; santini, j. m.; oremland, r. s.: arsenic and selenium in microbial metabolism. ann. rev. microbiol., 60, 2006, 107–130. torma, a. e. the role of thiobacillus ferrooxidans in hydrometallurgical processes. in: ghose, t.k. and fiechter, a. and blakebrough, n. (eds.) advances in biochemical engineering. springer berlin heidelberg, berlin, 1977, 1-37. závada, j., bouchal, t.: chemické metody zpracování nerostných surovin a odpadů, všb – tuo, ostrava, 2010, 99 pp. presented at the 2nd international conference “biotechnology and metals 2011”, september 22–23, 2011, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:12 utc nova biotechnologica et chimica 11-2 (2012) 125 doi 10.2478/v10296-012-0014-z © university of ss. cyril and methodius in trnava biosorption of copper, zinc and nickel from multi-ion solutions jana kaduková, hedviga horváthová technical university in košice, faculty of metallurgy, department of material science, letná 9, košice, 040 00, slovak republic (jana.kadukova@tuke.sk) abstract: the biosorption of cu(ii), zn(ii) and ni(ii) by microscopic green algae chlorella kessleri was investigated using batch experiments. biosorption studies with single and multi ion solutions were carried out to study the effect of several ions on the biosorption of selected metal. the influence of zinc and nickel on copper biosorption, copper and nickel on zinc biosorption and zinc and copper on nickel biosorption were investigated. the langmuir and freundlich model were used to describe the adsorption equilibrium of studied metals on chlorella kessleri biomass. based on the experimental results it was found that the presence of copper increased the biosorption capacity of zinc from 48.6 mg/g to 96.8 mg/g and nickel from 29.3 mg/g to 62.7 mg/g, respectively. however, the presence of nickel decreased the biosorption capacity of zinc from 48.6 mg/g to 31.7 mg/g. key words: adsorption isotherms, biosorption, chlorella kessleri, heavy metals, bioavailability 1. introduction environmental pollution is a very serious problem in the present time. the main sources of heavy metals contamination in wastewater derived from electroplating, metal finishing and metallurgical industry. metals like copper, zinc and nickel are present in almost all types of wastewaters. these metals are toxic even at low concentrations (kaduková and virčíková, 2003). several studies reported the biosorption of these metals from solution using different types of biosorbents. filamentous fungus rhizopus arrhizus have been used for effective biosorption of copper and nickel from single ion solutions (sag and arda, 2000). peanut shell biomass also showed good biosorption capacity for copper (anna and roman, 2011). phaseolus vulgaris immobilized in silica-gel had good biosorption capacity for nickel (akar and zerrin, 2009). zinc can be effectively removed by activated sludge or by penicillium simplicissimum biomass (chunping and min, 2010). bacterial strains bacillus cereus and pseudomonas sp. showed promising biosorption capacity for nickel and zinc (wei – chen and chieh – chen, 2008). the major problem in wastewater treatment is that it usually contains more than one metal. the presence of several metals in solution may have a synergistic or conversely antagonistic effect on their biosorption, which depends on many factors, such as the quantity of metal ions competing for the same binding sites, the concentration of metal ions, the nature and concentration of biosorbent (wang and yong, 2005; horváthová, kaduková, 2008; marešová et al., 2011). due to this fact it is very important to study the influence of several metals on their biosorption as well as to study the biosorption process itself. in terms of metal biosorption from multi ion solution systems several experimental studies have been reported up to now. due to the relatively small amount of information on the interaction among non-ferrous metal bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:59 utc 126 kaduková, j. et al. ions during biosorption process on green algae biomass the effect of copper, zinc and nickel on their biosorption from multi ion solutions was experimentally studied in this work. 2. materials and methods 2.1 biosorbent and model solutions preparation chlorella kessleri, an unicellular green alga, obtained from the institute of botany of slovak academy of sciences was used in this study. it was grown at 25°c, aerated and lighted by 4×40 w fluorescent tubes in liquid medium milieu bristol. the ph of the media was adjusted to 7.0 with 10% naoh. in the log phase of the growth, chlorella kessleri cells were cooled, centrifuged at 3000 rpm for 5 min, washed twice in distilled water and then dried at 100°c for 3 h. algal cells in the form of powder were used as a biosorbent. solutions of copper, zinc and nickel used for experiments were prepared by dissolving cuso4.5h2o, znso4.7h2o and niso4.7h2o in distilled water. solutions ph was adjusted using 10% h2so4 and 10% naoh. 2.2 metal biosorption experiments powdered algae were used for the biosorption experiments at the concentration 2 g/l. initial concentrations of copper, zinc and nickel in studied solutions ranged from 0 – 1000 mg/l. experiments were carried out according to the protocol for adsorption isotherms experiments (kaduková and virčíková, 2003). the ph value of used solutions was 5.0. these solutions were agitated on a shaker during 24 hours. after this time solution samples were withdrawn for metal analysis. the concentration of metal ions was measured by atomic absorption spectroscopy (varian aa20+). the metal uptake q was calculated from the mass balance equation as follows: m ccv q e )( 0 −= (1) where q the quantity of metal uptake by biomass [mg/g], c0 the initial metal concentration [mg/l], ce final (after sorption at equilibrium) metal concentration [mg/l], v the volume of solution [l] and m dry weight of the biomass added [g]. 2.3 biosorption isotherm modeling the modeling of the biosorption process by using chlorella kessleri was obtained by using different adsorption isotherms. in order to clarify the adsorption isotherms, langmuir and freundlich equations were chosen. the langmuir equation can be expressed as: e e e bc bcq q + = 1 max (2) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:59 utc nova biotechnologica et chimica 11-2 (2012) 127 where qe is the amount of metal ion sorbed on the biosorbent (mg/g) at equilibrium time, qm is monolayer capacity of the biosorbent (mg/g), b is the biosorption constant (l/mg), and ce is equilibrium concentration of metal ion in solution (mg/l). the freundlich equation can be expressed as: n efe ckq /1= (3) where kf and n are empirical freundlich constants and indicative of adsorption capacity and adsorption intensity, respectively, qe and ce are like the langmuir equation described above. the value of n is generally between 2 to 10. langmuir mathematical model for binary system solutions can be expressed as: ( )2211111max1 1/ eee cbcbcbqq ++= (4) ( )2211222max2 1/ eee cbcbcbqq ++= (5) 3. results and discussion 3.1. influence of zn2+ and ni2+ on copper biosorption the influence of zinc and nickel on copper biosorption is shown in the fig. 1. the presence of zn had a positive effect on the copper biosorption, the biosorption capacity of copper increased two times in the solution cu – zn in comparison with the biosorption capacity from the single copper solution. fig. 1. comparison of biosorption capacity of copper from one, two and three ion solutions using langmuir model. the positive effect of zn on cu biosorption was observed at higher equilibrium concentrations (above 70 mg/l). it is possible that at higher concentrations copper is bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:59 utc 128 kaduková, j. et al. forced to bind to various types of binding sites resulting in the increase of the biosorption capacity. on the contrary al-rub et al. (2004) observed 10% decrease of the copper biosorption capacity by alga chlorella vulgaris in system cu zn. synergic effect was reported by tien (2002) during cu2+ biosorption from the three-ion solution cu-cd-pb by alga eudorina elegans. the influence of nickel was negligible. in our case slight decrease of the biosorption capacity was observed for the three ion system cu-zn-ni. 3.2. influence of cu2+ and ni2+ on zinc biosorption significant, almost double, increase of the biosorption capacity was also observed in the presence of copper during zn biosorption (fig. 2). similarly as in the case of copper the increase was observed at higher equilibrium concentrations (above 290 mg/l). the influence of copper on zinc biosorption was negative at lower concentrations. the synergic effect of metal ions during biosorption is very rarely mentioned in literature so the detail explanation of that phenomenon is missing. probably after the easily available binding sites are occupied the metal ions are forced to bind on less available sites which would not be occupied in more favorable situation. the presence of nickel or nickel and copper significantly decreased the zinc biosorption capacity. in this case metal ions are probably competing for binding sites (fan et al., 2008). fig. 2. comparison of biosorption capacity of zinc from one, two and three ion solutions using langmuir model. significant inhibition of zinc biosorption by copper presence was found when cork biomass was used as biosorbent (chubar et al., 2004) as well as when the biomass of fucus spiralis (romera et al., 2008) or cyanobacteria oscillatoria angustissima (mohapatra and gupta, 2005) were used. similarly pipíška et al. (2010) found that zn and cd compete for the same binding sites on the rhytidiadelphus bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:59 utc nova biotechnologica et chimica 11-2 (2012) 129 squarrosus biomass surface resulting in equal biosorption efficiency. negative effect on zinc biosorption was found also for other ions such as cd2+ (remenárová et al., 2012). 3.3. influence of zn2+ and cu2+ on nickel biosorption the presence of copper had very positive effect also on the nickel biosorption (fig. 3). nickel biosorption capacity increased more than twice. presence of zinc slightly increases the biosorption capacity of nickel in the range of low concentration. on the other hand in the highest concentration range the effect of zinc on nickel biosorption is negligible. fig. 3. comparison of biosorption capacity of nickel from one, two and three ion solutions using langmuir model. mutual influence of metals on their biosorption probably depends on the biosorbent type, too. villaescusa et al. (2004) observed negative effect of copper (20% decrease of the capacity) on nickel biosorption when grape waste was used as the biosorbent. 3.4 equilibrium modeling of process biosorption the highest value of maximum biosorption capacity from single ion solutions was found for zinc, which is consistent with experimental results. based on the obtained experimental results the affinity constant bl was calculated using langmuir mathematical model. the experimental results showed that zinc had the highest affinity for the green alga chlorella kessleri. using the freundlich model similar results were obtained. obviously algae had the highest adsorption capacity for zinc from single ion solutions. study of the biosorption from multiple ion solutions is much more complicated than from single ion solutions because it is always necessary to include the influence bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:59 utc 130 kaduková, j. et al. of other ions present in the solution. in the case of copper – zinc solution maximum biosorption capacity was higher for copper. on the other hand, in the solution containing zinc and nickel, the maximum biosorption capacity was higher for zinc. the values of parameters calculated from the langmuir model for single and multiple ion solutions are shown in table 1 and 2. table 1. model constants for single solution of metals. isotherms model constants cu zn ni qmax [mg/g] 44.0 48.6 29.3 b [l/mg] 0.052 0.070 0.007 langmuir r2 0.976 0.971 0.921 kf [l/g] 10.1 12.9 2.21 1/n 0.230 0.213 0.363 freundlich r2 0.940 0.866 0.916 table 2. model constants for binary system solutions. langmuir qmax [mg/g] b1 [l/mg] b2 [l/mg] r 2 cu 90.3 0.0007 0.9887 cu + zn zn 96.8 0.0036 0.9898 cu 57.3 0.0007 0.9951 cu + ni ni 62.7 0.0063 0.9849 zn 31.7 -0.019 0.9959 zn + ni ni 28.5 0.0349 0.9785 maximum biosorption capacity for zinc from the system cu-zn is slightly higher than that of copper. zinc in zn + ni system has a slightly higher biosorption capacity in comparison with nickel. in the cu + ni solution system maximum biosorption capacities for studied metals were very similar. from the comparison of affinity constants for cu + zn solution it is obvious that the algae have higher affinity to zinc. surprisingly, algae have higher affinity to nickel in the other two studied systems. as in the previous cases, in the study of the mutual influence of three ions cu2+, zn2+ and ni2+ in multiple solution systems the adsorption isotherms was calculated. biosorption capacity of studied metals decreased in the order: cu > zn > ni. at the present time well known mathematical models do not reliably calculate the necessary parameters. therefore the modeling of three-and multi-ionic solutions still requires further study. 4. conclusions wastewater treatment is very important issue of today because of the increase of pollutant amount released into surface water followed by the pollution of groundwater. many studies are dedicated to the developing new wastewater treatment methods. among the suitable methods biological methods represents good alternative for wastewater treatment. copper, zinc and nickel present in the solution can influence their mutual biosorption significantly. interestingly the presence of copper had positive bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:59 utc nova biotechnologica et chimica 11-2 (2012) 131 effect on zinc and nickel biosorption capacity at higher equilibrium concentrations. also zinc increased the biosorption capacity of copper. more study would be necessary to explain the phenomenon because in general metal ions should compete for binding sites on the biosorbent surface resulting in the decrease of the biosorption capacity. the decrease was observed only in some of two-ion solutions but in three ion solution the decrease of the biosorption capacity was reported for all three ions. understanding of mutual influence of metals on their biosorption could significantly contribute to broader biosorption application in wastewater treatment. acknowledgement: authors thanks to ing. vladimír pencák for his help with the article preparation. this work was supported by vega 1/0235/12. references abu al-rub, f.a., el-naas, m.h., benyahia, f., ashour i.: biosorption of nickel on blank alginate beads, free and immobilized algal cells. process biochem., 39, 2004, 1767-1773. akar, t., zerrin, k.: enhanced biosorption of nickel (ii) ions by silica-gelimmobilized waste biomass: biosorption characteristics in batch and dynamic flow mode. j. hazard. mater., 163, 2009, 1134–1141. anna, w., roman, g.: biosorption of heavy metals from aqueous solutions onto peanut shell as a low-cost biosorbent. desalination, 265, 2011, 126–134. chubar, n., carvalho, j.r., correia m.j.n.: cork biomass as biosorbent for cu(ii), zn(ii) and ni(ii). colloid surface physicochem. eng. aspects 230, 2004, 57–65. chunping, y., min, l.: biosorption of zinc(ii) from aqueous solution by dried activated sludge. j. environ. sci., 22, 2010, 675–680. fan, h.j., yang, h.s., tsai, y.s., furuya, e.: prediction of individual freundlich isotherms from binary and ternary phenolic compounds mixtures. chemosphere 71, 2008, 886-893. horváthová, h., kaduková, j.: effect of nickel on copper and zinc biosorption. acta metallurg. slovaca, 14, 2008, 78–82. kaduková, j., virčíková, e.: mineral biotechnology iii, biosorption of metals from solution, 1st edition, všb-tu, ostrava, 2003. isbn 80-248-0244-9. marešová, j., pipíška, m., rozložník, m., horník, m., remenárová, l., augustín, j.: cobalt and strontium sorption by moss biosorbent: modeling of single and bingy metal systems. desalination, 266, 2011, 134–141. mohapatra, h., gupta, r.: concurrent sorption of zn(ii), cu(ii) and co(ii) by oscillatoria angustissima as a function of ph in binary and ternary metal solutions. bioresour. technol., 96, 2005, 1387–1398. pipíška, m., horník, m., remenárová, l. augustín, j., lesný, j: biosorption of cadmium, cobalt and zinc by moss rhytidiadelphus squarrosus in the single and binary component systems. acta chim. slov., 57, 2010, 163–172. remenárová, l., pipíška, m., horník, m., rozložník, m., augustín, j., lesný, j.: biosorption of cadmium and zinc by activated sludge from single bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:59 utc 132 kaduková, j. et al. and binary solutions: mechanism, equilibrium and experimental design study, j. taiwan inst. chem. eng., 43, 2012, 433–443. romera, e., gonzález, f., ballester, f., a., blázquez, m.l., muoz, j.a.: biosorption of heavy metals by fucus spiralis. bioresour. technol., 99, 2008, 4684-4693. sag, y., arda, y.: mono and multi-component biosorption of heavy metal ions on rhizopus arrhizus in a cfst. process biochem., 35, 2000, 787–799. tien, c.j.: biosorption of metal ions by freshwater algae with different surface characteristics. process biochem., 38, 2002, 605-613. villaescusa, i., fiol, n., martinez, m., miralles, n. et al.: removal of copper and nickel ions from aqueous solutions by grape stalks wastes. water res., 38, 2004, 992–10. wang, x., yong, q.: equilibrium sorption isotherms for cu2+ on rice bran. process biochem., 40, 2005, 677–680. wei – chen, k., chieh – chen, h.: biosorption of nickel, chromium and zinc by merpexpressing recombinant escherichia coli. j. hazard. mater., 158, 2008, 100 – 106. presented at the 2nd international conference “biotechnology and metals 2011”, september 22–23, 2011, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:59 utc microsoft word apv-pipiska fulltext.doc nova biotechnologica vii-i (2007) 23 sorption of cobalt and zinc from single and binary metal solutions by evernia prunastri martin pipíška, miroslav horník, ľuboš vrtoch, soňa šnircová, jozef augustín department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (pipiskam@ucm.sk) non-living lichen evernia prunastri was studied as biosorbent material for zinc and cobalt removal from single and binary metal solutions. sorption equilibrium of zn2+ and co2+ ions was reached within 1 hour. both cobalt and zinc biosorption was not ph dependent within the range ph 4-6 and negligible at ph 2. the experimental results were fitted to the langmuir, freundlich, redlich-peterson and langmuir-freundlich adsorption isotherms to obtain the characteristic parameters of each model. the langmuir, redlich-peterson and langmuir-freundlich isotherms were found to well represent the measured sorption data. according to the evaluation using the langmuir equation, the maximum sorption capacities of metal ions onto lichen biomass were 112 μmol/g zn and 97.2 μmol/g co from single metal solutions. e. prunastri exhibited preferential uptake of zinc from equimolar binary zn2+ co2+ mixtures within the range 50 – 4000 μm. even thought mutual interference was seen in all co-zn binary systems. to evaluate the two-metal sorption system, simple curves had to be replaced by three-dimensional sorption surface. these results can be used to elucidate the behavior of lichens as bioindicators of cobalt and zinc pollution in water and terrestrial ecosystems. key words: zinc, cobalt, evernia prunastri, sorption, equilibrium, isotherms 1. introduction sorption processes represent one of the possible mechanisms of interaction of toxic metals in contaminated aquatic systems. bioremoval of single species of metal ions is affected by several factors such as the specific surface properties of biosorbent, temperature, ph, initial metal ion concentration and biomass concentration. when several metals are present, many other parameters affect the sorption process. simple sorption isotherms are usually constructed as a result of studying equilibrium batch sorption behavior of different biosorbent materials. these curves enable quantitative evaluation of biosorption performance of these materials for only one metal (hammaini et al., 2003; aksu and dőnmez, 2006) when more than one metal at a time is present in a sorption system, evaluation, interpretation, and representation of biosorption results become much more complicated. the aim of this study was to investigate the ability of the lichen e. prunastri growing in slovak territory to sorb co2+ and zn2+ from single and binary metal solutions. co-zn system was chosen as representative of bivalent metals found in industrial effluents. a range of equilibrium adsorption isotherms were obtained to quantitatively describe cobalt and zinc uptake. 24 pipíška, m. et al. 2. materials and methods 2.1 biomass biomass of fruticose lichen e. prunastri were taken from the spruces (picea abis) grown in the forest of high tatras, slovak republic. the lichen was washed twice in deionized water and oven-dried at 45°c for 72 h. cut thallus sections of diameter 3-4 mm were used in experiments. 2.2 sorption studies sorption experiments were carried out in 10 ml solutions ranged from 50 to 4000 μm of zn2+, co2+ and zn2+-co2+, spiked with 65zn or 60co. the ph value was adjusted to 4.0. biomass (50 mg, d.w.) was added, and the content in flasks was agitated on a reciprocal shaker (120 rpm) for 4 h at 20°c. at the end of the experiments radioactivity of both lichen and liquid samples was measured. all experiments were carried out four times. if not otherwise stated, presented data are arithmetic mean values. to calculate the qmax values and the corresponding parameters of adsorption isotherms (tab. 1) non-linear regression analysis was performed by the nlreg® software (statistical analysis program, version 6.3 created by p.h. sherrod) and origin 7.0 professional (originlab corporation, northampton, usa). the 3-d sorption surfaces were obtained by plotting the experimental zn and co equilibrium concentrations ceq on the x and y axes, against the co, zn and total metal uptake qeq on the z axis. the statistica 7.0 (statsoft, inc., tulsa, usa) and nlreg® software were used for this purpose. table 1. adsorption isotherm models used in this work. isotherm equation equation no. adjustable parameters langmuir eq eq eq bc cbq q + = 1 max 1 qmax, b freundlich qeq= k ceq (1/n) 2 b, n redlich – peterson g eq eq eq cb ac q )(1+ = 3 a, b, g langmuir – freundlich ( ) n eq n eq eq bc bcq q /1 /1 max )(1+ = 4 qmax, b, n binary langmuir 2211 1max1 1 1 eqeq eq eq cbcb cqb q ++ = 5 qmax, b1,b2 2.3 speciation modeling prediction of the speciation of co and zn in the aqueous systems was performed using the visualminteq (version 2.52) program. this speciation model allows the calculation of the composition of solutions for specified conditions. nova biotechnologica vii-i (2007) 25 2.4 radiometric analysis for radiometric determination of 60co and 65zn in liquid samples and lichen biomass, gamma spectrometric scintillation detector 54bp54/2-x with well type crystal nai(tl) (scionix, netherlands) and data processing software scintivision32 (ortec, usa) were used. standardized 60cocl2 solution (5.181 mbq.ml -1, cocl2 20 mg.l-1 in 3 g.l-1 hcl) and 65zncl2 (0.8767 mbq.ml -1, zncl2 50 mg.l -1 in 3 g.l-1 hcl) obtained from alldeco inc., slovakia were used in all experiments. 3. results and discussion 3.1 metal uptake metals speciation in solution is important in sorption studies since the metal uptake depends on the solution ph. as can be calculated by visualminteq speciation program, cobalt and zinc in the single and binary solutions at ph 4 occur practically as free cations (>99,5 % co2+, >98 % zn2+) (data not shown). the time-course studies on the biosorption of cobalt and zinc ions were performed by contacting co and zn solutions with lichen biomass at ph 4.0 and 20 °c. biosorption of co2+ and zn2+ ions by e. prunastri shown in fig. 1 is a rapid process. maximum uptakes (approximately 90%) were reached within 1 hour and practically did not change during the next 24 hours. 0 5 10 15 20 25 0 20 40 60 80 100 b io so rp tio n [% ] t [h] zn2+ co2+ fig. 1. biosorption kinetics of co2+ (100 µmol/l, 60cocl2 89 kbq/l) and zn2+ (100 µmol/l, 65zncl2 70 kbq/l) by e. prunastri (5 g /l, d.w.) from single metal solutions at 20°c. initial ph 4.0; ph 4.2 after 24 hours. error bars represent standard deviation of the mean (n = 4). similar adsorption kinetics of cd2+, zn2+, pb2+ and cu2+ by lichen e. prunastri was observed by antonelli et al., (1998), where equilibrium was reached within a few minutes. the above mentioned results indicate that biosorption of the aforementioned cations was not dependent on metabolic activity. the biosorption process can be attributed to interactions of cations with anionic functional groups on the thallus surface. the thallus surface was supposed to contain ionogenic groups that generate a negative net charge (garty, 2001). 26 pipíška, m. et al. 3.2 sorption equilibrium in single metal solution analysis of equilibrium data on a specific mathematical equation is of significance for comparing different sorbents under different experimental conditions. four well known adsorption isotherm models: langmuir, freundlich, redlich-peterson and langmuir-freundlich (see equations in table 1) were applied for the analysis of the experimental data. these models use parameters that reflect the nature of the sorbent and can be used to compare biosorption performance. qmax represents the maximum sorption capacity, b is a constant related to the energy of adsorption. k and 1/n values are the freundlich constants referring to adsorption capacity and intensity of adsorption, respectively. a, b and g (0<g<1) are the redlich –peterson constants. 0 500 1000 1500 2000 2500 3000 3500 4000 0 20 40 60 80 100 120 experimental data langmuir freundlich redlich-peterson langmuir-freundlich q eq [μ m ol c o2 + /g ] c eq [μmol/l] a 0 500 1000 1500 2000 2500 3000 3500 0 20 40 60 80 100 120 q eq [μ m ol z n2 + /g ] c eq [μmol/l] experimental data langmuir freundlich redlich-peterson langmuir-freundlich b fig. 2. fit of the langmuir, freundlich, redlich-peterson and langmuir-freundlich isotherms of cobalt (a) and zinc (b) sorption by e. prunastri (5 g /l, d.w.) from single metal solutions at 20°c. initial ph 4.0; ph 4.2 after 4 hours. nova biotechnologica vii-i (2007) 27 we found, that the sorption of co2+ and zn2+ ions by e. prunastri increased with the increasing concentration of cocl2 and zncl2 in solutions (data not shown) and the equilibrium was reached within 2 hours. fig. 2 shows the experimental data fitted to the isotherm models for co (a) and zn (b) sorption by e. prunastri at ph 4 from single metal solutions. the obtained adjustable parameters are shown in table 2 with the corresponding coefficients of determination. the values of r2 are generally regarded as a measure of the goodness of fit of experimental data on the isotherm models (al-asheh et al., 2000). as can be seen from table 2, higher coefficients of determination r2 were obtained for langmuir-freundlich (r2 = 0.996 for co, 0.998 for zn), redlich-peterson (r2 = 0.995 for co, 0.995 for zn) and langmuir (r2 = 0.986 for co, 0.987 for zn) models compared to freundlich isotherm (r2 = 0.934 for co, 0.952 for zn). this shows that the langmuir-freundlich, redlich-peterson and langmuir isotherms are better fitted to the experimental data of co2+ and zn2+ sorption by e. prunastri in the concentration range studied and describe process well and quantitatively. however we draw attention to some published papers stressing that the application of adsorption models are not able to explain the biosorption mechanisms of complex biological systems (diniz and volesky, 2005; cordero et al. 2004). estimates of maximum sorption capacities qmax for co sorption obtained from langmuir and langmuir-freundlich isotherms were 97.2 and 107 μmol/g d.w., respectively (table 2). the qmax 112 and 127 μmol/g d.w. for zn sorption was obtained. this indicates higher affinity of e. prunastri for zn than co sorption from single metal solutions. the sorption capacity for cobalt and zinc by the lichen e. prunastri is of the same order of magnitude than that has been found using similar biosorbents such as aquatic mosses (martins et al., 2004), fungi (pal et al., 2006), foliose lichens (ohnuki et al., 2003) and wood fibers (saeed et al., 2005). table 2. adsorption isotherm models and corresponding parameters for co and zn sorption by e. prunastri from single metal solutions. model qmax [μmol/g] b [l/μmol] k [μmol/g] [l/μmol]1/n 1/n r2 langmuir co zn 97.2 ± 4.5 112 ± 5.2 0.01± 0.001 0.01 ± 0.001 0.986 0.987 freundlich co zn 11.0 ± 4.2 11.6 ± 3.6 0.28 ± 0.05 0.29 ± 0.04 0.934 0.952 langmuir -freundlich co zn 107 ± 6.6 127 ± 6.0 0.02 ± 0.005 0.03 ± 0.005 0.73 ± 0.08 0.60 ± 0.05 0.996 0.998 a [l/μmol] b g [l/μmol] redlich -peterson co zn 1.22 ± 0.36 2.25 ± 0.72 0.03 ± 0.01 0.06 ± 0.03 0.89 ± 0.04 0.86 ± 0.03 0.995 0.995 3.3 sorption equilibrium in binary metal solution water systems contain the whole spectrum of cations and anions competing for adsorption sites of solid phase. isotherms suitable for description of single component system are not suitable for prediction of ion equilibrium in multi-component system. 28 pipíška, m. et al. in the case of multi-component systems, evaluation and interpretation in 2d geometry is rather complicated. in such cases, 2d biosorption isotherms have to be replaced by 3d biosorption isotherm surfaces. this approach was successfully used by ma and tobin, (2003), hammaini et al., (2002, 2003) and fraile et al., (2005). in our sorption experiments co-zn binary system were tested. binary langmuir type equations (equations no. 6, 7, 8 in table 3) were used to fit the experimental data. parameters obtained by the application of these models are presented in table 3. total uptake q (co+zn) as a function of equilibrium concentration ceq of cobalt and zinc is presented in figure 3. continuous surface represents total metal uptake as predicted by the equation no. 6. experimental values of total metal uptake are shown as individual data points. at high total metal concentrations in solution sorbent easily reaches the saturation level demonstrated by the plateau of the sorption surface (figure 3). fig. 3. two-metal sorption isotherm surface corresponding to the equation no. 6 (tab. 3). the uptake as a sum of co + zn (μmol/g, d.w.) is plotted as a function of the equilibrium concentration of co and zn (μmol/l) on x and y axes. table 3. langmuir parameters derived from binary langmuir type equations for zn-co sorption from binary metal solutions by e. prunastri. model equation no. qmax [μmol/g] bco [l/μmol] bzn [l/μmol] eqznzneqcoco eqznzneqcoco cbcb cbcbq zncoq ++ + =+ 1 )( )( max 6 101 ± 3.4 0.005 ± 0.002 0.012 ± 0.004 eqznzneqcoco eqcoco cbcb cqb coq ++ = 1 )( max 7 93.1 ± 3.4 0.008 ± 0.002 0.013 ± 0.003 eqcocoeqznzn eqznzn cbcb cqb znq ++ = 1 )( max 8 115 ± 4.4 0.004 ± 0.001 0.006 ± 0.001 nova biotechnologica vii-i (2007) 29 fig. 4. two-metal sorption isotherm surface corresponding to equation 7 (tab. 3). the uptake of co (μmol/g, d.w.)is plotted as a function of the equilibrium concentration of co and zn (μmol/l). fig. 5. two-metal sorption isotherm surface corresponding to equation 8 (tab. 3). the uptake of zn (μmol/g, d.w.)is plotted as a function of the equilibrium concentration of co and zn (μmol/l). when metals are present in equimolar concentrations within the range 500 – 4 000 μmol/l, the cobalt to zinc concentration ratio sorbed by biomass is constant with the value [co]/[zn] = 0.64. this indicates higher affinity of metal binding sites of e. prunastri for zn2+ ions comparing with the affinity to co2+ ions. the presence of zn caused a significant decrease in co sorption capacity which can be seen from fig. 4. on the contrary, the presence of co caused only moderately 30 pipíška, m. et al. decrease of zn sorption capacity (fig. 5). these results also confirm the fact that e. prunastri biomass exhibited higher affinity for zn than for co. comparable higher affinity of chlorella vulgaris for zn sorption from zn – ni binary system was described by fraile et al., (2005). 4. conclusions the adsorption capacity of e. prunastri for cobalt and zinc is of the same order of magnitude than that has been found using similar biosorbents. according to the evaluation using the langmuir equation, the maximum sorption capacities of metal ions onto lichen biomass were 112 μmol/g zn and 97.2 μmol/g co from single metal solutions. sorption of cobalt and zinc from binary solutions was investigated using binary langmuir equations. e. prunastri exhibited higher affinity for zn. presented 3dsorption isotherms showed good agreement with experimental data. it can be concluded that differences in competition effect of individual metals are determine mainly by physico-chemical properties of metal ions, and not in differences in chemical composition of microbial biomass. chemical composition of biomass will determine the overall sorption capacity qmax. references aksu, z., dönmez, g.: binary biosorption of cadmium(ii) and nickel(ii) onto dried chlorella vulgaris: co-ion effect on mono-component isotherm parameters. process biochem., 41, 2006, p. 860-868. al-asheh, s., banat, f., al-omari, r., duvnjak, z. : predictions of binary sorption isotherms for the sorption of heavy metals by pine bark using single isotherm data. chemosphere, 41, 2000, p. 659-665. antonelli, m.l., ercole, p., campanella, l.: studies about the adsorption on lichen evernia prunastri by enthalpimetric measurements. talanta, 45, 1998, p. 1039-1047. cordero, b., lodeiro, p., herrero, r., sastre de vicente, m.e.: biosorption of cadmium by fucus spinalis. environ. chem., 1, 2004, p. 180-187. diniz, v., volesky, b.: effect of counterions on lanthanum biosorption by sargassum polycystum. water res., 39, 2005, p. 2229-2236. fraile, a., penche, s., gonzáles, f., blázquez, m.l., muñoz, j., ballester, a.: biosorption of copper, zinc, cadmium and nickel by chlorella vulgaris. chem. ecol., 21, 2005, p. 61-75. garty, j.: biomonitoring atmospheric heavy metals with lichens: theory and application. crit. rev. plant sci., 20, 2001, p. 309-371. hammaini, a., ballester, a., blázquez, m.l., gonzáles, f., muñoz, j.: effect of the presence of lead on the biosortpion of copper, cadmium and zinc by activated sludge. hydrometallurgy, 67, 2002, p. 109-116. hammaini, a., ballester, a., blázquez, m.l., gonzáles, f., muñoz, j.: simultaneous uptake of metals by activated sludge. minerals eng., 16, 2003, p. 723-729. nova biotechnologica vii-i (2007) 31 ma, w., tobin, j.m.: development of multimetal binding model and application to binary metal biosorption onto peat biomass. water res., 37, 2003, p. 3967-3977. martins, r.j.e., pardo, r., boaventura, r.a.r.: cadmium(ii) and zinc(ii) adsorption by the aquatic moss fontinalis antipyretica: effect of temperature, ph and water hardness. water res., 38, 2004, p. 693-699. ohnuki, t., sakamoto, f., kozai, n., sakai, t., kamiya, t., satoh t., oikawa, m.: micro-pixe study on sorption behavior of cobalt by lichen biomass. nucl. instrum. methods phys. res. b, 210, 2003, p. 407-411. pal, a., ghosh, s., paul, a.k.: biosorption of cobalt by fungi from serpentine soil of andaman. bioresour. technol. 97, 2006, p. 1253-1258. saeed, a., akhter, m.w., iqbal, m.: removal and recovery of heavy metals from aqueous solution using papaya wood as a new biosorbent. sep. purif. technol., 45, 2005, p. 25-31. microsoft word pisarcikova nb.doc nova biotechnologica vii-i (2007) 93 dch18c6 using strontium extraction selectivity investigation and sequential soil fractionation study gabriela pisarčíková1, lucia závodská2, juraj lesný1 1department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (pisarcig@ucm.sk) 2department of projecting & nuclear and radiation safety, ekosur, jaslovské bohunice, sk-919 31, slovak republic (zavodska@ekosur.sk) abstract: 85sr as radioindicator has been applied to strontium separation selectivity study using cisdicyclohexano-18-crown-6 (dch18c6) as extraction agent and picric acid as counter ion with the aim to contribute to the development of a rapid method of strontium extraction. the same radioindicator has been used for strontium fractionation study in chosen soils by application of tessierand bcr sequential extraction procedures. experimental results have shown, that the mentioned extraction system is applicable for ida determination of sr2+ in ∼1000-fold abundance of ca2+, ∼1000-fold abundance and mg2+, ∼10-fold abundance of k+ and ∼0.2-fold abundance of ba2+. for the soil samples chernozems developed on loess from the near vicinity of npp jaslovské bohunice the exchangeable strontium fraction reached as much as 50 – 60 %. key words: strontium, separation, solvent extraction, crown ethers 1. introduction strontium-90 is one of the most hazardous components of radioactive fallout. it is produced essentially by the 235u and 239pu nuclear fission reactions and has been emitted mainly to the atmosphere via nuclear weapon tests and nuclear reactor accidents. owing to its chemical and biochemical similarity to calcium, more than 99% of strontium is efficiently incorporated into bone tissue and teeth. characterized by long physicaland effective half-lives 90sr may cause damage to bone marrow by means of its βparticles (eβ = 0.546 mev) as well as β particles of its daughterradionuclide, namely 90y (eβ = 2.273 mev) (brun et al., 2003). numerous methods have been described for the determination of radiostrontium in biological and environmental samples. an essential step in all of these methods is the selective separation of strontium, both to remove radionuclides which may interfere with subsequent β-counting and to free it from the large quantities of inactive substances typically present. the most significant of these inert constituents is usually calcium, which presents a special problem because it follows strontium in most chemical reactions (bojanowski and skiba, 1990; horwitz et al., 1991). classical methods for the separation of strontium (fuming nitric acid method, cation-exchange method, extraction method using hdehp or tbp, etc.) are timeand labor-consuming, they namely include many stages of chemical sample preparation, 94 pisarčíková, g. et al. and, to carry out the measurements, the time of about 2 weeks is required to obtain the equilibrium of 90sr 90y mixtures (guogang, 1994; andryushchenko et al., 2003; jakopič and benedik, 2005). macrocyclic crown ethers are known as selective ligands for various ions (izatt et al, 1991, ghasemi et al., 2005). the use of macrocyclic polyethers for the separation of strontium has been proposed as well (blasius et al., 1985; horwitz et al., 1991). dicyclohexano-18-crown-6 forms with alkaline metaland alkaline-earth metal ions complexes of different stability. in this work the selectivity of sr2+ separation by means of sr-dch18c6 complex has been studied. the results of tessierand bcr sequential soil fractionation serve to assess the possibility of strontium separation from potentially 90sr-contamined soil samples. 2. material and methods 2.1 reagents all the chemicals used were of analytical reagent grade. the stock solution of the relevant crown ether in chloroform was prepared from dch18c6 a mixture of cissyn-cis and cis-anti-cis isomers purchased from across organics (ceel, belgium). aqueous solutions of picric acid were prepared using the relevant reagent (merck, darmstadt, germany). all aqueous solutions have been prepared using ultrapure deionized water (0.054 μs cm-1). solutions of interfering cations (ca2+, ba2+, mg2+, k+) were prepared from their commercial salts. sodium citrate and hydrochloric acid of proper concentrations were used as buffer solutions. the radioindicator etalon solution 85srcl2 of known specific activity (106.6 kbq g -1) and of known chemical concentration (20 mg dm-3 diluted in 3 g dm-3 hcl) was obtained from the czech metrology institute (prague, czech republic). the tessierand bcr sequential extractions have been performed using the normalized chemical reagents, namely mgcl2, naoac, nh4oac, hoac, nh2oh·hcl, hno3 and h2o2 (lachema, czech republic). 2.2 samples two soil samples, namely chernozems developed on loess (soil horizon 0 – 5 cm and 10 – 20 cm), and montmorillonite k-10 (aldrich) were investigated by sequential extractions. the soil samples were collected from near vicinity of npp jaslovské bohunice. they are typical ones for the relevant region. soil samples were air dried and sieved. only the size-fraction less than 2 mm has been taken into experiments. 2.3 extraction procedure solvent extraction experiments were performed in 20 cm3 glass vessels. as initial volumes of aqueous and organic phases 2.5 cm3 have been chosen. the extraction nova biotechnologica vii-i (2007) 95 mixtures were shaken using a laboratory shaker at 25°c. after the phases were allowed to separate 1 cm3 of both aqueous and organic phases was taken. using a gamma spectrometric detection assembly the counting rate in all of the resulting solutions has been measured. all analytical procedures have been carried out in three replicate experiments. 2.4 sequential extractions the fractional analyses have been performed by means of the tessierand bcr procedures (tessier et al., 1979; gleyzes et al., 2002). both of them were slightly modified at the beginning, namely by completing the given procedures by a starting extraction step with rainwater collected from the near vicinity of npp jaslovské bohunice. the value of the residual fraction was calculated from the difference of the starting count rate of the labelled solid sample and the sum of that ones of partial fractions. for bcr procedure, besides the rainfalland the residual fractions, only the exchangeable and the reducible ones have been measured because of the expected radioactivity fractionation. the chemical concentration of the used radioindicator was considered as carrier less. 2.5 radiometric analysis for radiometric determinations of 85sr in waterand organic phases the gamma spectrometric scintillation detector 54bp54/2-x with the well type crystal nai(tl) (scionix, netherlands) and the data processing software scintivision32 (ortec, usa) were used. the counting time 400 s was sufficient for obtaining data with the relative detection error < 2%. 3. results and discussion in order to establish the best extraction conditions a detailed study of the relevant separation parameters was performed. the parameters chosen were: phase contact time, extraction medium ph, concentration of crown ether, concentration of picric acid and the presence of the interfering cations. the results obtained are presented by means of the particular fractions of radioactivity in the organic phase. 3.1 the effect of the contact time to determine the degree of extraction and to establish the rate/kinetic of the investigated process we realized the shaking of aqueous and organic phases for different times, from 2 to 120 minutes. for these experiments the same volumes (2.5 cm3) of the phases were chosen. the results showed (fig. 1) that the separation was a very fast process which reached its equilibrium within a few minutes. on the basis of the corresponding measurements, a shaking time of 10 minutes was used for all of the subsequent experiments. 96 pisarčíková, g. et al. 3.2 the effect of counter-ion concentration the transfer of cationic crown ether complex species across the concerned extraction interphase requires the presence of a suitable counter anion in the separation system. the nature of the counter ion involved plays an important role strongly influencing the extraction efficiency. in respect to the function in question, large organophilic anions, like picrate, tetraphenyl borate and chloroacetate have been found to be quite effective (chuang and lo, 1995; kumar et al., 1997). in our experiments picrate was used. the complex formation equilibrium in chloroform is defined as m+aq + x aq + lorg m +lx-org where m+, xand l refer to the metal ion, counter anion and crown ether respectively (talanova et al., 1999; kikuchi and sakamoto, 2000). fig. 1 illustrates the obtained results. the extraction of strontium has been found more efficient when the concentration of picric acid increased. in our further experiments a concentration of 4.36.10-2 m picric acid was used. 0 20 40 60 80 100 120 4 6 8 10 12 14 16 18 20 22 24 ex tr ac tio n [% ] t [min] picric acid 4.36.10-3 m picric acid 4.36.10-2 m fig. 1. the effect of contact time and picric acid concentration on the strontium extraction (csr2+ = 1.25.10-4 m, cdch18c6 = 2.5.10-4 m, ph = 4.8). 3.3 the effect of ph the assessment of the optimal ph for the extraction of sr2+ using 2.5.10-4 m dch18c6 in chloroform was realized in presence of 4.36.10-3 m picrate ion in ph values ranged from 2.9 to 4.8. the effect of ph on the extraction of strontium is evident from the fig. 2. as it is evident from fig. 2, the extraction of sr2+ into organic phase is strongly ph dependent. sr2+ was most effectively extracted in a narrow ph range, namely from 3.6 to 3.8. an important limitation of each of crown ether based separation procedures is their ineffectiveness for highly acidic sample solutions. kimura et al., (1979) recommend for strontium extraction the ph range from 2.5 to 7. nova biotechnologica vii-i (2007) 97 2,6 2,8 3,0 3,2 3,4 3,6 3,8 4,0 4,2 4,4 4,6 4,8 5,0 12 14 16 18 20 22 ex tr ac tio n [% ] ph fig. 2. the effect of ph on the extraction of strontium (csr2+ = 1.25.10-4 m, cdch18c6 = 2.5.10-4 m, cc6h3n3o7 = 4.36.10-2 m). 3.4 the effect of interfering cations the extraction selectivity of strontium was investigated in presence of various metal ions. the choice of interfering cations was carried out considering the expected size correlation with dch18c6. 3,0 3,2 3,4 3,6 3,8 4,0 4,2 4,4 4,6 4,8 5,0 12 14 16 18 20 22 24 ex tr ac tio n [% ] ph 500 x ca and 500 x mg 1000 x ca and 1000 x mg without interfering cations fig. 3. the effect of presence ca2+, mg2+ on the extraction of strontium (csr2+ = 1.25.10-4 m, cdch18c6 = 2.5.10-4 m, cc6h3n3o7 = 4.36.10-2 m). our experiments show (fig. 3) that the presence of ca2+ and mg2+ has only a negligible influence on the strontium extraction even at 1000-fold abundance. however, the extraction of sr2+ decreased significantly in markedly lower abundance of k+ and ba2+ (fig. 4) namely, the presence of 50-fold concentration of ba2+ resulted in negligible strontium extraction. it should be noted, that the interference effects of 98 pisarčíková, g. et al. ba2+ ions are more apparent in comparison with the ca2+ and mg2+ ones. this feature is presumably due to the stronger impact of the favourable ionic radius of ba2+ to the cavity size of the dch18c6 than the adequate coincidence of ca2+ (mg2+). the relevant complexation size compatibility plays apparently a critical role. 2,2 2,4 2,6 2,8 3,0 3,2 3,4 3,6 3,8 4,0 4,2 4,4 4,6 4,8 5,0 2 4 6 8 10 12 14 16 18 20 22 24 ex tr ac tio n [% ] ph 1000 x ca, 1000 x mg, 10 x k, 0.2 x ba without interfering cations fig. 4. the effect of presence ba2+, ca2+, mg2+ and k+ on the extraction of strontium (csr2+ = 1.25.10-4 m, cdch18c6 = 2.5.10-4 m, cc6h3n3o7 = 4.36.10-2 m)). 3.5 sequential extractions both, the tessierand bcr procedures confirm the expected assumption that the main portion of soil strontium content (50 – 60%) is connected with the exchangeable fraction. in the same time montmorillonite k-10 showed a significantly different behaviour. this fact is evidently caused by highly homogenous surface of it on the contrary with the strongly heterogeneous soil surface. 1 2 3 4 5 0 10 20 30 40 50 60 70 80 90 100 bound to fe-m n oxides bound to carbonates exchangeable residualrainfall soil horizon 0-5 cm soil horizon 10-20 cm m ontm orillonite k-10 tessier sequential extraction d es or pt io n yi el d [% ] fig.5: 85sr fractionation in soil samples and in montmorillonite k-10 using tessier sequential extraction nova biotechnologica vii-i (2007) 99 1 2 3 4 0 10 20 30 40 50 60 70 80 90 100 reducibleexchangeable residualrainfall soil horizon 0-5 cm soil horizon 10-20 cm m ontm orillonite k-10 bcr sequential extraction d es or pt io n yi el d [% ] fig.6: 85sr fractionation in soil samples and in montmorillonite k-10 using bcr sequential extraction the newly introduced rainwater step resulted in similarly low values of strontium fractions (10 – 20%) for both of the applied procedures. the aim of this step was to contribute to the assessment of the strontium distribution to the environment immediately after a hypothetic nuclear accident. such a step serves to bring about a more realistic imitation of natural conditions for the related case. 4. conclusions the mixture of cis-syn-cis and cis-anti-cis isomers of dicyclohexano-18-crown-6 has been proved as a suitable and commercially available extraction reagent which in optimized reaction system offers an adequate tool for advantageous separation of strontium. by us experimentally specified optimal extraction conditions have been as follows: csr2+ ≥ 1 . 10 -4 m; chloroform as organic solvent; cdch18c6 : cc6h3n3o7 : csr2+ = 2 : 350 : 1; contact time 600 s; ph 3.6 – 3.8. using the above defined conditions, the selectivity of the process studied reached a suitable degree for its applicability in ida determination of sr2+, namely in its aqueous solutions in the above indicated concentrations, and in the same time in ∼1000-fold abundance of ca2+, ∼1000-fold abundance and mg2+, ∼10-fold abundance of k+ and ∼0.2-fold abundance of ba2+. both sequential extraction procedures confirmed that the main and quantitatively a not negligible deal of soil strontium is connected to the exchangeable soil fraction. the obtained results support the potential possibility of strontium separation from soil using dch18c6 for analytical purposes. references andryushchenko, a.y., blank, a.b., budakovsky, s.v., tarasenko, o.a., shevtsov, n.i.: sorption-scintillation determination of 90sr in natural water. anal. chim. acta, 480, 2003, 151-156. 100 pisarčíková, g. et al. blasius, e., klein, w., schön, u.: separation of strontium from nuclear waste solutions by solvent extraction with crown ethers. j. radioanal. nucl. chem., 89, 1985, 389-398. bojanowski, r., skiba, d.p.: determination of low-level 90sr in environmental materials: a novel approach to the classical method. j. radioanal. nucl. chem., 138, 1990, 207-218. brun, s., kergadallan, y., boursier b., fremy, j.m., janin, f.: methodology for determination of radiostrontium in milk: a review. lait, 83, 2003, 1-15. chuang, j.t., lo, j.g.: the solvent extraction of carrier-free 90y from 90sr with crown ether. j. radioanal. nucl. chem., 189, 1995, 207-317. ghasemi, j., nikrahi, a., niazi, a.: extraction-spectrophotometric determination of trace amounts of barium and strontium by 18-crown-6 and rose bengal using partial least squares. turk. j. chem., 29, 2005, 669-678. gleyzes, ch., tellier, s., astruc, m.: fractionation studies of trace elements in contaminated soils and sediments: a review of sequential extraction procedures. trends anal. chem., 21, 2002, 451-467. guogang, j.: a rapid and accurate method for the determination of strontium-90 in environmental soil. j. radioanal. nucl. chem., 185, 1994, 255-264. horwitz, e.p., dietz, m.l., fisher, d.e.: separation and preconcentration of strontium from biological, environmental, and nuclear waste samples by extraction chromatography using a crown ether. anal. chem., 63, 1991, 522525. izatt, r.m., pawlak, l., bradshaw, j.s., bruening, r.l.: thermodynamic and kinetic data for macrocycle interactions with cations and anions. chem. rev., 91, 1991, 1721-2085. jakopič, r., benedik, l.: tracer studies on sr resin and determination of 90sr in environmental samples. acta chim. slov., 52, 2005, 297–302. kikuchi, y., sakamoto, y.: complex formation of alkali metal ions with 18crown-6 and its derivates in 1,2-dichlorethane. anal. chim. acta, 403, 2000, 325332. kimura, t., iwashima, k., ishimori, t., hamada, t.: separation of strontium-89 and -90 from calcium in milk with a macrocyclic ether. anal. chem., 51, 1979, 1113-1116. kumar, a., mohapatra, p.k., pathak, p.n., manchanda, v.k.: dicyclohexano 18 crown 6 in butanol-octanol mixture: a promising extractant of sr(ii) from nitric acid medium. talanta, 45, 1997, 387-395. talanova, g.g., elkarim, n.s.a., talanov, v.s., hanes, r.e., hwang, h.-s., bartsch, r.a., rogers, r.d.: the “picrate effect” on extraction selectivities of aromatic group-containing crown ethers for alkali metal cations. j. am. chem. soc., 121, 1999, 11281-11290. tessier, a., campbell, p.g.c., bisson, m.: sequential extraction procedure for the speciation of particulate trace metals. anal. chem., 51, 1979, 844-851. nova biotechnol chim (2022) 21(2): e1247 doi: 10.36547/nbc.1247 1 nova biotechnologica et chimica new approaches in pretreatment methods of beech particles in enzymatic hydrolysis – effect of impregnation time and reactor selection andrej pažitný1,2, , albert russ1, štefan boháček1, michal halaj1, štefan šutý2, ida skotnicová2, vladimír ihnát3 1pulp and paper research institute, jsc, dúbravská cesta 14, bratislava 84104, slovakia 2institute of polymer materials, faculty of chemical and food technology, slovak university of technology, radlinského 9, bratislava 81237, slovakia 3slovak forest products research institute, dúbravská cesta 14, bratislava 84104, slovakia  corresponding author: pazitny@vupc.sk article info article history: received: 21st october 2021 accepted: 9th march 2022 keywords: beech particles dendromass enzymatic hydrolysis second-generation biofuel steam explosion steam extrusion abstract waste dendromass is one of the least used types of biomass and together with waste from the harvesting and processing of agricultural crops, belongs to the wastes that have a high potential for the second-generation ethanol production. mass concentration of biofuel added to fossil fuels is established by law and this concentration is determined within the reference values for each year. for example, for 2021 it was 8.0 % and for years from 2022 to 2030 the value will increase to 8.2 %. biofuels in the so-called reference value in the total fuel content have been mandatory since 2017, while the specific reference value is calculated from the energy content of the total amount of fuels placed on the market. this paper provides initial considerations and pilot results based on experimental data obtained in our workplace concerning the application potential of selected type of dendromass – beech particles from the point of view of their pretreatment by steam explosion and steam extrusion for potential production of liquid biofuels based on lignocellulosic biomass. we compared temperature courses of the mentioned pretreatment methods and pretreated samples were tested by enzymatic hydrolysis. enzymatic hydrolysis results showed that steam explosion and steam extrusion led to concentration enhancement of fermentable monosaccharides compared to original samples depending on used pretreatment method. introduction biomass and especially waste dendromass is one of the solid fuels within renewable energy sources. today it often replaces fossil fuels used in heating plants producing heat and hot water for households (plešingerová et al. 2020; nosek et al. 2021). in recent years, however, attention has focused not only on the production of biopellets and other fuel products based on dendromass, but also on research into the disintegration of dendromass through its pretreatment and hydrolysis, especially enzymatic hydrolysis (wang et al. 2013; pažitný et al. 2020a and 2020b). dendromass-based hydrolysates can then be used in the alcoholic fermentation process. the composition of hydrolysates depends on the structure of the used lignocellulosic material, and on the other hand, the resulting composition of the hydrolysates may affect the reformulation of the feedstock mixture, which is more suitable for the production of the second-generation biofuel. in essence, it is an optimization of the composition of mailto:pazitny@vupc.sk nova biotechnol chim (2022) 21(2): e1247 2 input lignocellulosic raw materials. in addition, information obtained during enzymatic hydrolysis process and also from the material composition optimization can be potentially used in enzymology and in the wood processing industry (pažitný 2019). the composition of different lignocellulosic materials may vary considerably between each other type (jørgensen et al. 2007; joshi et al. 2011; bertero et al. 2012; hazuchová et al. 2017; kucharska et al. 2018; pažitný et al. 2020b) and the chemical composition of individual wood species is variable also. the wood composition can also depend on whether wood is softwood or hardwood (prasad et al. 2007; iqbal et al. 2013; shahzadi et al. 2014; pažitný et al. 2020b). however, wood is essentially composed of lignin and hemicelluloses (côté 1968), which are tightly crosslinked and form a complex structure. in this way, the wood raw materials have a very strong and durable structure, while the rigid and highly resistant structure is caused in particular by the entanglement of lignin and hemicelluloses with cellulose (yang et al. 2019), as shown in fig. 1, in which a flowchart is depicted for organosolvolysis pretreatment of recalcitrant softwood including following processing steps up to the production of high value-added materials (chemicals, fuels and other value-added materials). the complex of the mentioned natural substances forms a solid and durable matrix, which prevents the release of sugar from cellulose (demartini et al. 2013; yang et al. 2020). fig. 1. flowchart for sugar and ultrafine organosolv lignin production from recalcitrant softwood (yang et al. 2020). except of organosolvolysis, a possible approach is also pretreatment by thermo-hydro-mechanical procedures (sandberg et al. 2013). so far, the most suitable solution seems to be steam explosion and steam extrusion, the so-called continuous steam explosion with similar mechanism such as in case of organosolvolysis. the mentioned pretreatment methods are very suitable and promising physicochemical methods that modify lignocellulosic materials with resistant nature (stankovská et al. 2018; pažitný et al. 2021). these methods were originally developed for other purposes e.g., for extrusion of plastics (huang and liao 2002) and food products (menis-henrique et al. 2020). in the group of thermo-hydro-mechanical pretreatment methods, explosive processes using water, but also ammonia (wyman et al. 2005) and sulfur dioxide (wang et al. 2018) are significantly promoted, especially in softwood pretreatment (galbe and zacchi 2007; hu and ragauskas 2012; jönsson and martín 2016). additionally, new more efficient processes such as alkaline extrusion are being developed, but so far it has been realized mainly on mechanically less resistant lignocellulosic materials such as wheat straw or similar lignocellulosic materials (thian hong 2013). although alkaline pretreatment methods are chemical pretreatment methods, their combination with thermo-hydromechanical pretreatment methods causes even more extensive disruption of the dendromass recalcitrant structure, with fractionation by chemical agents and shear forces. steam explosion is a type of chemo-thermo-mechanical pretreatment method, which causes decomposition of lignocellulosic materials by chemical modification by in situ formation of acids. the generated acids mainly cause depolymerisation of hemicelluloses, and mechanical disintegration occurs due to high nova biotechnol chim (2022) 21(2): e1247 3 shear forces acting on lignocellulosic material immediately after energy or pressure release. the disruption of the structure of the lignocellulosic fibers and their bulk density reduction during this process are caused by a sudden drop in pressure which occurs at the end of set residence time. compared to steam explosion and its chemical modifications, steam extrusion provides a continuous pretreatment with high shear and thermal stress, which is caused by rotational movement of a screw inserted in the extruder cylinder within a short time. in the field of dendromass pretreatment by steam extrusion method, very few published papers are probably due to the need to procure better and more mechanically resistant extruders. we assume that the main reason for not using dendromass in industrial scale production of liquid biofuels is, that the dendromass is processed for other purposes e.g., for mentioned solid fuel (biopellets) production. the most significant effect on the extrusion course has temperature or the temperature regime in which the extrusion is performed. for structurally simpler substances of organic origin, in particular based on lignocellulosic materials with relatively low molecular weight, lower extrusion temperature up to 140 ºc is recommended (li et al. 2021). in contrast, for more complex or chemically resistant substances, and in case of inorganic compounds, the extrusion temperature should be raised in the range from 150 ºc to 300 ºc (ji et al. 2021). the aim of this work was to study steam explosion and pilot steam extrusion with the inclusion of their machine preparation with respect to their temperature regimes, as well as the impregnation regimes of the selected lignocellulosic substrate with emphasis on different impregnation periods and concentration of released monosaccharides. experimental lignocellulosic materials beech particles (fagus sylvatica l.) with uneven size distribution were obtained from company ing. jozef fraňo drevovýroba (pezinok, slovakia). the beech particles were not ground on mills, but they were directly impregnated with deionized water. in case of the impregnation method of preparation of swollen particles for steam explosion, the impregnation time was up to 15 min at room temperature, and in case of impregnation method of preparation of swollen particles for steam extrusion this time was at least 12 – 24 h (overnight), also at room temperature. enzyme reagent enzyme mixture cellic ctec3, prepared as a stabilized enzyme complex for immediate use and supplied by novozymes a/s (bagsværd, denmark), was used for enzymatic hydrolysis. after pretreatment, this enzyme mixture was used to degrade the lignocellulosic feedstock to fermentable monosaccharides. enzyme activity was measured in the laboratory at 1,700 bhu (biomass hydrolysis units)/g of product. steam explosion and extrusion pretreatment of lignocellulosic materials beech particles impregnated for thermo-hydromechanical pretreatment were subjected to steam explosion at 200 ºc in a 2 l stainless steel batch reactor (amar equipments pvt. ltd., mumbai, india) and at the same temperature on a continuously working extruder (hungaromix agrárfejlesztő-fővállalkozó kft., komárom, hungary). in both cases of pretreatment, temperature regimes of the experiment were recorded. the temperature regime was recorded before the lignocellulosic substrate passed through the discharge valve in case of steam explosion. after the residence time of 10 min a sample of steam-treated dendromass was released from reactor by rapid depressurization of steam explosion reactor vessel. when a continuously working extruder for swollen particles was used, the temperature regime was measured at the nozzle of extruder. in contrast to steam explosion, the appropriate sample of dendromass began to load after reaching a temperature of 200 ºc at the nozzle, without any residence time. both laboratory experiments had a common parameter – the temperature was pre-set to 200 ºc. after steam explosion and continuous steam extrusion nova biotechnol chim (2022) 21(2): e1247 2 pretreatment were samples labelled as steam explosion pretreated (stexp) samples or continuous steam extrusion pretreated (c-stexp), respectively. the samples obtained by these pretreatment methods were subjected to enzymatic hydrolysis. non-pretreated (np) material as the original samples of dendromass was subjected to enzymatic hydrolysis, for comparison. enzymatic hydrolysis of lignocellulosic materials the beech substrates obtained by pretreatment were subjected to hydrolytic cleavage to monosaccharides. enzymatic hydrolysis of the nonpretreated and pretreated biomass particles with cellic ctec3 at a dose of 15 % w/w (g cellic ctec3/100 g cellulose) was carried out at 50 ºc, ph = 5.0 for 72 h and 12.5 % w/w of the samples. the ph was adjusted continuously during the process using 0.1 n sulphuric acid or 0.1 n sodium hydroxide. the hydrolysate samples were sampled after 24, 48 and 72 h, respectively. determination of monosaccharides in hydrolysates from pretreated dendromass concentration of monosaccharides and inhibitors was determined using the procedure of national renewable energy laboratory (sluiter et al. 2008). monosaccharides (glucose, xylose, and arabinose) and generated inhibitors (formic acid and acetic acid) were determined in hydrolysates by hplc method with rezex roa (organic acid) h+ column. the hplc system was supplied by chromservis sk (chromservis sk ltd., bratislava, slovak republic). refractive index detector ri 101 shodex was a part of the hplc system. it was used for detecting and quantitative analysis of monosaccharides – glucose, xylose, and arabinose (glu, xyl and ara, respectively) and inhibitors. hydrolysates were injected into the hplc system through smartline universal mounting bracket for manual injection valves. each sample was analysed three times. the mobile phase passing through column was 0.005 n sulphuric acid at a flow of 0.5 ml.min-1 and temperature set to 30 ºc. chromatography data processing was performed by the software clarity version 5.3.0.180 (dataapex ltd., prague, czech republic). results and discussion temperature regime of batch reactor for steam explosion as it shown in fig. 2, the temperature regime of the steam explosion reactor heater changed during the experiment with different intensity. 0 50 100 150 200 250 0 10 20 30 40 50 60 t e m p e ra tu re ( c ) time (minutes) rea ctor hea ting region residence time fig. 2. time course of recorded temperature of the steam explosion reactor heater provided with ceramic heating element. the steepest temperature increase was at the temperature of 158 ºc after 24 min. then the temperature rose more slowly and uniformly, and heating of the reactor heater filled with a sample of beech particles occurred in the reactor heating region in the temperature range from 24 ºc to 200 ºc i.e., for 49 min. the temperature was stabilized at the temperature of 200 ºc. this temperature was set to end the experiment or for expected steam explosion, respectively. however, the sample of the impregnated beech particles remained in the reactor at this temperature and appropriate pressure for 10 min before the steam explosion. the period of 10 min is called residence time. after residence time of 10 min, and 59 min after beginning of heating, the valve on the reactor was electronically opened and a sample of beech particles impregnated with water was subjected to a steam explosion at 200 ºc. 4 nova biotechnol chim (2022) 21(2): e1247 3 temperature regime of continuously working extruder for steam extrusion fig. 3 shows that the temperature regime of the heating element of continuously working steam extrusion reactor differs significantly from the temperature regime of the batch reactor for steam explosion. the reactor heating region had termination as early as 45 min, which was 4 min earlier compared to the reactor heating region for the batch reactor for steam explosion (fig. 3). 0 50 100 150 200 250 0 10 20 30 40 50 60 t e m p e ra tu re ( c ) time (minutes) rea ctor hea ting region residence time fig. 3. time course of recorded temperature of the continuously working steam extrusion reactor heater provided with metal heating element. the shortening of the heating period can be caused by a different electric power input, power, and type of heating element, but mainly the absence of the sample of beech particles impregnated with water during the heating phase of steam extrusion reactor i.e., in the reactor heating region of continuously working steam extrusion reactor (fig. 3). the steepest temperature increase was at the temperature of 153 ºc after 23 min. as in case of the batch reactor for the steam explosion, in this case the temperature rose more slowly and more uniformly. the heating of heating element of the steam extrusion reactor occurred in the reactor heating region in the temperature range from 24 ºc to 200 ºc i.e., for 45 min. the temperature stabilization occurred at a temperature of 200 ºc, which was set temperature for starting of addition of water-impregnated beech particles i.e., for continuous steam explosion. at the indicated temperature the sample was passed through the inner body of the reactor for 10 min. this period is analogically called the residence time, as in case of a discontinuous steam explosion. after that period of 10 min, and 55 min after beginning of heating, the sample consisting of heated and milled waterimpregnated beech particles was discharged through a nozzle with an internal slot size of 6 mm. the experiment was terminated in the beginning of the formation of blocking pieces, as the sample of beech particles was not passed through the nozzle, and it was necessary to terminate the experiment due to possible damage of the steam extrusion reactor and due to further sintering of the pretreated sample of the lignocellulosic substrate. hplc chromatograms of stexp and c-stexp samples of dendromass the aim of this work was also to study the chemical compositions of hydrolysates obtained after enzymatic hydrolysis of np, stexp and cstexp samples of water-impregnated beech particles. the resulting monosaccharides and their concentration in hydrolysates are affected strongly by the original chemical composition of dendromass particles and the pretreatment temperature (russ et al. 2016). however, removing hemicelluloses at high temperatures can effectively enhance cellulose digestibility (zhao et al. 2012). cellulose accessibility improvement for enzymes leads to high concentration of monomers due to dramatic changes in dendromass morphology after pretreatment aimed at enhancing the reactivity of dendromass. the monosaccharides obtained during enzymatic hydrolysis – glucose (glu), xylose (xyl) and arabinose (ara) were determined using hplc (high performance liquid chromatography) combined with external standard methods. these methods are used to perform an accurate qualitative and quantitative analysis of measured monosaccharides and inhibition products such as formic acid and acetic acid in hydrolysates from pretreated dendromass (llano et al. 2017). according to scientific papers (demčák et al. 2017; 2019; ibrahim et al. 2020) it can be assumed that chemical structure of dendromass such as beech wood particles is mainly composed from cellulose, hemicelluloses and also lignin. the mentioned monosaccharides were formed just from holocellulose – cellulose and hemicelluloses during enzymatic hydrolysis of pretreated waterimpregnated beech particles. the hydrolysates 5 nova biotechnol chim (2022) 21(2): e1247 2 obtained from dendromass hydrolysis had different concentration of monosaccharides based on the specific chemical composition and porous structure of np, stexp and c-stexp samples of pretreated beech particles. we also suppose some significant effect of the temperature regimes within two different reactors. hplc analysis showed that the monosaccharides in obtained hydrolysates had a different elution time (table 1). the elution time did not depend on concentration of hydrolyzed monosaccharides however, it depended on set-up and conditions of hplc method (pažitný et al. 2020b). table 1. elution time of analysed monosaccharides in hydrolysates. analysed component elution time [min] glucose 11.80 ± 0.01 xylose 12.60 ± 0.02 arabinose 13.85 ± 0.02 fig. 4. hplc chromatograms from hydrolyzates after 72 h of enzymatic hydrolysis of stexp and c-stexp samples pretreated at the temperature of 200 ºc (a – beech sample pretreated by steam explosion; b – beech sample pretreated by steam extrusion). fig. 4 demonstrates two hplc chromatograms from hydrolysates of stexp and c-stexp samples pretreated at the temperatures of 200 ºc. the hydrolysates based on pretreated dendromass were sampled after 72 h. all the chromatograms show the strong signal of glu monomer (elution time of 11.80 min) and the weaker signals of c5 monosaccharides such as xyl (elution time of 12.60 min) and ara (elution time of 13.85 min). significant reduction of monosaccharides can be seen in case of beech wood hydrolysates obtained from c-stexp compared to stexp at the same pretreatment temperature. since both reactors have a different temperature regime, this is also reflected in the influence of the temperature regime type on the formation of monosaccharides. additionally, various impregnation regimes also play a role. production and determination of monosaccharides the average values for concentrations of monosaccharides measured by the hplc method depend on enzymatic hydrolysis (eh) time and also on used pretreatment method. the qualitative hplc method revealed the monosaccharides of both cellulose and hemicelluloses. the hydrolysates obtained from pretreated beech particles contained monomers glu, xyl and ara depending on the used pretreatment method. the highest total monosaccharide (glu+xyl) concentration of 73.2 g.l-1 was obtained after enzymatic hydrolysis of beech particles pretreated by steam explosion at the temperature of 200 ºc (hydrolysis time of 72 h, stex temperature of 200 ºc). significantly lower concentration of monosaccharides was found in the enzymatic hydrolysates of beech particles pretreated by steam extrusion (c-stex) at the temperature of 200 ºc after enzymatic hydrolysis for 72 h (55.2 g.l-1). in case of np sample the total monosaccharide (glu+xyl) concentration was 6.0 g.l-1 on average. production and determination of inhibition products the main inhibitor for each hydrolysed sample was acetic acid which acted as a slight hydrolysis inhibitor (yang et al. 2011). concentration of total a b a 6 nova biotechnol chim (2022) 21(2): e1247 3 inhibition products ranged from 4.4 g.l-1 to 4.9 g.l-1 among all the hydrolysates obtained from pretreated beech particles. formic acid concentration was negligible and almost immeasurable. high concentration of acetic acid causes inhibition but a specific concentration may be useful in a specific pretreatment to coproduce xylooligosaccharides and fermentable sugars (lai et al. 2019). among both the inhibition products, formic acid is the stronger cellulase inhibitor due to complete inactivation of enzymes (panagiotou and olsson 2007; yang et al. 2011). however, formic acid had low concentration in all samples taken from hydrolysates of pretreated beech particles during enzymatic hydrolysis. 2g bioethanol production from dendromass the results showed that beech particles were a dendromass resource suitable for 2g bioethanol production. mentioned wooden material is good storable due to lower biodegradability when compared to agricultural wastes. studied dendromass resource utilization and its thermohydro-mechanical pretreatment in 2g bioethanol production require further research. temperature regimes in a batch and continuous reactor should be paid to the greatest attention. an important reason for the beech utilization in 2g bioethanol production is a large abundance within slovakia, but also the entire europe (guckland et al. 2009). conclusions dendromass-based lignocellulosic materials are highly abundant and their renewable potential exceeds transformation efficiency or utilizability of biomass from agricultural crops, given by their relatively low voluminosity. processing of waste dendromass at the mining site or waste obtained in the processing of wood raw materials in production plants provides raw material which is suitable for the production of fermentable monosaccharides. however, mechanical processing and impregnation of dendromass is followed by thermo-hydromechanical pretreatment. pretreatment of beech particles showed a significant impact of selected reactor type. the very important parameter is also impregnation period of selected dendromass substrate. beech particles that were pretreated at the temperature of 200 ºc provided the higher concentration of monosaccharides compared to non-pretreated sample. concentration of monosaccharides in hydrolysates was approximately twelve-fold for hydrolysates of water-impregnated beech particles within shorter period soaking (stex samples) and approximately nine-fold for hydrolysates of water-impregnated beech particles within long period soaking (cstex) compared to average hydrolysates of np sample of beech particles. concentration of inhibition products was very low in all cases. based on the obtained results, it may be considerably useful to process selected dendromass by thermohydro-mechanical pretreatment in 2g bioethanol production. acknowledgement this work was supported by the slovak research and development agency under the contract no. apvv-18-0240. conflict of interest the authors declare that they have no conflict of interest. references bertero m, puente gdl, sedran u (2012) fuels from biooils: bio-oil production from different residual sources, characterization and thermal 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enzymatic hydrolysis of lignocellulose. biofuels, bioprod. bioref. 6: 465-482. 9 microsoft word fargasova nb.doc nova biotechnologica vii-i (2007) 51 phytotoxity of waste waters with cr and ni agáta fargašová1, 2, katarína szárazová1 1department of ecosozology and physiotactics, faculty of natural sciences, comenius university in bratislava, mlynská dolina, bratislava, sk-842-15, slovak republic (fargasova@fns.uniba.sk) 2department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic abstract: the dry and fresh biomass and metal concentration (cr, ni) in roots and shoots of mustard (s. alba l.) seedlings was evaluated in laboratory experiments with three types of washing waste-waters from cutlery production line. all tested washing waters reduced root dry mass, where-as the dry mass of shoots was either not affected or it increased. the effect of tested washing waters was stronger on fresh mass production than on dry mass production. this indicates problems in water reception and translocation. while the accumulation of cr was higher in the roots, ni was distributed equally through the whole plant seedling. cr uptake in the roots and shoots was in average about 1.7 and 7.3 times, respectively, lower than that of ni. ni percentage uptake from washing waters in the roots and shoots was nearly equal and range from 10.2 to 15.8%. key words: cutlery washing waste-waters; cr; ni; phytotoxicity; mustard 1. introduction phytotoxicity assessment plays an important role in environmental monitoring and risk assessment of metal-contaminated places. since, only a few guidelines are available for the assessment of heavy metal phytotoxicity (römbke and moltman, 1996) quality-controlled toxicity data using standardized methods are actually quite rarely reported in the literature. efroymson et al. (1997) developed toxicological benchmarks for screening the effects of contaminants which have the potential to arouse concern. this included the effect of certain heavy metals on terrestrial plants, and they also reviewed phytotoxicity data derived from experiments conducted in nutrient culture and spiked soils. phytotoxicity tests generally use toxicological endpoints such as root and shoot growth, biomass production and germination percent. however, physiological responses of plants to toxic metals consist not only of growth and production inhibition, but also in changes intensity of various physiological parameters which are not standardized yet (hartleywhitaker et al., 2001). also, relationships between metal toxicity and metal tissue concentration have so far been poorly characterized. 2. material and methods mustard (sinapis alba l.) seeds were germinated in petri dishes with 17-cm diameter and filter paper and plastic net on the bottom (svetková and fargašová, 2007). tested washing waste-waters were used in five various 52 fargašová, a. and szárazová, k. concentrations and tap water (80 mg.l-1 ca, 27 mg.l-1 mg; ph = 7.3 ± 0.05) was used for their dilution. after 10 days growth the plants were divided into roots and shoots and fresh mass was immediately weighed. the plant material was then dried (t = 80 oc) to a constant weight. for metal accumulation plant samples (including control plants) were taken after 10 days of exposure. the seedlings were removed from tested waste-waters. different plant parts (roots, shoots) were separated manually and dried at 80 oc for 24 h. dried shoot and root tissues were mineralized by mixture of concentrated hno3/h2o2 (4/1, v/v) and heating in sealed teflon containers at 160 oc for 2 h in oven. the final solutions were analyzed for ni and cr concentration by et-aas (cr) and f-aas (ni) (aas; varian, spectr. aa, australia, gta 110, with zeeman 220 background correction). the tested samples were three different waste-waters from washing reservoirs of cutlery production line mainly polluted by heavy metals (cr, ni). the total metal and non-extractable organic compounds (nec) contents are shown in table 1. first two reservoirs (r1, r2) collected waste-waters from degreasing baths, where the cutleries are degreased from residual oils and furniture creams, while the third reservoir (r3) collected waters from the final cutlery washing pool. table 1. composition of tested washing waste-waters from cutlery production line sample cr (mg.l-1) ni (mg.l-1) neca (mg.l-1) r1 41.6 50.2 1.78 r2 18.8 6.52 2.24 r3 0.3 0.255 6.49 a nec non-polar extractable compound all phytotoxicity tests were carried out in triplicate and included a control. quality control data were considered acceptable to control charts and other established criteria. adstat 2.0 software was used for statistical evaluation. 3. results and discussion the overall adverse effect of cr and ni on the growth and development of plants may be serious impairment of the uptake of mineral nutrients and water, which leads to deficiency in the shoots. wilting of various crops and plant species due to cr toxicity has been reported (turner and rust, 1971), but little information is available on the exact effect of cr and ni on water relationships in higher plants. when the relationship between dry and fresh mass was determined here-in, the dry mass fraction was increased parallel with increasing of washing waters concentration. this indicates a reduction in water uptake and possibly also translocation through the plant. water content was reduced very rapidly in comparison to that in control seedlings, and this occurred mainly in the roots where water content varied with tested water concentrations (fig. 1.). water content in the shoots was not significantly reduced in the presence of tested waste-waters. it can be concluded that tested washing waste-waters with cr and ni inhibited water absorption by the root, but not water nova biotechnologica vii-i (2007) 53 translocation into the upper seedlings parts. these results agree with the chatterjee and chatterjee (2000) conclusion, that excess cr decreases the water potential and transpiration rates and increases diffusive resistance and relative water content in leaves of cauliflowers. fig. 1. relation between dry (dm) and fresh mass (fm) [%] and their polynomic trend lines after 10 days growth of s. alba seedlings in the presence of tested washing waste-waters from cutlery production line (s – shoot; r – root; c – control); mean of three determinations, standard deviation 6% or less. the results from the uptake of cr and ni from washing waste-waters from cutlery production line in the roots and shoots of s. alba seedlings are introduced in tab. 2. while the accumulation of cr was higher in the roots, ni was distributed equally through the whole plant seedlings. however, prasad (1998) observed that cr is accumulated rather in the shoots (stems and leaves) than in the roots, our results are in agreement with those introduced by carry et al. (1977). ni accumulates uniformly in roots and shoots (prasad, 1998) and this is in good agreement with our results. while cr is not considered as essential element for plant nutrition (sharma et al., 1995), ni is classified as trace and essential trace element (brown et al., 1987). cr was from tested washing waters accumulated in both plant parts in lower amount than ni, and its percentage uptake range from 6.8 to 8.7 and 1 to 2.5 % for roots and shoots, respectively. its percentage uptake in the roots and shoots was in average about 1.7 and 7.3 times, respectively, lower than that of ni. ni percentage uptake from washing waters in the roots to shoots was nearly equal and range from 10.2 to 15.8%. singh et al. (2004) described differences in the metal accumulation in the different parts of plants and suggested on different cellular mechanism of bioaccumulation and translocation of metals. the high accumulation of metals (cr, fe, zn and mn) particularly in the root tissues of h. annuus may be due to complexation 54 fargašová, a. and szárazová, k. of metals with the sulfhydryl groups resulting into less translocation of metals to upper part of the plant, which vary from one metal to another. similar conclusions can be done from our study of cr accumulation from washing waste-waters to roots and shoots of s. alba seedlings. cr translocation from roots to shoots of s. alba was in our study minimum as well as that introduced for cauliflower by chatterjee and chatterjee (2000). the reason of the high accumulation in roots of the plants could be because cr is immobilized in the vacuoles of the root cells, thus rendering it less toxic, which may be a natural toxicity response of the plant (shanker et al., 2004). table 2. uptake of nickel and chromium from washing waste-waters after 10 days growth into the roots and shoots of sinapis alba seedlings roots initial conc. in the waste-water (mg. l-1) cr ni reservoir cr ni conc. tissue (mg.g-1 dma) % uptake conc. tissue (mg.g-1 dma) % uptake r1 0.376 0.377 0.0258 6.8 0.0595 15.8 r2 0.312 0.130 0.0213 6.9 0.0148 11.4 r3 0.15 0.013 0.0130 8.7 0.0013 10.2 shoots initial conc. in the waste-water (mg.l-1) cr ni reservoir cr ni conc. tissue (mg.g-1 dma) % uptake conc. tissue (mg.g-1 dma) % uptake r1 0.520 0.628 0.0051 0.98 0.0873 13.9 r2 0.282 0.098 0.0041 1.45 0.0103 10.5 r3 0.15 0.013 0.0038 2.5 0.0014 10.4 a dm – dry mass; all the values are means of triplicates after reduction of control; standard deviation 6% or less resistance to ni and its translocation through plant depends on plant species. while some plants are introduced as ni hyperaccumulators other are very sensitive and introduced as non-accumulators (freeman et al., 2004). in the cytoplasm, high levels of free nickel are generally avoided by removal of the metal ions to the vacuoles and by the formation of complexes with organic acids. if the nickel level remains high, it inevitably binds organic macromolecules and denatures them. ni is transported to underground plant parts by the oxygen atoms either as metal complexes of organic acids or as hydrated cations (salt et al., 2002). while barman et al. (2000) introduced its higher accumulation in the roots of cyperus difformis l. and chenopodium ambrosiodes l., pandey and sharma (2002) observed in brassica oleracea l. plants higher nickel accumulation in shoots. this statement confirmed results obtained during accumulation tests with washing waste-water here-in. in previous study with ni accumulation in s. alba roots and shoots fargašová (1998) and fargašová and beinrohr (1998) confirmed higher ni accumulation in the shoots than in the roots when ni concentration in the shoots was about twice as high as nova biotechnologica vii-i (2007) 55 that in roots. this was not confirmed during our study when ni accumulation in the shoots was equal to that in the roots and no significant differences were confirmed in accumulated metal amounts from medium. 4. conclusions it is concluded from present study that washing waste-waters from cutlery production line are quite toxic to plants and they reduced biomass and influence water and metal translocation through the plant. on the basis of this study high toxicity of the presented waste-waters from the metal surface finishing was confirmed and justness of their liquidation as hazardous wastes by legally assigned persons was confirmed. acknowledgements: this study was supported by grants vega 1/4361/07 and kega 3/4183/06. references barman, s.c., sahu, r.k., bhargava, s. k., chaterjee, c.: distribution of heavy metals in wheat, mustard and weed grown in field irrigated with industrial effluents. bull. environ. contam. toxicol., 64, 2000, 489-596. brown, p.h., welch, r.m., cary, e.e.: nickel: a micronutrient essential for higher plants. plant physiol., 85, 1987, 801-803. chatterjee, j., chatterjee, c.: phytotoxicity of cobalt, chromium and copper in cauliflower. environ. pollut., 109, 2000, 69-74. carry, e.e., alloway, w.h., olsen, o.e.: control of chromium concentrations in food plants. i. absorpion and translocation of chromium by plants. j. agric. food chem., 25, 1977, 300-304. efroymson, r.a., will, m.e., suter, g.w., wootten, a.c.: toxicological benchmarks for screening contaminants of potential concern for effects on terrestrial plants, revision prepared for the us department of energy office of environmental management, 1997. fargašová, a.: root growth inhibition, photosynthetic pigments production, and metal accumulation in sinapis alba as the parameters for trace metals effect determination. bull. environ. contam. toxicol., 61, 1998, 762-769. fargašová, a., beinrohr, e.: metal-metal interactions in accumulation of v5+, ni2+, mo6+, mn2+ and cu2+ in underand above-ground parts of sinapis alba. chemosphere, 36, 1998, 1305-1317. freeman, j.l., persans, m.w., nieman, k., albrecht, c., peer, w., pickering, i.j., salt, d.e.: increased glutathione biosynthesis plays a role in nickel tolerance in thlaspi nickel hyperaccumulators. plant cell, 16, 2004, 21762191. hartley-whitaker, j., ainsworth, g., meharg, a.a.: copper and arsenate induced oxidative stress in holcus lanatus l. clones with differential sensitivity. plant cell environ., 24, 2001, 713-722. 56 fargašová, a. and szárazová, k. pandey, n., sharma, c.p.: effect of heavy metals co2+, ni2+ and cd2+ on growth and metabolism of cabbage. plant sci., 163, 2002, 753-758. prasad, m.n.v.: metal-biomolecule complexes in plants: occurrence, functions, and applications. analusis, 26, 1998, 25-28. römbke, j., moltman j.f.: applied ecotoxicology, crc press, boca raton, fl. 1996. salt, d.e., prince, r.c., pickering, i.j.: chemical speciation accumulated metals in plants: evidence from x-ray absorption spectroscopy. microchem. j., 71, 2002, 255–259. shanker, a.k., djanaguiraman, m., sudhagar, r., chandrashekar, c.n., pathmanabhan, g.: differential antioxidative response of ascorbate glutathione pathway enzymes and metabolites to chromium speciation stress in green gram (vigna radiate (l.) r wilczek, cv co 4) roots. plant sci., 166, 2004, 1035-1043. sharma, d.c., chatterjee, c., sharma, c.p.: chromium accumulation and its effects on wheat (triticum aestivum l. c.v. dh 2204) metabolism. plant sci., 111, 1995, 145-151. singh, s., sinha, s., saxena, r., pandey, k., bhatt, k.: translocation of metals and its effects in the tomato plants grown on various amendments of tannery waste: evidence for involvement of antioxidants. chemosphere, 57, 2004, 91-99. svetková, k., fargašová, a.: phytotoxicity of washing wastewaters from cutlery production line. bull. environ. contam. toxicol., 79, 2007, 109-113. turner, m.a., rust, r.h.: effects of chromium on growth and mineral nutrition of soybeans. soil sci. soc. am. proc., 35, 1971, 755-758. nova biotechnologica et chimica 12-1 (2013) 39 doi 10.2478/nbec-2013-0004 © university of ss. cyril and methodius in trnava actinomyces ruminicola g10 the rumen bacterium recovered from glycerol enriched cultivation media anna vandžurová, gabriel bódy, peter javorský, peter pristaš institute of animal physiology, slovak academy of sciences, šoltésovej 4-6, sk-040 01 košice, slovak republic (vandzurova@saske.sk) abstract: gradual increasing of glycerol concentration up to 10% using sheep ruminal fluid as an inoculum for in vitro cultivation was accompanied by significant changes in bacterial population as documented by dgge analysis. the resulting bacterial consortium was composed of three dominant bacteria with actinomyces related bacterium to be predominant. upon cultivation on media with glycerol as a sole carbon source a single bacterium was cultivated from this consortium. isolate g10 was found to be anaerobic, gram-positive rod-shaped bacterium. phylogenetic analysis based on 16s rrna gene sequences showed that g10 isolate is related to the actinomyces ruminicola species (97.7% of similarity). the role of rumen actinobacteria is largely unknown and their participation in glycerol utilization (tolerance) has not been described yet. the g10 bacterium and related consortium could be possibly used to improve glycerol tolerance and uptake by ruminants. key words: rumen bacteria, sheep, glycerol, actinomyces ruminicola 1. introduction biofuels are nowadays prospective replacement for the classic fossil energy sources which inventory is slowly declining. the most widely produced biofuel is a biodiesel, which is produced from vegetable oils and ethanol produced from plant polysaccharides derived from corn and sugar cane (patzek et al., 2004). during the biofuels production many secondary products are generated whose disposal is not easy. such products include glycerol, which occurs mainly in the production of biodiesel from rapeseed and flaxseed oil. excess glycerol can become environmental problems because it cannot be destroyed naturally in the environment, and its storage and processing is very costly therefore to be should ways to be processed further (arechederra and minteer, 2008). one of glycerol disposal options is its use as a source of carbon and energy source for the industrial microbiology. another possibility is the use of glycerol as a feed component for ruminants. the digestive tract of ruminants possesses the unique microbial ecosystem, which allows them to transform the plant food into microbial biomass. in vitro experiments have shown that the addition of glycerol to the diet has no deleterious effect on ruminants. it has been shown that the microorganisms present in the forestomach of ruminants can tolerate elevated concentrations of the glycerol in the feed, but only up to a certain concentration (rémond et al., 1993; parsons et al., 2009; drouillard, 2009; lee et al., 2011). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:46 utc 40 vandžurová, a. et al. the aim of our study was to characterize the effect of glycerol supplementation on composition of rumen microflora in vitro and to identify the bacterial species able to grow at high glycerol concentration. 2. materials and methods 2.1 origin sample, isolation and identification the sample of the ruminal fluid was taken from the digestive tract of sheep (slovenské merino breed) at conventional diet without any glycerol supplementation and analyzed for the presence of cultivable bacteria. to 1 ml of rumen fluid 10 ml of sterile cultivation m2 (hobson, 1969) medium were added in anaerobe box-bactron (sheldon manufacturing inc., usa) under atmosphere formed by an anaerobic gas mixture amg (anaerobic mixed gas) consisting of 5% carbon dioxide, 5% hydrogen and 90% nitrogen and cultivated for 48 hours at 37°c. resulting bacterial consortium was transferred (50μl) every 24 hours to liquid medium m2 with gradually increasing concentration of glycerol (1%, 5%, 7.5%, 10%). the bacterial population from the highest concentration of glycerol was inoculated on m2 agar plates without glycerol and cultivation was conducted under anaerobic conditions for 24-48h. after repeated subcultivations and control for the purity, the isolates were identified by gram staining and on the basis of colony characteristic. 2.2 dna isolation and pcr amplification the total dna from bacterial cultures was extracted by genelute™ bacterial genomic dna kit (sigma-aldrich, usa). isolated dna was used as a template for pcr amplification of 16s rrna gene fragments. all pcr reactions were performed in a 50 μl pcr mixture containing 1 μl of genomic of dna, 1 x pcr buffer, 2 mmol/l mgcl2, 1 μl of a 200 μmol/l of each dntp, 1,25 u platinum taq dna polymerase (invitrogen, ca usa) and 25 pmol each primer using mj mini thermal cycler (bio-rad laboratories, usa). in the first round of pcr universal primers fd1 (5´-aga gtt tga tcc tgg ctc ag-3´) and rp2 (5´-acg gct acc ttg tta cga ctt-3´) (weisburg et al., 1991) were used to amplify a 1500 bp region of 16s rdna gene. pcr conditions were as follows: 94°c for 5 min followed by 35 cycles of 94°c for 1 min, 52°c for 1 min, 72°c for 1 min 30 sec, 72°c for 5 min. the obtained 16s rdna fragments were subsequently used as a template for the second round of pcr using specific bacterial primers gc-clamp-968f (5´cgc ccg ggg cgc gcc ccg ggc ggg gcg ggg gca cgg ggg gaa cgc gaa gaa cct tac 3´) and 1401r (5´cgg tgt gta caa gac cc – 3´) (nübel et al., 1996). the cycling conditions were 94°c for 5 min, 9 cycles of 94°c for 1 min, 45°c for 1 min, 72°c for 1 min; 14 cycles of 94°c for 1 min, 60°c for 1 min, 72°c for 1 min followed by final extension at 72°c for 10 min. pcr products were detected by 1.2% agarose gel electrophoresis containing ethidium bromide. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:46 utc nova biotechnologica et chimica 12-1(2013) 41 2.3 sequence and phylogenetic analysis amplified pcr fragments (either amplified 16s rrna of g10 isolate or dgge band) were purified by purification kit (wizard sv gel and pcr clean-up system, promega) cloned into plasmid vector (instaclone pcr cloning kit, thermo scientific ) and subsequently sequenced using sanger didexy sequencing method using plasmid specific primers at gatc biotech sequencing facility (gatc biotech ag, konstanz, deutschland). 16s rrna sequences were subsequently subjected to blastn analysis at http://www.ncbi.nlm.nih.gov. the sequences of type strains of actinomyces spp. were downloaded from genbank database, aligned using clustalw algorithm, and phylogenetic tree was constructed using minimum-evolution method. for all phylogenetic analyses software mega 5 (tamura et al., 2011) was used. the sequence of 16s rrna gene of g10 isolate was deposited in the genbank database under the accession number kc866613. 2.4 dgge analysis pcr products generated with gc-clamp-968f and 1401r primers were subjected to dgge analysis. dgge was performed using dcodetm universal mutation detection system (bio-rad laboratories, hercules, ca usa). the round ii pcr reaction products in a total volume of 45 μl were loaded onto 8% (w/v) polyacrylamide gel (40% acrylamide-bis 37.5:1) in 1 x tae (40 mm tris, 20 mm acetate, 1mm edta) containing a linear denaturing gradient ranging from 30 60% denaturant (100% denaturant solution consists of 7 m urea and 40% formamide). electrophoresis was run for 17h at a constant voltage of 50v and a temperature 60°c. after electrophoresis, the gel was incubated for 20 min in ethidium bromide (0.5 μg/ml), rinsed for 20 min in distilled water, and photographed with uv transillumination using a gel logic imaging system (carestream, ny usa). 3. results and discussion the rumen fluid used in our work came from the rumen of sheep (slovenske merino breed) on regular diet (forage to concentrate ratio 70:30). by cultivation in the liquid m2 cultivation medium in the presence of increased concentration of glycerol (1%, 5%, 7.5%, 10%) substantial changes in the composition of bacterial population were observed using dgge method (fig. 1). variability of inoculum continually decreased and dominant species practically disappeared and were replaced by bacterial strains undetectable in original inoculum. gradually increased concentrations of glycerol significantly decreased variability of population and bacterial consortium growing at 10% glycerol consisted of three dominant bacterial species. no significant changes in the composition of bacterial community were observed upon repeated subculturing at 10% glycerol (data not shown). dominant species represented by band marked g10 (fig. 1) represented approximately 34% of the total of bacterial population. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:46 utc 42 vandžurová, a. et al. fig. 1. dgge analysis of ruminal fluid sheep inoculum growing in the presence of increasing glycerol concentrations. 1 mixed bacterial culture from the rumen of sheep; 2 bacterial culture growing at 1% glycerol; 3 bacterial culture growing at 5% glycerol; 4 bacterial culture growing at 7.5% glycerol; 5 bacterial culture growing at 10% glycerol. the arrow indicates the band corresponding to g10 isolate. sequence analysis of this band showed that this band shows the highest similarity to actinomyces ruminicola (96%). bacterial consortium growing at 10% glycerol was tested for the presence of cultivable bacteria. cultivation on m2 agar without glycerol yielded one type of isolate only. by gram staining and on the basis of cell morphology, the isolate was identified as gram-positive and rod-shaped bacterium. the isolate was designated as g10 and subsequently characterized. the complete identity was observed between sequence of g10 dgge band and 16s rrna sequence of g10 isolate. phylogenetic analysis showed that 16s rrna sequence of g10 isolate reveals the highest similarity (97.7% at 16s rrna level) to a. ruminicola, indicating that g10 isolate is possibly a representative of a new, yet undescribed anaerobic rumen bacterium belonging to the actinomyces genus. phylogenetic tree showed that our sequence form together with a. ruminicola species a separate branch within a. dentalis cluster (fig. 2). based on this comparison our sequences belong to the a. ruminicola species. the genus actinomyces consists of a heterogeneous group of anaerobic and facultative anaerobic, asporogenous, gram-positive, non-acid-fast, rod-shaped organisms. of about 40 species of this genus occur worldwide as commensals and/or pathogens of man and other animals (hansen et al., 2009). a. ruminicola species was originally isolated from the rumen of cattle. bacteria of a. ruminicola species hydrolyse xylan and starch, ferment some mono-, diand oligosaccharides and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:46 utc nova biotechnologica et chimica 12-1(2013) 43 produce formic, acetic and lactic acids as end fermentation products of glucose (an et al., 2006). the role of rumen actinobacteria or a. ruminicola is largely unknown and their participation in glycerol utilization (tolerance) has not been described yet. fig. 2. phylogenetic tree showing phylogenetic relatedness of a. ruminicola g10 isolate based on 16s ribosomal rna sequence comparison. the scale bar represents 0.01 substitutions per nucleotide site. the g10 bacterium and related consortium could be possibly used to improve glycerol tolerance and uptake by ruminants. research has shown that feeding glycerol up to a rate of 10% of the daily dietary dry matter has no effect on feed intake or performance in finishing beef cattle or lactating dairy cows (krueger et al., 2010). however, in most experiments fermentation capacity of the whole rumen microbial population was accessed without attempts to analyze the possible changes in rumen microbial population composition. predominant glycerol utilizing bacteria are not known and in feeding experiments in vivo it was shown that facultatively anaerobic bacteria appear to play little part in glycerol fermentation in the sheep rumen. amongst the most important members of the glycerol-fermenting flora are strict anaerobes of the group selenomonas ruminantium var. lactilytica (hobson and mann, 1961). abu et al., (2011) showed that glycerol substitution had no effects on ph, ammonia nitrogen concentration, and dry matter digestibility in vitro fermentations using cow rumen fluid as an inoculum. the numbers of butyrivibrio bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:46 utc 44 vandžurová, a. et al. fibrisolvens, clostridium proteoclasticum, and s. ruminantium decreased under high level glycerol concentration diet but no differences for ruminococcus albus and succinivibrio dextrinosolvens among diets with different glycerol concentrations were observed. authors suggest that substituting corn with glycerol at low level (up to 4%) had no adverse effects on fermentation, digestion or ruminal bacteria population. higher substitution levels, however, may adversely affect rumen fermentation through reducing fiber digestion, acetate production and bacterial populations. other experiments, both in vitro and in vivo, will be necessary to understand the effect of glycerol supplementation on rumen microbial population. 4. conclusions a bacterial consortium able to grow in the presence of 10% glycerol was prepared from the rumen sheep inoculum. the consortium is composed of 3 bacterial species from which single only, actinomyces ruminicola g10 is cultivable under in vitro conditions. acknowledgements: this publication is the result of the project no. 26220220152 implementation supported by the research & development operational programme funded by the erdf. references abu ghazaleh, a.a., abo el-nor, s., ibrahim, s.a.: the effect of replacing corn with glycerol on ruminal bacteria in continuous culture fermenters. j. anim. physiol. anim. nutr. (berl.), 95, 2011, 313-319. an, d., cai, s., dong, x.: actinomyces ruminicola sp. nov., isolated from cattle rumen. int. j. syst. evol. microbiol., 56, 2006, 2043-2048. arechederra, r.l., minteer, s.d.: complete oxidation of glycerol in an enzymatic biofuel cell. fuel cells: from fundamentals to systems., 9, 2008, 6369. drouillard, j.s.: glycerin as a feed for ruminants: using glycerin in highconcentrate diets. symposium: ruminant nutrition: glycerin as a feed for ruminants, kansas state university: manhattan, 2009; 392-393. hansen, j.m., fjeldsoe-nielsen, h., sulim, s., kemp, m., christensen, j.j.: actinomyces species: a danish survey on human infections and microbiological characteristics. open microbiol j., 3, 2009,113-120. hobson, p.n.: rumen bacteria. volume 3b. edited by norris jr, ribbons dw. london and new york, academic press. in methods in microbiology, 1969,133149. hobson, p.n., mann, s.o.: the isolation of glycerol-fermenting and lipolytic bacteria from the rumen of the sheep. j. gen. microbiol., 25, 1961, 227-240. krueger, n.a., anderson, r.c., tedeschi, l.o., callaway, t. r., edrington, t.s., nisbet, d.j.: evaluation of feeding glycerol on free-fatty acid production and fermentation kinetics of mixed ruminal microbes in vitro. bioresour. technol., 101, 2010, 8469-8472. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:46 utc nova biotechnologica et chimica 12-1(2013) 45 lee, s.y., lee, s.m., cho, y.b, kam, d.k., lee, s.ch., kim, ch.h., seo, s.: glycerol as a feed supplement for ruminants: in vitro fermentation characteristics and methane production. in animal feed science and technology, 166, 2011, 269274. nübel, u., engelen, b., felske, a., snaidr, j., wieshuber, a., amann, r.i.: sequence heterogeneities of genes encoding 16s rrnas in paenibacillus polymyxa detected by temperature gradient gel electrophoresis. j. bacteriol., 178, 1996, 5636-5643. parsons, g.l., shelor, m.k., drouillard, j.s.: performance and carcass traits of finishing heifers fed crude glycerin. j. anim. sci., 87, 2009, 653-657. patzek, t.w.: thermodynamics of the corn-ethanol biofuel cycle. crc crit. rev. plant. sci., 23, 2004, 519-567. rémond, b., souday, e., jouany, j.p.: in vitro and in vivo fermentation of glycerol by rumen microbes. j. anim. sci. biotechnol., 41, 1993, 121-132. tamura, k., peterson, d., peterson, n., stecher, g., nei, m., kumar, s.: mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. mol. biol. evol., 28, 2011, 2731-2739. weisburg, w.g., barns, s.m., pelltier, d.a., lane, d.j.: 16s ribosomal dna amplification for phylogenetic study. j. bacteriol., 173, 1991, 697-703. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:46 utc nova biotechnologica et chimica 13-1 (2014) 73   doi 10.2478/nbec-2014-0008 © university of ss. cyril and methodius in trnava application of ann for prediction of co2+, cd2+ and zn2+ ions uptake by r. squarrosus biomass in single and binary mixtures peter nemeček1, dáša kružlicová1, lucia remenárová2 1department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, j. herdu 2, trnava, (peter.nemecek@ucm.sk) 2department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, j. herdu 2, trnava, sk-917 01, slovak republic abstract: discharge of heavy metals into aquatic ecosystems has become a matter of concern over the last few decades. the search for new technologies involving the removal of toxic metals from wastewaters has directed the attention to biosorption, based on metal binding capacities of various biological materials. degree of sorbent affinity for the sorbate determines its distribution between the solid and liquid phases and this behavior can be described by adsorption isotherm models (freundlich and langmuir isotherm models) representing the classical approach. in this study, an artificial neural network (ann) was proposed to predict the sorption efficiency in single and binary component solutions of cd2+, zn2+ and co2+ ions by biosorbent prepared from biomass of moss rhytidiadelphus squarrosus. calculated non-linear ann models presented in this paper are advantageous for its capability of successful prediction, which can be problematic in the case of classical isotherm approach. quality of prediction was proved by strong agreement between calculated and measured data, expressed by the coefficient of determination in both, single and binary metal systems (r2= 0.996 and r2= 0.987, respectively). another important benefit of these models is necessity of significantly smaller amount of data (about 50%) for the model calculation. also, it is possible to calculate qeq for all studied metals by one combined ann model, which totally overcomes a classical isotherm approach. key words: heavy metal, biosorption, metal uptake, prediction, ann 1. introduction past two decades has witnessed a drastic increase in the quality and quantity of metal pollutants discharged into aqueous environmental sink. heavy metals are toxic to aquatic flora and fauna even in relatively low concentrations. some of these are capable of being assimilated, stored and concentrated by organisms. a special care needs to be taken in order to prevent the cumulative accumulation in the wastewater (mohan and singh, 2002). various treatment technologies have been developed for the purification of water and wastewater contaminated by heavy metals. the most commonly used methods for the removal of metal ions from industrial effluents include: chemical precipitation, solvent extraction, oxidation, reduction, dialysis, reverse osmosis, ion-exchange, etc. among these, adsorption has evolved as the front line of defense and especially for those, which cannot be removed by other techniques. selective adsorption utilizing biological materials, mineral oxides, activated carbon, or polymer resins, has bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:15 utc 74 nemeček, p. et al. generated much excitement among researchers, environmental engineers and scientists (mohapatra and gupta, 2005). a lot of works have been focused on the application of various adsorbents for the metal removal from the aqueous media, but only a few of manuscripts are dedicated to the competitive uptake from the binary or multiple aqueous solutions (see e.g. bayo, 2012; seo et al., 2008). as a matter of fact, it is more important to evaluate the simultaneous adsorption behavior and interactions involving two or more metal species since sole toxic metal species rarely exist in wastewater (li et al., 2011). so, it is necessary to extensively investigate the competitive binding of the heavy metal ions such as co2+, cd2+ and zn2+ onto a biosorbent thoroughly. in this research, r. squarrosus moss biomass has been used as a powerful biosorbent for the biosorption of metal ions. in the application of adsorption for purification of wastewater the solution will normally be a mixture of many compounds rather than a single one. the interactions of these compounds may mutually enhance or mutually inhibit adsorption capacity (ho and mckay, 1999). industrial effluents, however, far from being single-component, are complex solutions containing several metals simultaneously. in the biosorption of complex solutions, different metal ions may compete for the active sites present on the cell wall of the biomass. consequently, the preference of the biomass for some metals is an important issue, which is affected by the type of biomass, its physicochemical characteristics, its preparation or previous conditioning, and the nature of the metallic solution (rincón et al., 2005). in previous studies (remenárová et al., 2010; pipíška et al., 2010; pipíška et al., 2009) the effect of multisolute interactions on the capacity of r. squarrosus biomass was investigated using binary and ternary mixtures of co2+, cd2+ and zn2+ having pre-fixed ratios. the freundlich and langmuir isotherm models were applied to the equilibrium data. parameters of these isotherm models provided an insight into the sorption process, reflected the nature of the sorbent, surface properties as well as the degree of the affinity of the sorbents and could be used to compare biosorption performance (kafshgari et al., 2013). in addition to common approach, artificial neural networks (ann) were studied as a suitable method for estimating complex functions in order to evaluate the equilibrium data. ghosh et al. (2014) used ann and response surface methodology to develop predictive models for simulation and optimization of the biosorption process. tovar-gómez et al. (2013) applied the ann for prediction of biosorption in fixed-bed column. similarly, oguz and ersoy (2014) used ann modeling to investigate the cause-effect relationship in the bed studies of cobalt (ii) biosorption onto sunflower biomass. fagundes-klen et al. (2007) predicted biosorption equilibrium of the binary mixture of cadmium-zinc ions by the sargassum filipendula species. prakash et al. (2008) predicted biosorption efficiency of copper (ii) removal from single aqueous solution by taking into account the effect of initial copper concentration, ph and temperature and particle size of the adsorbent using ann. anns are computational systems, which mimic the computational abilities of biological systems by using a number of interconnected artificial neurons (fazlali bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:15 utc nova biotechnologica et chimica 13-1 (2014) 75   et al., 2013). considering the inherent ability of the anns to learn and recognize nonlinear and complex relations, they can be used in various fields of chemistry. the anns consist of a number of interconnected nodes arranged in layers corresponding to input layer, hidden layer and output layer. the hidden layers encode and arrange the information received from the input layer and deliver them to the output layer. each neuron of the network is connected with an associated weight, to the others via direct communication links, which finally provides a logical relationship between input and output parameters. the number of neurons for the input and output layers is usually determined by the number of input and target variables, but the number of neurons in the hidden layers is variable significant for optimization of the network. this work presents an ann model, trained by the back propagation algorithm, to predict the metal uptake (qeq) of co 2+, cd2+ and zn2+ by moss r. squarrosus in different mixtures. the main goal of this work was to test the capability of ann as a promising tool for modeling of biosorption behavior in single and binary metal solutions. study was divided into two parts: (1) first part was focused on development of an ann model capable to predict sorption behavior in single metal solutions; (2) second part was focused on the modeling of competitive sorption behavior in binary metal solutions. suitability of ann models was proved by comparison of calculated results with a classical approach of langmuir adsorption isotherms. 2. materials and methods 2.1 biosorbent preparation a biomass of moss r. squarrosus used in present study was collected from the forests of high tatras mountains, slovak republic. to remove the impurities, the biomass was washed twice in deionised water, oven-dried for 72 h at a maximal temperature 45°c to avoid the degradation of binding sites. after drying the biomass was milled and sieved. particle size 300 600 μm was used in biosorption experiments. 2.2 batch biosorption experiments in single and multi-metal systems batch biosorption experiments in single-metal systems were carried out in aqueous solutions containing cdcl2, cocl2 or zncl2 in concentration range 100 to 4000 μm and spiked with 109cdcl2, 65zncl2 or 60cocl2. biosorption experiments in binary metal systems were carried out in series of solutions (cd-zn, co-zn or cd-co) containing each metal in concentrations varying from 100 to 4000 μm and in various molar ratios 2:1, 1:1, 1:2. the ph was adjusted to 6.0 with 0.1 m naoh. biosorbent (2.5 g/l, d.w.) was added, and the content in erlenmeyer flasks was agitated on a reciprocal shaker (120 rpm) for 4 h at 20°c. at the end of each experiment biosorbent was filtered out, washed twice with deionised water and radioactivity of both moss biomass and liquid phase was measured. the metal uptake was calculated as ( ) m v ccq eqeq −= 0 (1) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:15 utc 76 nemeček, p. et al. where q is the uptake (μmol/g, d.w.), c0 and ceq is the initial and the final metal concentrations in solution (μmol/l) and m is the amount of dried biosorbent (given in grams). each experiment was repeated only twice due to total number of experiments (replication of all measurements is very time consuming). if two values of same experiment were significantly different from each other (α = 0.05), both were excluded from further data processing. 2.3 radiometric analysis for radiometric determination of 109cd, 60co and 65zn in liquid phase and moss biomass, gamma spectrometric scintillation detector 54bp54/2-x and 76bp76/3 with well type crystal nai(tl) (scionix, netherlands) and data processing software scintivision32 (ortec, usa) were used. standardized 109cdcl2 solution (3.857 mbq/ml, cdcl2 50 mg/l in 3 g/l hcl), 65zncl2 solution (0.8767 mbq/ml; 50 mg zncl2/l in 3 g/l hcl) and 60cocl2 solution (5.181 mbq/ml, cocl2 20 mg/l in 3 g/l hcl) were obtained from the czech institute of metrology, prague (czech republic). 2.4 experimental data analysis and isotherms calculations details about measured biosorption data have been already published in previous papers (e.g. remenárová et al., 2010). the langmuir (eq. 2) and fruendlich (eq. 3) adsorption models were used for describing equilibrium data in single metal systems. langmuir sorption isotherm models the monolayer coverage of the sorption surfaces and assumes that sorption occurs on a structurally homogeneous adsorbent and all the sorption sites are energetically identical. the linearized form of the langmuir equation is given by: eq eq eq bc cbq q + = 1 max (2) where qeq is the amount of metal ion sorbed per unit weight of sorbent (µmol/g), ceq the equilibrium concentration of the metal ion in the equilibrium solution (µmol/l), qmax is the maximum sorption capacity of the sorbent (µmol/g) and b is the affinity parameter. freundlich equation is derived to model for the multilayer sorption and for the sorption on heterogeneous surfaces. the logarithmic form of freundlich equation may be written as: )/1( n eqeq kcq = (3) where k is constant indicative of the relative sorption capacity of sorbent (µmol/g) and 1/n is the constant indicative of the intensity of the sorption process. sorption equilibrium in binary systems cd2+-co2+, zn2+-cd2+ and co2+zn2+ was described by the competitive langmuir model developed under the concept of original langmuir isotherm for single systems for description of binary equilibrium data. the final expression of competitive langmuir model is as follows: bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:15 utc nova biotechnologica et chimica 13-1 (2014) 77             [ ] [ ] [ ] [ ]21 1max 1 21 11 1 mecbmecb mecqb meq eqmeeqme eqmeme eq ++ = (4)             [ ] [ ] [ ] [ ]21 2max 2 21 22 1 mecbmecb mecqb meq eqmeeqme eqmeme eq ++ =   (5)   1 1 1 me me k b = and 2 2 1 me me k b = the total metal uptake in binary systems can be expressed as follows:   [ ] [ ] [ ] [ ] [ ] [ ] [ ]21 21 2121 21 21 1 mecbmecb mecbmecb meqmeqmemeq eqmeeqme eqmeeqme eqeqeq ++ + =+=+ (6) where qeq[me1] and qeq[me2] represent equilibrium sorption capacities of metals me1 and me2, qeq[me1+me2] is the sum of uptakes of the two metals, ceq[me1] and ceq[me2] represent equilibrium concentrations of metals remaining in solution and qmax is the maximum sorption capacity of metals in the binary component systems. parameters bme1 and bme2 represent affinity constants of langmuir model for the first and second metal ions (apiratikul and pavasant, 2006).   to calculate the maximum sorption capacities qmax values and the corresponding parameters of adsorption isotherms non-linear regression analysis was performed by the software origin 8.5 professional (originlab corporation, northampton, usa). 2.5 data description and processing the initial step in the ann modelling is compiling an adequate database to train the network and to evaluate its capacity for generalization. experimental and theoretical values were organized in the basic data matrix into the lines with respect to the metal and its metal uptake (qeq). the basic set of independent variables (columns) contained: type of metal (m) categorical variable with 3 levels zn, co and cd, its maximum sorption capacity (qmax) and its physico-chemical parameters: ratio of charge squared to ionic radius (ionind), covalent index (kind), ionization energy (ioneng), ionic radius (ionr), electronegativity (elneg). another group of variables were indicator variables of metals presented in analyzed solution (mzn, mcd, mco). the last group was oriented on the initial concentrations of the metals in the system (znc0, cdc0, coc0), initial concentration of metal with supplemented qeq value (c0), total concentration of all presented metals in solution (sumc0), ratio (wm) between the initial metal concentration (c0) and total concentration of all metals presented in the system (sumc0). in the case of the last group, all concentrations were adjusted by the speciation of the metal and ph values. in addition the "concentration variables" were transformed into the logarithmic form with the effort of better fitting the sorption behaviour. this provided a basic data matrix with 118 lines (cases) and 22 columns (variables). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:15 utc 78 nemeček, p. et al. the basic data matrix was reduced to contain data belonging to only single metal systems. this reduced data matrix contained only 21 cases (7 concentrations for 3 studied metals) and 22 original variables was used in modelling of single metal solutions by ann and the basic data matrix was used for binary metal systems. 2.6 ann model development anns with different architectures provide different outputs, so there is more chance to reach the optimal model with more developed and evaluated models. in this study, various numbers of neurons were used for the hidden layer as well as the activation functions of the hidden and output layer, and the optimal settings were evaluated. in order to prevent overfitting in anns training process, the data set was randomly separated into three parts, the training set, the test set and the validation set, with the population ratio 2:1:1. the best ann model was assessed by the calculation performances of these three sets, especially the validation set as the measure of model generalization (may et al., 2011). high number of variables at the anns input compared to the number of cases adversely influences the ann performance and generalization. thus very important step is reduction of the number of ann inputs. in presented research, the variables considered as the inputs of the network were selected according to the sensitivity analysis of the best developed models. sensitivity analysis, is a supplement of the anns output in software statistica 10, which provides sensitivity ratio for each input variable of selected model. by referring to sensitivity ratio value, the input variables can be ranked for their contribution to the output. the results with a value more than 1 represent major variables and the value less than 1 represents minor variables (montano et al., 2003). sensitivity analysis approach was successfully used to select an optimal set of input variables for ann calculation as well as their suitable representation (logarithmic form etc.). all presented ann outputs and developed models were performed by the statistica 10 (statsoft, tulsa, usa). 3. results and discussion 3.1 ann modeling of biosorption in single metal systems the first aim of the presented study was development of an ann model with multilayer perceptron architecture capable to predict metal uptake (qeq) in single metal system and compare obtained results with a classical approach – langmuir equilibrium isotherm. reduced data matrix was used for the ann calculations. the best neural network (fig. 1) configuration had five input neurons, namely c0, ionind and m (with 3 levels zn, co and cd), two hidden neurons and one output neuron (qeq). best ann model was trained using only eleven cases (training set) and five cases for internal validation (test set). excellent results were obtained by the regression analysis (tab. 1) of the training and test sets with the determination coefficients (0.986 and 0.999) and slopes (0.987 and 1.024) close to one and intercepts bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:15 utc nova biotechnologica et chimica 13-1 (2014) 79   (1.753 and 0.525) close to zero, respectively. the coefficient of determination (r2), slope and intercept values of the validation set (five cases) for the prediction of the qeq were 0.996; 1.053 and 3.942, respectively. fig. 2 shows fitting of the predicted values with the experimental results for each studied metal. figures were made as an addition to the classical isotherm approach. this was the main reason, why graphs were not constructed by the common ann principle (by training, test and validation subsets), but by the individual metals.   fig. 1. scheme of three-layer perceptron built for qeq prediction of single metal systems. in the case of categorical variable m, three sublevels are present in the brackets. results obtained by langmuir isotherms for each metal were very similar to those calculated by the ann model. the strong correlations between the calculated and experimental metal uptakes were proved for all studied metals. coefficients of determination for langmuir isotherm and ann model were very close to one for cobalt 0.914 vs. 0.997, for cadmium 0.994 vs. 0.992 and zinc 0.995 vs. 0.980, respectively. the resulting ann model was able to replace along three isotherms (for each of the studied metal). compared with a traditional approach of isotherm modelling was able not only to describe the sorption behavior, but in addition it could also predict the sorption efficiency what was confirmed by an independent validation.     0 50 100 150 200 250 300 0 50 100 150 200 250 300 zn co cd experimental qeq [μmol/g] calculated qeq [μmol/g] fig. 2. qeq values calculated by the ann model compared with the experimental data for each metal that offers an alternative view on prediction performance of biosorption. straight-line indicates the ideal scenario, where the calculated values are identical with the experimentally measured data. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:15 utc 80 nemeček, p. et al. achieved ann performance is also comparable with other authors. masood et al. (2012) used artificial neural network for prediction of biosorption of total chromium by bacillus sp. developed model used ph, time and initial metal concentration on the input. regression analysis of the results proved strong correlation between the calculated and the experimental values, r2= 0.971. for determination of the degree of effectiveness of a variable the sensitivity analysis was conducted using the proposal ann model. findings of the sensitivity analysis showed that initial ph was the most significant parameter for the prediction of removal efficiency. similarly, raj et al. (2013) highlighted the possibility of the prediction of sorption efficiency for the arsenic species from water bodies using leucaena leucocephala seed powder (llsp) in the range at which lab experiments have not been conducted.they have developed a single layer ann model (using biosorbent dosage, arsenic ion concentration, contact time and volume on the input) for the predicition of sorption efficiency of arsenic species using llsp. yetilmezsoy and demirel (2008) utilized ann for more complex tasks, which included also kinetic studies of pb2+ sorption on pistacia vera l. shells. developed model used sorbent dosage, initial concentration of pb2+, ph, temperature and contact time on the input. regression analysis of sorption efficiency results proved strong correlation between the calculated and the experimental values, r2= 0.936, slope b1= 0.896 and intercept b0= 8.46. in spite of the results are promising, the application of the complex nonlinear algorithm is not necessary. classical isotherm model approach is more than sufficient for description of the sorption behaviour in single component systems. in this phase of the research, the development of the ann model was more about methodical than practical importance. tab. 1. table shows the results of ann validation expressed by the regression analysis for each system and subset, where n represents the number of objects in subset, r2 is the coefficient of determination, b1 and b0 are the slope and intercept, respectively. system type subset n r2 b1 b0 training 11 0.998 1.024 0.525 testing 5 0.986 0.987 1.753 single metal systems validation 5 0.996 1.053 3.942 training 49 0.987 1.030 -2.532 testing 24 0.987 1.018 -2.889 binary metal systems validation 45 0.987 1.007 -0.881 3.2 ann modeling of biosorption in binary metal systems the second part of this study was oriented to the modeling of a competitive sorption behavior in binary metal systems (zn-cd, cd-co, co-zn). the basic data matrix was used containing the sorption data of studied metals in single systems as well as their combinations in binary metal systems. again, the anns results were bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:15 utc nova biotechnologica et chimica 13-1 (2014) 81   compared with the classical approach represented by competitive langmuir isotherm for binary systems. the best ann model has fourteen input neurons, five neurons in the hidden layer and one output neuron (fig. 3). the most important variables ordered by the sensitivity analysis were: metal indicator variables mco, mcd, mzn (each with two levels y present and n-not present), followed by the concentration variables in logarithmic form, initial concentrations of the metals c0co, c0cd, c0zn and the total concentration of all metals in the system sumc0, type of the predicted metal m (with 3 levels zn, co and cd), and ratio of charge squared to ionic radius ionind. distribution of the cases was 40% used in the training process, 20% in the test set for internal validation and 40% of data was in the validation set. regression analysis (fig. 4) confirmed the calculation accuracy of the final ann model, where the coefficients of determination were 0.997; 0.985 and 0.994 in the order training, test and validation set. slopes and intercepts were reasonably close to one and zero, respectively; detailed results for the training data 0.993 and 0.497; test data 0.983 and 1.651 and validation data 1.056 and -5.638 (tab. 1). fig. 3. scheme of three-layer perceptron built for qeq prediction of binary metal systems. this figure contains input categorical variables m, mcd, mco and mzn, which sublevels are shown in brackets. the langmuir isotherms for binary systems were used to compare the results of the classical approach with the obtained ann output. also in this case were evaluated the calculated metal uptakes with the experimental data by coefficients of determination for each binary system. for the system zinc-cobalt were obtained high values of the coefficients of determination in both cases; 0.992 for langmuir isotherms and 0.990 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:15 utc 82 nemeček, p. et al. for ann model. other two systems co-cd and zn-cd for both approaches had values a little smaller, but still very good. classical approach had 0.938 and 0.901 while ann model provided 0.938 and 0.946, in previously given order. chu and kim (2006) studied the competitive sorption of copper and cadmium on the microbial biosorbent. feed-forward ann model was developed for the description of the competitive sorption behavior in the binary metal system. the ann topology composed from the initial metal concentrations (cu and cd) and ph used as the inputs, ten hidden neurons and two output neurons (one neuron for each metal). developed model achieved the mean absolute relative errors 3.3% for cu and 3.5% for cd sorption prediction, which is a very good performance considering the simplicity of the model just 3 input neurons. 0 50 100 150 200 0 50 100 150 200 zn + co co + cd zn + cd experimental qeq [μmol/g] calculated qeq [μmol/g]   fig. 4. regression analysis of qeq values calculated from ann model, compared with the experimental data separately for each binary system. this graphical output offers an alternative view on prediction performance of biosorption. straight-line indicates ideal scenario, where the calculated values are identical with the experimental data. similarly, kabuba et al. (2012) used a neural network for prediction of the sorption of binary mixture of copper-cobalt ions. they found that experimental data of the single-component system and the binary mixture were well described by several isotherm models. however, these isotherm models were not able to predict the adsorption in binary mixture accurately. on the contrary, application of neural network showed that this technique is more efficient with the one using the adsorption isotherms. 4. conclusions it can be concluded that the approach of ann is efficient in modeling of complex biological phenomenon such as biosorption. the preliminary study on single systems proves high potential of anns in the modeling of experimental data of biosorption processes. developed models provide also several advantages compare to the classical isotherm approach: (1) one ann model can successfully describe the behavior of several metals (in this study three) while isotherm can always describe sorption behavior of only one metal; (2) in general, the ann model development requires bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:15 utc nova biotechnologica et chimica 13-1 (2014) 83   approximately 50% of original cases in contrast to isotherm approaches requiring most of the data; (3) qeq calculation for new (not measured) initial concentrations (in studied range) was proved as very accurate according to the validation set results; (4) anns allow to study different input variables and their influence on the target metal uptake using the sensitivity analysis. ann models presented in this paper are advantageous for its capability of successful prediction, which can be problematic in the case of classical isotherm approach. quality of prediction was proved by coefficient of determination between calculated and measured data in single (r2= 0.996) and binary metal systems (r2= 0.987). slightly lower r2-value for binary system was expected due to the effect of metal competition. acknowledgement: authors are greatly acknowledged to fund for research support of the university of ss. cyril and methodius, project no. fppv-34-2013; to the slovak research and development agency, project no. apvv-0014-11; and to the scientific grant agency of the ministry of education of the slovak republic and of slovak academy of sciences, project no. vega 1/0233/12 for the financial support. references apiratikul, r., pavasant, p.: sorption isotherm model for binary component sorption of copper, cadmium, and lead ions using dried green macroalga caulerpa lentillifera. j. chem. eng., 119, 2006, 135-145. bayo, j.: kinetic studies for cd(ii) biosorption from treated urban effluents by native grapefruit biomass (citrus paradisi l.): the competitive effect of pb(ii), cu(ii) and ni(ii). chem. eng. j., 191, 2012, 278– 287. fagundes-klen, m.r., ferri, p., martins, t.d., tavares, c.r.g., silva, e.a.: equilibrium study of the binary mxture of cadmium-zinc ions biosorption by 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37, slovak republic (zuzana.sramkova@stuba.sk) 2 plant production research centre, research institute of plant production; piešťany, sk-921 01, slovak republic (gregova@vurv.sk) abstract: bread wheat (triticum aestivum l.) plays a major role among the few crop species being extensively grown as staple food sources. as the human population grows, new methods and approaches must be found to attain wheat cultivars with improved characteristics. the challenge now is to produce higher-yielding varieties with good technological quality that are resistant or tolerant to a wide range of biotic and abiotic stresses. however, because of the critical nutritional status of human population, there is an urgent need for development of such wheat varieties that would be more nutritious (with improved protein, zinc, iron, etc. value), meeting our health demands. this article summarises present status in this field. keywords: biotic/abiotic factors, genetic transformation, grain yield, nutritional quality, plant breeding, wheat 1. introduction wheat (triticum spp.) is a self-pollinating annual plant, belonging to the family poaceae (grasses), tribe triticeae, genus triticum. according to different classifications, number of species in the genus varies from 5 to 27 (merezhko, 1998). the two main groups of commercial wheats are the durums (triticum durum l.) and bread wheats (triticum aestivum l.) with 28 and 42 chromosomes respectively. the wild species are still a valuable source of useful agronomic traits for the continued improvement of cultivated wheats. wide hybridization of wheat with grasses, coupled with cytogenetic manipulation of the hybrid material, has been instrumental in the genetic improvement of wheat. chromosome engineering methodologies based on the manipulation of pairing control mechanisms and induced translocations, have been employed to transfer into wheat specific disease and pest resistance genes from annual (e.g., rye) or perennial (e.g., thinopyrum spp., lophopyrum spp., and agropyron spp.) members of the tribe triticeae. the use of dna markers helps to identify desirable genotypes more precisely and facilitates gene transfer into wheat. the development of novel gene-transfer techniques that allow direct delivery of dna into regenerable embryogenic calli has opened up new avenues of alien-gene transfer into wheat cultivars. thus, transgenic bread and durum wheats have been produced. the application of transgenic technology has not only yielded herbicide-resistant wheats, but has also helped to improve grain quality by modifying the protein and starch profiles of the grain (regina et al., 2006; bicar et al., 2008). recently, biofortification of cereal crops with micronutrients (vitamins, minerals, etc.) using plant breeding and/or transgenic strategies has become of great interest. 28 šramková, z. et al. approaches to gene transfer are developing rapidly, and promise to become an integral part of plant breeding efforts. however, the new biotechnological tools will complement, not replace, conventional plant breeding (jauhar and chibbar, 2001; vasil, 2007). 2. history, cultivation, production and green revolution wheat in its present-day form has gone through a long and interesting evolution. the origin of the genus triticum (wheat) is found in asia, in the area known as the fertile crescent, and parts of africa, in the area that stretches from syria to kashmir, and southwards to ethiopia. the genetic relationships between einkorn and emmer indicate that the most likely site of domestication is near diyarbakir in turkey. the cultivation of wheat began to spread beyond the fertile crescent during the neolithic period. by 5,000 years ago, wheat had reached ethiopia, india, great britain, ireland and spain. a millennium later it reached china (dubčovský and dvořák, 2007). all the species belonging to the genus triticum can be divided into three basic groups, each distinguished by the number of chromosomes in the generative and vegetative cells. unfertilized egg cells contain 7, 14 or 21 chromosomes. in vegetative cells this number is doubled. consequently, diploid, tetraploid and hexaploid wheat species carry 2x7=14; 4x7=28 and 6x7=42 chromosomes, respectively (belderok et al., 2000). hexaploid wheat is believed to have arisen, when genomes of tetraploid wheat (t. turgidum, 2n=28, ab-genome) and aegilops squarrosa l. (also called triticum tauschii) were combined via amphidiploidisation (fig. 1). aegilops is a species without any economic value that grows as a weed on the borders of wheat fields in the near east and it has a somatic chromosome number of 14. these chromosomes belong to the d-genome. therefore, the genome of t. aestivum is called the abd-genome (dvořák, et al., 1998). the bread wheats (t. aestivum l.) encompass a wide range of different types classified largely by their growth habit and functionality. the various classes are combinations of winter or spring growth habit with white or red and hardor soft-textured kernels. since only the hexaploid wheat cultivars, possessing the d set of chromosomes, have the unique milling and baking properties, these desirable quality characteristics have been attributed preponderantly to the third genomic component (belderok et al., 2000). an important attribute of wheat is its adaptability to varied climatic conditions. although grown mostly in temperate climates (between latitudes 30° and 60° north and south) with an optimum growing temperature of 25°c (minimum and maximum temperatures of 3°c and 32°c), it can be grown from within the arctic circle to higher elevations near the equator, and from sea level to as much as 3000m above sea level. as such, it is one of the most widely cultivated crops with a short growing season, and a good yield per unit area. these attributes makes wheat one of the most important commodities in international trade (vasil, 2007). with 607 mmt (million metric tons) produced on 217 mha (million hectares) in 2007, wheat continues to be one of the largest food crops in terms of area of nova biotechnologica 9-1 (2009) 29 cultivation as well as production (faostat, 2007; table 1). the igc's (international grains council) current estimate for production in 2008 is 683 mmt (source: igc, http://www.igc.org.uk/; 2008). fig. 1. evolution of cultivated wheat: the diploid (2n = 14, aa) forms of t. monococcum (a) were naturally pollinated by weed species (2n = 14, bb). the subsequent genome duplication of hybrids by natural polyploidy gave rise to several wild and cultivated tetraploid species (2n = 28, aabb) like t. dicoccum (b) and t. durum; again, the natural pollination of the tetraploid t. dicoccum (b) by aegilops squarrosa (2n = 14, dd) gave rise to the hexaploid (2n = 42, aabbdd) species (c) (from fernandes et al., 2000). in the middle of the twentieth century, wheat breeders were faced serious problem: increases in productivity have fallen below the rate of population growth. at the request of the mexican government and the urging of the usa, a program to develop high-yielding and disease-resistant varieties of wheat started in mexico in 1944. norman borlaug and his team first developed disease-resistant varieties and then crossed these with the japanese dwarf variety norin 10, to produce semi-dwarf, disease-resistant and high-yielding varieties of wheat (hedden, 2003). these new varietieswhich made more grain and less stemformed the basis of the “green revolution”, which has allowed many countries (such as india, pakistan, china, etc.) to be self-sufficient and food production to keep pace with worldwide population growth (vasil, 2007). before the period of the green revolution, the farmers in slovakia cultivated tall historical wheat cultivars (landraces), with insufficient seed yield. later, wheat cultivars originated in czech republic and soviet union were adopted. from the year 1967, there were no slovak varieties grown in slovakia. from 1945 to 1970, sixteen breeding stations were established and slovak wheat breading began to progress rapidly. new, modern semi-dwarf wheat varieties with higher yields and improved resistance to pathogens replaced the tall traditional varieties like in almost all countries over the world. between the years 1976 and 2008, 47 wheat cultivars of slovak origin were included in the national list of released varieties (anonymous, 2008). 30 šramková, z. et al. wheat production will have to be doubled to 1200 mmt by the 2025 in order to meet increasing world demands and future needs. this increase must be brought about by improving productivity on land that is already under cultivation and not by bringing new land into use by destruction of forests, grasslands, etc. (vasil, 2003). such significant increases in yield are unlikely to be attained through only the traditional, or even the newly developed marker-assisted breeding methods, because neither the germplasm of wheat nor its close relatives is likely to contain the wide variety of genes that would be needed to meet future demands. therefore, new methods and approaches must be found to integrate useful genes from any organism (plants, animals or microorganisms) to attain the dramatic yields that would be required to meet future demands: (1) significantly reduce or eliminate productivity losses caused by pests, pathogens and weeds (oerke et al., 1994), and the substantial additional losses attributed to abiotic factors (drought, salinity, etc.) as well as post-harvest spoilage during storage. (2) fundamental improvement of the physiological capability or limit of the plant by increasing its photosynthetic efficiency and nutrient utilization in order to attain higher yields. moreover, the number of micronutrient-malnourished people is raising, therefore the improvement of nutritional quality of wheat as a staple crop must also be of interest to plant breeders. table 1. production quantity of the most important cereal crops. production quantity [million tonns]/area harvested [million hectars] year cereal crop 2005 2006 2007 maize 716/ 148 699/ 147 785/ 158 rice 632/ 155 644/ 156 651/ 157 wheat 626/ 220 598/ 214 607/ 217 source: faostat (http://www.faostat.fao.org) these objectives require the introduction of noveland in many instances alien and multiplegenes into commercial varieties of wheat by genetic transformation, and production of transgenic varieties with the desired attributes (vasil, 2003). however, high costs currently limit the implementation of functional genomics in breeding programs. genomics research will continue to enhance the efficiency and precision for crop improvement but will not totally replace conventional breeding and evaluation methods (varshney et al., 2007) 3. approaches to genetic improvement landraces of cereal crops contain an array of genes for agronomically important traits. these landraces fall in the primary gene pool with their respective crop species with which they can be easily crossed. complete or high pairing between chromosomes of the landrace and those of the crop plant facilitates alien gene transfer into the crop species (vasil, 2007). many wild relatives of wheat carry genes that offer superior traits, some of which have been incorporated into wheat via interspecific and intergeneric hybridization nova biotechnologica 9-1 (2009) 31 (jauhar and chibbar, 1999). chromosome pairing between chromosomes of the alien donor and those of cereal crops is the key to such gene introgressions. this process is relatively easy in diploid cereals. on the other hand, the hexaploid wheat has three genomes and there is genetic control of chromosome pairing so that only homologous partners pair to form bivalents. thus, wheat (both bread wheat and durum wheat) has a homologous pairing suppressor gene, ph1, which ensures diploid-like chromosome pairing and hence disomic inheritance. these characteristics are essential for the meiotic and reproductive stability of wheat. however, ph1 does not permit pairing between the wheat and alien chromosomes, thereby impeding alien gene transfer. methods of suppressing the activity of ph1 and hence promoting wheat-alien chromosome pairing are known (jauhar and chibbar, 1999). through wide hybridization coupled with manipulation of chromosome pairing, several desirable genes have been incorporated into wheat (jauhar and chibbar, 1999). thus, genes for resistance to leaf rust and barley yellow dwarf virus have been transferred from agropyron, thinopyrum and aegilops into wheat. jauhar and peterson (2000) produced scrab-resistant durum wheat germplasm by transferring in it segments of chromosomes from thinopyrum junceiforme. a better understanding of the factors limiting practical exploitation of exotic germplasm promise to transform existing, and accelerate the development of new, strategies for efficient and directed germplasm utilization (feuillet et al., 2008). plant protoplasts were an attractive target for transformation during the 1980s as it was already known that plants could be regenerated from protoplasts and that dna could be introduced into them by electroporation or by osmotic shock (by polyethylene glycol treatment). these methods are technically simple and inexpensive, often resulting in thousands of transformed micro-colonies in one experiment, with the possibility of producing many independent transformants. however, cell suspension cultures used for protoplast isolation are highly genotype-dependent, often prone to somaclonal variations and have a time-limited morphogenic competence. these disadvantages have restricted full use of the protoplast-based transformation technology, and the interest for the technique has declined for most cereals (repellin et al., 2001; vasil, 2007). although production of stably transformed cell lines of t. monococcum as well as t. aestivum was described (muller et al. 1996), none of these transformed lines were regenerated into plants. neither the fertility of the plants, nor the transmission of the transgenes to progeny, was demonstrated on the rare occasions when plants were regenerated from transformed protoplasts (he et al. 1994). during the past quarter century the unique and natural ability of the soil-borne crown gall bacteriumagrobacterium tumefaciensto transfer and integrate dna into the genome of wounded intact plant cells has been exploited extensively for the genetic transformation of higher plants (fig. 2). although agrobacterium as a phytopathogen is known to have an exceptionally wide host range, it does not generally infect monocots, particularly the economically important cereal crops. its inability to infect monocots in nature was the main reason for the widely accepted wisdom of the time that agrobacterium could not be used to transform cereals. this created the need to find alternative methods of transformation and led to the now 32 šramková, z. et al. universally-held view that embryogenic cultures are the best, and perhaps the only, source of regenerable cells in the poaceae (jauhar, 2006; lakshman, 2006). fig. 2. approaches usable in genetic improvement of wheat. a: agrobacterium-mediated transformation, b: biolistic method (www.ag.ndsu.edu/pubs/plantsci/crops/a1219-2.gif). one reason for the delay in cereal transformation by agrobacterium was the weak, or lack of woundresponse, from injured cereal tissues. the difficulties were overcome by co-cultivation of actively dividing embryogenic cells with supervirulent strains of agrobacterium tumefaciens in the presence of acetosyringone, a potent inducer of virulence genes (shrawat and lorz, 2006). the most commonly used method to deliver dna into plant tissues and callus is the high velocity bombardment of dnacoated microprojectiles (biolistics) (fig. 2). this technology of direct dna delivery, developed by sanford et al. (2000), overcame many of the problems inherent in the use of protoplasts. it rapidly became the method of choice for the transformation of cereals, particularly wheat (vasil and vasil, 2006). for optimal efficiency, a balance between the penetration power of the particles and the intensity of the wounds created in the target tissues must be attained. transgenic plants of wheat were produced for the first time in 1992, by the bombardment of embryogenic callus tissues with the plasmid pbargus, in which the expression of the gus reporter gene is driven by the maize adh1 promoter attached to adh 1 intron 1, and the expression of the selectable bar gene which confers resistance to the broad-spectrum herbicide basta is driven by the camv35s promoter attached to the maize adh1 intron 1 (vasil et al., 1992). further improvements in production of transgenic wheat plants were obtained by using other types of plasmids and promoters. although the transgenes are integrated at various sites in the genome, they do not cause any detectable chromosomal rearrangements, and their expression is more a function of the promoters used rather than the site of integration (vasil, 2007). nova biotechnologica 9-1 (2009) 33 agrobacterium-based systems and biolistic methods share common features. the same explant tissues can be used as targets, the transformation frequencies are comparable (stoger et al., 1998), and both techniques are genotype-dependent (lee et al., 1999). however, the perceived disadvantages of particle bombardment compared to agrobacterium-mediated transformation, i.e., the tendency to generate large transgene arrays containing rearranged and broken transgene copies, are not borne out by the recent detailed structural analysis of transgene loci produced by each of the methods. there is also little evidence for major differences in the levels of transgene instability and silencing when these transformation methods are compared in agriculturally important cereals (altpeter et al., 2005a) it is also important to keep in mind that one of the serious problems associated with agrobacterium-based transformation is the frequent vector backbone integration in transgenic lines of wheat and other plants, which is a serious impediment to gaining regulatory approval for environmental release (wu et al., 2006). irrespective of the method of transformation used, the transgenes are integrated at random sites in the host genome that may sometimes cause position effects, gene silencing, etc. sitespecific recombination has been proposed as a solution to this problem in wheat (srivastava et al., 1999). this technology also allows the removal of the selectable marker gene. 4. herbicide, pathogen and pests resistance weeds compete with crop plants for available nutrients and light energy, and thus reduce crop yields. actual worldwide crop losses to wheat productivity owing to weeds are estimated to be 12.3%, but as high as 23.9% without crop protection (oerke et al., 1994). with widespread and long-term herbicide use, and the fact that wheat has a single natural mechanism for degrading herbicides, many of the most noxious weeds found in wheat fields have over time developed resistances to the commonly used selective herbicides by evolving a similar mechanism (gressel, 1996). in general, production of herbicide-resistant crops has involved insertion of only one or two genes that encode inactivation of the herbicide by any of the following three mechanisms: (i) overproduction of a herbicide-sensitive biochemical target, (ii) structural alteration in biochemical target resulting in altered binding to the herbicide, or (iii) detoxification or degradation of the herbicide, before it reaches its target site in the plant cell (repellin et al., 2001). glyphosate is the active ingredient of herbicide roundup®. glyphosate-tolerant wheat plants were obtained showing in field trials neither any vegetative nor reproductive damage, nor any reduction in yield (zhou et al., 2003). recent advances have focused on development of highly efficient detoxifying enzyme that allows plants to resist glyphosate and provide improved protection against weeds when incorporated into wheat (johannes and zhao, 2006). metabolic inactivation has been used as a powerful and effective tool in engineering tolerance to the nonselective, broad-spectrum, post-emergent, contact herbicides commonly known as a basta, bialaphos, herbiace and glufosinate (vasil, 1996). they provide a high degree of human and environmental safety because they are non-toxic and are rapidly 34 šramková, z. et al. biodegraded, resulting in minimal residue persistence in soil. their herbicidal activity is due to l-phosphinothricin (ppt), a potent inhibitor of the biosynthetic enzyme glutamine synthetase (gs), which is involved in general nitrogen metabolism in plants. ppt is inactivated by the acetylating enzyme phosphinothricinn-acetyltransferase (pat). two similar genes, bar and pat, that encode pat, have been identified, cloned and used to transform wheat, thus making the transgenic wheat immune to ppt and making it possible to use basta for effective weed control in wheat fields. the transgene is transmitted to progeny in a mendelian fashion and has been shown to be stable under field conditions (vasil, 2007). a large number of fungal (such as rust caused by puccinia spp., smut and bunt caused by tilletia and ustilago spp., blotch caused by septoria spp., fusarium blight/scab, helminthosporium leaf blight, powdery mildew caused by blumeria graminis, etc.), bacterial (such as leaf streak caused by xanthomonas translucens) and more than 50 viral diseases are known to cause considerable worldwide damage to wheat production (curtis et al., 2002). losses of wheat production owing to pathogens are estimated to be 12,4%, but as high as 16.7% without crop protection (oerke et al., 1994). only limited protection against pathogens can be achieved by chemical treatments or cultural practices (curtis et al., 2002). resistance breeding is a continuing and difficult process as resistance in most cases appears to be under polygenic control, and even when resistant cultivars are developed, they do not provide long-term relief due to ever-evolving or mutating pathogens (friesen et al., 2006). only modest progress has been made in engineering resistance to major fungal and viral pathogens of wheat. in an early study, the coat protein gene of barley yellow mosaic virus was introduced into wheat but no information was provided about its effect on protection from any pathogens (karunaratne et al., 1996.). however, transgenic plants expressing the rnc70 gene expressing a mutant bacterial ribonuclease iii showed a high level of resistance to barley stripe mosaic virus (zhang et al., 2001). wheat plants transformed with the viral replicase gene nib or the coat protein gene of wheat streak mosaic virus showed a high level of resistance to inoculations with two strains of the virus, had milder symptoms and lower virus titer than the control plants but did not provide field resistance to the virus and yielded less than their parent cultivars (sharp et al., 2002). the pina gene could be considered as a potential and effective for the control of a wide range of plant pathogens by genetic engineering. luo et al. (2008) produced transformed durum wheat cultivars expressing the pina protein (puroindoline protein), which has been shown to have antimicrobial and antifungal activity. transgenic plants showed enhanced response to leaf rust in greenhouse and field. transgenic wheat plants stably expressing an antifungal barley seed class ii chitinase gene (pr3) showed increased resistance to powdery mildew, but even more significant protection was obtained with the introduction of the gene for an apoplastic ribosome-inactivation protein (rip) from barley (yahiaoui et al., 2006). the expression of genes for an antifungal protein from aspergilus giganteum and a barley class ii chitinase were shown to significantly reduce the formation of powdery mildew and leaf rust (oldach et al., 2001). altpeter et al. (2005b) found enhanced nova biotechnologica 9-1 (2009) 35 resistance against powdery mildew in wheat plants over-expressing the defence-related tapero peroxidase gene in shoot epidermis. expression of the antifungal protein kp4 from ustilago maydis-infecting virus resulted in increased endogenous resistance against stinking smut. partial protection against fusarium head blight has been reported in wheat plants expressing the f. sporotrichioides gene fstri101 (vasil, 2007). much work still needs to be done to produce wheat lines that are resistant to various pathogens that cause major losses in productivity. the mapping of the leaf rust resistance gene lr10, of fhb1, a major gene controlling fussarium head blight resistance (cuthbert et al., 2006; chen et al., 2007), and the identification and characterization of the stripe rust resistance gene yr34 should help in the isolation and sequencing of the relevant genes that can be of much use in the development of resistant varieties through marker-assisted breeding as well as genetic transformation (cuthbert et al., 2006; lin and chen, 2008; šliková et al., 2009). actual worldwide crop losses to wheat productivity owing to a variety of pests (aphids, hessian fly, locusts, beetles, moths, etc.) are estimated to be 9,3%, but as high as 11,3% without crop protection (oerke et al., 1994). pests-resistant crops are expected to increase crop yields and also to reduce the amount of agrochemicals used for crop protection. pests cause additional losses during post-harvest storage. unfortunately, research on introduction of pest resistance genes into wheat has so far been very limited, because breeding for these traits is time consuming and does not provide long-term protection as the pests develop biotypes which are able to overcome the resistance genes. future work should explore the possible benefits of introducing resistance genes from related as well as unrelated species, such as the recent characterization and expression study of a novel wheat gene (hfr-3) encoding a putative chitin-binding lectin that is associated with resistance against hessian fly, a major pest that causes considerable damage (giovanini et al., 2007). altpeter et al. (1999) introduced the barley trypsin inhibitor cme (bti-cme) into wheat. expression and functional integrity of bti-cme in transgenic seed were demonstrated, along with a significant reduction in the survival of the angoumois grain moth (sitotroga ceralella), a major pest of stored wheat grain, reared on transgenic seed expressing bti-cme. similarly, wheat plants expressing the gene encoding snowdrop lectin (galanthus nivalis agglutinin: gna) have been shown to decrease the fecundity, but not the survival, of the grain aphid sitobion avenae (repellin et al., 2001). akhtar et al. (2008) conducted experimental trials to evaluate the resistance of host wheat plants against rhopalosiphum padi l. (aphid) and only one variety v-9021 was found to show the highest level of resistance. 5. abiotic stress tolerance abiotic stresses (drought, salinity, flooding, excessively high or low temperatures, high levels of minerals such as salt, heavy metals, etc.) cause adverse affects on plant growth that can reduce crop productivity in wheat by more than 80% (bray et al., 2000). so far the most successful approach to these problems has been to exploit natural variation present either in the crop itself or in wild relatives (for example, salt 36 šramková, z. et al. tolerance in laphopyrum elongatum, aluminium tolerance in aegilops uniarisfata). recent advances in understanding the genetic control of abiotic stress tolerance, including the identification and coning of related genes, has encouraged research in engineering plants that can tolerate these stresses without any negative impact on their yield (seki et al., 2003). although drought and salt tolerant genes that are present in germplasm of wheat and other crops have been successfully used to obtain stresstolerant varieties, breeding for stress tolerance is time labor-intensive and complicated by the multigenic nature of stress tolerance and the need for the simultaneous selection of the related genes as the elimination of the undesirable genes (vasil, 2007). plants are known to respond to biotic as well as abiotic stresses by inducing stressresponsive genes. several stress-inducible genes and their products have been identified (yamaguchi and blumwald, 2005). manipulation of the transcription of stress responsive genes is increasingly being used to enhance stress tolerance, such as drought resistance and salt tolerance in rice (hu et al., 2006). kawaura et al. (2008) found out, that approximately 19% of wheat genes are salt responsive. although salt-responsive genes of wheat were grouped into 12 groups based on expression patterns, their functions in salt tolerance remain to be clarified. introduction of the aba-responsive barley gene hva1 into wheat improved growth under soil-water deficit conditions. further field evaluations of some of the transgenic lines showed greater plant biomass and grain yield in plants expressing the hva1 gene in comparison to the non-transformed controls (bahieldin et al., 2005). improved tolerance to salinity has been also reported in wheat plants expressing a vacuolar na+/h+ antiporter gene (huang et al., 2006). much more attention needs to be paid now to the development of wheat varieties that can provide high yields under drought and at higher temperatures in saline soils (umezawa et al., 2006). peleg et al. (2008a) performed a comprehensive survey of wild emmer populations from across aridity gradient in israel and revealed a wide phenotypic and allelic diversity for drought response, demonstrating the potential of the wild genepool for wheat improvement. these results exemplify the unique opportunities to exploit favourable alleles that were excluded from the domesticated genepool and may serve as a start point for introgression of promising qtls into elite cultivated materials via marker-assisted selection. improvement of frost tolerance (winter hardiness) is an important aim of wheat breeding programs (dörffling et al., 2009). miller et al. (2006) have identified several c-repeat-binding factors (cbf) located at the frost tolerance locus fr-a’’’2 in triticum monococcum. these can be useful in heat improvement programs in regions where considerable yield losses occur during severe winters. in many parts of world, excessive deforestation causes frequent devastating floods resulting in significant crop losses when plants are submerged in water for long periods of time. recently, sub1a gene has been identified in rice and its overexpression in a submergence-intolerant variety conferred enhanced submergence tolerance to the plants (xu et al., 2006). it would be worthwhile to transfer this gene, that is responsible for submergence tolerance in rice, into wheat by genetic transformation to test if it will have a similar effect in wheat varieties that are grown in flood-prone areas. nova biotechnologica 9-1 (2009) 37 natural genetic variability has been exploited to standing of aluminium tolerance in wheat. however, further understanding of the genetic and physiological factors that regulate aluminium tolerance is needed in order to develop a wider range of wheat varieties with a high level of aluminium tolerance. transgenic plants over-expressing citrate synthase or malate dehydrogenase genes have been shown to enhance aluminium tolerance (magalhaes, 2006). the gene (altm1) that regulates aluminium tolerance in wheat has been identified (zhou et al., 2007). 6. yield increasing yield of wheat can be improved by increasing seed number and/or weight, the latter by increasing the amount of starch, which is the most abundant component (more than 70% of seed weight) of wheat endosperm, or by regulation of endosperm development. starch synthesis in cereals is regulated by adp-glucose pyrophosphorylase (agp), that is likely involved in determination of seed sink strength (hannah and james, 2008). smidansky et al. (2002) found that transgenic lines expressing a modified maize sh2 gene (sh2r6hs), which encoded an altered agp large subunit, showed a 38% increase in seed weight/plant and a 31% increase in total biomass. seed weight of inferior spikelets can be improved in rice panicle by increasing activities of starch synthesizing enzymes (mohapatra et al., 2009) the number of tillers formed on each plant is among many factors that determine yield in wheat (and in rice), by influencing the number and size of panicles and seeds produced. monoculm1 (moc1), a gene that controls tillering in rice, has been identified (li et al., 2003). changing the architecture of the plant by the formation of more tillers and leaves which are spread out (sakamoto et al., 2006; kuraparthy et al., 2007) would expose a larger leaf surface for the capture of sunlight for increased photosynthesis, leading to improved productivity. genes that regulate the activity of the major plant hormones (gibberellins, auxins, cytokinins and brassinosteroids) have been shown to be involved in dwarfing and grain production (ashikari et al., 2005; sakamoto, 2006). the increasing understanding of the genetic control of plant architecture (shoot branching, plant height, inflorescence morphology, etc.), and the effect of phytohormones on yield is expected to play a major role in the development of high yielding crops (sakamoto, 2006; wang and li, 2006). the element silicon is present in significant but variable amounts in all plants. it has many useful functions, including enhancing resistance to lodging, pests and pathogens. the varied roles of the silicon transporter gene provide ‘‘a new strategy for producing crops with high resistance to multiple stresses by genetic modification of the root’s silicon uptake capacity’’ (ma et al., 2006). the other possible approaches for yield improvement include: changing c3 wheat into a c4 plant (which is much more efficient because of greatly reduced loss of carbon by photorespiration) or the production of wheat hybrids (heterosis or hybrid vigor has been used to obtain dramatic increases in crop yields for nearly 75 years, most prominently in maize and recently also in rice) (wang et al., 2005) 38 šramková, z. et al. regulation of flowering and its manipulation must remain an important part of the overall strategy to improve wheat productivity. several genes (vrn1, vrn2 and vrn3) that have been shown to be involved in the vernalization response (the requirement for a long period of exposure to low temperatures for flowering) in wheat can be manipulated by rna interference and other means to affect flowering time, or to convert winter wheats to spring wheats (yan et al. 2006). 7. improvement of grain quality for most traditional uses, wheat quality derives mainly from two interrelated characteristics: grain hardness and protein content. grain hardness is a heritable trait but it can be strongly affected by abnormal weather conditions such as excessive rainfall during the harvest period. protein content is weakly heritable and strongly dependent on environmental factors such as available soil nitrogen and moisture during the growing season (belderok et al., 2000). in addition, each end-use requires a specific 'quality' in the protein. durum wheat cultivars have the hardest grain texture and are usually high in protein content. they are especially suited to the production of pasta because of their highly vitreous grain (high milling yield of semolina), unique combination of storage proteins for good cooking quality of pasta, and high yellow pigment content required for attractive appearance of cooked product. all three characteristics are highly heritable and can be readily improved by conventional breeding. recent research has shown that the presence of γ-gliadin 45 is a reliable marker of good cooking quality. this marker is now used for screening early generation material in many durum wheat breeding programs. bread (also common or hexaploid) wheats cover a wide range of grain hardness and protein content. the hardest wheats of this class, generally highest in protein, are used for pan bread. common wheats of medium hardness and lower protein content are used for other types of bread and noodles. wheats with softest texture and lowest protein are used for cakes and cookies. in some end-uses, e.g., chinese-type noodles, starch quality is important together with protein quality; this feature should be taken into consideration in developing a screening strategy for wheats for this application. screening tests that reflect end-use requirements for most of the known products are available, and should be applied in testing wheats according to intended use (bushuk, 1998). an important factor, which affects negatively the grain quality, is sprouting. the germination causes an increase in alpha amylase, an enzyme that breaks down starch. sprouting can affect food made from wheat in many ways. it can reduce mixing strength, cause sticky dough, and affect loaf volume and shelf life. in pasta, sprouting can reduce shelf life, increase cooking loss, and produce softer cooked pasta. flour damaged by alpha-amylase holds less water when mixed and the dough absorbs less water during baking. “falling number” gives an indication of the amount of sprout damage that has occurred within a wheat sample. as the amount of enzyme activity increases, the falling number decreases. generally, a falling number value of 350 seconds or longer indicates a low enzyme activity and very sound wheat quality. as the pre-harvest sprouting (phs) of wheat greatly reduces the quality and economic value of grain, phs tolerance is one of the most important traits in wheat breeding. viviparous 1 (vp1) gene of maize is known to encode a transcription factor vp1 that nova biotechnologica 9-1 (2009) 39 controls seed germination. hexaploid wheat possesses three vp1 homoeologues (tavp1): tavp-a1, tavp-b1 and tavp-d1. the sequence analyses of cdnas revealed that some of tavp-a1 transcripts and tavp-d1 transcripts are spliced incorrectly, resulting in production of truncated or deleted proteins. most tavp-b1 transcripts are spliced correctly. the dual function of tavp-b1 was confirmed: the activation of em expression (required for aba-inducible expression) and the repression of α-amylase expression (utsugi et al., 2008). recently, a co-dominant sts marker of vp-b1 gene was developed and designated as vp1b3. statistical analysis indicated that vp1b3 was strongly associated with phs tolerance in the set of chinese wheat germplasm, suggesting that vp1b3 could be used as an efficient and reliable co-dominant marker in the evaluation of wheat germplasm for phs tolerance and marker-assisted breeding for phs tolerant cultivars (yang et al., 2008; xia et al., 2009). among all the cereals, only the flour of hexaploid wheat (triticum aestivum) is able to form dough that exhibits the rheological properties required for the production of leavened bread. this property results from the ability of wheat storage proteins, gliadins and glutenins, to form special protein complex known as gluten. biochemical and genetic evidence has demonstrated that high molecular weight glutenin subunit (hmw-gs) plays a major role in determining the viscoelastic properties thereby determining bread making qualities. the hmw glutenins are necessary to create strong dough, which is essential for making high quality, yeast-raised breads. the hmw-gs are further subdivided into high mr, x-type and low mr, y-type subunits. two hmw-gs alleles, which are inherited as tightly linked pairs encoding an x-type and a y-type subunit, are present at the glu-1 locus on the long arms of the homoeologous chromosomes 1a, 1b and 1d of all hexaploid bread wheat cultivars (payne, 1987). all cultivars of wheat, therefore, contain six hmw-gs genes, but only three, four or five subunits, because some of the genes are silent; the 1ay gene is silent in all cultivars. hmw-gs may represent up to 10% of the total seed protein, as each hmw-gs accounts for about 2% of the total extractable protein. the hmw-gs alleles 1ax1, 1ax2* and the 1dx5 + 1dy10 subunit pair are said to be associated with stronger doughs and better baking properties, and 1dx2 + 1dy12 pair with weaker doughs (vasil and anderson, 1997). the number and composition of hmw-gs present in a cultivar are closely related to the quality of its gluten or dough (payne, 1987). considerable progress has been achieved in research of the molecular properties of flour proteins that are required for highest bread quality. segregating breeding populations can be screened by electrophoresis or high performance liquid chromatography for the presence of desirable glutenin subunits. however, because of the tight linkage of the hmw-gs alleles, it is quite difficult to manipulate them by traditional breeding methods. determination of technological quality of wheat cultivars according to analysis of hmw-gs reveals a successful progress in slovak wheat breeding and its successes in creating cultivars with high bread-making quality. this can be seen particularly by allele distribution at glu-b1 and glu-d1 loci where hmw-gs having a negative impact on quality, mainly alleles at the glu-b1 6+8 locus and glu-d1 2+12 locus, occur at a substantially lower rate than in west european cultivars (gregová et al., 2007; šramková et al., 2008). 40 šramková, z. et al. a number of hmw-gs as well as low-molecular-weight glutenin subunit (lmwgs) genes have been introduced into bread and pasta wheat by genetic transformation (tosi et al., 2005; bregitzer et al., 2006; blechl et al., 2007). the hmw-gs 1ax1 gene which is known to be associated with superior bread making quality was introduced into the cultivar bobwhite lacking this gene (altpeter et al., 1996). the transgenic plants expressing the introduced hmw-gs were normal, fertile and showed mendelian segregation of the transgene. the effect of hmw glutenin subunits 1ax1 and/or 1dx5 on dough elasticity was demonstrated by introducing the corresponding genes into durum wheat and triticale; cereals with poor bread making properties. transgenic durum wheat producing one additional hmw glutenin, 1ax1 or 1dx5, gave an increased dough strength and stability, thus changing the properties of durum flour for both bread and pasta making (he et al., 1999). alvarez et al. (2000) introduced the hmw-gs genes 1ax1 and 1dx5 into a commercial cultivar of t. aestivum that already expresses five subunits. the overexpression of 1dx5 gene increases the contribution of the hmw-gs to a level of 22% of the total protein content. however, the overexpression of the 1dx5 gene was found to be associated with a dramatic increase in dough strength by making it too strong, and, therefore, unsuitable for use in conventional breadmaking (blechl et al., 2007). wheat landraces represent interesting biological material because of their genetic variability. some of them possess genes, not occurring in modern cultivars, although these genes can be valuable for improvement of their quality. thus, screening landraces for the novel hmw-gs alleles for further utilization has become a part of some breeding programs (gregová et al., 1999; gregová et al., 2004; gregová et al., 2006). tosi et al. (2005) reported the production of transgenic wheats expressing additional lmw subunits in the commercial durum wheat varieties. the gene constructs used in this work were derived from glu-a3 and glu-b3 loci. expression levels ranged up to those of the major endogenous lmw subunits. their incorporation into polymers was demonstrated. however, co-suppression of the major endogenous hmw subunit was observed, leading to reduced dough strength in comparison with line, which did not exhibit co-suppression, having increased strength. identification and characterisation of novel lmw-gs suggests that there is intensive research in this area and improvement of wheat quality is in progress (an et al. 2006; zhao et al. 2006; zhao et al. 2007). in near future, molecular approaches including genetic transformation and markerassisted selection will provide an opportunity for improving further the wheat processing qualities by introduction of genes associated with good bread-making qualities into a cultivar which is agronomically desirable but which has poor breadmaking qualities. this would avoid or minimize the necessity of blending flour from different cultivars in milling operations. 8. increasing of healthful components hunger and malnutrition are among the most devastating problems affecting a large part of the world’s population. the nutritional value of wheat is extremely nova biotechnologica 9-1 (2009) 41 important as it takes an important place among the few crop species being extensively grown as staple food sources. the importance of wheat is mainly due to the fact that its seed can be ground into flour, semolina, etc., which form the basic ingredients of bread and other bakery products, as well as pastas, and thus it presents the main source of nutrients such as proteins, carbohydrates, lipids, fibre and vitamins, to the most of the world population. however, the total content or absolute concentration, of a given nutrient in a food is not always a reliable indicator of its useful nutritional quality, because not all of the nutrients is absorbed (paredes-lópez and osunacastro, 2007). one of the continuing criticisms of modern crop varieties, that have been commercialized thus far, is that although they do have many desirable attributes, none of them offer any direct, tangible benefits to the consumer. this situation is changing rapidly with increasing understanding of the molecular and genetic control of various aspects of plant growth and development, which has made it possible to enhance the quality as well as the quantity of proteins/starches/oils, and the content of vitamins, essential amino acids, minerals and other healthful components of plants. although not much work of this nature has been carried out in wheat, there is no reason why the success which has been achieved in crops such as rice, maize and soybean, or demonstrated in model species such as arabidopsis, cannot be extended to wheat (vasil, 2007). protein quality is based on their amino acid composition (particularly their relative content of essential amino acids) and their digestibility. therefore, high quality proteins are those that are easily digested and contain the essential amino acids in quantities that correspond to human requirements. deficiency in certain amino acids reduces the availability of others present in abundance. in general, cereal proteins are low in lysine (1.5–4.5% vs. 5.5% of who recommendation), tryptophan (trp, 0.8– 2.0% vs. 1.0%), and threonine (thr, 2.7–3.9% vs. 4.0%). because of this deficiency, these essential amino acids (eaas) become the limiting amino acids in cereals. it is thus of economic and nutritional significance to enhance the eaas in plant proteins. in the past, plant geneticists and breeders have made much effort to improve the quality of plant proteins. natural mutations such as high-lysine corn and barley have been identified and developed into elite genotypes (bright and shewry, 1983). unfortunately, undesirable traits such as greater susceptibility to diseases and pests and lower yields were associated with these mutations. the correlation between nutritional quality and yield has been a serious issue over the years, since the two factors appear to be negatively correlated. this problem appears to have been overcome since the introduction of modifier genes (mo2 genes) that changed the opaque-2 phenotype of the maize seed, thus allowing wild-type-like seed characteristics to be maintained, resulting in normal yield but conserving the high lysine and high tryptophan concentrations (gaziola et al., 1999). these new maize lines have been designated qpm (quality protein maize) and several hybrids were produced and introduced into the market. however, the widespread use of these varieties has not been as fast as initially expected. apart from the qpm lines, it would appear that very little else in the way of high-lysine crops is available. perhaps recent legislation and general concern about the use of modified genetic organisms have been the major setback regarding the release of such crops (ferreira et al., 2005). 42 šramková, z. et al. for a long time there has been much interest in developing high-amylose wheat as a source of resistant starch (rs), which is one of the major sources of dietary fiber and its many related benefits (e.g., prevention of coronary heart disease, cancers of the colon and rectum, diabetes) to humans. suppression of starch branching enzyme ii (sbeiia and sbeiib) expression by rna interference was used to produce highamylose wheat which was shown to be healthful for rats (regina et al., 2006). the (1→3;1→4)-β-d-glucans, found exclusively in the cell walls of cereal and grass species, are important components of dietary fiber and highly beneficial in the prevention and treatment of serious human health conditions, including colorectal cancer, high serum cholesterol and cardiovascular diseases, obesity and non-insulindependent diabetes. β-d-glucans were shown to have immunostimulating activity (dalmo and bøgwald, 2008). genes responsible for (1→3;1→4)-β-d-glucan synthesis in grasses have been identified and provide an excellent opportunity to enhance the dietary fiber content of cereal and other food crops through transformation (burton et al., 2006). in attempting to enhance micronutrient levels in wheat through conventional plant breeding, it is important to identify genetic resources with high levels of the targeted compound, to consider the heritability of the targeted traits, to explore the availability of high throughput screening tools and to gain a better understanding of genotype by environment interactions (ortiz-monasterio et al., 2007). a well-known success of genetic transformation in cereals represents the development of the golden rice. it was first engineered with the insertion of the psy gene from daffodil (narcissus pseudonarcissus) and the bacterial phytoene desaturase (crti) gene from erwinia uredovora (ye et al., 2000). bacterial crti can catalyze three enzymatic steps from phytoene to all-trans-lycopene. the psy gene is under the control of an endosperm-specific glutelin promoter. to localize the product in plastids, crti was designed as a fusion with the ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) small subunit. an alternative construct was made by co-transformation with constructs carrying the psy/crti gene as described above and the lcy gene under the control of a glutelin promoter. by the latter approach, the carotenoid content of edible rice endosperm was 1.6 μg/g dry weight (ye et al., 2000). however, in 2005, golden rice2 was developed and the carotenoid content was increased up to 23fold (37 μg/g of dry weight) compared to the original golden rice. this content is close to a realistic level for palliating vad (vitamin a deficiency) in children (paine et al., 2005). expression of carotenoid biosynthetic genes in other cereals, such as wheat, requires further scientific investigation. ehrenbergerová et al. (2006) presented data indicating significant effects of cropping system, genotype, and year grown on the tocol levels in barley. the results suggest that a selective breeding program using the best genotypes would be beneficial to produce food barleys with higher levels of total tocols and tocotrienols. the international maize and wheat improvement center, along with its many partners, has identified several maize and wheat varieties with 25% to 30% higher grain iron and zinc concentrations. wild relatives of wheat have been found to contain some of the highest iron and zinc concentrations in the grains. backcrossing to bread nova biotechnologica 9-1 (2009) 43 wheat could result in highly nutritious cultivars (ozturk et al., 2006; peleg et al., 2008b). uauy et al. (2006) have characterized and cloned gpc-b1, a quantitative trait locus from wild emmer wheat that is associated with increased levels of grain protein, zinc and iron as a consequence of accelerated senescence and increased nutrient mobilization from leaves to the developing grains. in ancestral wild wheat the allele encodes a nac transcription factor (nam-b1), while only a non-functional nam-b1 allele is present in modern cultivated wheat varieties. silencing of the multiple nam homologues by rna interference in transgenic plants caused delayed maturation and reduced grain protein, iron and zinc content by more than 30%. the cloning of gpc-b1 provides a direct link between the regulation of senescence and nutrient remobilization and an entry point to characterize the genes regulating these two processes. this may contribute to their more efficient manipulation in crops and translate into food with enhanced nutritional value. phosphate, which is stored in the form of phytic acid in plant seeds (including wheat), is indigestible in monogastric animals including humans due to the fact that they lack phytases which degrade phytic acid in the digestive tract. transgenic wheat plants expressing the aspergillus niger phytase encoding gene phya accumulate phytase in their seed (brinch-pedersen et al., 2003; brinch-pedersen et al., 2006). further improvement in the expression and thermostability of phytases in transgenic wheat plants has the potential to increase the bioavailability of zn2+, ca2+ and fe2+ by breaking down their otherwise indigestible complexes with phytic acid. guttieri et al. (2007) identified a wheat mutant (lpa1-1) with reduced phytic acid phosphorus and increased inorganic phosphorus (pi). during germination there is a large decrease in phytine, which can make up to 80% of the total phosphate found in seeds (reddy et al., 1982) and a concomitant increase of pi suggesting that phytin acts as a storage pool of phosphate during germination. thus, it can be disputable whether the phytine decrease could result in growth disorders of the wheat plant and negatively affect all the metabolic reactions where phosphorus is a limiting factor (biosynthesis of nucleic acids, phospholipids, proteins and other energy-generating processes). however, genetically modified, low-phytic acid strains of maize having approximately 35% of the phytic acid content of wilde-type maize (wtm), did not exhibit substantial differences in growth as well as micronutrient and mineral concentrations when compared to wtm. moreover, absorption of iron from tortillas prepared from transgenic maize was nearly 50% greater than from normal tortillas (mendoza et al., 1998; shi et al., 2005). the world's agricultural community should adopt plant breeding and other genetic technologies to improve human health, and the world's nutrition and health communities should support these efforts. sustainable solutions to the enormous global problem of 'hidden hunger' will not come without employing agricultural approaches (welch and graham, 2004.). biofortification has the potential to contribute to increased micronutrient intakes and improved micronutrient status. the success of this strategy will require the collaboration between health and agriculture sectors (hotz and mcclafferty, 2007; cakmak, 2008). 44 šramková, z. et al. references akhtar, n., anwar, m. b., jilani, g., javed, h. , yasmin, s., begum, i.: resistance to foliage feeding aphid in wheat. pak. j. biol. sci., 11, 2008, 801-804. altpeter, f., baisakh, n., beachy, r., bock, r., capell, t., christou, p., daniell, h., datta, k., datta, s., dix, p.j., fauquet, c., juany, n., kohli, a., mooibroek, h., nicholson, l., nguyen, t.t., nugent, g., raemakers, k., romano, a., somers, d.a., stoger, e., nigel, t., visser, r.: particle bombardment and the genetic enhancement of crops: myths and realities. mol. breed., 15, 2005a, 305-327. altpeter, f., diaz ,i., mcauslane, h., gaddour, k., carbonero, p., vasil, i.k.: increased insect resistance in transgenic wheat stably expressing trypsin inhibitor cme. mol. breed., 5, 1999, 53-63. altpeter, f., varshney, a., abderhalden, o., douchkov, d., sautter, c., kumlehn, j., dudler, r., schweizer, p.: stable expression of a defense-related gene in wheat epidermis under transcriptional control of a novel promoter confers pathogen resistance. plant. mol. biol., 57, 2005b, 271-283. altpeter, f., vasil, v., srivastava, v., vasil, i.k.: integration and expression of the high-molecular-weight glutenin subunit 1ax1 into wheat. nat. biotechnol., 14, 1996, 1155–1159. alvarez, j.b., d'ovidio, r., lafiandra, d.: comparison of allelic x-type genes present at the glu-d1 locus in bread wheat by pcr and sequence analysis of their n-terminal domain. j. genet. breed., 51, 1997, 161-166. an, x., zhang, q., yan, y., li, q., zhang, y., wang, a., pei, y., tian, j., wang, h., hsam, s.l., zeller, f.j.: cloning and molecular characterization of three novel lmw-i glutenin subunit genes from cultivated einkorn (triticum monococcum l.). theor. appl. genet., 113, 2006, 383-95. anonymous: slovak national list of released varieties. at publishing, bratislava, 198, 2008, 25-26. ashikari, m., sakakibara, h., lin, s., yamamoto, t., takashi, t., nishimura, a., angeles, e.r., qian, q., kitano, h., matsuoka, m.: cytokinin oxidase regulates rice grain production. science, 309, 2005, 741-745. bahieldin, a., mafouz, h.t., eissa, h.f., saleh, o.m., ramadan, a.m., ahmed, i.a., dyer, w.e., elitriby, h.a., madkour, m.a.: field evaluation of transgenic wheat plants stably expressing the hva1 gene for drought tolerance. physiol. plant., 123, 2005, 421-427. belderok, b., mesdag, h., donner, d. a.: bread-making quality of wheat. springer. p. 3. 2000. bicar, e., h., woodman-clikeman, w., sangtong, v., peterson, j. m., yang, s. s., lee, m., scott, m. p. transgenic maize endosperm containing a milk protein has improved amino acid balance. transgenic res., 17, 2008, 59-71. blechl, a., lin, .j, nguyen, s., chan, r., anderson, o.d., dupont, f.m.: transgenic wheats with elevated levels of dx5 and/or dy10 high molecular nova biotechnologica 9-1 (2009) 45 weight glutenin subunits yield doughs with increased mixing strength and tolerance. j. cereal sci., 45, 2007, 172-183. bray, e.a., bailey-serres, j., weretilnyk, e. responses to abiotic stresses. 2000. in: buchanan, b., gruissem, w., jones, r. 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451-60. zhou, h., berg, j.d., blank, s.e., chay, c.a., chen, g., eskelen, s.r., fry, j.e., hoi, s., hu, t., isakson, p.j., lawton, m.b., metz, s.g., rempel, c.b., ryerson, d.k., sansone, a.p., shook, a.l., starke, r.j., tichota, j.m., valenti, s.a.: field efficacy assessment of transgenic roundup ready wheat. crop sci., 43, 2003, 1072-1075. zhou, l., bai, g., ma, h., carver, b.f. quantitative trait loci for aluminum resistance in wheat. mol. breed.,19, 2007, 153–161. microsoft word polivka nb.doc nova biotechnologica vii-i (2007) 69 bioeconomy and white biotechnology as a basic pillar of the lisbon strategy ľudovít polívka, eva ürgeová department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (ludovit.polivka@ucm.sk) abstract: the entrance into the new millennium is branded by intensive development of science and new technologies. life science and biotechnologies are widely recognize to be, after ict, the parallel wave of knowledge – based economy, creating new opportunities for our society and economies. this application is the basic object of lisbon strategy in europe. this new trends, to make full use of biotechnology for sustainable economy, is official titled as bioeconomy around the world. the characteristics of bioeconomy and utilization of industrial biotechnology are presented in this article. key words: bioeconomy, lisbon strategy, biotechnologies 1. introduction the development of the world society since the last decades of the past century and the beginning of a new millennium has been characterised by several specific factors (safranski, 2006): the process of globalisation has come into the next phase, defined as a stage of fast economic growth, yet accompanied by the world global problems. many of them cannot be resolved by individual nation-states acting alone: deterioration of soil, air and water pollutions, global warming, exploitation of natural resources, problems connected with the world population growth to name the most essential ones. contemporary and future economic and social growth is conditioned by the building of a knowledge-based economy. the lisbon strategy is a paradigm for europe. the knowledge and operative introduction of new scientific results into practice have become the basic pillars for further development. in connection with the environmental pollution, depletion of natural resources, accumulation of wastes and other negative anthropogenic activities, exigency of sustainability and sustainable development have come to the forefront of progress. sustainable development aims to reduce the environmental pollution and decrease energy and resources consumption. in order to be sustainable, new technologies must be economically feasible and socially responsible in addition to being environmental friendly – they must present a cost advantage, monetary or other, before they can be accepted by the industry. a new phase of scientific and technical revolution came after automatisation, regulation and robotisation started three decades ago. it is a stage of biological revolution, nanotechnology and ict (information and communication technologies). 70 polívka,ľ. and ürgeová, e. from this short characteristics of the contemporary and future development follows that the knowledge and new scientific results applicable in practice are motive forces for further growth of economy, decrease of environmental pollution and sustainment of natural resouces. 2. the lisbon strategy and european technology platforms the lisbon strategy, also known as the lisbon process, is an action and development plan for the european union. the main goal of this strategy is clear – to make the eu “the most competitive and dynamic knowledge–based economy capable of sustainable economic growth with more and better jobs and greater social cohesion” (http://www.svez.gov.si/en/higlights/ lisabon_strategy; 2000, http://www.europsky parlament.sk, 2004). in a knowledge-based society, the knowledge is understood as “the knowledge triangle which refers to the interaction between research, education and innovation”. the main priority areas are: investment in the network of knowledge (3 % of gdp) strengthening competitiveness in industry and entrepreneurial environment informative society increasing the labour market the eu commission is active in a number of initiatives which will give a boost to the bio-based economy. some of the most recent initiatives include the new seventh framework research programme; environmental technology action plan (etap); and european technology platforms. 2.1 european technology platforms a) the european commission has established a helpful new mechanism for fostering important areas where research, technology and development are key to addressing major economic, technological or societal challenges, the technology platforms (tp). these can enable the formation of strategic alliances to foster publicprivate partnerships between the research community, industry and policy makers. the intention is to stimulate effective investment in r&d, accelerate innovation and remove barriers to growth. at the same time, they provide an important output to national and eu policy makers. participation in the technology platform should include the research community, industry (including small and medium-sized enterprises – sme’s, and private research and technology transfer firms), public authorities (e.g., policy makers, regulators, and purchasers), the financial community, consumers, civil society groups, and other relevant stakeholders (http://www.bioeconomy.net/centre3europ.html, 2006). b) the expectation is that each technology platform should: • provide a common vision that contributes to coherent policy making, • overcome obstacles at all levels to accelerate market penetration of new technologies, nova biotechnologica vii-i (2007) 71 • stimulate knowledge and innovation, thereby increasing productivity and competitiveness and making the investment climate more attractive, • encourage public debate on risks and benefits to facilitate technology acceptance. to accent the importance of knowledge and continual application of the newest results of biotech research in the practice of economy, the conception of knowledgebased bioeconomy (kbbe) has been used since 2005. the content and meaning of kbbe are known as the “cologne papers”. the most important technological platforms are: eumat – engineering materials and technologies artemis – embedded computing systems plants – plants for the future suschem – sustainable chemistry food – food for life nanomedicine nanotechnology in medical applications ectp – european construction technology platform foresty – forest-based sector technology platform industrial safety – industrial safety etp europ – robotica bf tp – biofuel tp hfp – hydrogen and fuel cell technology platform wss tp – water supply and sanitation technology platform ftc – future textiles and clothing emobility – mobile and wireless communications c) the development of each technology platform consists of three phases (meadowerolt et al., 2005): 1. characterisation of the current situation and the vision for future determination of the participants of a platform – alliance of academic research, production, stakeholders, government, consumers. 2. strategic research agenda – research and development priorities from the medium to long-term, measures for the networking of research capacity and resources. 3. develop an action plan, encourage implementation time relations, barriers, public awareness, public acceptance. 3. biotechnology and bioeconomy the term “biotechnology” was first used by karoly ereky in 1919, to mean “all lines of work by which products are produced from raw materials with the aid of living organisms”. the definition has been broadened slightly to include producing things with the aid of substances from living organisms and some raw materials that are produced by living organisms themselves, and narrowed to focus on new technologies, rather than traditional production. there have been proposed several definitions of biotechnology up to the present time. their content has reflected the new achievements and knowledge of life sciences in a concrete period of time, at a particular stage of development. it is possible to find 72 polívka,ľ. and ürgeová, e. the basic philosophy of the word “biotechnology” – pragmatic aspects of science and technology, making use of our knowledge about living systems in practical applications. we have witnessed extraordinary advances in natural sciences in general, and life sciences in particular, especially since the middle of the last century. the most important achievements have affected the fields of genetics, molecular biology and biochemistry (elucidation of a chemical structure of genetic materials, understanding of chemical principles of heredity, solving of principal arrangement of genetic codes, mechanisms of synthesis and degradation of dna, and proteosynthesis). the growth of such knowledge has engendered the development of several sophisticated biochemical and genetic methods which enabled a new insight into the mystery of living cells (barnett, 2005). these methods have become the basic tools for genetic and biochemical modification of cells, or biological living systems. the most revolutionary of these methods became the recombinant dna technology, genetic engineering r-dna technology. it was firstly implemented by s. cohen and h. boyer in 1975. the new biosystems or their metabolites can be created by this method, incorporating segments of dna from one organism into the cells of another organism. the first biotech company was established in 1976 – genetech (gen–etic engineering technology). the first biotechnological products – human growth hormone and insulin, were produced between 1979 and 1980. the first genetic engineering of plants and mammalia was carried out in the eighth and ninth decades of the last century. the human genome project was launched in 1990. in connection with these discoveries and their real utilisation in practice, the character of definition of biotechnology was accepted. for the purpose of this article, the definition according to the agenda 21 is introduced. “biotechnology engineering, a knowledge–intensive field, is a set of enabling techniques for bringing about specific man–made changes in deoxyribonucleic acid or genetic material in plants, animals and microbial systems, leading to useful products and technologies.” from this point of view, biotechnology possesses a big potential for not only fermentation industries but for the whole economic sector. since the beginning of 1975, the development of biotechnology has been very rapid. the following table (tab. 1) shows a survey of the european biotechnology situation in the year 2004 compared to 2003 (ratzinger, 2005). tab 1. survey of the european biotechnology situation in the year 2004 compared to 2003 measure 2004 2003 companies 2163 2200 employed 96500 96000 in r&d 42500 41000 r&d spend €7.6 bn €7.6 bn revenue €21.5 bn €20.5 bn new firms 119 132 nova biotechnologica vii-i (2007) 73 biotechnologies are the basic pillar of several important european technological platforms, for example: it clearly follows from this scheme that: biotechnology has been applied in several sectors of economy biotechnology has become a driving force of sustainable economic growth meeting the goals of etp, in which biotechnologies are included, is determined by the new results of integrated scientific disciplines and bioscience. the new expression for infiltration of biotechnology into economic sectors – bioeconomy – has been used since 2003 (http://www.oecd.org, 2005). bioeconomy – bio-based economy is a term which (gavrilescu and chisti, 2005): 1. encapsulates our vision of the future society that is no longer wholly dependent on fossil fuel energy and non-renewable raw materials. 2. involves all industries, agricultural and economic sectors that produce, manage or otherwise exploit biological resources (including gmo’s) using environmentally acceptable technologies. very often the 4 “r’s” of sustainable bioeconomy are published: 1. replace fossil-based energy, chemicals and materials with renewable biomass through the new innovative technologies and appropriate policies. biotechnology engineering materials and technologies plants for the future sustainable chemistry food for life nanotechnology in medical application forestry future textiles and clothing biofuel hydrogen and fuel cells 74 polívka,ľ. and ürgeová, e. 2. reduce greenhouse gases and other emissions associated with biosystems by improved management strategies, technologies, and policies. 3. remove atmospheric co2 by developing new plant genotypes and improved management practices for building healthy, energy-rich ecosystems. 4. respond to global atmosphere and climate changes by implementing technologies and management strategies which are environmentally acceptable. looking to the future, new techniques in biotechnology (analytical method and the relating equipment, achievements of new scientific disciplines) will continue to converge with other technologies resulting in potentially large-scale changes in global economies in the next thirty years. from this point of view, bioeconomy is part of strategic development on the base of sustainability. 4. colours of biotechnology and white biotechnology the new system for designation of biotechnology activities – according to the colour type – was accepted at the beginning of a new millennium. according to the authors, the colour index may be a useful guide with further additions, as biotechnology and colours intertwine over time in promoting public perception and understanding of biotechnology applications for the cause of science, development, and the current and post-human future of humankind (dasilva, 2004). tab. 2 colour type of biotechnology and area of activities colour type area of biotech activities red health, medical diagnostics yellow food biotechnology, nutrition science blue aquaculture, coastal and marine biotech green agricultural, environmental biotechnology – biofuels, biofertilisers, bioremediation, geomicrobiology brown arid zone and desert biotechnology dark bioterrorism, biowarfare, biocrimes, anticrop warfare purple patents, publications, inventions, iprs white gene-based bioindustries gold bioinformatics, nanobiotechnology grey classical fermentation and bioprocess technology these colours designations are widely used at the present time. white biotechnology white or industrial biotechnology is the application of biotechnology in processing and production of chemicals, materials, and energy. the whole cell system, gmo’s, enzymes, tailorised enzymes, and synthetic cells – the products of synthetic and systems biology are used or will be used. white biotechnology is by nature a multidisciplinary area, combining the knowledge of different scientific disciplines. industrial biotechnology is the basic nova biotechnologica vii-i (2007) 75 pillar of the european technological platform – sustainable chemistry. together with materials technology and reaction, the process design forms a complete technological platform. it is estimated that biological processes will account for 10 – 20 % of production across the whole chemical industry by the end of 2010. the strategic research agenda includes the following main research areas: novel enzymes and microorganisms microbial genomics and bioinformatics metabolic engineering and modelling biocatalyst function and optimisation biocatalytic process design innovative fermentation science and engineering innovative down–stream processing fig. 1. three dimensions of sustainable development the action plan – policy measures to provide incentives to reduce barriers and to raise public awareness and encourage public acceptance of industrial biotechnology were published in august 2006. a vision for industrial biotechnology in europe – the year 2025 1. industrial biotechnology will enable a range of industries to manufacture products in an economically and environmentally sustainable way. 2. increasing numbers of chemicals and materials will be produced using biotechnology in one of its processing steps. biotechnological processes environment reduction: greenhouse gas emissions, wastes water, air & soil pollutions, use of non-renewable resources society employment: creation of new jobs, especially in rural areas innovation: new products, new technologies save valuable resources for future generations, improvement of quality of life economy cost reduction: raw materials technological and process costs investment additional revenues for new products, value added processes, growth of competition 76 polívka,ľ. and ürgeová, e. are used for producing chemicals and materials otherwise not accessible by conventional means or in a more efficient way. 3. biotechnology will allow an increasingly eco-efficient use of renewable resources as raw materials for industry. 4. biomass-derived energy based on biotechnology will be expected to cover an increasing amount of our energy needs. 5. rural bio-refineries could be a source of important chemical products as a raw material for chemicals. 6. green biotechnology will make a substantial contribution to the efficient production of raw materials. 7. european industry will be innovative and competitive, it will sustain cooperation and support between the academic community, industry, agriculture, and society. white biotechnology has the potential to improve industrial production along all three dimensions of sustainable development (fig. 1) (http://www.suschem.org , 2005). 5. conclusions the potential of white biotechnology is very promising and it is expected that industrial application of biotechnology will be a key technology contributing to the achievements of the lisbon strategy to make europe the most competitive and dynamic knowledge-based economy in the world on the principles of sustainability. the contemporary “bio-economisation” is a specific symptom characterising the processes of the globalisation of the world economy. references barnett, t. p. m.: a. bluepnint for action – a future worth creating. ed. g. p. putman s son, n.y. 2005 isbn 019 – 852 – 498 – 6 dasilva, e.: the colours of biotechnology: science, developnet and flumankind elektron. j. biotechnol 7, 3, 2004, 1-2 european technology platform, 2006, http://www.bio-economy.net/ centre3europ.html gavrilescu, m., chisti, y.: biotechnology – a sustainable alternative for chemical industry, biotechnol. adv., 23, 2005, 471-499 lisabon strategy, 2000, http://www.svez.gov.si/en/higlights/ lisabon_strategy, lisabonská strategia, 2004, http://www.europskyparlament.sk meadowerolt, j., farrel, k., spangerbery, j.: developing framework for sustainability in the eu. int. j. sustain. dev., 8, 2005, 3-9 ratzinger, j.: európa. ssv trnava, 2005, isbn 80 – 7162 – 570 – 1 safranski, r.: koľko globalizácie znesie človek ed. kaligram bratislava 2006; isbn 80 – 7149 – 858 – 8 the bioeconomy in 2030, apolicy agenda.oecd 2005, http://www.oecd.org the vision for 2025 and beyond, suschem, 2005, http://www.suschem.org 130 nova biotechnologica et chimica 13-2 (2014) doi 10.1515/nbec-2015-0003 © university of ss. cyril and methodius in trnava electrochemical studies of interactions between fe(ii)/fe(iii) and amino acids using ferrocene-modified carbon paste electrode jaroslav vatrál, roman boča department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (jvatral@gmail.com) abstract: the electrochemical behavior of an fe(ii)/fe(iii) redox couple in the presence of various selected amino acids has been studied using ferrocene-modified carbon paste electrode at ph = 7.4. because of fe(ii)/fe(iii) solubility issues at physiological ph, ferrocene was used as a source of iron. anodic oxidation of iron (ph = 7.2) occurred at 0.356 v and cathodic oxidation at 0.231 v, both vs ag|agcl. treatment of the voltammetric data showed that it was a purely diffusion-controlled reaction with the involvement of one electron. after addition of amino acids, potential shifts and current changes can be observed on the voltammograms. cyclic voltammetry experiments revealed the capability of amino acids to change the electrochemical behavior of the fe(ii)/fe(iii) redox couple. key words: iron, amino acid, cyclic voltammetry, ferrocene, carbon paste electrode 1. introduction iron has long been implicated in neurodegenerative disease through its redox transitions in vivo. the consequential generation of oxygen free radicals can further induce oxidative stress in tissues (winterbourn, 1995; lloyd et al., 1997; zatta, 2003; valko et al., 2005). abnormally high levels of iron and oxidative stress have been found in neurodegenerative disorders such as alzheimer and parkinson diseases, multiple system atrophy, and progressive supranuclear palsy (smith et al., 1997; sayre et al., 2000; arreguin et al., 2009). this evidence for misregulation of iron concentrations in neurodegenerative diseases highlights the need to understand interactions of endogenous biological important substances, like amino acids, with iron (garcía et al., 2012). the most important mammalian iron storage protein is ferritin, widely distributed in nature; it sequesters a large amount of iron in the protein interior. the diameter of the roughly spherical protein interior is about 6 nm and, when filled to capacity, the crystalline polymeric iron core can accommodate up to 4500 iron atoms (watt et al., 1985; jameson and linert, 1997; bou-abdallah, 2010). unfortunately, a limitation of the electrochemical approach, in the case of studying interactions between fe(ii)/fe(iii) and amino acids is that it cannot be applied at physiological ph values because of the insolubility of fe(ii)/fe(iii) above ph 4.0. carbon paste electrodes (cpes) are inherent part of electroanalytical chemistry due to their unique properties such easy surface renewing or applicability in inorganic, organic and biological analysis (švancara et al., 2012). carbon pastes can easily bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:26 utc nova biotechnologica et chimica 13-2 (2014) 131 be modified by addition of a solid compound and then, they are called “modified cpes”. ferrocene (fc) and its derivatives are widely used in electrochemistry because of their good stability in solution and rapid response to many substances (kamyabi and aghajanloo, 2009). fig. 1. selected amino acids classified according to their chemical properties. the aim of this study is to investigate a redox behavior of fe(ii)/fe(iii) in the presence of selected amino acids (fig. 1) using cyclic voltammetry in order to assess a degree of risk that the agent will interact with available iron sources in the human body including ferritin in the human brain. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:26 utc 132 vátral, j. and boča, r.. 2. material and methods 2.1 chemical and reagents all chemicals used in this work were of analytical grade (merck or sigma aldrich). stock solution of 0.01 m phosphate buffer saline (ph = 7.4) was used as a supporting electrolyte. before use it was diluted appropriately. 2.2 apparatus all measurements were performed with a computer-controlled electrochemical analyzer (model pgstat 302n, metrohm autolab) operated via the nova 1.9 software (metrohm autolab b.v.). a conventional three-electrode system was used with modified carbon paste electrode as a working electrode, ag/agcl reference electrode and a platinum wire counter electrode. digital ph meter model inolab ph 720 was applied for the preparation of the buffer solutions. 2.2 fabrication of modified electrode the unmodified carbon-paste mixture was prepared by mixing graphite powder with an appropriate amount of paraffin oil (mass ratio 65 : 35). the modified electrode was prepared by mixing unmodified composite with ferrocene (modifier mass fraction, w(fc) = 0.8 %) and then homogenized by spatula. the resultant modified carbon paste was packed into a piston-driven electrode holder. the electrode surface was renewed by smoothing on wet filter paper before starting a new set of experiments. 3. results and discussion electrochemical behavior of fe(ii)/fe(iii) in the presence of selected amino acids in phosphate buffer saline was studied by cyclic voltammetry. since the redox couple fe(ii)/fe(iii) is insoluble in aqueous solutions at physiological ph, ferrocene was used as a source of iron which was add to the carbon paste material as a modifier. fig. 2. shows the measured cyclic voltamograms of the fe(ii)/fe(iii) redox couple in the absence and presence of the 1 mm amino acids in a phosphate buffer saline solution (ph = 7.4) at a ferrocene (w(fc) = 0.8 %) modified cpe between -0.3 to 1.1 v (-0.3 to 1.3 v for trp and tyr) at scan rate 50 mv s-1. in the absence of amino acids the oxidation and reduction peak potentials at the ferrocene-modified cpe occurred at 0.356 and 0.231 v, respectively. under the identical conditions, the behavior of fe(ii)/fe(iii) redox couple was changed after addition of a selected aminoacid. the positive shift of the oxidation peak potential was observed in the case of all amino acids; the most significant differences were found for cysteine (0.388 v), tyrosine (0.419 v) and tryptophan (0.521 v). the negative shift of the reduction peak potential was observed almost in all cases except tryptophan (0.258 v). similar result were obtained by studying interactions of redox activity of the redox couple fe(ii)/fe(iii) in the presence of nicotine (bridgea et al., 2004). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:26 utc nova biotechnologica et chimica 13-2 (2014) 133 fig. 2. cyclic voltammograms of the fe(ii)/fe(iii) redox couple in the absence (dotted) and presence of the 1 mm amino acids in phosphate buffer saline (ph = 7.4) at a ferrocene-modified cpe (w(fc) = 0.8 %) at the scan rate 50 mv s-1. the amino acids were classified according to their chemical properties mentioned in fig. 1. before the addition of amino acids the oxidation peak current for the ferrocenemodified-cpe was 20.09 μa and the reduction peak was 20.40 μa. after the addition of amino acids a current change was observed. the most common sign of this change war the increasing intensity of the current unlike the study when aqueous fe(ii)/fe(iii) was used (bridgea et al., 2004). the oxidation peak current increased almost with all additions of the amino acids, specifically for ala (24.24 μa), arg (22.89 μa), asn bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:26 utc 134 vátral, j. and boča, r.. (28.15 μa), cys (28.08 μa), glu (26.62 μa), gly (27.23 μa), his (21.52 μa), phe (21.77 μa) and tyr (20.69 μa). however, a decrease of the current to the value of 17.92 μa, was observed for tryptophan along with the potential shifts. the reduction peak current increased with addition of ala (22.78 μa), arg (21.92 μa), asn (25.99 μa), glu (24.47 μa), gly (21.35 μa) and phe (21.32 μa) and decreased with addition of cys (18.97 μa), his (20.27 μa), trp (10.44 μa) and tyr (14.82 μa). again tryptophan influences this particular electrochemical parameter most significantly. in an ideal case the ratio of ipa/ipc approaches unity. as it can be seen from table 1, a value close to unity was obtained only in the absence of amino acids and with the addition of ala, arg, asn, glu, his, and phe. this indicates that the presence of the rest of the amino acids (cys, gly, trp, and tyr) modifies the electrochemical behavior of the redox couple from a reversible diffusion controlled reaction to a kinetically controlled reaction (bard and faulkner, 2001). the value of δep varied upon addition of amino acids, with the largest deviation being observed in the case of tyrosine and tryptophan. the value of peak to peak separation was not approximately 0.059 v, which indicates a reversible process but according to the measured δep our systems exhibited quasi-reversible behavior. table 1. electrochemical characteristics of the fe(ii)/fe(iii) redox couple in the presence and absence of selected amino acids, experimental conditions as in fig. 2. amino acid epa [v] epc [v] ipa/ipc δep [v] e1/2 [v] none 0.356 0.231 0.985 0.125 0.294 ala 0.359 0.227 1.064 0.132 0.293 arg 0.364 0.224 1.044 0.134 0.296 asn 0.363 0.229 1.083 0.168 0.304 cys 0.388 0.219 1.480 0.168 0.304 glu 0.360 0.226 1.088 0.134 0.293 gly 0.375 0.228 1.275 0.147 0.301 his 0.380 0.229 1.062 0.151 0.304 phe 0.373 0.222 1.021 0.151 0.297 trp 0.521 0.258 1.717 0.264 0.390 tyr 0.419 0.177 1.395 0.242 0.298 4. conclusions cyclic voltammetry experiments revealed an alteration in the electrochemical behavior of the fe(ii)/fe(iii) redox couple in the presence of selected amino acids. the oxidation and reduction potentials both shifted, generally becoming more positive and negative, respectively, while the current intensity increased or decreased. a value bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:26 utc nova biotechnologica et chimica 13-2 (2014) 135 for ipa/ipc close to unity was obtained only in the absence of amino acids and with the addition of ala, arg, asn, glu, his and phe. the value of δep varied upon addition of amino acids indicating that our systems exhibited quasi-reversible behavior. cyclic voltammetry experiments revealed the capability of amino acids to change the electrochemical behavior of the fe(ii)/fe(iii) redox couple. acknowledgement: slovak grant agencies (vega 1/0073/13, apvv-0014-11) are acknowledged for the financial support. thanks are also to the mobility (jv) supported by the project cost-cm1103 “structure-based drug design for diagnosis and treatment of neurological diseases: dissecting and modulating complex function in the monoaminergic systems of the brain”. references arreguin, s., nelson, p., padway, s., shirazi, m., pierpont, c.: dopamine complexes of iron in the etiology and pathogenesis of parkinson’s disease. j. inorg. biochem. 103, 2009, 87-93. bard, a. j., faulkner, l. r.: electrochemical methods: fundamentals and applications. john wiley & sons, new york, 2001. 833 pp. bou-abdallah, f.: the iron redox and hydrolysis chemistry of the ferritins. biochim. biophys. acta, 1800, 2010, 719-731. bridgea, m.h., williamsa, e., lyonsb, m.e.g., tiptonc, k.f., linert, w.: electrochemical investigation into the redox activity of fe(ii)/fe(iii) in the presence of nicotine and possible relations to neurodegenerative diseases. biochim. biophys. acta, 1690, 2004, 77-84. garcía, c.r., angelé-martínez, c., wilkes, j.a., wang, h.c., battin, e.e., brumaghim, j.l.: prevention of ironand copper-mediated dna damage by catecholamine and amino acid neurotransmitters, l-dopa, and curcumin: metal binding as a general antioxidant mechanism. dalton trans., 41, 2012, 64586467. jameson, g.n.l., linert, w.: iron release from horse-spleen ferritin by 6hydroxydopamine : a kinetic study. j. inorg. biochem., 67, 1997, 164-164. kamyabi, m.a., aghajanloo, f.: electrocatalytic response of dopamine at a carbon paste electrode modified with ferrocene. croat. chem. acta, 82(3), 2009, 599–606. lloyd, r.v., hanna, p.m., mason, r.p.: the origin of the hydroxyl radical oxygen in the fenton reaction. free rad. biol. med., 22, 1997, 885-888. sayre, l.m., perry, g., harris, p.l.r., liu, y., schubert, k.a., smith, m.a.: in situ oxidative catalysis by neurofibrillary tangles and senile plaques in alzheimer’s disease: a central role for bound transition metals. j. neurochem., 74, 2000, 270-279. smith, m.a., harris, p.l.r., sayre, l.m., perry, g.: iron accumulation in alzheimer disease is a source of redox-generated free radicals. proc. natl. acad. sci. usa, 94, 1997, 9866-9868. švancara, i., kalcher, k., walcarius, a., vytras, k.: electroanalysis with carbon paste electrodes, crc press, boca raton, 2012, 602 pp. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:26 utc 136 vátral, j. and boča, r.. valko, m., morri, h., cronin, m.t.d.: metals, toxicity and oxidative stress. curr. medicinal chem., 12, 2005, 1161-1208. watt, g.d., frankel, r.b., papaefthymiou g.c.: reduction of mammalian ferritin. proc. natl. acad. sci. usa, 82, 1985, 3640-3643. winterbourn, c.c.: toxicity of iron and hydrogen peroxide: the fenton reaction. toxicol. lett., 82/83, 1995, 969-974. zatta, p.: metal ions and neurodegenerative disorders, world scientific, singapore, 2003, 511 pp. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:26 utc nova biotechnol chim (2023) 22(1): e1429 doi: 10.34135/nbc.1429 1 nova biotechnologica et chimica molecular identification, antibacterial activity, and production of hydrolytic enzymes by halotolerant bacterium streptomyces sp. esm2-25 gtf strain isolated from extreme environment in north-east of algeria chérifa alliouch-kerboua1,, ghania bourzama1, djamila gacemi-kirane2, bernard la scola3,4 1laboratory of biochemistry and environmental toxicology, department of biochemistry, faculty of sciences, university of badji mokhtar, annaba 23000, algeria 2department of biochemistry, faculty of sciences, university of badji mokhtar, annaba 23000, algeria 3ihu-méditerranée infection, marseille, france 4aix-marseille university, ird, aphm, mephi, marseille, france  corresponding author: alliouch_cherifa@yahoo.fr article info article history: received: 14th october 2021 accepted: 20th october 2022 keywords: antibacterial activity biotechnological applications el-mellah lagoon halotolerant bacterium hydrolytic enzymes pathogenic bacteria abstract the aim of this study is to establish the taxonomic position of the halotolerant bacterium esm2-25 gtf strain isolated from the extreme environment of el-mellah lagoon water, which is situated in the city of el-kala in the northeast of algeria and to study its phenotypic characteristics, antibacterial activity against several pathogenic bacteria, and hydrolytic enzymes production. the novel bacterium esm2-25 gtf strain was isolated from water samples by the dilution agar plating method using the starch-casein medium, screened in vitro for its hydrolytic enzymes production and antibacterial activity. the phenotypic and molecular characteristics show that the strain esm2-25 gtf belongs to the genus streptomyces. however, the comparison of the morphological and physiological characteristics of the strain esm2-25 gtf with those of the nearest species showed significant differences. this strain showed an antibacterial activity against bacillus subtilis subsp. spizizenii cip 106094, escherichia coli atcc 29522, enterococcus faecalis atcc 29212, staphylococcus aureus atcc 29213 and s. aureus mrsa atcc 43300, while it is not active against pseudomonas aeruginosa atcc 27853. furthermore, the strain ecm2-25 gtf was able to produce different hydrolytic enzymes such as lipase, protease, amylase, catalase, and gelatinase, which have applications in the field of food and industry. the interesting antibacterial activity of the strain esm2-25 gtf against pathogenic bacteria and hydrolytic enzymes production indicate the importance of the exploitation of marine actinomycetes for biotechnological applications and the discovery of new antibacterial molecules and could encourage further research on the bioactive molecules secreted by this strain. © university of ss. cyril and methodius in trnava introduction late blowing is the major cause of spoilage in hard microbial resistance to antibiotics increased with the covid-19 pandemic, as although physical distancing played a positive role in decreasing transmission, it was countered by the overuse of broad-spectrum antibiotics to treat patients infected with sars-cov-2 (severe acute respiratory syndrome coronavirus 2) (rodríguez-baño et al. mailto:alliouch_cherifa@yahoo.fr nova biotechnol chim (2023) 22(1): e1429 2 2020; lai et al. 2021; ukuhor 2021). there is also the problem of the toxicity of certain drugs for the environment and human health (rowan et al. 2016; hasani et al. 2020; han et al. 2021). these factors represent a significant public health risk that requires active research into new, less toxic, naturally occurring antimicrobial molecules, especially as all efforts have currently been directed towards research into the covid 19 pandemic (marston et al. 2016; rodríguez-baño et al. 2020). actinomycetes are gram positive filamentous bacteria forming a mycelium and with a high percentage of g + c (lechevalier and lechevalier 1967; mazza et al. 2003). based on their ability to produce antibiotics, the enzymes and other bioactive substances of actinomycetes are considered the most useful and beneficial bioactive molecules producing living organisms for humanity (benedict 1953; cai 2009; mitousis et al. 2020). in fact, these microorganisms produce 45 % of the bioactive molecules (berdy 2005). among the actinomycetes, the genus streptomyces is the most important producer of bioactive molecules (kieser et al. 2000; hopwood 2007a; kovácsová et al. 2017; mahasneh et al. 2021) including antibacterial agents such as tetracyclines, antifungal agents such as amphotericin b, ancticancer drugs exemplified by adriamycin, the immunosuppressant tacrolimus, enzymes such as amylase, lipase, protease, cellulase, and chitinase (miyadoh 1993; hopwood 2007b; suthindhiran and kannabiran 2009; kumar et al. 2020). streptomyces species have been reported to produce nearly 80 % of the antibiotics known currently (miyadoh 1993; kieser et al. 2000). as in the case of the antibiotics produced by streptomyces, the extracellular enzymes produced from them could also be used instead of toxic products that are being used in various industries and affecting human health and the environment (kumar et al. 2020). scientists are also faced with a decline in the discovery of new bioactive molecules, due to the depletion of resources caused by the overexploitation of the natural environment (demain and sanchez 2009). the exploration of new and extreme environments that were previously unexplored would allow the discovery of new bioactive molecules. among these unexplored and extreme environments, the el-mellah lagoon located in the north-east of algeria as a part of the el-kala national park, illustrates a good example of wetlands in the mediterranean region. this brackish water lagoon is registered on the ramsar list of wetlands of international importance to be protected (boumezbeur and bouteldji 2005). the aim of the present work is to describe the phenotypic and molecular characteristics, as well as the antibacterial activity and hydrolytic enzymes production of a new marine bacterial isolate from the water of el-mellah lagoon. experimental isolation of the actinomycete strain water samples were collected from el-mellah lagoon of el kala (fig. 1) (northeast of algeria; latitude 8°20’e, longitude 36°54’n), kept at 4 °c and transferred immediately to the laboratory. strain ecm2-25 gtf was isolated using the dilution method after 14 days of incubation at 28 °c from water samples on starch-casein agar medium (10 g starch; 0.3 g casein; 2 g k2hpo4; 2 g kno3; 0.02 g caco3; 0.01 g feso4·7h2o; 0.05 g mgso4·7 h2o; 20 g agar; 1,000 ml distilled water; ph 7.2) (williams and cross 1971) on petri plates without any antibacterial agents. fig. 1. geographical localization of el-mellah lagoon (36◦54’n, 8◦20’e) (bensaâd-bendjedid et al. 2018). nova biotechnol chim (2023) 22(1): e1429 3 identification of the actinomycete strain morphological studies the observation of the micro-morphology of the strain esm2-25 gtf was carried out using a scanning electron microscope phillips xl30 esem-efei (europe nanoport, eindhoven, netherlands). the strain used for observation has been cultured for 14 days at 28 °c on isp2 medium (4.0 g yeast extract; 10 g bacto-malt extract; 4.0 g glucose; 1,000 ml distilled water; 20 g agar; ph 7.2) (boudjella et al. 2006; morakchi et al. 2009; alliouch-kerboua et al. 2015). cultural and physiological characteristics cultural characteristics of strain esm2-25gtf were characterized following the guidelines of the international streptomyces project (isp) (shirling and gottlieb 1966; mohamed et al. 2013). these characteristics were observed on the medium isp2 after 21 days of incubation at 28 °c. colors were determined according to isccnbs color charts (kenneth and judd 1976). the growth in the presence of nacl was evaluated according to larpent and larpent gourgaud (1997). the production of melanoid pigments was observed on isp7 agar medium (15 g glycerol; 0.5 g l-tyrosine; 1 g l-asparagine; 0.5 g k2hpo4; 0.5 g mgso4·7h2o; 0.5 g nacl; 0.01 g feso4·7h2o; 1 ml standard saline solution; 18 g agar; 1,000 ml distilled water; ph 7.2) as described by shirling and gottlieb (1966). genomic dna extraction dna extraction was performed directly on actinomycete colonies. the strain was inoculated on the blood horse agar medium and incubated at 28 °c for 14 days. a bacterial suspension (200 µl) was prepared with sterile ultrapure water for the esm2-25 gtf strain. after homogenization, the mixture was centrifuged, and the dna was recovered (supernatant) (bonnet et al. 2011; alliouch-kerboua et al. 2015). amplification and 16s rrna gene analysis for the sequencing of 16s rdna first, the amplification of the 16s gene was conducted using the kit phusion finnzymes hf buffer (finnzymes, keilaranta, finland) and two universal primers, 536f 5’-cag-cag-ccg-cgg-taa-tac-3’and rp2-5’acg-gct-acc-ttg-tta-ctt-3’. the reaction mixture contains for a final volume of 50 ml: pcr buffer 1 (10 mm trishcl, 20 µl kcl), 4 µl of 5hf buffer, 0.4 µl of dntp, 1 µl of each primer, 0.2 µl of taq polymerase, 2 µl of dna and 12.4 µl of qsp water to give a final volume of 20 µl. the amplification of the 16s rna gene is performed in an “ab applied biosystem 2720 thermal cycler” (life technologies, carlsbad, usa) with the following profile: an initial denaturation step at 98 °c for 30 s (98 °c for 30 s instead of 95 °c for 10 s, this increase corresponds to heat shock), followed by 35 cycles of amplification at 98 °c for 30 s, 54 °c for 30 s and 72 °c for 30 s. at the end of the cycles, a final step is to hold the reaction mixture at 72 °c for 10 min, which is then cooled to 4 °c (la scola and raoult 1998). the resulting amplifiers are detected by agarose gel electrophoresis and visualized under ultraviolet (uv) after addition of ethidium bromide. phylogenetic analysis the 16s rrna sequence was determined by a 3130x/genetic analyser (applied biosystems, hitachi, japan), with the same primers used. the 16s rrna sequences obtained were compared with sequences deposited in the genbank genomic bank using the "blast ncbi" program available on the website: https://blast.ncbi.nlm.nih.gov/blast.cgi. the phylogenetic tree was constructed via the maximum likelihood algorithm using the software mega version 5.2.2. (tamura et al. 2011). the rrna gene sequence of the strain esm25-gtf was aligned using muscle software with nucleotide sequences of representatives of the genus streptomyces obtained from genbank database (edgar 2004). the evolutionary distance matrices were made as described by kimura (1980). the tree topology of the maximum likelihood data was evaluated by bootstrap analysis https://blast.ncbi.nlm.nih.gov/blast.cgi nova biotechnol chim (2023) 22(1): e1429 2 with 1000 re-samplings (felsenstein 1981; felsenstein 1985). finally, the nucleotide sequence of the strain of the actinomycete was deposited in genbank (embl) under the accession number ok384567. screening of extracellular enzymes production for the detection of extracellular enzymes, the assays were performed on agar plates after incubation at 28 °c for 7 – 14 days. the starting culture used for enzyme screening was obtained by growing the streptomyces sp. esm2-25 gtf isolate on agar plates on isp2 medium after incubation at 28 °c for 7 days. all experiments were conducted according to the standard protocols described below. protease activity proteolytic activity of the isolate streptomyces sp. esm2-25 gtf was determined in skim milk based medium: (10 g skim milk; 3.6 g agar; 110 ml distilled water; ph 7.5). clear zones around the growth after 14 days indicated protease activity (gordon et al. 1974). amylase activity amylase activity was tested on a starch based medium: (10 g soluble starch; 100 ml nutrient agar; ph 7.5). the starch hydrolysis was observed by flooding plates with iodine-potassium iodide solution (lugol’s iodine solution) (1.0 g iodine; 5.0 g potassium iodide; 330 ml distilled water) and a clear zone around the colony showing hydrolysis of starch (gordon et smith 1953). gelatinase hydrolysis gelatin hydrolysis was screened using gelatinbased agar: (10g gelatin; 1g yeast extract; 20g agar; 1,000 ml distilled water; ph 7.2). after 7 days, plates were flooded with the following solution: 15 g hgcl2; 20 ml concentrated hcl; 100 ml distilled water. gelatinase hydrolysis was observed when clear zones appeared around colonies (gordon and smith 1953). lipolytic activity lipolytic activity was detected on tween 80 based medium: (10 ml tween 80; 1g nano 3; 5 g yeast extract; 50 ml salt solution; 0.1 g cacl2·h2o; 18g agar; 1,000 ml distilled water; ph 7.2). after 14 days a turbid or good-visible halo around the colonies indicated lipolysis (sierra 1957). hemolytic activity hemolytic activity was tested using columbia agar: (17 g polypeptone; 3 g pancreatic peptone; 3 g yeast extract; 1 g cornstarch; 5 g nacl; 13.5 g agar; 1,000 ml distilled water; 100 ml horse blood; ph 7.3). after 7 days, a clear zone around the colonies showed hemolytic activity (larpent and larpent gourgaud 1997). antibacterial activity the antibacterial activity of the strain esm2-25 gtf was studied by the cross-streak agar plate method. a longitudinal streak of the actinomycete strain was streaked on isp2 medium. after incubation at 28 °c for 7 days, the tested strains (escherichia coli atcc 29522, pseudomonas aeruginosa atcc 27853, staphylococcus aureus mrsa atcc 43300, enterococcus faecalis atcc 29212, bacillus subtilis subsp. spizizenii cip 106094, staphylococcus aureus atcc 29213) were inoculated in streaks crossing the actinomycete, then the plates were re-incubated at 28 °c for 24 h. antibacterial activity was evaluated by measuring the diameter of inhibition zone on both sides of the streak (boudjella et al. 2006). results identification of the strain esm2-25 gtf cultural and morphological characteristics of the strain the scanning electron micrograph of the strain esm2-25gtf (fig. 2b) showed long chains of 4 nova biotechnol chim (2023) 22(1): e1429 3 spores formed on aerial hyphae. these chains were straight to flexuous (rectiflexibles) and spiral (spirales). the spores had the smooth surface and an ovoid shape. no sclerotia, sporangia or flagellated spores were observed, and no fragmentation of substrate mycelium was formed. the cultural characteristics (fig. 2a) showed a whitish gray aerial mycelium with good growth on isp2 medium. the colony was elevated and spreading. the morphological and cultural characteristics of strain esm2-25 gtf were compared with those of known species of actinomycetes described in the in bergey’s manual of systematic bacteriology (whitman et al. 2012), and they strongly suggested that strain esm2-25 gtf belong to genus streptomyces. fig. 2. colonies of streptomyces sp. esm2-25 gtf grown on isp2 solid medium at 28 °c after 14 days of incubation (a). scanning electron micrographs of the chains of spores of streptomyces sp. esm2-25gtf in isp2 solid medium at 28 °c after 14 days of incubation (b). physiological characteristics of the isolated strain physiological characteristics listed in table 1 showed that isolate esm2-25 gtf is not able to assimilate citrate. the isolated strain can grow with 0 %, 4 %, 7 %, 10 %, and 13 % of nacl. the catalase and oxidase reaction were positive. in addition, this strain produces a light yellow melanoid pigment on the isp7 medium. table 1. physiological characteristics and extracellular enzymes production by the strain esm2-25gtf (+: growth or positive reaction of the test strain, -: no growth or negative reaction, ±: moderate growth). characteristics strain esm2-25gtf carbon utilization citrate extracellular enzymes starch casein tween 80 gelatin hemolysis tolerance to nacl: 0 % 4 % 7 % 10 % 13 % catalase oxidase melanoid pigment production tyrosine agar (isp7) + + + + +s + ± ± ± + + + light yelow phylogenetic analysis the alignment of the isolate esm2-25 gtf 16s rrna gene sequence (922 bp) with the 16s rrna retrieved from the genbank database has given 98 % similarity with streptomyces pluricolorescens nrrl b-2121t and 99 % similarity with streptomyces cinereorectus nbrc 15395t. the maximum likelihood tree shows the relationship between esm2-25 gtf, streptomyces pluricolorescens nrrl b-2121t, streptomyces cinereorectus nbrc 15395t and other representatives streptomyces. the position of esm2-25 gtf in the maximum likelihood phylogenetic tree is shown in fig. 3. the strain of actinomycete was deposited in genbank under the accession number ok384567. a b 1 1 1 5 nova biotechnol chim (2023) 22(1): e1429 3 fig. 3. maximum likelihood tree based on 16s rrna sequences showing relationship between streptomyces sp. esm2-25 gtf and related sequences obtained from "ncbi blast". bootstrap = 1,000. the scale bar indicates 0.1 substitution per site. extracellular enzymes production extracellular enzymes production listed in table 1 showed that isolate esm2-25 gtf degrades starch, casein, tween 80 and gelatin. this strain also shows a hemolytic activity. antibacterial activity the activity of the strain esm2-25gtf is shown in table 2. table 2. antibacterial activity of esm2-25gtf strain on isp2 solid medium. the diameters of the inhibition zones of target microorganisms are shown in millimetres (mm). target microorganisms inhibition zone diameter [mm] bacillus subtilis subsp. spizizenii cip 106094 escherichia coli atcc 29522 enterococcus faecalis atcc 29212 staphylococcus aureus atcc 29213 staphylococcus aureus mrsa atcc 43300 pseudomonas aeruginosa atcc 27853 41 35 34 18 3 0 the strain exhibited a strong antibacterial activity against bacillus subtilis subsp. spizizenii cip 106094, escherichia coli atcc 29522 and enterococcus faecalis atcc 29212, moderate activity against staphylococcus aureus atcc 29213, and low activity against s. aureus mrsa atcc 43300. it has no activity against pseudomonas aeruginosa atcc 27853. discussion today, microbial resistance constitutes major public health problems, expressed both at the hospital level by nosocomial infections, and at the community level (boucher et al. 2009). the causes of this microbial resistance are multiple and various, such as the frequency of the mutations, the number and the type of mutations required for the expression of the resistance, the spectrum of activity, the concentration of the drug, and also the antimicrobial abuse that promotes the transfer of resistance through vertical and horizontal transfer mechanisms (levy 2000). the covid 19 6 nova biotechnol chim (2023) 22(1): e1429 2 pandemic and coronavirus infections have increased the problem of microbial resistance due to the prescription of broad-spectrum antibiotics for coinfections (rawson et al. 2020). indeed, several microbes have been found in coinfections with coronavirus such as: escherichia coli, pseudomonas aeruginosa, multi-resistant staphylococcus aureus mrsa, enterococcus spp. which correspond to the strains chosen for testing the antibacterial activity of the strain esm2-25 gtf (rawson et al. 2020). despite their ability to heal, some medications, including antibiotics, are still harmful to health, because of their toxicity (demain and sanchez 2009). currently, microbial enzymes are replacing chemical catalysts in manufacturing of chemicals, food, leather, and pharmaceutical industries. moreover, due to the quick doubling time of microbes with special features compared to plants and animals, the microbial fermentation process may possibly meet the current market demand for the industrial enzyme (kumar and takagi 1999; hames-kocabas and uzel 2007; sathya and ushadevi 2014). there is also the problem of decreasing the discovery of new bioactive molecules, and the continuing and persistent demand for new biological sources of industrial enzymes that are competitive on the market compared to the nonbiological products, particularly in terms of cost and convenience of use, especially antibiotics and enzymes due to resource depletion, caused by overexploitation of normal environments (botella 2009; demain and sanchez 2009). scientists are faced with the challenges of discovering new antibiotics and new sources of enzymes from underused or untapped environments such as extreme environments (daviesbolorunduro et al. 2021). many new antibiotics such as dithiolopyrrolones, mzabimycins, mutactimycin pr, and 7-deoxy-8-omethyltetrangomycin have been reported from extremophilic actinomycetes isolated from algeria (lamari et al. 2002; zitouni et al. 2004; hadj rabia-boukhalfa et al. 2017; corral et al. 2019; tata et al. 2019). the extreme environment chosen for our study is el-mellah lagoon, which by its rich flora and fauna offers an ecosystem whose biodiversity makes it suitable for the development of new molecules of biotechnological interest. however, one of the most important characteristics of el-mellah lagoon is its membership of the marine domain, because of the existence of a channel linking it to the mediterranean sea. it has been established that actinomycetes inhabit marine environments, including the species of streptomyces, have the ability to produce different bioactive molecules from those produced in the terrestrial environment, including antibiotics and enzymes, which are of great interest to the scientific community (ramesh et al. 2009; manivasagan et al. 2013; sathya and ushadevi 2014). el-mellah lagoon is a brackish water lagoon with salinity ranging from 34.8 to 25.4 mg.l-1 depending on the season (chaoui et al. 2006). this makes it an extreme environment containing an extremophile microflora including actinomycetes offering the opportunity for the discovery of new bioactive molecules of therapeutic interest. several recent studies have highlighted the existence of marine streptomyces producing bioactive molecules (gebreyohannes et al. 2013; alliouchkerboua et al. 2015). many new halotolerant and halophile actinomycetes have been reported from different algerian ecological niches. among the halotolerant actinomycetes there is streptomyces sp. sy bs5 isolated from an arid region in south of algeria (souagui et al. 2015), nocardiopsis sp. hr-4 isolated from the salt-lake soil named sebkha of ain (djinni et al. 2019) and melghirimyces algeriensis sp. nov. isolated from soil of an algerian salt lake (addou et al. 2013). for halophilic actinomycetes we can mention actinopolyspora mzabensis sp.nov. isolated from a saharan soil sample collected from the mzab region of algerian sahara (meklat et al. 2013), actinoalloteichus hoggarensis sp. nov. isolated from tamanrasset an arid area in the south of algeria (hoggar) (boudjelal et al. 2015) and actinopolyspora biskrensis sp. nov. isolated from a saharan soil from biskra from northeast of algeria (saker et al. 2015). the study of macro-morphological characteristics showed that this isolate belongs to the series of gray, this color is specific to bacteria of the genus 7 nova biotechnol chim (2023) 22(1): e1429 3 streptomyces (tresner and backus 1963). the micro-morphological characterization showed that the isolate exhibits smooth spores, and spiral type and straight to flexuous that are typical of the genus streptomyces (dietz and mathews 1971). physiologically, the isolated strain was metabolically active. this activity was manifested by various degradation activities. however, given the brackish character of el-mellah lagoon and its impact on its microbial microflora, among the most important physiological characteristics in our study and which inevitably influences all the other physiological characteristics, it is "the tolerance to the different concentrations of nacl". for this reason, our first interest was focused on this character. in fact, by testing different nacl concentrations, it was found that our isolate tolerates nacl concentrations up to 10 %, with optimum growth at 0 % nacl concentration, making it halotolerant. the isolate showed the ability to produce different extracellular hydrolytic enzymes such as protease, amylase, lipase, and gelatinase. several studies have demonstrated production of these enzymes by marine halotolerant streptomyces (suthindhiran and kannabiran 2009; sathya and ushadevi 2014; tatar et al. 2014; ashraf et al. 2021). the hemolytic activity of streptomyces sp. esm225gtf suggests that this isolate could be clinically more important and must be investigated further for the cytotoxic and anticancer properties. suthindhiran and kannabiran (2009) also demonstrated the hemolytic activity of a moderately halophilic streptomyces sp. the isolated strain is able to produce melanoid pigment like halotolerant streptomyces reported in many other studies (sonya et al. 2005; mohamed et al. 2013). based on morphological and physiological characteristics, a first preliminary identification of the genus streptomyces could be made (shirling and gottlieb 1966; whitman et al. 2012). nevertheless, it is only by realizing the phylogenetic characterization that this observation has been confirmed. the genus streptomyces is the most abundant in nature, especially in soil, and its number is increasing (lucas et al. 2013). they also inhabit other terrestrial and aquatic niches (hopwood 2007a). the phylogenetic analysis was based on the 16s gene sequencing, which is characterized by its high stability (woese 1987). therefore, it is the most accurate and adapted molecular chronometer for this type of study (stackebrandt et al. 2002). indeed, the bacteria belonging to the genus streptomyces, have the notoriety of being very unstable genetically. the characters most affected by this phenomenon are aerial mycelium formation, spore production, antibiotic production and/or resistance, and the formation of extracellular enzymes, such as tyrosinase involved in pigmentation (leblond et al. 1990). these mutations are mainly due to chromosomal deletions and amplification of dna (leblond et al. 1990). for this reason, the 16s rrna gene sequence of the isolate esm2-25 gtf (922 bp) was amplified by pcr, sequenced, and submitted to genbank. the maximum likelihood tree (fig. 3) showed that streptomyces sp. esm225gtf (genbank accession number ok384567) forms a separate line in the phylogenetic tree, it is closest to streptomyces cinereorectus nbrc 15395t with a bootstrap value of 99 %, and to streptomyces pluricolorescens nrrl b-2121t with a bootstrap value of 98 %. contrary to our strain, streptomyces cinereorectus species nbrc 15395t and streptomyces pluricolorescens species nrrl b-2121t do not produce melanoid pigment. in addition, streptomyces pluricolorescens species nrrl b-2121t aerial mycelium was yellow or red color series and the substrate mycelium was grayed yellow to yellow brown. moreover, streptomyces cinereorectus species nbrc 15395t had short spore chains with up to 10 spores per chain. by relying on these results, the strain streptomyces sp. esm2-25 gtf could be a new species however, the dna-dna hybridization analysis is necessary to confirm this result. conclusion based on the results, the esm2-25 gtf strain showed an antibacterial activity against pathogenic bacteria: bacillus subtilis subsp. spizizenii cip 106094, escherichia coli atcc 29522 enterococcus faecalis atcc 29212, staphylococcus aureus atcc 29213, s. aureus mrsa atcc 43300, but it is not active against pseudomonas aeruginosa atcc 27853. our data 8 nova biotechnol chim (2023) 22(1): e1429 2 also shows that the esm2-25 gtf strain was able to produce various hydrolytic enzymes such as protease, amylase, lipase, and gelatinase. these promising results indicate the importance of the exploitation of marine actinomycetes for the discovery of new bioactive molecules, using cultural methods such as antibacterial molecules which are a tool for controlling bacterial infections, and extracellular enzymes with potential applications in the fields of biotechnology and industry. further investigations to extract, purify, and characterise the bioactive molecules are currently in progress. acknowledgments this study was partially funded by the department of biochemistry, faculty of sciences, university of badji mokhtar of annaba. the authors would like to thank the head of the department of biochemistry; branes zidane for its collaboration and all facilities provided. sincere thanks to technicians priscilla jardot and sonia bouchard from the faculty of medicine of marseille france for their invaluable help in the molecular identification of streptomyces sp. esm2-25 gtf. also, thank for aline bonnotte, jeannine lherminier and yves waché of the inra microscopy center / university of france burgundy, dimacell platform, umr1347 agroecology, bp 86510, f-21000 dijon, france for their assistance in carrying out electron microscopy. conflict of interests the authors declare that they have no conflict of interest. references addou an, schumann p, spröer c, bouanane-darenfed a, amarouche-yala s, hacene h, cayol j-l, fardeau, ml (2013) melghirimyces thermohalophilus sp. nov., a thermoactinomycete isolated from an algerian salt lake. int. j. syst. evol. microbiol. 63: 1717-1722. alliouch-kerboua c, kirane dg, la scola b (2015) activité antimicrobienne d’une 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received: 2nd january 2022 accepted: 30th september 2022 keywords: adverse effects advertising alcohol caffeine regulations taurine abstract energy drinks (eds) have been available at the global market for almost 100 years and today, they are advertised as ’tools’ boost energy, enhance physical performance and mental alertness. some of the main chemical components in eds include caffeine, ginseng, and taurine. the market and consumption of these beverages is growing exponentially, and this is becoming a public health problem due to the adverse effects associated with these drinks. the main objective of this article is to review important ingredients in popular eds in south africa and look at their molecular mechanisms of action and interaction with other compounds within the body. at the same time, the authors aim to review the global consumption pattern of eds among children and adolescents. finally, this review article will provide an overview the health risks associated with eds consumption. a literature review was conducted using scholarly databases. keywords such as energy drinks, adverse effects, advertising, alcohol, caffeine, taurine, and regulations were used. despite the claims of having significant benefits to mental and physical stamina, long-term consumption of eds could have detrimental public health implications; and could result in increasing rates of the central nervous system disorders and the cardiovascular complications. a knowledge gap exists on how the lack of education impacts the decision of consumers of eds and the parental guidance provided to children and adolescents in relation to the consumption of these beverages in south africa. to address the issue of excessive ed consumption, communities need to be made aware of the harmful effects of these beverages through health education and implementing strict regulatory measures through policy. © university of ss. cyril and methodius in trnava introduction energy drinks (eds) have been available at the global market for almost 100 years and today, they are advertised as ’tools’ boost energy, enhance physical performance and mental alertness. according to several reports, eds are one of the most popular supplement commodities in the united states second only multivitamins (froiland et al. 2004; hoffman 2010; campbell et al. 2013). eds have not only appealed to the adult population but to children and adolescents too, as we have seen an increase in the consumption rate within these population groups. in 2015 and focusing on mailto:r.tandlich@ru.ac.za.sk nova biotechnol chim (2023) 22(1): e1344 2 the patterns of the ed consumption, the european food safety authority commissioned survey-based research in 16 of the eu countries (european food safety authority 2015). among the adult population and the adolescent age groups (10 – 18 years old), as well as in the below-ten-year-old children, the results indicated that (< 10 years old) demographic 30 % of adults, 68 % of adolescents and 18 % of children were consuming eds (european food safety authority 2015). correlating results were observed in a survey study conducted in 43 high schools in ontario, which revealed that in a student population of 23,610; 73.6 % were reported to be consumers of eds (reid et al. 2015). in the recent years, novel findings have been published in scientific literature on the ed consumption and the related health effects. some of the most concerning, and potentially damaging public health ones, include the increased probability and risks from childhood obesity, the increased probability in the rise of early onset cardiovascular pathophysiological conditions, suicidal ideation, and tendency, as well as impaired cognitive function in children (usman and jawaid 2012; kammerer et al. 2014; park et al. 2016). in addition to these health risks, aggressive behaviour and mood disorders were reported in a study and linked to caffeine consumption in 15 – 16 years olds (soós et al. 2021). due to their potential effect of enhancing physical performance, eds have become increasingly appealing and popular to sport athletes. the distinction between eds and sports drinks (sd) must be noted and clearly defined. eds and sds are two different types of supplement beverages with their own specific performance enhancement claims and nutritional compositions. sds are targeted at individuals who play sports (athletes) or engage in high-performance activities. their aim is to provide hydration and electrolyte replenishment to the body with the intent to support it during a high-performance activity (campbell et al. 2013). carbohydrates are one of the molecules found in both eds and sds in different estimated quantities of around 12 g and 3 g respectively, this compound is the body’s primary source of energy. eds commonly contain caffeine as the main active ingredient, along with other ingredients such as ginseng, b vitamins, taurine, sugar, guarana, carnitine, green tea extracts and ginkgo biloba, which all have significant physiological effects on the body (heckman et al. 2010). the observed exponential growth of ed consumption has become a great health concern towards nutrition and dietary inadequacies; some of the factors promoting this growth is the rise of the food processing industry, targeted advertising, and the absence of independent regulatory bodies (higgins et al. 2010). eds entered the mainstream beverage industry first in the early 1960s and since then have grown exponentially into a multi-billion-dollar industry. bailey et al. (2014) carried out a study, using data from 160 countries, which showed that the annual consumption of eds was greater than 5.8 billion litres. in the united states alone, sales in 2015 were reported to be $13.4 billion, with the major ed companies at the helm of the market in terms of sales and advertising (richter 2014). the aggressive growth of the eds market is fuelled by advertising. one of the marketing strategies used to gain massive exposure is through providing financial support to well-known sports teams and sponsoring major events, such as sports competitions and music festivals as (macknight 2020; erdmann et al. 2021). the regulation of eds, including health information and content labelling is different across countries and the absence of strict and consistent regulatory principles fuels the aggressive marketing of these beverages (higgins et al. 2010). a knowledge gap exists on how the lack of education impacts the decision of consumers of eds and the parental guidance provided to children and adolescents in relation to the consumption of these beverages in south africa. this review article seeks to highlight the health risks associated with the chronic consumption of eds in children and adolescents. furthermore, interventions that can address and reduce the excessive consumption of these beverages will be discussed. experimental a systematic literature review was conducted using the following databases: cochrane library, ebsco host, google scholar, pubmed, scopus, and sciencedirect to search for relevant literature nova biotechnol chim (2023) 22(1): e1344 3 published from 1980 to 2022. only 9 publications were selected before the year 2005, 6 publications were selected between the year 2005 and 2009, the rest of the publications are from 2010 to 2022. we did not exclude articles based upon a date of publication due to lack of scientific research and literature in this area. keywords such as adverse effects, advertising, alcohol, caffeine, taurine, and regulations were used. furthermore, five different eds were bought, together with cola drink 1 and cola drink 2. the ingredients of these beverages as shown on the packaging labels were analysed and compared, particularly looking at the amounts of caffeine, taurine, and sucrose in each of them. using google search, we looked at the quantity of caffeine found in brewed coffee, expresso, decaf coffee, ice tea, and green tea. results and discussion eds are a rapidly growing market globally. they are popular among children, adolescents, and young adults. various drinks were analysed based on their labelled content; the emphasis was particularly placed on the amount of caffeine, taurine, and sugar in each of them. table 1. a list of popular energy drinks in south africa with the main ingredients (based on label analysis). energy drinks & soft drinks caffeine content [mg.100 ml-1] taurine concentration [%] sugar content [g.100 ml-1] 1. ed brand 1 32 not stated 6.0 2. ed brand 2 26 not stated 3.5 3. ed brand 3 32 0.4 11 (sucrose) 4. ed brand 4 30 0.4 3.7 5. ed brand 5 32 0.4 sugar free (contains sucralose) 6. cola drink 1 34 0 10.6 7. cola drink 2 37.6 0 40 table 1. above shows some of the popular eds consumed in south africa which are namely ed brand 1, ed brand 2, ed brand 3, ed brand 4, and ed brand 5. ed brands 1 and 2 are locally produced and owned. the average caffeine content of the listed eds is 32 mg.100 ml-1. an interesting observation made from the table is that the quantity of caffeine in cola drinks 1 and 2 which are 34 mg.100 ml-1and 37.6 mg.100 ml-1 respectively. this is greater than that of the eds. the sugar content varies between the eds and colas, with cola drink 2 containing more sugar (40 g.100.ml-1) than any one of the beverages. perhaps more research is needed to investigate the impacts of cola in comparison with those of eds, particularly looking at their caffeine content and the composition with other included ingredients. all the listed eds have taurine concentration of 0.4 %, except of ed brand 1 having zero taurine content. recently, ed brand 1 launched a new ed flavour which includes wormwood extracts as one of the ingredients. wormwood, scientifically called artemisia absinthium l., is known to have “antibacterial, anti-oxidant, anti-malarial, antiinflammatory, anti-depressant and hepatoprotective pharmacological activities amongst others” (ahamad et al. 2019; batiha et al. 2020). the longterm use of wormwood has been reported to cause neurotoxic effects and mental disorder called “absinthism”. these psychoactive and neurotoxic effects are due to the high concentrations of thujone which is a chemical substance found in a. absinthium and is known to be an antagonist of gamma-aminobutyric acid-a (gabaa) receptor (lachenmeier 2010; batiha et al. 2020). the impacts of a. absinthium, particularly thujone in combination with caffeine and other chemical substances found in eds still needs to be investigated. table 2. a list of different coffee and tea with their caffeine content. coffee and tea (4-ounce) caffeine [mg] 1. brewed coffee 45.4 2. expresso 240.4 3. decaf coffee 0.0 – 3.0 4. iced tea 12.5 5. green tea 9.0 nova biotechnol chim (2023) 22(1): e1344 2 table 2. a list of different coffee and tea with their caffeine contentabove shows the different caffeine quantities that were measured in coffee and tea that have been reported in literature. the concentrations of caffeine in brewed coffee and expresso are higher in values vs. the concentrations in eds listed in table 1. brewed coffee and expresso are made from coffee beans which contain caffeine, tannin, proteins, lipids, and carbohydrates (sharma 2020). although eds and coffee/expresso contain a similar compound “caffeine” as the main ingredient, what sets them apart are the added ingredients, which exhibit different mechanisms and effects in the body. tea contains four substances known to have a stimulating effect on the brain, which are caffeine, theophylline, theobromine, and l-theanine. like caffeine, theophylline and theobromine belong to a class of xanthines, and they both have physiological effects. however, the amounts found in a cup of tea are so small that the impact on the body can be considered negligible (gunnars 2022). according to the 2012 guidelines issued by the uk’s children food trust, it was recommended that children should avoid the consumption of food or drinks that contain caffeine, such as tea, coffee, cola, and other drinks (children’s food trust 2012). review of biochemical mechanism of action of the main ed ingredients caffeine caffeine is an alkaloid compound of the methylxanthine class, with its pharmacological mechanism of action including stimulation of the central nervous system (cns) (petre 2020). this class of drug is used clinically as a cns stimulant, diuretic, bronchodilator, and cardiotonic. there is some evidence in biochemical and pharmacological literature that xanthines block or antagonize adenosine receptors in the human body (bruns et al. 1980). adenosine is a neurotransmitter that causes the brain to relax which decreases the nerve cell activity and leads to drowsiness and tiredness (petre 2020). the formation of adenosine depends on the rate of atp synthesis and breakdown (bruns et al. 1980). caffeine and adenosine have a similar molecular structure (fig. 1), and therefore, the nerve cell recognizes caffeine as adenosine. the resulting binding causes the caffeine molecule to serve as an antagonist to the pharmacological activity of adenosine, which in turn leads to speeding up the nerve cell activity. the intracellular concentration of adenosine molecules rises as a consequence of the blockade of the receptors, thus activates noradrenaline neurons. the pituitary gland detects the cell activity and releases hormones that will activate the adrenal gland and therefore produce catecholamines (brain et al. 2007). caffeine blocks the activity of adenosine to open the brain’s blood vessels, which leads to constriction and decrease in cerebral blood flow (nehlig et al. 1992). therefore, some headache medicines contain a combination of aspirin and caffeine. (brain et al. 2007). an experimental study conducted by conlay et al. (1997) showed how caffeine can change the adenosine plasma level in rats that were exposed to 0.1 % caffeine. their adenosine plasma concentration increased from 0.32 ± 0.5 μm to 3.17 ± 0.30 μm when compared to the control group. fig. 1. chemical structures of caffeine and adenosine illustrating the similarity of their molecular structure. generated using chemdraw software application. experimental studies have demonstrated that caffeine consumption influences the cardiovascular system in that it acutely increases blood pressure, circulating concentrations of noradrenaline, inhibits ischemic preconditioning and impairs endothelium dependent vasodilation (nawrot et al. 2003; riksen et al. 2009; turnbull et al. 2017). a high intake of eds can make the veins firm which will contribute to heart disease. the commonly reported conditions are heart palpitations, increased heart rate, 4 nova biotechnol chim (2023) 22(1): e1344 3 hypertension, chest pain, and dysrhythmias (turnbull et al. 2017). an experimental research study found an association between myocardial infarction and ed consumption in healthy 17and 19-year-old boys (rath 2012). molecules of caffeine function as inhibitors of the adenosine receptors, which results in the enhancement of angiotensin ii, epinephrine, and catecholamines. the increased production of angiotensin causes the rise in systolic blood pressure, whilst the increased level of catecholamines, particularly epinephrine elevates the heart rate. the study conducted by alsunni (2015) showed that there is generally an increase in the heart rate and the blood pressure in human arteries once a patient has consumed a caffeinated drink. this is linked to the above mechanisms. moreover, caffeine acts as a weak non-selective phosphodiesterase (pde) inhibitor, whereas the cyclic-amp (camp) is catalysed by the campdependent phosphodiesterase (pde3) and this process leads to the change in concentration of camp inside the cell. the inhibition of this enzyme leads to the increase of camp concentration inside the cell, and this results in the increase of cardiac inotropy, chronotropy, and dromotropy (klabunde et al. 2020). caffeine is rapidly absorbed in the gut, reaching nearly 100 % bioavailability in the body and peak plasma concentration within 30 – 120 min. it is metabolized largely by cytochrome p-450 and specifically by the cyp 1a2 isozyme (babu et al. 2008). the major metabolites of caffeine are 3,7-dimethylxanthine (systematic name), which is trivially known as theobromine, and 1,3-dimethylxanthine (systematic name), which is trivially known as theophylline. both compounds/metabolites complement the pharmacological effect of caffeine, while also being biologically active in the human body in their own right (chen et al. 2017). caffeine elimination follows non-linear pharmacokinetics that is based on the michaelis-menten kinetics equation. the caffeine half-life falls inside the interval from 3 to 6 h (babu et al. 2008). taurine taurine has the systematic chemical name of 2-aminoethane-sulphonic acid (c2h7no3s). it is a β-amino acid, and its concentration is high in the heart and the skeletal muscles, as well as in other tissues, with the normal plasma concentration of 44 ± 8 μmol.l-1. although it falls under the group of amino acids, it does not take part in the formation of proteins (schaffer et al. 2010). taurine is a by-product of cysteine and methionine. it contains a sulfonate group and not a carboxyl group. this is how it differs from other amino acids hence it is considered as an amino sulfonic acid. the main route of taurine biosynthesis is illustrated below in fig. 2 (vitvitsky et al. 2011). in that figure, the molecule of the amino acid cysteine is converted into cysteinesulfinate by the action of cysteine dioxygenase. the next step in the pathway is catalysed by the cysteinesulfinic acid decarboxylase, with the reactant being cysteine sulfonate and the product being hypotaurine (vitvitsky et al. 2011). as a result of this enzymatic reaction sequence, the final step occurs when hypotaurine undergoes oxidation to form taurine (vitvitsky et al. 2011). when it comes to additional biochemical effects in the human body, taurine is an organic osmolyte that regulates cell volume and is involved in modulating free calcium concentration inside the cell. this amino sulfonic acid is found throughout the body but does not form components of proteins. it is found mostly in the pupil, heart, brain, retina, muscle tissue, and the heart (ripps et al. 2012). taurine has an influence on the homeostasis of calcium within the cell through the pathway of na+/ca2+ exchanger. it decreases glutamateinduced [ca2+]i accumulation in the cells by preventing the intracellular uptake of ca2+ and the molecular mechanism of this is the reverse mode of the na+/ca2+ exchanger (wu and prentice 2010). literature suggests that taurine also influences the metabolism of energy in cardiac tissue and its deficiency leads to an impairment of the respiratory chain (schaffer et al. 2016). fig. 2 above outlines the main steps of how taurine is formed: 1) cysteine is catalyzed by the enzyme cysteine dioxygenase and oxidized into cysteine sulfinate; 2) cysteinesulfinate decarboxylase 5 nova biotechnol chim (2023) 22(1): e1344 2 catalyzes cysteine sulfinate to form hypotaurine; 3) hypotaurine is oxidized to taurine, catalyzed by hypotaurine dehydrogenase. there can be another branch of hypotaurine synthesis, in which cysteamine is converted to hypotaurine by the ado enzyme activity (vitvitsky et al. 2011). fig. 2. a diagram illustrating the biosynthesis of taurine. generated using chemdraw software application (based on the article by vitvitsky et al., 2011). in addition, taurine has a molecular structure similar to gamma-aminobutyric acid (gaba) illustrated in fig. 3, and binds to gaba receptors where it potentiates gaba-ergic effects in the body (reyes-haro et al. 2014). usually, when drugs interact with the gaba receptor, they potentiate an anxiolytic effect, therefore it is possible that taurine may have the same effect as well. dombovy-johnson (2010) suggests that when taurine is combined with caffeine it may increase caffeine stimulant activity by interacting synergistically. this finding is supported by research done on the sleep-wake activity of the combination of taurine and caffeine on drosophila melanogaster, a species of fruit fly. the results have shown that caffeine increases locomotive activity and decreases sleep duration, whereas taurine decreases locomotive activity and increases sleep time. when taurine was added to caffeine it resulted in an inhibition of sleep with a greater effect than when caffeine was administered alone (lin 2010). this could be a result of the fact that taurine is known to influence the metabolism of energy. literature suggests that taurine deficiencymediated defects lead to a reduction in atp generation by impairing energy metabolism (schaffer et al. 2016). therefore, when the ratio of nadh(+h+)/nad+ is elevated, the resulting action leads to the inhibition of key dehydrogenases such as those in the kreb’s cycle and they include “α-ketoglutarate dehydrogenase, isocitrate dehydrogenase and citrate synthase” (schaffer and kim 2018). fig. 3. chemical structures of gaba and taurine illustrating similar molecular structure. generated using chemdraw software application. cuttitta et al. (2013) investigated the role of taurine in the cardiovascular system. in the study, gaba was identified as the major inhibitory neurotransmitter responsible for the regulation of the heart. this neurotransmitter is found in the peripheral tissues as well as the cns. the study was conducted on rabbits, and it was reported that gaba can suppress the release of noradrenaline which may lead to the regulation of vascular tone (schaffer and kim 2018). the selective permeability of the blood brain barrier restricts this inhibitory neurotransmitter from penetrating. 1 2 3 6 nova biotechnol chim (2023) 22(1): e1344 3 alternatively, taurine has more permeability than gaba, therefore there is no concentration change in the brain following iv administration of gaba. the study also indicated that iv administration of taurine exhibited a significant decrease in systolic blood pressure in an experiment that was performed on conscious adult rats. furthermore, it has been proven using an aortic ring preparation that taurine can act as a vasodilator. taurine as a neuromodulator protects the cns and reduces seizure episodes by agonizing the gabaa receptors which are found on the muscularis of the cerebral blood vessels and the aorta (cuttitta et al. 2013). there are similar reports that have been published by chan et al. (2013) and l’amoreaux et al. (2010) in recent years. in japan, taurine has been approved to treat congestive heart failure. the chronic administration of taurine reduces the action of noradrenaline and angiotensin ii, which decreases myocardial performance. taurine can decrease catecholamine overflow through the alteration in ca2+ transport, thus leading to the reduction of noradrenaline activity (schaffer and kim 2018). the therapeutic window period of taurine is at least 8 hours. this was proven in a study conducted by sun et al. (2012) on experimental stroke in rats however, data is still lacking on the oral administration of taurine in humans. the nh2 group in the covalent structure of taurine can undergo the maillard-type reactions (elzoghby et al. 2015). carbonyl groups are found in many primary metabolites in the human body and examples of those molecules include glucopyranose and ch3coh. furthermore, it has been illustrated that taurine-glucose reaction results in an antioxidant effect and there is a likelihood of hindering taurine effect against protein modification when it reacts with acetaldehyde (ogasawara et al. 1994). carbohydrates carbohydrates or sugars [cn(h2o)n] are molecular compounds that are the major source of metabolic energy in humans and mammals. the carbohydrate content of the human diet is usually derived from fructose corn syrup, glucose, and sucrose (alsunni 2015). lactose can also play a role here. as listed on the packaging labels, most eds contain sugar in the form of sucrose. the world health organization (who) has decreased the suggested amount of sugar that should be taken daily from 10 % to 5%, this is about 25 g of sugar per day. the sugar content in the eds falls within the recommended amount. the consumption of high sugar content for a long period is a risk factor for obesity which subsequently leads to diabetes. there is a strong link between the body mass index (bmi), diabetes and insulin resistance. in obese individuals, the building-up of insulin resistance is influenced by the increase in quantity of free fatty acids, cytokines, hormones, and other substances involved in the development of insulin resistance (al-goblan et al. 2014). an in vivo study that was performed on rodents showed that a high chronic consumption of fructose will result in obesity, high blood pressure, type 2 diabetes, hepatic and extrahepatic insulin resistance. in addition, the high intake of fructose can cause dyslipidemia and impair hepatic insulin sensitivity (tappy and lê 2010). therefore, the high usage of eds is an attribute that elevates the chances of developing type 2 diabetes and obesity (alsunni 2015). although the summary molecular formulas of fructose and glucose are the same (c6h12o6), their metabolism and absorption pathways have some differences. fructose absorption takes place in the gut via the glut-5 transporter facilitated diffusion (goran et al. 2013), whereas glucose absorption is mediated by the sglt1 co-transport mechanism. scientific reports in the literature have indicated that molecules of fructofuranose, does not trigger the secretion of insulin and leptin hormones (bray et al. 2004) instead, it is believed that it directly adds to weight gain and increases energy intake (goran et al. 2013). ragsdale et al. (2010) conducted a study which reported that there is an increase in blood sugar level after the consumption of eds; and this can be explained by the study performed by lee et al. (2005) which demonstrated that caffeine consumption leads to decrease in insulin sensitivity. alcohol and energy drinks the trend of mixing eds with alcohol is no longer seen as an abnormality, but more of a common 7 nova biotechnol chim (2023) 22(1): e1344 2 practice. both alcohol and eds have a diuretic and dehydrating effect therefore, they may decrease the body’s capability to metabolize alcohol (ferré and o’brien 2011). caffeine and alcohol combinations the underlying molecular mechanism of the interaction between alcohol and caffeine is unknown however, it is known that both can alter the neurotransmission of adenosine. in a review carried out by ferré and o’brien (2011) it is suggested that during alcohol consumption, caffeine blocks the adenosine al receptors and thus opposes the undesirable effects of alcohol. in addition, the interaction of dopamine d2 and adenosine a2a receptors can increase the effect of alcohol-induced dopamine release, and this can occur through the blockade of adenosine a2a receptors by caffeine. li et al. (2014) demonstrated that some eds can increase or decrease the activity of alcohol dehydrogenase (adh) and aldehyde dehydrogenase (aldh). the degradation happens when alcohol is ingested and broken down into acetaldehyde (toxic compound) by alcohol dehydrogenase and further metabolized to acetate (less active by-product) by aldehyde dehydrogenase (u.s. department of health & human services 2007). an increase in the activity of these enzymes results in rapid metabolization of ethanol or acetaldehyde (li et al. 2014). another study by wang et al. (2016) selected 20 ed beverages to assess their effect on ethanol and acetaldehyde levels in the blood; literature supporting this study suggests that eds increase the activity of alcohol dehydrogenase, leading to the increased toxic effects of alcohol in the body. similar findings were reported by rutledge et al. (2012). these cases highlight the dangers of mixing alcohol with eds. the effects of carbon dioxide on alcohol absorption there is evidence suggesting that carbonation, or addition of co2 to eds, may increase the alcohol absorption rate, which will overall lead to toxicity (attila and çakir 2011; velazquez et al. 2012). it has been hypothesized that the gas released into the gastric lumen by carbonated beverages causes distension of the stomach, which ultimately elevates the rates of gastric emptying. this is believed to have effects on alcohol absorption rates (roberts and robinson 2007). there have been many speculations on carbonation and its effect on alcohol absorption rate therefore, further research needs to be conducted in this aspect of study. the effects of carbon dioxide on the mucosal blood flow were looked at in a study conducted by siński et al. (2003) whereby the research was investigating if the gastric mucosal blood flow (gmbf) can be influenced by the stimulation of the central chemoreceptors in rats. the experiment was conducted in a hypercapnic-hyperoxic atmosphere with the following chemical components in it and with the following volume fractions present (%, v/v): co2 (10 %), n2 (40 %), and o2 (50 %). the application of this gas mixture resulted in the activation of the central chemoreceptors which in turn resulted in the decrease of the gmbf. findings from another research study by sherwood et al. (2015) supported dean’s theory of gastric co2 ventilation and proved the hypothesis that, hypercapnia stimulates gastric co2 production and ventilation, thus rendering the “gastroesophageal dysfunction”. health risks associated with chronic consumption of energy drinks among youth central nervous system effects despite having significant benefits for mental and physical stamina of some or all the abovementioned ed components, chronic consumption can be potentially dangerous to the cns. caffeinism is a pathophysiological condition which results from consuming excessive amount of caffeine of extended periods for time i.e., it is a medical condition which results from chronic abuse of caffeine by an individual. the condition presents itself in the cns and periphery in the form of anxiety, psychomotor alterations, sleep disturbances, mood disturbances, and the like. research has shown that abstaining from caffeine causes withdrawal syndrome as a result of prior over-consumption and dependence on the 8 nova biotechnol chim (2023) 22(1): e1344 3 substance. the withdrawal symptoms include headaches, nausea, vomiting, diarrhea, facial flushes, muscle twitching, restlessness, and cardiac arrhythmias. at the same time, caffeinism can only be clinically diagnosed with blood tests. these symptoms can present themselves as quickly as 24 h from prior ingestion however, it has been reported that reintroducing caffeine into the body alleviates these symptoms within an hour of consumption (doering et al. 2017). a long-term solution may be to gradually wear off caffeine until the individual is no longer dependent on it, or alternatively switch to a caffeine-free product. if an individual consumes a caffeine dose equal to or above 200 mg, they may develop the symptoms of caffeine toxicity (alsunni 2015). the most common cns side effects reported include anxiety, tremors, confusion, irritability, psychosis, agitation, seizures, and altered mental status (rath 2012; alsunni 2015). in addition, excessive chronic consumption of caffeine stimulates a “pronociceptive state of cortical hyperexcitability that can trigger or intensify headaches” (espinosa et al. 2017). caffeine induces four psychiatric disorders that are recognized by the diagnostic and statistical manual of mental disorders (dsm). this is a diagnostic guideline used by psychiatrists and clinicians to diagnose psychiatric illnesses, namely “caffeine intoxication, caffeine-induced sleep disorder, caffeine-related disorder, and caffeine-induced anxiety disorder” (bedi et al. 2014). park et al. (2016) sought out to study the association between ed consumption and mood disorders in korean adolescents. their findings revealed that ed consumption was statistically significantly linked to sleep problems, extreme stress, “suicidal ideation and a higher risk of suicide attempt” (park et al. 2016). depending on subjective sensitivity, ed consumption has been linked to events of memory loss, anxiety, and certain types of sleep disorder (daly et al. 1983; nehlig et al. 1992). this has consequentially translated to fatigue during class hours, resulting in behavioural problems and lower academic performance (dikici et al. 2013). the study by park et al. (2016) also suggested a positive association between the consumption of eds, soft drinks, and fast-food products. adolescents who consumed eds and fast-food products for 5 or more days a week were shown to be more susceptible to mental health risks and mood disorders. however, the study results did not indicate that there was a directional causality between ed consumption and mental illness. further to this point, it can be inferred that stress, anxiety, and sleep disorders could be seen as pathophysiological states that would encourage an adolescent to consume eds to resume a level of ‘functionality’. thus, a reverse causality is equally plausible. a cross-sectional study was conducted at a primary school in iceland, whereby a population-based survey was carried out with 11,267 boys and girls aged between 10 – 12 years participating (visram et al. 2016). in this study, the percentage of boys and girls who reported to have consumed eds daily were 7 % and 3 %, respectively. some of the symptoms that were experienced and communicated were headaches, stomach-aches, and insomnia. there is currently not enough scientific information on the risks associated with the chronic consumption of eds by children aged under 12 years, thus more research needs to be done in this age group. dikici et al. (2013) described a case report of a healthy adult male who was rushed to the emergency ward with epileptic seizure and ischemic stroke. this incident occurred following the consumption of three 250 ml eds with alcohol by the patient. there was no history of epilepsy in the patient or his family. another case report of an 18-year-old male patient was described by hernandez-huerta et al. (2017) where the patient on admission presented with a psychotic episode after consuming 6 cans of eds per day for 7 days. he did not have a history of any psychotic disorders, although his paternal family had a history of undefined mental disease. these case reports suggest that excessive consumption of caffeinated drinks may induce seizures and psychotic disorders. cardiovascular system effects according to who, cardio-vascular diseases (cvds) are the leading cause of global deaths with an estimation of 31 % deaths claimed globally in 2016. the chronic consumption of high-caffeinated beverages is a major concern due to effects they have on the cerebrovascular system. caffeine is 9 nova biotechnol chim (2023) 22(1): e1344 2 a sympathomimetic, thus its ingestion causes vasoconstriction and elevates blood pressure, the extent to which is dependent on the dose of caffeine, dosage form (capsule or liquid), and subjective sensitivity (grasser et al. 2016). doerner et al. (2015) studied the effect of ed consumption on myocardial contractility and their investigations found that there was an increased contractility of the left ventricle an hour after ingestion of caffeine and taurine mixture. in addition, observed an increase in diastolic pressure when caffeine was consumed alone. other such studies have confirmed the blood pressure elevating effect of caffeinated drinks, and all account conclude that ed consumption is directly associated with, at the very least, acute increases in blood pressure (cavka et al. 2015). nowak et al. (2018) on the other hand, reported a statistically significant increase in the diastolic blood pressure based on the results of a study on 68 young adolescents. with dependence being a very possible outcome of consistent use of and tolerance to caffeinated eds, it is possible for eds to become a dangerous dietary addition that in the long term, may potentiate events such as early onset hypertension and other cardiovascular illnesses such as tachycardia. cannon et al. (2001) wrote up and published a case report on the health outcomes of a 25-year-old australian woman, who developed intractable ventricular fibrillation, a condition where the heart quivers in beat rather than in rhythm, caused by a combination of their consumption of a “natural energy” guarana health drink and the underlying pre-existing mitral valve prolapse. the case was already studied by other authors, but it is mentioned here against due to its clinical significance (doering et al. 2017). the patient was discovered to have consumed a 55 ml squirt bottle of an ed with guarana and ginseng, and it was confirmed that she had no other sources of dietary caffeine apart from tea. during her autopsy, toxicologists detected caffeine in her aortic blood at a concentration of 19 mg.l-1, the equivalent of 15 – 20 cups of coffee and the link had been made to the natural ed consumed as it contained 10 g.l-1of caffeine. despite the patient having a pre-existing cardiac condition, the levels of caffeine in her blood including the post-mortem results, indicated the extremely high concentrations of caffeine from the consumed ed which potentiated a fatal cardiac arrhythmia (cannon et al. 2001). another case report of a 13-year-old female who was consuming eds every day for two consecutive weeks was described by mangi et al. (2017). the patient was presented to the hospital with chest pains, palpitations and her qt interval was prolonged. a genetic test was performed which confirmed that she had a long qt syndrome (lqts). this is a condition where the heart’s electronic system is disordered and takes a long to recharge after each heartbeat, resulting in a qt interval longer than normal. this disorder occurs when there is a defect in the ion channels and can lead to a potentially life-threatening cardiac arrhythmia. regulations and advertising legislation for caffeinated beverages in south africa the market for eds in south africa has been rising rapidly, with an estimated increase of 2 to 3 l per capita between the years 2009 and 2014 (stacey et al. 2017). advertising is one of the driving factors that influences consumption and spikes the volume of sales. stacey et al. (2017) studied the consumption of eds and the influence of marketing in south africa. their findings revealed that there is minimal scientific research on the advertisement and the consumption of caffeinated beverages in south africa. the government introduced a sugar tax on soft drinks with the aim to reduce their consumption. this proposal was published in 2016 and came into effect on the 1st of april 2018; where some manufacturers replaced the sugar with artificial sweeteners (zero sugar drinks), some reduced the sugar whilst others remained the same. the minister of health has amended the regulations of soft drinks, and the legislation that has been put into place requires manufacturers to display the following messages on the label of the container with capital letters of a height not less than 3.0 mm (south africa. department of health 2018): i) “high caffeine content”; ii) “not recommended for children under 12 years of age, pregnant women, persons sensitive to caffeine and not to be consumed as a mixture with alcohol beverages”. the manufacturers are also required to state the amount of caffeine in mg/serving size and mg/100 ml. the executive director of the bevsa 10 nova biotechnol chim (2023) 22(1): e1344 3 declared that all their members have signed a marketing code that prevents the marketing to children younger than 12 years as regulated (south africa, department of health 2018). although a marketing code has been signed, there still seems to be an aggressive marketing that attracts diverse children who are particularly below 12 years of age, tom. for example, one of the most popular eds is highly marketed to events such as the xgames, wrestling (wwe) and other extreme sports which attract a large adolescent demographic. suggested proposals for the public health management of ed consumption by children and adolescents in south africa according to curran and marczinski (2017) seventy percent of children, as well as around 40 % of teenagers, consumed caffeine doses in excess of the 3 mg.kg-1.day. such consumption levels can result in adverse caffeine effects just by consuming one ed together with other food sources. the results were calculated from an experiment where the following population groups; “5 – 12 years old (children), 13 – 19 years old (adolescents) and 20 – 24 years old (young men) had consumed a single retail unit of ed” (curran and marczinski 2017). these experimental results, along with the edsrelated scientific information mentioned in this article, show the seriousness of this (potential) health-related issues from the consumption of eds. as a result, policy interventions are needed to protect the health of children and adolescents against eds. given that caffeine is a psychostimulant drug with proven adverse effects upon high consumption, its inclusion in soft drinks should be treated the same way as other drugs, such as alcohol. the fact that the packaging label states that ed is “not recommended for children under 12” (curran and marczinski 2017), should propel the banning of its sales to children, and places such as schools and events organised for children should not allow eds to be sold there. stacey et al. (2017) studied the consumption of eds and the influence of marketing in south africa, their findings revealed that there is minimal scientific research on the advertisement and the consumption of caffeinated beverages in south africa. they further concluded that viewers, who are exposed to tv channels that advertise eds are likely to be consumers. how the viewers comprehend the message of the advertisement differs, especially when it comes to their (health) literacy levels. a child/south african citizen, who is proficient in english, will understand an advert about eds very differently when compared to their counterparts, whose mother tongue or major medium of communication is not english. the same applies to parents or guardians; most of these adverts, including the packaging labels are written in english and the comprehension may differ in accordance with the english-proficiency level or mother tongue of the speaker. therefore parents/guardians and their approach to their children ed consumption might differ. more focus should be placed on regulatory measures to update the ed packaging labels. specifically speaking about eds, the label should be designed and populated with information so that “all” children, who are 12 years or younger, including adolescents and parents/guardians, are able to understand the packaging label and avoid the negative side-effects of ed consumption. considering the education disparities that some parts of the population in south africa are subjected to, raises the potential challenge of the general and universal application of english as the language of the labels and medium to disseminate information about the ed health effects. this calls for more regulatory measures to be implemented to overcome such challenges. to address the challenges related to of the eds consumption by children and adolescents, educational health programmes and campaigns targeted at children, adolescents, adults (particularly parents and guardians) need to be initiated and rolled out at school and community levels (especially the lowincome parts of south africa). pamphlets and posters showing images of the harm caused by the chronic usage of eds can be distributed near and around social places. social media is one of the platforms that could be used to mostly reach adolescents with the ed-related public health campaigns. 11 nova biotechnol chim (2023) 22(1): e1344 2 conclusion in the current article, the authors sought to place a focus on some of the health risks associated with the chronic consumption of eds in children and adolescents. additionally, they propose interventions that can be implemented to address this issue. the exponential growth of the sales and consumption of eds needs to be matched with strict regulatory measures to protect vulnerable population groups from the associated health risks. eds are dangerous to the health of children and adolescents, therefore consumption of eds by this age group should be targeted for urgent public health interventions. south africa has one of the most unequal societies in the world and the country is still draped with inequalities inherited from the colonial times. socio-economic factors therefore play an important in public health. any intervention must be targeted for the local education and literacy levels have, as well as the links to the decision-making of consumers in relation to eds. the parental guidance provided to children and adolescents in relation to the consumption of these beverages in south africa. conflict of interest the authors declare that there is no conflict of interest. references ahamad j, mir sr, amin s (2019) a pharmacolognostic review on artemisia absinthium. int. res. j. pharm. 10: 25-31. al-goblan as, al-alfi ma, khan mz (2014) mechanism linking diabetes mellitus and obesity. diabetes metab. syndr. ob. 7: 587-591. alsunni aa (2015) energy drink consumption: beneficial and adverse health effects. int. j. health sci. 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tests jana kavuličová1, jana kaduková2, dana ivánová1 1department of chemistry, faculty of metallurgy, technical university in košice letná 9, košice, 040 11, slovak republic (jana.kavulicova@tuke.sk) 2department of material science, faculty of metallurgy, technical university in košice, letná 9, košice, 040 11, slovak republic (jana.kadukova@tuke.sk) abstract: the evaluation of the toxicity and stress caused by heavy metals on plants is very important part of the phytoremediation research. several physiological parameters can be used to assess the heavy metalinduced stress such as germination, plant growth and biomass production, photosynthetic pigments, antioxidant enzymes or antioxidants. published results of measured physiological parameters in plants exposed to metals were characterized from of the negative effects of metals point of view and compared with the experimental study of the metal (cu, cd, zn) toxicity in flax (linum usitatissimum) and china aster (callistephus chinensis) using the biochemical tests under the laboratory conditions. the germination and biomass production of c. chinensis significantly decreased with the increase of metal concentration which is considered a very typical response, however in l. usitatissimum slight stimulation of germination and biomass production at low metal content was observed. from the two studied plants c. chinensis expressed typical symptoms of the heavy metal toxicity including the decrease of total chlorophyll, chlorophylls a, b. on the contrary, heavy metal ions affected positively the physiological parameters of l. usitatissimum when low metal concentrations were added for example slight increase of the chlorophyll concentration in leaves was recorded. the decrease of the chlorophyll content was observed only at the high metal content. on the other hand, the typical response of plants on the heavy metal stress – the increase in peroxidase activity – was observed only for l. usitatissimum but not for c. chinensis that in all other tests showed significant toxicity symptoms. so if only one physiological parameter would be considered incorrect interpretation could be concluded. with the increase of phytoremediation practical applications the more systematic tests of heavy metal stress are necessary to help scientists working in that field correctly interpret their results and understand the plant behaviour. key words: phytoremediation, heavy metals, stress assessment, toxicity, linum usitatissimum, callistephus chinensis 1. introduction at present, phytoremediation is a method very often used to remove heavy metals from contaminated soils. high heavy metal concentrations in soils could be very toxic for physiology of plants and induce stress in plants (macfarlane and burchett, 2001; zhou and qiu, 2005). in general, two responses to metal stress can be distinguished – metal sensitivity and metal tolerance. sensitivity to metals results in injury or death of plants. resistance means that in spite of metal toxicity plant responses in a way enabling it to survive high concentrations of metals, and to produce the next generation of plants (orcutt and nilsen, 2000; kadukova and kavulicova, 2010). technologists dealing with phytoremediation often do not consider the biochemical aspects of the heavy metal-induced stress in plants. therefore it is necessary to know not only the ability of plants to accumulate metals but also their bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:51 utc 102 kavuličová, j. et al. ability to alleviate stress (taulavuori et al., 2005). there are a lot of results concerning the effect of elevated metal concentrations on physiological state of plants published in scientific literature, however, this information are often contradictory. the aim of our study was to summarize and compare results of measured parameters in plants exposed to metals published in scientific literature – the toxic effects induced by heavy metals on plant physiology using biochemical tests. combination of selected physiological parameters, such as germination, plant growth and biomass production, content of photosynthetic pigments, antioxidant enzymes or antioxidants from the negative effects of metals point of view expressing the influence on photosynthesis and cell oxidative status can be desirable for stress evaluation. these published results are compared with our results of physiological parameters measurement in linum usitatissimum and callistephus chinensis grown at the presence of cu, zn, and cd. 2. materials and methods 2.1. experimental set-up l. usitatissimum and c. chinensis seeds were germinated and grown under laboratory conditions in plastic pots (four seeds per each pot) filled with organic substrate (weight of substrate in each pot was 1 130g). the organic substrate used for plants was produced in slovakia with the trade name florcom. the main constituents were white sphagnum peat and black sphagnum peat blended with the nutrients. substrate ph (h2o) was 5.5 – 7.0, electrical conductivity 0.8 ms/cm, humidity max. 65%. plants were watered two times per week with approximately 200 ml tap water. at the beginning of the experiment plants were divided into three groups. five pots each with four seeds were used as controls with uncontaminated soil substrate and 10 pots (two groups) were used for the treatment with cu2+, cd2+ and zn2+ ions in contaminated soils. soil substrate in the second group (named low metal content) was contaminated with amount of the metal ions 144.3, 220.0 and 0.9 mg.kg-1 of dry weight of substrate per pot for cu2+, zn2+ and cd2+ ions, respectively. the third group was contaminated with five times higher concentrations of cu2+, cd2+ and zn2+ ions than in the second group and named high metal content. cu2+, cd2+ and zn2+ ions were separately added into each pot as an aqueous solutions of cuso4, cd(no3)2 and zn(no3)2 in one dose on the first day of the experiment. 2.2. measurement of physiological parameters germination – seeds of l. usitatissimum and c. chinensis were sown in the soil substrates mentioned above. seeds were placed under the surface of the soil substrate in pots under the laboratory conditions, five replicates for each pot, and five pots for each treatment. the pots were watered with tap water daily till seed germination. only daylight was used for illuminating. temperature was measured once a day and was in the range of 20 – 23°c. plant length – the length of plants was measured every week regularly. at the end of the experiment all plants were excavated from all the sets of treatments, partitioned bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:51 utc nova biotechnologica et chimica 11-2 (2012) 103 into shoots and roots, carefully washed with distilled water and oven dried at 80°c for 24 h, to determine the biomass (g plant−1) of each plant part. the chlorophyll content – the chlorophyll content was measured at the tenth week of experiment according to harborne (1984). 0.05 g of fresh leaves was taken from each plant randomly, washed with distilled water and small cuttings were homogenized in 80% acetone. after double centrifugation (14 000 g, 1 min), direct determination of the absorbance of the supernatant was carried out at wavelengths of 663 and 646 nm. guaiacol peroxidase (gpx) activity – for measuring the gpx activity according to the modified method of erdelský and frič (1979) 0.5 g of fresh plant material was homogenised in 4 ml of cold 0.05m phosphate buffer (ph 5.8). the homogenate was centrifuged at 14 000 g for 20 min. the oxidation of guaiacol by peroxidase from the plant extract was observed by the monitoring of absorbance increase during 3 minutes of reaction. the change of absorbance per minute was used to quantify the amount of enzyme in the mixture using the extinction coefficient for tetraguaiacol ε = 26.6 μm-1.cm-1. enzyme activity unit was expressed as the change in absorbance per minute (∆a470/min). 3. results and discussion 3.1. germination germination test is a basic method to assess heavy metals toxic effects on plants (di salvatore et al., 2008). seed germination and the early seedling growth are more sensitive to metal stress because some of the defence mechanisms have not developed and it is also strongly related to the seed coat permeability to metal ions (liu et al., 2005). germination test gives the picture about the metal toxicity but it is also important for the practical application of plants in phytoremediation under the field conditions. negative effects (delay in germination, lower germination rate, inhibition of growth of roots and shoots, no germination, detrimental effect on germination) of metals exposure on seed germination have been often reported by several authors (di salvatore et al., 2008; wierzbicka and obidzińska, 1998; rout et al., 2000; abedin and meharg, 2002; rahoui et al., 2010). some authors observed decreased germination only at higher metal content but at low metal content stimulation of germination was determined (espen et al., 1997; li et al., 2007). among other factors which affected seed germination can be involved – replacing filter paper with agar resulting in improved sensitivity of germination, combination of several metals invokes detrimental effect on germination, adaptation of plants at germination, support of germination by less toxic metal (e.g. zn) and differences in plant species. in our study it also was verified slight stimulation of germination at low metal content in l. usitatissimum, whereas china aster was more sensitive to the metal toxicity than flax (fig. 1). the negative effect of metals on germination also depends on the kind of the metal and the metal form. kind of metal salt has strong effect on germination and can influence the toxicity test results significantly. in general, nitrates alleviate negative effect of metal on germination (kadukova and kavulicova, 2010). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:51 utc 104 kavuličová, j. et al. 0 20 40 60 80 100 120 control low metal content high metal content treatment g er m in at io n [% ] l.ussitatissimum c.chinensis fig. 1. germination ability of l. usitatissimum and c. chinensis at cu, zn, and cd treatment. data are given as means (± s.e.) of five replicates. 3.2. reduction in growth the reduction in plant growth caused by heavy metal toxic effects on plants can be determined as reduced growth rate or decreased biomass production (begonia et al., 1998). negative effects of metals exposure on reduction in growth have been often observed by several authors (arduini et al., 2004; guo et al., 2007; vernay et al., 2008; cao et al., 2009; ozturk et al., 2010). some of them (arduini et al., 2004; cao et al., 2009; ozturk et al., 2010) determined that low metal content has stimulative effect on biomass production. for example in the case of l. usitatissimum, presence of low cu, cd and zn concentrations stimulated the biomass production (fig. 2a) but the same metal concentrations decreased the biomass production in c. chinensis (fig. 2b). it shows that plants of c. chinensis are more sensitive to the metal toxicity than plants of l. usitatissimum. a) 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 control low metal content high metal content treatment b io m as s dr y w ei gh t [g p la nt -1 ] shoots roots b) 0 2 4 6 8 10 12 control low metal content high metal content treatment b io m as s dr y w ei gh t [g p la nt -1 ] shoots roots fig. 2. the biomass dry weight of l. usitatissimum (a) and c. chinensis (b). data are given as means (± s.e.) of five replicates. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:51 utc nova biotechnologica et chimica 11-2 (2012) 105 the influence of metal on plant growth and biomass production significantly depends on the plant species and metal concentration. according to the comparison of published results (milone et al., 2003; arduini et al., 2004; fayiga et al., 2004; an 2006; rooney et al., 2007) it is visible that metal salt form does not influence plant growth and biomass production as significantly as it is in the case of germination. although some positive effects of metals in the form of nitrates were observed in comparison with the negative effects of the same metal in the form of sulphates or chlorides. however, the negative effect of metal cation usually predominates over the effect of anion (kadukova and kavulicova, 2010). considering this fact, we can conclude that this parameter – reduction in growth is not definitive physiological parameter for metal induced stress evaluation. 3.3. photosynthetic pigments a common response of plants to metal stress is a decrease of photosynthetic pigments (chlorophylls and carotenoids) in leaves of plants (monteiro et al., 2009). the reduction at the levels of total chlorophyll, chlorophylls a, b and carotenoids on exposure to heavy metals has been observed in many species treated with different metals (macfarlane and burchett, 2001; ekmekçi et al., 2008; ghnaya et al., 2009; ekmekçi et al., 2008; monferrán et al., 2009). however, some authors observed no changes or slight increase of photosynthetic pigments in leaves at low metal content (zhou and qiu, 2005; gupta and sinha, 2009). in our study, total chlorophyll, chlorophyll a and chlorophyll b contents were not different from the controls for all plants of l. usitatissimum under investigation (fig. 3a). on the other hand, the chlorophyll content in the leaves of c. chinensis was slightly decreased by the metal mixture in comparison with controls (fig. 3b). a) 0 0,5 1 1,5 2 2,5 control low metal content high metal content treatment c hl or op hy ll co nt en t [m g g -1 ] total ch ch a ch b b) 0 0,5 1 1,5 2 2,5 control low metal content high metal content treatment c hl or op hy ll co nt en t [m g g -1 ] total ch ch a ch b fig. 3. the chlorophyll content in the fresh leaves of l. usitatissimum (a) and c. chinensis (b) (per g of fresh weight of the leaves). data are given as means (± s.e.) of three replicates. it is obvious that chlorophylls seem to be more sensitive to metals than carotenoids. due to the ability of several metals to substitute the mg in the chlorophyll bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:51 utc 106 kavuličová, j. et al. molecule colour changes are not necessary to be observed after metal addition although the chlorophyll is non-functional. to evaluate the overall state of photosynthetic system the measurement of chlorophyll a fluorescence can give more precise view about the chlorophyll (küpper et al., 2000). according to published results it is possible to conclude that the influence of the metal on the chlorophyll content is not uniform and depends on several factors, such as plant species, kind of metal salt and its concentrations, time of exposure and presence of other chemicals or the specific mixture of the metals (kadukova and kavulicova, 2010). 3.4. antioxidant enzymes and antioxidants the formation of reactive oxygen species (ros) causing oxidative damage to plants is one of the typical heavy metals toxicity symptoms in plants. to combat the oxidative damage plants have the antioxidant defense systems comprising of enzymes catalases (cat), peroxidases (pod), superoxide dismutases (sod) and antioxidants such as ascorbate (asc), glutathione (gsh), α-tocopherol, flavonoids which remove, neutralize and scavenge ros (schützendübel and polle, 2002). measurement of the activity of antioxidant enzymes – catalases (cat), peroxidases (pod), superoxide dismutases (sod) can provide useful information about the stress level in plants. amongst various enzymes involved in scavenging of the ros activity, guaiacol peroxidase (gpx) plays a decisive role to evaluate the intensity of stress in plants (verma and dubey, 2003). significant increase in peroxidase activity on exposure to heavy metals has been observed in many plant species treated with different metals (baccouch et al., 1998; macfarlane and burchett, 2001; león et al., 2002; boominathan and doran, 2003; verma and dubey, 2003). in our study the content of guaiacol peroxidase of l. usitatissimum from the low and high metal treatment increased 1.5 and 3.5 times, respectively in comparison with the controls (fig. 4a). a) 0 20 40 60 80 100 120 140 160 180 control low metal content high metal content treatment s pe ci fic g p x a ct iv ity [ u m g -1 ] b) 0 1 2 3 4 5 6 7 8 9 control low metal content high metal content treatment s pe ci fic g p x a ct iv ity [ u m g -1 ] fig. 4. the specific gpx activity in l. usitatissimum (a) and c. chinensis (b) under different treatment. data are given as means (± s.e.) of three replicates. in some cases the enzyme activity levels do not change or even can be decreased in the presence of metals (león et al., 2002; zhang et al., 2005). for example, the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:51 utc nova biotechnologica et chimica 11-2 (2012) 107 specific activity of gpx of c. chinensis was found to be from 1.1 to 4.3 times lower in the plants from all treatments (fig. 4b). low activity of enzyme may not denote the low stress level. it can be connected with the fact that under extreme conditions of metal stress, plants may be too weak to produce enough antioxidant enzymes to protect it (fayiga et al., 2004). some authors determined different changes in enzyme activity in different plant organs in the same plant species (mazhoudi et al., 1997; shah et al., 2001). amongst several antioxidants gsh along with asc is involved in plant ascorbateglutathione cycle and under the metal stress their levels usually increase, however, their contents depend on ion species, metal concentration and plant species. the increase at the levels of gsh and asc on exposure to heavy metals has been observed in many plant species (romero-puertas et al., 2007; sun et al., 2007; pongrac et al., 2009). glutathione redox state is regarded as a good biomarker of the cellular redox state in metal stress. the decrease gsh/gssg (gssg glutathione disulfide) ratio indicates elevated sensitivity to metal stress in plant cells. αtocopherol and flavonoids are antioxidants and their increased contents can result in the protection of plants from oxidative stress. due to complex metal stress evaluation in plants it would be necessary to assess several parameters – antioxidant enzymes and antioxidants together and separately for tolerant and sensitive plant species. 4. conclusions phytoremediation is a potential remediation technology for cleaning-up polluted soils and water. research related to this relatively new and promise technology needs to be widened by better understanding heavy metal-induced stress in plants. symptoms of stress in plants depend on the particular metal (yet metal combination can act differently), plant species, but also on preliminary adaptation and other factors. the evaluation of heavy metal toxicity in plants using the biochemical tests is rather complex process. the influence of metals on physiological parameters is not uniform and changes of these parameters are difficult to be generalized. the combination of measurements of various physiological parameters is necessary to evaluate the metal stress in plants. there is still the demand to assess several selected key parameters (including their meaning) together which express the metal influence on photosynthesis and the degree of oxidative stress. it is desirable to prepare a standard protocol for clear stress evaluation for the researchers engaged in the field of phytoremediation without background in plant physiology. acknowledgement: this work was supported by slovak agency – project vega 1/0235/12. references abedin, m.j., meharg, a.a: relative toxicity of 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under cadmium stress. j. hazard. mater., 178, 2010, 1128-1131. romero-puertas, m.c., corpas, f.j., rodríguez-serrano, m., gómez, m., del río, l.a., sandalio, l.m.: differential expression and regulation of antioxidative enzymes by cadmium in pea plants. j. plant physiol., 164, 2007, 1346-1357. rooney, c.p., zhao, f.-j. mcgrath, s.p.: phytotoxicity of nickel in a range of european soils: influence of soil properties, ni solubility and speciation. environ. pollut., 145, 2007, 596-605. rout, g.r., samantaray, s., das, p.: effects of chromium and nickel on germination and growth in tolerant and non-tolerant populations of echinochloa colona (l.). chemosphere, 40, 2000, 855-859. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:51 utc 110 kavuličová, j. et al. schützendübel, a., polle, a. plant responses to abiotic stresses: heavy metal induced oxidative stress and protection by mycorrhization. j. exp. bot., 53, 2002, 1351-1365. shah, k., kumar, r.g., 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763-771. wierzbicka, m., obidzińska, j.: the effect of lead on seed imbibition and germination in different plant species. plant sci., 137, 1998, 155-171. zhang, h., jiang, y., he, z., ma, m.: cadmium accumulation and oxidative burst in garlic (allium sativum). j. plant physiol., 162, 2005, 977-984. zhou, w., qiu, b.: effects of cadmium hyperaccumulation on physiological characteristics of sedum alfredii hance (crassulaceae). plant sci., 169, 2005, 737-745. presented at the 2nd international conference “biotechnology and metals 2011”, september 22–23, 2011, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:51 utc nova biotechnologica et chimica 12-1 (2013) 1 doi 10.2478/nbec-2013-0001 © university of ss. cyril and methodius in trnava examination of the chemical composition of propolis ix. solid phase extraction of coumarins from propolis by using molecularly imprinted polymer katarína hroboňová1, jozef lehotay1,2, jozef čižmárik3 1institute of analytical chemistry, faculty of chemical and food technology, slovak university of technology, radlinského 9, bratislava, sk-812 37, slovak republic (katarina.hrobonova@stuba.sk) 2faculty of natural sciences, university of ss. cyril and methodius, j. herdu 2, trnava, sk 917 01, slovak republic 3department of pharmaceutical chemistry, faculty of pharmacy, comenius university, odbojárov 10, bratislava, sk-832 32, slovak republic abstract: the group selective molecularly imprinted polymers (mips) for coumarins, including umbelliferone, herniarin, 4-methylumbelliferone, scoparone were developed. using umbelliferone as the template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as linking agent, chloroform as porogen and bulk polymerization as synthetic method, the mips were synthesized and characterized with rebinding experiments. the characteristics of mips were evaluated by chromatographic method and frontal analysis, and demonstrating good selectivity and high binding capacity (269 µg of umbelliferone per 100 mg of polymer). the group selective mip was used as sorbent for the spe pretreatment of coumarins from propolis extracts prior to hplc analysis. analysis of the samples showed good recoveries (>70 %). the limits of quantitation (loqs) for studied compounds were 0.3-10 ng.ml-1 (determined for fluorescence detection). key words: molecularly imprinted polymer, umbelliferone, solid phase extraction, propolis, hplc 1. introduction propolis is a bee product which has lots of functional properties. it is a darkcoloured resinous natural substance collected by honeybees from leaf buds and the exudates of various tree species. propolis’s chemical composition is quite variable and complex depending on the provenance of the sample which is closely related with the flora surrounding the hive, the geographic and climatic characteristics at the site, honey bee species, etc.. raw propolis is composed of resin (consisting of flavonoids, phenolic acids, …), wax, essential oils, pollen and other organic compounds (terpenoids, steroids, aromatic alcohols, aliphatic acids and esters, coumarins, sugars, amino acids, ...). in addition, more than 300 compounds, mainly polyphenols, have been identified as constituents of propolis from different sources. propolis has been used extensively in folk medicine since it possesses various biological properties, such as antiseptic, antifungal, antibacterial, antiviral, anti-inflammatory, anaesthethic and antioxidant properties. (silici et al., 2005; sforcin et al., 2011). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:18 utc 2 hroboňová, k. et al. coumarins (benzo-α-pyrone derivatives) constitute an important group of natural compounds. many of their analogues are found to be biologically active and have been proven to exhibit pharmacological activity. chemical and pharmaceutical studies have revealed that important compounds in propolis samples are the coumarins, mainly including simple coumarins. umbelliferone (fig. 1) has been reported to have antioxidant and antimicrobial properties (jurd et al., 1971). several analytical methods can be used for the determination of simple coumarins. the recent methods for the analysis of coumarins are summarized in table 1. the cleaning step prior to analysis is necessary to separate target analytes from the other propolis constituents. the most widely used sample preparation techniques (clean-up and pre-concentration) are soxhlet extraction, solvent extraction and solid phase extraction (spe) of the propolis extract obtained. the typical spe sorbents often lack selectivity towards the target analyte and this is problematic for the selective extraction of analytes from complex matrices. molecularly imprinted polymers (mips) are tailor-made materials with a pre-defined selectivity for a given analyte or closely related compounds for which they were synthesised. these materials are obtained by polymerizing functional and cross-linking monomers around a template molecule, which lead to a highly cross-linked three-dimensional network polymer. the monomers are selected according to their ability to interact with the functional groups of the template molecule. after polymerisation, the template molecule is extracted and binding sites having their shape, size, and functionalities complementary to the target analyte are established. the resulting imprinted polymers are stable, robust and resistant to a wide range of ph, solvents, and temperature (tamayo et al., 2007). in previous work (hroboňová et al., 2013) the liquid-solid extraction with stirring, ultrasonic assisted extraction and accelerated solvent extraction (ase) methods were tested for sample preparations of propolis. the yields of umbelliferone and scoparone were higher by using of ultrasonic assisted extraction and by using of ase method in comparison with stirring support extraction. in the present work the molecularly imprinted polymer was applied as a solid phase sorbent for mip-spe cleaning of propolis extracts prior to hplc analysis. umbelliferone was used as the template for imprints formation. methacrylic acid was used as the monomer, and chloroform as the porogen. the binding capacity and the selectivity of polymer, evaluated on the basis of structurally related compounds, were studied. 2. materials and methods 2.1 chemicals and samples umbelliferone (7-hydroxycoumarin), 4-methylumbelliferone, herniarin, scoparone, and coumarin (fig. 1) were purchased from sigma-aldrich. acetonitrile, methanol, ethanol, chloroform (all hplc grade), acetone, acetic acid, 2,2′-azobis(2methylpropionitrile) (2,2′-azobisisobutyronitrile, aibn), methacrylic acid (maa), and ethylene glycol dimethacrylate (egdma) (all analytical grade) were purchased from merck. before use, the maa and egdma were distilled at reduced pressure to remove stabiliser from monomers. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:18 utc nova biotechnologica et chimica 12-1 (2013) 3 table 1. summary of the recent analytical methods for separation of coumarins. analytical method separated compounds conditions lod [ng·ml-1] reference uplc umbelliferone, herniarin sp: c18/beh mp: methanol/0.1% formic acid dt: ms/ms 0.1 nováková et al., 2010 hplc esculin, esculetin sp: c18/diamonsil; mp: acetonitrile/0.5% acetic acid (12/88 v/v) dt: uv (340 nm) zhou et al., 2011 hplc daphnetin, umbelliferone, 7-methoxycoumarin sp: c18/zorbax sb; mp: acetonitrile/0.5% acetic acid, gradient elution dt: dad (325 nm), msesi 40-84 su et al., 2009 hplc umbelliferone, 4-hydroxycoumarin, scopoletin sp: ods-bp/daisopak-120, mp: methanol/50 mmol·l-1 phosphate buffer (ph 5) dt: uv (286, 323 nm), fl (320/450 nm) 12.1-35.6 (uv) 0.06-12.5 (fl) ikeda et al., 2009 2d tlc ubelliferone, aescuketin, coumarin, scopoletin sp: cn silica mp i: acetonitrile/water (30/70 v/v) mp ii: ethyl acetate/nhexane (35/65 v/v) ciesla et al., 2009 ce esculin, esculetin 5 mmol·l-1 borate buffer ph 9.4/methanol (80/20 v/v) dt: laser-induced fluorescence 1.8 1.4 wang et al., 2007 cec herniarin, umbelliferone sp: hypersil scx/c18 mp: 50 mmol phosphate buffer ph 2.8 /acetonitrile (50/50 v/v) 0.03 fonseca et al., 2007 mekc coumarin sp: fused-silica capillary, 20 kv mp: 20 mmol·l-1 tetraborate buffer ph 9.4 + 20 mmol·l-1 sodium dodecylsulfate, dt: dad (200 nm) 0.01 micke et al., 2003 spt umbelliferone λmax 324 nm, 500 malík et al., 2012 sf umbelliferone, 4-methylumbelliferone, coumarin, scopoletin λexcit 340 nm, λem 425 nm fernández izquierdo et al., 2000 uplc – ultra pressure liquid chromatography, hplc – high performance liquid chromatography, 2d tlc two-dimensional thin layer chromatography, ce – capillary electrophoresis, cec capillary electrochromatography, mekc micellar electrokinetic chromatography, spt – spectrophotometry, sf – spectrofluorimetry, sp – stationary phase, mp – mobile phase, dt – detection, dad – diode array detection, uv – ultraviolet light, ms – mass spectrometry, esi – electrospray. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:18 utc 4 hroboňová, k. et al. the working solutions were prepared from stock standard solutions of umbelliferone, 4-methylumbelliferone, herniarin, scoparone and coumarin in methanol (c = 1.0 mg·ml-1) by appropriate dilution with water. the solutions were filtered through a 0.45 mm nylon membrane filter. fig. 1. chemical structures of umbelliferone and related compounds. propolis preparative without alcohol (sample i) was directly analysed by hplc or applied on mip-spe. for the preparation propolis tincture (sample ii) the portion 1 g of propolis was extracted with 40 ml of pure ethanol for three days at temperature 22 °c. the extract was filtered through a 0.45 µm nylon membrane filter and volume of 20 μl was injected into the liquid chromatograph or applied on mip-spe (2 ml of extract was loaded into the conditioned mip-spe cartridge and processed as described above for the mip-spe experiment). 2.2 hplc analysis experiments were performed on a hewlett packard (series 1100) hplc system equipped with a quaternary solvent delivery system, an injection valve (rheodyne) with 20 µl injection loop, diode array and fluorescence detector. chromatographic separations were performed on an analytical column lichrospher 100 rp18 (250 × 4 mm i.d., 5 µm). the mobile phase for gradient elution consisted of a mixture of acetic acid (0.3 vol %)/acetonitrile (10/90 v/v) (a) and acetonitrile (b). the flow rate was 1.0 ml·min−1 and the column temperature kept constant at 22 °c. the monitored wavelength of 323 nm was used for the spectrophotometric detection or 320 nm (λex) and 450 (λem) for fluorescence detection. gradient profile of mobile phase is shown in table 2. table 2. gradient profile of mobile phase. time [min] % a % b 0 100 0 35 44 56 40 0 100 45 0 100 r o r r r o 2 3 4 1 r1 r2 r3 r4 umbelliferone h h oh h coumarin h h h h 4-methylumbelliferone ch3 h oh h herniarin h h ch3o h scoparone h ch3o ch3o h bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:18 utc nova biotechnologica et chimica 12-1 (2013) 5 2.3 preparation of mips and nips umbelliferone (0.1 g, 0.7 mmol), maa (0.25 g, 2.9 mmol), egdma (2.34 g, (11.75 mmol) and aibn (0.03 g, 0.18 mmol) were added to a glass tube containing 7.5 ml of chloroform. the polymerisation mixture was kept in a water bath at 60 °c for 24 h. the resulting bulk rigid polymers were crushed and sieved through a 40 µm sieve. fine particles were removed by flotation in acetone and the final product was dried under diminished pressure at 60 °c for 1 h. the template molecule was extracted from the mip by soxhlet extraction with 100 ml of methnol/acetic acid (9/1 v/v) for 24 h. the non-imprinted polymer (nip) was prepared following the same procedure as for mip but without the template molecules during polymerisation. 2.4 determination of polymer binding capacity for the binding capacity studies, 0.3 g of the polymer mip or nip was transferred into a glass column and washed with methanol prior to use. the column was equilibrated by washing with umbelliferone solvent and subsequently the solution of umbelliferone was applied. the flow-rate was 0.2 ml·min-1. the eluent was monitored by uv detector at 323 nm. the breakthrough curves of each mip and nip were measured for umbelliferone solutions prepared in methanol, acetonitrile, methanol/water (50/50 v/v), acetonitrile/water (50/50) and water at a spiking level of 100 µg·ml−1. 2.5 mip-spe experiments the mip-spe column was prepared by packing the spe cartridge with the mip or nip material (0.1 g). the material was fixed by inserting polyethylene frits at the top and at the bottom. the prepared cartridge was conditioned by washing with 3 ml of methanol and 2 ml of water. next, the working solution of umbelliferone (0.5 ml, c = 10 µg·ml−1) or 2 ml of propolis tincture was applied at 0.5 ml·min−1 flow-rate. the column was washed with 2 ml of water and dried under diminished pressure for 10 min. finally, elution of the umbelliferone was performed by passing the elution solvent (2 ml of methanol or methnol/acetic acid (90/10 v/v)) through the column at a flow-rate of 0.5 ml·min−1. all the fractions (each of the volume of 0.5 ml) from the sample loading, washing and elution step were collected, solvent evaporated to dryness (diminished pressure, 30 °c), dissolved in acetonitrile (250 µl) and analysed by hplc. 3. results and discussion 3.1 preparation of molecularly imprinted polymer the non-covalent approach is the most frequently used method of preparing mip, in which the binding sites are formed by self-assembly between the template and the monomer followed by a cross-linked co-polymerization. to perform this non-covalent bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:18 utc 6 hroboňová, k. et al. mip approach, we selected the methacrylic acid as monomer. the structure of umbelliferone includes functional groups that may form hydrogen bonds with the monomer. in order to favour hydrogen bonding between the template and the monomer, a nonprotic solvent, chloroform, was chosen as the porogen solvent. 3.2 characterization of molecularly imprinted polymer the sorbents were evaluated by determining the sorption capacity of polymers for umbelliferone dissolved in different solvents, by evaluating the imprinting effects for mip and nip polymers, by evaluating the selectivity of the polymer, and by optimizing the mip-spe procedure. for determination of binding capacity of polymers the solutions of methanol, methanol/water (50/50 v/v), acetonitrile, acetonitrile/watwer (50/50 v/v) and water spiked with 100 µg·ml−1 umbelliferone were percolated on the mip. the same experiment was carried out in parallel on the nip (non-imprinted polymer) in order to evaluate non-specific interactions during the retention process. the binding capacities of the prepared polymers were examined by the breakthrough curves method and corresponded to the maximum amount of umbelliferone that can be retained on it without its release monitored by uv detector. typical break through curves used for the determination of binding capacities of polymers are shown in fig. 2. in the case of methanol/water (50/50 v/v) as umbelliferone solvent, the breakthrough values were observed at 76 min (mip) and 18 min (nip), which corresponds to the polymer binding capacity of 401 µg and 132 µg of umbelliferone per 100 mg of polymer. [ i ] 0 20 40 60 80 [mv] v ol ta ge ( v ) 0 20 40 60 80 100 fig. 2. the break through curves for determination of binding capacities mip and nip. conditions: column – mip/nip (0.3 g), solvent used for capacity studies umbelliferone (concentration 100 μg·ml-1) in methanol/water (50/50 v/v), flow rate 0.2 ml·min-1, spectrophotometric detection at 323 nm. in the next step, the influence of the nature of the umbelliferone solvent on the binding capacity of prepared polymers was studied. the results presented in fig. 3 nip mip t (min) v ol ta ge (v ) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:18 utc nova biotechnologica et chimica 12-1 (2013) 7 show that the solvent used in the capacity determination process plays an important role in the sorption of umbelliferone on polymer. the nature of the solvent for this step probably influences the relative swelling of the polymer which led to changes in the binding cavities and could also be important for solvation processes. a significant imprinted effect was observed for the mixture methanol/water (50/50 v/v) as umbelliferone solvent (the highest value of specific capacity (mip capacity minus nip capacity). the specific binding capacity of mip prepared in chloroform porogen was 269 µg of umbelliferone per 100 mg of polymer. when the water was selected as the solvent for determining the binding capacity, the umbelliferone was strongly retained on both mips and nips but no significant difference in binding capacities (data not shown) was observed between mip and nip. retention in this case was probably due to non-specific interactions. therefore, considering the highest values of specific binding capacity obtained for methanol/water (50/50 v/v) solvent, the experiments with real samples were performed in this medium. me oh me oh /h 2o me cn me cn /h 2o nip mip0 100 200 300 400 b in di ng c ap ac ity /( µg o f um be llif er on e pe r1 00 m g of po ly m er ) fig. 3. the binding capacities of mip and nip obtained for different umbelliferone solvents. solvents ratio (50/50 v/v); n=3. 3.3 retention behaviour of structural analogous on the mip the retention of compounds structurally related to umbelliferone, namely coumarin, herniarin, scoparone, 4-methylumbelliferone, was study to evaluate the selectivity of prepared mip. for this purpose 0.1 g portions of the polymer were stirred in 10 ml of mixture methanol/water (50/50 v/v) (blank) and mixture methanol/water (50/50 v/v) spiked with structural analogous (c = 10 µg·ml−1) for 1 h. subsequently, the polymer was filtered, washed with water and dried. the suspensions were prepared in methanol/acetic acid (90/10 v/v) (1 mg·ml−1 of polymer). the absorption signals at 323 nm in uv spectra of the supernatant, measured in the wavelength interval of 210–400 nm indicated that the polymer prepared for umbelliferone was selective for the template molecule and three structurally related compounds tested (scoparone, herniarin, 4-methylumbelliferone), while the selectivity bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:18 utc 8 hroboňová, k. et al. for the underivatized molecule of coumarin was low. these results showed that mip could be used as group recognition sorbent. 3.4 method development of the mip-spe procedure since the main aim of this work was to incorporate the mip prepared to umbelliferone into spe pretreatment, the polymer was packed into cartridges as described in the experimental section (part 2.5) and used in the off-line procedure. to optimize the extraction, the wash step and the elution step were varied to achieve selective extraction. in the wash step, the clean-up solvent suppressed the non-specific interactions without disrupting the selective interactions between the mip and the target molecule, and the interfering compounds which were bound non-specifically to the mip were removed. the washing solvents were optimized to obtain maximal recovery of the analyte (tested for umbelliferone) on the nip and minimal recovery on the mip. solvents including water and methanol/water (1/1 v/v) were tested. the best results were obtained when washing was performed with 2 ml of water, whereas some umbelliferone was released when the mixture methanol/water (1/1 v/v) was used. the final step was to select a suitable solvent for umbelliferone desorption from the mip. the elution solvents including methanol and methanol/acetic acid (9/1 v/v) were tested. mixture methanol/acetic acid (9/1 v/v) gave rise to the best recovery (80 %), when the amount of solvent used being 1.0 ml. poor recovery (58 %) was found with methanol as elution solvent. an increase in the volumes of these elution solvents to 1.5 ml enhanced the results to 92 % (methanol/acetic acid (9/1 v/v)) or 70 % (methanol). the volume of 2 ml of methanol/acetic acid (9/1 v/v) was used for the elution of umbelliferone from real samples. 3.5 application of mip-spe to propolis samples to demonstrate the potential of the polymer for the sample clean-up of complex matrices, the molecularly imprinted polymer was applied for the extraction of coumarins from propolis tincture. the mip-spe extracts were analysed by hplc with fluorescent detection. three aliquots of sample i were spiked with 0, 10 and 100 ng·ml−1 of umbelliferone and analysed by the proposed mip-spe procedure. the recoveries of the extraction on mip were determined by comparing the results obtained for the elution fractions with the results of the sample analysis without mip-spe treatment. the recovery values obtained for the analyses of three replicates of umbelliferone spiked propolis preparative were more than 77 % with rsd less than 10 %. the results demonstrate the suitability of the prepared polymer for the mip-spe clean-up of the complex matrix. the propolis tincture prepared from crude propolis material (sample ii) is very complex matrix and the obtained recoveries were about 50 %. the chromatograms of propolis extracts without and after application of the mipspe procedure are shown in fig. 4. in the case of the propolis samples tested, the presence of the coumarin derivative scoparone over the lod was detected along with umbelliferone. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:18 utc nova biotechnologica et chimica 12-1 (2013) 9 a min0 5 10 15 20 25 lu 0 50 100 150 200 250 fld1 a, ex=320, em=450 (katarina\kupr000077.d) 1 umbelliferone 2 4-methylumbelliferone 3 scoparone 4 herniarin b c min0 5 10 15 20 25 lu 0 10 20 30 40 50 60 fld1 a, ex=320, em=450 (katarina\kupr000146.d) min0 5 10 15 20 25 lu 0 10 20 30 40 fld1 a, ex=320, em=450 (katarina\kupr000150.d) sample ii fig. 4. hplc chromatograms of reference compounds (a), extract of propolis sample without (b) and after (c) mip-spe. chromatographic conditions: part 2.2 3.6 assay validation the method was validated over the concentration range 0.3-1000 ng·ml−1. the regression analysis showed that there was a linear dependence (r = 0.985). using a signal-to-noise ratio of 3 and 10, the limit of detection (lod) for umbelliferone and scoparone were 0.1 and 5.0 ng·ml−1 and limit of quantification (loq) 0.3 and 10 ng·ml−1 respectively (determined for fluorescence detection). the values showed that the hplc method is suitable for the determination of target analytes at the concentration levels presented in the propolis samples. the intra-day and inter-day precision of the mip-spe procedure was evaluated by determining umbelliferone in the propolis sample i. each assay was repeated three times a day over three different days. the values of the intra-day and inter-day precision expressed by the rsd values were 10% and 18%, respectively. the precision of the hplc determination of umbelliferone was determined at for a concentration of 100 ng·ml−1. the values of the intra-day and inter-day precision expressed by the rsd were 3.0 % and 5.0 %, respectively. the results showed good repeatability of the method developed. 2 1 3 4 1 3 3 1 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:18 utc 10 hroboňová, k. et al. 4. conclusion a group selective molecularly imprinted polymer was prepared using umbelliferone as template. the results indicated that the polymer exhibits highly selective binding for coumarins. a specific binding capacity of 269 µg per 100 mg of polymer was determined. the natures of the sample solvent were studied to achieve the maximal sorption capacity. the developed off-line spe extraction method provided enhanced sample clean-up and adequate recovery from propolis samples. acknowledgements: this study was financially supported by the scientific grant agency of the ministry of education, science, research and sport of the slovak republic and of the slovak academy of sciences (grant vega no. 1/0164/11). references cieśla, ł., waksmundzka-hajnos, m.: two-dimensional thin-layer chromatography in the analysis of secondary plant metabolites. j. chromatogr. a, 1216, 2009, 1035–1052. fernández izquierdo, m.e., quesada granados, j., villalón mir, m., lópez martinez, m.c.: comparison of methods for determining coumarins in distilled beverages. food chem., 70, 2000, 251–258. fonseca, f.n., tavares, m.f.m., horváth, c.: capillary electrochromatography of selected phenolic compounds of chamomilla recutita. j. chromatogr. a, 1154, 2007, 390–399. hroboňová, k., čižmárik, j., lehotay, j., spišská, ľ.: examination of the chemical composition of propolis viii. extraction of coumarins from propolis samples. (in slovak) farm. obzor, 82, 2013, 17-21. ikeda, r., wada, m., nishigaki, t., nakashima, k.: quantitation of coumarin derivatives in noni (morinda citrifilia) and their contribution of guenching effect on reactive oxygen species. food chemistry, 113, 2009, 11691172. jurd, l., king, a. d., jr., mihara, k.: antimicrobial properties of umbelliferone derivatives. phytochemistry, 10, 1971, 2965–2970. malik, a., kushnoor, a., saini, v., singhal, s., kumar, s., yadav, y.c.: analytical methods development of nutraceutical: umbelliferone. pharma science monitor. int. j. pharm. sci., 3, 2012, 67–73. micke, g.a., moraes, e.p., farah, j.p.s., tavares, m.f.m.: assessing the separation of neutral plant secondary metabolites by micellar electrokinetic chromatography. j. chromatogr. a, 1004, 2003, 131–143. nováková, l., vildová, a., mateus, p.j., goncalves, t., solich, p.: development and application of uhplc – ms/ms method for the determination of phenolic compounds in chamomile flowers and chamomile tea extracts. talanta, 82, 2010, 1271 – 1280. sforcin, j.m., bankova, v.: propolis: is there a potential for the development of new drugs. rewiev. j. ethnopharmacology, 133, 2011, 253-260. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:18 utc nova biotechnologica et chimica 12-1 (2013) 11 silici, s., kutluca, s.: chemical composition and antibacterial activity of propolis collected by three different races of honeybees in the same region. j. ethnopharmacol., 99, 2005, 69-73. su, j., zhang, ch., zhang, w., shen, y., li, h., liu, r., zhang, x., hu, x., zhang, w.: qualitative and quantitative determination of the major coumarins in zushima by high performance liquid chromatography with diode array detector and mass spectrometry. j. chromatogr. a, 1216, 2009, 2111 – 2117. tamayo, f.g., turiel, e., martín-esteban, a.: molecularly imprinted polymers for solid-phase extraction and solid-phase microextraction: recent developments and future trends. j. chromatogr. a, 1152, 2007, 32–40. wang, w., tang, j., wang, s., zhou, l., hu, z.: method development for the determination of coumarin compounds by capillary electrophoresis with indirect laser-induced fluorescence detection. j. chromatogr. a, 1148, 2007, 108–114. zhou, t., xiao, x., li, g., zong-wei, c.: study of polyethylene glycol as green solvent in the microwave – assisted extraction of flavone and coumarin compounds from medicinal plants. j. chromatogr. a, 1218, 2011, 3608 – 3615. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:18 utc microsoft word ivanisova et al. 2012.doc nova biotechnologica et chimica 11-1 (2012) 45 doi 10.2478/v10296-012-0005-0 ©university of ss. cyril and methodius in trnava antioxidant activity of milling fractions of selected cereals eva ivanišová1, miroslav ondrejovič2, stanislav šilhár2 1department of storing and processing plant products, slovak university of agriculture, tr. a. hlinku 2, nitra, sk-949 76, slovak republic (eva.ivanisova@post.sk) 2department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (miroslav.ondrejovic@ucm.sk) abstract: the aim of this study was to evaluate antioxidant potential of four milling fractions of selected cereals grew in the year 2009 and 2010. free radical scavenging activity of samples was measured using dpph assay and reducing power was determined using frap assay. secondary, total phenolic and flavonoid content of cereal extracts was evaluated. we found that flour fractions (break flour and reduction flour) showed the lower proportion of the total antioxidant potential than bran fractions (fine bran and coarse bran), which was balanced in observed years. extract from barley had the highest values of antioxidant activity and polyphenol content. keywords: cereals, milling fractions, polyphenols, antioxidant activity 1. introduction in many countries, cereals are main ingredient for human foods or animal feeds (castro-rubio et al., 2006). epidemiological studies indicate that the consumption of whole-grain and whole-grain products is related to reduction in total mortality, coronary heart disease mortality, diabetes and cancer incidence (serpen et al., 2008). these beneficial effects are attributed to the bioactive factors in cereal grain such as non digestible carbohydrates and phytochemicals. the important part of phytochemicals with low molecular weight present in cereal grain is group of antioxidants such as tocopherols, lignans, flavonoids and phenolic acids. antioxidants are defined as molecules that, at low concentration and specific assay conditions, can delay or prevent oxidation of an oxidizable substrate (vaher et al., 2010). higher concentrations of these compounds are found in the outer layers of the kernel which constitute the bran (kim et al., 2006). cereal grains are rich in phenolic acids and saponins, while phytoestrogens and flavonoid are presented in small quantities (dordević et al., 2010). studies have shown that dietary phenolics have high antioxidant activity, which may contribute to their health benefits. in cereals, the predominant phenolic acid is ferulic acid, representing up to 90 % of total polyphenols. other phenolic acids including vanilic, syringic, chlorogenic, p-coumaric, m-coumaric and oh-cinnamic acid have also been reported in cereals (hosseinian and mazza, 2009). total amount of polyphenols in cereals is highly variable both in whole grain and in bran and also depends on the cereal variety and milling procedure (adom et al., 2005). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:06 utc 46 ivanišová, e. et al. the main objective of the present work was to evaluate antioxidant potential of selected cereals by free radical scavenging activity (dpph) and the ferric ion reducing antioxidant power (frap), and its distribution into the dry milling fractions. in addition, the content of flavonoids and phenolics was also determined, in order to refer unutilized potential of naturally occurring antioxidants in cereals, which leaving in the form of bran during the production of flour. 2. materials and methods 2.1 plant material cereals such as wheat (torysa, ppri), oat (cacko, depof), spelt wheat (roquir, ppri), triticale (kandar, ppri), rye (dankovské nové, ppri), barley (ezer, depof) were grown in the years 2009 and 2010 on a field nursery at department of environmental protection and organic farming (depof) spišská belá (sk) and on a field at plant production research institute (ppri) piešťany (sk). 2.2 sample preparation the samples were milled by laboratory mill (brabender quadrumat senior) gaining four milling products: break flour (mf i.), reduction flour (mf ii.), fine bran (mf iii.), and coarse bran (mf iv.). the milling fractions were extracted with methanol in ratio 1:80 (w/v) for 20 hours at laboratory temperature. after centrifugation at 3000 g (himac ct 6e, hitachi ltd., japan,) for 20 min, the supernatant was evaporated at 40 °c. residue was solubilised in methanol to a final volume 2 ml. 2.3 free radical scavenging activity free radical scavenging activity of samples was measured using the 2,2-difenyl-1picrylhydrazyl (dpph) according to the procedures described by yen and chen (1995). the extracts (25 µl) were mixed with 100 µl of dpph solution (0.012 g dpph in 100 ml methanol) and incubated for 10 min at laboratory temperature. after them, reaction mixture absorbance was determined at 550 nm using biotek microplate reader (elx800). free radical scavenging activity of the samples was expressed as mg of trolox equivalent antioxidant capacity per g of dry matter (mg teac/g dm). 2.4 reducing power reducing power of samples was determined according to the procedure by oyanizu (1986). the mixture of cereal extract (20 µl), phosphate buffered saline (50 µl, ph 6.6) and 1 % potassium ferricyanide (50 µl) was incubated at 50 °c for 20 min, then rapidly cooled, mixed with 50 µl of 10 % trichloacetic acid, and centrifugated at 11 000 g (eppendorf minispin) for 10 min. 50 µl of the supernatant was mixed with 50 µl of distilled water and 10 µl of 0.1 % ferric chloride. the absorbance at 700 nm using biotek microplate reader (elx800) was detected. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:06 utc nova biotechnologica et chimica 11-1 (2012) 47 reducing power was expressed as mg of trolox equivalent antioxidant capacity per g of dry matter (mg teac/ g dm). 2.5 total phenolic content total phenolic content of cereal extracts was measured photometrically, using the modified folin-ciocalteu method as described by singleton et al., (1965). each cereal extract (0.1 ml) was mixed with 0.1 ml of the folin-ciocalteau reagent and 1 ml of 20 % sodium carbonate, and centrifuged at 11 000 g (eppendorf minispin) for 10 min. supernatant (240 µl) was used for measuring the absorbance at 700 nm using biotek microplate reader (elx800). the total phenolics content was expressed as mg of gallic acid equivalent (gae) per g dry matter (dm). 2.6 total flavonoid content total flavonoid was determined using the modified method by quettierdeleu et al., (2000). cereal extract (0.1 ml) was mixed with 20 μl of 5 % methanolic solution of aluminum chloride and centrifuged at 11 000 g (eppendorf minispin) for 10 min. supernatant (120 µl) was used for measured the absorbance at 405 nm on a biotek microplate reader (elx800). the total flavonoid content was expressed as mg of quercetin equivalent (qe) per g dry matter (dm). 3. results and discussion 3.1 yield of milling fraction the wheat and rye are main commodities of milling industry for production various type of flours. the oat, barley, triticale and spelt are used mainly as additives and for production of flour (muchová et al., 2011). the main component of cereal grain is endosperm, which constitutes about 83 % of grain, and is source of white flour during the milling. bran represents 14.5 % of grain, and they are part of whole meal flour during the milling, but nowadays bran are often removed from flour, and used for animal feed. germ constitutes about 2.5 % of grain and from flour is often removed (muchová et al., 2011). the purpose of milling is to separate bran and germ from endosperm so as to extract as much flour as possible, at minimum operating cost, while maintaining high and consistent flour quality (campbell et al., 2007). the moisture of selected cereals was in range 12.29 – 13.42 % and these values corresponding with limit values for cereals (max. 14 %). the samples of wheat, spelt, rye and triticale showed higher yields of flour fractions than bran fractions, and sample of oat and barley showed higher yield of bran fractions, what is caused mainly by different structure of grains. a comparison of results from different studies can be difficult due to variability in milling conditions. 3.2 free radical scavenging activity dpph• is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. the reductions capability of dpph• is bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:06 utc 48 ivanišová, e. et al. determined by the decrease in its absorbance induced by antioxidant (liu & yao, 2007). the scavenging effect of cereal extracts in flour milling fractions on dpph radical decreased in the year 2009 in the order: barley (26%) > spelt (19%) > oat (16%) > triticale (14%) > rye (13%) > wheat (12%), and in the year 2010 in the order: barley (29%) > spelt (22%) > oat (15%) > rye (13%) > triticale (12%) > wheat (9%). the scavenging effect of cereal extracts in bran milling fractions on dpph radical decreased in the year 2009 in the order: barley (22%) > triticale (17%) > rye (17%) > wheat (16%) > oat (14%) > spelt (14%), and in the year 2010 in the order: barley (20%) > triticale (18%) > wheat (17%) > oat (15%) > rye (15%) > spelt (14%) (fig. 1 and 2). these results indicated that all extracts had a noticeable effect on scavenging of free radicals. the higher activities of all extracts were measured in bran (mf iii. and mf iv.). bran is a composite material made of several layers, such as pericarp, testa and aleurone (hemery et al., 2011). the main components of the fine bran (mf iii.) are aleurone layer and the germ. pericarp is dominant in coarse bran (mf iv.) (schnürer, 1991). it is known that cereals bran are a rich source of fatty acids and several substances, such as tocopherol, vitamins, and phenolic compounds, possessing antioxidant properties (prisenžňáková et al., 2010). the extract from barley had the strongest scavenging activity in flour milling fractions. from the literature it is known, that the barley is an excellent source of natural antioxidant either for food preservation (to inhibit lipid oxidation), or for disease prevention (fardet et al., 2008). liu and yao (2007) determined scavenging activity of barley seed extracts and found strong activity, which was dominant in 70 % acetone extracts and shown similar activity to bht at the amount of 200 µg. in bran fractions, extracts from rye and wheat showed the strongest activity. high activity was also determined in flour extracts of spelt and in bran extract of triticale. in a recent study, hosseinian and mazza (2009) described triticale bran as a potential new source of antioxidant compounds. data variation in the scavenging activity of cereals in the year 2009 and 2010 is to be expected, as many factors such as genetics, agrotechnical processes and environmental conditions can influence the presence of antioxidant compounds. in addition, a comparison of results from different studies can be difficult due to variability in the experimental conditions amongst the method used (alvarezjubete et al., 2010). 3.3 reducing power for measurement of the reductive ability, the fe3+ fe2+ transformation in the presence of cereal extracts (year 2009 and 2010) was investigated. reductive capabilities of cereal extracts are shown fig. 3 and 4. increase in absorbance of the reaction mixture indicated the reducing power of the samples. reducing power of flour milling fractions of cereal extracts in the year 2009 exhibited the following order: barley (33%) > spelt (21%) > oat (15%) > wheat (11%) > rye (10%) > triticale (9%), and in the year 2010 exhibited the following order: barley (50%) > spelt (16%) > oat (13%) > rye (10%) > wheat (7%) > triticale (3%). reducing power of bran milling fractions of cereal extracts in the year 2009 exhibited the following order: barley bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:06 utc nova biotechnologica et chimica 11-1 (2012) 49 fig. 1. free radical scavenging activity of cereal extracts (in the year 2009) expressed as mg of trolox equivalent antioxidant capacity per g of dry matter (mg teac/ g dm), (i. mf – break flour, ii. mf – reduction flour, iii. mf – fine bran, iv. mf – coarse bran). fig. 2. free radical scavenging activity of cereal extracts (in the year 2010) expressed as mg of trolox equivalent antioxidant capacity per g of dry matter (mg teac/ g dm), (i. mf – break flour, ii. mf – reduction flour, iii. mf – fine bran, iv. mf – coarse bran). (38%) > rye (15%) > triticale (14%) > oat (14%) > wheat (13%) > spelt (6%), and in the year 2010 exhibited the following order: barley (32%) > triticale (16%) > wheat (16%) > oat (14%) > rye (13%) > spelt (9%). the higher activities of all extracts were measured in bran (mf iii. and mf iv.). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:06 utc 50 ivanišová, e. et al. the reducing capacity of a compound may serve as significant indicator of its potential antioxidant activity (liu and yao, 2007). the reducing properties are generally associated with the presence of reductones (pin-der, 1998). it is reported that the antioxidant action of reductones is based on the breaking of the free radical fig. 3. reducing power of cereal extracts (in the year 2009) expressed mg of trolox equivalent antioxidant capacity per g of dry matter (mg teac/ g dm), (i. mf – break flour, ii. mf – reduction flour, iii. mf – fine bran, iv. mf – coarse bran). fig. 4. reducing power of cereal extracts (in the year 2010) expressed as mg of trolox equivalent antioxidant capacity per g of dry matter (mg teac/ g dm), (i. mf – break flour, ii. mf – reduction flour, iii. mf – fine bran, iv. mf – coarse bran). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:06 utc nova biotechnologica et chimica 11-1 (2012) 51 chain by donating a hydrogen atom, or reacting with certain precursors of peroxide to prevent peroxide formation. it is presented that the phenolic compounds in cereals may act in a similar fashion as reductones by donating electrons and reacting with free radicals to convert them to more stable products and terminating the free radical chain reaction (liu and yao, 2007). the data presented here indicates that the marked reducing power of cereal extracts seems to be the result of their antioxidant activity. the extract from barley had the strongest reducing power in both years in all fractions. liu and yao (2007) determined reducing power of different solvent extracts of barley and found that 70 % methanol extract exhibit the highest activity. zhao et al. (2008) measured reducing power of malting barley extracts. they confirmed strong antioxidant activity of barley. the high activities were also determined in flour fractions of spelt, and in bran fractions of triticale and rye. zieliński et al, (2007) reported that rye is an excellent raw material for healthy and tasty foods. the ray grain contains a large variety of substances, especially those that are biologically active and demonstrate antioxidant properties, which include free radical-scavengers, reducing agents, and potential complexes of prooxidant metals and quenchers of the formation of singlet oxygen. 3.4 cereal polyphenols phenolics are compounds with one or more aromatic ring and one or more hydroxyl groups. structurally, phenolics in cereals can be subdivided into acids derived from either benzoic acid or cinnamic acid. vanillic and salicylic acids are derivates of benzoic acid while ferulic acid, the dominant phenolic acid in cereals, and caffeic acid are derivates of cinnamic acid (abdel-aal et al., 2001). phenolic acids are predominantly found in the outer bran layer. flavonoids are one group of phenolics, which consists of two aromatic rings linked by 3 carbons that usually in an oxygenated heterocycle ring. in cereals flavonoids are located mainly in the pericarp (dykes and rooney, 2007) but their content is very low (peterson, 2001). kim et al. (2006) reported that wheat is a good source of phenolic acid and flavonoids. king (1962) isolated two related flavone glycosides from wheat germ. three major flavones, apigenin, luteolin and tricin were identified in oat flour (peterson, 2001). in barley grains are dominant catechin and epicatechin (shahidi and naczk, 2004). the majority of phenolics in cereals are insoluble and bound by ester and ether linkages with polysaccharides, such as arabinoxylan and lignin, in the cell wall (liyana-pathirana and shahidi, 2006), while a smaller portion is soluble (stalikas, 2007). the bran layer is highly stratified not only in phenolic composition, but also in the degree of ester and ether bonds and the compounds to which the phenolics are cross-linked (verma et al., 2009). several studies have shown that methanol is an effective solvent in extracting phenolics and other polar substances from cereals (ragaee et al., 2006). in this study, methanol extracts from cereals were used for the determination of phenolic (mg gae/g) and flavonoid content (mg qe/g dm). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:06 utc 52 ivanišová, e. et al. 3.4.1 total phenolic content the total phenolic content was determined by the folin-ciocalteau assay. the results (year 2009 and 2010) are presented in tab. 1 and 2. it is evident that bran has higher content of total phenolic than flour. this is not surprising because it is well known that phenolic compounds are concentrated in the bran fractions of cereals that are removed during the milling of cereals into white flour (kim et al., 2006; vaher et al., 2010). abdel-aal et al., (2001) investigated the distribution of phenolic acids in wheat milling fractions. about 73 % of grain phenolic acids were found in the bran, but only 5 % in first and second milling fraction. table 1. the phenolic contents (mg gae/g dm) of milling fractions from cereals grew in the year 2009. milling fraction sample i. ii. iii. iv. barley 31.4 ± 1.2 62.1 ± 0.7 161.0 ± 4.1 291.7 ± 3.5 wheat 17.6 ± 0.1 19.7 ± 0.5 159.1 ± 2.6 128.2 ± 0.3 oat 31.3 ± 0.2 39.1 ± 0.7 99.2 ± 2.2 61.3 ± 2.7 spelt 40.4 ± 0.7 36.4 ± 0.3 66.4 ± 1.0 151.0 ± 3.7 rye 17.6 ± 0.3 30.3 ± 0.6 95.9 ± 1.6 159.5 ± 12.6 triticale 11.8 ± 0.5 16.3 ± 1.0 178.0 ± 1.7 133.7 ± 3.4 i. – bread flour, ii. – reduction flour, iii. – fine bran, iv. – coarse bran table 2. the phenolic contents (mg gae/g dm) of milling fractions from cereals grew in the year 2010. milling fraction sample i. ii. iii. iv. barley 32.4 ± 1.1 53.7 ± 0.1 111.2 ± 1.4 232.9 ± 3.6 wheat 9.2 ± 0.6 12.3 ± 0 130.7 ± 3.2 190.3 ± 3.4 oat 29.3 ± 0.6 35.1 ± 2.3 121.8 ± 3.6 53.6 ± 1.7 spelt 38.7 ± 1.3 47.6 ± 3.4 71.1 ± 0.7 168.7 ± 3.3 rye 23.6 ± 0.4 30.9 ± 0.1 110.1 ± 3.6 153.9 ± 4.1 triticale 10.7 ± 1.0 9.2 ± 0.5 122.5 ± 1.6 155.9 ± 1.6 i. – bread flour, ii. – reduction flour, iii. – fine bran, iv. – coarse bran total phenolic content of cereal extracts in flour milling fraction in the year 2009 decreased in the order: barley > (26 %) > spelt (22 %) > oat (20 %) > rye (14 %) > wheat (11 %) > triticale (7 %), and in the year 2010 in the order: barley > (26 %) > spelt (26 %) > oat (19 %) > rye (15 %) > wheat (8 %) > triticale (6 %). total phenolic content of cereal extracts in bran milling fraction in the year 2009 decreased in this order in the order: barley (27 %) > triticale (18 %) > wheat (17 %) > rye (15 %) > spelt (13 %) > oat (10 %), and in the year 2010 in the order: barley > (21 %) > wheat (20 %) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:06 utc nova biotechnologica et chimica 11-1 (2012) 53 > triticale (17 %) > rye (16 %) > spelt (15 %) > oat (11 %). the results showed that phenolics were found in all cereal extracts, but bran fractions content higher values of phenolics. the sample of barley and spelt in flour fractions showed higher amounts of phenolics than extract from oat and wheat. in bran fractions were the highest amounts of phenolics determined in sample of barley, wheat and triticale. a comparison of results from different studies can be difficult, because in our study cereals were milling into four milling fraction, while in other works, sample of cereals were milling into flour and bran. 3.4.2 total flavonoid content the total flavonoid content of cereal extracts is shown in tab. 3 and 4. higher concentration was found in bran (mf iii. and mf iv.), but in smaller amounts than the total phenolic content. bran flavonoids may be important for the miller because bran is introduced into flour during the milling process. increasing amounts of bran will decrease the grade of the flour (feng et al., 1988). in flour milling fractions of cereal extracts content of flavonoid in the year 2009 decreased in the order: barley (33%) > oat (28%) > spelt (14%) > rye (14%) > triticale (5%) > wheat (5%), and in the year 2010 in the order: oat (31%) > barley (25%) > spelt (16%) > rye (15%) > triticale (6%) > wheat (6%). in bran milling fractions of cereal extracts content of flavonoid in the year 2009 decreased in the order: rye (22%) > wheat (20%) > oat (19%) > barley (16%) > spelt (12%) > triticale (11%), and in the year 2010 in the order: wheat (20%) > rye (20%) > oat (19%) > barley (17%) > spelt (12%) > triticale (12%). table 3. the flavonoid contents (mg qe/ g dm) of milling fractions from cereals grew in the year 2009. milling fraction sample i. ii. iii. iv. barley 0.75 ± 0.04 1.02 ± 0.03 1.08 ± 0.07 2.14 ± 0.07 wheat 0.12 ± 0.01 0.16 ± 0.01 0.93 ± 0.15 3.07 ± 0.06 oat 0.64 ± 0.04 0.84 ± 0.05 2.60 ± 0.02 1.23 ± 0.05 spelt 0.33 ± 0.03 0.42 ± 0.04 0.81 ± 0.02 1.55 ± 0.04 rye 0.24 ± 0.04 0.51 ± 0.02 2.39 ± 0.02 1.91 ± 0.03 triticale 0.11 ± 0.02 0.18 ± 0.02 1.01 ± 0.02 1.24 ± 0.04 i. – bread flour, ii. – reduction flour, iii. – fine bran, iv. – coarse bran from literature is known that flavonoids are concentrated mainly in pericarp. our results shown that flavonoids are presented also in flour fractions; this is very important information, because products from endosperm are basic in human nutrition. in flour milling fractions, high amount of total flavonoid showed extract of barley and oat. oat is a source of many compounds that exhibit antioxidant activity, but is consumed in considerably lower quantities worldwide than wheat (peterson, 2001). in bran milling fractions high amount of total flavonoid was determined in bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:06 utc 54 ivanišová, e. et al. extract of rye and wheat. these results indicate that the flavonoids of cereals were mostly concentrated in the outer layer of grains. adom and liu (2002) found similar results for cereal grains including wheat and oat. table 4. the flavonoid contents (mg qe/ g dm) of milling fractions from cereals grew in the year 2010. milling fraction sample i. ii. iii. iv. barley 0.62 ± 0.04 0.71 ± 0.02 1.24 ± 0.04 2.35 ± 0.05 wheat 0.12 ± 0.02 0.17 ± 0.02 1.11 ± 0.02 3.20 ± 0.02 oat 0.73 ± 0.04 0.89 ± 0.04 2.65 ± 0.05 1.30 ± 0.02 spelt 0.34 ± 0.04 0.49 ± 0.01 0.92 ± 0.05 1.68 ± 0.02 rye 0.29 ± 0.02 0.55 ± 0.04 2.32 ± 0.04 2.00 ± 0.01 triticale 0.16 ± 0.04 0.24 ± 0.04 1.02 ± 0.06 1.56 ± 0.05 i. – bread flour, ii. – reduction flour, iii. – fine bran, iv. – coarse bran 4. conclusion in this article, we prepared and evaluated milling fractions from selected cereals. antioxidant activities were determined by dpph and frap assay and total phenolic and flavonoid content was also determined. we found that flour fractions (break flour and reduction flour) showed the lower proportion of the total antioxidant potential, which was balanced in observed years. bran fractions (fine bran and coarse bran) showed higher antioxidant activity, but 30 – 80 % of these fractions are unused in food industry, they are used mainly as animal feed. extract from barley showed the highest values in all methods in observed years. it is evident, that bran fractions can be evaluated in the future and used for fortification of flours. references abdel-aal, e.s.m., hucl, p., sosulski, f. w., graf, r., gillott, r., pietrzak, l.: screening spring wheat for midge resistance in relation to ferulic acid content. j. agric. food chem., 49, 2001, 3559-3566. adom, k. k., liu, r. h.: antioxidant activity of grains. j. agric. food chem., 50, 2002, 6182-6187. adom, k.k., sorrells, m.e., rui, h.l.: phytochemicals and antioxidant activity of milled fractions different wheat varieties. j. agric. food chem., 53, 2005, 22972306. alvare-jubete, l., wijngaard, h., arendt, e.k., gallagher, e.: polyphenol composition and in vitro anioxidant activity of amaranth, quinoa, buckwheat and wheat as affected by sprouting and baking. food chem., 119, 2010, 770-778. campbell, g.m., fangi, c., muhamad, i.i.: on predicting roller milling performance vi: effect of kernel hardness and shape on the particle size bereitgestellt von slovenská poľnohospodárska knižnica | 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can be opened with acrobat and adobe reader 5.0 and later.) >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [2834.650 2834.650] >> setpagedevice instructions to authors submitting papers for the 32 nova biotechnologica et chimica 14-1 (2015) doi 10.1515/nbec-2015-0011  university of ss. cyril and methodius in trnava selective recovery of copper from solutions after bioleaching electronic waste joanna willner, agnieszka fornalczyk, mariola saternus silesian university of technology, faculty of materials engineering and metallurgy, institute of metals technology, krasińskiego 8, katowice 40-019, poland (joanna.willner@polsl.pl) abstract: research on selective extraction of copper from solution after bioleaching grounded printed circuit boards (pcbs) using lix 860n-ic were conducted. the effect of lix 860n-ic concentration, phase ratio and influence of initial ph value of aqueous phase on the extraction of copper and iron was examined. it was found that the extraction rate of copper increases with the lix 860n-ic concentration. best results of cu extraction (98 %) were achieved with extractant concentration of 5 % and ph 1.9. higher ph value of aqueous phase (ph=2.4) is conducive to the simultaneous effect of fe co-extraction. key words: solvent extraction, copper, solution after bioleaching, electronic waste. 1. introduction over the past decades, there has been an increasing interest in biological methods and possibilities of their applications in metals recovery. biological leaching has been conducted in the presence of variety of microorganisms and in a wide range of wastecarrying base, precious metals as well as hazardous substances, such as: fly ashes (zn, al, cd, cu, ni, cr, pb, mn, fe), tannery sludge (cr), jewellery waste and automobile catalytic converter (au, ag, pt) spent lithium-ion or ni-cd batteries (li, co, ni, cd). much attention is paid to the growing group of electronic waste, which are examples of complex material, containing a mixture of various metals, their alloys and also plastics and ceramics (cui and zhang, 2008; pant et al., 2012; willner and fornalczyk, 2012; velgosova et al., 2013; willner and fornalczyk, 2013). as a result of bioleaching electronic waste, a multi-component solution is obtained, containing various metal ions, of which copper is a predominant component. due to the presence of additional metal cations, especially iron cations, direct recovery of copper (i.e. by electrolysis) is much more hindered. in this case, an effective method that allows the separation of desired metal (cu) from their mixture may be solvent extraction (panda et al., 2012; gotfryd and pietek, 2013). over the past few years (bio)leaching–solvent extraction–electrowinning (bl–sx–ew) process has been conducted in hydrometallurgical production of copper from low grade ores. about 20–25 % of the world's total copper production is obtained through solvent extraction followed by electrowinning processes (munoz and dreisinger, 2007; panda et al., 2012). in practice during hydrometallurgical process of copper production, sulfuric acid solution for leaching raw material is used. post-extraction of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:52 utc nova biotechnologica et chimica 14-1 (2015) 33 the raffinate is subjected to solid/liquid separation (sedimentation and filtration) and final extractive treatment of the solution is conducted. next copper is recovered as a stripped liquor (moderately acidic copper sulfate) and is intended for electrolysis to obtain copper cathodes. simultaneously, acid regeneration is carried out and the received regenerated electrolyte is used again in cycles as a stripping factor (gotfryd and pietek, 2013). there are very few publications that refer to recovery of metals from solutions after bioleaching e-waste (chenglong et al., 2010). the purpose of the study was to identify the selectivity and efficiency of lix 860n-ic relatively to copper from polymetallic solution after bioleaching electronic waste. 2. materials and methods bioleaching was conducted on printed circuit boards (pcbs) derived from spent mobile phones. pcbs were shredded with the cutting mill to a particle size < 0.5 mm. the strain of acidithiobacillus ferrooxidans bacteria, isolated from ferruginous mineral waters in poland was used in the study (pacholewski and pacholewska, 2001). the culture was maintained in silverman/lundgren (9k) medium, containing (g/l): (nh4)2so4 3.0; kcl 0.1; k2hpo4 0.5; mgso4 7h2o 0.5; ca(no3)2 0.01; feso4 7h2o 44.2; fe(ii) – 9,0 g/l. bioleaching was carried out in erlenmeyer flasks (300 ml) by using a rotary shaker (130 rpm) at 20-22 ˚c. the samples of the waste were 1 g, the volume of solution was 100 ml, whereas the quantities of bacteria culture were 10 % (v/v) in experiments. solutions were filtered, combined and then subjected to solvent extraction. content of metals in sample was determined by atomic adsorption spectrometry (aas, solaar m6-unicam). composition of post-bioleaching solution is presented in table 1. table 1. concentration of metals in solution after bioleaching pcbs. metal cu fe sn pb ni concentration (g/l) 4.525 3.437 0.061 0.0005 0.0695 the reagents from the hydroxy oxime group have exceptional selectivity of copper extraction from acidic sulphate solutions in the presence of iron (iii) (gotfryd et al. 2002; szymanowski, 2003). therefore the organic solvent such as lix 860nic (5-nonilsalicylaldoxime) obtained from the supplier (basf, ireland), was used in studies. the extractant was diluted in the range 2.5-20 % with exxsol d80 (dissolvent). experiments were carried out in separatory funnel by contacting desired volume of the organic (o) and aqueous phase (a) by manual mixing for 5 and 10 minutes. different phase ratios o:a were used from 1:3 to 3:1, and initial ph of aqueous solutions was 2.4 and 1.9. concentration of cu and fe ions was determined by aas. stages of studies are illustrated in figure 1.the metal ion concentration in the organic phase was determined from the difference between concentration of metal before and after extraction. the efficiency of metal ions extraction process (e) was expressed in the following way: bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:52 utc 34 willner, j. et al. %100 0     ww oo vc vc e (1) where: co – concentration of metal ions after extraction in organic phase, g/l; cw0 – initial concentration of metal ions in the aqueous phase, g/l; vo – the volumes of the organic phase, ml; vw the volumes of the aqueous phase, ml. fig. 1. schematic diagram of experimental stages during solvent extraction of copper from solution after bioleaching pcbs. 3. results and discussion a preliminary study was carried out due to the possibility of extractant reaction with other cations present in the solution and the progress of solvent extraction in a non-specific manner. the aim was to initially determine used extractant selectivity towards copper extraction from multi-component solution after bioleaching electronic waste. preliminary tests of extraction copper from polymetallic solutions were conducted for 5 and 10 min at ph 2.4 and phase ratio o:a = 1. the results of these tests (data not illustrated) showed that the process of extraction of cu occurred very quickly. within 5 minutes, 99.6 % of cu was extracted to the organic phase. increasing the mixing time up to 10 minutes had not improved the efficiency of this process. due to the presence of iron ions in solution and possibility of it transition from the aqueous to the organic phase, phenomenon of co-extraction of iron was traced. within 5 minutes, 14 % extraction rate of fe from aqueous to the organic phase was recorded. the amount of iron in the organic phase was greater when the mixing time of phases lasted 10 minutes. together with copper, 24.9 % of fe was co-extracted to organic phase. this effect combined with the lack of efficiency of copper extraction, confirmed the need to shorten the time of mixing and reducing the ph of aqueous solution. further studies on identifying efficiency of selective extraction of copper were carried out in shorter time (5 minutes) and at lower ph. copper extraction was carried out at different concentrations of lix 860n-ic (2.520 %) and at ph 1.9, phase ratio o:a = 1 and contact time 5 min. as shown in fig. 2, copper rate extraction increased from 58.0 % to 98.3 % as the concentration of lix 860n-ic increased from 2.5 % to 5.0 %. beyond 5.0 % extraction of cu seems to be plateau. simultaneously co-extraction of fe was observed when concentration of extractive reagent raised. the optimal concentration of lix 860n-ic is suggested to be about 5.0 %. accompanying co-extraction of iron (14.6 % in preliminary tests) is a result of ph solution, which value was 2.4. effect of co-extraction of fe (in the range bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:52 utc nova biotechnologica et chimica 14-1 (2015) 35 1.9 % 5.2 %) was significantly reduced at ph=1.9. similar observations of elimination (or reduction) of the iron effect co-extraction at lower ph value was observed by panda et al. (2012) and chenglong et al. (2010). at this stage of the study the ph value of the aqueous phase (ph = 1.9) as a result of acidification of solution during bioleaching, was adopted as initial ph value and intended for further solvent extraction. however, further research on the influence of the ph on extraction efficiency and fe co-extraction should be carried out. fig. 2. effect of lix 860n-ic concentration on copper and iron extraction rate (ph = 1.9, o:a = 1, contact time 5 min). fig. 3. influence of phase ratio (o:a) on copper and iron extraction rate (ph = 1.9, lix 860n-ic 5 %, contact time 5 min). fig. 3 presents influence of phase ratio (o:a) on the cu and fe extraction. extraction rates of copper increased from 26.4 % to 98.3 % with phase ratio in the range of 1:3 to 1:1. above the ratio o:a = 1 extraction of cu maintained a slight increase. simultaneously with copper, a slight increase of iron was observed in the adopted phase ratios (a:o = 1:3 to 3:1). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:52 utc 36 willner, j. et al. 4. conclusions on the basis of conducted research involving the solvent extraction of copper from solutions after bioleaching electronic waste, it was found that: (i) the extraction rate of copper increases with the lix 860n-ic concentration; (ii) in the adopted experimental conditions best results were achieved when concentration of reagent was 5 % and proper phase ratio was o:a = 1:1; (iii) the increase of ph aqueous phase (ph = 2.4) was accompanied by the effect of fe co-extraction (24.9 %) and (iv) extraction of cu at the level of 98.3 % was obtained at ph = 1.9. at the same time, the lowest degree transition into the organic phase of fe amounting 1.9 % was simultaneously achieved. acknowledgements: the work was taken within the statutory research of silesian university of technology, poland. the authors are grateful to basf (ireland) for supplying organic solvent lix 860n-ic used in the research. the first author also wishes to thank mr leszek gotfryd, phd from institute of non-ferrous metals in gliwice poland, for scientific support. references chenglong, z.; jingwei, w.; jianfeng, b.; wenxiong, m.: production of high purity copper from bio-leaching solutions of waste printed circuit boards. proceedings of the 4th international conference on bioinformatics and biomedical engineering, china, chengdu, june 18-20, 2010, 1-4. gotfryd, l., steczkowski, j., anyszkiewicz, k., kwarciński, m., becker, k.: ekstrakcja jonowymienna w procesach metalurgicznych przemysłu metali nieżelaznych. rudy metale, 47, 2002, 607-610. gotfryd, l., pietek, g.: contaminants of post leaching copper solutions and their behavior during extraction with industrial extractants. physicochem. probl. miner. process, 49, 2013, 133-143. cui, j., zhang, l.: metallurgical recovery of metals from electronic waste: a review. j. hazard. mater., 158, 2008, 228-256. munoz, j.a., dreisinger, d.b., cooper, w.c., young, s.k.: silver catalyzed bioleaching of low grade ores. part i. shake flask tests. hydrometallurgy, 88, 2007, 3-18. pacholewski, a., pacholewska, m.: naturalne zdolności do utleniania związków żelaza (ii) przez bakterie żelazowe ze źródła wody mineralnej łomniczanka. współczesne problemy hydrogeologii, x, 2001, 389-396. panda s., parhi p.k., pradhan n., mohapatra u.b., sukla l.b., park k.h.: extraction of copper from bacterial leach liquor of a low grade chalcopyrite test heap using lix 984n-c. hydrometallurgy, 121-124, 2012, 116-119. szymanowski j.: wybrane fizykochemiczne aspekty wydzielania jonów metali. membrany teoria i praktyka zeszyt i. redaktor: romuald wódzki, fundacja rozwoju wydziału chemii, uniwersytet mikołaja kopernika, toruń 2003. velgosova, o., kadukova, j., marcincakova, r., palfy, p., trpcevska, j.: influence of h2so4 and ferric iron on cd bioleaching from spent ni-cd batteries. waste manage., 33, 2013, 456-461. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:52 utc nova biotechnologica et chimica 14-1 (2015) 37 willner, j., fornalczyk, a.: electronic scraps as a source of precious metals, chem. rev., 91, 2012, 517-523. willner, j., fornalczyk, a.: extraction of metals from electronic waste by bacterial leaching. environ. protect. eng., 39, 2013, 198-208. presented at the 3rd international conference “biotechnology and metals 2014”, september 17-19, 2014, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:52 utc microsoft word sák et al. final.doc 162 nova biotechnologica et chimica 13-2 (2014) doi 10.1515/nbec-2015-0006  university of ss. cyril and methodius in trnava elicitation of phenolic compounds in cell culture of vitis vinifera l. by phaeomoniella chlamydospora martin sák1,2, ivana dokupilová1,2, daniel mihálik3,4, jana lakatošová1,2, marcela gubišová3,5, ján kraic3,6* 1research institute of viticulture and enology, hlavná ulica 326, sk-90041, rovinka, slovak republic 2faculty of chemical and food technology, slovak university of technology, radlinského 9, sk-81237 bratislava, slovak republic 3research institute of plant production, bratislavská cesta 122, 921 68, piešťany, slovak republic 4institute of high mountain biology, university of žilina, sk-05956 tatranská javorina 7, slovak republic 5department of botany and genetics, faculty of natural sciences, constantine the philosopher university, tr. a. hlinku 1, sk-94974 nitra, slovak republic 6department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius, j. herdu 2, sk-91701 trnava, slovak republic (kraic@vurv.sk) abstract: the in vitro cell cultures of vitis vinifera l. cv. st. laurent were treated with two elicitors synthetic methyl jasmonate and natural, prepared from grapevine plant infected with the phaeomoniella chlamydospora, the agent causing the esca disease of grapevine. efficiency of phenolic compounds production after elicitation of cell culture was analysed immediately after treatment (15 min, 30 min, 60 min) and later (after 24, 48, and 72 hours). the cell growth and content of phenolic compounds (+)-catechin, (-)-epicatechin, p-coumaric acid, syringaldehyde, rutin, vanillic acid, and trans-resveratrol were analysed in cultivated cells as well as in cultivation medium. pch-treatment increased production of total polyphenols the most significantly 15 min after the elicitation and in optimal time was 2.86 times higher than in nonelicited culture and 1.44 times higher than in meja induced cell culture. abstract: vitis vinifera l., phaeomoniella chlamydospora, phenolics, cell culture, elicitation 1. introduction plant cells cultivated in vitro could potentially be competitive systems for effective production of marketable secondary metabolites possessing biological activities which can not be produced by microorganisms or by chemical synthesis. production of such molecules and compounds has been demonstrated in different plant species (mulabagal and tsay, 2004) as well as by in vitro cultivation of organs, tissues, or cells (reviewed by rao and ravishankar, 2002). an overproduction of some secondary metabolites, in the comparison with level in intact plants, was achieved in in vitro systems, e.g. production of rosmarinic acid in cell cultures of salvia officinalis l. and coleus blumei (ulbrich et al., 1985; hippolyte et al., 1992), antraquinones in cell cultures of morinda citrifolia l. (zenk et al., 1975). the cell cultures in vitro have been used advantageously in the production of substances such as vincristine, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:35 utc nova biotechnologica et chimica 13-2 (2014) 163 vinblastin, taxol, camptothecin, ellipticine, and others (rao and ravishankar, 2002). the most attractive compounds among them, from the commercial point of view, are plant-derived pharmaceuticals (xu et al., 2011; abdullahil baque et al., 2012) and food additives (jiménez-aparicio et al., 1999; matkowski, 2008). the plant biotechnology tools are instruments also for industrial exploitation of plant cell cultures, but the economical point of view is very important and always predominant. moreover, efficient in vitro production of secondary metabolites needs high effective systems with optimized cultivation conditions, methods of extraction of secondary metabolism products as well as specific factors improving production such as biotic and abiotic elicitors (dörnenburg and knorr, 1995; zhao et al., 2005), or simulation of nutritional deficiency of cells (tavares et al., 2013). the grapevine (vitis vinifera l.), especially ripe berries, contain many phenolic compounds. liang et al. (2011) identified 36 polyphenols including anthocyanins, flavanols, flavonols, hydroxycinnamic derivatives, and hydroxybenzoic acid, and 48 different polyphenols in berries of wild vitis species (liang et al., 2012). grapevine phytochemicals consumed in berries and wine are associated with the positive health benefits and relevant dietary style (iriti and faoro, 2006). they contain compounds with antioxidant and antibacterial activities, compounds decreasing cholesterol level, protecting against reactive oxygen species inducing dna damages, modulating glucose uptake, and anticancer activities (williamson and carughi, 2010; delgado adámez et al., 2012; apostolou et al., 2013). aqueous extracts from grapevine leaves also contain phenolic compounds with antioxidant activity (fernandes et al., 2013). production of secondary metabolites in the grapevine cell cultures in vitro has been presented in many studies and most of these experiments have been focused to improvement of in vitro production by adding of precursors, by genetic transformation of plants, or by elicitation. application of specific precursors and genetic transformations are expensive and laborious, therefore the most common approach is the induction of defence mechanisms by elicitors which are signalling triggers of secondary metabolite formation (mulabagal and tsay, 2004). zhang et al. (2002) induced biosynthesis of anthocyanins by irradiation and by jasmonic acid, cai et al. (2012) enhanced production of anthocyanins and resveratrol by indanoyl isoleucine, n-linolenoyl-l-glutamine, and insect saliva. saw et al. (2012) stimulated synthesis of anthocyanins by ethephon and pulsed electric field. also other elicitors were tested, well-known and frequently used are synthetic methyl jasmonate and salicylic acid. infections of plants by pathogens induce upregulation of plant defence mechanisms and production of anti-pathogen phenolic compounds. the fungal pathogen phaeomoniella chlamydospora (pch), associated with the esca-disease, also increased accumulation of phenolic components in infected young grapevine plants (martin et al., 2009). bruno and sparapano (2006) applied three esca-disease associated fungi phaeomoniella chlamydospora, togninia minima, and fomitiporia mediterranea as elicitors in dual in vitro cultivation with grapevine calluses. each fungus reduced growth of calluses but only t. minima induced higher production of total phenols in callus cultures. new, effective, and especially natural elicitors could improve production of secondary metabolites in vitro. therefore escoriaza et al. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:35 utc 164 sák, m. et al. (2013) treated grapevine (cv. chardonnay) callus and cell cultures by the fungus phaeoacremonium parasiticum associated with disease known as "hoja de malvón". the main response of plant tissue and cell defensive mechanism to the pathogen attack was increased terpene synthase activity and the production of nerolidon through de novo synthesis. nerolidon is able to retard the fungal growth. the broader spectrum of elicitors, the more their combinations could be used. the species-specific elicitors are very perspective and also desired for the production of secondary metabolites used for plant protection. natural elicitors should be also easily available for industrial (pharmaceutical, nutritional) applications. therefore the aims of this study were to: i) study response of grapevine cell culture in vitro to the speciesspecific natural elicitor prepared from phaeomoniella chlamydospora, ii) analyse time course of polyphenols production in elicited cell cultures, and iii) compare production of secondary metabolites in cell cultures treated with this elicitor and with widely used methyl jasmonate. 2. material and methods the callus cultures were initiated from sterile leaf segments of grapevine (vitis vinifera l.) cultivar st. laurent. the leaf segments of size 1 cm2 were cultivated on the murashige and skoog medium (1962) with salt concentration reduced to one half (½ ms), containing 3 % sucrose, 0.7 % (w/v) agar, 0.1 mg/l naa, and 0.2 mg/l bap, ph 5.8, at 25±1°c under a photoperiod 16 h light/8 h dark and were transferred to fresh cultivation medium every 30 days. the cell cultures have been initiated from calluses in 100 ml erlenmeyer flasks containing 40 ml of ½ ms liquid medium and cultivated with shaking (120 rpm) under the same conditions as callus cultures. elicitor from the p. chlamydospora (pch) was prepared from the cortex of grapevine plant infected by this fungus according to the procedure described by yu et al. (2001). carbohydrate concentration was determined by the orcinol-sulfuric acid method (francois et al., 1962). one-month old cell cultures were elicited by adding either of pch (0.2 g/l) or meja (0.18 g/l) and cultivated for 1 hour at 50°c. three parallel experiments for each cultivation were carried. reagents of analytical grade were used for hplc analysis including standards (+)catechin, vanillic acid, (-)-epicatechin, p-coumaric acid, syringaldehyd, rutin, and trans-resveratrol (sigma-aldrich, st. louis, usa). reference standard solutions of polyphenols were prepared in 50 % aqueous methanol and stored at 4°c in the dark. other used reagents were evans blue, fluorescein diacetate (fda), methyl jasmonate (meja), (sigma-aldrich, st. louis, usa), 1-naphthaleneacetic acid (naa), 6benzylaminopurine (bap), sucrose, agar, yeast extract, bacto-peptone, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, sodium dodecylsulphate (sds) (sigma-aldrich, st. louis, usa). water was prepared by the direct-q® 3 uv water purification system (merck millipore, darmstadt, germany). polyphenols were extracted from the cultivation medium as well as from cells according to cai et al. (2011) and liu et al. (2010) and analysed by hplc (agilent 1200 series system with photo-diode-array detector, operated under the agilent chemstation software). the plant cell cultures were separated from cultivation bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:35 utc nova biotechnologica et chimica 13-2 (2014) 165 medium by vacuum filtration and fresh weight was determined. dry cell weight was determined after freezing and lyophilisation. homogenised dry cells were extracted with ethyl acetate and methanol (1:1, v/v) in the ratio 1:10 (w/v) overnight at the room temperature. supernatant obtained after centrifugation (10 min at 10°c and 10 000 rpm) was collected and sediment was extracted for the second time. pooled supernatants were evaporated by vacuum and diluted in 1.5 ml of methanol. ten microliters of internal standard was added to samples for better identification. samples were filtered with syringe membrane filters (0.45 µm) before hplc analysis. twenty microliters of sample were injected into the supelcosiltm lc-18 column (250 mm × 4,6 mm, 5 μm particle size) warmed up to 30°c. program of hplc measurements was carried out according to rodriguez-bernaldo de quirós et al. (2009). total content of selected phenolic compounds was established as a sum of all quantified phenolic compounds. 3. results and discussion the cell cultures of vitis vinifera (cv. st. laurent) were elicited with the commercially produced and commonly available meja or by elicitor prepared from grapevine plant infected by the fungus phaeomoniella chlamydospora. this pathogen acts as an inducer of oxidative burst also in grapevine in vitro cell culture (lima and dias, 2012). both used elicitors induced change in the colour of cell cultures from green to brown and inhibited cell growth as reported by tassoni et al. (2005). the non-elicited (control) cell cultures had a higher mass of growing cells measured as fresh weight of cells. growth of cells decreased during the first 24 hours in mejaand pch-treated cell cultures and during the first 4 hours in non-elicited culture. after maximal decreasing of growth, i.e. 24 hours after elicitation, cell growth increased in both elicited cultures, nevertheless did not reach levels of non-elicited control culture (fig. 1). fresh weight of cells in culture elicited by meja after 72 hours of cultivation was similar to non-elicited culture. the lower fresh weight of cells elicited by pch was caused probably by polypeptides secreted by p. chlamydospora inhibiting plant cell activities and growth (luini et al., 2010). p. chlamydospora cultivated in vitro produced large amounts of extracellular polysaccharides containing pullulans and addition of crude extract or filtrate from culture to grapevine calluses reduced their growth by 85 % (sparapano et al., 2000). another group of chemical compounds produced by p. chlamydospora are extracellular enzymes also inhibiting growth of calluses (santos et al., 2006). the ph of the cultivation medium measured in samples for polyphenol analysis was almost constant in non-elicited sample (difference was ± 0.01) but decreased in average by 0.15 after each day of cultivation in elicited cultures. it can also reduce fresh cell weight as was described by tassoni et al. (2005) after using of meja. the total contents of polyphenols produced in cell cultures in vitro were determined immediately after treatment by elicitors (15 min, 1 hour, and 4 hours after) and later (1, 2, and 3 days after), respectively (fig. 1). the total content of polyphenols in non-elicited cultures increased continuously during 3 days but only moderately. the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:35 utc 166 sák, m. et al. impact of both elicitors on polyphenols production was different. their accumulation culminated 48 hours after meja addition then declined. the addition of the pch elicited the highest polyphenolic production immediately (15 60 min) after treatment, after 4 hours were minimal, then increased again and 72 hours after treatment polyphenols production was higher than producing meja-elicited cells. elicitation with the pch had a biphasic mode of production with two maxima immediately after elicitation and 3 days after. this could relate to a biphasic model of oxidative burst induced by extract of p. chlamydospora in grapevine cell suspension (lima et al., 2012), although the cultivar vinhão used in their study responded slightly differently and they analysed other phenolic compounds – viniferin-type and piceid-type stilbenes. time of accumulation and intensity of oxidative burst induced by pathogen can be very diverse, depending on plant species, used plant system, and resistance or tolerance level of specific cultivar. fig. 1. the effect of pch and meja elicitors on cell growth (curves with symbols ▲, □, ○) and the sum of detected phenolic compounds (columns). individual polyphenols determined in cultivation medium were: (+)-catechin, (-)epicatechin, vanillic acid, p-coumaric acid, syringaldehyde, trans-resveratrol, and rutin (table 1). significant differences in content of generated polyphenols were among the non-elicited and meja-elicited culture, between non-elicited and pch-elicited cultures, as well as between mejaand pch-elicited cultures. polyphenols in the cultivation medium in non-elicited cultures were not detected. very low quantity of polyphenols was released by meja-treated (vanillic acid, p-coumaric acid, and trans-resveratrol, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:35 utc nova biotechnologica et chimica 13-2 (2014) 167 totally 1.27 mg/l) and pch-treated cells (p-coumaric acid and trans-resveratrol, totally 0.53 mg/l) into cultivation medium 72 hours after elicitation. the vanillic acid and trans-resveratrol were present only in the cultivation medium. table 1. concentration of phenolics (±sd = standard deviation) in cultivation medium (a) (mg/l) and cultivated cells (b) (mg/g fw) (n.d. = not detected). time [hours] vanillic acida p-coumaric acida meja pch meja pch 0.25 n.d. n.d. 0.07±0.01 0.07±0.02 1 n.d. n.d. 0.05±0.004 0.05±0.02 4 n.d. n.d. n.d. 0.05±0.04 24 0.33±0.02 n.d. n.d. 0.14±0.08 48 0.49±0.03 0.32±0.19 0.06±0.005 0.15±0.05 72 0.73±0.05 0.34±0.20 0.09±0.01 0.19±0.06 control after 72 hours n.d. n.d. time [hours] trans-resveratrola (+)-catechinb meja pch meja pch 0.25 n.d. n.d. 0.58±0.03 2.74±0.18 1 0.36±0.004 n.d. 0.58±0.03 2.40±0.16 4 0.41±0.01 n.d. 0.66±0.04 0.46±0.02 24 0.43±0.01 n.d. 0.83±0.05 0.79±0.05 48 0.45±0.01 n.d. 1.48±0.05 0.92±0.05 72 0.45±0.01 n.d. 0.99±0.06 1.34±0.07 control after 72 hours n.d. 0.82±0.05 time [hours] (-)-epicatechinb p-coumaric acidb meja pch meja pch 0.25 0.43±0.01 0.73±0.03 n.d. 0.08±0.01 1 0.38±0.01 0.70±0.03 n.d. 0.07±0.01 4 0.43±0.01 0.26±0.01 n.d. 0.05±0.004 24 0.48±0.01 0.44±0.01 n.d. n.d. 48 2.05±0.02 0.69±0.02 n.d. 0.04±0.004 72 0.73±0.02 1.16±0.03 n.d. 0.08±0.01 control after 72 hours 0.37±0.012 0.06±0.004 time [hours] rutinb syringaldehydeb meja pch meja pch 0.25 0.59±0.03 3.10±0.20 0.39±0.03 1.53±0.01 1 0.69±0.04 1.99±0.13 0.30±0.02 1.76±0.12 4 0.66±0.04 0.37±0.02 0.44±0.03 0.27±0.02 24 1.11±0.07 0.54±0.03 0.73±0.05 0.72±0.05 48 1.32±0.05 0.64±0.03 0.84±0.06 1.62±0.11 72 0.72±0.04 1.01±0.05 0.96±0.07 2.77±0.19 control after 72 hours 0.69±0.04 0.92±0.06 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:35 utc 168 sák, m. et al. the non-elicited cells contained (+)-catechin, (-)-epicatechin, p-coumaric acid, rutin, and syringaldehyde, totally 2.86 mg of phenolic compounds per gram of fresh weight of cell biomass (mg/g fwb). addition of meja increased polyphenolic production, except the p-coumaric acid. the total amount of other four polyphenols at time of maximal production (48 hours after elicitation) was 5.69 mg/g fwb. the addition of pch increased level of monitored polyphenols after 48 hours to 3.91 mg/g fwb, after 72 hours to 6.36 mg/g fwb, but the most significantly immediately, i.e. 15 min after elicitation, to 8.18 mg/g fwb (especially content of rutin to 3.1 mg/g fwb). production of total polyphenols elicited by pch at time of maximal production was 2.9 times higher than in non-elicited culture and 1.4 times higher than in meja-induced culture. water content in cultivated cells in our cell cultures was 89.7 %. lima et al. (2012) increased production of other types of phenolic compounds – piceid-type and viniferin-type stilbenes by meja and pch elicitors, in comparison to non-treated control, 9-fold and 20-fold, respectively, but grapevine genotypes and evaluated phenolic compounds were different in their experiments. polyphenols are synthesised during the normal development of grapevine plant. the basic effect of genotype on polyphenols production documented liang et al. (2011) in european vitis vinifera l. germplasm as well as in accessions of wild vitis species (liang et al., 2011). the plant genotype determines this production essentially nevertheless their synthesis correlates with response to different environmental biotic and abiotic factors. plant tissues and cells cultivated in vitro, moreover affected by elicitors, can respond more sensitively. extract from the p. chlamydospora used in our study represented effective elicitor for polyphenols production in grapevine cell cultivation system in vitro and the vitis cell cultures might be a practicable system for biotechnological production of valuable compounds. the advantage of the pch-originated elicitor is also lower price and less health hazard during operation. 4. conclusions the present paper demonstrates enhancement of polyphenols production by application of synthetic and native elicitors – methyl jasmonate and extract from the grapevine pathogen phaeomoniella chlamydospora, respectively. different progress in elicitation of polyphenols production in cell culture was observed. elicitation with meja culminated 48 hours after addition pch elicited the highest polyphenols production immediately after treatment and again 72 hours later. finally, it could be concluded that elicitation by pch was more effective than by meja. acknowledgments: this study was supported by the project no. apvv 0550-11 funded by the slovak research and development agency. references abdullahil baque, m., moh, s.-h., lee, e.-j., zhong, j.-j., paek, k.-y.: production of biomass and useful compounds from adventitious roots of high-value added medicinal plants using bioreactor. biotechnol. adv., 30, 2012, 1255-1267. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:35 utc nova biotechnologica et chimica 13-2 (2014) 169 apostolou, a., stagos, d., galitsiou, e., spyrou, a., haroutounian, s., portesis, n., trizoglou, i., wallace hayes, a., tsatsakis, a.m., kouretas, d.: assessment of polyphenolic content, antioxidant activity, protection against ros-induced dna damage and anticancer activity of vitis vinifera stem extracts. food chem. toxicol., 61, 2013, 60-68. bruno, g., sparapano, g.: effects of three esca-associated fungi on vitis vinifera l.: i. characterization of secondary metabolites in culture media and host responses to the pathogens in calli. physiol. mol. plant pathol., 69, 2006, 209-223. cai, z., knorr, d., smetanska, i.: enhanced anthocyanins and resveratrol production in vitis vinifera cell suspension culture by indanoyl-isoleucine, nlinolenoyl-l glutamine and insect saliva. enzyme microb. technol., 50, 2012, 2934. cai, z., riedel, h., saw, n.m.m.t, mewis, i., reineke, k., knorr, d., smetanska, i.: effects of elicitors and high hydrostatic pressure on secondary metabolism of vitis vinifera suspension culture. process biochem., 46, 2011, 14111416. delgado adámez, j., gamero samino, e., valdés sánchez, e., gonzález-gómez, d.: in vitro estimation of the antibacterial activity and antioxidant capacity of aqueous extracts from grape-seeds (vitis vinifera l.). food control, 24, 2012, 136-141. dörnenburg, h., knorr, d.: strategies for the improvement of secondary metabolite production in plant cell cultures. enzyme microb. technol., 17, 1995, 674-684. escoriaza, g., sansberro, p., garcía-lampasona, s., gatica, m., bottini, r., piccoli, p.: in vitro cultures of vitis vinifera l. cv. chardonnay synthesize the phytoalexin nerolidol upon infection by phaeoacremonium parasiticum. phytopath. mediterranea, 52, 2013, 289-297. fernandes, f., ramalhosa, e., pires, p., verdial, j., valentão, p., andrade, p., bento, a., pereira, j.a.: vitis vinifera leaves towards bioactivity. ind. crops prod., 43, 2013, 434-440. francois, c., marshall, r.d., neuberger, a., carbohydrates in proteins. 4. the determination of mannose in hen’s-egg albumin by radioisotope dilution. j. biochem., 83, 1962, 335-341. hippolyte, i., marin, b., baccou, j.c., jonard, r.: growth and rosmarinic acid production in cell suspension cultures of salvia officinalis l. plant cell rep., 11, 1992, 109-112. iriti, m., faoro, f.: grape phytochemicals: a bouquet of old and new nutraceuticals for human health. med. hypotheses, 67, 2006, 833-838. jiménez-aparicio, a., gutiérrez-lópez, g.: production of food related colorants by culture of plant cells. in: shahidi, f., kolodziejczyk, p., whitaker, j.r., lopez munguia, a., fuller, g. 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(eds.) secondary metabolism of plant cell culture. springer-verlag, berlin, 1985, 293-303. williamson, g., carughi, a.: polyphenol content and health benefits of raisins. nutr. res., 30, 2010, 511-519. xu, j., ge, x., dolana, m.c.: towards high-yield production of pharmaceutical proteins with plant cell suspension cultures. biotechnol. adv., 29, 2011, 278-299. yu, l.-j., lan,w.-z., qin, w.-m., xu, h.-b., effects of salicylic acid on fungal elicitor-induced membrane-lipid peroxidation and taxol production in cell suspension cultures of taxus chinensis. process biochem., 37, 2001, 477-482. zenk, m.h., ei-shagi, h., schulte, u., anthraquinone production by cell suspension cultures of morinda citrifolia. planta medica suppl., 75, 1975, 79-101. zhang, w., curtin, c., kikuchi, m., franco, c.: integration of jasmonic acid and light irradiation for enhancement of anthocyanin biosynthesis in vitis vinifera suspension cultures. plant sci., 162, 2002, 459-468. zhao, j., lawrence, t., davis, c., verpoorte, r.: elicitor signal transduction leading to production of plant secondary metabolites. biotechnol. adv., 23, 2005, 283-333. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:35 utc microsoft word sadowski nbc 2015 corected.doc 52 nova biotechnologica et chimica 14-1 (2015) doi 10.1515/nbec-2015-0014 © university of ss. cyril and methodius in trnava hydrodynamic study of column bioleaching processes zygmunt sadowski, joanna a. baranska department of chemical engineering, wroclaw university of technology, faculty of chemistry, wybrzeze wyspianskiego 27,wroclaw 50-370,poland (zygmunt.sadowski@pwr.edu.pl) abstract: the modelling of flow leaching solution through the porous media has been considered. the heap bioleaching process can be tested using the column experimental equipment. this equipment was employed to the hydrodynamic studies of copper ore bioleaching. the copper ore (black shale ore) with the support, inertial materials (glass small balls and polyethylene beads) was used to the bioleaching tests. the packed beds were various composition, the ore/support ratio was changed. the correlation between the bed porosity and bioleaching kinetics, and copper recovery was investigated. key words: heap bioleaching, packed bed porous media, bioleaching, acidithiobacillus ferrooxidans, ergun equation, support material 1. introduction the hydraulic properties of porous media are important in heap or dump bioleaching processes. there is necessary to improve understanding of heap bioleaching process to achieve more effective metal recovery. the leaching solution flow through a porous medium (heap) can be described using soil liquid hydrodynamic (clement et al., 1998) or chemical engineering theory (li et al., 2001; henderson et al., 2010; nemec and lever, 2005). the two-dimensional model for a heap bioleaching of copper ore has been developed (casas et al., 1998). the main attention was focused on the gas transport through the heap. when the bed permeability was higher, air convection was the predominant mechanism. the darcy’s law has been used to a fluid transport description in a porous medium. the leaching solution flow through a porous medium is the essence of leaching process. the leaching solution in the porous bed can move to the button of heap and stagnant attached to the pore walls. the bioleaching ore dissolution is occurred inside the stagnant part of solution (sheikhzadeh et al., 2005). understanding of hydrodynamic and transport phenomena in a porous packed bed is essential for the heap bioleaching. the liquid flow is considered to be governed by both the gravitation and capillary forces. a part of liquid is holdup between the adjacent void cells. the transport of microbial cell is mainly connected with the leaching solution migration through the porous media. mathematical model of microbial cells transport in unsaturated porous media is described by a simplified form of advection-dispersion equation (tufenkji, 2007). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:03 utc nova biotechnologica et chimica 14-1 (2015) 53 r x c x c d t c − ∂ ∂ − ∂ ∂ = ∂ ∂ ν 2 2 (1) where c is the microbial cells concentration in the leaching solution at a distance x and time t. d is the hydrodynamic dispersion coefficient, and ν is the interstitial microbe velocity. r is the retardation factor. the microbial cells retardation factor is controlled by the cell attach to the surface of particles and the release from this surface. this equation can be derived from basic mass balance principles. in eq. 1, only hydrodynamic dispersion of microbial cells is considered. however, the overall transport process should take into consideration attachment and detachment of cells to the solid surface, and death of microorganisms. the attachment and detachment processes can be described by using the classical colloid filtration theory (tufenkji and elimelech, 2004). the bacteria attachment to the mineral surface depends on the potential energies of london-van der waals attraction and electrical double layer repulsion. the extended drejaguinlandau-verwey-overbeek (dlvo) colloid stability theory can be used to quantitative predicting the condition to detach bacteria cell to the pore surface. the bacteria cells adhering to the pore surface of porous bed can be detached by the hydrodynamic forces. the immobilization of microbial cells is correlated with the biofilm creation on the pore surface. the adsorption of biopolymer on the surface gives the foundations for the biofilm creation. the data from column bioleaching experiments are presented that the effect of biofilm growth inside the porous media is important for leaching process (bergendahl et al., 2000). a number of experimental studies have been conducted to prove the biofilm and microcolony model and they influence on bioleaching processes (rockhold et al., 2002; hernandez-lopez et al., 2011). darcy’s law is commonly used to describe one-dimensional liquid flow through uniform incompressible porous bed. generally, darcy’s law is presented as follows: l p dt d a μ ε ϕ ν δ = 1 (2) where: ν is the liquid velocity, a is the cross sectional area, φ is the permeability of porous bed, δp is the pressure drop, μ is the liquid viscosity, l is the thickness of porous bed. for non-overlapped spherical particles, the permeability (ε) is related to the drag coefficient (cd). drcπ ν ε 6 = (3) where: r is the sphere radius. the permeability of the porous media (φ) is estimated using carman-kozeny’s equation (henderson et al., 2010). the carman-kozeny’s equation can be written as: bm c 3 0 ε ϕ = (4) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:03 utc 54 sadowski, z. and baranska, j.a. where: c0 is a kozeny’s coefficient and mb is the specific surface of the porous bed. leaching liquid flowing through porous abundance is wasting the energy. the viscous energy loss is connected with the liquid velocity and the inertial energy loss is proportional to the velocity squared. 2νν ba l p += δ (5) where: δp is the pressure loss, l is the bed height, ν is the liquid velocity (averaged over the flow cross-section), and a and b are tow empirical parameters (a = 150; b = 1.75). for the laminar flow eq. 5 has a form: ( ) 2 322 175.1150 ν εϕ ερ ν ϕ μ pp ddl p − += δ (6) where φdp gives the diameter of the equivalent-volume sphere. the equation (6) is called “ergun equation”. the objective of this study was to investigate the effect of hydrodynamic parameters of porous bed in a packed column bioreactor on the bioleaching process of black shale copper ore. 2. materials and methods 2.1 bacteria strain and growth condition column bioleaching experiments were conducted with acidithiobacillus ferrooxidans strain. bacteria strain was isolated from the acid mine drainage located at the old pyrite mine (lower silesia, poland). strain was grown in 250 ml erlenmeyer flasks containing 150 ml of silvermann-lundgren medium (9k). 2.2 ore and support materials the black shale ore sample used throughout this study was provided by lubin concentrator (kghm polish copper) as middling. black shale is composed of claydolomite matter saturated with organic matter and with finely dispersed sulphide mineral particles. this material was a multi-mineral resource in which the copper sulphides were mainly chalcocite, covellite, bornite and chalcopyrite. in the flotation circuit of lubin concentrator, the black shale is partially concentrated at middlings (tailing from 1st cleaning flotation step). the average size of middling was 42.15 μm. according to xrd analysis (table 1), the bioleaching material contains 2.5 % of copper in the form of sulphide. two different types of support particles have been employed. the support materials used in this study were glass small bolls and polyethylene beads. these materials have different surface properties. the surface of glass bolls is hydrophilic but the surface of polyethylene beads is hydrophobic. the pictures of support materials are presented at fig. 1a and 1b. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:03 utc nova biotechnologica et chimica 14-1 (2015) 55 table 1. chemical composition of shale sample elements unites concentrations cu % 2.5 fe % 1.75 ni [g/t] 374 as % 0.08 ag [g/t] 175 stotal % 2.93 ctotal % 14.46 fig. 1. glass small bolls (a) and polyethylene beads (b). glass bolls have equal diameter 1.5 mm. the size of polyethylene beads was the range of 2 to 5 mm. the calculated surface area of this beads was 0, 08 m2/g. 2.3 column bioleaching experiments column leaching experiments were conducted in small column (37 cm/5.0 cm). the leaching solution was passed through the column by gravity. it was collected in the thermostatic bath and recycled to the top the column by peristaltic pump. the liquid layer was kept on the top of column. the bypass construction provides both a constant liquid layer and constant hydrostatic pressure. this liquid layer was aerated. the column was charged with 80 g of black shale material. at the next column experiments, the ratio black shale ore to support material was changed. the leaching solution (200 ml) was pumped from the feed container to the top of the column. the gas (air) was also introduced to the liquid layer at the top of column, where a layer of leaching solution was created during the leaching tests. the particle size distribution has been obtained by powder analysis using a laser dyfractiometer mastersizer 2000 (malvern instruments g.b). during the experiments fe2+, fe3+ ions and protein concentrations, as well as ph and eh were measured. ferrous iron concentration in solution was determined by spectrophometric methods. the concentration of copper was determined using atomic spectroscopy method. the bacterial population was controlled by protein analysis. 3. results and discussion the considerations regarding the optimum construction of heap and column packed bed are complex; therefore, an intensive study is needed. it is known, that the bed a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:03 utc 56 sadowski, z. and baranska, j.a. porosity increase the more if the shape of particles deviates from the spherical shape. in addition the particle size and packing procedure have a strong effect on the hydroprocessing conditions (nemec and levec, 2005). when the ore particles were used, the porosity of packed bed was relatively narrow. in such case, the flow through packed bed can be regulated by the support addition. bioleaching in column with recirculation of the leaching liquid is a lab-scale simulation of heap bioleaching processes (long et al., 2004; lizama et al., 2005). the bed permeability is an essential process parameter, in the case of column bioleaching. the particle size distribution of shale sample (fig. 2) shows that this material contains a lot of fine particles. the amount of fine particles is a key parameter for the modelling of flow through packed bed. it was recommended that the fine fraction should be less than 10 % (lin et al., 2004; 2005). fig. 2. particle size distribution of black shale sample. the present work shows the influence of two supports (glass small bolls and polyethylene beads) on the hydrodynamic parameter. to obtain a wide variety of bed porosity different quantity of support materials were employed. the leaching solution flow inside the heap or column can be divided into saturated and unsaturated. as mentioned before, understanding of the liquid flow phenomena inside the packed bed is important to enhance the bioleaching performance. the milli-ct scanner has been used to determine the pore structure of the packed column before and after bioleaching. the application of this sophisticated technique gives an opportunity to establish a fundamental relationship between pore microstructure and effective transport coefficient (lin et al., 2004; 2005). the velocity of liquid flowing through the porous bed (v) can be described by the following equation: ( )21 hhg −= ϕεν (7) where: φ is a permeability of the porous bed, εg is the gas (air) holdup in the pores, h1 is the upper level of liquid, and h2 is the bottom of bed. the porosity is an important parameter of porous bed at the macroscopic scale. it is defined as the pore volume of a representative sample divided by its bulk volume. the specific surface of a porous bed, here denoted as the bed surface, is defined as the interstitial surface area of the porous bed. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:03 utc nova biotechnologica et chimica 14-1 (2015) 57 the porous bed can be treated as a bundle of capillary tubes of equal length. hydraulic radius is defined for straight pipes as: i i h p a r = (8) where: ai is the cross-sectional flow area and pi the wetted parameter (niven, 2002). a local reynolds number is defined using as a scale length the square root of across-sectional area of the pore as follows: μ νϕ 2 1 a re = (9) at lower local reynolds number the flow of leaching solution is laminar. the local reynolds number is usually less than one (sederman et al., 1998). the experimental tests were conducted using a column reactor with a different composition of bed. the liquid flows were measured by such porous beds. the flow measurements were monitored at the time when the liquid flow did not change. this means that the deposit was porous in a natural way, unified, and sections of transport channels are not changed during the measurements. on the basis of data on the composition of the deposits, and the flow of liquid parameters calculated deposits such as porous: the degree of dispersibility deposits, deposits of hydraulic diameter and the number of reynolds with a kind of liquid flow by deposit. the calculated data are shown in tables 2 and 3. table 2. the flow of liquids through porous shale ore deposit (fixed) with a different amount of polyethylene beads fittings. no. polyethylene beads weight [g] black shale weight [g] liquid flow [ml/s] porous bed height [cm] liquid layer height [cm] 1 0 80 0.050 5.6 15.0 2 15 80 0.034 6.4 13.2 3 30 80 0.036 8.0 13.0 4 50 80 0.020 9.0 10.0 table 3. porous bed characteristics. no. porous bed porosity calculated flow 10-3 surface area [m2/g] particles medium size [µm] bed dispersion degree hydraulic diameter 10-3 reynolds number 1 0.5542 3.98 4.128 34.143 3.414 64.9 0.3048 2 0.4298 2.71 3.4888 387.18 26.328 4.40 1.8401 3 0.4195 2.86 3.0240 643.86 46.358 2.606 3.1721 4 0.3387 1.59 2.5711 873.04 34.921 1.552 2.0991 at the next series of experience, the weight of porous deposits was chanded using a different quantity of shale ore and polyethylene beads. the data collected by such flows of deposits are shown in table 4 and 5. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:03 utc 58 sadowski, z. and baranska, j.a. table 4. the flow of liquids through porous deposit with a fixed mass of shale ore and polyethylene beads. table 5. porous bed characteristic. no. porous bed porosity calculated flow 10-3 surface area [m2/g] particles medium size [μm] bed dispersion hydraulic diameter 10-3 reynolds number 1 0.5542 3.98 4.128 34.143 3.41 64.9 0.3048 2 0.4748 3.90 3.369 453.36 37.94 4.905 3.3658 3 0.4386 24.44 2.61 872.59 425.8 2.725 39.987 4 0.3928 68.23 1.598 1 431.55 153.24 1.757 160.86 similar experiences were carried out by replacing the polyethylene fittings by glass balls. the results obtained are presented in table 6 and 7. table 6. the flow of liquids through porous deposit with a fixed mass (80 g) and various quantities of ingredients. no. glass bolls weight [g] black shale weight [g] liquid flow [ml/s] porous bed height [cm] liquid layer height [cm] 1 0 80 0.050 5.6 15.0 2 15 65 0.051 4.5 14.3 3 30 50 0.054 4.6 14.5 4 50 30 0.117 4.0 15.5 table 7. porous bed characteristic. no. porous bed porosity calculated flow 10-3 surface area [m2/g] particles medium size [μm] bed dispersion hydraulic diameter 10-3 reynolds number 1 0.5542 3.98 4.128 34.143 3.41 64.9 0.3048 2 0.4126 4.06 3.376 74.61 7.61 2212 0.5157 3 0.4276 4.30 2.625 115.09 12.43 14.86 0.8646 4 0.3451 9.31 1.623 169.05 39.56 8.165 2.405 the following conclusions canbe inferred from the data presented in tables: adding to the finely ground black shale ore polyethylene beads or small glass beads increases the degree of deposit dispersion. the systematic reduction of the hydraulic diameter of poroud bed isevident. generelly, reynolds number has a value not exceeding the value 10. it is supported the idea of a laminar flow inside the porous deposit. the exception is the flow of liquid on a large number of polyethylene beads in a porous bed (table 5). for this case, the value of reynolds are larger than 10 (40 and 160), which may suggest picking off like strug liquid from the surface of the beads. this no. polyethylene beads weight [g] black shale weight [g] liquid flow [ml/s] porous bed height [cm] liquid layer height [cm] 1 0 80 0.050 5.6 15.0 2 15 65 0.049 6.0 13.6 3 30 50 0.307 6.5 14.0 4 50 30 0.857 8.5 8.8 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:03 utc nova biotechnologica et chimica 14-1 (2015) 59 phenomenon should be explained by the varying degrees of surface wettability of polyethylene surface and glass surface. polyethylene surface is a surface with a low surface energy, and unlike to the glass surface is hydrophobic. the difference in degree of hydrophobicity of infill material will affect cell adhesion micro-organisms to these areas. this hypothesis will be tested in future experiments. the changes in copper recovery values during 14 days of bioleaching experiments are shown at fig. 3. at first, substantial increase of metal recovery was obtained when the supported materials were added to the column bead. the results presented at fig. 3 indicate that the copper recovery was very similar to glass beads and polyethylene beads. 0 2 4 6 8 10 12 14 20 30 40 50 60 70 80 90 100 [% ] e xt ra ct io n time [days] bioleaching without support bioleaching with glass beads bioleaching with polyethylene beads fig. 3. bioleaching behaviours of shale with and without supported materials (copper recovery during bioleaching). before the bioleaching, the permeability was estimated to be on the order of 14.10-4 cm2. this value was reduced to about 4.10-4 cm2 after bioleaching. 4. conclusions in this paper, we have proposed a new approach to the column bioleaching of fine black shale ore. the development procedure was able to improve the copper recovery by adopting two support materials. these materials were glass small beads and polyethylene beads. it was shown that the addition of these materials to the porous bed, caused an improvement of hydrodynamic parameters. it is important to note that using the conventional leaching method it is not possible to obtain the copper recovery on the level of 35 %. nomenclature a – the cross section area [m2] a – the empirical parameter (a = 150) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:03 utc 60 sadowski, z. and baranska, j.a. c0 – the kozeny’s coefficient cd – the drag coefficient c – microbial cells concentration [cells/volume] d – the hydrodynamic dispersion coefficient dp – the diameter of particle [m] h1,h2 – upper and button levels [m] l – the bed height [cm] mb – the specific surface of the porous bed [m 2/g] pi – the wetted parameter δp – the pressure drop [pa] rh – the hydraulic radius [m] re – the reynold’s number r – the sphere radius [m] t – time [s] x – the distance [m] greek symbols ε – the porosity εg – the gas holdup µ – the kinetic viscosity [m2/s] φ – the permeability of porous bed [m2] acknowledgement: the work described in this paper had a financial support from eit+ company wroclaw at the frame of eit+ nanomat project. references bergendahl, j., grasso, d.: prediction of colloid detachment in a model porous media: hydrodynamics. chem. eng. sci., 55, 2000, 1523-1532. casas, m.j., martinez, j., moreno, l., vargas, t.: bioleaching model of a copper-sulfide ore bed in heap and dump configurations. metall. mater. trans. b, 29b, 1998, 899-909. clement, p.t., sun, y., hooker, s.b., peteren, n.j.: modelling multispecies reactive transport in ground water. ground water monit. r., 18, 1998, 79-92. henderson, n., brettas, c.j., sacco, f.w.: a three-parameter kozenycaman generalized equation for fractal porous media. chem. eng. sci., 65, 2010, 4432-4442. hernandez-lopez, f.m., ortiz, c., bonilla, a.c., gironas, j., munoz, f.j.: modelling changes to the hydrodynamic characteristics of agglomerated copper tailings. hydrometallurgy, 109, 2011, 175-180. li, m., bando, y., tsuge, t., yasuda, k., nakamura, m.: analysis of liquid distribution in non-uniformly packed trickle bed with single phase flow. chem. eng. sci., 56, 2001, 5969-5976. lin, c.l., miller, d. j.: pore structure analysis of particle beds for fluid transport simulation during filtration. int. j. miner. process., 73, 2004, 281-294. lin, c.l., miller, d.j., garcia, c.: saturated flow characteristics in column leaching as described by lb simulation. miner. eng., 18, 2005, 1045-1051. lizama, m.h.: a kinetic description of percolation bioleaching. miner. eng., 17, 2004, 23-32. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:03 utc nova biotechnologica et chimica 14-1 (2015) 61 lizama, m.h., harlamovs, r.j., mckay, j.d., dai, z.: heap leaching kinetics are proportional to the irrigation rate divided by heap height. miner. eng., 18, 2005, 623-630. long, z., huang, y., cai, z., cong, w., ouyang, f.: kinetics of continuous ferrous ion oxidation by acidithiobacillus ferrooxidans immobilized in poly(vinyl alcohol) cryogel carries. hydrometallurgy, 74, 2004, 181-187. nemec, d., levec, j.: flow through packed bed reactors: 1. single-phase flow, chem. eng. sci., 60, 2005, 6947-6957. niven, k.r.: physical insight into the ergun and wen & ya equation for fluid flow in packed and fluidised bed. chem. eng. sci., 57, 527-534. rockhold, l.m., yarwood, r.r., niemet, r.m., bottomley, j.p.., selker, s.j., consideration for modelling bacterial-induced changes in hydraulic properties of variably saturated porous media. adv. water resour., 25, 2002, 477495. sederman, j.a., johns, l.m., alexander, p., gladden, f.l., structureflow correlations in packed beds. chem. eng. sci., 53, 1998, 2117-2128. sheikhzadeh, a.g., mehrabian, a.m., mansouri, h.s., sarrafi, a.: computational modelling of unsaturated flow of liquid in heap leaching – using the results of column tests to calibrate the model. inter. j. heat mass transfer, 48, 2005, 279-292. tufenkji, n.: modelling microbial transport in porous media. traditional approaches and recent development. adv. water resour., 30, 2007, 1455-1469. tufenkji, n., elimelech, m.: deviation from the classical colloid filtration theory in the presence of repulsive dlvo interaction. langmuir, 20, 2004, 1081810828. presented at the 3rd international conference “biotechnology and metals 2014”, september 17-19, 2014, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:03 utc microsoft word gasparova nb.doc nova biotechnologica vii-i (2007) 115 effect of 2-[3-(trifluoromethyl)phenyl]4h-furo[3,2-b]pyrrole-5-carboxhydrazides on photosynthetic processes katarína kráľová1, renata gašparová2, martin moncman2 1institute of chemistry, faculty of natural sciences, comenius university, mlynská dolina ch-2, sk-842 15 bratislava, slovak republic (katarina.kralova@fns.uniba.sk) 2department of chemistry, faculty of natural sciences, university of ss. cyril and methodius, j. herdu 2, sk-917 01 trnava, slovak republic (gasparor@ucm.sk) abstract: a new series of carboxhydrazides 6-8 was synthesized under microwave irradiation by reaction of carboxhydrazide 1 with heterocyclic aldehydes 2-4 in the presence of p-toluenesulfonic acid in ethanol. nbenzoylcarboxhydrazide 9 was prepared by reaction of 1 with benzoylchlorid 5 in thf at room temperature. the effects of 6-9 on inhibition of photosynthetic electron transport in spinach chloroplasts and chlorophyll content in the antialgal suspensions of chlorella vulgaris were investigated. keywords: 2-[3-(trifluoromethyl)phenyl]furo[3,2-b]pyrrole-5-carboxhydrazide, chlorella vulgaris, spinach chloroplasts, photosynthetic electron transport 1. introduction carboxhydrazides and their derivatives represent an interesting class of compounds which exhibits antimicrobial (el-shaaer et al., 1998), antifungal (dutta et al., 1986), analgesic and anti-inflammatory (santos et al., 1997) activities. the presence of trifluoromethyl group in the molecule leads often to the biologically active compounds. many heterocycles bearing the trifluoromethyl group possess antiprotozoal (navarrete-vazquez et al., 2006), antimalarial (madrid et al., 2005) or antibacterial (wolfart et al., 2004) activities. substituted furans are structural units in natural products and pharmaceuticals (krutošíková, 1996) and have been widely used as synthetic intermediates (lipshutz, 1986). 5-substituted furan-2-carboxaldehydes and some of their derivatives show antibacterial (shindhar et al., 1980) or antiviral (shigetaka et al., 1970) activities the present study is a follow-up paper to our previous research dealing with the synthesis and reactions of furo[3,2-b]pyrrole system and the study of its biological activity (gašparová et al., 2005; moncman, 2006). 2. materials and methods 2.1 chemistry scheme 1 shows the synthetic pathway to hydrazides 6-9. 116 kráľová, k. et al. o n hn o nh2 a o h r o n hn o n a o n o h co2me r1 o n h hn o n o n co2me r 8a-8c r =ch3, ch2-o-ch3, ch2-ph 1 4 f3c f3c f3c 6a 6f a =o, r =4-cl, 3-cf3, 4-no2, 4-ch3, 4-i, 4-br 7 a = s, r = h 2 a =o, r = aryl 3 a = s, r = h scheme 1 h h 6 r cocl o n hn o nh f3c h o 5 9 thf pyridin 2.1.1 general procedure for synthesis of 6-8 the mixture carboxhydrazide 1 (0.3 g, 1 mmol), 5-arylfuran-2-carboxaldehyde 2 (or thiophen-2-carboxaldehyde 3, methyl 2-formylfuro[3,2-b]pyrrole-5-carboxylates 4) (0.21 g, 1 mmol) and catalytic amount of p-toluenesulfonic acid in ethanol (5 cm3) was irradiated in microwave oven at 90w for 0.5-2 min. after cooling, the solid product was filtered off, dried and crystallized from ethanol to give 51-87 % yields of products. 2.1.2 synthesis of 9 the solution of carboxhydrazide 1 (0.5 g, 1.6 mmol) in thf (5 cm3) and catalytic amount of pyridin was cooled in the ice bath to 0 °c. then benzoylchlorid 5 (0.18g, 1.3 mmol) was added and the reaction mixture was stirred at room temperature for 5 h. the solid product was filtered off, dried and crystallized from ethanol to give 60 % of product. 2.2 study of inhibition of photosynthetic electron transport in spinach chloroplasts spinach chloroplasts were prepared according to walker (1980). the effect of the compounds 5-7 on the inhibition of photosynthetic electron transport (pet) in nova biotechnologica vii-i (2007) 117 spinach chloroplasts was investigated spectrophotometrically in the presence of electron acceptor 2,6-dichlorophenol indophenol (dcpip) (30 μmol.dm−3). before measurements, the chloroplasts were resuspended in phosphate buffer (20 mmol.dm−3; ph = 7.2) containing 5 mmol.dm−3 mgcl2 and 15 mmol.dm −3 nacl. the chlorophyll content in the suspension was adjusted to 30 mg.dm−3. samples were irradiated at 25 °c with a halogen lamp (250 w) at a distance of 1 dm. a 4 cm water filter was used to prevent overheating of the samples. the pet-inhibitory activity of the compounds studied was expressed in term of ic50 values as their negative logarithms thus, corresponding to molar concentrations of inhibitors causing a 50% decrease of oxygen evolution rate (oer) with respect to the untreated control sample. due to lower aqueous solubility of the compounds studied, these were dissolved in dimethyl sulfoxide. the effect of dmso on oer in the suspensions of spinach chloroplasts was in the range of experimental error and could be neglected. 2.3 study of chlorophyll content in chlorella vulgaris the algae chlorella vulgaris were statically cultivated (photoperiod: 16h light / 8h dark; illumination: 5 000 lx; temperature: 23 ±1 °c) in liquid cultivation medium (ph = 7.2) (kráľová et al., 1998). the effect of the compounds applied at three stepwise increasing concentrations (10, 50 and 100 μmol.dm−3) on the total chlorophyll content of algal suspension was determined after 7 days of cultivation spectrophotometrically (kontron uvikon 800) and after extraction into methanol according to wellburn, 1994. the chlorophyll content in the suspensions at the beginning of cultivation was 0.1 mg.dm−3. the effect of the compounds studied and applied in the concentration range 1–100 μmol.dm−3 on the content of chlorophyll in the suspensions was expressed as the percentage from the corresponding value obtained for the control (table 2). 3. results and discussion 3.1 study of inhibition of photosynthetic electron transport in spinach chloroplasts hill and scarisbrick (1940) showed that isolated chloroplasts and chloroplast fragments could release o2 in the light if they were given a suitable acceptor for the electrons being removed from h2o. dcpip (2,6-dichlorophenol indophenol) is often used as an synthetic electron acceptor for this reaction for measuring oxygen evolution rate (oer) in isolated plant chloroplasts (e.g. šeršeň et al., 1990). pet-inhibitory activity is exhibited by many compounds possessing x = c-nh group with a sp2 hybridized carbon atom i.e. ureas, triazines or anilides (kráľová et al., 1999; miletin et al., 2001). due to formation of hydrogen bonds between this group and the target proteins in photosynthetic centers of thylakoid membranes, changes in protein conformation may occur resulting in inhibition of photosynthetic electron transport (draber et al., 1991). 118 kráľová, k. et al. table 1. inhibition of photosynthetic electron transport in spinach chloroplasts. compound r log(1 / ic50) [mol.dm-3] ic50 [mmol.dm-3] 6a 4-cl 3.8975 0.127 6b 3-cf3 4.1513 0.071 6c 4-no2 4.0244 0.095 6d 4-ch3 2.8936 1.278 6e 4-i 3.7615 0. 173 6f 4-br 3.0702 0.851 7 3.2194 0.604 8a ch3 2.2948 5.180 8b ch2och3 2.2857 5.178 8c ch2ph 3.2857 0.518 9 3.6583 2.220 ic50 values of the standards, used for testing of herbicidal as well as antialgal activity (kubicová et al., 2003; conrad et al., 1993), related to inhibition of photosynthetic electron transport in plant chloroplasts determined for these herbicides that act in the photosystem 2, varied in the range 0.25 – 0.79 μmol.dm-3 for atrazine (2chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine), 0.40 – 3.98 μmol.dm-3 for simazine (2-chloro-4,6-bis(ethylamino)-1,3,5-triazine) and 0.032-0.200 μmol.dm-3 for diurone (1-(3,4-dichlorophenyl)-3,3-dimethylurea), as described by fedtke (1982). in comparison with these values, carboxhydrazides 6 – 9 showed relatively low inhibitory effect on photosynthetic electron transport (pet) in spinach chloroplasts (table 1). the most effective inhibitors were compounds 6a (r = 4-cl), 6b (r = cf3), 6c (r = 4-no2) and 6e (r = 4-i). the influence of the electron acceptor properties of r substituent on pet-inhibitory activity was not significant. 3.2 study of chlorophyll content in chlorella vulgaris in the concentration range (1 – 100 μmol.dm-3) the majority of carboxhydrazides 6 – 9 only slightly reduced chlorophyll content in statically cultivated algal suspensions of chlorella vulgaris (table 2). the most effective inhibitors were compounds 6c (r = 4-no2) a 6e (r = 4-i). table 2. effect of compounds 6-8 on chlorophyll content in algal suspensions of chlorella vulgaris compound r concentration (μmol.dm-3) concentration of chlorophyll (mg .dm-3) average in 10 – 100 μm % of control 0 7.333 control 100 5.561 50 6.424 6a 4-cl 10 6.342 84.3±4.8 continued on next page nova biotechnologica vii-i (2007) 119 compound r concentration (μmol.dm-3) concentration of chlorophyll (mg .dm-3) average in 10 – 100 μm % of control 100 6.610 50 6.629 6b 3-cf3 10 6.836 91.3±1.7 100 1.877 50 2.609 6c 4-no2 10 5.539 30.9* 100 6.516 50 6.691 6d 4-ch3 10 6.964 91.7±3.1 100 3.552 50 4.979 6e 4-i 10 5.741 100* 100 5.917 50 5.909 6f 4-br 10 6.379 82.8±3.7 100 7.217 50 6.682 7 10 5.917 90.1±8.9 100 5.714 50 6.570 8a ch3 10 6.946 87.4±7.6 100 4.655 50 6.317 8b ch2och3 10 6.222 78.2±4.3 100 6.958 50 6.400 8c ch2ph 10 6.409 93.2±5.1 100 5.833 50 8.564 9 10 6.267 81.7±3.3 * ic50 in μm 4. conclusions n′-{[5-(r-phenyl)furan-2-yl]methylene}-2-[3-(trifluoromethyl)phenyl]-4h-furo [3,2-b] pyrrole-5-carboxhydrazides 6a – 6f, n′-[(thiophen-2-yl)methylene]-2-[3(trifluoromethyl) phenyl] 4h – furo [3,2-b] pyrrole – 5 carboxhydrazide 7, n′ {[5 (methoxycarbonyl) – 4 r1 – furo [3,2-b] pyrrol – 2 yl] methylene} – 2 [3 (trifluoromethyl) phenyl]-4h-furo[3,2-b]pyrrole-5-carboxhydrazides 8a – 8c and n'benzoyl-2-[3-(trifluoromethyl)phenyl]-4h-furo[3,2-b]pyrrole-5-carboxhydrazide 9 showed relative-ly low inhibitory effect on the photosynthetic electron transport of spinach chloroplasts and only slightly reduced chlorophyll content in statically cultivated algal suspensions of chlorella vulgaris. the most effective inhibitors of the pet were compounds 6a (r = 4-cl), 6b (r = cf3), 6c (r = 4-no2) and 6e (r = 4-i). carboxhydrazides 6c (r = 4-no2) a 6e (r = 4-i) were proved as the best inhibitors of the chlorophyll content in suspensions of chlorella vulgaris. 120 kráľová, k. et al. acknowledgement: the authors are grateful for financial support to the slovak research and development agency by way of project no. apvt-20-005204 and to the vega grant agency of slovak ministry of education 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adsorption of zn (ii) ions from refinery wastewater by sulfuric acidmodified bentonite: kinetic and isotherm studies dihia bellache1,2,, karim moussaceb2, jean-claude bollinger3, farouk boudrahem4 1department of process engineering, faculty of science and applied science, university of bouira, 10000 bouira, algeria 2laboratory of materials technology and process engineering), faculty of technology, a/mira bejaia university, targaouzemour road, 06000 bejaia, algeria 3water soil environment research group, university of limoges, 87060 limoges, france 4environmental engineering laboratory, faculty of technology, a/mira bejaia university, targa-ouzemour road, 06000 bejaia, algeria  corresponding author: d.bellache@univ-bouira.dz article info article history: received: 17th november 2021 accepted: 7th february 2022 keywords: adsorption bentonite isotherm kinetic wastewater zinc abstract a local bentonite clay from maghnia (algeria) was activated with chemical method characterized and tested for its ability to adsorb zinc (ii) from refinery wastewater. batch experiments were conducted to study the effects of the main parameters such as contact time, initial metal concentration and agitation speed on the adsorption of zn (ii) by local bentonite clay. experiences have led to the following results: an adsorption rate of the order of 98 % with operating conditions of ph = 4.5, agitation speed of 400 rpm and temperature of 25 ºc. pseudo-first-order, pseudo-second-order and intraparticle diffusion models were used to analyze the kinetic data obtained at different concentrations. the pseudo-second-order kinetic model agrees very well with the experimental results. in order to determine the best-fit isotherm, in the studied concentration range of zn(ii) at 25 ºc, the experimental equilibrium data were analyzed using tow adsorption isotherm models: langmuir and freundlich models, these two models give a good fit. introduction a number of industries, such as metal plating facilities, mining operations, tanneries, nuclear power plants, fertilizers and battery productions, often discharges heavy metals, this can lead to the contamination of freshwater and marine environment (low and lee 2000). metal ions in water can occur naturally from anthropogenic sources and from leaching of ore deposits, which mainly include solid waste disposal and industrial effluents. the levels of heavy metals in water system have substantially increased over time with rapid development of industrial activities (nouri et al. 2006). the main sources of zinc in the environment are the manufacturing of brass and bronze alloys and galvanization (arias and sen 2009). it is also utilized in paints, rubber, plastics, cosmetics, and pharmaceuticals. zinc is an essential element for life and act as micronutrient when present in trace amounts, the maximum daily dose not to be exceeded is 40 mg for an adult. however, high zinc can cause eminent health problems, such as stomach cramps, vomiting, skin irritations, anemia, and nausea (zwain 2012). mailto:d.bellache@univ-bouira.dz nova biotechnol chim (2022) 21(2): e1267 2 various methods are available to remove and isolate these heavy metals from water and wastewater such as ion exchange, chemical precipitation, membrane filtration, adsorption, and electrochemical treatment technologies (fu and wang 2011). adsorption is very promising because of the advantages of easy operation, low cost, and possibility of metal recovery (dang et al. 2021). these methods have proven their ability in removing heavy metals from wastewater; however, several factors need to be considered when choosing the appropriate method. adsorption has been proven to have better performance compared to other methods as mentioned in many studies (kamari et al. 2014) the adsorption ability of clay is caused by a net negative charge on the structure of fine grain silicate minerals. this negative charge is neutralized by the adsorption of positively charged species, giving clay the ability to attract and hold cations such as heavy metals. the large surface area of clays (up to 800 m2.g-1) also contributes to the high adsorption capacity, there are three basic classes of clays: kaolinite, micas (such as illite), and smectites (e.g., montmorillonite). of the three species, montmorillonite clays have the smallest crystals, the largest surface area, and the highest cation exchange capacity. thus, montmorillonite clays would be expected to have the highest adsorption capacity (haider et al. 2014). studies such as those conducted by (bellir et al. 2013; sen et al. 2013; vasconcelos et al. 2013) have investigated the potential of bentonite for the removal of zinc ion. although the results involving zinc removal by bentonite are significant and promising, the properties of adsorbents for optimizing the conditions of the process need to be better understood. this study explored the feasibility of utilizing an algerian activated bentonite for the adsorption of zinc from refinery wastewater. various parameters affecting adsorption process, such as contact time, initial zinc concentration, adsorbent dosages, agitation speed and the medium temperature were investigated. the kinetic adsorption results have been analyzed using both pseudo-first-order and pseudo-second-order kinetics models. the mechanism of adsorption process has been explained based on intra-particle diffusion model. finally, the langmuir and freundlich models were applied for the analysis of the adsorption equilibrium. experimental characteristics of refinery wastewater the main physicochemical parameters on the quality of refinery wastewater are shown in table 1. the determination of these parameters is performed according to standard analytical methods (rodier et al. 2009). based on this table, the physicochemical characteristics of the refinery water are within the limits of wastewater standards, except cod and chloride and zinc concentrations that exceed current standards. table 1. physical-chemical characteristics of refinery wastewater and the oms limit values of wastewater parameters. cod – chemical oxygen; tss – total suspended solids. characteristic of raw bentonite in this study, bentonite obtained from maghnia (north-western part of algeria) and supplied by enof “national company of useful and nonferrous products, algeria” was used as an adsorbent. its chemical and physico-chemical characteristics are summarized in table 2 and table 3. preparation of acid-activated bentonite the na-bentonite was prepared with a procedure similar to already reported (khalaf et al. 1997). 30 g of crude bentonite was mixed with 1 l of 1 m nacl solution and stirred for 24 h. after three successive treatments, the homoionic bentonite was parametres values normes ph 7.07 6,5 – 8,5 conductivitym s/cm 40.89 2,8 turbidity (ntu) 85.6 5 – 30 tss [mg.l-1] 387.8 30 cod [mg.o2.l -1] 1601.24 120 chloride [mg.l-1] 2098.03 200 sulfate [mg.l-1] 33.18 250 chromium (vi) [mg.l-1] 0.02 0,1 lead [mg.l-1] 0.04 0,5 zinc [mg.l-1] 6.7 3 nova biotechnol chim (2022) 21(2): e1267 3 dialyzed in deionized water until it was free of chloride. then it was separated by centrifugation to eliminate all other solid phases (quartz, and calcite) boutahala and tedjar 1993). in a conical flask, 50 g of purified bentonite and 250 ml of sulfuric acid solution (0.5 m and 1 m). the mixture was homogenized and allowed to stand at room temperature for 24 h. the resulting activated clay was centrifuged and washed with distilled water several times until it was free of so4 2− as indicated by the agno3 test and was then dried at 100 – 107 ºc for 24 h. acid-activated bentonite with different concentration of sulfuric acid at 0.5 m end 1 m are noted respectively b05 and b1, was then stored for further use in the adsorption tests. table 2. chemical composition of bentonite sample. constituent sio2 mgo al2o3 k2o cao f2o3 na2o tio2 as weight [%] 69.4 1.1 14.7 0.8 0.3 1.2 0.5 0.2 0.05 loss of ignitions = 11 table 3. physico-chemical properties of bentonite. surface area [m².g-1] cec [eqg-1] ph exchangeable cation [meq/100g] ca2+ mg2+ na+ k+ 80 0.97 6.2 30.6 12.8 36.2 9.5 batch adsorption study the ability of bentonite to adsorb zn(ii) ions from refinery wastewater was studied under various optimized conditions of concentration of metals, and contact time, adsorbent dosage and strength speed. batch adsorption experiments were carried in 250 cm3 erlenmeyer flasks with known amount of the adsorbent and 100 ml of solution, with ph = 4.5, the initial ph of the solutions was adjusted with 0.1 m hno3 or 0.1 m naoh to the desired value. the erlenmeyer were kept at the desired temperature under constant agitation. after 70 min, the suspensions were centrifuged and the solutions were analyzed for zn(ii) ions by atomic absorption spectrometry aa-6501f. the amount of adsorbed at any time, qt (mg.g -1), was calculated using eq. 1: (1) where, ci and ct are the initial and liquid-phase concentrations at any time t of zinc solution (mg.l1), respectively, v is the volume of wastewater (l) and m is the mass (g) of the adsorbent used. the removal efficiency, e (%) of the system, is eq. 2 (boudrahem et al. 2011): (2) results and discussion adsorbent characterization the ftir spectra of natural bentonite and acid activated bentonite were taken in the range of 4,000 – 400 cm-1 (shimadzu ftir-8400, shimadzu corp., tokyo, japan). ftir spectra are illustrated in fig. 1 and 2. positions and assignment of the vibrational bands of this clay are shown in table 4. the broad and slight bands at 3,627 and 3,435 cm-1 are due to the o–h stretching vibration of the silanol (si–oh) groups from the solid and h–o–h vibration of the water molecules adsorbed on the silicate surface. the band at 1,634.5 cm-1 reflects the bending h–o–h bond of water molecules, which is retained in the silicate matrix. the most intensive and sharp band at 1,039 cm-1 represents the si–o–si groups of the tetrahedral sheets. where the bands around 515 and 523 cm-1 is are ascribed to si–o–al (where al is the octahedral cation) and to si–o–si bending vibration. furthermore, the sharp bands at 790 with reflexion at 778.4 cm-1 confirm the presence of quartz admixtures in the sample (tomic et al. 2011). nova biotechnol chim (2022) 21(2): e1267 2 ftir spectroscopy is very sensitive to modification of the clay structure upon acid treatment, as illustrated in fig. 1. in ftir-spectrum of the acid activated bentonite, weakening of absorption band intensity at 3,437 and 1,639 cm−1 is marked (fig. 2). it corresponds to the process of removal of interlayer water (schrader and loeb 1992). absorption peaks between 3,435 and 3,627 cm-1 are due to stretching bands of the oh groups. 4000 3000 2000 1000 1,4 1,2 1,0 0,8 0,6 0,4 a ( % ) wave number(cm -1 ) fig. 1. ftir spectrum of raw bentonite. 4000 3500 3000 2500 2000 1500 1000 500 2 4 6 8 10 12 14 16 18 20 22 24 1 m 0.5 m a (% ) wave number(cm -1 ) fig. 2. ftir spectrum of acid activated bentonites. while the band at 1,639 cm-1 corresponds to the oh deformation of water to observe natural bentonite and acid activated bentonite, but the peak intensities of both the acid-activated bentonites are lower than that of natural bentonite. absorption band reduction at 3,627 cm-1 indicates development of dehydroxylation process. this is believed to occur as a result of acid activation of bentonite (ozcan and ozcan 2004). in addition, the transformation of the tetrahedral sheet was found at 781 cm-1 and 783 cm-1 for b1 and b05 activated bentonites respectively. the acid activation leads to the formation of amorphous silica, indicated by the increased intensity of the peak, which may expose more adsorption sites (komandel et al. 1990). table 4. ftir band assignments of raw and activated bentonites. maxima [cm-1] assignements 3,627 et 3,435 –oh stretching 3,443 –oh stretching, hydratation 1,637 et 1,639 –oh stretching, hydratation 1,039 et 1,038 si–o stretching (in plane) 523 et 515 si–o–al bending 477 et 458 si-o-si 790 et 799 quatrz effect of contact time and initial zn (ii) concentration on the adsorption fig. 3 represents a plot of the amount of zinc metal ion adsorbed (mg.g-1) versus contact time for znb05 and zn-b1 system at different initial metal ion concentration range. 0 20 40 60 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 3.5 mg/l 4.2 mg/l 5,7 mg/l 6.3 mg/l 6.7 mg/l q t( m g g -1 ) t(min) 0 20 40 60 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 3.5 mg/l 4.2 mg/l 5,7 mg/l 6.3 mg/l 6.7 mg/l q e (m g g -1 ) t(min) fig. 3. effect of contact time on zn (ii) metal ion adsorption by b05 (a) and b1 (b) for different initial zn (ii) ions concentration. conditions: ph = 4.5; adsorbent dose = 1 g.l-1; agitation speed = 400 rpm and t = 25 ºc. a a b b05 b1 4 nova biotechnol chim (2022) 21(2): e1267 3 from these plots, it is found that the amount of adsorption i.e. mg of adsorbate per gram of adsorbent increases with increasing of contact time at all initial metal ion concentrations and equilibrium is attained within 50 min for both the systems further it was observed that the amount of metal ion uptake, qt (mg.g -1) is increased with increase in initial metal ion concentration. the increase in adsorption is more pronounced for znb1 system compared to zn-b05 system the difference in the zinc sorption onto the bentonites may be due to the difference in the mineralogical compositions and associated cations in the exchangeable sites (sheta et al. 2003). these kinetic experiments clearly indicate that the adsorption of zn (ii) on acid activated clay surface is a two-step process: a rapid adsorption of metal ions to the external surface is followed by possible slow intraparticle diffusion in the interior of the particles (sen and sarzali 2008). this two-stage process is also due to the presence of two different types of binding sites on the adsorbents. the two stage sorption mechanism with the first rapid and quantitatively predominant and the second slower and quantitatively insignificant, has been extensively reported in literature (yang et al. 2010). effect of agitation speed the experiments were undertaken with different agitation speeds of (200, 400, 600,800 and 1,000) rpm keeping constant the other process variables. fig. 4 show that the high amount of zn(ii) ions adsorbed at equilibrium (5.5 and 3.1 mg.g-1 for b1 and b05, respectively) is obtained with an agitation speed of 400 rpm for both adsorbents. the adsorbed amount of zn(ii) increases with an increase of the agitation speed from (200 to 400) rpm, and at higher stirring speed (> 400 rpm), the sorbed amount of zn(ii) decreases. when increasing the agitation speed (< 400 rpm), the diffusion rate of metal ions from the bulk liquid to the liquid boundary layer surrounding sorbent particles becomes higher because of an enhancement of turbulence and a decrease of the thickness of the liquid boundary layer. at higher stirring speeds (> 400 rpm), the decrease of the sorbed amount of zn(ii) is attributed to the rejection of the adsorbent part, which is found plated against the internal walls of the reactor (boudrahem et al. 2011). 0 200 400 600 800 1000 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 q t (m gg -1 ) agitation speed(rpm) b05 b1 fig. 4. effect of agitation speed on the sorption of zinc by both activated clay b1 and b05. conditions: ph = 4.5; initial concentration of zn(ii) = 6.7 mg.l-1 and t = 25 ºc. effect of adsorbent dosage the effect of adsorbent dosage on the adsorption of zinc (ii) was studied at different dosages in the solution at ph 4.5. as shown in fig. 5, the adsorption percentage of zinc (ii) removed ions increased as the dosage of bentonite increased from 1.0 to 3.0 g.l-1. this may be explained by the metal ions competing for limiting adsorption sites at a lower bentonite dosage. 0 10 20 30 40 50 60 0 20 40 60 80 100 e (% ) t(min) 1g/l 2g/l 3g/l fig. 5. effect of dosage of b1 activated bentonite on the sorption of zinc. conditions: ph = 4.5; initial concentration of zn(ii) = 6.7 mg.l-1 and temperature = 25 ºc. the increase in the adsorption percentage with an increase in adsorbent dosage was due to an increase in active sites on the adsorbent, thus facilitating the 5 nova biotechnol chim (2022) 21(2): e1267 2 penetration of metal ions to the sorption sites (sarı et al. 2007a; 2007b). these observations agree with others reported in the literature for the adsorption of metals ions by different materials (mishra and patel 2009). effect of the medium temperature fig. 6 shows that the effect of temperature on the retention of zn(ii) ions is explained by the fact that an increase in temperature leads to a moderately considerable increase in the adsorption capacity, and which becomes increasingly less effective after 15 min of contact, the elimination rate curves for different temperatures become superimposed. the increase in temperature provides energy for the ionic particles which in turn allows them to surpass the repulsion forces with the supports up to a certain limit, beyond which the temperature becomes ineffective “state of saturation”. it can be seen from the analysis of fig. 6 that the bentonite removal rate reaches approximately 82 % for all temperatures studied. 0 10 20 30 40 50 60 0 20 40 60 80 25°c 35°c 45°c e (% ) t(min) fig. 6. effect of dosage of b1 activated bentonite on the sorption of zinc. conditions: ph = 4.5; initial concentration of zn(ii) = 6.7 mg.l-1, adsorbent dose = 1 g.l-1, agitation speed = 400 rpm. adsorption kinetics the kinetics of adsorption describes the rate of metal ions uptake on activated bentonites and this rate controls the equilibrium time. the kinetics of adsorbate uptake is required for selecting optimum operating conditions for the full-scale batch process (gupta et al. 1997). the kinetic parameter, which is helpful for the prediction of adsorption rate, gives important information for designing and modelling the processes. the kinetics of the adsorption data was analyzed using different kinetic models, such as pseudo-first-order and pseudo-second-order models. pseudo-first-order model the kinetic data were treated with the lagergren first-order model eq. 3 (lagergren 1898): (3) where k1 is the adsorption rate constant for the first order adsorption, qtis the amount of heavy metal adsorbed at time t (mg.g-1) and qe is the amount of heavy metal adsorbed at saturation (mg.g-1). the integration of the eq. 3 gives the following expression eq. 4: (4) where c1 is the integration constant for first order reaction kinetic. if it is supposed that q = 0 at t = 0, then (eq. 5): (5) values of adsorption rate constant k1 for the zinc(ii) adsorption onto acid activated bentonites (b1, b05) were determined from the straight-line plot of against t. the data were fitted with a poor correlation coefficient (table 5), indicating that the rate of removal of zinc onto both acid-activated bentonites does not follow the pseudo first-order equation. pseudo-second-order reaction kinetic adsorption data was also evaluated according to the pseudo second-order reaction kinetic proposed by ho and mckay (ho and mckay 1998; eq. 6): (6) 6 nova biotechnol chim (2022) 21(2): e1267 3 where k2 is the second order reaction constant. if eq. 6 is integrated, the following expression is obtained (eq. 7): (7) in eq. 7, c2 is the integration constant of the second order reaction kinetic. with an algorithmic arrangement, the following statement is formed (eq. 8): (8) the pseudo second order model can be determined experimentally by plotting t/qt against t, and the constants qe and k2 obtained from the slope and intercept. the pseudo second order kinetic parameters and the r2 value obtained are given in table 5. this model can thus be applied to the adsorption data, as indicated by the good r2 value obtained. this implies that the rate-limiting step is a chemisorption involving valency forces caused by sharing or exchange of electrons between sorbent and sorbate species in solution (unlu and ersoz 2006). the pseudo second order model have been reported to give very good fits to experimental data by many researchers (sarı et al. 2007a; 2007b). table 5. calculated kinetic parameters for pseudo first-order and second order for the adsorption of zinc onto acid activated bentonites. adsorbent concentration [mg.l-1] qeexp [mg.g-1] k [g.mg-1.min-1] qe cal [mg.g-1] r2 p se u d o fi r st o r d e r k in e ti c b05 3.500 1.920 0.085 3.414 0.911 4.200 2.250 0.088 3.857 0.916 6.700 3.100 0.079 4.275 0.909 b1 3.500 3.120 0.066 3.421 0.955 4.200 3.680 0.07 3.849 0.968 6.700 5.500 0.084 6.61 0.915 p se u d o se c o n d o r d e r k in e ti c b05 3.500 1.920 0.001 5.649 0.781 4.200 2.250 0.004 4.587 0.939 6.700 3.100 0.010 4.424 0.994 b1 3.500 3.120 0.0107 4.366 0.975 4.200 3.680 0.0123 4.807 0.990 6.700 5.500 0.012 6.666 0.998 adsorption mechanism generally, any sorption process can be described by the following three steps: (i) film or surface diffusion, (ii) intraparticle or pore diffusion and (iii) sorption on the interior sites of the sorbent. since the last step is very rapid, it is assumed that it does not influence the overall kinetics. the rate of adsorption process, therefore, will be controlled by either film diffusion or intraparticle diffusion depending on which step is slower. the weber– morris intraparticle diffusion model has often been used to determine if intraparticle diffusion is the rate-limiting step. the intraparticle diffusion equation can be written by following eq. 9: (9) where kd is the intraparticle diffusion constant (mg.g-1.min-1/2). according to this model, the plot of qt versus the square root of time t 1/2 should be linear if intraparticle diffusion is involved in the adsorption process and if the plot passes through the origin, then intraparticle diffusion is the sole rate-limiting step. it has also been suggested that in instances when the plot is multilinear two or more steps govern the adsorption process (boparai and joseph 2011). linear plots of the intraparticle diffusion model are shown in fig. 7 and table 6. for both b05 and b1 acid activated bentonite, the adsorption process can be divided into three stages; the initial first-shape portion of the curve corresponds to the external surface adsorption stage or instantaneous adsorption stage. the second, gradual linear portion corresponds to intraparticle diffusion, and the final represents the equilibrium stage. 7 nova biotechnol chim (2022) 21(2): e1267 2 therefore, zn(ii) adsorption on both acid activated bentonites can be considered as the combination of surface adsorption and pore-filling by diffusing. fig. 7. intraparticle diffusion plot for zn (ii) adsorption onto acid activated bentonites; condition: ph = 4.5; agitation speed = 400 rpm and t = 25 ºc. table 6. calculated parameters of intraparticle diffusion for the adsorption of zinc onto acid activated bentonites. adsorbents concentration [mg.l-1] kd1 [g.mg-1z.min-1/2] r12 kd2 [g.mg-1.min-1/2] r22 b05 3.500 0.311 0.954 0.159 0.963 4.200 0.362 0.987 0.200 0.975 6.700 0.586 0.993 0.252 0.993 b1 3.500 0.480 0.943 0.306 0.998 4.200 0.604 0.963 0.299 0.997 6.700 0.967 0.999 0.523 0.981 adsorption langmuir isotherm the theoretical langmuir sorption isotherm is based on the assumption that the maximum adsorption occurs when a saturated monolayer of solute molecules is present on the adsorbent surface, the energy of adsorption is constant and there is no migration of adsorbate molecules in the surface plane. the langmuir isotherm model is expressed as follows eq. 10 (langmuir 1918): (10) where kl is the langmuir constant (l.mg -1) and qmax is the maximum adsorption capacity (mg.g -1). the linearized langmuir equation is (eq. 11): (11) the langmuir constants, qm (maximum adsorption capacity) and kl, can be obtained from plots between ce/qe versus ce, with fixed initial conditions. overall, langmuir isotherm model has a high regression coefficient (r2) for both the systems. the amount of metal adsorbed at equilibrium, adjusted by langmuir model, was 8.4 mg.g-1 for the b1 activated clay and 5.64 mg.g-1 for the b05 activated clay. table 7. langmuir parameters obtained from langmuir plots. adsorbent t [ºc] qmax [mg.g-1] kl [mg-1] r2 rl [mg.l-1] 3.5 4.2 6.7 b05 25°c 5.64 0.33 0.994 0.46 0.41 0.31 b1 25°c 8.40 1.52 0.996 0.15 0.13 0.08 b a b05 b1 8 nova biotechnol chim (2022) 21(2): e1267 3 the essential characteristics of the langmuir isotherm can be expressed in terms of dimensionless constant separation factor or equilibrium parameter, rl which is defined by eq. 12 (hall et al. 1966): (12) the dimensionless separation factor (rl) was calculated, based on langmuir constant kl and the initial zinc concentration presented in table 7 for both the systems. these rl values indicates favorable adsorption as it lie in the range < 0 <rl< 1 (sen and dustin 2011). adsorption freundlich isotherm in 1906, freundlich presented the earliest known sorption isotherm equation (veli and alyuz 2007). this empirical model can be applied to non-ideal sorption on heterogeneous surfaces as well as multilayer sorption and is expressed by the following equation (eq. 13): (13) where ce (mg.l -1) is the equilibrium concentration and qe (mg.g -1) is the amount adsorbed of zinc per unit mass of the adsorbent. the constant n is the freundlich equation exponent that represents the parameter characterizing quasi-gaussian energetic heterogeneity of the adsorption surface (bansal and goyal 2005). kf (mg1-(1/n).l1/n .g-1) is the freundlich constant indicative of the relative adsorption capacity of the adsorbent. the freundlich exponent, n, should have values lying in the range of 1 – 10 for classification as favorable adsorption (h.m.f. 1906). the linear form of this model is given by eq. 14: (14) where kf and n can be calculated from the intercept and slope between ln (qe) and ln (ce) which are shown in table 8 for both the systems. the value of r2 obtained confirms the applicability of the freundlich isotherm to the sorption process. the n values are greater than one, which may ndicate that the adsorption of metal ions by the activated bentonite is favorable (el-geundi 1990). table 8. freundlich parameters obtained from freundlich plots. adsorbant t [ºc] n kf r2 b05 25 1.79 0.414 0.994 b1 25 2.07 1.61 0.999 the comparison of the experimental values with the values of qe obtained by both models is shown in fig. 8. as it is seen from this figure, langmuir isotherms usually fitted better with the experimental data rather than freundlich isotherms. in addition, some other studies showed that langmuir and freundlich isotherms correspond well with the experimental results of some heavy metals (bereket et al. 1997). several researchers have studied the adsorption capacity of different sorbents. the comparison of the activated bentonite with various adsorbents, in terms of adsorption capacity for zinc (ii) ions from aqueous solution at laboratory, is given in table 9. 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 q e (m g g -1 ) c e (mgl -1 ) % lagmuir % freundlich % exr 25°c 0,0 0,5 1,0 0 1 2 3 4 5 6 % lagmuir % freundlich % exr 25°c q e (m g g -1 ) c e (mgl -1 ) fig. 8. sorption isotherms for zinc onto activated bentonites. conditions: ph = 4.5; agitation speed = 400 rpm; agitation time = 60 min and temperature = 25 ºc. b a b05 b1 9 nova biotechnol chim (2022) 21(2): e1267 3 several researchers have studied the adsorption capacity of different sorbents. the comparison of the activated bentonite with various adsorbents, in terms of adsorption capacity for zinc (ii) ions from aqueous solution at laboratory, is given in table 9. table 9. values of maximum sorption capacities of some sorbent for zinc (ii). sorbent modifying agent [s] zn(ii) [mg.l-1] qm [mg.g-1] source bentonite — 300 52.91 (tunali and akar 2006) botrytis cinerea naoh 100 12.98 (agrawal et al. 2004) sea nodule residue hydrochloric acid 200 32.46 (kargi and cikla 2006) conclusion the present study has evaluated the zinc (ii) ions removal potential of an algerian chemically treated bentonite; the obtained results can be summarized as follows: in batch adsorption studies, the amount of metal ion zn (ii) adsorption on both clay minerals was found to increase with increase in initial metal ion concentration, contact time and in the mass of clay. the amount of zn(ii) ions adsorbed by both materials increases with increasing initial zinc concentration and contact time. the adsorption capacity of activated bentonite at 1m h2so4 (b1) is higher than that of activated bentonite at 0.5m h2so4 (b05) stirring rate does not affect the amount of zn(ii) ions adsorbed from 400 rpm for both adsorbents; zinc removal rate increases with increasing adsorbent mass. the study of the influence of temperature on adsorption showed that the temperature has no influence on the zinc removal rate. high adsorption capacity of acid activates bentonite (b1) compared to acid activate bentonite (b05). kinetic experiments clearly indicated that sorption of zn (ii) on both activated bentonite is a two steps process: a rapid adsorption of metal ion to the external surface followed by intraparticle diffusion into the interior of adsorbent, which has also confirmed by intraparticle diffusion model. overall, the kinetic studies revealed that adsorption process followed the pseudo-secondorder kinetics model. the langmuir and freundlich isotherms models were applicable. references agrawal a, sahu kk, pandey bd (2004) removal of zinc from aqueous solutions using sea nodule residue. colloids surf. a 3: 133-140. arias f, sen tk (2009) removal of zinc metal ion (zn2+) from its aqueous solution by kaolin clay mineral: a kinetic and equilibrium study, colloids surf. a. 348: 100108. bansal rc, goyal m (2005) activated carbon adsorption. crc press, boca raton, usa, 520 p. bellir 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226-233 yang c, wang j, lei m (2010) biosorption of zinc (ii) from aqueous solution by dried activated sludge. j. environ. sci. 22: 675-680. zwain hm (2012) comprehensive study on wastewater treatment using low cost adsorbent. lap lambert academic publishing, saarbrucken, germany, 124 p. 11 nova biotechnologica et chimica 13-1 (2014) 21 doi 10.2478/nbec-2014-0003 © university of ss. cyril and methodius in trnava a new approach to identification of biomarkers for early cancer stage detection bogusław buszewski1, joanna rudnicka1, marta walczak2, tadeusz jezierski2 1department of environmental chemistry and bioanalytics, faculty of chemistry, nicolaus copernicus university, 7 gagarin st, 87-100 toruń, poland (bbusz@chem.umk.pl) 2institute of genetics and animal breeding pas, department of animal behavior jastrzebiec, postepu 36a, 05-552 magdalenka, poland abstract: gas chromatography and mass spectrometry (gc/ms) was applied for determination of concentrations volatile organic compounds present in human breath samples. the technique allows to rapid determination compounds in human air, at the level of parts per billion. it showed linear correlations ranging from 0.83-234.05 ppb, limit detection in the range of 0.31-0.75 ppb and precision, expressed as rsd, was less then 10.00%. moreover, trained dogs are able to discriminate breath samples of patients with diagnosed cancer disease. we found positive correlation between dog indications and content of ethyl acetate and 2pentanone in breath (r=0.85 and r=0.97, respectively). key words: volatile organic compounds, breath analysis, gas chromatography-mass spectrometry, canine olfaction 1. introduction lung cancer belongs to most often of malicious tumors being a main cause death both men and women in industrialized countries. predominant factor of lung cancer is active and passive smoking because cigarette smoke contains about 200 substances with influence carcinogenic and mutagenic. other common initiator cause of lung cancer belongs to exposure to radon, cadmium, arsenic, beryllium, asbestos (mcwilliams et al., 2002; baker, 2006). lung cancer is classified into two broad groups: small cell lung carcinoma (sclc) (20-25% frequency of occurrence) and non-small cell lung carcinoma (nsclc) (70-75%). the letter category includes adenocarcinomas (25-30%), squamous cell (30-35%) and large cell carcinomas (1015%). this classification takes into account histological type of lung cancer as well as facilitates method of treatment and prognosis of disease (collins et. al., 2007; yao et al., 2010). in the recent years scientific interest to search of non-invasive technique, painless and agreeable for patients which would facilitate diagnosis of early-stage of lung cancer without necessary using invasive medical routine has been increased. exemplary denouement could be analysis of breath which has numerous advantages in comparison with traditional diagnostic methods (prado, et al., 2005; libardoni, et al., 2006). the determination of volatile organic compounds (vocs) in exhaled breath air can provide valuable information about the condition of human health. presence of voc in breath air at the trace levels makes breath analysis difficult, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:59 utc 22 buszewski, b. et al. therefore, as the sampling technique, solid phase microextraction (spme) (yu, et al., 2005) and thermal desorption (td) (hryniuk, et al., 2009) are mainly applied. after sampling of vocs, identification of the components is performed most often by using: gas chromatography-mass spectrometry (gc/ms) (deng, et al., 2004), selected ion flow tube-mass spectrometry (sift-ms) (spanel, et al., 1996), proton transfer reaction-mass spectrometry (ptr-ms) (warneke, et al., 1996). an innovative, unconventional method is applied the dog smell to detect changes in carcinoma. in dogs and other macrosmatic animal species (e.g. rats, pigs) that are characterized by a well developed sense of smell, the area of olfactory epithelium in the nasal cavity is much larger, compared to in microsmatic species, including humans (fig. 1, buszewski et al., 2012). dogs could be used for the detection of different kinds of neoplastic diseases, e.g. melanoma, lung, breast, prostate, or ovarian cancer (horvath, et al., 2008; pickel, et al., 2004; willis, et al., 2004; mcculloch, et al., 2006). sniffer dogs’ employment has some advantages when compared to contemporary analytical methods for the identification of vocs such as chromatography or mass spectrometry (gc/ms). in this research work, experiments which involved using trained dogs in order to detect odour markers of lung cancer in breath samples are presented. additionally, the results obtained by dogs recognition are compared with those obtained by the gc/ms. fig. 1. canine sense of smell (buszewski et al., 2012). 2. materials and methods 2.1 materials and reagents the gc-tof/ms analysis was performed on gas chromatograph 7890 a (agilent, waldbronn, germany) coupled with spectrometer trutof (leco, st. joseph, mi, usa) equipped with cp-porabond-q (varian inc., middelburg, the netherlands) 25 m × 0.25 m × 3 μm column. oven temperature program was as follows: initial 40 °c held for 2 min, then ramped at 10 °c/min to 140 °c and the ramped at 5 °c/min to 270 °c and held for 5 min. the temperature of the split-splitless injector was 200 °c. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:59 utc nova biotechnologica et chimica 13-1 (2014) 23 acquisition was performed at mass range m/z 30 – 300, acquisition rate 30 spectra/ sec. spectra were collected at electron ionization (ei) 70 ev, both ion source and line transfer temperatures were set to 200 °c. the acquisition of chromatographic data was performed by means of chroma tof software (leco). carboxen/polydimethylsiloxane coated fibre (supelco) was used for the spme method. all the chromatographic standards (aldehydes, alcohols, hydrocarbons and ketones) were purchased from sigma–aldrich (steinheim, germany). 2.2 breath collection breath samples were collected from 44 healthy volunteers and 29 patients with lung cancer (including 18 people with sclc and 11 with nsclc and volunteers). breath samples were collected in department of lung diseases, collegium medicum, toruń. the study was approved by the local ethic commission. in each patient’s case, a questionnaire on cancer and its stage was filled. data such as age, sex, other diseases, prescribed drugs, smoking habits, and information about previously consumed meal were also collected. the alveolar breath samples were collected by means of a co2 controlled sampler (department of anesthesiology and general intensive care, innsbruck medical university, innsbruck, austria) in a 1 l tedlar bags. before collection of breath, all bags were cleaned by flushing with argon gas and then filled with argon and heated at 60 °c for 12 h to remove any contaminations. afterwards, a 200 ml sample was transferred into second bag. the spme fibre was introduced into the bag through the septum and exposed to a sample. after 15 min. of extraction, the spme fibre was desorbed in the hot gc injector port for 2 min at 200 °c. ambient air samples were taken for blank measurement. 2.3 standard preparation a gaseous standard was prepared by injecting 1 or 3 μl of liquid compounds into 1 l glass bulb. the liquid was then evaporated. next, the mixture was subsequently diluted in tedlar bags filled with nitrogen to obtain concentration in the range 0.5 – 200 ppb. 2.4 experiments with the use of sniffing dogs in tests were used male dogs (german shepherd mix) that successfully underwent a three-phase training in the scent line-up were used for the lung cancer detection. the dogs were 20-22 months old. the experimental procedure and keeping conditions for the dogs were approved by the local ethical commission for animal experimentation in warsaw. for testing with the use of dogs, breath odour samples were collected from the same donors and at the same time as for the gc-ms analysis. the breath samples for the canine training and detection were taken by exhaling 2-3 times through disposable polypropylene sampling tubes (defencetek, pretoria, south africa) 15 cm long and 3 cm in diameter. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:59 utc 24 buszewski, b. et al. in order to test the samples, the removable insertions were taken out of the tubes and put into sterile polypropylene boxes covered with hole-punched lids to prevent a direct contact of a dog’s nose with the insertion, salivation, etc. one breath odour sample taken from patients with lung cancer was placed in a line-up together with four samples from healthy volunteers (controls). the dogs were taught to indicate the lung cancer sample by the sitting-down response in front of the sample. the trials with dogs were repeated approximately 30 times on different days. the positions of odour samples in the line-up were randomly changed for every trail. in order to prevent suggesting answers to the dogs during detection, the experimenter was invisible to the dog, and the dog handler was not aware of the cancer sample position in the line-up. 3. results and discussion 3.1 validation of the method the relative standard deviation (rsd) was in the range from 3.3 % to 9.5 % for hydrocarbons, alcohols, aldehydes, ketones and aromatic compounds. a calibration curve was linear for aliphatic hydrocarbons (pentane, hexane, 2-methylpentane, 3methylpentane) in the range 0.9-150.0 ppb, for alcohols (1-propanol, 2-propanol) 1.6 163.5 ppb, for aldehydes (butanal, propanal, 2-methylpropanal) 1.3 170.4 ppb, for ketones (acetone, 2-butanone, 2-pentanone) 1.3 166.4 ppb, for aromatic compounds (benzene, toluene, ethylbenzene, o-xylene) 1.00 165.61 ppb. regression coefficient values were high reaching at least 0.991. the lowest lod values obtained for hydrocarbons and aromatic compounds varied from 0.31 to 0.49 ppb. 3.2 exhaled air all the compounds detected in breath samples were compared to ambient air samples, and only those compounds showing concentration values at least 10 % higher than those in ambient air. the concentration of pentane, which is regarded to be an oxidative stress marker, ranged from 6.8 to 14.3 ppb in the case of healthy people and from 0.7 to 17.5 ppb in the case of patients with cancer. furan and its derivatives are considered to be smoking status markers. 1-propanol was observed in breath of patients with cancer at concentration values in the range 4.37 – 13.15. moreover, 2propanol was found in healthy and ill people’s breath samples. additionally, its concentrations in exhaled and ambient air were on a similar level. the original data from vocs measurement did not meet the normality assumption of parametric anova and even log transformation did not managed to produce a normal distribution for some compounds. therefore, the alternative nonparametric anova (kruskal–wallis test) was used to verify the null hypothesis assuming that three studied groups (cancer patients, control smokers, and control of non-smokers) came from the same population. this measures the probability that a random observation from one group is the same as a random observation from another group. the value of the χ2 test confirms the significance of an observation obtained in the kruskal–wallis test. for 12 substances the hypothesis of uniform concentrations of vocs in patients’ breath and in that of healthy controls could be rejected. for butanal, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:59 utc nova biotechnologica et chimica 13-1 (2014) 25 2-butanone, ethyl acetate, ethyl benzene, 2-pentanone, 1-propanol, and 2-propanol the tendency of greater concentration in the breath of cancer patients than in controls was found to be significant at p < 0.001. 3.3 dog experiments detection sensitivity and specificity were calculated by use of a yes/no response criterion toward each sniffed sample in the line-up, i.e. at 50 % probability of getting the correct response by chance. to evaluate differences in dogs’ indications between cancer and control samples, and the results obtained from particular dogs, the chi2 test was used. the dogs indicated correctly the pattern of breath samples from lung cancer patients with detection sensitivity and specificity of 82.2 % and 82.4 %, respectively. false positive indications toward healthy controls amounted to 17.8 % of trials. the differences between dogs’ indications of cancer samples vs. controls were highly significant (χ2 = 1056, p < 0.000). there were significant differences between dogs in detection sensitivity (χ2 = 25.17, d.f = 1, p < 0.001) but no significant differences in detection specificity. 3.4 correlation between a dog’s indications and chemical analysis the data concerning the dogs’ indications were analysed. we tried to find the correlation between the vocs in exhaled air and the dogs’ indications. two data sets, for control people (n = 49) and for patients (n = 29), contained the percentage of a dog’s indications and the chosen compounds. for the patients group, positive pearson’s correlations between the dogs’ indications and vocs content in breath air exhibited significant positive or negative tendency, e.g. ethyl acetate and 2-pentanone positively correlated with the dog’s indications positively (r = 0.85 and r =0.97, respectively), whereas for acetonitrile, propanal, and 1-propanol, the contents were negatively correlated with the dog’s indications (r = -0.78, r = -0.87 and r = -0.98 respectively). fig. 2. classification compounds by pca. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:59 utc 26 buszewski, b. et al. two multivariate methods, i.e. factor analysis (fa) and principal component analysis (pca), were applied for data calculation. data of each dimension were standardised within the individuals and, to obtain a meaningful structure of principal components, the number of factors has been finally limited to two. factor analysis with varimax rotation was carried out as a first approach to the datasets. these two factors explained over 80% of the total variation. factor 1 gave positive signs of loading coefficients for ethanol, isobutane, butane, isoprene, pentane, and benzene, whilst negative signs were apparent for carbon disulfide, 2-butanone and toluene. a principal component analysis (pca) was also used for the classification to show the relations between vocs and the dog’s indications towards two components (fig. 2). 4. conclusions trained dogs are able to discriminate breath samples of patients with diagnosed cancer disease from those of healthy donors at a „better than by chance” rate. the canine method has the following advantages: dog training and testing is relatively simple and inexpensive in comparison to analytical equipment application (gc/ms) and relatively high and easily interpretable detection sensitivity and specificity in relation to pattern samples were ascertained. acknowledgement: this work was supported by grant sensormed (2012-2015). references baker, r.r.: smoke generation inside a burning cigarette: modifying combustion to develop cigarettes that may be less hazardous to health. prog. energy combust. sci., 32, 2006, 373-385. buszewski, b., rudnicka, j., ligor, t., walczak, m., jezierski, t., amann, a., analytical and unconventional methods of cancer detection using odor. trac, 38, 2012, 1-12. collins, l.g., haines, c., perkel, r., enck, r.e.: lung cancer: diagnosis and mamagement. am. fam. physician, 75, 2006, 56-63. deng, c., zhang, j., xiaofeng, y., zhang, w., zhang, x.: determination of acetone in human breath by gas chromatography-mass spectrometry and solid phase microextraction with on fiber derivatization. j. chromatogr. b, 810, 2004, 269-275. horvath, g., jarverud, g.a., jarverud, s., horvath, i.: human ovarian carcinomas detected by specific odor. integr. cancer ther., 7, 2008, 76-80. hryniuk, a., ross b.m.: detection of acetone and isoprene in human breath using a combination of thermal desorption and selected ion flow tube mass spectrometry. int. j. mass spectrom., 285, 2009, 26-30. libardoni, m., stevens, p.t., waite, j., sacks, r.: analysis of human breath samples with a multi-bed sorption trap and comprehensive two-dimensional gas chromatography (gc×gc). j. chromatogr. b, 842, 2006, 13-21. mcculloch, m., jezierski, t., broffman, m., hubbard, a., turner, k., janecki, t., diagnostic accuracy of canine scent detection in earlyand latestage lung and breast cancers. integr. cancer ther., 5, 2006, 30-39. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:59 utc nova biotechnologica et chimica 13-1 (2014) 27 mcwilliams, a., lam, s.: new approaches to lung cancer prevention. curr. oncol. rep., 4, 2002, 487-494. pickel, d., manucy, g.p., walker, d.b., hall, s.b., walker, j.c.: evidence for canine olfactory detection of melanoma. appl. anim. behav. sci., 89, 2004, 107-116. prado, c., martin, p., periago, j.f.: application of solid phase microextraction and gas chromatography-mass spectrometry to the determination of volatile organic compounds in end-exhaled breath samples. j. chromatogr. a, 1011, 2003, 125-134. spanel, p., rolfe, p., rajan, b., smith, d.: the selected ion flow tube (sift)—a novel technique for biological monitoring. ann. occup. hyg., 40, 1996, 615–626. warneke, c., kuczynski, j., hansel, a., jordan, a., vogel, w., lindinger, w.: proton transfer reaction mass spectrometry (ptr-ms): propanol in human breath. int. j. mass spectrom. ion processes, 154, 1996, 61-70. willis, c.m., church, s.m., guest, c.m., cook, w.a., mccarthy, n., bransbury, a.j., church, m.r.t., church j.c.t.: olfactory detection of human bladder cancer by dogs: proof of principle study, brit. med. j., 329, 2004, 712-715. yao, s., ventura, d., petribois c.: analytical characterization of cell– asbestos fiber interactions in lung pathogenesis. anal. bioanal. chem., 397, 2010, 2079-2089. yu, h., xu l., wang, p.: solid phase microextraction for analysis of alkanes and aromatic hydrocarbons in human breath. j. chromatogr. b, 826, 2005, 69-74. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:59 utc microsoft word mocak 2012.doc nova biotechnologica et chimica 11-1 (2012) 11 doi 10.2478/v10296-012-0002-3 ©university of ss. cyril and methodius in trnava chemometrics in medicine and pharmacy ján mocák department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, nam. j. herdu 2, trnava, sk-917 01, slovakia (jan.mocak@ucm.sk) abstract: this minireview summarizes the basic ways of application of chemometrics in medicine and pharmacy. it brings a collection of applications of chemometric used for the solution of diverse practical problems, e.g. exploitation of biologically active species, effective use of biomarkers, advancement of clinical diagnosis, monitoring of the patient’s state and prediction of its perspectives, drug design or classification of toxic chemical substances. the aim of this contribution is a brief presentation of versatile potentialities of contemporary chemometrical techniques and relevant software. they are exemplified by typical cases from literature as well as by own research results of the chemometrics group at department of chemistry, the university of ss. cyril & methodius in trnava. key words: chemometrics, biological activity, diagnosis prediction, drug design, qspr, proteochemometrics 1. introduction chemometrics put to use statistics, applied mathematics and computer science to extract and manage chemical information, which brings benefits to many experimental life sciences at and even behind the borders of chemistry in medicine, pharmacy, biology, etc. it is a data-driven interdisciplinary science suitable for solving diverse applications. an important advantage of chemometrics and its methods is versatility, facilitated by its high level of abstraction, characteristic for the scientific disciplines extensively utilizing mathematics. when processing various data of everyday life, assembled in medicine, pharmacy, food control, environmental monitoring, etc., very similar algorithms and analogous ways of the data processing and evaluation are implemented for rather different investigated objects explored by means of miscellaneous techniques. by chemometrics, both descriptive as well as predictive issues of life sciences are solved. in descriptive applications, properties of the investigated systems are modeled in order to learn the underlying relationships and the system structure, which leads to the model identification, composition and understanding. in predictive applications, numerous system properties are utilized in the elaborated model with the intent of predicting the target properties, wanted features or behaviour of interest. in this study an overview of the main directions of chemometric applications in medicine and pharmacy is propounded. as a part of it, the results of our own research activities oriented to medicinal and pharmaceutical chemistry are added and its impact upon several practical problems is discussed. 2. materials and implemented software synthetic work is described in detail in the cited literature. the synthesis of the series of compounds, which exhibit considerable antimycobacterial activity, was bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:54 utc 12 mocák, j. performed at the collaborating faculty of pharmacy, charles university, hradec králové, czech republic. the series of various types of phenylcarbamic acid derivatives were prepared at department of pharmaceutical chemistry, faculty of pharmacy, comenius university, bratislava. all prepared compounds were characterized by their ir and nmr spectra and elemental analysis. molecular properties and the quantum chemistry descriptors of the investigated compounds were calculated by means of the contemporary commercial software packages acd/labs, chemdraw, hyperchem, dragon or directly on the vccl web site (virtual computational chemistry laboratory). determination of tumour markers in blood and pleural exudate was carried out in the institute of tuberculosis and respiratory diseases in poprad. determinations of cardiovascular markers, accomplishment of basic human biochemical tests, monitoring of the biochemical responses to the statine treatment and determination of glycated haemoglobin were performed at the collaborating analytical diagnostic laboratory, in prešov. the children’s hypertension data were assembled at the 2nd department of paediatrics, faculty of medicine, comenius university, bratislava. for the chemometric data processing and evaluation software packages statistica, spss, statgraphics and sas were employed. 3. domains of chemometrics applications 3.1 chemometric aids in medicine and pharmacy present automated laboratory instruments in biological/medical research produce a vast volume of measurement data, which are difficult to absorb and interpret. therefore a challenging task of powerful mathematical and statistical methods of chemometrics is to reduce them and reveal all useful information. an important role of chemometrics in medicine was known a long time ago, shortly after it was widespread among life sciences (boyd, 1986). there exist several areas of medicine as well as pharmacy where the aid of chemometrics is essential and indispensable: (a) quality control of laboratory results, laboratory measurement standardization. (b) decision about diagnosis, confirming or excluding a diagnosis. (c) optimal clinical interpretation of laboratory tests in disease monitoring. (d) detection of significant change in patient’s condition during medical treatment and clinical care, prediction of the future medical state of the patient. (e) drug synthesis, development and design, tools for drug discovery, structure-activity relationships, drug mechanism. (f) chemometrics in metabolomics, proteochemometrics a new bioinformatics technology. (g) identification of dopes and toxic substances; expert systems for toxicity classification. (h) prediction of bio-properties of various remedies, herbal medicine, and functional foods. we have searched the application possibilities in many of the above mentioned issues and tried to provide several typical case studies (mocak et al., 2011). nevertheless, a more systematic approach is described in the following sub-chapters. 3.2 quality control of laboratory tests, measurement standardization a common way of evaluating the biochemical state of “normality” or “abnormality” is based on the statistically derived reference intervals, which serve bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:54 utc nova biotechnologica et chimica 11-1 (2012) 13 as the standards by which the laboratory test results are judged. careful design of experimental protocol is the key in carrying out any evaluation of clinical diagnostic value (boyd, 1997). reference interval development has classically relied on concepts elaborated by the international federation of clinical chemistry expert panel on reference values during the 1980s. recent approaches to defining the reference values and decision limits are discussed in the papers (ceriotti et al., 2008; boyd, 2010). the obvious way of assessment of the diagnostic value of biochemical markers is by means of sensitivity, specificity, efficiency and predictive values, which have long been used as the indices of test accuracy (balla et al., 2004a). however, newer methods such as receiver operating characteristic curve (roc) analysis or logistic regression analysis are more robust indicators that overcome many limitations of the traditional indices. their successful implementation is discussed in further text. an important part of quality control is statistical comparison of agreement of laboratory tests. selection of an optimal measurement method is usually based on comparison of a newly developed procedure with the traditional one. in such a case both compared methods represent random variables as neither of them is error-free. consequently, the use of ordinary standard least squares regression method is not appropriate since the basic assumption about the error-free independent variable is violated, which may cause critical errors when using standard ways of regression (mocak et al., 2003). advanced regression modelling in such a case requires the use of advanced techniques like deming, orthogonal or passing-bablok regressions (islamčevič, 2004; mrazova et al., 2008a). first two techniques consider the specific variance value of each variable (their ratio is assumed unity in orthogonal regression), the third one is a non-metric, robust, rank dependent technique, nowadays frequently used in medicinal chemistry. the application of the mentioned advanced regression techniques was applied to ensure correct measurements of glycated haemoglobin in blood. it is an important task since glycohaemoglobin hba1c is the best marker of the glycemic state in patients with diabetes and enables its long-term assessment (mrazova et al., 2008b). for measurement of glycated haemoglobin in blood two automatic analyzers were used: advia 1200 (chemistry systems bayer) based on the ngsp (u.s. national glycohaemoglobin standardization program) calibration assay and hitachi 912 (roche diagnostic systems) calibrated according to the ifcc (the international federation of clinical chemistry working group) reference method. a thorough chemometrical data treatment by deming, orthogonal and passing-bablok regressions revealed (mrazova et al., 2010a) that the ngsp reference system gives systematically higher results of glycated haemoglobin than the ifcc reference system. if the recalculation of the ifcc into ngsp values is made via master equation, the regression intercept is adjusted to zero as expected, but the resulting straight-line deviates by the slope. in conclusion, further harmonisation of the two reference systems is demanded and more experimental data from the accredited laboratories are needed (mrazova et al., 2010b). similar advanced regression approach was successfully applied to determination of low-density lipoprotein cholesterol (ldlc), which serves as a marker bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:54 utc 14 mocák, j. for hypertension and progression of cardiovascular diseases (islamčevič, 2004). theoretical background of all used regression methods is there described. 3.3 medical diagnosis confirmation or prediction, assessment of effectiveness of laboratory tests advanced implementation of chemometric and statistical algorithms facilitates clarifying of many important practical applications in medicine. it was found that an appropriate exploitation of multivariate statistics, e.g. the outputs of principal component analysis, techniques of discriminant analysis, logistic regression, together with the standard statistical tools like correlation analysis, analysis of variance (anova) as well as the roc curves (mocak et al., 2005; balla et al., 2004a; balla and mocak, 2008), may enable a better medical diagnosis confirmation and/or prediction. the area under the roc curve is the best global indicator of the test accuracy (boyd, 1997). logistic regression analysis allows the diagnostic information from several tests to be evaluated simultaneously. in addition, it provides a straightforward method for calculating likelihood ratios. likelihood ratios are useful for interpreting the test results in the individual patient because they provide a convenient means to directly determine predictive value without having to calculate sensitivity and specificity for a given decision limit. it is common that the physician used to evaluate all performed laboratory tests sequentially and then it depends on his/her knowledge how these results are composed into final decision about diagnosis. on the contrary, our new approach is based on a simultaneous use of several selected laboratory tests (balla et al., 2004b). utilization of biochemical markers as the chemometrical descriptors and their optimal combination into a newly calculated multicomponent marker should provide an optimally weighted collective diagnostic information. this effective new approach was applied in the treatment of cardiovascular diseases (balla et al., 2004b; mocak et al., 2005). in this case, with regard to diagnosis the most effective were the multicomponent markers composed of eight individual cardiovascular markers directly measured in the clinical laboratory. as manifested by the roc curve areas, the best combination was provided by logit the output of logistic regression, the second best was the first discriminant function (as the most informative output of discriminant analysis), linearly combined from all eight individual markers. according to the roc curve area, the composed multicomponent markers exhibited the best diagnostic effectiveness also in diagnosing lung malignity, where cea and cyfra tumour markers in the blood as well as in pleural exudate were originally measured (mocak and balla, 2003; kavkova et al., 2007; mrazova et al., 2008c; balla and mocak, 2008). in this case the differentiation from benign pulmonary diseases is highly demanded (kavkova et al., 2007). again, the best muticomponent was logit, followed by the first principal component (the most informative output of principal component analysis) and first discriminant function; the roc curve area for cea in exudate, found as the best individual marker, was significantly smaller (mrazova et al., 2009; mocak, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:54 utc nova biotechnologica et chimica 11-1 (2012) 15 2010a). we have confirmed that the calculation of the composed multicomponent marker from the originally measured individual markers is simple and can be easily implemented into the hospital information system as a new effective diagnostic tool. 3.4 monitoring of the health state of the patients further task of chemometrics is its assistance in detecting the changes in patient’s condition concerning progress of the disease and reaction to the medical treatment. another target of chemometrics is determination of patient prognosis prediction of the future medical state of a patient on the basis of present laboratory test results and clinical care. several typical examples are shortly discussed in the next paragraphs. a complex monitoring of the health state of the patients during a long term treatment by some drugs involves simultaneous evaluation of numerous biochemical tests. a good example of chemometrical processing of the laboratory test results was described in (durcekova et al., 2009a) where the state of the probands before and one year after statin administration was studied. statins are effective inhibitors of 3-hydroxy-3-methylglutaryl coenzyme a reductase and represent a major advance in prevention and treatment of cardiovascular diseases. their benefit is caused by the lipids suppression. on the other hand, it is important to find whether the hypolipidemic effect of statins is not accompanied by the unfavourable impact on some human body organ. before, during and after the statin administration the level of many biochemical tests is therefore determined and statistical evaluation of the changes is performed. the results obtained in (durcekova et al., 2009b; 2011) unambiguously confirmed the positive changes in the lipid metabolism of the patients with cardiovascular risk. the multivariate data processing techniques allow clear visualisation of the statin administration effect. roc curves, box-plots and discriminant analysis exhibit a distinct discrimination of the patients’ samples into two categories before and one year after the statin treatment. analysis of variance reveals that four biochemical tests are capable to differentiate the statin treatment also with regard to the patient gender: total cholesterol, low-density lipoprotein cholesterol, triacylglycerols and aterogenity index. a substantial outcome of the achieved results is a very high diagnostic effectiveness of three composed multicomponent variables, calculated by linear combination of individual laboratory tests. changes in the kidney and hepatic test values caused by the statin uptake were found not statistically significant (durcekova et al., 2011). hypertension is not only a serious disease of adults, but as demonstrated in recent years, it is now increasingly encountered in children. the progress of this latent disease leads to damage and subsequent failure of vital organs. diagnosis of hypertension in children is very difficult. the problems with the blood pressure measurement require a long-term monitoring of the state of the patient via biochemical tests leading the physician to diagnose. chemometrical approach evaluates the impact of all risk factors; the applied multidimensional classification techniques (artificial neural networks, kth nearest neighbour and general discriminant analysis) enable quantification of the hypertension risk in children (mrazova et al., 2012). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:54 utc 16 mocák, j. another interesting study involves a combination of hplc and chemometrics data evaluation concerning determination of morphine and its two main metabolites, morphine-3-glucuronide (m3g) and morphine-6-glucuronide (m6g), in the serum of oncological patients (netriova et al., 2006). the results are important for appropriate analgesic treatment of the patients under palliative care. in the mentioned work it was clearly manifested that a higher m3g/m6g ratio is reflected in a stronger patient’s dissatisfaction with the morphine treatment. it was found that the morphine and morphine-3-glucuronide concentrations depend on the patient’s gender, with the men values higher than the corresponding women ones. since the morphine-6-glucuronide serum concentration is about the same for men and women, the m3g/m6g ratio is in general higher for men than for women. in addition to the specific role of the patient gender it was also found a remarkable influence of the tumour location on the serum concentration of morphine and its glucuronide metabolites, and consequently the m3g/m6g concentration ratio. 3.5 drug design, structure-activity and structure-property relationships multivariate chemometric models can be used also for prediction of bio-properties from chemical data obtained either from various instrumental measurements or by calculation of the descriptors derived from the molecular structure. drug development (drews, 2000) is a tedious and resource demanding process consisting of several steps. chemometrics plays important role especially in the starting step – in the search of the compounds with a strong activity against the originators of diseases. in the last decades the qsar (quantitative structure – activity relationships) became an important tool in drug discovery and in toxicology. the goal of qsar is investigation how to predict the biological activity of a set of compounds in demand and elucidation of the specific action of the administrated drug; it deals with generalization of relations between the chemical and biological properties of the examined set of chemically resembling derivatives. there exist numerous publications devoted to this attractive topic, two recent books were published by (puzyn et al., 2010; merz et al., 2010). some examples of qsar studies are discussed in the next paragraph. in the second revised edition of the book (todeschini and consonni, 2009a; 2009b) the entire relevant literature over the previous six years was surveyed. volume i contains an alphabetical listing of more than 3300 descriptors and related terms for chemoinformatic analysis of chemical compound properties, while the second volume lists over 6000 references selected from 450 journals. with regard to high antimycobacterial activity, important mainly in investigation of new strongly active antituberculotic drugs, we have explored qsar of two sets of compounds: (1) n-benzylsalicylamide derivatives (nemecek et al., 2006; 2009) and 3-phenyl-2h-1,3-benzoxazin-2,4(3h)-dione (nemecek et al., 2012). crucial for a successful prediction of biological activity of the studied set of compounds is reduction of the descriptor number and finding an optimal set of descriptors, which is due to their mutual dependence not a simple task. for this purpose several tools are customarily used: (a) the correlation analysis applied to all descriptors, (b) sensitivity bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:54 utc nova biotechnologica et chimica 11-1 (2012) 17 analysis, or (c) in the recent time most popular genetic algorithms. after selection the descriptors with the strongest correlation to biological activity (the activity against the investigated strains of mycobacteria), the structures of new derivatives with the strongest effect were found with the predicted lowest minimal inhibition concentration (mic). since a part of the mentioned studies was performed exclusively with the calculated molecular descriptors and the simulated 1h and 13c nmr signals, the mic prediction was possible without the need of a previous synthesis. in this way, the collection of the best structures represents effective and economical way of the first step in drug design. thank to the development of chemoinformatics, public availability of bioactivity databases (like pubchem, chem-bl, drugbank, etc.) and web-based tools for drug discovery and toxicology assessment, several open access web sites are available for effective qsar calculations (http://www.vvclab.org, http://www.ochem.eu, http://www.opentox.org, http://www.collaborativedrug.com, http://qsardb.org, http://qsardb.jrc.it, http://chembench.mml.unc.edu, http://pharmaexpert.ru, etc.). molecular structure is often related to further properties in addition to biological ones and the area of interest in qsar modelling has been expanded from activity towards properties. qspr (quantitative structure – property relationships) connects the knowledge from biology, chemistry, physics, mathematics, and computer sciences with the aim of exploring properties of substances and processes. the novel characteristics of organic, metal organic and coordination compounds can be calculated by qspr and applied in practice. qspr is applicable to a vast number of different properties. the calculation procedures utilizable in these cases are similar to those known in qsar. the qspr methodology is based at first on finding the correlations between a desired property already experimentally measured for a series of molecules and then using these correlations to predict the same property for additional molecules, including the compounds that have not yet been synthesized. even for these calculations the open access web systems are available (e.g. http://www.qsprthesaurus.eu). when chromatographic properties are concerned, instead of qspr the specific term qsrr (quantitative structure – retention relationships) is used. at present, the qsrr studies are more and more widespread since they contribute to a deeper understanding the relations between the structure of the chromatographically separated species and the stationary and mobile phases (kaliszan, 1997; kaliszan 2007; héberger, 2007). in our qsrr (quantitative structure – retention relationships) studies the hplc retention factor was used as the target variable. retention factor is in general very closely related to lipophilicity, which seems to be the descriptor most influencing biological activity. a very successful prediction of the pbod retention factors were published in (nemecek et al., 2010a; 2010b). successful qsrr model was recently elucidated also for esters of alkoxyphenylcarbamic acid (durcekova et al., 2010a; 2012), which exhibit a strong anaesthetical activity. details about the factors most influencing the surface anaesthetical activity and infiltration anaesthetical activity are described in (durcekova et al., 2010b). for prediction of anaesthetical activity the artificial neural network models were employed; further properties bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:54 utc 18 mocák, j. of the investigated set of compounds were well characterized by principal component analysis, cluster analysis and discrimination analysis. a unique use of the whole chromatogram instead of individual peaks for fingerprinting of various phytochemicals was described by (komsta, 2012). the chromatographic fingerprinting technique is essentially a kind of high-throughput and integral tools to explore complex and multivariate data. it was used e.g. to investigate the complexity of herbal medicines where confirmation or identification the most important chemical components is needed (liang et al., 2006; ni et al., 2007); further seven similar papers are cited in the review of (komsta, 2012). a special approach to calculate new precise values of many important physicochemical properties for a homologous series of organic compounds is based on mathematical recurrence relations, which can be easily computed even in a common spreadsheet, e.g. microsoft excel (mocak and rabarova, 2010b). 3.6 chemometrics in metabolomics metabolomics concerns the systematic study of the unique chemical fingerprints that specific cellular processes leave behind the study of their small-molecule metabolite profiles. metlin metabolomics web database contained in 2001 over 40,000 metabolites. it is connected mainly to the development of gas chromatography mass spectrometry techniques; another crucial instrumental technique in metabolomics is high-resolution nmr spectroscopy. recent very sensitive 1h nmr metabolomic studies became to be widespread in analytical chemistry, e.g. in papers (sands, et al., 2009; alves et al., 2009; robinette et al., 2009; pearce et al., 2009; maher et al., 2008; cloarec et al., 2005). gc/ms and nmr techniques together with multivariate chemometrics are capable to characterize the metabolic profiles of biofluids, understand the mechanism of patogenesis and uncover potential biomarkers of disease progression. chemometrics constitutes an integral part of the metabolomic technology by providing organized multivariate statistical approaches for modelling and interpreting changes in the metabolic profiles (bruce et al., 2008; madsen et al., 2010; eliasson et al., 2011). combinations of „omics“ investigations (transcriptomic, proteomic, metabolomic) are increasingly applied to get comprehensive understanding of biological systems. they allow a more holistic approach to understanding the complexity of human biochemistry and biomolecular medicine. proteochemometrics is a bioinformatic approach to protein engineering, novel drug design, target analysis and functional genomics analysis (lapinsh et al., 2001; wikberg 2004). proteochemometrics combines chemical organic synthesis and design with molecular biological molecular pharmacological analysis and with bioinformatics/mathematical modeling. using proteochemometrics the detailed maps can be built, down to the physico-chemical and structural level, exhibiting the interactions of organic molecules with macromolecules in a cell and these maps may be directly used for the design of new molecules with desired properties. many further useful details can be found on the web site of uppsala university (http://www.proteochemometrics.org). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:54 utc nova biotechnologica et chimica 11-1 (2012) 19 3.7 toxicity assessment and prediction in the last two decades a great deal of effort has been put into the study of the relationships between a compound’s structure and its toxicity (cronin et al., 2003; gallegos, 2006). attempts have been made to classify chemicals according to the mechanism of their toxicity and to screen them for their environmental risk assessment. most of the qsar applications to toxicities have been developed for congeneric sets, but non-congeneric sets of compounds have also been documented (cronin and dearden, 1995a; 1995b; 1995c; 1995d). it is necessary to note that many qsar toxicity applications have been called qstr (quantitative structure – toxicity relationships). numerous calculated descriptors and recommended software packages are discussed e.g. in (katritzky and tatham, 2001). the european policy for evaluation of chemicals has lead to a controversy with regard to the need of additional animal safety testing. in order to save time and resources, alternative in vitro or in silico tests for the assessment of toxic effects of chemicals were suggested. in toxicology, besides in vitro models the computerassisted prediction models are used as predictive tools especially to elucidate acute, chronic and/or organ toxicities (simon-hettich and steger-hartmann, 2006). computer-assisted prediction tools play an important role mainly in the assessment of chemicals for which no data is available or for which toxicological testing is impractical due to the lack of availability of sufficient compounds for testing (e.g. impurities in pharmaceuticals). eu and oecd have produced several documents concerning toxicity prediction, e.g. (worth et al., 2005; vračko et al., 2006). there exist many applications concerning toxic substances where chemometrics plays an important explaining role, e.g. in expert systems for toxicity classification, in identification of drugs and toxic substances in complex mixtures and in forensic chemistry (garkani-nejad, 2004). in analytical procedures dealing with the analytes containing toxic components very important are also the rules of good laboratory practice and exact evaluation of the measurement results according to metrological principles. two examples of quality assurance application are in the papers of borošová et al. (2001; 2002). 3.8 further chemometrics applications another area demanding prediction of bio-properties from chemical data is the study of bioactive compounds in food. the presence of specific molecules which have a positive pharmacological effect when consumed in sufficient quantities represents the basic benefit of functional food (mcgoverin et al., 2010). functional foods are foods or dietary ingredients that provide a health benefit beyond basic nutrition. however, the legal framework within the european union now demands that any claim about the nutritional or physiological effects of a product must be scientifically demonstrated. an example of chemometric study of metabolomics in functional food is described in (lundstedt et al., 2010) where a combined intake of soybean and grapefruit was analyzed with respect to both pharmacological and physiological effects. resulting multivariate models showed that this diet induced bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:54 utc 20 mocák, j. decrease of lactate, cholesterols and triglycerides; further biotransformation details are there also discussed. food commodities are the objects of so many chemometrics application that they cannot be included here and will be described and discussed in another review. another special attention should be paid to the details of data processing like data pre-treatment, normalization, denoising, baseline removal, warping, then to the proper choice and combination of chemometrics tools and implementation of pertinent software. despite of their importance they are outside the scope of this paper. nevertheless, at least one recent excellent book should be mentioned in this connection (brereton, 2007) where not only older as well as newest techniques of chemometrics are clarified but chapter 10 is directly devoted to biological and medical applications of chemometrics. 4. conclusions chemometrical processing of the experimentally measured data and/or the use of diverse calculated molecular descriptors represents at present a substantial aid to medicine and pharmacy. chemometric methods and specialized software packages enable (a) quality control of laboratory tests and standardization of measurements in laboratory medicine, (b) assessment of effectiveness of laboratory tests used for confirmation as well as prediction of clinical diagnosis, (c) long-term monitoring of the disease, success or failure in medical treatment and assessment of effectiveness of the applied drugs, (d) targeted design of new potentially high effective drugs using structure – activity relationships, (e) prediction of desired property of the investigated group of substances by qspr studies, (f) deeper insight into chemical processes involving metabolites, which can be utilized in deeper knowledge of the disease but also in more effective drug design, (g) elucidation of toxicity of chemical substances related to their structure and molecular properties, (h) prediction of bio-properties from chemical data in food. progressive chemometric tools exhibit a notable role in advancement of theoretical cognition in all mentioned fields. acknowledgement: this study is supported by the grant vega 1/0233/12. collaboration of all co-authors of the cited own publications is highly acknowledged. references alves, a.c., rantalainen, m., holmes, e., nicholson, j.k., ebbels, t.m.: analytic properties of statistical total correlation spectroscopy based information recovery in 1h nmr metabolic data sets. anal. chem., 81, 2009, 2075-2084. balla, b., mocak, j., varmusova, e., kavkova, d., tudik, i.: assessment of effectivity of laboratory methods (in slovak). chem. listy, 97, 2004a, 333-338. balla, b., mocak, j., pivovarnikova, h., balla, j.: comparative study of cardiovascular markers data by various techniques of multivariate analysis. chemom. intell. lab. systems, 72, 2004b, 259-267. bereitgestellt von slovenská poľnohospodárska knižnica | 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(2012) 25 todeschini, r., consonni, v.: molecular descriptors for chemoinformatics: vol. i: alphabetical listing. 2nd ed., wiley-vch, weinheim, 2009. todeschini, r., consonni, v.: molecular descriptors for chemoinformatics: vol. ii: appendices, references. 2nd ed., wiley-vch, weinheim, 2009. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:54 utc << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (iso coated) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.3 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /perceptual /detectblends true /detectcurves 0.1000 /colorconversionstrategy /srgb /dothumbnails true /embedallfonts true /embedopentype false 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can be opened with acrobat and adobe reader 5.0 and later.) >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [2834.650 2834.650] >> setpagedevice microsoft word hrasna_et_al 2012.doc nova biotechnologica et chimica 11-1 (2012) 73 doi 10.2478/v10296-012-0008-x ©university of ss. cyril and methodius in trnava synthesis, complex compounds and antimicrobial activity of some derivatives of furo[3,2-c]pyridine and their starting compounds martin hrašna1, eva ürgeová2, alžbeta krutošíková1 1department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (alzbeta.krutosikova@ucm.sk) 2department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic abstract: some [3,2-c]pyridine derivatives were synthesized. 3-(furan-2-yl)propenoic acid (1a) was prepared from furan-2-carbaldehyde under the perkin’s conditions. obtained acid was converted to the corresponding azide 3, which in turn was cyclized to give furo[3,2-c]pyridin-4(5h)-one (4a). the reaction of pyridone 4a with phosphorus oxychloride rendered the chloroderivative 7a, which was treated in the condition of suzuki coupling reaction with boronic acid to give 4-phenylfuro[3,2-c]pyridine (8e) and an unexpected product 10. some title compounds have shown moderate to good antimicrobial activity against tested bacteria xanthomonas sp., erwinia amylovora, and filamentous fungi pyrenophora avenae, fusarium graminearum. keywords: furo[3,2-c]pyridines, nucleophilic substitution, coupling reaction, 1h and 13c nmr spectra, biological activity 1. introduction compounds with 2-trifluoromethyl group in molecule have interest biological activity. for example 2-(trifluoromethyl)benzimidazoles are known as an important class of compounds due to their wide range of biological activity acting as antiviral, antifungal, antibacterial and anticancer drugs (navarrete-vázquez et al., 2006). studies about the antiparasitic activity of 2-(trifluoromethyl)benzimidazole derivatives have shown high potential as antiprotozoal agents (navarretevázquez et al., 2001). the main features of these compounds are the application of the 2-trifluoromethyl group in order to enhance solubility and absorption properties and therefore antiparasitic activity (navarrete-vázquez et al., 2003). some 4substituted furo[3,2-b]pyrrole-5-carbohydrazides bearing the 3(trifluoromethyl)phenyl group at the c-2 position were prepared and their effect on the chlorophyll content in alga suspensions of chlorella vulgaris and the inhibition of photosynthetic electron transport in spinach chloroplasts were studied (gašparová et al., 2008). a variety of fused pyridines have been studied for a long time in the field of the chemistry of heterocyclic compounds (sherman 1996; 2008). furopyridines are very similar to such skeletons as quinoline and isoquinoline which are present in many bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:17 utc 74 hrašna, m. et al. compounds possessing biological activity. it was reported that some pharmacophores with potential antipsychotic activity contain the thienoand furo[3,2-c]pyridine ring systems (new et al., 1989). by studying (kennis et al., 2000) of biological activity of tetrahydrobenzofuropyridines and benzothienopyridines was determined that these two groups of compounds are a part of compounds which show a high affinity in face of subtypes of receptors α1 and α2. this fact incites chemists about more complete explication of the structure and functions of these systems. biological activity of the copper(ii) and cobalt(ii) 3-methylsufanylnicotinate complexes with furopyridines against various strains of bacteria and filamentous fungi has been investigated (segľa et al., 2008; 2009). for a long time we have been interested in studying of the synthesis and reactivity of various furo[3,2-c]pyridines (bobošík et al., 1995; krutošíková and sleziak 1996; mojumdar et al., 2005; 2009; gajdoš et al., 2006; búdová et al., 2006; bradiaková et al., 2008; 2009; tarabová et al., 2010). this type of the fused heterocycles can be readily coordinated to metal centers through n-donor atom. a few from these compounds were used as ligands in the preparation of coordination compounds with transition metals cu(ii), co(ii) and ni(ii). the spectral, magnetic, thermal properties, coordination chemistry and x-ray analysis of these compounds have already been outlined (krutošíková et al., 2001; miklovič et al., 2004; baran et al., 2005; titiš et al., 2007; vrábel et al., 2007a; 2007b; 2007c; boča and titiš 2008). in the past were published synthesis 2-methyl and 2-aryl substituted furo[3,2c]pyridine-4(5h)-thiones by reaction of corresponding 2-substituted furo[3,2c]pyridine-4(5h)-ones with phosphorus pentasulfide (bobošík et al., 1995; krutošíková and sleziak 1996). methylation of the 2-methylfuro[3,2c]pyridine-4(5h)-thione and 1-benzofuro[3,2-c]pyridine-1(2h)-thione with methyl iodide afforded 4-methylsufanylfuro[3,2-c]pyridine or 1-methylsulfanyl-1benzofuro[3,2-c]pyridine but methylation corresponding pyridones gave n-methyl compounds (bobošík et al., 1995; tarabová et al., 2010). the 4-substituted furo[3,2-c]pyridines were prepared by various methods. one method used as the starting compounds of the 4-chlorofuro[3,2-c]pyridines in which the chlorine atom was substituted with several nucleophiles (bobošík et al., 1995; búdová et al., 2006; bradiaková et al., 2009). the reaction with sodium alkoxides in appropriate alcohol afforded the corresponding 4-alkoxyfuro[3,2c]pyridines (búdová et al., 2006). similarly, utilization of sodium methyltiolate gave methylsufanyl derivatives (bobošík et al., 1995). chloro derivatives were converted to 4-amino-substituted furo[3,2-c]pyridines by nucleophilic substitution chlorine atom with some heterocyclic secondary amines as piperidine, morpholine or pyrrolidine (búdová et al., 2006). the nucleophilic reactions of 1chloro[1]benzofuro[3,2-c]pyridine with secondary heterocyclic amines proceeded analogously (mojumdar et al., 2009; tarabová et al., 2010). suzuki coupling reaction was realized with 1-chloro[1]benzofuro[3,2-c]pyridine and phenylboronic acid or pyridin-3-ylboronic acid in the presence of pd(pph3)4 catalyst in dichloromethane 1-phenyl[1]benzofuro[3,2-c]pyridine and 1-(pyridin-3yl)[1]benzofuro[3,2-c]pyridine were formed (tarabová et al., 2010). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:17 utc nova biotechnologica et chimica 11-1 (2012) 75 in the paper (bencková and krutošíková 1999, mojumdar et al., 2009) are described the 4-cyanofuro[3,2-c]pyridine and 1-cyano[1]benzofuro[3,2c]pyridine derivatives via n-oxides by reissert-henze reaction. the alkaline hydrolysis of the cyano derivatives raised the corresponding furo[3,2c]pyridinecarboxylic acids and their amides were prepared by hydrolysis in acid conditions. the aim of this work was to study the reaction of 4-chlorofuro[3,2-c]pyridine with boronic acid in the condition of suzuki coupling reaction. the main target of this work were syntheses of the earlier known derivatives of furo[3,2-c]pyridine and their starting compounds for testing their antimicrobial activities. 2. material and methods 2.1 chemical, physical methods and instruments melting points were determined using kofler hot plate. all solvents were distilled and dried before use. all reagents were commercially available and were used without purification. elemental analyses were determined using an eager 300 at institute of inorganic chemistry, technology and materials, stu in bratislava. ir spectra were taken on a ftir nicolet nexus 470 spectrophotometer using kbr pellets (0.5 mg in 300 mg kbr) in region 4000 – 400 cm-1 at institute of physical chemistry and chemical physics, stu in bratislava. for interpretation of ir spectra following abbreviations are used s = strong band (a value of transmittance: 0-35%), m = medium band (a value of transmittance: 36-50%) w = weak (a value of transmittance: over 50%). 1h nmr spectra were measured in dmso-d6 using varian inova 600 (for 1h 599.782 mhz and for 13c 150.830 mhz) spectrometer, at 25 °c, at institute of analytical chemistry, department of nmr and ms spectroscopy, stu in bratislava. chemical shifts (δ-scale) are quoted in parts per million and following abbreviations are used: s = singlet; d = doublet; coupling constants (j) are given in hz. 2.2 phytopathogene microorganism in our work phytopathogene bacteria xanthomonas sp. ccm 2888 from czech collection of microorganism of masaryk university brno and erwinia amylovora cppb a203 obtained from the collection of phytopathogenic bacteria and referential antidotes (cppb) at the crop research institute in prague-ruzyně, czech republic were used. bacteria were kept as a stock culture in the refrigerator at the temperature of 8 °c. isolates of the filamentous fungi pyrenophora avenae, fusarium graminearum were provided by the institute of the plants production from piešťany as pure cultures. obtained isolates were reinoculated, and stored at temperature 6 ± 2 °c. 2.3 testing the biocide effect of synthesized compounds the antimicrobial activity of synthesized compounds was determined in vitro against a variety of phytopathogenic microorganism. antimicrobial activities were bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:17 utc 76 hrašna, m. et al. tested by the standard plate diffusion method (piddock, 1989) and zones of inhibition were measured in mm. the biocide effect was compared with the effect of 1.2 % w/v solution of tmtd (tetramethylthiuram disulfide), active substance of commercial pesticides. the solution of compounds (15 μl) in dmso (respectively methanol) with concentration 200, 100, 50 and 25 mg.l-1 was placed to metal cylinders on the surface of a media inoculated with the tested microorganism. the plates were incubated at 25 °c, and clear zones developed around cylinders indicated the inhibition of microbial growth. the size of the zone of inhibition caused by diffusion of agent into agar is directly related to the degree of susceptibility of an organism. if the tested sample shows microbicidal activity, inhibition zone will appear on agar plate. tetramethyl tiuram disulfide (tmtd) (1.2% w/w methanol solution) was used as a standard of antimicrobial activity. tested plates were inoculated with 1 ml of microbial suspension (106–107 cfu.ml-1). sterile cylinders were placed onto the plates and filled by 15 μl of the solution (dmso, methanol respectively) of the tested compounds. plates were incubated at 25 °c for 24 h and 4 d, respectively. the zone inhibition diameter (in millimeters) was recorded. two parallel tests of the compounds inhibition activity were made. 3. results and discussion 3.1 chemistry 3-(furan-2-yl)propenoic acid (1a) was prepared from furan-2-carbaldehyde under the perkin’s conditions. the acid 1a was converted to the corresponding azide 3a, which was cyclized by heating in diphenyl ether to furo[3,2-c]pyridine-4(5h)-one (4a). the compound 4a was aromatized with phosphorus oxychloride to chloroderivative 7a which was treated in the condition of suzuki coupling reaction with boronic acid to give 4-phenylfuro[3,2-c]pyridine (8e) and an unexpected product 4-(furo[3,2c]pyridine-4-yl)furo[3,2-c]pyridine (10) (fig. 1). the acids 1b-1d were synthesized by condensation of the appropriate carbaldehyde with malonic acid under knoevenagel conditions. the compounds 4b-4f were prepared analogously by cyclisation of appropriated azides (sleziak and krutošíková, 1996; gajdoš et al., 2006; mojumdar et al., 2009) reaction of 4b and 4d with phosphorus pentasulfide led to corresponding thiones 5a, 5b, which were methylated in ptc conditions giving 6a and 6b (bradiaková et al., 2009; tarabová et al., 2010). 2methyl[1]benzofuro[3,2-c]pyridine-1-one (4e) and 5-metyl-2-[3-(trifluoromethyl)phenyl]furo[3,2-c]pyridine-4(5h)-one (4f) were obtained by reaction of 4b, 4d with nah and then methylated with methyl iodide. the compound 4a was aromatized with phosphorus oxychloride to chloroderivative 7a. analogously were synthesized chloroderivatives 7b-7d and they are described in ref. (sleziak and krutošíková, 1996; bradiaková et al., 2008; gajdoš et al., 2006) refluxing of appropriate chloroderivatives 7b and 7d with secondary heterocyclic amines gave 8a-8d (bradiaková et al., 2009; mojumdar et al., 2009). the compounds 8f-8j were prepared according (tarabová et al., 2010; bradiaková et al., 2009; bencková and krutošíková 1999). n-oxides 9a, 9b were prepared as it is described in (bradiaková et al., 2009). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:17 utc nova biotechnologica et chimica 11-1 (2012) 77 fig. 1. synthesis and reaction of 4-chlorofuro[3,2-c]pyridine. 4-phenylfuro[3,2-c]pyridine (8e). the mixture of 4-chlorofuro[3,2-c]pyridine (7a) (0.767 g; 5 mmol), phenyl boronic acid (0.945 g; 8 mmol), water solution of sodium carbonate (2 m, 7.5 ml) and pd(pph3)4 (0.345 g, 0.3 mmol) in 1.2dimethoxyethane (9 ml) was heated at 80 °c for 6 h. then the reaction mixture was poured into mixture dichloromethane and ice water (1:1). the separate organic layer was washed with water and brine, dried with magnesium sulfate, the solvent was evaporated in vacuum. then from the residue the product 8e after purification by column chromatography was isolated (silica gel, eluted with chcl3). yield 0.2 g, 20.5%, white crystals, m.p. 99-100 °c (methanol); rf = 0.2 (chcl3). anal. calcd. for c13h9no: c, 79.98; h, 4.65; n, 7.17. found: c, 80.31; h, 4.55; n, 7.23%. 1h nmr (dmso-d6), δ: 8.56 (d, 1h, 3j(7,6) = 5.8 hz, h-7), 8.19 (d, 1h, 3j(2,3) = 1.77 hz, h-2), 8.01 (d, 2h, 3j(2´,3´) = 7.8 hz, h-2´, h-6´), 7.67 (d 1h, h-6), 7.55 (dd, 2h, h-3´, h-5´), 7.49 (d 2h, 3j(4´,3´) = 7.2 hz, h-4´), 7.32 (d, 1h, h-3). 13c nmr (dmso-d6), δ: 159.75 (c-7a), 151.88 (c-4), 147.2 (c-2), 144.15 (c-6), 138.61 (c-1´), 128.98 (c-4´), 128.69 (c-3´, c-5´), 128.19 (c-2´, c-6´), 121.44 (c-4´), 106.12 (c-7), 105.58 (c-3). ir / cm-1: 3088s; 3038s; 1602s; 1495m; 1412s; 1260s; 1112m; 1056s; 992w; 818s; 702s; 686m. 4-(furo[3,2-c]pyridin-4-yl)furo[3,2-c]pyridine (10). yield 12.7%, m.p. 160-165 °c (methanol). anal. calcd. for c13h9no: c, 71.18; h, 3.41; n, 11.86. found: c, 71.13; h, 3.78; n, 11.48%. 1h nmr (dmso-d6), δ: 8.68 (d, 2h, 3j(6,7) = 6.0 hz, h-6, h-6´), 8.21 (d, 2h, 3j(2,3) = 2.4 hz, h-2, h-2´), 7.86 (dd 2h, 3j(3,2) = 2.4 hz, 5j(3,7) = 0.6 hz, h-3, h-3´), 7.78 (dd 2h, 3j(7,6) = 6.0 hz, 5j(7,3) = 0.6 hz, h-7, h-7´). 13c nmr (dmso-d6), δ: 160.0 (c-7a, c-7´a), 150.5 (c-4, c-4´), 147.2 (c-2, c-2´), 143.7 (c-6, c-6´), 122.8 (c-3a, c-3´a), 107.9 (c-3, c-3´), 107.4 (c-7, c-7´). ir/ (cm-1): 3472m; 3453m; 3173w; 3164w; 3140m; 1597m; 1567w; 1532w; 1443m; 1400m; 1317w; 1299w; 1263w; 1194w; 1160w; 1121w; 1111w; 1043w; 1013s; 903w; 879m; 816m; 794w; 783m; 749s; 625w; 607w; 590w; 466w; 425w. suzuki coupling reaction was realized with 4-chlorofuro[3,2-c]pyridine (7a) and phenylboronic acid in the presence of pd(pph3)4 catalyst in dichloromethane, in which 4-phenylfuro[3,2-c]pyridine (8e) and 4-(furo[3,2-c]pyridine-4-yl)furo[3,2-c]pyridine (10) were formed (fig. 1). the compounds 8e and 10 were purified on a silica gel column. their structures were determined by 1h and 13c nmr spectra. the low yield of 8e (20.2%) comparing with 1-phenyl[1]benzofuro[3,2-c]pyridine (8f) (59%) o ch=ch-co2ho ch=o ch3co2na clco2et nan3 o ch=ch-con3 δ o nh o pocl3 o n cl phb(oh)2 pd(pph3)4o n n o + ph2o (ch3co)2o 1a 3 4a 7a 8e10 o n 1 2 3 4 5 6 7 1´ 2´ 3´ 4´ 1 2 3 3a 3a4 6 7 7a7a 5 1´ 2´ 3´ 4´ 5´ 6´ 7´ 3´a 7´a bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:17 utc 78 hrašna, m. et al. (tarabová et al., 2010) can be explained by the higher [1]benzofuro[3,2c]pyridine system stability. the mechanism of the compound 10 formation is for us until now confused. 3.2 antimicrobial activity the structures whose biological activity was studied are presented in fig. 2. fig. 2. structures of compounds, in which biological activity was studied. antimicrobial activity of tested compounds 1a-d (characterized by mic) is summarized in table 1. it was noticed that the tested compounds 1 inhibited growth of microorganism with the lowest concentration 25 mg.l-1, except the compound 1d, in o n o o nh s o r1 r r2 co2h r1 r compound r r1 1a h h 1b 3-cf3c6h4 h 1c ch3 h 1d ch=ch-ch=ch o co2 r1 r 2 cu o co2 r1 r 2 co compound r r1 2a h h 2b ch=ch-ch=ch h 2c ch3 h compound r r1 2d h h 2e ch=ch-ch=ch h 2f ch3 h o con3 3a compound r r1 r2 4a h h h 4b 3-cf3c6h4 h h 4c 4-no2c6h4 h h 4d ch=ch-ch=ch h 4e 3-cf3c6h4 h ch3 4f ch=ch-ch=ch ch3 r r1 compound r r1 5a 3-cf3c6h4 h 5b ch=ch-ch=ch o n sch3 r r1 compound r r1 6a 3-cf3c6h4 h 6b ch=ch-ch=ch compound r r1 7a h h 7b 3-cf3c6h4 h 7c 4-ch3oc6h4 h 7d ch=ch-ch=ch o n cl r r1 o r n r1 r 2 compound r r1 r2 8a 3-cf3c6h4 h piperidin-1-yl 8b 3-cf3c6h4 h morpholin-4-yl 8c ch=ch-ch=ch piperidin-1-yl 8d ch=ch-ch=ch morpholin-4-yl 8e h h phenyl 8f ch=ch-ch=ch phenyl 8g h h co2h 8h h h cn 8i h h conh2 8j 3-cf3c6h4 h co2h o n o r compound r 9a h 9b 3-cf3c6h4 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:17 utc nova biotechnologica et chimica 11-1 (2012) 79 which the antifungal effect was repressed. substitution on a furan ring did not influence on antifungal and antibacterial effect. table 1. antimicrobial activity of tested compounds 1a-1d characterized by mic [mg.l-1]. bacteria filamentous fungi xanthomonas sp. e. amylovora f. graminearum p. avenae compound øa mic ø a mic ø a mic ø a mic 1a 3.3 ± 1.3 25 1,6 ± 0.1 25 3.3 ± 0.0 25 1.5 ± 0.0 25 1b 3.3 ± 2.3 25 1,6 ± 0.4 25 1.0 ± 0.0 25 3.0 ± 1.3 25 1c 2.8 ± 0.3 25 2.0 ± 0.5 25 2.4 ± 0.1 25 2.0 ± 0.5 25 1d 2.8 ± 1.0 25 1.0 ± 0.0 25 0 n/a 3. 0 ± 0.0 25 tmtd 0.8 ± 0.2 100 1.0 ± 0.0 200 4.6 ± 0.1 200 3.4 ± 0.9 200 a zone inhibition diameter expressed in mm over 24 h, 4 d respectively, ± sd mic minimal inhibition concentration, n/a compound does not show antimicrobial effect antimicrobial activities of tested compounds 2a-2f are summarized in table 2. in comparison, the effect of some of this compounds is lower (mic 50-200 mg.l-1) than tested compounds of group 1a-1d. the complexes of copper(ii) and cobalt(ii) shown similar antibacterial effects but the antifungal effects are various. in comparison, copper(ii) complexes 2a-2c are more effective on f. graminearum than cobalt(ii) complexes. cobalt(ii) complexes 2d-2f are more effective on p. avenae. sensitivity of microorganism on compounds 2a-2f decreased in the order: e. amylovora > xanthomonas sp. > p. avenae > f. graminearum. table 2. antimicrobial activity of tested compounds 2a-2f characterized by mic [mg.l-1]. bacteria filamentous fungi xanthomonas. sp. e. amylovora f. graminearum p. avenae compound øa mic ø a mic ø a mic ø a mic 2a 1.5 ± 0.5 25 4.5 ± 1.5 25 1.0 ± 0.0 25 1.0 ± 0.0 50 2b 1.0 ± 0.0 25 1.0 ± 0.0 25 1.3 ± 0.0 50 1.5 ± 0.0 25 2c 1.3 ± 0.0 25 4.3 ± 0.0 25 4.3 ± 0.0 25 2.5 ± 0.0 100 2d 5.3 ± 0.0 200 2.0 ± 0.0 25 0 n/a 4.5 ± 0.0 25 2e 2.0 ± 1.0 25 1.3 ± 0.3 25 0 n/a 2.0 ± 0.0 25 2f 2.0 ± 1.0 25 2.8 ± 0.8 25 3.0 ± 0.0 25 1.5 ± 0.5 25 tmtd 0.8 ± 0.2 100 1.0 ± 0.0 200 4.6 ± 0.1 200 3.4 ± 0.9 200 a zone inhibition diameter expressed in mm over 24 h, 4 d respectively, ± sd mic minimal inhibition concentration, n/a compound does not show antimicrobial effect the azide 3a inhibited the growth of bacteria xanthomonas sp. and e. amylovora (mic 25 mg.l-1). the same effect was registered on filamentous fungi f. graminearum and p. avenae with mic 25 mg.l-1. in comparison, all of tested bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:17 utc 80 hrašna, m. et al. microorganisms are more sensitive to azide 3a than to previous group of complexes 2a-2f. table 3. antimicrobial activity of 3a characterized by mic [mg.l-1]. bacteria filamentous fungi xanthomonas sp. e. amylovora f. graminearum p. avenae compound øa micb ø a mic ø a mic ø a mic 3a 1.5 ± 0.3 25 2.3 ± 1.0 25 2.3 ± 0.4 25 1.0 ± 0.0 25 tmtd 0.8 ± 0.2 100 1.0 ± 0.0 200 4.6 ± 0.1 200 3.4 ± 0.9 200 a zone inhibition diameter expressed in mm over 24 h, 4 d respectively, ± sd mic minimal inhibition concentration from the results we can assumed, that the derivatives of 3-(furan-2 yl)propenoic acid have potential in the development of the new pesticides, effective on bacterial as well as fungal affections of the plants. antimicrobial activities of compounds 4a-4f are summarized in table 4. in comparison, these compounds are less sensitive to filamentous fungi than in previous groups of compounds 1-3. inhibition of the growth of filamentous fungi p. avenae is registered only by pyridones 4d and 4e. the rest of microorganisms react sensitively on pyridones 4a-4f, except pyridone 4b (xanthomonas sp., mic 100 mg.l-1), 4c (e. amylovora, mic 100 mg.l-1). pyridone 4e has not antifungal effect on f. graminearum. table 4. antimicrobial activity of pyridones 4a-4f characterized by mic [mg.l-1]. bacteria filamentous fungi xanthomonas sp. e. amylovora f. graminearum p. avenae compound øa mic ø a mic ø a mic ø a mic 4a 3. 0 ± 0.0 25 3.0 ± 0.0 25 1.0 ± 0.0 25 0 n/a 4b 2.0 ± 0.0 100 1.0 ± 0.0 25 2.5 ± 0.2 25 0 n/a 4c 1.0 ± 0.0 25 3.0 ± 0.0 100 4.5 ± 0.0 25 0 n/a 4d 4.2 ± 0.8 25 3.8 ± 0.0 25 4.0 ± 0.0 25 2.0 ± 0.0 50 4e 3.0 ± 0.0 25 4.0 ± 0.0 25 0 n/a 1.0 ± 0.0 25 4f 2.5 ± 0.5 25 1.0 ± 0.0 25 2.8 ± 0.8 25 0 n/a tmtd 0.8 ± 0.2 100 1.0 ± 0.0 200 4.6 ± 0.1 200 3.4 ± 0.9 200 a zone inhibition diameter expressed in mm over 24 h, 4 d respectively, ± sd mic minimal inhibition concentration, n/a compound does not show antimicrobial effect antimicrobial activities of pyridine-thiones 5a a 5b are summarized in table 5. in comparison, pyridine-thiones are more effective on all microorganisms than in groups 2 and 4. both of these compounds have the same effect on microorganisms xanthomonas sp., e. amylovora, and p. avenae. f. graminearum reacts sensitively on pyridine-thione 5b (mic 25 mg.l-1). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:17 utc nova biotechnologica et chimica 11-1 (2012) 81 table 5. antimicrobial activity of 5a,b characterized by mic [mg.l-1]. bacteria filamentous fungi xanthomonas sp. e. amylovora f. graminearum p. avenae compound øa mic ø a mic ø a mic ø a mic 5a 3.0 ± 0.0 25 1.0 ± 0.0 25 3.5 ± 0.0 50 3.5 ± 0.0 25 5b 2.5 ± 0.5 25 2.5 ± 0.0 25 2.3 ± 0.0 25 3.0 ± 0.0 25 tmtd 0.8 ± 0.2 100 1.0 ± 0.0 200 4.6 ± 0.1 200 3.4 ± 0.9 200 a zone inhibtion diameter expressed in mm over 24 h, 4 d respectively, ± sd mic minimal inhibition concentration antimicrobial effects of chloro derivatives 7a-7d are summarized in table 6. compounds of this group showed various antimicrobial effects. filamentous fungi are the most sensitive on chloro derivatives 7a a 7b (mic 25 mg.l-1). the biggest effect on bacteria xanthomonas sp. and e. amylovora has chloro derivative 7c (mic 25 mg.l-1). table 6. antimicrobial activity of compounds 7a-7d characterized by mic [mg.l-1]. bacteria filamentous fungi xanthomonas sp. e. amylovora f. graminearum p. avenae compound øa mic ø a mic ø a mic ø a mic 7a 3.0 ± 0.0 50 2.3 ± 1.3 25 2.5 ± 0.5 25 3.8 ± 0.0 25 7b 2.7 ± 0.0 25 3.7 ± 0.0 50 3.3 ± 0.0 25 3.0 ± 0.0 25 7c 3.0 ± 0.5 25 2.8 ± 0.0 25 2.0 ± 0.0 100 1.5 ± 0.0 100 7d 2.5 ± 0.2 50 1.0 ± 0.0 100 4.0 ± 0.0 100 1.0 ± 0.0 50 tmtd 0.8 ± 0.2 100 1.0 ± 0.0 200 4.6 ± 0.1 200 3.4 ± 0.9 200 a zone inhibition diameter expressed in mm over 24 h, 4 d respectively, ± sd mic minimal inhibition concentration antimicrobial activities of 8a-8j are summarized in table 7. in comparison, this group of compounds 8a-8j has lower antimicrobial effects than previous groups. compounds 8a and 8f inhibited the growth of bacteria by higher concentrations (mic 50 mg.l-1) and they are less effective. compounds 8h and 8i with mic 25 mg.l-1 have antifungal effect on f. graminearum, the effect of compounds 8c (mic 100 mg.l-1), 8f (mic 200 mg.l-1) and 8g (50 mg.l-1) is registered also. sensitivity of microorganisms by these compounds decreased in the order: e. amylovora > xanthomonas sp. > p. avenae > f. graminearum. the antimicrobial activities of tested n-oxides 9a, 9b are summarized in table 8. compounds 9a and 9b showed the lowest antimicrobial effect of all tested compounds. inhibition of the growth of bacteria xanthomonas sp. and e. amylovora is stronger for n-oxide 9b, which inhibits the growth of filamentous fungi p. avenae (mic 100 mg.l-1) too. however, this compound has not antifungal effect on f. graminearum. antifungal effect is not shown by n-oxides 9a. n-oxide 9b show bigger antimicrobial effect. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:17 utc 82 hrašna, m. et al. table 7. antimicrobial activity of compounds 8a-8j characterized by mic [mg.l-1]. bacteria filamentous fungi xanthomonas sp. e. amylovora f. graminearum p. avenae compound øa mic ø a mic ø a mic ø a mic 8a 3.0 ± 0.0 50 4.0 ± 0.0 50 0 n/a 2.0 ± 0.0 100 8b 4.6 ± 0.2 50 2.5 ± 0.0 25 0 n/a 2.0 ± 0.0 25 8c 3.6 ± 0.0 25 3.5 ± 0,0 25 1.5 ± 0.5 100 4.3 ± 0.0 50 8d 2.2 ± 0.2 25 3.3 ± 0.3 25 0 n/a 3.5 ± 1.5 50 8e 0.3 ± 0.0 25 6.7 ± 0.0 25 0 n/a 4.9 ± 0.9 25 8f 2.7 ± 0.0 50 3.3 ± 0.0 50 10.3 ± 0.0 200 2.0 ± 0.0 25 8g 1.5 ± 0.3 25 2.3 ± 0.0 25 5.0 ± 1.0 50 1.7 ± 0.2 25 8h 1.7 ± 0.2 25 2.3 ± 0.3 25 1.0 ± 0.5 25 1.8 ± 0.3 25 8i 2.7 ± 0.0 25 2.4 ± 0.2 25 3.1 ± 0.4 25 1.8 ± 0.2 25 8j 3.3 ± 0.0 25 2.3 ± 0.0 25 0 n/a 2.0 ± 0.0 100 tmtd 0.8 ± 0.2 100 1.0 ± 0.0 200 4.6 ± 0.1 200 3.4 ± 0.9 200 a zone inhibition diameter expressed in mm over 24 h, 4 d respectively, ± sd mic minimal inhibition concentration, n/a compound does not show antimicrobial effect table 8. antimicrobial activity of tested compounds 9a, 9b characterized by mic [mg.l-1]. bacteria filamentous fungi xanthomonas sp. e. amylovora f.graminearum p. avenae compound øa mic ø a mic ø a mic ø a mic 9a 2.3 ± 0.8 50 1.0 ± 0.0 200 0 n/a 0 n/a 9b 2.3 ± 1.3 25 1.0 ± 0.0 50 0 n/a 3.0 ± 0.0 100 tmtd 0.8 ± 0.2 100 1.0 ± 0.0 200 4.6 ± 0.1 200 3.4 ± 0.9 200 a zone inhibition diameter expressed in mm over 24 h, 4 d respectively, ± sd mic minimal inhibition concentration, n/a compound does not show antimicrobial effect. 4. conclusions in summary, it was found that during study of the reaction of 4-chlorofuro[3,2c]pyridine with boronic acid in the condition of suzuki coupling reaction were formed two products. the main expecting product 4-phenylfuro[3,2-c]pyridine and the unexpected 4-(furo[3,2-c]pyridine-4-yl)furo[3,2-c]pyridine. the antimicrobial testing shown that earlier known synthesized derivatives of furo[3,2-c]pyridine and their starting compounds have various potential to inhibit the growth of phytopathogene microorganisms. in general it is shown, that the bacterial strains are more sensitive on these compounds than the fungal strains. acknowledgements: this work was supported by the grants vega 1/233/12. nmr experimental part of this work was facilitated by support of slovak national research and development program no. 2003sp200280203. the authors are grateful to prof. a. gatial for ir spectra and dr. n. pronayová for nmr spectra measurements. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:17 utc nova biotechnologica et chimica 11-1 (2012) 83 references baran, p., boča, m., boča, r., krutošíková, a., miklovič, j., pelikán, j., titiš, j.: structural characterization, spectral and properties of isothiocyanate nickel(ii) complexes with furopyridine derivatives. polyhedron, 24, 2005, 1510-1516. bencková, m., krutošíková, a.: 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acta cryst., 2007b, e63, m2427-2428. vrábel, v., švorc, ľ., bradiaková, i., kožíšek, j., krutošíková, a.: 2-[3-(trifluoromethyl)phenyl]furo[3,2-c]pyridine. acta cryst., 2007c, e63, o4516. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:17 utc << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (iso coated) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.3 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /perceptual /detectblends true /detectcurves 0.1000 /colorconversionstrategy /srgb /dothumbnails true /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage 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can be opened with acrobat and adobe reader 5.0 and later.) >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [2834.650 2834.650] >> setpagedevice microsoft word manuscript _sestinová_proofread.doc 78 nova biotechnologica et chimica 14-1 (2015) doi 10.1515/nbec-2015-0017 © university of ss. cyril and methodius in trnava effect of environmental load on the toxicity of bottom sediments oľga šestinová, lenka findoráková, silvia dolinská, jozef hančuľák, tomislav špaldon, erika fedorová institute of geotechnics, slovak academy of sciences,watsonova 45, kosice, slovak republic (sestinova@saske.sk) abstract: this study is devoted to ecotoxicity tests, terrestrial plant test (modification of oecd 208), phytotoxkit microbiotest on sinapis alba and chronic tests of earthworm (eisenia veneta), modification of oecd guidelines for the testing of chemicals 317, bioaccumulation in terrestrial oligochaetes on polluted sediments. earthworms can accelerate the removal of contaminants from soil. the study materials are river sediments, which were obtained from a monitoring station the water reservoir the ružín no.1 particularly, the river hornád, hnilec and sample from sludge bed rudňany. the samples of sediment were used to assess of the potential phytotoxic effect of heavy metals on higher plants. total mortality was established in earthworms using chronic toxicity test after 7 and 28 exposure days. based on the phytotoxicity testing, phytotoxic effects of the metals contaminated sediments from the sludge bed rudňany on s. alba seeds was observed. the largest concentration differences were recorded in the sample r7 after 7 days earthworms exposure. the earthworms mortality was not influenced by sediment neither after 7 nor 28 exposure days the spectra of samples h, ho and r showed broad peak at 1 419 1 512 cm-1 characteristic for carbonate radical. in the spectra of the samples (r and r7) the vibration of c-h groups at 2 926 and 2 921 cm-1, respectively were also observed, demonstrating the presence of organic matter. our research will continue with determination of metals concentration in earthworms. key words: heavy metals, sediments, phytotoxkit test, test of earthworm, xrf, ftir 1. introduction ecotoxicity testing of complex materials, intended for application on soil is necessary because bioassays (compared with pure chemical assessments) reveal possible interactions between the pollutants in a mixture and integrate the effects of the environmental matrix and bioavailability (phytotoxkit microbiotest, bioaccumulation in terrestrial oligochaetes). negative effects of contaminants on the ecosystems and humans are characterized by their environmental toxicity. earthworms are often used as terrestrial model organisms for ecotoxicity testing, because of their importance for the structure and function of soil ecosystems (piola et al., 2009). ecological tests, such as avoidance behaviour, based on the earthworm's ability to detect a toxicant and move away, may be sensitive and fast (levequea et al., 2014). earthworms change the physical and chemical properties of soil by mixing it with organic material and through their burrowing they improve aeration and render contaminants available for microorganisms. the presence of earthworms in contaminated soil indicate that they can survive a wide range of different organic contaminants, such as pesticides, herbicides, polycyclic aromatic hydrocarbons (pahs), polychlorinated biphenyls (pcbs), and crude oil, at least when concentrations of the contaminant are not too high. the improvement of the soil due to their activity bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:13 utc nova biotechnologica et chimica 14-1 (2015) 79 and the microorganisms in their digestive track can contribute to the accelerated removal of contaminants from soil, but sometimes their casts adsorb the pollutant so that its dissipation is delayed (camposa et al., 2014). effect assessment using ecotoxicological bioassays with organisms at different trophic levels can provide valuable pieces of information on the risk of chemical substances in the ecosystem (nemcová et al., 2013). sediments in aquatic ecosystems are often contaminated with metals as a result of natural background or anthropogenic activities. anthropogenic activities, especially mining activity are source of heavy metals contamination in central spiš. the highest concentrations of these pollutants are concentrated to the sediments water reservoirs of ružín no.1, branches of the hornád and hnilec rivers. numerous precious metals, including fe and cu ores, were exploited in the smolník area, and cu, fe, hg, and ba ores were mined in the rudňany area (macingova et al., 2012; jencarova et al., 2013). one of the mercury polluted area is sludge bed rudňany, which is contaminated from the former mining activities. additionally, cu ores from home production and import (87 %) were treated in krompachy (junaková and bálintová, 2012). these activities, as the long-term monitoring of the metal content in the area has confirmed, influenced the quality of the bottom sediments in the water reservoir ružín no.1 and sludge bed rudňany. these mining operations and the metallurgical processing of complex metals negatively impacted this region. 2. material and methods 2.1 characterization of sample this study is devoted to two tests of ecotoxicity, terrestrial plant test: phytotoxkit microbiotest with sinapis alba and chronic tests of earthworm (eisenia veneta). ecotoxicity and bioavailability levels of heavy metals were studied in contaminated sediments collected in eastern slovakia. the materials are river sediments, which were obtained from the water reservoir the ružín no.1 particularly, the river hornád (ho), hnilec (h) and sample from sludge bed rudňany (r). the sediments were sampled in october/2012 at a depth of 50 cm. the samples were first dried at room temperature, and the sediments were thoroughly mechanically homogenized immediately prior to the experiments and then quartered. the sediments consist of sand, silt and clay fractions. the sediment reference material lgc6187 (crm) was used as control soil (c) in experiments. the x-ray fluorescence spectrometry was used to determine concentrations of metals (cu, zn, as, pb, cr, ni, cd and hg) in sediment fraction 63 μm. on the basis of obtained xrf and ftir results, the sediment contamination was investigated. one way of sediment utilization could be its application into agglomerate using the sintering process which is used for processing of fine-grained materials (legemza et al., 2011; findorák et al., 2014). 2.2 x-ray fluorescence spectrometry the concentration of heavy metals in sediments and sediments after 7 and 28 days earthworms exposure was measured through the use of spectro xepos x-ray bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:13 utc 80 šestinová, o. et al. fluorescence spectrometer (range of elements: na(11)-u(92), spectro analytical instruments, germany). the sample material for xrf measurement was dried and homogenized, then powder sample (5 g) was homogenized with clariant micropowder c (1 g) and then pressed with 15 tons to pellet with 32 mm diameter. 2.3 infrared spectroscopy the infrared spectra were recorded on ftir spectrometer bruker tensor 27, (germany) equipped with dtgs kbr detector. for each sample 64 scans were measured in the 4 000-400 cm-1 spectral range in the transmission mode with a resolution of 4 cm-1. the kbr pressed-disc technique was used for preparing a solid sample for routine scanning of the spectra. samples of approximately 0.1 mg were dispersed in 200 mg of kbr to record optimal spectra in the regions 4 000-400 cm-1. a diameter of the pellets, pressed from samples, was 10 mm. for ftir measurements total samples and samples after 7 and 28 days earthworms exposure were used. 2.4 toxicity testing with phytotoxkit phytotoxkit is an alternative test procedure that enables determination of the biological effects of chemical compounds on plants. the sediment was covered with the filter plate and ten seeds of s. alba were placed on top of the filter in a single row. after closing the test plates with the transparent cover, they were placed vertically and incubated for 72 h at 25 °c. three replicates were performed for each sample set. root growth inhibition was measured after 72 h. the test was considered to be valid if the number of germinated seeds in the control was at least 90 %. pictures of the test plates were analyzed with the free image analysis program image tools. the sediment samples were used to evaluate potential phytotoxic effects using the parameters of the percentage inhibition of seed germination (isg) and percentage inhibition of root growth (irg) in the test sediment. 2.5 toxicity testing with earthworms the experiments were carried out as described in the oecd guidelines 317 for the testing of chemicals relating to environmental fate, tests of mortality. contact bioassays are important for testing the ecotoxicity of solid materials (kobetičová et al., 2010). the reaction to the earthworm (e. veneta) was used for chronic tests in the sediments. the earthworms (e. veneta) were purchased from a local supplier. prior to the start of the experiment, the earthworms were allowed to acclimatize for one week in the experimental conditions. the adult worms were used in the tests. the ph of the sediment samples was in the range 6.25 7.35. three replicates were performed for each test (of the sediment 100 g dry weight) with ten earthworms added to each boxes. then distilled water was added for purpose to obtain 30 % moisture of sediment. after that, the boxes with sediments were kept for 7 and 28 days at 20 °c. a small part of chippings oats was added every week in each box as a source of food as recommended. the results were evaluated as the percentage inhibition of mortality and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:13 utc nova biotechnologica et chimica 14-1 (2015) 81 compared to the control soil. total mortality was observed at earthworms after 7 and 28 exposure days. 3. results and discussion table 1 summarizes the results of the chemical analyses of the metals in the sediments, revealing significant contamination with cu and as for sample hnilec (h) and with all the metals for sample rudňany (r) according to the laws of the methodological instruction of the ministry of environment of the slovak republic no. 549/1998-2 for assessment of risks from pollution of sediments of streams and water reservoirs. table 1. concentrations of heavy metals in the total sediments h, ho, r, c and in sediments after 7 and 28 days earthworms exposure (h7, h28, ho7, ho28, r7, r28, c7, c28). concentration (mg/kg d. w.) cu zn as pb cr ni cd hg h 408.5 ± 2.2 399.5 ± 12.3 56.7 ± 1.4 94.3 ± 1.3 54.3 ± 4.2 45.8 ± 2.9 2.8 ± 0.3 2.6 ± 0.3 h7 397.7 ± 4.7 397.4 ± 5.4 49.5 ± 4.6 90.3 ± 1.0 53.3 ± 9.6 46.0 ± 3.9 2.1 ± 0.1 2.1 ± 0.2 h28 404.1 ± 3.2 404.0 ± 15.9 54.8 ± 2.1 93.0 ± 2.2 61.8 ± 8.9 45.6 ± 2.5 2.5 ± 0.4 2.5 ± 0.2 ho 161.2 ± 1.9 263.0 ± 14.9 32.8 ± 3 47.1 ± 1.9 95.9 ± 3.8 76.5 ± 6.6 6.5 ± 0.7 6.7 ± 0.4 ho7 150.1 ± 0.9 264.5 ± 6.7 35.6 ± 2.8 50.1 ± 4.3 82.9 ± 6.4 67.9 ± 4.8 4.2 ± 0.3 5.9 ± 0.3 ho28 144.0 ± 2.2 255.6 ± 8.6 34.5 ± 2,1 49.2 ± 2.4 84.9 ± 8.3 67.6 ± 4.5 3.8 ± 0.4 5.7 ± 0.3 r 1429 ± 5.7 1853 ± 31.8 133.5 ± 5.4 645.5 ± 9.1 481.7 ± 16.4 155.1 ± 4.7 67.7 ± 2.1 188.5 ± 1.1 r7 1269 ± 24.5 1574 ± 17.5 124.7 ± 6.3 547.9 ± 6.7 359.1 ± 17.3 136.9 ± 9.5 58.5 ± 3.9 137.9 ± 8.4 r28 1415 ± 33.3 1756 ± 21.2 144.4 ± 9.6 617.9 ± 10.0 476.7 ± 13.6 150.4 ± 4.2 63.7 ± 2.6 187.0 ± 4.9 c <0.5 ± 0.1 8.0 ± 1.2 0.7 ± 0.3 31.7 ± 0.9 175.5 ± 4,3 7.4 ± 0.9 8.4 ± 1.1 <0.5 ± 0.2 c7 <0.5 ± 0.2 5.9 ± 1.5 0.2 ± 0.4 21.4 ± 1.0 86.7 ± 7.4 6.3 ± 0.7 4.3 ± 1.0 0.2 ± 0.1 c28 <0.5 ± 0.2 7.1 ± 0.2 0.4 ± 0.3 20.1 ± 0.9 95.0 ± 4.9 8.4 ± 0.3 9.6 ± 1.2 0.4 ± 0.3 norm used for comparison (mg/kg d.w.) tv 36 140 29 85 100 35 0.8 0.3 mpc 73 620 55 530 380 44 12 10 iv 190 720 55 530 380 210 12 10 norm no. 549/1998-2: tv-target value (negligible risk), mpc–maximum permissible concentration (max. tolerable risk), iv-intervention value (serious risk), control soil (c, c7, c28), sediment of hnilec (h, h7, h28) and hornád (ho, ho7, ho28) river, sludge bed of rudňany (r, r7, r28), ± sd bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:13 utc 82 šestinová, o. et al. from the obtained xrf results it is evident (shown in table 1) that in all study samples without sample h was recorded decrease in metal concentrations after 7 days earthworms exposure, mainly the concentration of as, cd for sample h7; cu, cr, ni cd, hg for sample ho7; cu, zn, as, pb, cr, ni, cd, hg for sample r7; zn, as, pb, cr, hg for sample c7. other concentrations were higher than basic concentration. after 28 days earthworm exposure decrease of concentrations cu, as, cd for sample h28; cu, zn, cr, ni, cd for sample ho28; cu, zn, pb, cr, hg for sample r28; cu, zn, as, pb, cr for sample c28 was found. the largest concentration differences were recorded in the sample r7 after 7 days earthworms exposure. it was found that earthworms in some cases caused decrease of metals concentration in contaminated sample. the earthworms mortality was not influenced by sediment neither after 7 nor 28 exposure days. the ftir spectra of studied sediments (total samples and after 7 and 28 days earthworm exposure) are shown in fig.1. the wavenumbers and associated vibration types of dominant minerals are given in table 2 3. it was found that the absorption bands in the range 3 620-3 695cm-1 were due to asymmetric valence vibration of structurally bound oh group from clay minerals. the peaks around 3 423 and 1 630cm-1 correspond to the h-o-h stretching and bending vibrations of the adsorbed water, respectively. in the spectra of the samples (r and r7) the vibration of c-h groups at 2 926 and 2 921cm-1, respectively were also observed, demonstrating the presence of organic matter (farmer, 1974; madejová, 2003). but in the sample r28 the vibration around 2 920cm-1 was missing. this is probably related to the viability and metabolic activity of earthworms. fig. 1. the ftir spectra of the total sediments: h, ho, r and k (control soil) and in sediments after 7 and 28 days earthworms exposure (h7, h28, ho7, ho28, r7, r28, k7, k28). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:13 utc nova biotechnologica et chimica 14-1 (2015) 83 the spectra of samples h, ho and r showed broad peak at 1 419-1 512cm-1 characteristic for carbonate radical. the main absorption band for sio2 connected with the stretching vibration of bridging oxygen atoms is known to be in the range 1 0001 100 cm-1 and the position of the peak depend on the structural arrangement of the oxygen atom (green et al., 2001). the absorption bands located at 790 and 690 cm-1 are characteristic bands of network si-o-si, which correspond to quartz. at 529, 530 and 533 cm-1, respectively was found the absorption band, which corresponds to stretching vibration of si-o-si. in ftir spectrum the characteristic bands of carbonate vibrations at 1 418 and 1 023 cm-1 were also seen (carnin et al., 2012), or it could be organic proportion. to recognize the mineral species the positive identification of many characteristic bands of individual minerals as possible in the whole 4 000-400 cm-1 range is needed though the oh stretching region which is perhaps the most important diagnostic part of the spectrum. the results of table 2 and 3 confirm the presence of functional groups that are part of the toxic substances. table 2. characteristic vibrations (ν/cm-1) of the studied sediments. ν(aloh) ν (h2o) ν (c-h) calcite and dolomite carbonate δ(h2o) ν (c-h) ν (c-c) h 3 623 3 400 2 365 1 637 1 419 h7 3 620 3 369 2 360 1 642 h28 3 623 3 394 2 365 1 632 1 387 ho 3 627 3 301 2 365 1 648 1 512 ho7 3 623 3 400 2 350 1 637 1 423 ho28 3 623 3 410 2 354 1 637 1 434 r 3 623 3 431 2926 2506 1 626 1 419 r7 3 623 3 420 2921 2506 2 360 1 616 1 419 r28 3 623 3 405 2506 2 360 1 419 c 3 695 3 618 c7 3 695 3 618 c28 3 695 3 618 table 3. the characteristic vibrations (ν/cm-1) of studied sediments. ν (sio) δ(alaloh) si-o-si ν (siosi) δ(siosi) δ(sioal) h 1 023 800 696 529 462 h7 1 013 753 687 529 467 h28 1 008 747 685 529 462 ho 1 023 784 685 530 467 ho7 1 018 789 685 530 467 ho28 1 008 789 691 530 462 r 1 023 862 607 529 472 r7 867 607 529 472 r28 1 018 862 618 529 472 c 1 105 913 777 684 533 460 c7 1 105 913 777 684 533 c28 1 105 913 777 684 533 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:13 utc 84 šestinová, o. et al. according to fig. 2 the percentage inhibition of seed germination (isg) was 11.6 – 47.1 % in the h and ho samples and 28.5–60 % in the r. the test is considered to be valid if the number of germinated seeds in the control was at least 90 %. the percentage inhibition of root growth (irg) was 9.7–48.1 % in the h and ho samples and 29.7–70.1 % in the r respectively, which was higher than the inhibition of seed germination (isg) especially of the sediments from sludge bed rudňany. the isg (%) was 10.9-47.2 % in the h-28 and ho-28 samples and 24.7-50.9 % in the r-28 after 28 exposure days of earthworms. the irg (%) was 9.8–44.8 % in the h-28 and ho-28 samples and 28.9–66.2 % in the r-28. according to the phytotoxkit microbiotest, the experimental concentration at which growth inhibition rises above 50 % after 72 hours can be considered as the effective concentration 72/ec50. the inhibition of the germination rate differs significantly from the controls in our case. based on the phytotoxicity testing, phytotoxic effects of the metals contaminated sediments from the sludge bed rudňany on s. alba seeds was observed. fig. 2. indexes isg and irg of the s. alba in the six series of the sediments (h and h-28), (ho and ho-28), (r and r-28) and (control and control-28). 4. conclusions this study was aimed to obtain detailed information about the total metals concentrations, mineral species, organic matter and percentage of earthworms mortality after 7 and 28 exposure days in river sediments (hnilec and hornád) and sludge bed rudňany. the results of the chemical analysis of the metals in the sediments, show significant contamination with all the metals for sample rudňany. it was found that earthworms in some cases caused decline of metals concentration in contaminated samples. ftir spectroscopy confirmed in samples of r and r7 the presence of vibrations of c-h groups at 2 926 and 2 921 cm-1. it demonstrates the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:13 utc nova biotechnologica et chimica 14-1 (2015) 85 presence of organic matter. the absence of that vibration in sample r28 probably related to the viability and metabolic activity of earthworms. the results of our study confirmed that no mortality was observed in the studied sediments. based on the phytotoxicity tests, no phytotoxic effects of the metal contaminated sediments from the water reservoir ružin no.i, hnilec (h, h-28) and hornád (ho, ho-28) on s. alba seeds was observed. potential phytotoxic effects of the metals contaminated sediments from the sludge bed rudňany (r, r-28) on s. alba seeds was observed. the inhibition of the germination rate (phytotoxic and mortality tests) of sludge bed rudňany was probably the result of their high contamination by heavy metals and of their physico-chemical properties. this is the first study to use e. veneta as an indicator species to assess the risk of sediment contamination by heavy metals. however, more toxicity data for various species are needed to evaluate the environmental risks of heavy metals in sediments. our research will continue with determination of metals concentration in earthworms. the obtained results might be useful in the planning of future sediment utilization. acknowledgements: the authors greatly acknowledge the fellowship support of vega-slovak grant agency for science (no-2/0194/15) and apvv-slovak research and development agency (no-0252-10). references camposa, j.r., dendoovenb, l., alvarez-bernalc, d., contrerasramosd, s.m.: potential of earthworms to accelerate removal of organic contaminants from soil. appl. soil ecol., 79, 2014, 10-25. carnin, r.l.p., folgueras, m.v., luvizâo, r.r., correia, s.l., da cunha, c.j., dungan, r.s.: use of an integrated approach to characterize the physicochemical properties of foundry green sands. thermochim. acta, 543, 2012, 150-155. green, m.l., gusev, e.p., degraeve, r. garfunkel, e.l.: ultrathin (<4 nm) sio2 and si-o-n gate dielectric layers for silicon microelectronics: understanding the processing, structure, and physical and electrical limits. j. appl. phys., 90, 2001, 2057-2121. jenčárová, j., luptáková, a., kupka, d.: the possibilities of sulphatereducing bacteria use in mine drainage waters remediation. inżynieria mineralna – j. pol. mineral eng. soc., 14, 2013, 47-51. junáková, n., bálintová, m.: assessment of nutrient concentration in reservoir bottom sediments. 20th international congress of chemical and process engineering chisa 2012, procedia engineering 42, praha, czech republic, 2012, 165-170. farmer, v.c.: the infrared spectra of minerals. mineralogical society, monograph 4, london, 1974, 539 pp. findorák, r., fröhlichová, m., legemza, j.: potential of ilmenite sand application in the iron ore materials agglomeration. metalurgija, 53, 2014, 9-12. kobetičová, k., hofman, j., holoubek, i.: ecotoxicity of wastes in avoidance tests with enchytraeus albidus, enchytraeus crypticus and eisenia fetida (oligochaeta). waste manage., 30, 2010, 558-564. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:13 utc 86 šestinová, o. et al. legemza, j., fröhlichová,, m., findorák, r., bakaj, f.: the simulating of production the agglomerate in laboratory sintering pan. acta metall. slovaca, 17, 2011, 245-252. levequea, t., capowiezd, y., schrecke, e., xionga, t., foucault, y., camille, d.: earthworm bioturbation influences the phytoavailability of metals released by particles in cultivated soils. environ. pollut., 191, 2014, 199206. mačingová, e., luptáková, a., schütz, t.: sulphates removal from acid mine drainage. modern management of mine producing, geology and environmental protection sgem 2012: proceedings from 12th international multidisciplinary scientific geoconference, albena, bulgaria, 2012, 17-23. madejová, j.: ftir techniques in clay mineral studies. vib. spectrosc., 31, 2003, 1-10. nemcová, b., mikulašková, h., bednarová, i., beklová, m., pikula, j.: impact of platinum group elements on the soil invertebrate folsomia candida. neuroendocrinol. lett., 34, 2013, 5-10. piola, l., fuchs, j., oneto, m.l., basack, s., giménez, r., massaro, r., papa, j.c., kesten, e., casabé, n.: biomarkers for the assessment of chlorpyrifos effects on earthworms and on soil functional parameters. pesqui. agropecu. bras., 44, 2009, 874-880. presented at the 3rd international conference “biotechnology and metals 2014”, september 17-19, 2014, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:13 utc nova biotechnol chim (2023) 22(1): e1519 doi: 10.34135/nbc.1519 1 nova biotechnologica et chimica zizyphus lotus and ruta chalepensis essential oils for combating antimicrobial resistance in pathogenic clinical bacteria and fungi nour el houda bekkar1,, boumediene meddah1, bahadir keskin2, pascal sonnet3 1laboratory of bioconversion, microbiological engineering and health safety, faculty of life and nature sciences, university mustapha stambouli of mascara, mascara 29000, algeria 2department of chemistry, faculty of arts & science, yildiz technical university, istanbul tr34210, turkey 3agir laboratory: infectious agents, resistance and chemotherapy, ea4294 ufr of pharmacy, picardie jules verne university, amiens, france  corresponding author: nourelhouda.bekkar@univ-mascara.dz article info article history: received: 14th october 2021 accepted: 20th october 2022 keywords: antimicrobial activity essential oil gc-ms ruta chalepensis zizyphus lotus abstract the antimicrobial activities of essential oils (eos) isolated from zizyphus lotus (zl) and ruta chalepensis (rc) harvested in oran (north-west algeria) were assessed against pathogenic clinical bacteria and fungi. the eos were isolated using the steam distillation process, the phenolic and flavonoid contents were determined using colorimetric methods, and the chemical composition was carried out using gc-ms analysis. the antimicrobial activity was evaluated using agar disc diffusion and microdilution methods. the evaluation of the synergistic effect using the combination of z. lotus (zleo) and r. chalepensis essential oils (rceo) was done using the checkerboard assay. effective extraction yields were determined for both plants, with an actual amount in rc than zl. concentrations of 8.47 ± 0 mg gae/g de and 8.56 ± 0.154 mg ce/g de of total phenols were determined in zleo and rceo, respectively. thus, a chemotype of diisooctyl-phthalate (80.343 %) was determined in zleo and the 2-undecanone (13.236 %) in rc. both plant eos exhibited important antimicrobial activity against the selected multidrug-resistant human pathogens. the most potent effect was estimated against proteus mirabilis, salmonella enterica subsp. arizonae, and hafnia alvei with growth inhibition zone diameters of 24.06 ± 0.12, 40.1 ± 0.1 and 40.16 ± 0.15 mm using zleo, respectively. also, essential anti-candida activity was estimated. zleo and rceo did not exhibit either synergistic or additive effects, with fractional inhibition concentration index values greater than 2. both plants exhibited significant antimicrobial effects alone, while in combination they did not exhibit a synergistic effect but an antagonistic one. therefore, zleo and rceo can be developed as natural antimicrobial agents in the medical and food industries to combat antimicrobial resistance. © university of ss. cyril and methodius in trnava introduction the emergence of antimicrobial resistance in most reanimation services, hospitals, and food-borne pathogens is considered a global health concern. mailto:nourelhouda.bekkar@univ-mascara.dz nova biotechnol chim (2023) 22(1): e1519 2 the bacterial and fungal strains developed increasing resistance to many antibiotics, making the therapeutic application less efficient for treating microbial infections. besides, the significant side effects of these drugs and the dysbiosis phenomena that appeared after the consumption of antibiotics induce essential consequences for human health. so, this allowed us to search for more safe alternative products, especially in the world of medicinal and aromatic plants. an emergent interest in using natural antibacterial and antifungal molecules, such as plant essential oils, as potent alternative antimicrobials for combating the antimicrobial resistance in various pathogenic clinical strains responsible for severe human microbial infections. the algerian medicinal plants are known for their contents of bioactive components, and various recent studies have elucidated the antimicrobial effects of eos and phenolic extracts of a wild variety of medicinal plants (ayad et al. 2022; bennacer et al. 2022). among the medicinal plants of high interest in traditional medicine in algeria and used by the local population are zizyphus lotus l. (rhamnaceae) and ruta chalepensis l. (rutaceae) species. both plants contain an immense variety of bioactive substances with various biological properties. recent studies have demonstrated that zizyphus bioactive components exert various biological activities, such as memory enhancement effects (zhang et al. 2014). furthermore, the ziziphus species have mainly been used in traditional medicine to treat severe diseases such as respiratory problems, scabies, pimples, and inflammation of the mouth and gums. the flavonoids, saponins, and fatty acids isolated from ziziphus species are responsible for the plant's sedative effects (xie et al. 2012). moreover, z. lotus is used in many fields, including nutrition, cosmetics, and healthcare. this plant is widely used in the treatment of urinary tract infections, digestive disorders, and intestinal microbial infections. it is used as a hypoglycemic, hypotensive, antidiarrheal, and anti-ulcer agent in stomach diseases (bnouham et al. 2002; borgi et al. 2007). various studies have reported the anticancer and anti-inflammatory properties of this plant. it also exhibited potent antifungal activity, a gastroprotective effect against helicobacter pylori and was used as an analgesic, as well as having a potent antibacterial effect (wahida et al. 2007; borgi et al. 2008; benammar et al. 2010; bakhtaoui et al. 2014; el hachimi et al. 2016; bencheikh et al. 2019). ghalem et al. (2014), marmouzi et al. (2019), and bencheikh et al. (2021) elucidated the antioxidant and nephroprotective effects of z. lotus. referring to the literature, most of the research is about the biological properties of phenolic extracts of z. lotus, while limited studies demonstrate the chemical composition and the antimicrobial effects of z. lotus essential oils, that is why we are interested in selecting this plant for the isolation of potent antimicrobials that can be used as alternatives to antibiotics. r. chalepensis is a common species in algeria and is intensely interested in international traditional medicine. this plant is known for its richness in therapeutic compounds, especially inessential oils (2-undecanone) (gonzález-trujano et al. 2006; mejri et al. 2010). recent studies mentioned that r. chalepensis leaf extract is known for its essential antioxidant and hypoglycemic activities (loizzo et al. 2017). althaher et al. (2020) demonstrated the cytotoxic effect of r. chalepensis essential oils on human mcf-7, t47d, and caco-2 cancer cell lines. the essential oils isolated from the aerial parts of this plant exert an antifungal effect against aspergillus sp., fusarium culmorum, fusarium pseudograminearum, fusarium proliferatum, and fusarium graminearum (bouajaj et al. 2014). according to amdouni et al. (2016), the r. chalepensis eos also exert an antibacterial effect against pseudomonas aeruginosa, staphylococcus aureus, and escherichia coli. gonzález-trujano et al. (2021) have mentioned the multiuse of ruta chalepensis to treat various disorders such as fever, rheumatism, mental disorders, menstrual problems, anxiety, and epilepsy problems. this is the first time that the antimicrobial effect of the essential oils of z. lotus and r. chalepensis collected from the oran-tafraoui region in western algeria has been reported. no studies on the chemical composition of zleo in volatile bioactive components have been reported. the present study aimed to determine the chemical https://www.google.com/search?client=firefox-b-d&q=pseudomonas+aeruginosa&spell=1&sa=x&ved=2ahukewilg-s504l5ahup-yukhxfkdgkqkeeckab6bagcedu nova biotechnol chim (2023) 22(1): e1519 3 composition and chemotypes of eos isolated from z. lotus leaves and r. chalepensis aerial parts using gc-ms analysis and their antimicrobial effects against pathogenic clinical bacteria and fungi for combating antimicrobial resistance. experimental plant material the wild z. lotus leaves and r. chalepensis aerial parts were collected in july and april 2017, respectively, during their flowering stages, from the tafraoui region in oran (north-west algeria). furthermore, both plants were identified by the botanist pr. righi k. from the department of biology of mascara university, algeria. clinical microbial strains the antimicrobial effect was assessed using pathogenic clinical isolates, including grampositive bacteria (staphylococcus aureus, streptococcus pyogenes, and enterococcus faecalis), gram-negative bacteria (enteropathogenic escherichia coli, salmonella enterica subsp. arizonae, proteus mirabilis, hafnia alvei), and pathogenic microscopic fungi (candida albicans). all these microbial strains were isolated from different clinical samples. stool specimens from gastroenteritis patients, oral cavity samples from the periodontal pocket of patients with periodontal disease, and urine samples from patients with urinary tract infections. on the other hand, the microbial identification was carried out in the laboratory of microbiology of meslem taib hospital in mascara, algeria and in the laboratory of bioconversion, microbiological engineering, and health safety department of biology, university mustapha stambouli of mascara, algeria. antibiotics susceptibility test the antibiotic susceptibility testing was carried out using the agar disc diffusion method, following the clsi (2015). susceptibility to various antibiotics was tested: penicillin family: penicillin g (10 µg/disc), amoxycillin (25 µg/disc), oxacillin (5 µg/disc), aminoglycosides: neomycin (30 µg/disc), polymyxin e: colistin (10 µg/disc), macrolide: spiramycin (100 µg/disc), streptogramin a: pristinamycin (15 µg/disc), nitroquinolone: nitroxolin (20 µg/disc) for bacteria, and azole antifungal: fluconazole (25 µg/disc) for c. albicans strain. according to the fms-ac (2013) and the fmsac/eucast (2018) guidelines, the growth inhibition zone diameters were interpreted using the critical diameters mentioned in table 1. the antibiotic resistance profiles allowed us to qualify the isolated clinical microbial strains as pathogenic, multidrug-resistant strains. table 1. antimicrobial resistance profile of clinical isolates. clinical isolates sp amx pt ni n ox ct p p-g fca s. aureus 0 r 12 r 0 r 18 i 15 i 0 r 12 r 10 r 0 r nt s. pyogenes 0 r 15 r 0 r 22 i 15 i 0 r 12 r 0 r 0 r nt e. faecalis 23 s 19 i 20 i 12 i 0 r 0 r 0 r 21 i 0 s nt e. coli 0 r 0 r 0 r 20 i 15 r 0 r 11 r 0 r 0 r nt p. mirabilis 0 r 15 r 0 r 14 i 16 r 0 r 0 r 10 r 0 r nt s. enterica subsp. arizonae 0 r 0 r 0 r 22 i 18 s 0 r 13 r 0 r 0 r nt h. alvei 0 r 0 r 0 r 20 i 20 s 0 r 13 r 0 r 0 r nt c. albicans 0 r 0 r 0 r 0 r 0 r 0 r 0 r 0 r 0 r 0 r sp – spiramycin; amx – amoxycillin; pt – pristinamycin; ni – nitroxolin; n – neomycin; ox – oxacillin; ct – colistin; p – penicillin-g; fca – fluconazole; r – resistant; s – sensitive; i – intermediate sensitivity; nt – not tested. essential oils extraction the extraction of essential oils (eos) was carried out using steam distillation. briefly, each 45 g of the fresh leaves of z. lotus and the aerial parts of r. chalepensis were subjected to steam distillation in distilled water for three consecutive hours according to the current european pharmacopoeia nova biotechnol chim (2023) 22(1): e1519 2 (2007). the obtained eos were collected and stored at 4 °c in brown, sealed glass vials until used. this process was done in triplicate, and the extraction yield ( ) was expressed as the weight of the eo volume (g) on the weight (g) of the plant used. physicochemical index determination different physicochemical indices of the eos were determined: relative density at 20 °c (nf iso 279 1999), refractive index (nf iso 280 1999), specific optical rotation (nf iso 592 1999), acid index (nf iso 1242 1999), ester index (nf iso 709 2002), and miscibility with ethanol (nf iso 875 1999). determination of total phenolics content (tpc) the total phenolic content (tpc) was determined according to boizot and charpentier (2006). in brief, 200 μl of each eo sample (zleo and rceo) at a concentration of 1 mg.ml-1 was mixed with 1 ml of folin ciocalteu reagent and 800 µl of sodium carbonate na2co3 solution (7.5 %). the mixture was incubated at room temperature in the dark for 10 min, and the absorbance was determined at 735 nm using a spectrophotometer (jenway, 6400 spectrophotometer). gallic acid (ga) (0.05 – 0.2 mg.ml-1) was used as a standard: .the tpc was expressed as gallic acid equivalents (gae) per gram of dry extract (mg gae/g de). determinations of tpc were performed in triplicate. results were expressed as mean ±sd. determination of flavonoids contents (tfc) the total flavonoid content (tfc) was determined according to samatha et al. (2012). briefly, 1 ml of 2 % aluminum trichloride (alcl3) in methanol was mixed with 1 ml of the eo samples. the absorbance values were determined at 430 nm after 40 min against a blank. quercetin (q) (0.05 to 0.25 mg.ml-1) was used as a standard: .the tfc of the extracts was expressed as mg of quercetin per gram of dry extract (mg qe/g de). determinations of tpc were performed in triplicate. results were expressed as mean ± sd. gas chromatography-mass spectroscopy analysis (gc-ms) the identification and quantification of volatile bioactive compounds from z. lotus and r. chalepensis essential oils was carried out using a shimadzu gas chromatograph (gc), agilent gc model 7890b equipped with the hp 5977a mass spectrometer. analytical conditions: agilent 1227062 db-waxn capillary column (dimension: 60m, id: 250 micrometers, 0.25-micrometer film thickness). helium was used as a carrier gas with a linear velocity in a column of 19 cm.s-1 and 37.862 psi of pressure. the carrier gas split flow was about 250 ml.min-1, with a split ratio: of 100 : 1, and septum purge flow of 3 ml.min-1. the oven temperature program was between 70 and 260 °c with an equilibration time of 1 min and was then maintained at 250 °c. the injection in split mode of 2 µl of the substance to be analyzed was carried out using a microsyringe of 10 µl. the components were identified by comparing their relative retention times and mass spectra with the data from the library of eo constituents (wiley, mass-finder, and adams gc/ms libraries). the percentage composition determination was based on peak area normalization without correction factors. antimicrobial activity the agar disc diffusion method was applied to determine the antimicrobial potency of eos from z. lotus and r. chalepensis. muller-hinton agar (mha) plates were spread by adjusted microbial culture suspensions (0.5 mcfarland), and therefore, sterile discs were impregnated in 20 μl of each eo solution. the eos of 200 mg.ml-1 concentration were dissolved in the dimethyl sulfoxide 10 % (dmso) that was used as a negative control. the discs were aseptically deposited on the inoculated plates. after 2 h at 4°c, plates were incubated at 37 °c for 24 h, and the antimicrobial effect was expressed by measuring the diameters of the microbial growth inhibition zones (ø). each assay was carried out in triplicate. the eos effectiveness 4 nova biotechnol chim (2023) 22(1): e1519 3 assessment was determined as follows: ø < 8mm: resistant, 9 mm < ø < 14 mm: sensitive, 15 mm< ø < 19 mm: very sensitive, ø >20 mm: extremely sensitive (ponce et al. 2003). the microdilution method was performed to determine the minimum inhibitory (mic), bactericidal (mbc), and fungicidal (mfc) concentrations. the assays were done in sterile 96well microplates and examined according to the method described by chandrasekaran and venkatesalu (2004). briefly, 50 µl of mueller hinton and sabouraud broth (for bacteria and yeast tests, respectively) was distributed aseptically in all the sterile microplates' wells. subsequently, 50 µl of each eo sample of both tested plants, at a concentration of 200 mg.ml-1 was added to the first well, and then serial dilutions were obtained to achieve a final concentration of 1.56 mg.ml-1. after that, 50 μl of the adjusted microbial suspensions (0.5 mcfarland) were inoculated in each microplate well. the microplates were incubated at 37 °c, and microbial growth kinetics were measured by reading the optical density at 620 nm for bacteria and 450 nm for fungi at 0-4-18 – 48, and 72 h, using a microplate absorbance reader (tecan spectra ii microplate reader). the microbial tests were prepared in triplicate, and the results were expressed as log germs.ml-1 obtained for each plant extract concentration. checkerboard method during this study, we evaluated the synergistic, additive, or antagonistic effects using the combinations between both plants’ eos: zleo/rceo. the synergistic interaction of both antimicrobial drugs was quantified after the determination of the mic values for each plant eo (previously determined) by calculating the index of fractional inhibitory concentrations (fici or ∑fic), which are the lowest concentrations of the antimicrobial drugs in combination, inhibiting completely the growth of the microbial strains tested. a total of 50 µl of sterile mueller hinton broth was distributed aseptically in all the sterile cupules of the microplates. the first eo solution of z. lotus was serially diluted along the abscissa, while the eo of r. chalepensis was diluted along the ordinate. an inoculum equal to 0.5 mcfarland turbidity standards was prepared for each culture in sterile saline water. each suspension cupule was inoculated with 50 µl of the microbial culture, and the microplates were incubated at 37 °c for 18 h. the value of the association was measured using the fic in the cupules in which the microbial growth is inhibited and considered an effective mic for the combination (orhan et al. 2005). the ∑fics were calculated as follows eq. 1: (1) where, fic a = mic of drug a (ppe or eo of z. lotus) in the combination/mic of drug a (z. lotus extract) alone, and fic b = mic of drug b (ppe or eo of r. chalepensis) in the combination/mic of drug b (r. chalepensis extract) alone. the combination is considered synergistic when the fici is ≤ 0.5, additive when the 0.5 <fici ≤ 1, indifference: 1 <fici≤ 4 and antagonism: fici ˃4 (siqueira et al. 2021). statistical analysis replicates were prepared for all experiments. the results were given as means and standard deviations (mean ± sd). the means were compared using one-way and multivariate analysis of variance (anova). the differences between individual means were significant at p <0.05. results and discussion extraction yield and quantitative determination of polyphenols results of the extraction yields and the quantitative determination of polyphenol and flavonoid content in the essential oils of z. lotus and r. chalepensis are shown in table 2. statistical analysis showed significant differences between the yields of both plants eos, while the highest yield was obtained for rceo: 8.65 ± 0.025 % compared to z. lotus: 4.57 ± 0.015 % (table 2). yields of zleo and rceo were higher than reported in other studies. the eo yield from r. chalepensis (8.65 ± 0.025%) originated from the tafraoui region in oran (north-west algeria) was higher than that reported in other studies on the 5 nova biotechnol chim (2023) 22(1): e1519 2 same plant species originated from different regions of algeria e.g., from remchi region (1.17 %), naama region (0.19 %) (benammara et al. 2006), while from sidi bel abbes region was 7.23 % (boumediene 2014). according to attou (2011), the eo yield of r. chalepensis harvested in ain temouchent in algeria was 0.42 %, while in tlemcen it was around 2.21 % (benammra et al. 2006), and in the range 0.28 – 0.78 % (merghache et al. 2009). table 2. extraction yields and quantitative determination of polyphenols in the essential oils of z. lotus and r. chalepensis. p <0.05 (significant). plant extracts yield [%] tpc tfc zleo 4.57 ±0.015 8.47 ±0 3.33 ±0.26 rceo 8.65 ±0.025 8.56 ±0.154 7.07 ±0 tpc – total phenol content; tfc – total flavonoid content. compared to our results, a recent study by bekkar et al. (2021) reported that the eo yields were 4.16 ± 0.036 % in z. lotus leaves and 4.99 ± 0.86 % in r. chalepensis aerial parts from mascara in western algeria. in addition, the harvest region has an immense influence on medicinal plant essential oil yield. each area is characterized by climatic conditions (precipitation and temperature), which influence the physical qualities of medicinal plants, as well as soil type, moisture retention, and ph. other ecological factors can intervene in the development of the plant species: altitude, the harvest period, the plant part, the technique, as well as the extraction of the bioactive substances period, which influences not only the yield but also the plant extract chemical composition (lucchesi 2005; pinto et al. 2006; zouari et al. 2012). comparison of our results with other studies on the same plants harvested from other regions outside algeria. according to daoudi et al. (2016) and ali et al. (2013), the eo yield of r. chalepensis from the meknes region was estimated at 1 % and 0.84 %, respectively. on the other hand, the essential oil yield of r. chalepensis aerial parts (add region) was 0.27 % (dob et al. 2008). while in tunisia it was 5.51 % (mejri et al. 2010). moreover, the eo yield of r. chalepensis in the current study was higher than that of z. lotus. also, z. lotus and r. chalepensis from the tafraoui region in oran contain significant concentrations (p <0.05) of phenol (8.47 mg gae/g de) and (8.56 ± 0.154 mg gae/g de), respectively. at the same time, rceo was richer in flavonoids (7.07 ± 0 mg qe/g de) than zleo (3.33 ± 0.26 mg qe/g de). althaher et al. (2020) determined a tpc of 5 ± 0.005 mg gae/g of oil extract and a tfc of 34.09 ± 0.140 mg qe/g oil. in addition, it has been reported that the polyphenol content was much higher in both plant phenolic extracts than the essential oils (bekkar et al. 2021), which indicates that the polyphenolic extracts are considered plant drugs, representing a rich and potent reservoir of phenols than eos. physicochemical index of essential oils the physicochemical results of both plants’ eos carried out according to the french association for standardization (1996) method are mentioned in table 3. the fsm-ac (2013) recommends an eo density between 0.895 – 0.920 for very highquality eos, so, referring to our results, z. lotus and r. chalepensis eo samples are of good quality. another parameter that allows us to determine the purity of our eo samples is the refractive index. the fas standard recommends a refractive index of 1.495 for high-quality eos and 1.513 for lesserquality oils. indeed, in the present study, we determined an ri for zleo 1.441, and for rceo (4.426) (table 3). so, according to the results of this study, it appears that our eo samples of z. lotus and r. chalepensis are of very good quality. however, a high acid index for rceo was determined by comparing it with the zleo sample, for which a value of 0.933 was obtained. these results indicate that the eo of z. lotus contains fewer free acids compared to r. chalepensis. a high ester index also determines the quality of our eo samples. 6 nova biotechnol chim (2023) 22(1): e1519 3 table 3. physicochemical characteristics of z. lotus and r. chalepensis essential oils. physicochemical index plant essential oils rd20°c ri ors ai ei em zleo 0.6822 1.441 +7.5° 0.933 19.64 1v/1v rceo 0.7945 1.426 +10° 4.45 16.85 1v/2v rd 20°c – relative density at 20 °c; ri – refractive index; ors – specific optical rotation; ai – acid index; ei – ester index, em – miscibility with ethanol. according to dummortier (2006), higher ei values indicate the essential quality of eo samples. for comparison, the study conducted by alloun (2013) on r. chalepensis eo reports physicochemical characteristics with a density of 0.804 and a refractive index of 1.430. thus, merghache et al. (2009) proved that the physicochemical properties of r. chalepensis eo vary according to the harvest period and region. in the literature, no study has been described on z. lotus eo. therefore, no comparison has been made with previous studies. gas chromatography-mass spectroscopy analysis (gc-ms) the gc-ms analysis was used to determine the chemical composition of volatile bioactive compounds in the eo samples prepared from z. lotus leaves and r. chalepensis aerial parts harvested from the tafraoui region, oran, in western algeria. results are shown in table 4 and table 5. a total of 14 volatile components were identified for z. lotus and 12 compounds for r. chalepensis, representing 91.33 % and 58.84 % of the total eos, respectively. the chromatographic analysis allowed us to determine the majority chemotype of di-isooctyl phthalate (80.343 %) for zleo, while 2-undecanone was the major component of rceo (13.236 %), followed by chalepensine (12.547 %), 2-nonanol acetate (9.128 %), 2-nonanone (6.405%) and 2-undecanol acetate (4.411%) (table 4 and 5). other minor compounds were also identified and quantified in zleo: di-methylene sulfoxide (7.287 %), linalol 1.025, thioureatetramethyle (1.151%), thymol (0.292 %), linalyl acetate (0.110 %), 2-nonanone (0.059 %), pcymene (0.178 %) and ɤ-terpinene (0.060 %) (table 4). table 4. chemical composition of the essential oil of zizyphus lotus leaves harvested in tafraoui region-oran. n° rt volatil bioactive components [%] 1 11.190 ɤ-terpinene 0.060 2 12.075 p-cymene 0.178 3 14.485 methyl-heptenone 0.077 4 16.295 2-nonanone 0.059 5 17.580 1,1'-bicyclohexyl, cas no 92-51-3 0.409 6 20.974 linalol 1.025 7 21.257 linalyl acetate 0.110 8 21.776 di-methylene sulfoxide 7.287 10 22.524 2-undecanone methylene nonyl ketone 0.197 11 31.760 thiourea -tetramethyle 1.151 12 40.813 thymol 0.292 13 44.271 diethyl phthalate -dep 0.138 14 56.774 diisooctyl phthalate 80.343 components identified 91.326 n° – number; rt– retention time. table 5. chemical composition of the essential oil of ruta chalepensis aerial parts harvested in tafraoui region-oran. n° rt volatil bioactive components [%] 1 15.443 2-octanol acetate 0.229 2 16.291 2-nonanone 6.405 3 16.735 tetra-decane 0.655 4 18.586 2-nonanol acetate 9.128 5 19.501 2-decanone 0.784 6 20.25 2-nonanol 2.524 7 22.531 2-undecanone 13.236 8 24.389 2-undecanol acetate 4.411 9 26.086 2-undecanol 2.544 10 28.837 2-tridecanone 0.892 11 31.814 thiourea -tetramethyl 5.481 12 61.122 chalepensine 12.547 components identified 58.84 n°– number; rt– retention time. to the best of our knowledge, this is the second report on the leaf essential oils of z. lotus collected from western algeria, but from another region, for the determination of the harvest region influences 7 nova biotechnol chim (2023) 22(1): e1519 2 on the chemical profile of bioactive substances in the plant phenolic extracts and essential oils. moreover, limited studies have been reported on the biological properties of z. lotus essential oils. in contrast, most of the research is directed to this plant's polyphenolic extracts. for this, we were very interested in studying the biological properties of z. lotus essential oils. the study carried out by ourzeddine et al. (2017) on the chemical composition and the antioxidant activity of the fruit’s essential oil of z. lotus, harvested from the ouled fadhel region of batna (eastern algeria) showed that ethyl hexadecanoate (12 %), decanoic acid (11 %), ethyl dodecanoate (9.4 %), ethyl hexadeca-9-enoate (7.9 %), dodecanoic acid (6.5 %), ethyl tetradecanoate (6.1 %), tetradecanoic acid (5 %), ethyl decanoate (4.8 %), octanoic acid (3.1 %), ethyl undecanoate (2.8 %), nonanoic acid (2.4 %) and undecanoic acid (2.1 %) are the predominant components in the eo sample. another study by ghannadi et al. (2003) on the volatile constituents of z. lotus eos from iran showed different chemical profiles: geranyl acetone (14 %), hexadecanoate (10 %), inethyloctadecanoate (9.9%), farnesyl acetone c (9.9 %), hexadecanol (9.7 %), and ethyl-octadecanoate (8 %) which were found to be the main constituents. thus, bekkar et al. (2021) determined a majority chemotype of di-isooctyl-phthalate for z. lotus eos and 2-undecanone for r. chalepensis harvested from the el-mamounia region in mascara (western algeria), which is in agreement with the results of the present study however, a difference was recorded in the eos diversity in bioactive components, where the plants harvested from mascara in western algeria gave a chemical profile very rich in these compounds compared to the plants harvested from oran. by comparing our results with previous studies of mejri et al. (2010) and merghache et al. (2009) on the essential oil chemical composition of r. chalepensis collected from tunisia and western algeria (tlemcen), respectively, showed that the eos chemical profile is very variable, with 2-undecanone always being the predominant component (69.23 % and 43.71 %, respectively). thus, in their study, rustaiyan et al. (2002) showed that rceo is dominated by 2-undecanone (52.5 %). haddouchi et al. (2013) showed the predominance of 2-nonanone and 2-undecanone: 32.79 % and 32.58 % respectively, followed by 1nonene at 13.95 % and ɑ-limonene at 5.27 % in the eo of the plant harvested at ain temouchent, algeria. in jordan, the chemical composition of essential oil from r. chalepensis was rich in nonterpenoid aromatic compounds (80.32 %), and the major identified compounds were 2-nonanone (19.45%), methyl hexadecanoate (4.04 %), and 4,5dimethoxy-6-prop-2-enyl-1,3-benzodioxole (1.87 %) (althaher et al. 2020). antimicrobial activity tests results of the antimicrobial activity assessments are shown in table 6, fig. 1 and 2. a potent antimicrobial effect was recorded on all bacterial strains, and significant antifungal activity on c. albicans, with diameters of the microbial growth inhibition zones exceeding 10 mm. the largest inhibition diameters were recorded using zleo: 40.16 ± 0.15mm, 40.1 ± 0.1mm and 24.06 ± 0.12mm against h. alvei, s. enterica subsp. arizonae, and p. mirabilis, respectively, with extremely high susceptibility (table 6). in addition, our results indicated that the essential oils of these plants are more effective in inhibiting the growth of major pathogenic bacteria and yeast isolated from gastroenteritis, urinary tract infections, and periodontitis diseases, which may justify the use of z. lotus and r. chalepensis in the treatment of these pathologies. regarding the antibiotic susceptibility testing, zleo and rceo were more active against the clinical strains however, the inhibitory potency of z. lotus eos was more important than r. chalepensis eo. z. lotus and r. chalepensis eos were more efficient against all gram-positive, gram-negative bacteria, and c. albicans with its significant effects (p <0.05), while s. aureus and e. faecalis among the gram-positive bacteria were the most susceptible to zleo and rceo respectively. among the gram-negative bacteria, h. alvei, s. enterica subsp. arizonae, and p. mirabilis were the most sensitive to zleo with the qualification as extremely high susceptibility of these germs to zleo regarding the important diameters of the growth inhibition zones measured (table 6). 8 nova biotechnol chim (2023) 22(1): e1519 3 the most effective effect of rceo was recorded against s. enterica subsp. arizonae and c. albicans, with diameters of growth inhibition zones of 17.13 ± 0.15mm and 14.06 ± 0.12 respectively (table 6). so, an important anti-candida activity was detected. however, both plants’ eos did not exert any antibacterial effect on s. pyogenes (table 6), which explains the high-frequency resistance of this clinical strain to antimicrobials. for gramnegative bacteria, some strains were distinguished by a very high sensitivity compared to others, as shown by the case of s. enterica subsp. arizonae and h. alvei, whose inhibition diameters were much higher and exceeded 30 mm by applying z. lotus and r. chalepensis eos compared to the antibacterial effect of r. chalepensis. table 6. antimicrobial activity of essential oils of zizyphus lotus and ruta chalepensis harvested in tafraoui region oran against pathogenic clinical germs. the values are presented as the mean of three replicates ± the standard deviation. p <0.05 (significant). clinical strains diameters of growth inhibition zones [ø mm] zleo cmizleo rceo cmirceo s. aureus 14.06 ± 0.12s 100 10.06 ± 0.12s 100 s. pyogenes ne 100 ne 100 e. faecalis 10.03 ± 0.06s 25 13.1 ± 0.1s 100 e. coli (epec) 15 ± 0.1hs 100 9.16 ± 0.15s 50 p. mirabilis 24.06 ± 0.12ehs 100 11.06 ± 0.12s 50 s. enterica subsp. arizonae 40.1 ± 0.1ehs 50 17.13 ± 0.15hs 25 h. alvei 40.16 ± 0.15ehs 50 10.03 ± 0.06s 50 c. albicans 15.03 ± 0.06hs 25 14.06 ± 0.12s 25 ø (mm) – diameters of growth inhibition zone in millimeter; ne – no effect; r – resistance (ø < 8mm); s – sensitivity (9 mm < ø < 14 mm); hs – high susceptibility (15 mm < ø <19 mm); ehs – extremely high susceptibility (ø >20 mm). all these results were completed by quantitatively determining an important antimicrobial parameter, the minimum inhibitory concentration (mic). the results of the mic values for each clinical strain are mentioned in table 6. the curves of the microbial growth kinetics in the presence of z. lotus and r. chalepensis eos are mentioned in fig. 1 and 2. an important decrease in microbial cell concentration was detected after the 4th hour, expressed by the decrease in the log germs/ml number (fig. 1 and 2). the inhibitory properties of the essential oils of z. lotus and r. chalepensis against all the microbial strains were determined, with the lowest mic values of 25 mg.ml-1 against e. faecalis and c. albicans using the zleo and against s. enterica subsp. arizonae and c. albicans using the rceo (table 6). the bactericidal and fungicidal effects could be explained by the abundant richness of z. lotus leaves in diisooctylphthalate, the major component in zleo, and r. chalepensis aerial part in 2-undecanone, the major volatile compound in rceo. numerous studies have demonstrated the antimicrobial effect of r. chalepensis essential oils with the 2-undecanone chemotype (marami et al. 2021; nahar et al. 2021). our results agreed with those of other studies (belkassam et al. 2011; zellagui et al. 2012; alloun 2013; arámbula et al. 2019). our results also agree with those of daoudi et al. (2016) on the antibacterial effect exerted by the essential oil of r. chalepensis aerial part on s. aureus and p. mirabilis. however, the results showed that the rceo has no effect on e. coli, which does not agree with our results. during the current study, an important antibacterial effect was recorded on this bacterial strain by applying the eo of this plant. thus, our results agree with those mentioned in another study that showed that the r. chalepensis eo has antibacterial activity against salmonella, e. coli, and s. aureus (amdouni et al. 2016). the inhibitory effect of z. lotus and r. chalepensis could be explained by the most abundant richness of these plants eos in secondary metabolites, in particular antimicrobial compounds, which justified specific uses of these medicinal plants in the treatment of several infectious diseases (garcíalafuente et al. 2009; ben-bnina et al. 2010; 9 nova biotechnol chim (2023) 22(1): e1519 2 haddouchi et al. 2013; boual et al. 2015; hammi et al. 2015). fig. 1. antimicrobial effect of zizyphus lotus essential oil on clinical strains isolated from the different biological samples (p <0.05). c1-c8 – concentrations. c1 – 200 mg.ml-1; c2 – 100 mg.ml-1; c3 – 50 mg.ml1; c4 – 25 mg.ml-1; c5 – 12.5 mg.ml-1; c6 – 6.25 mg.ml-1; c7 – 3.13 mg.ml-1; c8 – 1.56 mg.ml-1; t – control test. a – staphylococcus aureus; b – streptococcus pyogenes; c – enterococcus faecalis; d – enteropathogenic escherichia coli; e – proteus mirabilis; f – salmonella enterica subsp. arizonae; g – hafnia alvei; h – candida albicans. 10 nova biotechnol chim (2023) 22(1): e1519 3 fig. 2. antimicrobial effect of ruta chalepensis essential oil on clinical strains isolated from the different biological samples (p <0.05). c1-c8 – concentrations; c1 – 200 mg.ml-1; c2 – 100 mg.ml-1; c3 – 50 mg.ml1; c4 – 25 mg.ml-1; c5 – 12.5 mg.ml-1; c6 – 6.25 mg.ml-1; c7 – 3.13 mg.ml-1; c8 – 1.56 mg.ml-1; t – control test. a – staphylococcus aureus; b – streptococcus pyogenes; c – enterococcus faecalis; d – enteropathogenic escherichia coli; e – proteus mirabilis; f – salmonella enterica subsp. arizonae; g – hafnia alvei; h – candida albicans. 11 nova biotechnol chim (2023) 22(1): e1519 2 checkerboard method the individual and combination minimum inhibitory concentrations, the individual fractional inhibitory concentrations, the index inhibitory fractional concentrations, and the fic index of z. lotus and r. chalepensis eos combinations against the different microbial strains tested are mentioned in table 7. no synergistic effect was determined by applying the combination of z. lotus and r. chalepensis eos against the different microbial strains tested. this combination exerted an antagonistic effect against the microbial strains tested, with ficindex values greater than 4 (ficindex˃4). while an indifference interaction was recorded against s. aureus, s. pyogenes, and h. alvei using both plants eos (table 7). these results have enabled us to demonstrate the ineffectiveness of plant essential oils used in combination. in some cases, associations between antimicrobial drugs can be used to broaden their action spectrum on pathogenic germs. various studies have shown the effectiveness of plant extracts and essential oils combinations in the synergistic effect expression against pathogenic bacteria. amirouche and belkolai (2013) demonstrated that a combination of sage and tea tree essential oils works synergistically against s. aureus. however, it was determined during this study that the effect of the eo combinations is lower compared to the effect exerted by the eos of each plant alone. therefore, z. lotus and r. chalepensis cannot be used in combination due to the antagonism effect exerted on the different microbial strains tested. thus, the combination of these eos can limit and reduce the action spectrum of pathogenic bacteria and yeasts. table 7. ficindex of the combinations of z. lotus and r. chalepensis essential oils on the different microbial strains tested. clinical strains essential oils: zleo/rceo individual mics mics in combination individual fic ficindex s. aureus 100/100 200/200 2/2 4i s. pyogenes 100/100 200/200 2/2 4i e. faecalis 25/100 200/200 8/2 10a e. coli 100/50 200/200 2/4 6a p. mirabilis 100/50 200/200 2/4 6a s. enterica subsp. arizonae 50/50 200/200 4/4 8a h. alvei 50/50 100/100 2/2 4i c. albicans 25/25 200/200 8/8 16a a – antagonism; i – indifference. conclusion z. lotus and r. chalepensis eos are potent antimicrobials with a comprehensive action spectrum against pathogenic microbial strains. however, both plants' eos combinations were less effective against all the tested microbial strains, with antagonism effects. so, the impact of each plant eo alone is considered more important in inducing the antimicrobial effect than using of eo combinations in both plants. z. lotus and r. chalepensis essential oils could be used in the medical and food industries as potent antimicrobials for combating antimicrobial resistance. conflicts of interest the authors declare that they have no conflict of interest. acknowledgment the authors thank the university of mascara (algeria) for providing financial support to achieve this work, to the laboratory of bioconversion, microbiological engineering and health safety of mascara university in algeria for providing support in completing this study. to pr. bahadir keskin in the department of chemistry, faculty of arts & science, yildiz technical university, tr34210 istanbul, turkey for his encouragement and the interest he gave to completing a vital part of this study. 12 nova biotechnol chim (2023) 22(1): e1519 3 references ali a, demirci b, tuba kiyan h, bernier ur, tsikolia m, wedge de, khan ia, husnu can başer k, tabanca n (2013) biting deterrence, repellency, and larvicidal activity of ruta chalepensis (sapindales: rutaceae) essential oil and its major individual constituents against mosquitoes. j. med. entomol. 50: 1267-1274. alloun k (2013) chemical composition and antioxidant and antimicrobial activity of essential oils of dill (anethum graveolens l.), sage (saliva officinalis l.) and mountain rue (ruta montana l.). magister thesis, national school of agronomic, el-harrach, algiers. althaher a, oran s, bustanji y (2020) phytochemical analysis, in vitro assessment of antioxidant properties and cytotoxic potential of ruta chalepensis l. essential oil. j. essent. oil-bear plants. 23: 1409-1421. amdouni t, ben abdallah s, msilini n, merck f, chebbi m, lachaal m, karray-bouraoui n, ouerghi z, fernandez x (2016) effect of salt stress on the antimicrobial activity of ruta chalepensis essential oils. acta physiol. plant. 38: 113. amirouche r, belkolai f (2013) in vitro effect of the association of essential oils of salvia officinalis, melaleuca alternifolia and two major compounds on bacteria. engineer thesis, faculty of nature and life sciences, mira a university, bejaia. arámbula ci, diaz ce, garcia mi (2019) performance, chemical composition and antibacterial activity of the essential oil of ruta chalepensis and origanum vulgare. j. phys. conf. ser. 1386: 012059. attou a (2011) contribution to the phytochemical study and biological activities of the extracts of the plant ruta chalepensis (fidjel) from the region of ain témouchent. abou bekr belkaid university, tlemcen, p. 119. ayad n, benaraba r, hemida h, abdellah f (2022) biological activities of phenolic extracts from artemisia herba-alba asso grown in western algeria. eur. j. biol. res. 12: 46-61. bakhtaoui fz, lakmichi h, megraud f, chait a, gadhi cea (2014) gastro-protective, anti-helicobacter pylori and, antioxidant properties of moroccan zizyphus lotus l. j. appl. pharm. sci. 4: 81-87. bekkar neh, meddah b, keskin b, sonnet p (2021) oral acute toxicity, influence on the gastrointestinal microbiota and in vivo anti-salmonellosis effect of zizyphus lotus (l.) and ruta chalepensis (l.) essential oils. j. appl. biotechnol. rep. 8: 13-26. belkassam a, zellagui a, gherraf n, lahouel m, rhouati s (2011) essential oil composition of algerian ruta montana (clus.) l and its antibacterial effects on microorganisms responsible for respiratory infections. adv. nat. appl. sci. 5: 264-268. ben bnina e, hammami s, daamii remadi m, ben jannet h, mighri z (2010) chemical composition and antimicrobial effects of tunisian ruta chalepensis l. essential oils. j. soc. chim. tunisie 12: 1-9. benammar c, hichami a, yessoufou a, simonin am, belarbi m, allali h (2010) zizyphus lotus l. 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sulphites, acidity and ethanol content. low concentrations of heavy metals and sulphite contents are determined by making use of stripping chronopotentiometry. for the measurement of acid and ethanol content thin-layer coulometric titration is used. key words: wine analysis, flow-through coulometry, heavy metals, sulphite, acids, ethanol 1. introduction wine is a traditional beverage consumed all over the world. its quality depends not only on the sensoric characteristics but also on the presence, or better absence of undesirable, mostly allergic or even toxic substances, such as heavy metals, high sulphite contents. acid and ethanol contents significantly influence the taste and long term stability of wines. electrochemical methods provide a useful tool for simple, fast and economical determination of numerous species in wine samples. incorporating the measuring cell into a flow system it is possible to provide the analysis on full automated manner, controlled by a pc or microprocessor. electrochemical flow-through cells specially designed for automatic and virtually maintenance-free operation facilitate the measurement of various species in wine samples: heavy metals (cd, pb, hg), semimetals (as, se), anions (sulphite, chloride), organics (ethanol, ascorbic acid, tartric acid). the measurement of heavy metals is based on the deposition/stripping procedure, where the metal ions are electrochemically deposited on the electrode surface and then are stripped by constant current whereas the duration of the stripping is measured. this mode enables the measurement of low metal concentrations, down to the μg/l contents. sulphite (as free so2 or total sulphite) can be measured in a similar way, just making use of a special gold coated electrode adsorbing so2 molecules. the interfering effect of other species, especially those of chloride ions is eliminated by making use of an on-line silicone separator. the procedure was successfully applied for beer samples (dvorak et al., 2006). the acid content is determined by direct reduction of h+ ions 102 beinrohr, e. et al. to hydrogen in an encapsulated gold or platinum electrode, whereas ethanol is measured through its electrochemical oxidation to acetaldehyde on a platinum electrode coated with a catalytic surface. 2. experimental flow-through electrochemical measurements were carried out by an electrochemical analyser ecaflow 150 (istran, s.r.o., bratislava, slovakia), equipped with two solenoid valves, a peristaltic pump, 1 mm inned diameter ptfe tubing and a microprocessor controlled potentiostat/galvanostat. the block diagram of the system is depicted in fig. 1. fig. 1. block diagram of the flow-through analyser ecaflow (beinrohr et al., 1999). the pump can be positioned downstream or upstream to the cell. the manifold is an option and may comprise a mixing chamber, in-line filter, etc. the flow rate is controlled by the tube diameter of the peristaltic pump and by its revolution rate. the signals were recorded and evaluated by the memory-mapping technique (thomsen et al., 1994). compact flow-through electrochemical cell of type 353c and 104 with pt auxiliary and ag/agcl reference electrodes were used (istran, s.r.o., bratislava, slovakia). the working electrodes used were of the type of e-56l or e104l (zn, cd, pb, cu), e-t/ax (sulphite), e-t/pt (acids, ethanol) (all of istran, s.r.o., bratislava, slovakia). the measurement consisted of two main steps: i) the background signal was measured first by means of the blank sample, ii) then, the sample or standard solution was measured. the background signal was then subtracted from this signal yielding a true background corrected net signal (manova et al., 2007). nova biotechnologica vii-i (2007) 103 analytical-reagent grade chemical were used in all experiments. deionised and degassed water was used for the preparation of all solutions. 3. results and discussion 3.1 heavy metals low concentration of heavy metals can be measured electrochemically after a preconcentration step ensuring the deposition of sufficient amount of analyte species on electrode. then, the deposit can be dissolved (“stripped”), whereas the duration of the stripping is measured (stripping chronopotentiometry). reasonable signal-tobackground ratios can be achieved even for very low concentrations. the measurement of zn, cd, pb and cu is usually done on mercury or mercury-coated electrodes, where corresponding amalgams are formed (m denotes the corresponding metal): deposition: m2+ + 2 e= m(hg) stripping: m(hg) – 2 e= m2+ fig. 2. stripping chronopotentiograms of simultaneous measurement of zn, cd, pb, and cu (signals from left to the right) in a red wine sample by the technique of standard additions. sample diluted 10-times in 0.1 mol/l hcl, carrier electrolyte 0.1 mol/l na2so4 with acetate buffer (ph 4.8), electrode e-56l, deposition at – 4 ma, stripping with 0.2 ma, sample volume 1 ml. the former method makes use of hanging mercury electrode, the latter a thin layer of mercury, usually on carbon surface. both approaches are less suitable for routine use, especially where mercury material or mercury plating solutions are not welcomed. a reasonable way is immobilise the mercury film into a compact electrode which is 104 beinrohr, e. et al. the used until fouling and then is returned to the producer for discharge. in this study a porous glassy carbon electrode with mercury and nafion® coatings was used. the nafion® film significantly enhances the stability of the mercury coating and eliminates interferences from many organic substances. moreover, owing to its hydrophilic properties, bubbles are the easily removed from electrode bulk which enhances the signal reproducibility. however, the matrix of wine samples is complex and may interfere. to minimise this, the sample should be sufficiently diluted (lastincova et al., 2006). a typical recording of a red wine analysis is depicted in fig. 2. 3.2 sulphites sulphites are usually determined by the modified monier-williams method (aoac, 2000) which is tedious and time consuming. gold electrode materials may adsorb sulphur dioxide, which can be used for simple and fast sulphite determination in foods and beverages. here, the sample is acidified to convert sulphites to sulphur dioxide, which is the collected on a suitable gold electrode. the adsorbed species are the galvanostatically oxidised, presumably to sulphate and the duration of this step is measured and evaluated. there are some interferences, the most serious one is associated with chloride anions. adsorption may be a reason for this, which passivates the electrode surface. a simple way to minimise this is to utilise the easy diffusion of sulphur dioxide through a silicone membrane, whereas the interfering chloride ions remain in the original solution. a thin-wall silicone tube in a form of a submersible coil was used for this: the sample was acidified, the silicone coil was immersed into it and a trapping solution was pumped through the tube into the measuring cell. obviously, the calibration should be done in the same fashion. total sulphite content is determined after hydrolysing the sample on adding sodium hydroxide solution. then, the released sulphite together with the free one is measured in the same way as free sulphite. 3.3 ethanol ethanol can directly be oxidised electrochemically on platinum electrode, which is the principle of its potential use in fuel cells (iwasita, 2002). the product of this oxidation is acetaldehyde up to carbon dioxide, depending on the conditions. the electrode surface should exhibit some catalytic properties towards ethanol oxidation. a simple way to achieve this is to coat the electrode with a thin layer of nafion®. a typical signal of ethanol measurement is depicted in fig. 3. table 1. analysis of wine samples for sulphite (beinrohr et al., 2006) sample free so2, mg/l total so2, mg/l müller thurgau (slovakia) 45.1 ± 3.1 88.4 ± 4.5 rizling (slovakia) 54.3 ± 4.3 115 ± 3.7 cabernet sauvignon (slovakia) 35.1 ± 2.4 79.6 ± 5.7 chiraz (california) 56.2 ± 4.2 125 ± 4.3 vino di tavola (italy) 31.5 ± 2.6 82.6 ± 3.5 nova biotechnologica vii-i (2007) 105 fig. 3. oxidation peak of ethanol in a diluted white wine sample (lower curves) and that for an ethanol standard in water (0.0032 %, v/v, higher peak). electrolyte 0.1 mol/l na2so4, electrode e-ca/pt, oxidation current 5 μa. 3.4 acidity acid-base titrations can easily be carried out electrochemically by making use of thin-layer cell arrangements. the neutralisation of hydroxonium ions is done by their direct electrochemical reduction to neutral hydrogen: 2 h3o + + 2 e= h2 + 2 h2o the sample should contain a neutral electrolyte not comprising species which are reduced at similar potentials as the hydroxonium ions. the potential of the reduction depends mainly on dissociation constant (-s) of the acid, the lower is the dissociation constant the more negative is the potential of the reduction and vice versa. at very low dissociation constants (below 10-7), the reduction peak of hydroxonium ions coalesce with the reduction of water. dissolved oxygen is reduced as well and may contribute to the hydroxonium ion content if the concentration of the former is higher than that of the latter (about 10-4 mol/l at room temperature). anyway, it is possible to measure simultaneously strong and weak acids as it is known in volumetric titrations. the acidity of wines is expressed as tartaric acid content and is determined by volumetric titration either to a predefined ph value (e.g. ph 7) or to an indicator’s colour change. 4. conclusions flow-through coulometry has proved to be a suitable method for the determination of numerous species in wine samples. its main advantage is simplicity, low costs and 106 beinrohr, e. et al. available instrumentation. however, only electrochemically active species can be measured and the elimination of possible interferences may be a problem, especially in wine samples with high sugar and carbon dioxide contents. the latter problem can be solved by proper sample preparation, e.g. by sample dilution and ultrasonic degassing. anyway, the presented method may become a reasonable alternative or complement to spectroscopic and chromatographic methods in food and beverage analysis. acknowledgement: the financial support of the slovak ministry of education (applied research grant) is greatly appreciated. references aoac official method 990.28.: sulfites in foods, optimized monier-williams method. aoac official method of analysis (2000) 47.3.43 p29. beinrohr, e cakrt, m., dzurov, j., jurica, l., broekaert, j. a. c.: simultaneous calibrationless determination of zinc, cadmium, lead and copper by flow-through stripping chronopotentiometry. electroanalysis, 11, 1999, 1137-1144. beinrohr, e., manova, a., strelec, m., dzurov, j.: jednoduche a rychle stanovenie siricitanov vo vinach (simple and fast determination of sulphites in wine). chemagazin, 16, 2006, 6-7. dvorak, j., dostalek, p., sterba, k., cejka, p., kellner, v., culik, j., beinrohr, e.: determination of total sulphur dioxide in beer samples by flowthrough chronopotentiometry. j. inst. brew., 112, 2006, 308-313. iwasita, t.: the electrocatalysis of ethanol oxidation. proceedings of the 3rd lamnet workshop, brasil, 2-4 december, 2002, 76-83. lastincova, j., cavojcova, i., pospisilova l., beinrohr, e.: proceedings of the xxxth oiv world congress, budapest, hungary, 10-16 june, 2007. manova, a., humenikova, s., strelec, m., beinrohr, e.: determination of chromium(vi) and total chromium in water by in-electrode coulometric titration in a porous glassy carbon electrode. microchim. acta, 159, 2007, 41-47. thomsen, k. n., skov, h. j., dam, m.: a flexible instrument for voltammetry, amperometry and stripping potentiometry. anal. chim. acta, 293, 1994, 1-8. microsoft word schmidtszakal nb.doc nova biotechnologica vii-i (2007) 57 the application of copper and zinc containing ion-exchanged synthesised zeolite in agricultural plant growing rezső schmidt, pál szakál university of west hungary faculty of agricultural and food sciences 9200 mosonmagyaróvár, vár, hungary (schmidtr@mtk.nyme.hu) abstract: foliar application of copper and zinc containing ion exchanged synthesised „zeolon-p4a” type zeolite was used in experiments. foliar application in winter wheat experiments proved that plants had a better uptake and utilisation of copper and zinc bound on zeolite. as a result of the treatments yields and four quality parameters increased. key words: zeolite, copper, zinc, retard, protein, gluten, baking quality index 1. introduction winter wheat (triticum aestivum l.) is grown on most of the arable land of hungary. quality loss causes an even greater problem in maintaining export competitiveness. this problem is due to weather conditions but mainly to nutrient supply deficiency (kádár, 1992). qualitative and quantitative plant growing can only be achieved if it is combined with proper plant nutrition. besides the three macro elements (n, p, k) essential micro elements are inevitable. because of their function in the enzyme activity we have to give priority to the essential microelements copper and zinc in the nutrient supply of soil and plants. their lack greatly influences both the quantity and the quality of plant growing. winter wheat gives an especially sensitive response to these microelements. 30% of hungary’s soils show copper deficiency, and 60-70% of zinc. the lack of these microelements can be restored through the soil or the foliage (szakál et. al.; 2000, 2003). in order to deliver microelements copper salts are mainly used for soil treatment and different microelement complexes are used for foliar treatments for the sake of better utilisation and better uptake. in our experiments we studied the influence of copper and zinc ion exchanged synthesised zeolite on winter wheat. plants leaves ensure nutrient uptake for the development of plants. photosynthesis and the regulation of transpiration are the primary tasks of foliage. because of their structure leaves can uptake nutrients under certain conditions and to a certain extent only. foliar nutrient supply can only be successful if we find the proper compounds and conditions. the plant controls ions reaching the cell walls by the turgor pressure in the skin tissues. nutrient ions passing through the cell walls are adsorbed and get into the protoplasm. the movements of ions taken up through the leaves differ from each other greatly. the advantage of nutrient uptake through the leaves is that it gets very quickly and directly to the leaf cells, where they are utilised. 58 schmidt, r. and szakál, p. 1.1. microelements copper and zinc the average zinc content of the earth’ crust is about 70 mg/kg, and its copper content about 55 mg/kg. only a very small part of them is in a form that is absorbable by plants. the copper and zinc content of hungary’s arable land is very limited: the upper cultivated layer contains 90-450 kg of zinc and 12-10kg of copper per hectare. about 1 %of them is in an available form (győri, 1962). copper has a stronger complex forming capacity than zinc, and because of its higher adsorption energy a small part of it is in an available form. primary function of copper and zinc is that through its positive charge it can contact electron rich parts of small and large molecules, mainly proteins being present in living organisms. metal ions can be bound to proteins in different ways: metallo-enzymes: if metal ions are strongly bound to side chains of aminoacids, enzyme-activating: if metal ions are not a part of the structure but their presence is inevitable to induce enzyme activity. the role of essential microelements copper and zinc was proved in forming of enzymes by more than 200 enzymes (spiro, 1983). copper can be found in high amounts in the granum and chloroplast. its lack hinders nitrogen uptake and protein synthesis. it plays an important role in transpiration metabolism and electron transport. copper plays a role in the redox processes in cells, and zinc in h-transportation reactions (kőrös, 1980). as a result of acid rains metal ions leach out and hinder enzyme activity, which directly influences plant development (dessler and börlitz, 1986). the relationship between auxin and zinc is known since 1940. zinc directly influences plant growth, enhances the activity of rna polymerase and glutamine-acid dehydrogenase in nitrifying micro-organisms and improves the ammonia-n metabolism. an abundant supply of zinc contributes to triptophan synthesis in plants. 1.2. influence of copper and zinc compounds on flour quality hungarian and foreign researchers have tested the influence of copper and zinc on flour quality for a long time. publications on the application of chelates and other complexes are already available. flyn et al. (1987) observed an increased protein content and yield in their experiments. the same results achieved peterson et al. (1986), han and shepherd (1991). higher copper and zinc content resulted in higher protein content. the department of soil management and the department of chemistry at the faculty of agriculture and food sciences of the university of west hungary carried out experiments with copper and zinc complexes of different ligands and applied them in soil or foliar treatments for more than 20 years (schmidt et al., 1999, barkóczi et al., 2000, szakál et al., 2003). these experiments delivered especially good results with the application of copper-tetramine and zinc-carbohydrate complexes in foliar treatments. the best results were achieved when the experiments were carried out in the phenological phase of flowering. we produced and used synthesised ion-exchanged copper-zeolite in our experiments, which is not commonly nova biotechnologica vii-i (2007) 59 used in agriculture. with the use of ion-exchanged zeolite we proved that a slowrelease process was taking place and the compound had a positive effect on raw protein content and baking quality index. zeolites are crystal alkalior alkali-earth metal-aluminium-hydro-silicates. the trivalent al containing tetrahedrons have one negative charge, therefore the positive charged metal ions neutralise it. the smallest units of zeolites are the cells, which can be explained by the following formula: mx/n [(alo2)x(sio2)y] wh2o, where m cation is of n valent, w is the number of water molecules, (y+x) is the number of tetrahedrons in the elemental cell. y/x i.e. rate of si/al important feature of zeolites can range from one to infinity, e.g. the zeolite structured silicates practically do not contain any al. the rate of 1:1 is theoretically the lowest limit, as in zeolites alo4 tetrahedrons cannot connect with each other directly but through sio4 tetrahedrons. in zeolite structured metal silicates the rate of si/m can be very low otherwise it will damage the structure. we call second generation zeolites those which are synthesised with the use of ionic or neutral organic compounds, mostly amines or quaternary ammonium salts, which have a leading role in structure-forming owing to their structure controlling effect. 1.3. ion exchange of zeolites from physically and chemically point of view ion exchange is balancing process, which can be described by ion exchanging isotherms. the balance of ion exchange can be described by the following formula: zba za s + zab zb z = zba za z + zab zb s where zb, za means the cation charge, sub-index s means concentrations of balance in the solution, z on the zeolite. synthesised zeolites generally contain na ions. through ion exchange of na-ions by other ions the pore size of zeolite changes. through exchange of ions of larger pore size it will decrease (e.g. the pore diameter of na-lta decreases from 0.4 nm to 0.3 nm). if the pore diameter of the entering ion is smaller the phenomenon will turn round (e.g. in case of calcium ions the pore entry is 0.5 nm). fig. 1. change of zeolite pore sizes affected by cations (kiricsi and hannus, 2007.) 60 schmidt, r. and szakál, p. exchange of ions is size selective. ion exchange is carried out in water medium. ions are there in hydrated form. hydrated forms are bigger in size, therefore they cannot always penetrate into the structure of zeolite; in dehydrated form ion exchange is easy. ion exchange is carried out at a temperature of 80 °c, hydrate membrane is smaller. 2. material and methods experiments on winter wheat were launched in four repetitions randomized blocks design on plots of 10m2. foliar treatments were introduced at the end of tillering on danube alluvial soil. treatments were done with high pressure hand sprayers. we took samples and measured yield and chemical composition. the zinc content of the applied zinc-zeolite was 2.8 m%, and copper content of copper-zeolite was 4.4 m%. zeolites were mixed and applied in the form of water-suspension. plots were harvested with plot-combine harvester. 2.1 effect of copper and zinc on the yield as a result of copper ion exchanged synthesised zeolite and zinc ion-exchanged synthesised zeolite treatments yields increased in both cases. the most considerable yield increase was produced by copper-zeolite treatments. based on the copper-zeolite experiments we can state that as a result of copper-zeolite treatments yields increased significantly, but zinc-zeolite treatments produced only a little rise in yield. 4,00 4,20 4,40 4,60 4,80 5,00 5,20 0,0 1,0 2,0 3,0 dose kg/ha yi el d t/h a cu-zeolit zn-zeolit fig. 2. effect of zn zeolite and cu-zeolite on the yield 2.2 effect of copper-zeolite, zinc-zeolite on raw protein content as a result of copper-zeolite the raw protein content increased significantly but at a lower rate, than due to zinc-treatment. as an effect of zinc-treatment the raw protein content increased continuously. no toxic effect could be shown even at a dose of 2 kg/ha. nova biotechnologica vii-i (2007) 61 12,00 12,50 13,00 13,50 14,00 14,50 0,0 1,0 2,0 3,0 dose kg/ha ra w p ro te in % cu-zeolit zn-zeolit fig. 3. effect of zn-zeolite and cu-zeolite on raw protein 3. conclusions experiments of foliar treatments were carried out with ion-exchanged zeolite on winter wheat for three years. the experiments were launched on small plots of calcareous danube alluvial soil. on average of three years copper treatments were effective, if yield increase was set as a goal. as a result of zinc-zeolite the increase of raw protein was more favourable than that of copper-zeolite treatment. references barkóczi, m., szakál, p., schmidt, r.: mikroelem-tartalmú hulladékok mikroelem-tartalmának zeolittal történő kinyerése és annak felhasználása növénytáplálási célra. xvi. országos környezetvédelmi konferencia és szakkiállítás. proceeding. 2002, 240-247. dessler, h.g., börlitz, s.: modellversuche zur auswaschung von lonen aus pflanzenteilen durch „saueren regen”. mengen und spurelementen arbeitstagung, karl, mras universitat, leipzig. 1968. győri, d.: a mg, zn,m cu, mo co mikroelemek eloszlása és vegyületformái néhány talajtípusban. mta agrátud. oszt. közll., 21, 1962, 1-2. han, b., shepherd, k.w.: the correlations between ljmw gluten in subunits and gliadins and their effects on bread-making quality in the progeny of two wheats. scientia agricultura sinica, 24, 1991, 19-25. kádár, i. : a növénytáplálás alapelvei és módszerei. mta taki. budapest, 1992, 0-75. peterson, c.j., johnson, v.a., mattern. p.j.: influence of cultivar and environment on mineral and protein concentrations of wheat flour, bran, and grain. cereal chem., 63, 1986, 183-186. schmidt, r., barkóczi, m., szakál, p., horak, o., lesny, j.: hulladékból előállított fém-komplexek mezőgazdasági újrahasznosítása. xiii. országos környezetvédelmi konferencia. kiadvány, 1999, 206-214. spiro, t.g.: metal ions in biology. zinc enzymes, new work. 1983 62 schmidt, r. and szakál, p. szakál, p., schmidt, r., lesny, j., vegh, d., horak, o., tölgyessy, j.: application of ion exchanged copperand zinc containing zeolites in agriculture. analytical and environmental conference, mosonmagyaróvár, 2000, 109-113. szakál, p., schmidt, r., barkóczi, m., kalocsai, r., giczi, zs.: the effect of copper containing ion exchanged zeolite on the yield and protein content of winter wheat. trace elements in the food chain. bessenyei györgy könyvkiadó, 2003, 237-238. szakál, p., schmidt, r., barkóczi, m., kalocsai, r., giczi, zs.: the effect of copper containing ion exchanged zeolite on the yield and protein content of winter wheat. trace elements in the food chain. bessenyei györgy könyvkiadó, 2003, 237-238. nova biotechnologica et chimica 11-2 (2012) 133 doi 10.2478/v10296-012-0015-y © university of ss. cyril and methodius in trnava study of precipitating methods for elimination of heavy metals from acid mine drainage alena luptáková1, stefano ubaldini2 , eva mačingová1, ingrida kotuličová1 1institute of geotechnics of slovak academy of sciences, watsonova 45, košice, sk-043 53, slovak republic (luptakal@saske.sk) 2institute of environmental geology and geoengineering, cnr, roma, italy (stefano.ubaldini@igag.cnr.it) abstract: the submitted paper deals with the study of combination of chemical and biological-chemical methods for the heavy metals elimination from the acid mine drainage. the experiments were carried out at the laboratory scale using a synthetic solution with similar properties to the real sample of acid mine drainage, originating from the zinc mine located in tùnel kingsmill outflow of the rio yaulì (district of yauli – perù). the successive repetition of the metal precipitations as hydroxides (chemical method) and sulphides (biological-chemical method) at the various acid mine drainage ph was the basis of the examined processes. for the hydrogen sulphide production the sulphate-reducing bacteria of genus desulfovibrio were used. results confirmed the precipitation of fe, as, al and mn in the hydroxide form; cu and zn in the form of sulphides. for the selective removal of cu, zn, al and mn the excellent results were received. selective precipitation of fe and as was not so successful since the co-precipitation of fe and as was later determined as a main mechanism of the precipitate forming. keywords: acid mine drainage, metals, selective sequential precipitation 1. introduction acid mine drainage (amd) represents the major environmental problem especially associated with mining activities. the basis of the amd generation is the oxidation of pyrite and other sulphidic minerals exposed to both oxygen and water. each amd has a specific composition, but always contains high concentration of the sulphuric acid, dissolved heavy metals, sulphates, iron precipitates and their ph values can be very low. consequently amd must be collected and treated before it can be discharged (younger et al., 2002; kadukova and stofko, 2009). various methods are used for the treatment of amd but any of them have not been applied under the commercial-scale. chemical and biological-chemical methods are mostly studied with the aim to eliminate metals, sulphates and achievement the neutral ph (prascakova, 2005; kaksonen and puhakka, 2007; jencarová, 2008; kavulicova et al., 2009; ivanova et al., 2010). in general two strategies active and passive technologies for treating amd are used: (skousen et al., 1998; balintova and luptakova, 2012). in the conventional treatment system alkaline materials and other chemicals are added to the amd to neutralize it and enhance hydroxide precipitation. gradually the ph increases and the metal ions precipitation (for example as hydroxides) are achieved. each metal in aqueous solution contributes to the amd acidity. additionally, individual metals precipitate at specific bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:02 utc 134 luptáková, a. et al. ph levels. for example, hydroxides of fe3+ precipitate at about ph 3 and of al3+ at ph ranging from 3.7 to 4.5. divalent metal ions precipitate in the alkaline range, ni2+ at about ph 8, fe2+ at ph ranging from 8 to 9 and zn2+ at more than ph 9 (skousen et al., 1998). the precipitation process is affected not only by reaction time but also by the thermodynamic considerations. the general importance of ph in an amd system is not the only important factor in metal removal but also ionic strength, temperature, redox potential, and concentrations of suitable complexing agents (e.g. humic substances) and interactions of the precipitated solids play a significant role. the treatments using alkaline materials involve some serious limitations of application and effectiveness. they usually result in production of unstable metal hydroxides mixture, also leading to a greater disposal expense. the high operating costs and production of a bulky sludge, which must be disposed, are the disadvantages of the traditional chemical treatment. the research and development of the appropriate combinations of the chemical and biological-chemical methods for the metals selective recovery from amd suggested the interesting solution of problems concerning the amd treatment. these methods constitute the possibility of metals recovery in a suitable form for commercial or industrial utilization. the combination of the metal precipitation using the sodium hydroxide (chemical methods) and metal precipitation using the bacterially produced hydrogen sulphide (biological-chemical method) presents the base of the selective sequential precipitation (ssp) (tabak et al., 2003). the base of ssp is the evidence that metals in aqueous solution precipitate by addition of sodium hydroxide solution or hydrogen sulphide at specific ph levels. the acid-base titration by the sodium hydroxide solution is a simple and convenient method for the suitable ph determination of the metals selective precipitation from aqueous solution. the issue of alkalimetry is the titration curve (the vertical part shows the process ohions neutralizing h+ ions; the horizontal part indicates ohions precipitating metal ions into metal hydroxides, which will act as a buffer keeping the constant ph for the short time until the specific metal will completely precipitate). when ph reaches at the certain level the metal ions will precipitate and be eliminated from the water. ssp is the environmentally friendly way for the metals and metalloids elimination from amd. the main objective of the paper was to interpret the ssp process that is the combination of chemical and biological-chemical methods as a way of chosen metals (fe, as, cu, al, zn and mn) separation from the model solution of peruvian amd. 2. materials and methods 2.1 synthetic solution of acid mine drainage the experiments were carried out at the laboratory scale using a synthetic solution of amd coming from the zinc mine located in tùnel kingsmill outlet of the rio yaulì (district of yauli – perù). the synthetic solution with the similar properties to the real sample of amd was prepared. reagents with a high analytical degree of purity were used (rpe carlo erba). based on the metal concentrations in the real amd sample, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:02 utc nova biotechnologica et chimica 11-2 (2012) 135 the model solution with the same metal concentrations was prepared. the corresponding salts were weighed and dissolved in deionized water. the solution ph value was adjusted to 3 using 5 m naoh. a little amount of h2o2 was added to the synthetic solution with the aim to get fe, as and mn in the form of fe3+, as5+ and mn2+. the annual average metals concentration of peruvian amd is outlined in table 1. table 1. concentration of chosen elements of amd sample from perù with the annual average ph value 3.1. 2.2 chemical and biological-chemical methods combination the successive repetition of the metal precipitation as hydroxides (chemical method) and sulphides (biological-chemical method) at the acid mine drainage various ph were the fundamental of the examined processes. this selective sequential precipitation (ssp) was realized in two principal steps: 1 – addition of 0,2m naoh solution by the automatic titrator titralab 850; 2 – addition of bacterially produced h2s by the nitrogen gas continuous transfer from the cultivation tank. the determination of suitable ph values for selective precipitation of metals from studied model solution was performed in the previous experiments by the acid-base titration – alkalimetry using automatic titrator titralab 850 in connection with pc program titramaster 85. the strains of srb (genus desulfovibrio) were used for the hydrogen sulphide production. bacteria were isolated from the potable mineral water (luptakova et al., 2002; macingova, 2010). at the end of ssp respective steps the formed precipitates of the individual metals were removed by filtration from the amd solution. in the filtrates the metal concentrations were analysed by atomic absorption spectrometry (aas). 3. results and discussions table 2 illustrates particular steps of the experimental procedure and obtained results of the selective sequential precipitation of heavy metals from the amd synthetic solution. table 2. metals precipitation by naoh and bacterially produced h2s. 1-step 2-step 3-step 4-step 5-step ph 3.0→4.5 4.5→3.9 3.9→5.8 5.8→5.0 5.0→10.5 precipitating agent naoh h2s naoh h2s naoh removed metals (precipitates) fe, as cu al zn mn in the 1-step of process the addition of 0.2 m naoh into the amd solution from initial ph 3.0 to ph 4.5 by the automatic titrator was realized. the addition was element al cd cu zn as mn fe ca mg concentration (mg.l-1) 7.80 0.16 10.80 69.15 1.96 62.35 127.86 381 49.50 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:02 utc 136 luptáková, a. et al. performed slowly (0.1ml/min) and with the solution magnetic agitation. formation of the orange precipitates was observed at ph around 3.5. figure 1 outlines coprecipitation of as and fe. this behaviour is in accordance with the results of many authors since the arsenic compounds are known to have the high affinity to adsorb on the iron hydroxide (kaksonen and puhakka, 2007). 0 20 40 60 80 100 3 3,2 3,4 3,6 3,8 4 4,2 4,4 ph f e, a l, z n, c u an d m n (m g/ l) 0 0,5 1 1,5 2 2,5 fe al zn mn c u as a s (m g/ l) fig. 1. co-precipitation of as and fe during the 1-step of ssp process. bacterially produced h2s was transferred by means of the nitrogen gas continuous flow from the bacteria cultivation tank into the filtrate (after the removal of as and fe hydroxides by filtration) (2-step). this step lasted for 30 minutes. the brown precipitates were observed shortly after 5 minutes. the ph of amd solution decreased from 4.5 to 3.9 under the influence of the hydrogen sulphide (kaksonen and puhakka, 2007). cu was probably eliminated in the form of sulphide (table 3). table 3. precipitation of cu by bacterially produced h2s (2-step of ssp process) cu zn al mn time (min) ph (mg.ml-1) 0 4.5 9.25 78 3,8 70.5 5 4.0 < 0.03 77,9 3,9 69.9 10 3.9 < 0.03 77,8 3,8 69.5 15 3.8 < 0.03 77,6 3,8 69.4 20 3.8 < 0.03 77,9 3,7 69.6 25 3.8 < 0.03 77,8 3,8 69.5 30 3.9 < 0.03 77,7 3,7 69.4 after the removal of cu precipitates by filtration, 0.2 m naoh solution was added into the filtrate by the automatic titrator until ph of 5.8 was reached (3-step). the precipitation of al was detected (fig. 2). white al precipitates were obtained after filtration. bacterially produced h2s was again added into the remaining filtrate (4step). the white precipitates were observed after 10 minutes. the decrease of amd solution ph values from 5.8 to 5.0 was probably due to the influence of the hydrogen sulphide (table 3). the white precipitates were removed and the last step of the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:02 utc nova biotechnologica et chimica 11-2 (2012) 137 process, the addition of 0.2 m naoh into the filtrate with consequent ph increase from 5.0 to 10.5, followed. the precipitation of mn was identified (fig. 3.). 0 20 40 60 80 3,5 4 4,5 5 5,5 6 6,5 ph z n a m n (m g/ l) 0 1 2 3 4 5 zn mn al a l (m g/ l) fig. 2. precipitation of al during the 1-step of ssp process. table 4. precipitation of zn by bacterially produced h2s (4-step of ssp process). zn mn time (min) ph (mg.ml-1) 0 5.8 75.4 69.8 5 5.4 22.6 69.6 10 5.2 0.6 69.5 15 5.1 0.6 69.4 20 5.0 0.6 69.6 25 5.2 0.5 69.5 30 5.0 0.5 69.4 4. conclusions the combination of chemical and biological-chemical precipitating methods for the heavy metals elimination from the acid mine drainage was studied. the chemical method was realised by the metal precipitation using the sodium hydroxide and the biological-chemical method using the bacterially produced hydrogen sulphide. their suitable combination presents the basis of the selective sequential precipitation (ssp). achieved results demonstrated the possibility of heavy metals removal from the synthetic solution simulating the composition of acid mine drainage coming from the zinc mine located in tùnel kingsmill outlet of the rio yaulì (district of yauli – perù). in ssp process fe3+, as5+, al3+ and mn2+ precipitated in the form of hydroxides and cu2+ and zn2+ in the form of sulfides. excellent results were received for the removal of cu, zn, al and mn. selective precipitation of fe and as was not possible because of the co-precipitation of fe and as. acknowledgements: work was supported by: joint project n.1 of the cnr/sav (2010/2012); project cnr rstl n. 0042.0005; joint project n.2 of the cnr/concytec (perù) (2009/2011); srda-0252-10 and the scientific grant agency no. 2/0166/11. 0 20 40 60 80 5 7 9 11 ph m n (m g/ l) fig. 3. precipitation of mn during the 5-step of ssp process. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:02 utc 138 luptáková, a. et al. references balintova, m., luptakova, a.: treatment of acid mine drainage. technical university in kosice, faculty of civil engineering, 2012, 131 pp. isbn 978-80553-0868-5. (in slovak) ivanova, d., horvathova, h., kadukova, j., kavulicova, j.: stability of immobilized biosorbents and its influence on biosorption of copper. nova biotechnol., 10, 2010, 45-51. issn 1337-8783. jenčárová, j.: the bacterially preparation of iron sulphides. acta metallurg. slovaca. 14, 2008, 106-109. kadukova, j., stofko, m.: utilization of algae for pollution elimination, chapter 5, algae: nutrition, energy source and environmental control (ed. kristian n. hagen), nova publishers, new york, 2009, 57-87. isbn 978-1-60692-008-4. kaksonen, a.h., puhakka, j.a.: sulfate reduction based bioprocesses for the treatment of acid mine drainage and the recovery of metals, eng. life sci., 7, 6, 2007, 541–564. kavulicova, j., kadukova, j., podracky, j., ivanova, d.: effect of metals on oxidative stress in linum usitatissimum. proceedings of the 1st international conference biotechnology and metals 2009. košice, slovak republic, september 24-25, 2009, 57-60. isbn 978-80-553-0236-2 luptakova, a., kusnierova m., fecko, p.: mineral biotechnology ii. – sulfuretum in nature and industry, vsb -tu ostrava, ostrava, czech republic, 2002. isbn 80-248-0114-0. macingova, e.: application possibilities of the bioremediation methods for the elimination of the environmental and industrial loads. technical university of košice, ph.d. thesis, 2010, p.101. prascakova, m.: adsorption of heavy metals on biologically activated brown coal sludge, acta montan. slovaca, 10, 2005, 161-163. skousen, j., rose, a., geidel, g., foreman, j., evans, r., hellier, w.: a handbook of technologies for avoidance and reclamation of acid mine drainage, west virginia university, morgantown, wv: nmlrc, 1998. tabak, h.h., scharp, r., burckle, j., kawahara, f.k., govind, r.: advances in biotreatment of acid mine drainage and biorecovery of metals: metal precipitation for recovery and recycle. biodegradation , 14, 2003, 423-436. younger, p.l., banwart, s.a., hedin, r.s.: mine water: hydrology, pollution, remediation. in: environmental pollution, vol.5, eds. alloway, b.j., trevors, j.t., kluwer academic publishers, dordrecht, netherlands, 2002. presented at the 2nd international conference “biotechnology and metals 2011”, september 22–23, 2011, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:02 utc microsoft word fristak and soja_2015.doc 104 nova biotechnologica et chimica 14-1 (2015) doi 10.1515/nbec-2015-0020 © university of ss. cyril and methodius in trnava effect of wood-based biochar and sewage sludge amendments for soil phosphorus availability vladimír frišták, gerhard soja department of health & environment, ait austrian institute of technology gmbh, tulln, 3430, austria (vladimir.fristak@ait.ac.at) abstract: this study investigated the effects of two biochars (pyrolysed wood chips and garden clippings) on phosphorus (p) availability in a heavy-metal contaminated soil poor in phosphorus. short-term 14-days incubation experiments were conducted to study how applications of biochars at different rates (1 and 5 %) in combination with (1:1) and without dried sewage sludge from a municipal waste water treatment plant (wwtp) affected the content of soil extractable p. for p-availability analyses deionized water, calcium acetate lactate (cal), mehlich 3 and olsen extraction protocols were applied. in addition, the content of total and mobile forms of potentially toxic heavy metals (pthm) was studied. application of both biochars caused a significant decrease of pthm available forms in sewage sludge amended soil samples. the concentration of total and available p increased with higher biochar and sewage sludge application rates. key words: biochar, sewage sludge, availability, heavy metals, phosphorus, soil 1. introduction phosphorus represents a non-substitutable element that serves vital functions in all living organisms. however, the remaining natural resources of this important macronutrient have rather limited availability and decreasing quality (egle et al., 2014). a considerable amount of phosphorus (p) is removed from field soils by harvesting crops and another significant amount of phosphorus is stored in residual plant biomass with low use efficiency (zhai et al., 2015). a continued large demand for this plant nutrient in agriculture will lead to a foreseeable depletion of natural resources (van vuuren et al., 2010). the search for alternatives for rock phosphate as a basic raw material for p fertilizers has become a burning issue in recent discussions about global food production. the processing of phosphorus-rich municipal and agricultural wastes could contribute to the closing of the geochemical phosphorus cycle and to the conservation of the remaining rock phosphates. sewage sludge as a product of municipal waste water treatment plants represents a heterogeneous mixture of beneficial and hazardous compounds, micro-/macroelements and organic matter (frišták et al., 2014a). concentrations and availability of heavy metals and other contaminants from sewage sludge restrict the agricultural applications as fertilizers (šuňovská et al., 2013). however, the abundant availability of such phosphorus-rich material opens a new option in the search for effective recovery of the valuable sewage sludge components with nutrient effects when used as soil amendment. several studies have shown that the application of biochar as high porous material produced by pyrolysis of lignocellulosic biomass can positively influence basic soil bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:20 utc nova biotechnologica et chimica 14-1 (2015) 105 properties, such as ph, water holding capacity, microbial diversity or content of available nutrients (lehmann et al., 2015). thermo-chemical conversion processes provide the key for the transfer of phosphorus from waste materials to stabilized products. additionally, pyrolysis as a carbonization technology converts biomass thermochemically into stable, recalcitrant organic carbon compounds that can serve as nutrient carriers to be applied as fertilizer for agricultural soils (glaser et al., 2015). the phosphorus content of biochar can increase by 2or 3-fold during the pyrolysis process as the phosphorus in the biomass is enriched with inorganic phosphates and pyrophosphates (atkinson et al., 2010). reversibility of phosphorus binding represents the crucial parameter of biochar utilization as an appropriate fertilizer. the combination of pyrolysed plant residues with sewage sludge might on the one hand enrich nutrient-poor biochars and on the other hand provide a slow release of the nutrients to avoid a rapid leaching. therefore the main aim of our study was to assess the effectivity of wood-based biochar and its mixture with municipal sewage sludge with respect to phosphorus availability. 2. materials and methods 2.1 soil samples top soil samples (0-20 cm) of a mixed type cambisol and rendzina were collected from locality arnoldstein (as; fig. 1), austria (46º33'09.5"n, 13º41'21.5"e). the soil samples were air-dried, homogenised and sieved through a 2 mm mesh sieve. fig. 1. sampling sites of studied soil and sewage sludge. 2.2 biochar and sewage sludge amendments biochar samples were produced in slow pyrolysis process from two different feedstocks: beech wood chips (bc a) and garden green waste residues (bc b). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:20 utc 106 frišták, v. and soja, g. procedure and production conditions were described in our previous work (frišták et al., 2014b). for soil experiments the particle size 0.5 1 mm was used. municipal sewage sludge (ss) was obtained from aeration tank of waste water treatment plant (wwtp) in tulln an der donau (fig. 1). after sampling, the sludge was filtered, 2 times rinsed with deionized water, oven dried at 60 °c and 24 h, ground and sieved. for soil experiments also the particle size fraction of 0.5 1 mm was used. 2.3 amendments characterization the active and potential ph values of bc a, bc b and ss were measured after stirring the biochars and sewage sludge with deionized water and 1.0 mol l-1 kcl (ratio 1:2.5) for 1 h and stabilization for 1 h (inolab ph level 2p, weilheim, germany). the electrical conductivities (ec) of bc a, bc b and ss were measured in 1:10 deionised water extracts after 24 h mixing (inolab ph level 2p, weilheim, germany). the anion exchange capacities (aec) of biochars and sewage sludge were determined using bromide as an index anion (lawrinenko, 2014). surface areas (sa) were estimated by the titration method with naoh according to melichová and hromada (2013) and confirmed by methylene-blue adsorption method (elgeundi et al., 2014). the total c, n and h contents of biochars were measured by an elemental analyzer (chns-o ea 1108, carlo erba instruments, italy). initial concentrations of cd, cu and pb as models of potentially toxic heavy metals (pthm) in biochar and sludge samples were determined after hno3 – h2o2 digestion (enders and lehmann, 2012) by icp-ms. total p content was measured in the same digests by malachite green microplate uv-vis method. 2.4 experimental design for incorporation into the experimental soil 1 and 5 % (w/w) amendments of bc a, bc b, ss, bca + ss (1:1), bcb + ss (1:1) were applied. for comparison the commercial fertilizer superphosphate (sp, 42 % p2o5) was used. amended soil samples were incubated at 19 ± 2 °c and 65 % water content for 14 days under greenhouse conditions. 2.5 soil samples characterization and p determination the amended soil samples were characterized by determination of active and potential ph, electrical conductivity (ec), anion exchange capacity (aec), surface area (sa), total and water extractable content of cd, cu, pb, total and available forms of phosphorus. for determination labile forms calcium-acetate-lactate (cal), deionized water (dw), mehlich 3 (m3) and olsen (ol) extraction protocols were used. for total phosphorus content modified hno3 – h2o2 digestion method (enders and lehmann, 2012) was applied. for determination of heavy metals in soil extracts the mass spectrometry with inductively coupled plasma (icp-ms, perkin elmer, elan drce 9000, usa) and atomic absorption spectrometry with flame atomization (faas, aa 400, perkin elmer, usa) was used and malachite green microplate uv-vis method was used for phosphorus determination in soil extracts. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:20 utc nova biotechnologica et chimica 14-1 (2015) 107 3. results and discussion 3.1 characterization of soil and soil amendments the physico-chemical properties of the studied soil sample from arnoldstein (as), two biochar (bca, bcb) and sludge amendments (ss) were analysed and are summarised in table 1. levels of ph, aec and sa are major determinants of the behaviour of organic and inorganic contaminants in soil (kabata–pendias, 2011). nevertheless, ph value affects not only xenobiotic availability, but also controls the process of microand macronutrient uptake by plant root systems. the determination of actual and potential ph confirmed a slightly acidic character of the studied soil and dried sewage sludge. a similar character of dried ss was also found in our previous study (frišták et al., 2014a). table 1. physico-chemical characteristics of the studied soil (as), biochar amendmends (bca, bcb) and sewage sludge amendment (ss). intervals denote standard deviations (n=4). bca bcb ss as phact 8.78 ± 0.02 9.03 ± 0.01 6.94 ± 0.01 6.29 ± 0.02 phpot 8.46 ± 0.02 8.77 ± 0.02 6.71 ± 0.01 5.45 ± 0.03 ec (ms cm-1) 0.54 ± 0.03 1.67 ± 0.03 0.87 ± 0.05 0.05 ± 0.04 aec (cmol kg-1) 0.63 ± 0.01 0.73 ± 0.01 2.89 ± 0.01 1.05 ± 0.01 sa (m2 g-1) 28.04 ± 1.55 31.79 ± 1.98 3.87 ± 0.12 18.02 ± 0.54 c (%) 80.30 ± 1.02 79.78 ± 2.41 n.a. n.a. h (%) 1.60 ± 0.08 1.59 ± 0.05 n.a. n.a. n (%) 0.40 ± 0.01 0.65 ± 0.01 n.a. n.a. ptot * (mg kg -1) 2 153 ± 89 6197 ± 369 34 685 ± 488 196 ± 6 cd 0.20 ± 0.01 0.21 ± 0.01 0.56 ± 0.02 9.60 ± 0.58 cu 15.70 ± 0.85 21.00 ± 1.15 360.02 ± 10.54 104.19 ± 4.78 pb 2.10 ± 0.15 0.80 ± 0.01 35.27 ± 2.47 1 607.40 ± 98.45 *ptot = total concentration of phosphorus both biochar amendments showed an alkaline reaction which was related to their production conditions and type of feedstock. wood-based biochars tend to have a higher ph compared to other types of biomass such as crop residues or manures (gal et al., 2015). the values of aec decreased in the order ss > as > bc b > bc a. the very low ability of biochar-based amendments to exchange the anionic forms was caused by the negative character of the surface charges. the feedstock quality and content of mineral components (ca, na, fe) can catalyse the formation of oxygen functional groups which are responsible for the negative charges of biochar-based amendments (gaskin et al., 2008). for the increase of aes, surface modifications with certain feor al-oxides, -hydroxides or -chlorides may be effective (morenojiménez et al., 2012; zhang and gao, 2013). the determination of sa values confirmed the more porous character of biochar materials compared to sewage sludge. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:20 utc 108 frišták, v. and soja, g. the parameter sa is crucial for assessing the volume and distribution of microand mesopores in carbon-based material (galamboš et al., 2015). the determination of selected pthm (cd, cu and pb) revealed 20 times higher total concentrations in municipal sewage sludge and 85 times higher concentrations in the studied soil compared to biochar-based amendments. the high level of pthm in the soil sample as was related to the specific sampling site that was located in an area of historic mining and ore processing. similar results were described by horak and frieslhanl (2007). the determination of total p confirmed phosphorus deficiency in as soil. the concentration of p in potential amendments increased in the order bc a < bc b < ss. our analysis confirmed that sewage sludge as a heterogeneous mixture of chemical substances including plant nutrients represents a phosphorus-rich material with a potentially fertilizing effect. 4000 3500 3000 2500 2000 1500 1000 500 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 t ( % ) ν (cm-1) ss bc a bc b fig. 2. ft-ir spectra of bc a, bc b and ss. the spectral analysis in the infrared region belongs to the most important structural qualitative and semiquantitative analyses (de la rosa et al., 2014). ftir spectra of bca, bcb and ss (fig. 2) showed typical shape and peaks of functional groups. the spectrum of ss compared to biochars is more complicated, reflecting the complex character of dried sludge biomass obtained from the activation tank of a municipal wwtp. despite the complexity of spectra some characteristic peaks can be recognized that reflect the relative concentration of functional groups. the shape and position of wide peaks at σ 3 295 cm-1 for ss and at 3 420 cm-1 for bc a represent self-associated –oh groups. this peak was not recognized in bc b spectrum. narrow peaks at σ 2 930 cm-1 and 2 812 cm-1 are due to c-h stretching vibration in ch, ch2 and ch3 functional groups in ss sample. strong peaks in the vicinity of σ bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:20 utc nova biotechnologica et chimica 14-1 (2015) 109 1 650-1 695 cm-1 and 1 550-1 530 cm-1 were attributed to asymmetric and symmetric stretching vibration of c=o, c-c and c-n groups in bca, bcb and ss. the broad peak at σ 1 045 cm-1 reflects the c-h bending in the sludge amendment. similar peaks were recognized and in detail described in study of frišták et al. (2014a) and de la rosa et al. (2014). 3.2 effect of soil amendments incorporation the short-term incubation experiments after incorporation of 1 % and 5 % (w/w) bc a, bc b and ss amendments into soil as led to significant differences in physicochemical properties and structural characteristics of the substrate (table 2-3). pedological analyses revealed a decrease of soil ph in the order superphosphate > ss > bc a + ss > bc b + ss > bc a > bc b for both application rates. table 2. physico-chemical characteristics of amended soil with 1 % (w/w) bc a, bc b, ss, bc a+ss, bc b+ss and sp after short-term incubation (14 d, 19 °c, 65 % water content, greenhouse conditions). intervals denote standard deviations (n=4). bca bcb ss bca+ss bcb+ss sp phact 6.61 ± 0.01 6.64 ± 0.00 6.30 ± 0.02 6.58 ± 0.01 6.60 ± 0.02 6.18 ± 0.01 phpot 5.61 ± 0.01 5.79 ± 0.02 6.08 ± 0.02 5.61 ± 0.00 5.64 ± 0.02 5.42 ± 0.02 ec (ms cm-1) 0.06 ± 0.00 0.07 ± 0.00 0.05 ± 0.00 0.08 ± 0.00 0.11 ± 0.01 0.18 ± 0.01 aec (cmol kg-1) 0.99 ± 0.01 0.98 ± 0.02 1.59 ± 0.02 1.12 ± 0.01 1.19 ± 0.00 1.08 ± 0.03 sa (m2 g-1) 22.45 ± 0.65 26.74 ± 0.45 18.58 ± 0.21 20.12 ± 0.38 23.87 ± 0.44 18.41 ± 0.41 ptot * (mg kg -1) 205 ± 8 219 ± 5 498 ± 5 364 ± 4 381 ± 4 822 ± 9 ∑ pthm **(mg kg-1) 1 721 1 721 1 739 1 727 1 728 1 721 *ptot = total concentration of phosphorus ** ∑ pthm = cdtot +cutot+pbtot an increase of soil alkalinity following biochar amendment has been reported for many soil types (farrell et al., 2014; stewart et al., 2013; xu et al., 2013). as mentioned before, this effect is due to the alkaline character of biochar because of the presence of negatively charged phenolic, carboxyl and hydroxyl functional groups on surfaces (brewer et al., 2012). these groups create active sites for binding h+ ions from soil solution and increase alkalinity of soil matrix. additionally stewart et al. (2013) described the significant role of biochar composition such as carbonates and silicates in binding and removal of h+ ions from soil solutions. our study confirmed the positive effect of bc a and bc b on sa values of amended soils caused by the porous character of amendments. municipal sewage sludge as a soil amendment bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:20 utc 110 frišták, v. and soja, g. decreased values of soil ph but on the other side increased aec. generally, the composition of sewage sludge from municipal wwtp is variable concerning their chemical and biochemical components (šuňovská et al., 2013); this can result in higher concentrations of exchangeable anions compared to pyrolysis products. additionally, soil samples amended by ss showed an increase of total p but also in selected pthm concentrations. this fact is very often the limitation for the deployment of sewage sludge in agricultural soil management. for this reason we investigated the potential effect of mixed biochar/sewage sludge additives. table 3. physico-chemical characteristics of amended soil with 5 % (w/w) bc a, bc b, ss, bc a+ss, bc b+ss and sp after short-term incubation (14 d, 19 °c, 65 % water content, greenhouse conditions). intervals denote standard deviations (n=4). bca bcb ss bca+ss bcb+ss sp phact 6.72 ± 0.02 6.92 ± 0.02 6.39 ± 0.03 6.62 ± 0.01 6.62 ± 0.02 6.16 ± 0.00 phpot 5.72 ± 0.01 6.13 ± 0.01 6.12 ± 0.01 5.70 ± 0.02 6.12 ± 0.05 5.32 ± 0.02 ec (ms cm-1) 0.09 ± 0.0 0.13 ± 0.01 0.09 ± 0.00 0.08 ± 0.01 0.12 ± 0.01 0.48 ± 0.02 aec (cmol kg-1) 0.95 ± 0.01 0.92 ± 0.04 1.98 ± 0.12 1.19 ± 0.04 1.24 ± 0.09 1.02 ± 0.04 sa (m2 g-1) 22.45 ± 1.55 26.74 ± 1.01 19.68 ± 1.17 21.04 ± 0.98 23.41 ± 0.91 18.49 ± 0.28 ptot * (mg kg -1) 253 ± 14 321 ± 9 1 525 ± 87 621 ± 17 649 ± 24 1031 ± 61 ∑ pthm ** (mg kg-1) 1 735 1 737 1 826 1 801 1 803 1 720 * ptot = total concentration of phosphorus ** ∑ pthm = cdtot +cutot+pbtot mixed amendments of both biochar types and sewage sludge in both application rates showed a significant increase of total p concentrations compared to biochar only. on the other hand application of 1 % or 5 % bc a + ss and bc b + ss decreased the total pthm concentration in comparison with single ss amendment. however, for better assessment of this effect the extractable and available forms of p and pthm were quantified. application of 4 different extraction agents (calcium-acetate-lactate (cal), deionized water (dw), mehlich 3 (m3) and olsen (ol) extraction mixture) revealed differences in the labile and removable fractions of phosphorus in the amended soil samples. for all extraction protocols the same trend was confirmed (fig. 3, 4). extractable p concentration increased in the order bc b < bc a < control < bc b + ss < bc a + ss < ss < sp for both application rates. for better explanation of the obtained results about the effects of the main soil characteristics on total and available p concentrations, a correlation analysis was performed. in the case of bc a amendment the pearson correlation coefficients bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:20 utc nova biotechnologica et chimica 14-1 (2015) 111 calculated at α = 0.05 (tab. 4) showed a statistical significance for the positive correlation of total p with ec and a negative correlation with aec, sa and available σ pthm, obtained by cacl2 extraction. 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 olm3dw cal 1% bca 1% bcb 1% ss 1% ss+bca 1% ss+bcb 1% p 2 o 5 control p ex tr ac ta bl e / p to ta l * 1 00 ( % ) fig. 3. labile fractions of p extracted by calcium-acetate-lactate (cal), deionized water (dw), mehlich 3 (m3) and olsen (ol) extraction protocols from arnoldstein soil amended with 1 % bc a, bc b, ss, bc a + ss, bc b + ss and sp (p2o5). the error bars show the mean of standard deviation (n=4). 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 olm3dw cal 5% bca 5% bcb 5% ss 5% ss+bca 5% ss+bcb 5% p 2 o 5 control p ex tr ac ta bl e / p to ta l * 1 00 (% ) fig. 4. labile fractions of p extracted by calcium-acetate-lactate (cal), deionized water (dw), mehlich 3 (m3) and olsen (ol) extraction protocols from arnoldstein soil amended with 5 % bc a, bc b, ss, bc a + ss, bc b + ss and sp (p2o5). the error bars show the mean of standard deviation (n=4). for soil samples amended by bc b, the positive correlation between total p and ec was significant. similarly to bc a, also for bc b a significantly negative correlation between total p and available σ pthm was confirmed. ss as a soil amendment showed significances of the positive correlations between total p and ec, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:20 utc 112 frišták, v. and soja, g. total p and total σ pthm and also p total and available σ pthm. additionally, also the positive correlation of available p with total σ pthm and available σ pthm was significant. soil samples amended with mixed bc a/ss and bc b/ss exhibited a positive correlation between total p and total σ pthm and a negative one between total p and available σ pthm. for superphosphate fertilizer as soil amendment there was a significantly positive correlation between total p and ec. table 4. pearson correlation coefficients (r) between soil characteristics (a ph, b ec, c aec, d sa, e ∑ pthm (cdtot +cutot+pbtot), f 0.01 m cacl2 extractable ∑ pthm) and total and available phosphorus content in amended soil samples. level of significance α = 0.05. a b c d e f bca ptot* 0.41 0.95 -0.86 -0.95 0.23 -0.96 pavail** 0.57 0.76 -0.78 -0.63 0.31 -0.63 bcb ptot* 0.42 0.88 -0.57 -0.67 0.26 -0.92 pavail** 0.53 0.73 -0.28 -0.55 0.36 -0.53 ss ptot* 0.48 0.87 -0.17 -0.45 0.98 0.89 pavail** 0.37 0.38 -0.13 -0.37 0.95 0.98 bca+ss ptot* 0.54 0.58 -0.78 -0.69 0.87 -0.48 pavail** 0.51 0.14 -0.71 -0.68 0.81 -0.42 bcb+ss ptot* 0.62 0.60 -0.81 -0.78 0.88 -0.89 pavail** 0.60 0.59 -0.80 -0.79 0.80 -0.58 sp ptot* 0.74 0.89 0.12 0.18 0.41 0.23 pavail** 0.78 0.77 0.11 0.21 0.32 0.23 * ptot –total concentration of phosphorus in soil samples ** pavail – cal-extractable concentration of phosphorus in soil samples the obtained results showed the relations between increasing p levels and decreasing levels of available pthm when mixed biochar/sewage sludge amendments were applied. zhao et al. (2014) highlighted the effect of maize straw-derived biochar on soil phosphorus leaching from amended alluvial and red soils. the authors confirmed the increase of labile forms of soil p after biochar incorporation and short-term incubation. on the other hand, the application of a single woody biochar amendment could improve soil p retention that in parallel decreased the eutrophication risk to aquatic system (zhang et al., 2014). laird et al. (2010) also showed the reducing effect of a typical wood-derived biochar to migrations of soil nutrients in typical midwestern agricultural soils. however, for detailed assessments of the biochar effect on soil p levels in terms of feedstock material, feedstock pre-treatment, combinations of feedstocks, processing conditions, and biochar application rates further studies will be required. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:20 utc nova biotechnologica et chimica 14-1 (2015) 113 4. conclusions this paper illuminates the utilization of biochar as a potential tool for soil phosphorus management. in short-term incubation conditions, the application of woody biochar, sewage sludge from municipal waste water treatment plants and its mixtures with a studied soil could significantly alter the soil physico-chemical characteristics and available p levels. our study confirmed the applicability of two types of biochar (wood chips-derived biochar and garden residues-derived biochar) for reducing leachable phosphorus and for decreasing eutrophication risk. the combinations of biochar and sewage sludge used as soil amendments confirmed their ability to increase soil p levels and to decrease heavy metal availability. acknowledgment: the authors thank wolfgang friesl-hanl for soil sampling and the austrian ffg for financial support of the research studio ferti-mine, project nr. 844744. references atkinson, c.j., fitzgerald, j.d., hipps, n.a.: potential mechanisms for achieving agricultural benefits from biochar application to temperate soils: a review. plant soil, 337, 2010, 1-18. brewer, c.e., hu, y.y., schmidt-rohr, k., loynachan, t.e., laird, d.a., brown, r.c.: influence of extent of pyrolysis on corn stover fast pyrolysis biochar and soil properties. soil biol. biochem., 41, 2012, 1115-1122. de la rosa, ch., paneque, m., miller, a.z., knicker, h.: relating physical and chemical properties of four different biochars and their application rate to biomass production of lolium pereme on a calcic cambisol during a pot experiment of 79 days. sci. total environ., 499, 2014, 175-184. egle, l., zoboli, o., thaler, s., rechberger, h., zesser, m.: austrian p budget as a basis for resource optimization. resour. conserv. recy., 83, 2014, 152162. el-geundi, m.s., ashourm, e.a., abobeah, r.m.a, shehata n.: determination of specific surface area of natural clay by comparative methods. int. j. sci. engineer. technol. res., 3, 2014, 2100-2104. enders, a., lehmann, j.: comparison of wet-digestion and dry-ashing methods for total elemental analysis of biochar. commun. soil sci. plan., 43, 2012, 10421052. farrell, m., macdonald, l.m., butler, g., chirino-valle, i., condron, l.m.: biochar and fertilizer applications influence phosphorus fractionation and wheat yield. biol. fertil. soils, 50, 2014, 169-178. frišták, v., pipíška, m., valovčiaková, m., lesný, j., rozložník, m.: monitoring 60co activity for the characterization of the sorption process of co2+ ions in municipal activated sludge. j. radioanal. nucl. chem., 299, 2014a, 16071614. frišták, v., friesl-hanl, w., pipíška, m., richveisová micháleková, b., soja, g..: the response of artificial aging to sorption bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:20 utc 114 frišták, v. and soja, g. properties of biochar for potentially toxic heavy metals. nova biotechnol. chim., 13, 2014b, 137-147. galamboš, m., daňo, m., víglašová, e., krivosudský, l., rosskopfová, o., novák, i., berek, d., rajec, p.: effect of competing anions on pertechnetate adsorption by activated carbon. j. radioanal. nucl. chem., 304, 2015, 1219-1224. gaskin, j.w., steiner, c., harris, k., das, k.c., bibens, b.: effect of lowtemperature pyrolysis conditions on biochar for agricultural use. transactions of asabe, 56, 2008, 2061-2069. glaser, b.,wiedner, k., seelig, s., schmidt, h.p., gerber, h.: biochar organic fertilizers from natural resources as substitute for mineral fertilizers. agron. sustain. dev., 35, 2015, 667-678. horak, o., friesl-hanl, w.: soil additives immobilising heavy metals in contaminated soils. nova biotechnol., 7, 2007, 5-9. kabata-pendias, a.: trace elements in soils and plants, fourth edition, crc press, florida, boca raton, usa, 2011, 548 pp. laird, d.a., fleming, p., davis, d.d., horton, r., wang, b.q., karlen, d.l.: impact of biochar amendments on the quality of a typical midwestern agricultural soil. geoderma, 158, 2010, 443-449. lawrinenko, m.: anion exchange capacity of biochar. graduate theses and dissertations. paper 13685, 2014. lehmann, j., joseph, s.: biochar for environmental management: science, technology and implementation. earthscan from routledge: london, uk, 2015, 944 pp. melichová, z., hromada, l.: adsorption of pb2+ and cu2+ ions from aqueous solutions on natural bentonite. pol. j. environ. stud., 22, 2012, 457-464. moreno-jimenez, e., esteban, e., penalosa, j.m.: reviewing the fate of arsenic in the soil-plant system: from basic knowledge to the real applications. rev. environ. contam. t., 215, 2012, 1-37. stewart, c.e., zheng, j., botte, j., cortrufo, f.: co-generated fast pyrolysis biochar mitigates greenhouse gas emissions and increases carbon sequestration in temperate soils. global change. biol. bioenergy, 5, 2013, 153164. šuňovská, a., horník, m., pipíška, m., lesný, j., augustín, j., hostin, s.: characterization of soil additive derived from sewage sludge. nova biotechnol. chim., 12, 2013, 141-153. van vuuren, d.p., bouwman, a.f., beusen, a.h.w.: phosphorus demand for the 1970-2100 period: a scenario analysis of resource depletion. glob. environ. chem., 20, 2010, 428-439. xu, g., wei, l.l., sun, j.n., shao, h.b., chang, s.x.: what is more important for enhancing nutrient bioavailability with biochar application into a sandy soil: direct or indirect mechanism? ecol. eng., 52, 2013, 119-124. zhai, l., zhuoma, c.j., liu, j., wang, h., ren, t., gai, x., xi, b. liu, h.: short-term effects of maize residue biochar on phosphorus availability in two soils with different phosphorus sorption capacities. biol. fertil. soils, 51, 2015, 113122. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:20 utc nova biotechnologica et chimica 14-1 (2015) 115 zhang, b., gao, b.: removal of arsenic, methylene blue, and phosphate by biochar/alooh nanocomposite. chem. eng. j., 226, 2013, 286-292. zhang, j.h., lin, q.m., zhao, x.r.: the hydrochar characters of municipal sewage sludge under different hydrothermal temperatures and durations. j. integr. agric., 13, 2014, 471-482. zhao, x.r., li, d., kong, j., lin, q.m.: does biochar addition influence the change points of soil phosphorus leaching? j. integr. agric., 13, 2014, 499-506. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:20 utc microsoft word horak nb.doc nova biotechnologica vii-i (2007) 5 soil additives immobilising heavy metals in contaminated soils othmar horak, wolfgang friesl-hanl austrian research centers gmbh – arc, div. biogenetics and environmental resources, 2444 seibersdorf, austria abstract: addition of iron oxides, lime, clay minerals and other substances can be used to decrease the plant availability of toxic heavy metals such as pb, zn, and cd. extractability and consequently plant concentrations may be reduced in some cases by more than 50%. the assessment of remediation processes is supported by biomonitoring methods in the field with plantago lanceolata and in the greenhouse by barley test experiments, in combination with extraction by ammonium nitrate. key words: heavy metals, soil amendments, in situ remediation, biomonitoring methods. 1. introduction heavy metal pollution has received considerable attention as one of the most important environmental problems in industrialized countries. major sources of heavy metal input to ecosystems are mining, smelting, metallurgical industries, sludge disposal and agricultural practices. amelioration and rehabilitation of polluted soils can be achieved by in situ treatment, such as phytoremediation or immobilization of metals in soil. the latter technique is subject of research in the austrian reaearch centers and some of the most interesting results are presented in this paper. immobilisation of metals in soils is aimed to significant reduction of their bioavailability by liming and addition of adsorptive materials such as iron oxides, clay minerals, zeolites and organic matter. experiments are conducted mainly with soils from arnoldstein (carinthia), contaminated with pb, zn, and cd by smelter emissions. cu-contaminated soils were taken from brixlegg (tyrol, metal processing plant) and from vineyards in south tyrol. 2. materials and methods batch experiments were carried out with two soils of different ph and contamination levels (pb, zn, cd) for screening 15 soil amendments. in each case, 100 g of soil were mixed with the additives and exposed one month. afterwards, mobile heavy metal fractions were extracted with 1 m nh4no3 and subsequently analysed. plant experiments were conducted in a greenhouse with the two soils in 5 kg-pots and two barley cultivars of different accumulation properties for zn and cd. seven different treatments with combined additives (gravel sludge, iron oxides and lime) were tested for effectiveness to immobilize heavy metals and to reduce their uptake by plants. after a growth period of 99 days barley was harvested and metal analysis was carried out in straw and grains. 6 horak, o. and friesl, w. soil amendments were also tested under field conditions in the contaminated area near arnoldstein where experimental plots were arranged on two sites with barley and a third with pasture vegetation. a detailed description of the experimental methods is given by friesl et al., 2006. a bioavailability test system was designed for monitoring the heavy metal status of soils. short term growth experiments were carried out in the greenhouse with different soils in plastic pots of quadratic cross section and a volume of 1 litre. spring barley (“messina”) was sown in 4 replications, cut after a growth period of 18 days, oven dried and analyzed for heavy metals. analytical methods: soils were air-dried and the 2 mm fraction was separated by passing a screen. the fine soil was digested by aqua regia for total heavy metal analysis. mobile metal fractions were extracted with 1 m ammoniumnitrate (20 g of soil, 50 ml solution) by a shaking procedure of 2 hours. organic carbon was determined with a carlo erba element analyzer and ph was measured in 0.01 m cacl2. plant material was digested by a mixture of concentrated nitric and perchloric acid (5 + 1 volume parts). all heavy metal analyses were carried out by icp-oes or flame-aas, depending on concentration. soil extracts with 1 m nh4no3 were measured by icp-ms. 3. results and discussion some results of the batch experiments are presented in fig. 1. soil b, one of the two soils used in this experiment, had a ph of 6.1, a clay content of 12% and total concentrations (aqua regia) of cd = 4.75 mg.kg-1, zn = 500 mg.kg-1, and pb = 950 mg.kg-1. the amount of additives was generally 2% (w/w), 1.28% for tri, 3.8% for tbs and 0.5% for the dolomite and lime treatments. greatest reductions in mobile cd were achieved with syz (70%), fer (55%), and ca (45%). similar data were obtained for zn. gs reduced cd, pb, and zn by 19%, 27%, and 31%. mobile metal concentrations were increased significantly by addition of tri as a result of decreasing ph. in a second batch experiment dual applications of amendments were tested. a combination of gso + fer, for example, reduced the extractability of cd, zn, and pb by 39%, 42%, and 55%, respectively. all results of the batch experiments are presented in detail by friesl et al. (2006). the amendments giving the most promising results were used afterwards in the plant experiments. in fig. 2, as an example of the pot experiments, the cdconcentration in barley is presented. besides some of the amendments, shown in fig. 1, red mud (rm), a by-product of bauxite processing was used. it consists of about 15 – 17 % al2o3, 12 – 14 % sio2, 39 – 43 % fe2o3, and 8 – 10 % na2o. rm was successfully tested in previous experiments (friesl et al., 2003; 2004). the pot experiments were conducted with the two barley cultivars “hellana” and “bodega”, which were found to differ significantly in foliar accumulation of cd. in fig.1, the first block represents the control treatment of hellana (co-h). the results show the difference between the two cultivars and a significant reduction of the cd accumulation in the cultivar “b”, mainly in the treatments with iron oxides. the most effective treatment was that with combined addition of ferrihydrite + gravel nova biotechnologica vii-i (2007) 7 sludge, decreasing the cd-content in grain from 0.38 mg.kg-1 to 0.14 mg.kg-1. zn accumulation in barley was not influenced by the treatments while for pb a reduction between 20 and 33% could be observed. fig. 1. results of a batch experiment with soil from the experimental site b. cd-concentrations extractable by 1m ammoniumnitrate versus soil treatment. treatments: co (control), bio (compost), goe (goethite), fer (ferrihydrite), gso, gs (gravel sludge), bm, bm2 brick meal), cli (clinoptilolite), syz (synthetic zeolite), tri (triplesuperphosphate), tbs (thomasphosphate) do1, do2 (dolomite), cal (calcite ), ca (lime fertilize) cd in grain (pot soil b) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 bp -c o -h bp -c o -b bp -f er bp -g s+ fe r bp -g s+ r m bp -g s+ ca bp -f er + g s+ bi o m g k g -1 a d b c ccc fig. 2. results of a pot experiment with soil from the experimental site ii (soil b). cd-concentrations in barley grains versus soil treatment. rm = red mud. 0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 0,20 (b )-c o (b )-b io (b )-g oe (b )-f er (b )-g so (b )-g s (b )-b m1 (b )-b m2 (b )-c li (b )-s yz (b )-t ri (b )-t bs (b )-d o1 (b )-d o2 (b )-c al (b )-c a m g k g -1 70 %55 % 45 %20 % 8 horak, o. and friesl, w. the assessment of heavy metal bioavailability is very important in connection with the decision for soil remediation on the one hand and monitoring of the progress of remediation on the other hand. on pasture sites plantago lanceolata may be used as an indicator plant for available metals. for all elements the concentration in the tissue of the plant shows a relationship with soil ph, which is the main parameter influencing the solubility of the metals (horak et al., 2006 a). table 1 gives an example of monitoring pb-concentrations on different sites in arnoldstein, compared to an uncontaminated site in vienna. pb is generally not accumulating to high concentrations in aboveground plant parts (low bf). in plants growing on highly polluted soil of experimental site a (further soil data are shown in table 2) the limit for animal feed (eu guideline 1999/29 eg) will be exceeded. table 1. lead in plantago lanceolata and corresponding soils (mg.kg-1 dm), ph-values and bioconcentration-factor soil, indication ph soil (mg.kg-1) plant (mg.kg-1) bf exp. plot b, control 5.15 970 4.6 0.005 exp. plot b, limed 6.69 914 2.0 0.002 orchard, stossau 6.47 739 3.6 0.005 pasture, stossau 5.25 503 12.0 0.023 close to exp. plot a 6.81 5177 49.0 0.010 vienna, garden 6.80 35 0.3 0.008 table 2. data from the barley test experiment. total and mobile (nh4no3 extractable) heavy metal concentrations in soils, compared with corresponding barley concentrations. soil element total mg.kg-1 mobile mg.kg-1 plant mg.kg -1 zn 973 1.11 483 arnoldstein a pb 2983 1.083 98 cd 15.1 0.196 8.4 zn 524 0.38 127 arnoldstein b pb 912 0.185 2.48 cd 5.5 0.054 1.93 arnoldstein 3 zn 1573 0.88 190 treatment pb 3553 0.236 2.4 red mud *) cd 10.1 0.034 0.59 zn 2133 35.0 1064 stossau 1 d pb 1383 1.20 24 cd 14.7 0.54 8.8 seibersdorf zn 76 0.03 46 uncontaminated pb 16.8 <0.01 0.35 soil cd 0.30 0.008 0.04 *) red mud (iron oxide) was added in spring 1999 table 2 shows data from the barley test experiments. there is a relationship between the mobile metal fraction and the concentration in the barley plants. the long term effect of treatment with iron oxides can be observed in soil arnoldstein 3. this nova biotechnologica vii-i (2007) 9 soil was taken from a container experiment conducted under outdoor conditions since 1987 in the experimental field of austrian research centers. cu-polluted vineyard soils may be generally problematic for growing other crops as a consequence of the high plant toxicity of this element. in the barley test the cu concentration in plant tissue was found in a range between 25 and 40 mg.kg-1, which is above the plant toxicity level of 20 mg.kg-1. results are given in more detail by horak et al., 2006 b. 4. conclusions soil amendments with combined addition of iron oxides and lime may be a suitable method to reduce the mobility and hence the plant uptake of toxic heavy metals. the sustainability of this treatment is confirmed by results from the long term container experiments (friesl et al., 2004). the basic reaction of metal immobilization is the specific adsorption on hydrated surfaces of the iron oxides, which is most effective in the ph-range of >6.5. references friesl, w., lombi, e., horak, o., wenzel, w.w.: immobilisation of heavy metals in soils using inorganic amendments in a greenhouse study. j. plant nutr. soil sci. 166, 2003, 191 – 196. friesl, w., horak, o., wenzel, w.w.: immobilisation of heavy metals in soils by the application of bauxite residues: pot experiments under field conditions. j. plant nutr. soil sci. 167, 2004, 54 – 59. friesl, w., friedl, j., platzer, k., horak, o., gerzabek, m.h.: remediation of contaminated agricultural soils near a former pb/zn smelter in austria: batch, pot and field experiments. environ. pollut., 144, 2006, 40 – 50. horak, o., friesl, w., zwerger i.: heavy metal contamination in the surroundings of a former pb/zn smelter in arnoldstein (austria): monitoring of bioavailable metal fractions in soil. in: mihály szilágyi, klára szentmihályi (eds.), trace elements in the food chain, budapest, may 25 – 27, 2006. horak, o., friesl, w., stimpfl, e.: ein kombiniertes testverfahren zur ermittlung der pflanzenverfügbarkeit von schwermetallen. 118. vdlufakongress, freiburg (d) 19. – 22. sept. 2006. microsoft word hornik nb.doc nova biotechnologica vii-i (2007) 33 determination of long distance transport of cs+, co2+ and zn2+ ions in vascular plants by autoradiography and gamma-spectrometry miroslav horník, martin pipíška, jana sekáčová, jozef augustín department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (hornikm@ucm.sk) abstract: heavy metals and radionuclides can enter the food chain via cereals and vegetables grown in contaminated soils. in the case of microelements such as zinc, studies have not focused only to assessing its environmental risk, but also to enhancing its uptake by plants as an important growth-limiting factor. in our study, digitalized autoradiograms of whole plants of celery (apium graveolens l.), tobacco (nicotiana tabacum l.) and sunflower (helianthus annuus l.) grown in hydroponic nutrient media spiked with 137cscl, 60cocl2 and 65zncl2 were used for quantitative determination of uptake, long-distance transport and distribution of cs+, co2+ and zn2+ ions in plant tissues. results from autoradiography and gammaspectrometry of plants showed, that cesium was translocated to aboveground part of the plants with the shoot-to-root ratio 1.0 : 0.6. on the contrary, cobalt and zinc were more immobilized in roots, with the shoot-to-root ratio up to 1.0 : 3.8. the highest concentration of cesium, cobalt and zinc, expressed in specific radioactivity per unit of leaf surface (bq/cm2) was found in top, rapidly growing leaves, the lowest concentration in the oldest leaves in low position. detection limits 3, 2 and 14 bq/cm2 by using x-ray film for 137cs, 60co and 65zn, respectively were obtained. these data correspond to detection limits 10.5 pg cs+/cm2, 7.2 pg co2+/cm2 and 785 pg zn2+/cm2 at specific radioactivity of commercially available 137cscl, 60cocl2 and 65zncl2. resolutions 1-2 mm was sufficient for visualization of metal uptake and distribution in roots, stalks, leaves and leaf venation. obtained data are part of quantitative study of uptake and translocation of both low level-radioactive contamination in plants and microelements applied as fertilizers. keywords: cobalt, cesium, zinc, distribution, vascular plants, autoradiography 1. introduction heavy metals are present in soils and aqueous streams as both natural components or as a result of human activity i.e. metal-rich mine tailings, metal smelting, electroplanting, energy and fuel production, intensive agriculture, etc. (raskin et al., 1994). radionuclides exist in the environment either naturally or artificially by aboveground nuclear testing, nuclear accidents and nuclear power generation (zhu and shaw, 2000). cesium 137cs (τ = 30.17 y) is produced during the fission of actinides or the neutron bombardment of 133cs or 136ba (white and broadley, 2000). the behavior of cs in soils resembles that of k and, even in soils with high clay content, cs is available for uptake by plants since cs-fixation tends towards a reversible steady-state. cobalt usually occurs in the environment in association with other metals such as copper, nickel, manganese and arsenic. background soil cobalt levels range from 1.6 34 horník, m. et al. to 21.5 mg.kg-1 (perez-espinoza et al., 2005). cobalt 60co (τ = 5.27 y) is also present in low-level radioactive wastes. although zinc is an essential nutrient as a trace element for animals, plants, and microorganisms, it is toxic to these organisms when present at supranormal concentrations. zinc is a trace element essential for cell proliferation and differentiation. it is a structural constituent of many enzymes and proteins, including metabolic enzymes, transcription factors, and cellular signaling proteins. there is considerable interest in remediation of sites contaminated by these elements using extraction by plants that do not enter the human food chain. several comprehensive reviews have been written, summarizing many important aspects of this plant-based technology (lasat, 2002; dushenkov, 2003; suresh and ravishankar, 2004; pilon-smits, 2005; leduc and terry, 2005; marmiroli et al., 2006). the ecological problems related to heavy metals and radionuclides are not dependent only on their total content and radioactivity in the soil, but rather on their form of bonding and therefore their bioavailability. therefore, several studies have been conducted using seedlings or adult plant, which have been cultivated in hydroponic conditions (see e.g. page and feller, 2005; soudek et al., 2006). in our study, digitalization of autoradiograms of whole plants of celery (apium graveolens l.), tobacco (nicotiana tabacum l.) and sunflower (helianthus annuus l.) after cultivation in hydroponic nutrient media spiked with 137cscl, 60cocl2 and 65zncl2 was used as the method for quantitative determination of uptake, long-distance transport and distribution of cs+, co2+ and zn2+ ions in the plant tissues. 2. materials and methods 2.1 plant material seeds of celery (a. graveolens l.), tobacco (n. tabacum l.) and sunflower (h. annuus l.) were germinated and grown in pots filled with granulated perlite as an inert carrier and watered with diluted hydroponic medium according to hoagland (1920) in day/night period 12/12 h (illumination with 2 tubes brilliant daylight 6000 k, 1 300 lm and tropic sun – 4 700 k, 1 000 lm, sera) at 22±2°c. after 5 weeks (for celery and sunflower) or 8 weeks (for tobacco), seedlings were gently removed from perlite and roots were washed free of any adhering perlite fragments by distilled water. plants were pre-cultivated for one week in vessels with a cover to protect plant roots against lights in 25 % hoagland medium without perlite support. 2.2 bioaccumulation experiments for experiments selected plants were cultivated at 22±2°c in 25 % hoagland medium supplemented with 10 µmol.l-1 cscl, 10 µmol.l-1 cocl2 or 5 µmol.l -1 zncl2 and spiked with 137cs, 60co or 65zn at illumination 2 000 lx with 12/12 h day/night cycle. in time intervals aliquot samples of cultivation media were measured for nova biotechnologica vii-i (2007) 35 determination of remaining radioactivity by gamma-spectrometry. the amounts of transpired water were determined by measuring the weight changes of cultivation vessels. the amount of water lost by plant transpiration was replenished with fresh 25 % hoagland medium to initial solution volume. at the end of experiments plants were harvested and roots carefully rinsed in distilled water. in roots, stalks and leaves incorporated radioactivity was measured by gamma-spectrometry and topography visualized by autoradiography as well. 2.3 autoradiography plants were pressed between two filter papers and air-dried for 7 days at 20°c. 137cs, 60co and 65zn in dried plant were detected by autoradiography by exposing xray films (hr-gb 100 nif, fujifilm, jp) for 40 days at 20°c. developed films were scanned and data were converted to colour scale gradient by software photoshop cs2 ver. 9.0 (adobe, usa). 2.4 radiometric analysis gamma-spectrometric scintillation detector 54bp54/2-x with well type crystal nai(tl) (scionix, nl) and data processing software scintivision32 (ortec, usa) were used for 137cs, 60co and 65zn determination in plant and solution. counting time 600 s allowed obtaining data with measurement error <2 %, which do not reflect other source of errors. standardized 137cscl (5.723 mbq.ml-1; 20 mg.l-1 cscl + 3 g.l-1 hcl), 60cocl2 (5.571 mbq.ml -1; 20 mg.l-1 cocl2 + 3 g.l -1 hcl) and 65zncl2 (0.892 mbq.ml-1; 50 mg.l-1 zncl2 + 3 g.l -1 hcl) were obtained from cmi (cz). 3. results and discussion kinetics of cesium and cobalt bioaccumulation by celery (a. graveolens l.), tobacco (n. tabacum l.) and sunflower (h. annuus l.) hydroponically growing in nutrient media spiked with 137cscl and 60cocl2 were described in our previous papers (hornik et al., 2005; baratova et al., 2006; vrtoch et al., 2006; vrtoch et al., 2007). we found, that bioaccumulation of 137cs and 60co by these plants was proportional to the transpiration rate. the aim of this paper is to characterize long-distance transport of 137cs+, 60co2+ and 65zn2+ in celery, tobacco and sunflower. radiometric analysis showed, that cesium accumulated by celery and tobacco from nutrient solution containing sub-inhibitory concentrations 10 µmol.l-1 and 2.4 µmol.l1cscl was transported from roots to shoots. the highest cesium concentration was found in top leaves. (fig. 1, fig. 2). similar pattern of cesium transport in sunflower was observed in our previous paper (hornik et al., 2005). shoot-to-root concentration ratio was within the range 1: 0.60 – 1: 0.88 (tab. 1). concentration of cesium expressed in specific radioactivity per unit of leaf surface (bq/cm2) was 11936 horník, m. et al. times higher in top, rapidly growing celery leaves than in the oldest celery leaves and 12-times higher in top tobacco leaves (fig. 3). fig. 1: autoradiographic visualization of 137cs, 65co and 65zn distribution in celery (a. graveolens l.) after 8 days cultivation in 25 % hoagland medium. initial concentrations c0 = 10 μmol.l-1 cscl, 10 μmol.l-1 cocl2 or 5 μmol.l-1 zncl2, spiked with 167.6 kbq.l-1 137cscl, 91 kbq.l-1 60cocl2 or 68 kbq.l-1 65zncl2. uptake: cs – 37 %; co – 97 %; zn – 40 %. numbering of leaves in the order: 1. oldest leaves, 6. youngest leaves. 60co 137cs 65zn max min 1 2 3 4 5 6 1 2 3 4 5 6 6 nova biotechnologica vii-i (2007) 37 fig. 2: autoradiographic visualization of 137cs and 65co distribution in tobacco (n. tabacum l.) after 9 days cultivation in 25 % hoagland medium. initial concentrations c0 = 2.4 μmol.l-1 cscl or 25 μmol.l-1 cocl2, spiked with 283 kbq.l-1 137cscl or 29.7 kbq.l-1 60cocl2. uptake: cs – 65 %; co – 65 %. numbering of leaves in the order: 1. oldest leaves, 8. youngest leaves. long-distance transport of both cobalt and zinc in celery, tobacco and sunflower cultivated in nutrient media in sub-inhibitory concentration 0.385 – 25 µmol.l-1 cocl2 or 5 µmol.l-1 zncl2 was significantly lower than in the case of cesium. cobalt and zinc were mainly trapped in roots. shoot-to-root concentration ratio up to 1 : 3.78 was observed (tab. 1). concentration of cobalt and zinc, expressed in specific radioactivity per unit of leaf surface (bq/cm2) of celery, tobacco and sunflower was approx. 50times higher in top, younger leaves than in the oldest leaves (fig. 3). 1 2 3 4 5 6 0 100 200 300 400 500 600 250 0 50 100 150 65z n [b q/cm 2] 60c o [b q/cm 2] 13 7 c s [b q/ cm 2 ] leaf position on stems 137cs 60co 65zn 200 250 0 50 100 150 200 15 0 3 6 9 12 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 leaf position on stems 60 c o [b q/ cm 2 ] 0 20 40 60 80 100 120 140 137c s [b q/cm 2] fig. 3: specific radioactivity of 137cs, 60co and 75zn accumulated in leaves of celery (a) and tobacco (b), calculated for leaf surface (bq/cm2). numbering of leaves from top to root. 60co 137cs a b 1 2 3 4 5 6 1 2 3 4 5 6 7 8 max min 38 horník, m. et al. mobility of monovalent cesium in tobacco, celery and sunflower plants is higher than mobility of bivalent zinc and cobalt. this fact is in an agreement with well described transport mechanisms of cesium, comparable with transport mechanisms of essential potassium k+ ions (white and broadley, 2000). cesium, with the exception of some synthetic compounds such as crown ethers, does not form stable organic complexes (dozol et al., 2004). on the other hand, cobalt and zinc can react with low and high molecular organic components of plants (owen et al., 2002). salt et al. (1999) found, that zn was mostly complexed to histidine in roots, transported as zn2+ in the xylem sap, and complexed to organic acids in leaves. in dependence on ph value, zinc and cobalt can form charged species by the reaction with co2 and oh ions (dang et al., 1996; günther and kastenholz, 2005). page and feller (2005) showed that zinc was translocated from root to young wheat leaves within 50 days nearly uniformly in all leaves of the shoot. on the contrary, prevailing part of cobalt even after 50 days remained in root and transport to all leaves including apex leaves was negligible. mobility of zinc and cobalt in vascular plants within time intervals, comparable with duration of vegetation period, will be the topic of our next study. tab. 1: distribution of cesium, cobalt and zinc in shoots and roots of celery (a. graveolens l.), tobacco (n. tabacum l.) and sunflower (h. annuus l.) cultivated in 25 % hoagland medium spiked with 137cscl 60cocl2, or 65zncl2. shoot-to-root ratio nuclide celery 1 tobacco 2 sunflower 3 137cs 1.0 : 0.60 1.0 : 0.88 1.0 : 0.64 60co 1.0 : 3.78 1.0 : 1.02 1.0 : 2.03 65zn 1.0 : 1.29 – – 1 after 8 day cultivation, c0 = 10 μmol.l-1 cscl (168 kbq.l-1), c0 = 10 μmol.l-1 cocl2 (91 kbq.l-1) or c0 = 5 μmol.l-1 zncl2 (68 kbq.l-1) 2 after 9 day cultivation, c0 = 2.4 μmol.l-1 cscl (283 kbq.l-1) or c0 = 25 μmol.l-1 cocl2 (29.7 kbq.l-1) 3 after 9 day cultivation, c0 = 1.25 μmol.l-1 cscl (25 kbq.l-1) or c0 = 0.385 μmol.l-1 cocl2 (25 kbq.l-1). see also hornik et al., (2005). detection limits obtained by autoradiography of the whole plants was 3, 2 and 14 bq/cm2 for 137cs, 60co and 65zn, respectively. it corresponds to detection limits 10.5 pg cs/cm2, 7.2 pg co/cm2 and 785 pg zn/cm2 at specific radioactivities of commercially available 137cscl, 60cocl2 and 65zncl2. resolution 1-2 mm was sufficient for visualization of distribution in roots, stalks, leaves and leaf venation. 4. conclusions autoradiography and gamma-spectrometry of celery (apium graveolens l.), tobacco (nicotiana tabacum l.) and sunflower (helianthus annuus l.) after nova biotechnologica vii-i (2007) 39 cultivation in hydroponic nutrient media spiked with 137cscl, 60cocl2 and 65zncl2 was used for quantitative determination of uptake and long-distance transport of cs+, co2+ and zn2+ ions in the plant tissues. cesium was traslocated to the aboveground part of the plants with the shoot-to-root ratio 1.0: 0.6. on the contrary, cobalt and zinc was more immobilized in roots, with the shoot-to-root ratio up to 1.0: 3.8. the highest concentration of cesium, cobalt and zinc expressed in specific radioactivity per unit of leaf surface (bq/cm2) was found in top, rapidly growing leaves, the lowest concentration in the oldest leaves in low position. detection limits 3, 2 and 14 bq/cm2 by using x-ray film for 137cs, 60co and 65zn, respectively were obtained. it corresponds to detection limits 10.5 pg cs+/cm2, 7.2 pg co2+/cm2 and 785 pg zn2+/cm2 at specific radioactivity of commercially available 137cscl, 60cocl2 and 65zncl2. resolution 1-2 mm was sufficient for visualization of distribution in roots, stalks, leaves and leaf venation. obtained data are part of quantitative study of uptake and translocation of both low-level radioactive contamination in plants and microelements applied as fertilizers. references baratova, z., sekacova, j., hornik, m., pipiska, m., augustin, j.: bioaccumulation of 137cs and 60co by mustard sinapis alba l. and celery apium graveolens l.. nova biotechnol., vi-i, 2006, 7-18. dang, y.p., tiller, k.g., dalal, r.c., edwards, d.g.: zinc speciation in soil solutions of vertisols. aust. j. soil res., 34, 1996, 369-383. dozol, j.f., dozol, m., macias, r.m.: extraction of strontium and cesium by dicarbollides, crown ethers and functionalized calixarenes. j. incl. phenom. macrocyclic chem., 38, 2000, 1-22. dushenkov, s.: trends in phytoremediation of radionuclides. plant soil, 249, 2003, 167-175. günther, k., kastenholz, b.: speciation of zinc. in: r. cornelis; h. crews; j. caruso; k.g. heumann (eds.) handbook of elemental speciation ii: species in the environment, food, medicine and occupational health. john wiley & son, new york, 2005, 488-506. hoagland, d.r.: optimum nutrient solutions for plants. science, 52, 1920, 562564. hornik, m., pipiska, m., vrtoch, l., augustin, j., lesny, j.: bioaccumulation of 137cs and 60co by helianthus annuus. nukleonika, 50(1), 2005, s49-s52. lasat, m.m.: phytoextraction of toxic metals – a review of biological mechanisms. j. environ. qual., 31, 2002, 109-120. leduc, d.l., terry, n.: phytoremediation of toxic trace elements in soil and water. j. ind. microbiol. biotechnol., 32, 2005, 514-520. marmiroli, n., marmiroli, m., maestri, e.: phytoremediation and phytotechnologies: a review for the present and future. in: i. twardowska, allen, h.e., häggblom, m.m., stefaniak, s. (eds.) soil and water 40 horník, m. et al. pollution monitoring, protection and remediation, springer, dordrecht, the netherlands, 2006, 403-415. owen, m., grill, e., golan-golghirsh, a., kutchan, t.m., zenk, m.h.: increase of free cysteine and citric acid in plant cells exposed to cobalt ions. phytochem., 60, 2002, 467-474. page, v., feller, u.: selective transport of zinc, manganese, nickel, cobalt and cadmium in the root system and transfer to the leaves in young wheat plants. ann. bot., 96, 2005, 425-434. perez-espinoza, a., moral, r., moreno-caselles, j., cortés, a., perez-murcia, m.d., gómez, i.: co phytoavailability for tomato in amended calcareous soils. bioresour. technol., 96, 2005, 649-655. pilon-smits, e.a.h.: phytoremediation. ann. rev. plant biol., 56, 2005, 15-39. raskin, i., kumar, p.b.a.n., dushenkov, s., salt, d.e.: bioconcentration of heavy metals by plants. curr. opin. biotechnol., 5, 1994, 285-290. salt, d.e., prince, r.c., baker, a.j.m., raskin, i., pickering, i.j.: zinc ligands in the metal hyperaccumulator thlaspi caerulescens as determined using x-ray absorption spectroscopy. environ. sci. technol., 33, 1999, 713-717. soudek, p., tykva, r., vankova, r., vanek, t.: accumulation of radioiodine from aqueous-solution by sunflower (helianthus annuus l.) at the laboratory scale. environ. exp. bot., 57(3), 2006, 220-225. suresh, b., ravishankar, g.a.: phytoremediation – a novel and promising approach for environmental clean-up. crit. rev. biotechnol., 24(2-3), 2004, 97124. vrtoch, l., hornik, m., pipiska, m., augustin, j.: radiocesium bioaccumulation by hydroponically cultivated tobacco plant (nicotiana tabacum l.). nova biotechnol., vi-i, 2006, 19-28. vrtoch, l., pipiska, m., hornik, m., augustin, j.: bioaccumulation of 60co2+ ions in tobacco plants. nova biotechnol., vii-i, 2007, 41-49. white, p.j., broadley, m.r.: mechanisms of caesium uptake by plants. new phytol., 147, 2000, 241-256. zhu, y.g., shaw, g.: soil contamination with radionuclides and potential remediation. chemosphere, 41, 2000, 121-128. microsoft word szabová nb.doc nova biotechnologica vii-1 (2007) 77 long-term effects of a chemical decontamination procedure on the corrosion state of the heat exchanger tubes of steam generators andrea szabó nagy1, kálmán varga2, bernadett baja2, zoltán németh2, dezső oravetz3, zoltán homonnay4, ernő kuzmann4, jános schunk5 1department of physics and chemistry, university of istván széchenyi, h-9026 győr, hungary (nszaboa@sze.hu) 2institute of environmental engineering and radiochemistry, university of pannonia, h-8201 veszprém, p. o. box: 158, hungary (vargakl@almos.vein.hu) 3 institute of materials engineering, university of pannonia 4institute of chemistry, eötvös loránd university, h-1117 budapest, hungary 5paks npp ltd., h-7031 paks, p. o. box: 71, hungary abstract: our previous studies have revealed that a ”hybrid” structure of the amorphous and crystalline phases is formed in the outermost surface region of the austenitic stainless steel tubes of steam generators (sgs) as an undesired consequence of the industrial application of the ap-citrox (ap: alkaline permanganate; citrox: citric and oxalic acid) decontamination technology. the formation of this mobile oxide-layer increased the amount of the corrosion products in the primary circuit significantly, resulting in magnetite deposition on fuel assemblies. owing to the fact that there is no investigation method available for the in-situ monitoring of the inner surfaces of heat exchanger tubes, a research project based on sampling as well as on ex-situ electrochemical and surface analytical measurements was elaborated. within the frame of this project, comprehensive investigation of the general corrosion state and metallographic features of 36 stainless steel specimens, cut out from various locations of the 21 steam generators of the paks npp in the time period of 2000-2007 has been performed. the present work gives a brief overview on the general corrosion state of the heat exchanger tubes of sgs, concerning the long-term effects of the ap-citrox procedure on the chemical composition and structure of the protective oxide-layer. key words: steam generators, decontamination, voltammetry, sem-edx, cems 1. introduction in some soviet-made vver-type pressurized water reactors different versions of the so-called ap-citrox method (ap: alkaline permanganate; citrox: citric and oxalic acid) have been widely used for the chemical decontamination of the austenitic stainless steel piping of steam generators (sgs) (varga, 2004; varga et al., 2001, 2002, 2004). during the time period of 1993-2001 chemical decontamination of 24 sgs in the reactor blocks 1-3 of the paks npp (hungary) were carried out by a nonregenerative version of ap-citrox technology, sometimes in 2 or 3 consecutive cycles. the applied version of the ap-citrox procedure is an eight-step process, 78 szabó, a. et al. including an oxidizing pre-treatment of the surface with alkaline potassium permanganate followed by washing with a concentrated mixture of citric and oxalic acids to remove the contaminated surface layer (varga et al., 2001, 2002, 2004). our previous studies have revealed that a ”hybrid” structure of the amorphous and crystalline phases is formed in the outermost surface region of the austenitic stainless steel tubes as an undesired consequence of the industrial application of the apcitrox technology (varga, 2004; varga et al., 2004; szabó et al., 2006; radó et al., 2006; varga et al., 2006; homonnay et al., 2006). the formation of this mobile oxide-layer increased the amount of the corrosion products in the primary circuit significantly, resulting in magnetite deposition on fuel assemblies. as deposits blocked the cooling channels, the flow rate of water coolant through the reactor core decreased. consequently, the power capacity of three nuclear reactor units had to be reduced, and full core fuel replacement became necessary. in the light of the above events, the present work gives a brief overview on the general corrosion state of the heat exchanger tubes of sgs, concerning the long-term effects of the ap-citrox procedure on the chemical composition and structure of the protective oxide-layer. owing to the fact that there is no investigation method available for the in-situ monitoring of the inner and outer surfaces of heat exchanger tubes, a research project based on sampling as well as on ex-situ electrochemical and surface analytical measurements was elaborated. within the frame of this project, comprehensive investigation of the general corrosion state and metallographic features of 36 stainless steel specimens, cut out from various locations of the 21 steam generators of the paks npp in the time period of 2000-2007 has been performed. in this paper we report some voltammetric, sem-edx, and cems results that reveal the corrosion properties, morphology and chemical composition of the inner surfaces of heat exchanger tubes. 2. materials and methods the experiments have been performed on 36 austenitic stainless steel specimens (type: 08x18h10t (gost 5632-61) which corresponds to aisi 321 and din 1.4541; outer diameter: 16 mm, average wall thickness: 1.6 mm) originating from different sgs of the paks npp. the main characteristics of the specimens obtained from the same sgs at various sampling date are given in table 1. the surface decontamination procedure of the heat exchanger tubes was carried out at paks npp according to a nonregenerative version of the ap-citrox technology (varga et al., 2001, 2002, 2004). the passivity of the tube samples was studied by potentiostatic polarization method. the experiments were carried out by the means of a voltalab 40 (radiometer) type electrochemical measuring system controlled by pc. the measurements were carried out in boric acid solution (c=12 g·dm-3) in argon gas atmosphere (99.999 v/v % ar). the schematic of the measuring system, the detailed experimental procedure and the determination of the corrosion parameters have been described in our earlier papers (varga et al., 2001; szabó et al., 2006; varga et al., 2006; homonnay et al., 2006). nova biotechnologica vii-1 (2007) 79 the morphology and chemical composition of the oxide layer developed on the inner surfaces of the 36 stainless steel specimens were studied by scanning electron microscopy (sem), equipped with an energy dispersive x-ray microanalyzer (edx) (type: jeol jsm-50a, controlled with röntec edr 288 software). in addition, the metallographic cross sections of tube specimens were also studied (szabó et al., 2006; radó et al., 2006; varga et al., 2006; homonnay et al., 2006). in order to determine the phase composition of the surface oxide layers, the samples were measured by conversion electron mössbauer spectroscopy (cems). the cems spectra were recorded at room temperature with a conventional mössbauer spectrometer (wissel) in constant acceleration mode and a 57co(rh) source provided the γ-rays (szabó et al., 2006; varga et al., 2006; homonnay et al., 2006). table 1. characteristic features of tube samples obtained from the same steam generators at various sampling date. number of sg sample year of decontamination time period between the last decontamination and the sampling [year] year of sampling year of investigation i/1 1996, 1997 4 2001 2001 i/2 1996,1997, 2001 4 2005 2005 ii/1 1996 1 1997 2003 ii/2 1996 4 2000 2000 ii/3 1996, 2001 4 2005 2005 iii/1 1999, 2001 1 2002 2002 iii/2 1999, 2001 3 2004 2004 iii/3 1999, 2001 5 2006 2006 iv/1 2001 1 2002 2002 iv/2 2001 5 2006 2006 v/1 2001 0 2001 2001 v/2 2001 4 2005 2005 vi/1 1993 7 2000 2000 vi/2 1993, 2001 3 2004 2004 3. results and discussion 3.1 general corrosion state and morphology as illustration, potentiostatic polarization curves measured in boric acid solution at the inner surface of the stainless steel specimens cut out from the same steam generators of the paks npp at various sampling date are shown in figs. 1-2. as may be seen in figs. 1-2, the inner surfaces of the samples have a passive character in a wide potential interval next to the corrosion potential. the average corrosion rates for the samples, independently of the time period spent between the last decontamination and the sampling are very low (vc ≤ 3.5 μm/year). the measured corrosion rates are as low as that of the inactive reference sample and even better than the literature data which were measured for the stainless steels type 08x18h10t and aisi 321 in aqueous solutions (0.3 4 μm/year) (geraszimov and monahov, 1991; lister 2003, and references therein). the results imply that the average corrosion 80 szabó, a. et al. -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 potenciál / v lg i / a c m -2 ii/2 -6 -7 -5 -8 -9 ii/1 ii//3 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 potenciál / v lg i / a c m -2 iii/3 -6 -7 -5 -8 -9 iii/1 iii/2 rate of decontaminated steel surfaces in the long run exhibits favorable trend under normal operation conditions of vver-type nuclear reactor. the morphology and chemical composition of the inner surfaces of the specimens were studied by sem-edx method. some typical sem micrographs of the steel samples studied are shown in fig. 3. on the surfaces of samples never-decontaminated a thin film of the grown-on oxides with excellent protective character can be detected. cracks in these oxide-films can not be identified; however, large amounts of lessadherent crystalline phases (presumably magnetite, hematite and non-stoichiometric mixed oxide of spinel structure) are found on the top of the grown-on oxide-layer. it is of special interest to note that some large crystals can be observed sparsely on the surface of the samples decontaminated immediately before the cutting procedure, too. the protective oxide-layers formed on the surfaces of the samples decontaminated a few (3-4) years before the sampling are compact, thick (up to 11 µm), and contain many cracks and scattered deep failures. the surface region exhibits amorphous character and are rich in chromium and nickel. 3.2 phase analysis of the surface oxide-layers due to the absorption of the conversion electrons, information can be obtained from the ∼300 nm thick layer of the sample surfaces. the percent rates refer to the iron content involved in the indicated phases relative to the total iron content of the sample detectable by the conversion electrons. on analyzing the cems spectra it has become obvious that a common phase on the inner surface of all tube specimens is the amorphous iron-hydroxide (fe(oh)3 and/or feo(oh)). its dominance is most characteristic for the decontaminated samples, especially for those steel tubes which were decontaminated by the apcitrox procedure a few years before the sampling. at the same time, it is difficult to understand the slight amount of amorphous fe(oh)3 detected on the samples neverdecontaminated. this conspicuous oxide-layer may most likely be formed in either the transient and shut-down periods of the reactor operation or under the storage conditions of the steel specimens (as a consequence of the humid air). fig. 1. potentiostatic polarization curves measured at the inner surface of the samples no. ii in boric acid solution (c = 12 g·dm-3). scan rate: 10 mv·min-1. fig. 2. potentiostatic polarization curves measured at the inner surface of the samples no. iii in boric acid solution (c = 12 g·dm-3). scan rate: 10 mv·min-1. nova biotechnologica vii-1 (2007) 81 in accordance with the literature data (see e.g. szabó et al., 2006; varga et al., 2006 and references cited therein), magnetite and/or hematite are dominant on the inner surfaces of samples that were never subjected to decontamination. magnetite is represented by two sextets in the mössbauer spectra due to two different cationic sites for iron in the inverse spinel structure, while hematite can be described with one sextet only. the mössbauer parameters obtained for these phases (especially for magnetite) were slightly different from the literature data on the pure compounds, which can be attributed to the effect of crand ni-substitution. it is especially noteworthy that the approximate intensity ratio of the two sextets of magnetite is normally ih:il = 1:2, where ih is the intensity of the sextet with higher magnetic field (representing fe 3+ at the tetrahedral site of the unit cell of magnetite fe3+tet(fe 3+fe2+)octo4) and il is the intensity of the sextet with lower magnetic field (representing fe3+ and fe2+ coupled by rapid electron hopping at the octahedral site), but we have found in our samples roughly the opposite ratio. since it is well known that cr3+ and ni2+ is ready to substitute for iron at the octahedral site, it is logical to assume such substitution in our case, on the surface of a cr-ni-steel. a formal calculation results in a possible composition of fe3+tet(fe 3+ 1/4cr 3+ 3/4fe 2+ 1/4ni 2+ 3/4)octo4 for ih:il = 2:1. fig. 3. the long-term effects of the ap-citrox procedure on the surface structure and morphology of the heat exchanger tubes of sgs. it is important to return to the detection efficiency of the different phases by cems. since it is experimentally demonstrated (szabó et al., 2006; varga et al., chemical decontamination by ap-citrox technology under normal operating conditions sg no. i/1 sg. no. i/2 sg no. v/2 sg no. v/1 sg never subjected to decontamination 4 years 4 years 4 years 82 szabó, a. et al. 2006) that, as a tendency, the spinel phases (substituted magnetite) directly cover the bulk steel, and amorphous fe(oh)3 may be formed only on the outermost surface; the amount of the former is underestimated, while that of the latter is overestimated. the relative amount of the spinel phase can be even further reduced (may become practically invisible at the statistics of the particular mössbauer spectrum) if the substitution of fe by cr and ni is very significant. 4. conclusions within the frame of a comprehensive program to qualify the general corrosion state of heat exchanger tubes, corrosion and metallographic features of 36 specimens originating from different sgs of the paks npp were studied by electrochemical (voltammetry) and surface analyzing (sem-edx, cems) methods. despite the limited number of tube samples, some general conclusions can be drawn. some adverse effects (general attack, formation of “hybrid” layer with accelerated corrosion rate and greater mobility) have been detected promptly after applying the ap-citrox procedure. process restrictions and modifications to minimize corrosion damages should be defined, and there is ample data available now for utilities to select a citrox based process for particular application. it is, however, of special importance to highlight that significant beneficial change in the corrosion properties, mobility and chemical composition of the inner oxide layers of heat exchanger tubes can be observed in the time period of 2000-2007, as a consequence of the normal operation of the vver type reactors. acknowledgements this work was supported by the paks npp co. ltd (paks, hungary) and the hungarian science foundation (grant no. t047219). references geraszimov v. v., monahov sz.: materiali jadernoj techniki, atomizdat, moskva, 1981. (hungarian translation). homonnay, z., kuzmann, e., stichleutner, s., varga, k., németh, z., szabó, a., radó, k., makó, k. é.,kövér, l., cserny, i., varga, d., tóth, j., schunk, j., tilky, p., patek, g., oszvald, f.: comprehensive investigation of the corrosion state of the heat exchanger tubes of steam generators. part ii. chemical composition and structure of tube surfaces. j. nucl. mater., 348, 2006, 191-204. lister, d. h.: some aspects of corrosion in cooling water systems and their effects on corrosion product transport. proceedings of the eurocorr 2003, budapest, hungary, 28 september – 2 october, 2003. radó, k., németh, z., varga, k., schunk, j., kőrösi, f.: fundamental issues of the effective decontamination of the steam generators of paks npp. j. radioanal. nucl. chem., 268(2), 2006, 313-322. nova biotechnologica vii-1 (2007) 83 szabó, a., varga, k., németh, z., radó, k., oravetz, d., makó, k. é., homonnay, z., kuzmann, e., tilky, p., schunk, j., patek, g.: effect of a chemical decontamination procedure on the corrosion state of the heat exchanger tubes of steam generators. corrosion sci., 48, 2006, 2727-2749. varga, k.: the role of interfacial phenomena in the contamination and decontamination of nuclear reactors. in: radiotracer studies of interfaces (ed. by g. horányi), interface science and technology, elsevier b.v., amsterdam, 2004, vol. 3, 313-358. varga, k., németh, z., somlai, j., varga, i., szánthó, r., borszéki, j., halmos, p., schunk, j., tilky, p.: hydrodynamics of the effectiveness of the ap-citrox decontamination technology. j. radioanal. nucl. chem., 254(3), 2002, 589-596. varga, k., baradlai, p., hirschberg, g., németh, z., oravetz, d., schunk, j., tilky, p.: corrosion behaviour of stainless steel surfaces formed upon chemical decontamination. electrochim. acta, 46, 2001, 3783-3790. varga, k., németh, z., szabó, a., radó, k., oravetz, d., makó, k. é., homonnay, z., kuzmann, e., stichleutner, s., tilky, p., schunk, j., oszvald, f., patek, g.: comprehensive investigation of the corrosion state of the heat exchanger tubes of steam generators. proceedings of the wano-mc workshop: ”experiences and techniques of sg decontamination in vver plants”, paks npp, paks, hungary, april 5-9, 2004. varga, k., németh, z., homonnay, z., szabó, a., radó, k., oravetz, d., schunk, j., tilky, p., oszvald, f.: comprehensive investigation of the corrosion state of the heat exchanger tubes of steam generators. part i. general corrosion state and morphology. j. nucl. mater., 348, 2006, 181-190. microsoft word chmelova nbc 2015i.doc nova biotechnologica et chimica 14-2 (2015) 191 doi 10.1515/nbec-2015-0026 © university of ss. cyril and methodius in trnava effect of metal ions on triphenylmethane dye decolorization by laccase from trametes versicolor daniela chmelová, miroslav ondrejovič department of biotechnologies, faculty of natural sciences, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (daniela.chmelova@ucm.sk) abstract: the aim of this study was investigate the influence of different metal ions on laccase activity and triphenylmethane dye decolorization by laccase from white-rot fungus trametes versicolor. laccase activity was inhibited by monovalent ions (li+, na+, k+ and ag+) but the presence of divalent ions increased laccase activity at the concentration of 10 mmol/l. the effect of metal ions on decolorization of triphenylmethane dyes with different structures namely bromochlorophenol blue, bromophenol blue, bromocresol blue and phenol red was tested. the presence of metal ions (na+, k+, mg2+, ca2+, ba2+, mn2+, zn2+) slightly decreased triphenylmethane dye decolorization by laccase from t. versicolor except na+ and mg2+, which caused the increase of decolorization for all tested dyes. decolorization of selected dyes showed that the presence of low-molecular-weight compounds is necessary for effective decolorization. hydroxybenzotriazole (hbt) is the most frequently used. although hbt belongs to most frequently used redox mediator and generally increase decolorization efficiency, so its presence decreased decolorization percentage of bromophenol blue and bromochlorophenol blue, the influence of metal ions to dye decolorization by laccase has the similar course with or without presence of redox mediator hbt. key words: laccase activity, trametes versicolor, triphenylmethane dyes, decolorization, metal ions, hydroxybenzotriazole 1. introduction synthetic dyes with different heterocyclic, anthraquinone, triphenylmethane or azo-based chemical structures are extensively used in industrial applications. triphenylmethane dyes are widely used in various industrial sectors (dying of nylon, wool, silk or cotton, colouring of plastics, fats, oils or waxes, dermatological agents) and therefore are the main pollutants in wastewater (thakur, 2006; casas et al., 2009; yan et al., 2014). therefore, it is necessary to find ecologically effective biological removal of these solutions because chemical or physical methods are largely ineffective (robinson et al., 2001). biological methods have many advantages such as environmental friendly or production of less sludge (casas et al., 2009). currently, one of the possible alternatives for biodegradation of synthetic dyes is the use of white-rot fungi which produce extracellular ligninolytic enzymes, especially laccases, which can oxidize wide spectrum of phenolic and non-phenolic substrates including synthetic dyes (casas et al., 2009; levin et al., 2010; ashrafi et al., 2013; saroj et al., 2014). laccases (phenoloxidases, ec 1.10.3.2) are oxidases which belong to multi-copper oxidases family. these enzymes can oxidize various organic pollutants, using molecular oxygen as the final electron acceptor (barreca et al., 2003; bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:10 utc 192 chmelová, d. and ondrejovič, m. camarero et al., 2005). laccases can be used for triphenylmethane dye degradation. however, in terms of the practical applications of laccases to degradation dyes, such as triphenylmethane dye-containing wastewater, there are still many problems to overcome. wastewater typically contains high levels of salts, metal ions and organic compounds which could decrease laccase activity (grassi et al., 2011). therefore, there is a need for laccase with high tolerance to different ions or compounds which are present in the industrial wastewater. although several studies have been investigated on laccase/mediator system of synthetic dye decolorization (liu et al., 2004; grassi et al., 2011; chen and ting, 2015), little attention has been focused on the effect of metal ions on laccase-catalysed dye decolorization (murugesam et al., 2009). preliminary screening of laccase produced by different white-rot fungi was based on the literature. from the group of white-rot fungi, trametes species are the most important sources for laccase with interesting properties (grassi et al., 2011; yan et al., 2014). namely trametes versicolor is reported to be an excellent producer of laccase (birhanli and yesilada, 2010; souza et al., 2011; piscitelli et al., 2011; wang et al., 2013). the aims of this study were to determine the effect of metal ions on laccase activity and to investigate triphenylmethane dye decolorization by laccase from t. versicolor with metal ions. 2. materials and methods 2.1 chemicals, dye and the enzyme hydroxybenzotriazole (hbt), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (abts) and four synthetic dyes were obtained from sigma aldrich (usa). all other reagents and chemicals were of the highest purity available and were obtained from mikrochem (slovak republic). the extracellular laccase from trametes versicolor was purchased from sigma aldrich. 2.2 effect metal ions on laccase activity the effect of metal ions (li+, na+, k+, ag+, mg2+, ca2+, ba2+, mn2+, cu2+, zn2+, sn2+ and fe3+) on laccase activity was determine in concentrations 1, 5 and 10 mmol/l with abts (1 mmol/l) in phosphate buffer (100 mmol/l; ph 5.0) after 10 minutes. 2.3 decolorization of triphenylmethane dyes by laccase triphenylmethane dyes used for decolorization experiments were solubilized in 50 mmol/l phosphate buffer (ph 5.0). the reaction mixture (200 µl) contained phosphate buffer with dye (50 mg/l; 150 µl) and laccase solution (0.1 u/ml; 50 µl) with/without presence of inorganic ions (1 mmol/l) and redox mediator hbt (1 mmol/l). the reaction was initiated by enzyme addition. reaction mixture was incubated at 20 °c and analysed every 24 hours. absorbance was measured at bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:10 utc nova biotechnologica et chimica 14-2 (2015) 193 different wavelength depending on used synthetic dye. the percentage decolorization was calculated according to following formula: 2.4 determination of laccase activity laccase activity was determined by oxidation of abts (shin et al., 1987). the assay mixture contained 150 µl of 50 mmol/l phosphate buffer (ph 5.0) with 1 mmol/l abts and 50 µl of enzyme extracts. oxidation of abts was monitored by measuring of absorbance at 405 nm. activity of laccase was expressed in unit (u) as the amount of enzymes able to oxidation of 1μmol of abts per minute. 2.5 statistical analysis all experiments were realized in triplicate. the obtained data were evaluated by excel (microsoft, 2010). 3. results and discussion 3.1 effect of metal ions on laccase activity metal ions are considered to be potential laccase inhibitors because these ions bind to the enzymes and de/stabilize the protein and they change the enzyme activity (zhao et al., 2012; hu et al., 2014). textile wastewater usually contains a high concentration of metal ions such as cu2+, zn2+ or na+ which come from dye production process or the dye molecules (grassi et al., 2011; yan et al., 2014). the first step was evaluation of the effect of some potential inhibitors such as li+, na+, k+, ag+, mg2+, ca2+, ba2+, mn2+, cu2+, zn2+, sn 2+ and fe3+ ions on laccase activity. the influence of inorganic inhibitors to laccase expressed as percent of residual laccase activity is summarized in table 1. laccase from t. versicolor seem to be tolerant to metal ions which are generally present in wastewater (table 1). the results revealed that the most of the metal ions did not inhibit the laccase activity up to 1 mmol/l. however, laccase activity was strongly inhibited to various extents by the usual inhibitors, such as fe3+. monovalent ions such as li+, na+, k+ and ag+ inhibited laccase activity at the higher concentration (5 and 10 mmol/l). fang et al. (2012) found that ions bind near t1 site of laccase and acts as competitive inhibitors of electron donors by blocking the access of substrate to the t1 site or inhibiting electron transfer. divalent ions have positive effect to residual laccase activity. however, several metal ions (cu2+, mn2+, zn2+, mg2+) can induct conformational modification of the enzyme and stimulate decomposition of the trimer complex (substrate, enzyme, metal ion) as evidenced by non-competitive inhibition model (duggleby, 1979). but bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:10 utc 194 chmelová, d. and ondrejovič, m. their competition with cu2+ contained in the laccase catalytic site in the electron transport system probably changed into a cooperative relationship (lu et al., 2012). this synergistic effect on substrate had probably increased residual laccase activity. these results are better than the influence of metal ions on activity of laccases isolated from ganoderma lucidum (murugesam et al., 2009), trametes trogii (grassi et al., 2011) or russula virescens (zhu et al., 2013). it can be assumed that these divalent metal ions can act as inorganic redox mediators. table 1: the effect of potential inorganic inhibitors on laccase activity. residual laccase activity (%) inhibitor concentration (mmol/l) potential inhibitors 0 1 5 10 li+ 100.0 110.3 ± 3.1 105.1 ± 2.5 86.9 ± 3.1 na+ 100.0 96.6 ± 7.5 78.4 ± 3.3 64.2 ± 0.9 k+ 100.0 104.8 ± 9.0 89.4 ± 3.7 47.6 ± 0.6 ag+ 100.0 103.1 ± 1.5 95.5 ± 3.1 94.3 ± 4.7 mg2+ 100.0 109.6 ± 4.9 103.8 ± 0.4 116.3 ± 1.1 ca2+ 100.0 93.9 ± 0.9 119.7 ± 5.3 107.1 ± 5.2 ba2+ 100.0 103.5 ± 1.8 149.0 ± 4.9 139.2 ± 6.3 mn2+ 100.0 143.7 ± 4.0 123.9 ± 2.1 134.4 ± 2.1 cu2+ 100.0 105.8 ± 3.7 93.4 ± 3.8 210.0 ± 4.0 zn2+ 100.0 121.2 ± 1.5 104.1 ± 5.1 107.2 ± 2.1 sn2+ 100.0 101.5 ± 1.9 99.7 ± 4.9 99.6 ± 2.6 fe3+ 100.0 86.2 ± 2.2 34.8 ± 2.0 4.7 ± 3.4 3.2 effect of metal ions on laccase-catalyzed decolorization of triphenylmethane dyes determination of metal ion influence on laccase dye decolorization is essential to potential negative effect on laccase activity and decolorization process. in this study, triphenylmethane dyes were decolorized by laccase from trametes versicolor without or with metal ions. these dyes exhibit significant structural variability and are commonly used in textile or chemical industry (fig. 1). fig. 1. structure of selected triphenylmethane dyes. phenol red bromocresol purple bromophenol blue bromochlorophenol blue bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:10 utc nova biotechnologica et chimica 14-2 (2015) 195 purified commercial laccase ability of decolorization was expressed as percentage decrease of absorbance of selected triphenylmethane dye. dye decolorization of laccase was carried out at ph 5.0 and 20 °c with respect to the conditions of laccase activity determination (data not shown) for 7 days. decolorization of triphenylmethane dyes can be observed in vis kinetic spectrum (fig. 2) showing the decrease maximum absorbance by laccase oxidation. fig. 2. kinetics of selected triphenylmethane dye decolorization (a – phenol red, b – bromocresol purple, c – bromophenol blue and d – bromochlorophenol blue) by laccase from trametes versicolor during 7 days at ph 5.0 and 20 °c. decolorization efficiency may be attributed to the molecular structure of triphenylmethane dye (liu et al., 2004). the presence of electron-withdrawing groups such as -cooh, -so3h, -no2 groups can decrease dye degradation while the presence of electron-donating groups such as -oh, -ch3, och3, -nh2, -n(ch3)2, nh-coch3 can increase dye degradation (suzuki et al., 2001; chen and ting, 2015). the highest efficiency of laccase was observed at bromocresol purple decolorization (97.8 %), further bromophenol blue (19.9 %), phenol red (18.2 %) and bromochlorophenol blue (15.1 %) (fig. 2). the presence of halogen groups in the bromophenol blue (19.9 ± 0.4 %) and bromochlorophenol blue (15.1 ± 0.8 %) structure decrease of laccase-catalysed decolorization (chivukula and renganathan, 1995). the presence of chloro anions in bromochlorophenol blue structure led probably to decrease of the decolorization efficiency (fig. 2). although bromocresol purple similarly contains bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:10 utc 196 chmelová, d. and ondrejovič, m. halogen groups of the aromatic rings so the presence of electron-donating groups (-ch3) makes this dye less resistant to oxidative degradation by laccase (97.8 ± 0.4 %). surprisingly, phenol red degradation (18.2 ± 0.8 %) was comparable to bromophenol blue and bromochlorophenol blue, although phenol red is structurally similar to the monomers of lignin which are commonly laccase substrate and moreover, phenol red dye contains hydroxyl groups on its benzene rings. the change in peak pattern for triphenylmethane dyes with the high percent of decolorization based on the adsorption spectrum (400-700 nm) suggest the biodegradation potential for bromocresol purple and phenol red (fig. 3). these results suggest that the destruction of chromophoric groups would lead to dye degradation (chen and ting, 2015). small structural differences between selected triphenylmethane dyes affect their decolorization. therefore, dyeing effluents are serious problem for environment, determination of metal ion influence on laccase dye decolorization is essential. the waste water contains different concentration of metal ions in the range from 0.5 to 100 ppm (sancey et al., 2011; anirudhan and sreekumari, 2011). table 2: the effect of metal ions (10 mmol/l) on laccase mediated decolorization of selected triphenylmethane dyes during 7 days at 5.0 and 20 °c. phenol red bromocresol purple bromophenol blue bromochlorophenol blue na+ 20.2 ± 0.9 87.7 ± 0.7 34.9 ± 0.4 18.4 ± 1.5 k+ 13.7 ± 1.1 83.3 ± 0.5 17.1 ± 0.2 6.6 ± 0.6 mg2+ 9.9 ± 0.4 89.2 ± 0.2 16.5 ± 1.1 5.5 ± 2.4 ca2+ 10.2 ± 0.4 87.5 ± 0.5 15.0 ± 0.7 5.8 ± 0.5 ba2+ 8.6 ± 2.3 83.6 ± 0.1 15.8 ± 0.6 4.6 ± 0.3 mn2+ 10.7 ± 0.3 87.5 ± 0.3 31.9 ± 1.5 6.3 ± 1.2 zn2+ 17.6 ± 0.9 84.6 ± 0.5 39.6 ± 0.5 28.7 ± 1.0 control 18.2 ± 0.8 97.8 ± 0.4 19.9 ± 0.4 15.1 ± 0.8 wastewater with higher metal ion concentrations is usually considered dangerous for environment and must be treated using physical or chemical methods. in our experiments, we have worked with lower limit of this metal ion concentration range (10 mmol/l). based on the literature studying the influence of metal ion on enzymatic dye decolorization (liu et al., 2004; murugesan et al., 2009; grassi et al., 2011; chen and ting, 2015), the effect of selected metal ions on dye decolorization, namely phenol red, bromocresol purple, bromophenol blue and bromochlorophenol blue was observed and the results are shown in table 2. among the seven metal ions normally found in dyeing effluents, na+ and zn2+ have caused slightly increase of decolorization efficiency of selected synthetic dyes, except bromocresol purple with the comparable decolorization ability to the control (table 2). although the presence of monovalent ions such as li+, na+, k+ and ag+ rapidly bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:10 utc nova biotechnologica et chimica 14-2 (2015) 197 decreased laccase activities (table 1), the presence of these ions, especially na+ had slightly stimulatory effect on triphenylmethane dye decolorization (table 2). these results suggest that laccase from t. versicolor is resistant to sodium chloride, which is usually primary salt found in wastewater (zilly et al., 2011) similarly, murugesan et al. (2009) described that decolorization of laccase from ganoderma lucidum was positively enhanced with zn2+ ions in concentration 10 mmol/l. in the presence of other metal ions, bromocresol purple and bromophenol blue decolorization were minimally affected. decolorization of phenol red and bromochlorophenol blue was partially inhibited in the presence of metal ions, except na+ and zn2+. murugesan et al. (2009) found that the increasing the concentration of metal ions from 1 to 10 mmol/l negatively enhanced decolorization efficiency of laccases isolated from g. lucidum. the above results suggest that t. versicolor laccase is useful for synthetic dye degradation from wastewater. based on the results obtained above, we have verified the possibility of the increasing of decolorization efficiency by laccase in the presence of low-molecularweight compounds known as redox mediators. the most commonly used synthetic mediator is hydroxybenzotriazole (hbt) which appears to be very effective mediator (grassi et al., 2011; ashrafi et al., 2013; shankar and nill, 2015). table 3: the effect of metal ions (10 mmol/l) on laccase mediated decolorization of selected triphenylmethane dyes in the presence of hydroxybenzotriazole (1 mol/l) during 7 days at 5.0 and 20 °c. phenol red bromocresol purple bromophenol blue bromochlorophenol blue na+ 33.1 ± 1.8 89.9 ± 0.5 14.5 ± 2.0 14.0 ± 1.2 k+ 13.2 ± 0.2 87.4 ± 0.5 5.9 ± 0.3 6.3 ± 0.6 mg2+ 7.4 ± 2.9 87.7 ± 0.8 2.9 ± 0.5 3.1 ± 0.1 ca2+ 4.4 ± 0.8 88.0 ± 0.1 2.3 ± 0.4 2.9 ± 0.4 ba2+ 3.1 ± 1.5 85.9 ± 0.5 3.9 ± 0.5 2.9 ± 0.3 mn2+ 15.6 ± 3.7 90.8 ± 0.3 6.5 ± 0.2 7.1 ± 0.1 zn2+ 22.0 ± 1.4 90.6 ± 0.4 13.1 ± 1.1 11.6 ± 0.3 control 28.2 ± 0.7 99.6 ± 0.2 15.1 ± 0.3 12.0 ± 0.7 the effect of hbt on dye decolorization by laccase from t. versicolor showed an increase of decolorization efficiency of phenol red (28.2 %) and bromocresol purple (99.6 %). bromophenol blue (15.1 %) and bromochlorophenol blue (12.0 %) decolorization was inhibit by the presence of hbt in reaction (table 3). laccase oxidizes redox mediators and creates unstable radicals. in the case of hbt, putative n-oh radicals are generate (morozova et al., 2007; moreira et al., 2014). these radicals act by mechanism different from enzymatic one, namely radical mechanism. it is possible that radicals forming by mediator oxidation by laccase, cause triphenylmethane dye decolorization. the effect of metal ions on decolorization efficiencies of selected triphenylmethane dyes had the similar course as in the reaction without redox mediator hbt (table 2). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:10 utc 198 chmelová, d. and ondrejovič, m. 4. conclusion in this work, we studied the influence of metal ions on laccase activity and enzymatic dye decolorization of selected triphenylmethane dyes. the results showed that monovalent ions had negative effect on laccase activity while divalent ions positively affected laccase activity. experimental data indicated that the triphenylmethane dye degradation was depended on dye structure and the presence of methyl group of aromatic rings positively enhanced dye oxidation by laccase from trametes versicolor. the presence of na+ and zn2+ stimulated laccase decolorization efficiency and caused the increase of decolorization percent. acknowledgement: this work was supported by the grant no. fppv-32-2015. 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ind. microbiol. biotechnol., 31, 2004, 127-132. moreira, s., milagres, a.m.f., solange, i. m.: reactive dyes and textile effluent decolorization by a mediator system of salt-tolerant laccase from peniophora cinerea. sep. purif. technol., 135, 2014, 183-189. morozova, o.v., shumakovich, g.p., shleev, s.v., yaropolov, y.i.: laccase-mediator systems and their applications: a review. appl. biochem. microbiol., 43, 2007, 523-535. murugesam, k., kim, y.m., jeon, j.r., chang, y.s.: effect of metal ions on reactive dye decolorization of laccase from ganoderma lucidum. j. hazardous mater., 2009, 168, 523-529. piscitelli, a., giardina, p., lettera, v., pezzalla, c., sannia, g., faraco, v.: induction and transcriptional regulation of laccases in fungi. curr. genomics, 12, 2011, 104-112. robinson, t., chandran, b., nigam, p.: studies on the production of enzymes by white-rot fungi for the decolourisation of textile dyes. enzyme microb. technol., 29, 2001, 575-579. sancey, b., trunfio, g., charles, j., minary, j.f., gavoille, s., badot, p.m., crini, g.: heavy metal removal from industrial effluents by sorption on cross-linked starch: chemical study and impact on water toxicity. j. environ. manage., 92, 2011, 765-772. saroj, s., kumar, k., pareek, n., prasad, r., singh, r.p.: biodegradation of azo dyes acid red 183, direct blue 15 and direct red 75 by the isolate penicillium oxalicum sar-3. chemosphere, 107, 2014, 240-248. shankar, s., nill, s.: effect of metal ions and redox mediators on decolorization of synthetic dyes by crude laccase from a novel white rot fungus peniophora sp. (nfcci-2131). appl. biochem. biotechnol., 175, 2015, 635-647. shin, t., murao, s., matsumura, e.: a chromogenic oxidative coupling reaction of laccase: application for laccase and angiotensin i converting enzyme assay. anal. biochem., 166, 1987, 380-388. souza, e.s., sampaio, i.d., freire, a.k.d., da silva, b.k.s., sobrinho, a.d., lima, a.m., souza, j.v.b.: production of trametes versicolor laccase by solid state fermentation using a fixed-bed bioreactor. j. food agric. environ., 9, 2011, 55-58. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:10 utc 200 chmelová, d. and ondrejovič, m. suzuki, t., timofei, s., kurunczi, l., dietze, u., schűűrmann, g.: correlation of aerobic biodegradability of sulfonated azo dyes with the chemical structure. chemosphere, 45, 2001, 1-19. thakur, i.s.: industrial biotechnology: problems and remedies, i k international publishing house, 2006, 336 pp. wang, f., ma, a.z., guo, c., zhuang, g.q., liu, c.z.: ultrasound-intensified laccase production from trametes versicolor. ultrason. sonochem., 20, 2013, 118124. yan, j., niu, j., chen, d., chen, y., irbis, c.: screening of trametes strains for efficient decolorization of malachite green at high temperatures and ionic concentrations. int. biodeterior. biodegradation, 87, 2014, 109-115. zhang, c., zhang, s., diao, h., zhao, h., zhu, h., lu, f., lu, z.: purification and characterization of a temperature and ph stable laccase from the spores of bacillus vallismortis fmb-103 and its application in the degradation of malachite green, j. agric. food chem., 61, 2013, 5468-5473. zhao, d., zhang, x., cui, d., zhao, m.: characterisation of a novel white laccase from the deuteromycete fungus myrothecium verrucaria nf-05 and its decolourisation of dyes. plos one, 7, 2012, 38817. zhu, m.j.z., du, f., zhang, g.q., wang, h.x., ng, t.b.: purification a laccase exhibiting dye decolorizing ability from an edible mushroom russula virescens. int. biodeterior. biodegradation, 82, 2013, 33-39. zilly, a., coelho-moreira, j. s., marques de souza, c.g., carvaial, a.e., koehnlein, e.a., peralta, r.m.: influence of nacl and na2so4 on the kinetics and dye decolorization ability of crude laccase from ganoderma lucidum. int. biodeterior. biodegradation, 65, 2011, 340-344. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:10 utc nova biotechnologica et chimica 11-2 (2012) 111 doi 10.2478/v10296-012-0012-1 © university of ss. cyril and methodius in trnava toxicity testing of sediments oľga šestinová, lenka findoráková, jozef hančuľák institute of geotechnics, slovak academy of sciences, kosice, slovak republic (sestinova@saske.sk) abstract: this study presents the results of the testing toxicity of the contaminated sediments from the water reservoir of ružín no.i deposit (slovak republic) by using phytotoxkit tests (microbiotests inc., belgium). the phytotoxkit system is a screening tool used for a variety of toxicity testing applications. the advantages of this toxicity bioassay are its speed, relative simplicity and low cost compared to chemical analysis and many other biotests. evaluation of sediments phytotoxicity was based on the testing of seed germination and the assesment of the root growth decrease of the plant sinapis alba which allows to complete the assays after only 3 days of incubacion. chemical analysis of the sediment samples involved determination of heavy metal (cu, zn, ni, as, sb and hg) concentration. no potential phytotoxic effect of heavy metals in contaminated sediments was observed in the majority of tested seeds of sinapis alba.   key words: phytotoxkit test, sinapis alba, sediments, heavy metals 1. introduction the phytotoxkit microbiotest is very flexible method to determine the impact of pollutants on higher plants and can be applied to different types of plant seeds for testing the sediments in comparison to the control soil. the effect on seed germination and root growth of mustard (sinapis alba) in the first stadium of development is tested. mustard root is thin; shoot is erect, hairy and up to 150 mm with clear green leaves. seeds are relatively big in comparison with other brassicaceae species, yellow or white yellow and round-shaped. they reach to 1.5 – 4 mm in diameter. after germination, the simple root with hypocotyls grows up (zlamalova gargosova et al., 2011). phytotoxkit is the alternative test using modification of oecd 208 standards, (iso 11269-2) soil quality, method for the measurement of inhibition of root growth. due to complex relationships, a combination of toxicological and biological assays with physicochemical data is needed to interpret and characterize contaminated sites (plaza, et al., 2010). environmental toxicity is the major problem of industrially contaminated region, where the water reservoir of the ružín no.i is situated. mining operations with the metallurgical processing of complex metals and copper ores left negative effects in this region. the ružín no.i water reservoir is situated in the hornád river. the filling process of the reservoir began in 1969. during 40 years its volume was diminished by 10 millions m3. bottom sediments are contaminated by heavy metals, namely hg, cu, zn, cd, pb, cr, co, sb and as. the metals present in the reservoir originate from the localities formerly used for mining activities and thus they represent ecological load mainly at the inputs into reservoir (bobro, 2006). enhanced contents of heavy metals restrain direct application of sediments in agriculture, building and field bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:54 utc 112 šestinová, o. et al. engineering. for this reason it is important to consider their potential ecotoxicity and search for suitable ways of bottom sediments decontamination. the paper aimed to carry out the ecotoxicological bioassays of ružin sediments and evaluate their potential ecotoxicity. the germination is the most sensitive parameter for expression the low toxicity effects (zucconi et al.. 1981; giannis et al. 2008). environmental contamination resulting from the extensive use of metals and semi-metals in industry, agriculture and in manufactured products has magnified the treat of toxicity for plants, animals and society (giannis, et al., 2008; selim, et al., 1999). sediments are anaerobic and in most cases the heavy metals are strongly bound as sulphides or carbonates. sulphides are formed as a result of microbiological reduction of sulphate under anaerobic conditions. plant activities influence the turnover of organic matter, which may have a decreasing effect on the metal ion mobility (bortone, et al., 2007). the different species of heavy metals show various mobility degrees, various forms of transport in water and various bioavailabilities for plants and animals as well. for example, in hong kong, agricultural waste, particularly pig manure, have been shown to be the major contributor to stream pollution (tiquia, 1996). similarly the application of immature compost as soil fertilizer and conditioner causes negative effects on seed germination and plant growth and development, (tiquia, 1996; zucconi, 1981). 2. materials and methods phytotoxkit microbiotest (microbiotests inc., belgium) was performed on fresh contaminated sediments collected from the depth of 10-30 cm from the ružin no.i water reservoir, branch of the hornád river (1/2011 estuary hornád-hnilec) and the hnilec river (2/2011 fishing house). the samples of bottom sediments were sampled into glass bottles by sample device „multisampler“. sampling was realized in year 2011 and total content of heavy metals (copper, zinc, nickel, arsenic, antimonite and mercury) were determined. finally, the samples were dried, quartered, sieved under 0.1 mm. total heavy metal content (cu, zn, ni, as, sb and hg) in the sediments was determined after hot mineralization in a mixture of acids using the atomic absorption spectroscopy method (aas). three replicates were done for each sample set. phytotoxkit is the alternative test procedure, which allows determining the biological effect of chemical compounds on plants. the sediment was covered with the filter plate, and ten seeds of sinapis alba were placed on top of the filter in one row. after closing the test plates with the transparent cover, the plates were placed vertically and incubated for 72 h at 25°c (fig.1). 3. results and discussion the active sediments ph is presented in table 1. the 1/2011 and 2/2011 samples were slightly alkaline. heavy metal contents (table 1.) exceeded legal standards bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:54 utc nova biotechnologica et chimica 11-2 (2012) 113 allowed by law no.111/2010 (no. 188/2003) on application sludge and bottom sediment in agriculture fields) in the case of as, sb and hg. fig. 1 phytotoxkit test in the incubator. the root growth inhibition was measured after 72 hours (fig.2). the test was considered to be valid if the number of germinated seeds in control was at least 90% seeds. pictures of the test plates (fig.3) were analyzed with the free image analysis program (image tools). evaluation of the sediment phytotoxicity was based on the decrease of seed germination and root growth of the sinapis alba seeds in comparison to the control soil. table 1. physicochemical properties of the sediment samples from the ružin no.i water reservoir, branch of the hornád river (1/2011 estuary hornád-hnilec) and the hnilec river (2/2011 fishing house). parameters: samples: 1/2011 2/2011 dry mass (%) 39 40 ph (h2o) 8 7 orp (mv) 264 221 toc (%) 10 8 total heavy metal contents (mg.kg-1) cu 175 300 zn 376 360 ni 161 77 as 35 46 sb 90 58 hg 7 1 legislative no.111/2010 l (mg.kg-1) cu zn ni as sb hg 1000 2500 300 20 10 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:54 utc 114 šestinová, o. et al. fig. 2 phytotoxkit microbiotest – a comparison of seed germination in the samples 1/2011 (branch of the hornád river ) and 2/2011 ( branch of the hnilec river ) with control soil. fig. 3 calibration of length measurements ( in mm ) of the germinated seeds, program (image tools). the sediment samples were used to evaluate the potential phytotoxic effect using the parameters percentage inhibition of seed germination (isg) and percentage inhibition of root growth (irg) in test sediment: 100(%) ×= cs in germinated seedsof number sedimentin germinated seedsof numbercs in germinated seedsof number isg 100(%) ×= mm) (in cs in lenght root mean mm) (in sedimentin length root mean mm) (in cs in length root mean irg where cs represents control soil. the percentage inhibition of seed germination (isg) was 11.5 – 33.1% and 12.5 – 33% in the 1/2011-hornad and 2/2011hnilec samples, respectively. the percentage of the inhibition of root growth (irg) was higher in comparison with the inhibition of seed germination. it was 10.2 25.2% and 18.6 42.5% in the 1/2011hornád and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:54 utc nova biotechnologica et chimica 11-2 (2012) 115 2/2011hnilec sediments, respectively (figure 4-5). over 90% of seeds in control samples were germinated. according to the phytotoxkit microbiotest the experimental concentration in which growth inhibition is tightly above 50% after 72 hours can be considered to be effective concentration 72/ec50. both the inhibitions of germination rate were not much different from the controls. 0 5 10 15 20 25 30 35 1 2 3 4 5 6 series is k [% ] hornád hnilec fig. 4. the inhibition of seed germination (isg %) of the sinapis alba in the six series of sediments. 0 5 10 15 20 25 30 35 40 45 1 2 3 4 5 6 series ir k [ % ] hornád hnilec fig. 5. the inhibition of root growth (irg %) of the germinated seeds of the sinapis alba in the six series of sediments. 4. conclusions the seed germination and root elongation technique was used because it is an easy and inexpensive screening test. based on the experimental results of the phytotoxicity bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:54 utc 116 šestinová, o. et al. testing no potential phytotoxic effect of heavy metal contaminated sediments from ružin no.i water reservoir on tested sinapis alba seeds was observed. according to the assays carried out it was found that even 1.5 – 2 times higher as, sb and hg concentrations in sediments in comparison with legal standards did not have phytotoxic effects on tested sinapis alba seeds. acknowledgement: this work was supported by the slovak research and development agency (no 0252-10, no -51-02 7705) and by the slovak grant agency for science vega (grant no2/0187/11). references bobro, m.: záverečná správa z prieskumu kvality a kvantity nánosov a eróznych procesov v povodí hornádu a hnilca po priehradný profil vd ružín i v r. 20022005, košice, ústav geotechniky sav, 2006 (in slovak). borone, g., palumbo, l.: sustainable management of sediment resources. sediment and dredged material treatment, elsevier, vol. 2, 2007, 207 pp. giannis, a., gidarakos, e., skouta, a.: transport of cadmium and assessment of phytotoxicity after electrokinetic remediation. j. environ. manage., 86, 2008, 535-544. plaza, g.a., jawecki, g.n., pinyakong, o., illmer, p., margesin, r.: ecotoxicological and microbiological characterizatio of soils from heavy-matal and hydrocarboncontaminated sites. environ. monit. assess., 163, 2010, 477488. selim, h. d., iskandar, i. k.: fate and transport of heavy metals in the vadose zone. lewis publishers, florida, 1999. tiquia, s. m., tam, n. f y., hodgkiss, i. j .: effects of composting on phytotoxicity of spent pig-manure awdust litter. environ. pollut., 93, 1996, 249256. zlamalova gargosova, h., vavrova m., dolezalova weissmannova, h., mravcova, l., vydrova, l., zouhar, l.: the use of methods of environmental analysis and ecotoxicological tests in the evaluation of wastewater. waste water-evaluation and management. rijeka, croatia: intech, 2011, 3-30. zucconi, f., forte, m., manoco, m., bertoldi, m.: biological evaluation of compost maturity. bio cycle, 22, 1981, 27-29. presented at the 2nd international conference “biotechnology and metals 2011”, september 22–23, 2011, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:54 utc nova biotechnologica et chimica 12-1 (2013) 56 doi 10.2478/nbec-2013-0006 © university of ss. cyril and methodius in trnava utilization of the spin symmetry in fitting the magnetic data for large exchange clusters roman boča department of chemistry, university of ss. cyril and methodius, sk-917 01 trnava, slovak republic (roman.boca@stuba.sk) abstract: the heisenberg hamiltonian appropriate to exchange clusters commutes with the square of the total spin ant its third component. therefore in studying the exchange coupled clusters of medium/high nuclearity the spin quantum number s can be utilized in factoring of large interaction matrices (dimension of which is 104 105). then the blocks of much lower size can be diagonalized using the desktop computers. to this end, the eigenvalues form the partition function z(t,b) from which all thermodynamic properties, including the magnetization m(b,t0) and the magnetic susceptibility χ(t,b0), can be reconstructed. the matrix elements of the interaction operators in the coupled basis set of spin kets have been generated with the help of the irreducible tensor operators for a loop for s = smin until s = smax. in addition to the modelling of energy levels for different topologies, a fitting of magnetic data is exemplified by a number of examples like [fe6] and [mn3cr4] systems. key words: spin symmetry, exchange clusters, fe(iii) complexes, magnetic data 1. introduction synthesis, structural and spectral characterization of polynuclear homoor heteronuclear complexes represents one of the most rapidly developing areas of the coordination chemistry. of various properties the magnetic behavior represents a centre of interest since new architecture of complexes led to discovery of new magnetic phenomena that possess a great potential for technical exploitation. reports on the medium-sized metal complexes build of 4 to 8 (or even more) metal centers are rather numerous. the magnetic data of them (the temperature dependence of the magnetic susceptibility and eventually the field dependence of the magnetization) used to be reported for them. however, a complete understanding of those data requires a fixing of magnetic parameters (at least the exchange coupling constants jab, and the magnetogyric-ratio ga) by an appropriate fitting procedure. the principal problem associated with a computational approach lies in the size of the interaction matrices. the heisenberg hamiltonian appropriate to the exchange coupled system reads exˆ ( ) n ab a b a b h j < = − ⋅∑ s s (1) and it generates an interaction matrix exˆlkh l h k= in the basis set of spin kets. the size of such a matrix grows rapidly with the number of constituent spins, i.e. 1 (2 1) (2 1) n n a a a m s s = = + → +∏ (2) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:52 utc nova biotechnologica et chimica 12-1(2013) 57 for instance, in the hexanuclear [feiii6] complex one has to consider m = 6 6 = 46656 spin kets leading to the same number of magnetic energy levels. 2. results and discussion the heisenberg exchange hamiltonian commutes with the total spin of the system ex 2ˆˆ , 0h s⎡ ⎤ =⎣ ⎦ , ex ˆˆ , 0zh s⎡ ⎤ =⎣ ⎦ (3) therefore a set of eigenstates common for ex 2ˆ ˆˆ{ , , }zh s s operators exists; this means that the states spanning different total spin are orthogonal ex 1/ 2 exˆ ˆ: ... : ... (2 1) : ... : ...lk s s m mh l s m h k sm s l s h k sδ δ − ′ ′′ ′= = + (4) it allows a factoring of the interaction matrix into blocks of a much lower size min min ex max max 0 ... 0 0 0 1 ... 0 0 ... ... ... ... ... 0 0 ... 1 0 0 0 ... 0 s s s s s s s s ⎛ ⎞= ⎜ ⎟ ⎜ ⎟= + ⎜ ⎟ ⎜ ⎟→ ⎜ ⎟ = −⎜ ⎟ ⎜ ⎟ ⎜ ⎟=⎝ ⎠ h (5) which can be treated (diagonalized) independently. table 1 illustrates the effect of the s-blocking for systems with the genuine spins sa = 5/2, e.g. for the [fe iii n] clusters. the matrix elements of the interaction operators in the coupled basis set of spin kets 1 2 12 1...3 1...( , ,... ), ( , ,..., ),n nk s s s s s s s= have been generated with the help of the irreducible tensor operators (boča, 1999) for a loop for s = smin until s = smax. the intermediate spins (is) are abbreviated as 1...n ns s= % and the full set of is is ( )ns% . the general form of such matrix elements utilizes a consecutive decoupling of spins until the elementary spin operators with the help of the 9j-symbols (recoupling coefficients of the angular momenta) according to the formula 1 2 2 3 1 ex 1 2 1 2 1 2 2 3 3 1 1 ... ... 1 1/ 2 1 1 1 1 1 1 1 1 1 1 ˆ... ( ) ... ( ) ˆ ( ) [ ( ) ( )... ( ) ] [(2 1)(2 1)(2 1)] n n n n n n k n n n n n k k k k k k i i in i i i i i i i i i i s s s s s h s s s s s s t s s g k k k k k k k k s s k s s k s s k s s k − − − − + + + + + + = + + + ′ ′ = × ⎧ ⎫′ ⎪ ⎪ ′× + + + ⎨ ⎬ ⎪ ′⎩ ⎭ ∑ ∑ ∏ % % % % % r % % % %% % %% % %% % ˆ ( ) ii k i i s t s s ⎪ r (6) the only non-zero proportionality factor accounting for the relationship between the scalar and tensor product of spin operators is bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:52 utc 58 boča, r. 1 2 2 3 3 1 1[ ( ) ( )... ( ) ] 3n n n ijg k k k k k k k k j− − =% % % (7) where the tensor ranks are 1i jk k= = for i j≠ , 0fk = for ,f i j≠ , and 0nk k= =% . the matrix elements of the elementary spin operators are 1/ 2 0 ˆ ( ) (2 1) ff k f f f s t s s s= = + r , 1/ 21ˆ ( ) [ ( 1)(2 1)]ff k f f f f fs t s s s s s= = + + r (8) with 1 2( ) ...f ns s s s= . table 1. effect of the s-blocking for an systems with the genuine spins sa =5/2. an system magnetic states, all m zerofield states numerousity n(s) from the lowest spin, smin = 0 or 1/2, to the highest spin smax = nsa a3 216 27 2, 4, 6, 5, 4, 3, 2, 1 a4 1296 146 6, 15, 21, 24, 24, 21, 15, 10, 6, 3, 1 a5 7776 780 45, 84, 111, 120, 115, 100, 79, 56, 35, 20, 10, 4, 1 a6 46656 4332 111, 315, 475, 575, 609, 581, 505, 405, 300, 204, 126, 70, 35, 15, 5, 1 a7 279936 24017 1050, 1974, 2666, 3060, 3150, 2975, 2604, 2121, 1610, 1140, 750, 455, 252, 126, 56, 21, 6, 1 a8 1679616 135954 2666, 7700, 11900, 14875, 16429, 16576, 15520, 13600, 11200, 8680, 6328, 4333, 2779, 1660, 916, 462, 210, 84, 28, 7, 1 a9 10077696 767394 26775, 50904, 70146, 83000, 88900, 88200, 82005, 71904, 59661, 46920, 34980, 24696, 16478, 10360, 6111, 3360, 1707, 792, 330, 120, 36, 8, 1 the calculated zero-field energy levels for a variety of topologies are presented in table 2. it can be seen that the energy spectrum drastically depends upon the connectivity of centers (octahedron, trigonal prism, ring, chain, a star). the main computational problem arises when the magnetic field is applied: the matrix elements of the zeeman term in the basis set of the coupled kets are offdiagonal in the total spin number. there is one exception: when all g-factors are equal, then the off-diagonal matrix elements of the zeeman operator vanish exactly. this is really a fortunate case, since then the zeeman contributions can be simply added to the roots of the zero-field hamiltonian 0 b iso( , ) ( ) ss b s g bmε ε μ= + (9) then the magnetic functions can be exactly expressed with the help of the true partition function mol a 1 1 m n t z = , 2a 0mol 2 12 1 ( ) n t z t kt z μ χ = −% (10) the terms entering the magnetisation and the differential magnetic susceptibility are bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:52 utc nova biotechnologica et chimica 12-1(2013) 59 max min bexp( / ) exp[( ) / ] s s s i s s i s s m s z kt n j gbm ktε μ + = =− = − = −∑ ∑ ∑ (11) max min 1 b bexp( / ) exp[( ) / ] s s s i i s s s i s s m s t kt g m n j gbm kt b ∂ε ε μ μ ∂ + = =− ⎛ ⎞ = − − = −⎜ ⎟ ⎝ ⎠ ∑ ∑ ∑ (12) max min 2 2 2 2 b bexp( / ) ( ) exp[( ) / ] s s s i i s s s i s s m s t kt g m n j gbm kt b ∂ε ε μ μ ∂ + = =− ⎛ ⎞ = − = −⎜ ⎟ ⎝ ⎠ ∑ ∑ ∑ (13) with ( 1) / 2sn s s= + . table 2. modelling of the zero-field energy levels for hexanuclear spin systems, sa = 5/2, j/hc = −1 cm−1. uniform interactions j(15×), rotational band octahedron, jc(12×), jt2(3×) = 0 trigonal prism, jb(6×) = ja(3×), ja2(6×) = 0 spin 0 1 2 3 4 5 6 7 8 9 101112131415 ε / cm -1 0 20 40 60 80 100 120 spin 0 1 2 3 4 5 6 7 8 9 101112131415 ε / cm -1 0 20 40 60 80 100 120 spin 0 1 2 3 4 5 6 7 8 9 101112131415 ε / cm -1 0 20 40 60 80 100 120 ring, jr(6×) chain, jn(5×) ab5, star, jc(5×) spin 0 1 2 3 4 5 6 7 8 9 101112131415 ε / cm -1 0 10 20 30 40 50 60 70 80 spin 0 1 2 3 4 5 6 7 8 9 101112131415 ε / cm -1 0 10 20 30 40 50 60 70 80 spin 0 1 2 3 4 5 6 7 8 9 101112131415 ε/ cm -1 0 10 20 30 40 50 60 70 80 the approach described above has been applied to a number of large-spin clusters (boča, 2009). the first example refers to a hexanuclear fe(iii) complex (fig. 1a) possessing a low symmetry (kusserow et al., 2013). the structure of the cluster bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:52 utc 60 boča, r. implies that at least three different coupling constants need be considered. the experimental magnetic data are displayed in fig. 2 along with the calculated ones as they resulted from the fitting procedure. the results show three strong coupling paths of an antiferromagnetic nature (s = 0 ground state). a b fig 1. structure of the polynuclear complexes under study. the second example refers to a heteronuclear [mnii3cr iii 4] complex (fig. 1b) for which the size of the problem is 3 46 4 55296m = = . the zero field states zf 5737m = are factored according to the total spin between s = 1/2 and 27/2 as follows: 326, 661, 852, 915, 862, 726, 550, 375, 228, 122, 56, 21, 6, and 1 (i.e. 326 doublets, 661 quartets, etc). for the complex [mn3cr4(ncs)6(htea)6] the magnetic data are displayed in fig. 3. with the optimized set of magnetic parameters the ground state of the complex is s = 15/2 (fifteen unpaired electrons). this rationalizes why the magnetization per particle taken at t = 2.0 k saturates to the value of mmol/na = 15 μb (semenaka et al., 2010). s p in 0 1 2 3 4 5 6 7 8 9 1 01 11 21 31 41 5 ε / cm -1 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0 6 0 0 0 t / k 0 50 100 150 200 250 300 μ e ff / μ b 0 2 4 6 8 t / k 0 50 100 150 200 250 300 χ m ol /( 10 9 m 3 m ol −1 ) 0 50 100 150 200 250 fig. 2. the zero-field energy spectrum, and the magnetic functions for the complex [feiii6o3(otbu)5(oipr)7]; solid line – calculated with j1/hc = −28.7, j2/hc = −110.6, j3/hc = −49.3 cm−1, geff = 2.163; r(χ) = 0.0046. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:52 utc nova biotechnologica et chimica 12-1(2013) 61 spin, s 0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5 13.5 e ne rg y/ cm −1 0 20 40 60 80 100 120 b/t 0 1 2 3 4 5 m m ol /( n a μ b ) 0 5 10 15 20 t/k 0 50 100 150 200 250 300 μ e ff. /μ b 0 5 10 15 0 5 10 15 20 25 30 35 40 χ m ol /1 06 m 3 m ol -1 0 50 100 150 200 b = 0.1 t t = 2.0 k fig. 3. the zero-field energy spectrum, and the magnetic functions for the complex [mnii3criii4(ncs)6(htea)6]; solid line – calculated with jmn-cr/hc = +0.43 cm-1, jcr-cr/hc = -4.75 cm-1, jmnmn/hc = +1.78 cm-1, geff = 1.878; r(χ) = 0.045, r(m) = 0.049. 3. conclusions it has been demonstrated that the fitting of magnetic data (susceptibility and magnetization) for the medium-sized metal complexes and clusters can be effectively done by utilizing the spin symmetry. this leads to the factoring of the largedimensional interaction matrix of the order of 105 – 106 to the eigenvalue problems of much lower dimensions (102 103) that can be performed even at desk computers. acknowledgements: slovak grant agencies (vega 1/0233/12, apvv-0014-11) are acknowledged for the financial support. references boča, r.: theoretical foundations of molecular magnetism, elsevier, amsterdam 1999. boča, r.: program polymagnet-09. slovak university of technology, bratislava, 2009. kusserow, m., nahorska, m., mroziński, j., boča, r., spandl, j. 2013, to be published. semenaka, v. v., nesterova, o. v., kokozay, v. n., zybatyuk, r. i., shishkin, o. v., boča, r., shevchenko, d. v., huang, p., styring, s.: direct synthesis of an heterometallic {mn ii 3cr iii 4} wheel by decomposition of reineckes salt. dalton trans., 39, 2010, 2334-2349. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:52 utc nova biotechnol chim (2022) 21(1): e1387 doi: 10.36547/nbc.1387 1 nova biotechnologica et chimica the influence of inoculation and drought on the diversity of fungal communities in the roots of tomato plants katarína ondreičková1, , marcela gubišová1, katarína hrčková1, martina hudcovicová1, jozef gubiš1, miroslav horník2, silvia dulanská3,4 1national agricultural and food centre – research institute of plant production, bratislavská cesta 122, piešťany 92168, slovak republic 2department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava 91701, slovak republic 3institute of medical physics, biophysics, informatics and telemedicine, faculty of medicine, comenius university, sasinkova 2, bratislava 81372, slovak republic 4faculty of public health, slovak medical university, limbová 12, bratislava 83303, slovak republic  corresponding author: katarina.ondreickova@nppc.sk article info article history: received: 25th january 2022 accepted: 1st february 2022 keywords: alpha diversity arbuscular mycorrhiza fungal diversity plant stress pot experiment abstract arbuscular mycorrhizal fungi (amf) are microorganisms with very important functions in agricultural systems. since arbuscular mycorrhiza is one of the most common types of mycorrhizae, the diversity of amf is very varied. their diversity can be influenced by various biotic and abiotic factors. of these, drought is one of the most common abiotic stresses in agriculture. in this study, we evaluated the influence of drought and inoculation with three species of amf (rhizoglomus irregulare, funneliformis mosseae and f. caledonium) on the fungal genetic diversity in the roots of tomato plants (solanum lycopersicum l.) using partial 18s rrna gene in molecular fingerprinting method. two conserved primer pairs ns1– ns4 and ns31–am1 in nested pcr were used. the second primer pair is specific for am fungi from glomeromycota, but also amplifies dna from ascomycota and basidiomycota to a very small extent. drought caused a decrease in fungal alpha diversity in tomato roots of non-inoculated plants. on the other hand, an increase of this diversity due to drought in inoculated plants was observed. based on principal component analysis, a statistically significant shift in the composition of fungal communities in non-inoculated and inoculated plants due to drought was not detected. at the same time, the most variable fungal communities were in control well-watered and non-inoculated plants, but this variation was not significant. introduction arbuscular mycorrhizal fungi (amf) which form a symbiosis with approximately 80 % of terrestrial plant species have an important role in processes such as increasing mobilization and nutrient transfer, plant tolerance to root pathogens, production of plant growth hormones, plant biodiversity, adaptation of the plant to adverse environmental conditions, the absorption capacity of plant roots, and many others (garg and chandel 2010). adverse external conditions for plants whose effect amf can decrease are drought, salinity, heavy metal toxicity, soil acidification, or heat (finlay 2008). of these, drought is one of the world's most abundant abiotic stress (zhang et al. 2018). many studies have been done to examine the effect of drought on the plants inoculated with am mailto:katarina.ondreickova@nppc.sk nova biotechnol chim (2022) 21(1): e1387 2 fungi. however, the effect of drought on the diversity of amf as such was much less studied (bahadur et al. 2019) and how amf respond to this stress in agricultural ecosystems is not fully known (millar and bennett 2016). some studies have not found the impact of drought on amf communities (furze et al. 2017; maitra et al. 2019; kozjek et al. 2021). other studies found a shift in their diversity (deveautour et al. 2018; 2020), or others even found an increase in amf biomass (karlowsky et al. 2018; mackie et al. 2019; kundel et al. 2020). for this reason, our work aimed to investigate the effect of drought as well as the effect of inoculation of tomato plants with three amf species on the fungal diversity in the roots of such plants. when comparing two sets of samples (inoculated and non-inoculated plants) it could be expected that the drought simulation would have a stronger effect on the genetic diversity of fungi in the roots of noninoculated than inoculated plants. experimental pot experiment and amf inoculation in the experiment, tomato plants (solanum lycopersicum l.) of variety karla were used. the soil substrate was mixed from topsoil and sand in proportion 3 : 1. inoculation was done during seed sowing with a commercially available inoculum inoq advantage (inoq gmbh, germany) containing a mixture of 3 species of am fungi: rhizoglomus irregulare, funneliformis mosseae and funneliformis caledonium with a concentration of 550 × 104 mycorrhizal units.ml-1. the concentrated inoculum was diluted at 1 : 50 in autoclaved, finely homogenized soil and applied to a seed hole at a dose of 0.5 ml of inoculum per seed. the control group of plants was without inoculation. the plants were cultivated under greenhouse conditions in pots with a diameter of 22 cm, 4 pots per variant with 3 plants per pot. for the first 8 weeks (until the flowering stage), a standard irrigation was applied to keep the substrate humidity at a level of 25 % of volumetric water content. then the control and inoculated plants were divided into 2 groups, the first one being irrigated with the standard dose of water (3 times a week at a dose of 500 – 500 – 1000 ml of water per container), the second group with a half dose of water (which corresponded to 12 % of volumetric water content). the cultivation of plants under this regime took place for 4 weeks. dna extraction and molecular analysis root samples were obtained by carefully removing all three tomato plants from the soil from each pot, gently removing the soil from the roots, and placing the entire three roots in a sterile container. therefore, one sample was composed of roots from three plants cultivated in one pot. in this way, root samples from individual treatments (each treatment consisted of 4 replicates, which formed separate samples for subsequent analyses) were collected in the following order: 4 pots = 4 separate root samples from non-inoculated well-watered control plants, 4 samples from non-inoculated droughtaffected plants, 4 samples from inoculated wellwatered control plants, and 4 samples from inoculated drought-affected plants. after root collection, the samples were transferred to the laboratory, washed 3 times with sterile water, and thoroughly dried. dna was extracted from 100 mg of dry roots using the modified ctab protocol. one hundred ml of each root sample was homogenized to a powder with a mortar and pestle using liquid nitrogen. seven hundred μl of 2x ctab solution (2 m nacl, 0.2 m tris, 50 mm edta, 2 % ctab) and 3 μl of 2-mercaptoethanol were added to the pulverized root samples, mixed and thoroughly vortexed, and incubated in a water bath for 30 min at 60 °c. after incubation, the mixture was cooled to room temperature and centrifugated for 5 min at 14,000 × g. the supernatant was transferred to a new tube to which 5 μl of rnase a was added and incubated for 20 min at 37 °c. an equal volume of chloroform/isoamyl alcohol (24 : 1) was added. the mixture was vortexed for 5 s and centrifugated for 1 min at 14,000 × g to separate the phases. the upper aqueous phase was transferred to a new tube. this step with chloroform/isoamyl alcohol was repeated until the upper phase was clear. dna was precipitated by adding 0.7 volume of chilled isopropanol, incubated for 15 min at -20 °c and centrifugated for 10 min at 14,000 × g. supernatant was discarded without upsetting the nova biotechnol chim (2022) 21(1): e1387 3 pellet, which was subsequently washed with 500 µl of ice-cold 70 % ethanol. the ethanol was removed, and the samples were dried at room temperature until residual ethanol was completely removed. the dna was dissolved in 50 μl of preheated te buffer (10 mm tris, ph 8.0, 1 mm edta). the quantity and purity of dna were measured spectrophotometrically with nanodrop1000 spectrophotometer (thermo fisher scientific inc., waltham, ma, usa), samples were diluted to the same final concentration (25 ng.μl-1), and stored at -20 °c. molecular analysis of partial fungal 18s rrna gene using two conserved primer pairs ns1–ns4 (lee et al. 2008) and ns31–am1 (mummey et al. 2005) in terminal restriction fragment length polymorphism (t-rflp) was performed according to our previous study (ondreičková et al. 2019). the second primer pair is specific for am fungi (glomeromycota); however, it should be noted that also amplify dna from ascomycota and basidiomycota to a very small extent (clapp et al. 1995, 1999; ondreičková et al. 2019). data analysis and statistical evaluation outputs from t-rflp analysis in the form of electrophoregrams were analysed by the peak scanner 2 (applied biosystems, thermo fisher scientific inc., wilmington, usa), and operational taxonomic units (otus) in range 60 – 550 bp were used for the evaluation. only peaks above the threshold of 50 fluorescence units were considered. the venn diagrams were constructed using venny 2.1 online program (oliveros 2007-2015). statistically significant differences among samples were tested using t-test at the 95 % confidence interval for the means using the software statgraphics x64 (statpoint technologies, inc., warrenton, usa). alpha diversity indices were calculated from standardized profiles of individual samples using the number and height of peaks in each profile as representations of the number and relative abundance of phylotypes. fungal communities in different samples were compared from t-rflp profiles using the height of fluorescence in individual otus. these data were subsequently used for the principal component analysis (pca) using the scores of the first two principal components. analysis of similarities (anosim) was used to determine if significant effects occurred among samples using 2 factors – inoculation and drought (two-way anosim), and only 1 factor – all principal component values (one-way anosim) with euclidean distance measure with 9999 permutations. alpha diversity indices, pca, and anosim were evaluated by using the past (paleontological statistics) software version 3.19 (hammer and harper 2006). results fungal alpha diversity most fungal species were detected in the roots of non-inoculated control tomato plants and the least in non-inoculated drought-affected plants (56 vs 27, respectively; fig. 1a). fig. 1. venn diagrams constructed from t-rflp data representing the number of shared and unique otus between fungal communities from the roots of non-inoculated control and non-inoculated drought-affected tomato plants (a) and inoculated control and inoculated drought-affected tomato plants (b). nova biotechnol chim (2022) 21(1): e1387 3 in non-inoculated plants, the fungal species richness in the roots of drought-affected plants was reduced by 51.8 % compared to the control samples watered properly. the opposite effect was observed in inoculated plants. the increase of 41.9 % of fungal species was in the roots of drought-affected tomato plants compared to controls. roots of control plants contained 31 fungal species, while 44 were in drought-affected plants (fig. 1b). from this number of fungal species in the roots of noninoculated and inoculated plants, in both cases approximately the same percentage of common species was detected between controls and droughtaffected plants (31.7 % vs 29.3 %, respectively; fig. 1). fungal alpha-diversity between control and drought-affected non-inoculated and inoculated plants was evaluated using 10 different diversity indices (fig. 2). statistically significant differences between control and drought-affected plants were detected in two indices, margalef and fisher alpha, in non-inoculated tomato plants (t-test, α = 0.05). control plants, as well as drought-affected plants without and with inoculation, showed exactly opposite values. when the index value was higher in the non-inoculated controls, it was lower in the inoculated controls and vice versa. the same trend was observed in drought-affected plants without and with inoculation. fig. 2. the values of genetic diversity indices between fungal communities in the roots of non-inoculated control and noninoculated drought-affected tomato plants (a) and inoculated control and inoculated drought-affected tomato plants (b). displayed values represent the average values calculated from four replicates; bar represents the standard deviation; and * represents statistically significant difference (t-test, α = 0.05). impact of drought and inoculation on the fungal genetic diversity based on the principal component analysis (pca), fungal community from the roots of one noninoculated control sample represented the most different community from other non-inoculated samples (fig. 3a). in this case, one control sample is in the far right of the pca graph, with the horizontal axis expressing up to 84.16 % variability. non-inoculated drought-affected samples showed very similar fungal communities based on the pca graph. on the other hand, fungal communities from the roots of inoculated droughtaffected tomato plants are more different (fig. 3b) than those from non-inoculated drought-affected tomato plants. in particular, one such sample was separated from the others by pc1 and this axis 4 nova biotechnol chim (2022) 21(1): e1387 2 represented only 48.92 % variability. at the same time, pc1 mutual separated 2 and 2 inoculated control samples from each other on the opposite sides of this graph, which means that these samples represent two types of fungal communities. based on two-way anosim analysis, inoculation with amf and drought did not statistically significantly affect the fungal communities in the roots of tomato plants (p = 0.3456, p = 0.4827, respectively). fig. 3. the principal component analysis constructed from trflp fluorescent data of fungal communities from the roots of non-inoculated (a) and inoculated tomato plants (b). pca graphs explained a total of 90.71 % and 73.79 % (respectively) of the variability in the data. matrix plots generated from data of all principal components (pcs) provide a comprehensive view of these values and were different for noninoculated and inoculated plants (fig. 4). the value of pc1 from non-inoculated control sample 1 (abbreviated as c1 in fig. 4a) was in the red spectrum. this pc1 value was crucial for the separation of this control sample from other samples and therefore was in the right part of the pca graph (fig. 3a). as mentioned above, the pca result from the inoculated plants showed that the control plants contained two types of fungal communities in their roots. their values of the principal components also correspond to this (fig. 4b). these fungal communities of control samples 1 and 4 (abbreviated as ca1 and ca4 in fig. 4b) formed the first community type in the lower left part of the pca graph, and control samples 2 and 3 (abbreviated as ca2 and ca3 in fig. 4b) formed the second community type located in the right part of the graph in fig. 3b. different values for pc1 and pc2 which characterize inoculated drought-affected sample 3 (abbreviated as ad3 in fig. 4b) determine its separation from other drought-affected samples in the upper right part of the pca graph in fig. 3b. however, when comparing all the values of the principal components, not only pc1 and pc2, which are shown in the pca graphs in fig. 3, statistical differences between controls and drought-affected samples were not detected in noninoculated and inoculated plants (anosim, α = 0.05; fig. 4). fig. 4. matrix plots generated from data of all principal components from pca from non-inoculated (a) and inoculated tomato plants (b). c1-c4 represent non-inoculated control samples; d1-d4 represent non-inoculated droughtaffected samples; ca1-ca4 represent inoculated control samples; and ad1-ad4 represent inoculated drought-affected samples. p represents the values obtained from all pcs using analysis of similarities (anosim) with euclidean similarity index. 5 nova biotechnol chim (2022) 21(1): e1387 3 discussion the results of this study represent an ecological view of the fungal communities in the roots of tomato plants under the influence of drought and inoculation with three species of arbuscular mycorrhizal fungi. a positive effect of inoculation with rhizoglomus irregulare, funneliformis mosseae, and f. caledonium of tomato plants in drought condition on the overall communities of mainly am fungi in the roots of these plants was observed. this effect was manifested as an increase in species diversity as well as in the values of diversity indices (fig. 1 and 2). however, this alpha-diversity increase in inoculated plants was not statistically significant. on the other hand, root fungal alpha-diversity was decreased in droughtaffected non-inoculated tomato plants. andreojimenez et al. (2019) in their study observed the opposite trend in the rice root fungal microbiota community in comparison with our non-inoculated samples. their shannon diversity index was higher under drought for all 15 rice cultivars tested and was strongly statistically significant. also, they detected that these differences between treatments were rice cultivar dependent. however, in contrast to our results especially from glomeromycota and to a limited extend from ascomycota and basidiomycota (clapp et al. 1995, 1999; ondreičková et al. 2019), the statistical significance which was detected by the above mentioned andreo-jimenez et al. (2019) was noticed from the entire diversity of the fungal community including ascomycota, basidiomycota, chytridiomycota, zygomycota, glomeromycota, and unclassified fungi. though, based on diversity richness and shannon index at otu level between control and drought samples, they detected no significant effects on amf community structure in glomeromycota. additionally, alguacil et al. (2021) observed that drought did not significantly affect amf community richness and shannonwiener diversity index at otu level in the rhizosphere samples from helianthemum syriacum and gypsophila struthium. in the study of emery et al. (2022), drought as a single factor was also not significant for amf otu richness from soil associated with two cultivars of switchgrass. on the other hand, they detected that drought treatment had a significant direct effect on amf root colonization in switchgrass, and reduced root colonization by 6 %. an experiment similar to our was performed by he et al. (2019) inoculating the trifoliate orange with f. mosseae. they observed that the fungal richness in the roots of control noninoculated trifoliate orange plants was higher than in the roots of drought-affected non-inoculated plants. however, in the plants inoculated with f. mosseae, the fungal richness in the roots of control and drought-affected plants was the same. in addition, they detected statistically higher otus and more variable diversity in the roots than in the rhizosphere soil. in their experiment, environmental factors such as inoculation with f. mosseae and drought had a much less impact on fungal diversity in the rhizosphere than in the roots. conclusion inoculation of tomato (solanum lycopersicum l.) plants with arbuscular mycorrhizal fungi increased, but not significantly, fungal richness and alphadiversity in the roots of drought-affected plants. drought in non-inoculated plants did not cause a shift in the composition of the fungal community. in inoculated plants, the shift caused by drought was observed only in one sample. it was also observed that control well-watered, non-inoculated plants showed higher variability in fungal community composition than in drought-affected plants. although fungal richness and the increase in alpha-diversity due to inoculation with amf were not significant, the use of amf as an inoculant appears to be an appropriate way to prevent the effects of drought on root fungal diversity. acknowledgements this work was supported by the slovak research and development agency (contracts no. apvv-17-0150, apvv15-0098, apvv-14-0055) and by the operational programme integrated infrastructure (project "innovation of primary agricultural production for sustainable agriculture" itms: 313011t540) co-financed by the european regional development fund. conflict of interest the authors declare that they have no conflict of interest. 6 nova biotechnol chim (2022) 21(1): e1387 2 references alguacil mdm, schlaeppi k, lópez-garcía á, van der 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composition as assessed by t-rflp analysis. plant soil. 271: 83-90. oliveros jc (2007-2015) venny. an interactive tool for comparing lists with venn's diagrams. available online: http://bioinfogp.cnb.csic.es/tools/venny/index.html ondreičková k, gubišová m, piliarová m, horník m, matušinský p, gubiš j, klčová l, hudcovicová m, kraic j (2019) responses of rhizosphere fungal communities to the sewage sludge application into the soil. microorganisms 7: 505. zhang f, zou yn, wu qs (2018) quantitative estimation of water uptake by mycorrhizal extraradical hyphae in citrus under drought stress. sci. hortic. 229: 132-136. 7 http://bioinfogp.cnb.csic.es/tools/venny/index.html 48 nova biotechnologica et chimica 13-1 (2014) doi 10.2478/nbec-2014-0006 © university of ss. cyril and methodius in trnava magnetic deposits of iron oxides in the human brain miroslava makohusová,a viera mrázová,a martin kopáni,b roman bočaa adepartment of chemistry, university of ss. cyril and methodius in trnava, sk-917 01 trnava, slovakia (roman.boca@stuba.sk) binstitute of medical physics, biophysics, informatics and telemedicine, faculty of medicine, comenius university, bratislava, slovakia abstract: deposits of iron oxides in the human brain (globus pallidus) are visible under electron microscopy as object of regular and or/irregular shape but giving sharp diffraction patterns in the transmission mode. the squid magnetometry reveals that the magnetization curves decline form an ideal langevin function due to the dominating diamagnetism of organic tissue. the fitting procedure yields the quantitative characteristics of the overall magnetization curves that were further processed by statistical multivariate methods. key words: human brain, electron microscopy, iron oxides, hematite, magnetite, maghemite, squid magnetometry 1. introduction several studies confirm that iron oxides appear as mineral deposits in various areas of the human brain (kirschvink et al., 1992). the deposits differ in the size (typically from 10 – 50 nm) and composition given by the content of fe(iii) and fe(ii). these deposits differ from the ferritin inorganic core (6 nm in size) in many physical characteristics. the ferritin (e.g. the horse spleen ferritin, as a reference) is an iron storage protein containing ca 4500 atoms of fe(iii) in the form of the ferrihydrite and magnetically it behaves as a paramagnet until tc = 16 k. below this critical temperature it behaves as a superparamagnet that possesses the out-of-phase (imaginary) susceptibility component measured in the ac (alternating current) mode. moreover it exhibits a magnetic hysteresis which, however, escapes above tc so that it is absent at the room temperature. [ferrihydrite is hexagonal with the unit cell parameters a = 2.96 and c = 9.4 å, but variable water content formulated as 5fe2o3⋅9h2o, fe5ho8⋅4h2o, fe5o3(oh)9, fe2o3⋅2feooh⋅2.6h2o, feooh⋅0.4h2o, and also fe2o3⋅0.5h2o.] in the previous work (kirschvink et al., 1992) the deposits of iron oxides in cerebellum were identified as nanocrystals of magnetite, fe3o4 (fe iio⋅feiii2o3), possessing the cubic unit cell. this mineral is a ferrimagnet at the room temperature. another magnetic mineral is maghemite, γ-fe2o3, that again possesses the cubic unit cell with the structure derived from magnetite with fe(ii) deficient vacancies. maghemite nanoparticles are non-toxic and biocompatible so that they found application in biomedicine through their manipulation with external magnetic field (pankhurst et al., 2003; gubin et al., 2009). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:08 utc nova biotechnologica et chimica 13-1(2014) 49 single crystals of hematite, α-fe2o3, have been detected recently in basal ganglia (region globus pallidus) by means of the tem mode of the electron microscopy (boča et al., 2013). hematite possesses the hexagonal unit cell. in bulk it is an antiferromagnetic material below tc = 250 k and a canted antiferromagnet (weak ferromagnet) above this (morin) temperature; above the néel temperature tn = 948 k it behaves as a paramagnet. in the other words, bulk hematite is non-magnetic at the room temperature. hematite nanoparticles have different magnetic properties when compared to bulk as with lowering the particle size the morin transition decreases. notice, hematite is the most stable iron oxide from the point of view of thermodynamics. 2. experimental section samples from the human brain were extracted post mortem at the authorized department in accordance with helsinki declaration (only the age and gender of the donors is available). fresh samples were fixed in 10 % formaldehyde for 24 hours and embedded in paraffin blocks, cut by microtome to 5 μm thin sections and mounted on gelatin-coated slides. samples without cover glass were analysed by scanning electron microscope (jxa 840 a, jeol) with the accelerating voltage of 20 kv. the samples for transmission electron microscopy were fixed in 3% solution of glutar(di)aldehyde (serva, heidelberg) for two hours and buffered by phosphate (ph 7.2 – 7.4), embedded into durcupan acm (fluka ag, busch) as recommended by the manufacturer and cut by ultramicrotome (c. reichert, vienna) to the thickness of 200 nm. noncontrasted ultrathin sections were mounted on nickel grids and investigated by transmission electron microscope with acceleration voltage of 120 kv. fresh tissues were lyophilized and the resulting samples were obtained in a form of a powder. attention was paid to avoid manipulations with magnetisable instruments and environment. the superconducting quantum interference device (squid, quantum design, mpms-xl7) has been used for measurements of the magnetic moment of the sample in the sensitive rso mode. powder samples were encapsulated into gelatin-made sample holders. magnetization measurements were performed at t = 2.0 and 4.6 k, respectively, in the field-decreasing mode starting from b = 7.0 t. the susceptibility measurements between 2 and 300 k were done at the small applied field of b = 0.1 t. 3. results and discussion photographs of the hematite deposits found in globus pallidus are presented in fig. 1. miller indices in the tem mode unambiguously confirm the hexagonal unit cell characteristic for the hematite. this finding is in contrast with the generally accepted statement (cornell et al., 2003): “with the exception of hematite, all the major oxides are found in living organisms. the absence of hematite suggests that biological environments do not provide suitable conditions for the formation of this oxide.” unexpected is also the size of the deposits that exceeds 1000 nm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:08 utc 50 makohusová, m. et al. fig. 1. hematite deposits in the human brain of irregular and regular shape along with tem diffractograms. marker – 1000 nm. temperature evolution of the magnetic susceptibility of 16 samples is presented in the form of a product function χt in fig. 2. deviation from the straight line with zero slope means that the curie law is not obeyed. class i is represented by 9 samples exhibiting a dominating diamagnetic signal for which the χt product decreases with increasing temperature. class iii contains 3 samples showing the positive magnetic susceptibility in the whole temperature region and consequently the χt product function possesses a positive slope. class-ii covers 4 samples that are intermediate between class-i and class-iii. the magnetization curves for the samples under study are presented in fig. 3. these records arise from the combination of the classical magnetization curve according to the langevin function ( ) [coth( ) 1/ ]l x x x= − and a superimposed diamagnetic background (χdia) owing to the organic tissue mass pf para pf dia 0 ext( , ) (1 ) ( / )m b t w m w bχ μ= ⋅ + − ⋅ (1) with para fe a b( ) [coth( ) 1/ ]m m n x xμ μ= ⋅ ⋅ − (2) for the argument b ext int b( ) /x b b k tμ μ= + (3) here μ – magnetic moment in units of bohr magneton, mfe – the molar mass of iron, wpf – mass fraction of the paramagnetic species, and physical constants adopt their usual meaning. the internal magnetic field is parameterized by the weiss field constant (w) as bint = w⋅m and causes the existence of the remnant magnetization and consequently the magnetic hysteresis. the above equations need be solved by an iterative procedure. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:08 utc nova biotechnologica et chimica 13-1(2014) 51 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -4 -3 -2 -1 0 1 0 10 20 30 -0.2 0.0 0.2 sample 1 150 200 250 -3 -2 -1 0 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -4 -3 -2 -1 0 1 0 10 20 30 -0.2 0.0 0.2 sample 2 150 200 250 -4 -3 -2 -1 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -4 -3 -2 -1 0 1 0 10 20 30 -0.2 0.0 0.2 sample 3 150 200 250 -4 -3 -2 -1 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -4 -3 -2 -1 0 1 0 10 20 30 -0.2 0.0 0.2 sample 4 150 200 250 -4 -3 -2 -1 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -4 -3 -2 -1 0 1 0 10 20 30 0.0 0.2 0.4 sample 5 150 200 250 -4 -3 -2 -1 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -4 -3 -2 -1 0 1 0 10 20 30 -0.2 0.0 0.2 sample 6 150 200 250 -3 -2 -1 0 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -4 -3 -2 -1 0 1 0 10 20 30 -0.2 0.0 0.2 sample 13 150 200 250 -3 -2 -1 0 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -4 -3 -2 -1 0 1 0 10 20 30 -0.2 0.0 0.2 sample 14 150 200 250 -4 -3 -2 -1 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -3 -2 -1 0 1 2 0 10 20 30 -0.2 0.0 0.2 sample 15 150 200 250 -3 -2 -1 0 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -2 -1 0 1 2 3 4 5 0 10 20 30 0.0 0.2 0.4 0.6 sample 16 150 200 250 0 1 2 3 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -2 -1 0 1 2 3 4 5 0 10 20 30 0.0 0.2 0.4 0.6 sample 7 150 200 250 0 1 2 3 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) 0 10 20 30 0 10 20 30 0 1 2 3 sample 8 150 200 250 10 15 20 25 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -5 -4 -3 -2 -1 0 1 2 3 0 10 20 30 0.0 0.2 0.4 sample 9 150 200 250 -4 -3 -2 -1 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -5 -4 -3 -2 -1 0 1 2 3 0 10 20 30 0.0 0.2 0.4 sample 10 150 200 250 -3 -2 -1 0 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -5 -4 -3 -2 -1 0 1 2 3 0 10 20 30 0.0 0.2 0.4 sample 11 150 200 250 -3 -2 -1 0 t/k 0 100 200 300 χ t /( 10 −6 m 3 kg −1 k ) -1 0 1 2 0 10 20 30 0.2 0.4 0.6 sample 12 250 300 0 1 fig. 2. mass magnetic susceptibility in form of the product function χt vs t and its high-temperature fit. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:08 utc 52 makohusová, m. et al. b/t 0 1 2 3 4 5 6 7 -30 0 30 60 90 m m as s/ (1 0− 3 j t −1 k g− 1 ) t = 2.0 k t = 4.6 k sample 1 0.00 0.01 0 1 2 3 b/t 0 1 2 3 4 5 6 7 -30 0 30 60 90 m m as s/ (1 0− 3 j t −1 k g− 1 ) t = 2.0 k t = 4.6 k sample 2 0.00 0.01 0 1 b/t 0 1 2 3 4 5 6 7 -30 0 30 60 90 m m as s/ (1 0− 3 j t −1 k g− 1 ) t = 2.0 k t = 4.6 k sample 3 0.00 0.01 0 1 2 3 b/t 0 1 2 3 4 5 6 7 -30 0 30 60 90 m m as s/ (1 0− 3 j t −1 k g− 1 ) t = 2.0 k t = 4.6 k sample 4 0.00 0.01 0 1 2 3 b/t 0 1 2 3 4 5 6 7 -30 0 30 60 90 m m as s/ (1 0− 3 j t −1 k g− 1 ) t = 2.0 k t = 4.6 k sample 5 0.00 0.01 0 1 2 3 b/t 0 1 2 3 4 5 6 7 -30 0 30 60 90 m m as s/ (1 0− 3 j t −1 k g− 1 ) t = 2.0 k sample 6 -0.02 0.00 0.02 -2 0 2 b/t 0 1 2 3 4 5 6 7 -30 0 30 60 90 m m as s/ (1 0− 3 j t −1 k g− 1 ) t = 2.0 k t = 4.6 k sample 13 0.00 0.01 0 1 2 3 b/t 0 1 2 3 4 5 6 7 -30 0 30 60 90 m m as s/ (1 0− 3 j t −1 k g− 1 ) t = 2.0 k t = 4.6 k sample 14 0.00 0.01 0 1 b/t 0 1 2 3 4 5 6 7 -40 -20 0 20 40 60 80 m m as s/( 10 −3 j t −1 k g− 1 ) t = 2.0 k t = 4.6 k sample 15 0.00 0.01 0 1 2 3 b/t 0 1 2 3 4 5 6 7 -40 -20 0 20 40 60 80 m m as s/( 10 −3 j t −1 k g− 1 ) t = 2.0 k t = 4.6 k sample 16 0.00 0.01 0 1 2 3 b/t 0 1 2 3 4 5 6 7 -40 -20 0 20 40 60 80 m m as s/( 10 −3 j t −1 k g− 1 ) t = 2.0 k sample 7 0.00 0.01 0 1 2 3 b/t 0 1 2 3 4 5 6 7 -20 0 20 40 60 80 m m as s/( 10 −3 j t −1 k g− 1 ) t = 2.0 k sample 8 -0.01 0.00 0.01 -5 0 5 t = 4.6 k b/t 0 1 2 3 4 5 6 7 -40 -20 0 20 40 60 80 m m as s/ (1 0− 3 j t −1 k g− 1 ) t = 2.0 k t = 4.6 k sample 9 0.00 0.01 0 1 2 3 b/t 0 1 2 3 4 5 6 7 -40 -20 0 20 40 60 80 m m as s/ (1 0− 3 j t −1 k g− 1 ) t = 2.0 k t = 4.6 k sample 10 0.00 0.01 0 1 2 3 b/t 0 1 2 3 4 5 6 7 -40 -20 0 20 40 60 80 m m as s/ (1 0− 3 j t −1 k g− 1 ) t = 2.0 k t = 4.6 k sample 11 0.00 0.01 0 1 b/t 0 1 2 3 4 5 6 7 -40 -20 0 20 40 60 80 m m as s/ (1 0− 3 j t −1 k g− 1 ) t = 2.0 k sample 12 0.00 0.01 0 1 2 3 4 5 fig. 3. mass magnetization and its fit for t = 2.0 k (solid line) with the modified langevin function. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:08 utc nova biotechnologica et chimica 13-1(2014) 53 the fitting procedure applied to data referring to t = 2.0 gave the parameters that are listed in table 1. a great win is that in prevailing cases the model describes correctly the magnetization curves including the remnant magnetization. as a result, four variables were determined: the mass fraction of the paramagnetic species (wpf), diamagnetic background (χdia), mean magnetic moment (μ), and the weiss-field constant (w). also the χt product function in the interval t = 150 – 250 k has been fitted by a straight line χt = c + αt and the data are presented in table 1. the data shows that the mass fraction of the magnetic iron in the lyophilized samples under study is of the order of 10−4. the observed paramagnetic contribution is due to the minor fraction of paramagnetic metals (cu2+, fe2+ in hemoglobin), ferritin, and dioxygen molecules; the ferromagnetic contribution arises from the strong signal of γ-fe2o3 and fe3o4 deposits, respectively. nanoparticles of α-fe2o3 could also contribute through the uncompensated magnetic moments of the surface atoms. table 1. parameters of the susceptibility and magnetization for samples under study. a sample class age c α / 10-3 wpf /10−6 background (χdia/μ0)×10−3 μ/μb w / 10−3 remanence mr / 10-3 2.0 k 4.6 k abbr. age c a wpf dia m fw rm2 rm4 1 i 52 0.27 -8.1 106.0 -8.74 6.03 0.41 0.52 0.35 2 i 61 0.25 -14.3 267.4 0 3.25 0.48 0.22 0.23 3 i 65 0.31 -13.9 127.6 -9.25 4.17 2.35 1.21 0.90 4 i 69 0.34 -12.5 190.5 -8.76 4.22 1.66 1.31 0.93 5 i 85 0.64 -14.3 79.4 -8.13 5.26 4.08 2.75 2.24 6 w i 69 0.11 -8.9 190.0 0 4.32 0.47 0.24 0.24 7 iii 53 0.099 6.4 127.6 -17.14 6.14 1.10 1.82 1.29 8 iii 58 0.23 87.8 81.8 -4.90 4.59 9.09 0 0 9 ii 38 0.34 -16.1 120.1 -7.36 4.83 1.12 0.80 0.66 10 ii 66 0.44 -7.0 162.7 -9.22 4.98 1.57 1.76 1.34 11 ii 76 0.14 -7.1 70.4 -9.51 5.51 0.76 0.46 0.37 12 w ii 83 3.33 -10.2 72.0 -9.38 7.51 1.87 3.24 2.78 13 i 40 0.27 -7.3 89.2 -9.15 5.99 0.57 0.61 0.44 14 i 60 0.12 -12.6 41.8 -5.09 5.88 0.21 0.097 0.072 15 i 64 0.19 -4.7 24.3 -7.52 7.86 1.31 0.91 0.68 16 iii 73 0.35 7.2 122.4 -14.4 6.32 0.84 1.81 1.31 a curie constant c in m3 kg-1 k [si], temperature-independent susceptibility (tis) α in m3 kg-1 [si], remnant magnetization mr in j t-1 kg-1 [si], mass fraction of the paramagnetic fraction wpf – dimensionless, weiss field w in [si], magnetic moment μ in bohr magnetons μb, background diamagnetism (χdia/μ0) in [si]. w women. the quantitative characteristics of the fitted magnetization and susceptibility were processed by statistical multivariate methods: correlation (c), cluster analysis (ca), principal component analysis (pca), and factor analysis (fa) dealing with 9 variables (age, c, a, wpf, dia, m, fw, rm2 and rm4). with exception of samples no 8 and 12, the ca shows three mostly separated clusters containing 6, 6, and 2 members, respectively, according to their similarity (squared euclidean distance) – fig. 4. sample no 8 resembles features of a bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:08 utc 54 makohusová, m. et al. metamagnet and possesses some characteristics (α, w) unlike to the others; therefore this sample was deleted from further statistical processing. also the sample no 2 is very different from the others (see fig. 2). after this omission, the samples are a bit rearranged into two distant clusters as seen from fig. 4 – right. 0 30 60 90 120 150 d is ta nc e 1 23 4 5 67 89 1011 1213 14 15 16 0 20 40 60 80 100 120 d is ta nc e 1 2 3 4 56 79 101113 14 15 16 fig. 4. dendogram of the cluster analysis (wards method, metric – squared euclidean). left – all data, right – selected data. the pca generates a new set of variables through a linear combination of the original set (fig. 5). new components bear maximum information about the variability of data. the two principal components account 73 % of the variance in the data (43.6 and 29.4 %, respectively). lines are used to reflect the variables of the dataset, and circles show the observations. the biplot in figure 5 separates variables into three groups according to their strong interrelations: 1 – {age, c, fw, rm4, rm2}, 2 – {m, a}, and 3 – {dia, wpf}. the relationship between group 1 and group 2, and between group 1 and group 3 is weak. the correlation between group 2 and group 3 is strongnegative. the length of the lines approximates the variances of the variables, the longer the line, the higher is the variance. c wpf m rm4 2 6 14 9 4 3 5 16 7 15 1 11 13 -3 0 3 6 component 1 -3 -2 -1 0 1 2 3 c om po ne nt 2 age a dia fw rm2 10 component 1 c om po ne nt 2 -3 0 3 6 -3 -2 -1 0 1 2 3 cluster 1 2 fig. 5. biplot according to the principal component analysis. the samples 5, 10, 3, 4, 16 and 7 (forming the cluster no 1) have higher values of the variables from the group 1. the sample 2 has the highest value of dia and wpf, the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:08 utc nova biotechnologica et chimica 13-1(2014) 55 samples 15, 7 and 16 has the high values of m and a. points that are close together correspond to observations that have similar values of the variables. the fa reconstructs the original variables from a new, fictitious set with the same aim as pca (fig 6, after varimax rotation). it is seen that the variables {age, c, fw, rm4, rm2} are closely related whereas other groups are formed by {m, a} and {dia, wpf} variables. c wpf m rm4 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 factor 1 -1 -0.5 0 0.5 1 f ac to r 2 age a dia fw rm2 fig. 6. biplot according to the factor analysis. the correlation coefficients between pairs of variables are listed in table 2. two remnant magnetizations (rm2 and rm4) are strongly interrelated, as expected: r = 0.99. they correlate with the weiss field constant (fw), r > 0.80, and also with the susceptibility parameter (c), r > 0.70. the last variable adopts function of the curie constant in the formula χ = c/t + α whereas α is the temperature-independent term reflecting either the diamagnetism of the organic tissue (α < 0, in class-i and ii) and/or ferromagnetism (α >> 0, in class-iii). rm2 also negatively correlates with diamagnetic background dia. temperature-independent susceptibility a is positively correlated with mean magnetic moment m, and negatively correlated with diamagnetic background dia. the mass fraction of the paramagnetic species wpf negatively correlates with the mean magnetic moment m. these results are in agreement with those from the pca. table 2. correlation coefficients using 14 datapoints a age c a wpf dia m fw rm2 c 0.3083 a 0.0459 -0.2848 wpf 0.0120 0.0631 -0.1539 dia 0.0450 -0.1106 -0.6939 0.3670 m -0.0947 -0.1833 0.5795 -0.8134 -0.5052 fw 0.5031 0.7783 -0.2480 -0.1178 -0.1981 -0.1078 rm2 0.4194 0.7143 0.2982 -0.0799 -0.6052 0.1330 0.8029 rm4 0.4484 0.7528 0.2275 -0.0726 -0.5323 0.0933 0.8407 0.9942 a colored (italic) cells indicate statistically significant correlations at the 95.0% confidence level. the correlation analysis reveals that the age of the donors (weakly) correlates with variables that characterize ferromagnetism of iron-oxide deposits. such correlations are visualized by plots in fig. 7. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:08 utc 56 makohusová, m. et al. 30 40 50 60 70 80 90 age 0 1 2 3 r m 2 30 40 50 60 70 80 90 age 0 1 2 3 4 5 f w fig. 7. linear correlations of characteristics of the magnetic deposits with age of the donors: left – remnant magnetization rm2 = –0.51 + 0.025*age, weiss field fw = -1.22 + 0.030*age. confidence and prediction intervals are shown at 95 % probability level. 4. conclusions it has been demonstrated that in basal ganglia of the human brain some deposits of iron oxides are accumulated. microcrystals of hematite were detected by the electron microscopy (tem). the squid magnetometry reflects diamagnetism of the organic tissue, non-magnetic deposits like hematite, paramagnetic centers including ferritin, and ferro(ferri) magnetic deposits as maghemite. it was found that the individual characteristics of the diamagnetism (a, dia), paramagnetism (c, wpf), and ferro(ferri)magnetism (fw, rm2, rm4) are mutually interrelated. the most important finding is that the content of the magnetic deposits (wpf) does not correlate with the age of the donors. however, the parameters of the ferro(ferri)magnetism fw and rm2 (rm4) correlate with the age: on aging of the donors the ferromagnetic productivity of the deposits increases. acknowledgements: slovak grant agencies (vega 1/0233/12, vega 1/0220/12 and apvv-0014-11) are acknowledged for the financial support. references boča, r., kopáni, m., miglierini, m., čaplovičová, m., mrázová, v., dlháň, ľ.: magnetic and non-magnetic iron-oxide deposits in basal ganglia. in costa, a., villalba, e. (eds.), volume 12: horizons in neuroscience research. nova science publishers, inc., new york, 2013, 135-214. cornel, r. m., schwertmann, u.: the iron oxides: structure, properties, reactions, occurrences and uses, wiley-vch, weinheim, 2003, 703 pp. gubin, s. p. (ed.): magnetic nanoparticles, wiley-vch, weinheim, 2009. kirschvink, j. l., kobayashi-kirschvink, a., woodford, b. j.: magnetite biomineralization in the human brain. proc. natl. acad. sci. usa, 89, 1992, 7683-7687. pankhurst, q. a., connolly, j., jones, s. k., dobson, j.: applications of magnetic nanoparticles in biomedicine. j. phys. d: appl. phys., 36, 2003, 167-181. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:08 utc microsoft word remenarova 9-1 2009.doc nova biotechnologica 9-1 (2009) 53 sorption of cationic dyes from aqueous solutions by moss rhytidiadelphus squarrosus: kinetics and equilibrium studies lucia remenárová1, martin pipíška1,2, miroslav horník1,2, jozef augustín1,2 1department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (pipiskam@ucm.sk) 2consortium for environmental biotechnology and environmental chemistry, hlavná 418, špačince, sk-919 51, slovak republic abstract: with the aim to investigate sorption properties of natural sorbent prepared from moss rhytidiadelphus squarrosus we elucidated biosorption of cationic dyes malachite green (bg4), auramine o (by2) and thioflavine t (by1) from aqueous solutions. the removal of dyes by moss biosorbent was found to be rapid at an initial stage and the equilibrium was reached within 1-2 hours. the pseudo-n-order kinetic model was successfully applied to the kinetic data and the order of adsorption reaction was calculated in the range from 1.7 to 2.6. the value of rate constant kn' ranged from 0.001 to 0.039 [min-1]/[μmol/g]1-n. the equilibrium data were fitted to the adsorption isotherms. the freundlich isotherm was found to represent the measured sorption data of bg4, by1 and by2 well. the maximum sorption capacities of moss biomass from single dye solutions calculated by langmuir equation were 354 μmol/g for bg4, 310 μmol/g for by1 and 382 μmol/g for by2. these results showed that the prepared biomass presents low-cost, natural and easy available sorbent which may be potentially used for removal of dyes from environment and also may be an alternative to more costly materials such as activated carbon. key words: malachite green, thioflavine t, auramine o, rhytidiadelphus squarrosus, sorption kinetics, sorption equilibrium 1. introduction contamination of water sources by many organic pollutants is a major factor of global environmental pollution for the number of years. dyes represent one of the problematic groups. two main sources of dye pollution are the textile and dyestuff manufacturing industries. color is the first contaminant to be recognized in wastewater and the presence of very small amounts of dyes in water is highly visible and undesirable (crini and badot, 2008; iqbal and ashiq, 2007; aksu, 2005). the presence of synthetic dyes in the aquatic environment has been of great concern because of their potential health hazards associated with the carcinogenic, mutagenic, allergenic and toxic natures as well as negative effects on the photosynthetic activity in aquatic life. adsorption techniques employing solid adsorbents are effective methods for water decontamination. most commercial systems currently use activated carbon and organic resins as adsorbents to remove dye in wastewater because of their excellent adsorption 54 remenárová l. et al. abilities. a large variety of non-conventional adsorbent materials have been also proposed and studied for their ability to remove dyes (crini, 2006; 2008). sorption systems have been investigated to assess their suitability for application in the field of water pollution control. the cost and performance of a product or the mode of application are always of concern to control process efficiency. therefore the sorption capacity and required contact time are two of the most important parameters to understand (ho et al., 2000). the aim of this study was to realize biosorption of cationic dyes from aqueous solutions and to consider sorption properties of natural sorbent prepared from moss rhytidiadelphus squarrosus. experimental kinetics data were analyzed using a pseudo-n-order kinetic equation. the equilibrium data have been analyzed using langmuir and freundlich isotherms and the characteristics parameters of each isotherm have been determined. 2. materials and methods 2.1 chemicals in sorption experiments cationic dyes malachite green (bg4, basic green 4 – malachite green oxalate, mr 927, c.i. 42 000, merck, d); auramine o (by2, basic yellow 2, mr 304, c.i. 41 000, aldrich, d) and thioflavine t (by1, basic yellow 1, mr 319, c.i. 49 005, fluka, d) were used. the stock solutions of dyes were prepared in deionized water. 2.2 biosorbent preparation moss r. squarrosus was collected from forests in high tatras mountains, slovak republic. before using in experiments the biomass was thoroughly washed in deionized water, oven-dried for 72 h at a maximum of 45 °c to avoid degradation of binding sites and pulverized in blade homogenizer. size analysis showed the following separation of particles size: >600 μm – 5 %; 600-300 μm – 49 %; <300 μm – 46%. fraction <300 μm was used in sorption experiments. 2.3 sorption kinetics batch sorption experiments were carried out in erlenmayer flasks containing 20 ml bg4, by1 and by2 solutions with defined concentrations. ph value was adjusted to 4.0 with 0.1 m hcl. biomass (~30 or 40 mg, d.w.) was added and flasks were agitated on a reciprocal shaker (250 rpm) at 25 ºc. samples were taken from individual flasks in intervals 5, 10, 20, 40, 120, 240 and 1440 min and analyzed using varian cary 50 uv-vis spectrophotometer by measuring the optical densities of dyes at their respective maximum absorbance wavelengths. these are 617, 412 and 435 nm for bg4, by1 and by2, respectively. if not otherwise stated, presented data are the arithmetic mean values. nova biotechnologica 9-1 (2009) 55 2.4 sorption equilibrium all experiments were carried out in erlenmayer flasks with 20 ml of bg4, by1 and by2 solutions with initial concentration from 50 to 400 mg/l. ph value was adjusted to 4.0 with 0.1 m hcl. biomass (20 mg, d.w.) was added and the flasks were agitated on a reciprocal shaker (250 rpm) for 4 h at 25ºc. at the equilibrium, samples were taken and analyzed spectrophotometrically. if not otherwise stated, presented data are arithmetic mean values. 2.5 non-linear regression analysis to calculate parameters of pseudo-n-order model, the qmax values and the corresponding parameters of adsorption isotherms non-linear regression analysis was performed by the nlsf (origin’s nonlinear least square fitter) using program microcal origin 8.0 professional (originlab corporation, northampton, usa) with automatic initialization of parameters and graphpad prism 5.0 software (graphpad software inc., usa). 3. results and discussion 3.1 biosorption kinetics the time-course studies on the biosorption of cationic dyes by1, by2 and bg4 were performed by contacting the dye solutions with moss biosorbent at ph 4.0 and 25 °c. biosorption of by1, by2 and bg4 by r. squarrosus is faster at initial stage, during occupation of high affinity sites (fig. 1.) maximum uptake, approx. 95% at biosorbent concentration 2.0 g/l and initial bg4 concentration c0 = 122 μmol/l was reached within 60 min. in the case of by1 (c0 = 307 μmol/l) and by2 (c0 = 339 μmol/l) uptake reached aprox. 80% within the first 90 min at biosorbent concentration 1.5 g/l. after this time there was no considerable increase during the next 23 hours. crini et al. (2007) also observed a very fast sorption of bg4 by cyclodextrin-based adsorbent. the largest amount of bg4 was adsorbed to the polymer within the first 90 min. rapid sorption of by2 by poly-(γ-glutamic acid) was confirmed by inbarajn et al. (2006). maximum sorption was accomplished within 12 min indicating a rapid surface adsorption of by2 on poly-(γ-glutamic acid) occurred. the mechanism of a short-term dye uptake by biosorbents is generally regarded as an abiotic process. it is known that biomass contains ionogenic groups such as carboxyl, phenolic and alcoholic hydroxyl, and phosphate that generate a negative net charge. basic dyes can be ionized in solution to form positive charge and chemical reaction through electrostatic interaction may occur between anionic groups of moss biosorbent and cations of dyes molecules (crini and peindy, 2006). biosorption kinetics describing the pollutant uptake rate is one of the important characteristics defining the efficiency of sorption and feasibility of (bio)sorbents for their use in water pollution control. in almost all previous biosorption kinetic studies, 56 remenárová l. et al. both pseudo-first (1) and pseudo-second (2) order kinetic equations were directly chosen to fit biosorption data without any explanation about the rational behind. )('1 teq t qqk dt dq −= (1) 2'2 )( teq t qqk dt dq −= (2) liu and liu (2008) pointed out that, there is no reason and need to preset biosorption kinetics to be the first or the second order unless biosorption mechanisms are known. considering the complexity of biosorption process and fact that the various mechanisms would be involved őzer (2007) and liu and wang (2008) the direct calculation of rate constant and order of the biosorption reaction recommended as a more appropriate method. for these reasons the experimental data obtained by nonequilibrium conditions in our work were analyzed using a pseudo-n-order (3) model proposed by őzer (2007) and liu and shen (2008): nteqn t qqk dt dq )(' −= (3) where n is the reaction order determined from biosorption data and kn is a rate constant. the integrated form of equation (3) has the following form: [ ] nneqneqt qtknqq −−+−−= 1 1 )1(')1( (4) where qt is the amount of sorbate sorbed at time t, qeq represents sorbate sorption at equilibrium. 0 200 400 600 800 1000 1200 1400 1600 0 20 40 60 80 100 120 140 160 180 200 by1 by2 bg4 pseudo-n-order kinetic model pseudo-n-order kinetic model pseudo-n-order kinetic modelq t [ μ m ol /g ] t [min] fig. 1. kinetics of by1 ( ), by2 ( ) and bg4 ( ) biosorption by r. squarrosus at ph 4 and 25 °c. experimental data fitted to pseudo-n-order kinetic model. data shown in table 1. nova biotechnologica 9-1 (2009) 57 plot of pseudo-n-order kinetics model (4) for the biosorption of dyes by moss biosorbent is shown in fig. 1. the model parameters determined by non-linear regression analysis are shown in table 1. the calculated qeq cal values from the model are in good agreement with experimental qeq exp values and the correlation coefficients for the pseudo-n-order kinetic plots are very high. the respective reaction order for bg4, by1 and by2 biosorption was estimated as 2.3, 1.7 and 2.6. the values of rate constant kn′ ranged from 0.001 to 0.039 [min -1]/[μmol /g]1-n. from the presented results it is evident that pseudo-n-order model provides a satisfactory description of biosorption experimental data compared to prediction by equation (1) and (2) (data not shown). table 1. pseudo-n-order kinetics parameters for bg4, by1 and by2 sorption by r. squarrosus at ph 4 and 25°c from aqueous solutions obtained by using non-linear regression analysis. dye c0 [μmol/l] kn ′ [min-1]/[μmol/g]1-n qeq cal [μmol/g] d.w. n r2 qeq exp [μmol/g] d.w. bg4 * 122 0.014 65.0 ± 0.3 2.3 1.0 65 by1 ** 307 0.039 163 ± 2 1.7 0.998 168 by2 ** 339 0.001 195 ± 4 2.6 0.996 195 * biosorbent concentration = 2.0 g/l ** biosorbent concentration = 1.5 g/l 3.2 sorption equilibrium in single dye solutions two well known adsorption isotherm models, namely langmuir and freundlich ones, were applied for the analysis of the experimental data (table 2). table 2. adsorption isotherm models for single sorption systems in linear and non-linear forms used in this work. isotherm non-linear form linear form langmuir eq eq eq bc cbq q + = 1 max maxmax 11 bq c qq c eq eq eq += freundlich qeq= k ceq (1/n) log(qeq) = log k + 1/n log (ceq) these models use parameters that reflect the nature of the sorbent and can be used to compare biosorption performance. qmax represents the maximum sorption capacity, b is a constant related to the energy of adsorption. k and 1/n values are the freundlich constants referring to adsorption capacity and intensity of adsorption, respectively. simplicity and easy interpretability are some of the important reasons for the extensive use of these models. moreover, linear regression has been frequently used to evaluate the model parameters. however, transformations of nonlinear equations into linear 58 remenárová l. et al. forms usually result in parameter estimation error and distort the fit (kumar and sivanesan, 2006). for this reason, nonlinear methods for parameters estimation were used in our work. we have found, that sorption of cationic dyes by1, by2 and bg4 by r. squarrosus increased with the increasing concentration of dyes in solutions (data not shown) and the equilibrium was reached within 1-2 h. figure 2 shows the experimental data fitted to the isotherm models for by1, by2 and bg4 sorption by r. squarrosus at ph 4 from single dye solutions. the obtained adjustable parameters are shown in table 3 with the corresponding coefficients of determination. estimated maximum sorption capacity qmax for by1 biosorption obtained from langmuir isotherm was 310 ± 10 µmol/g d.w. (table 3). the qmax 382 ± 11 µmol/g d.w. for by2 was obtained. the maximum sorption capacity qmax for bg4 biosorption obtained from langmuir isotherm was 354 ± 33 µmol/g d.w. this indicates a higher sorption capacity of r. squarrosus for by2 in comparison with by1 and bg4. freundlich parameter 1/n is the heterogeneity factor with the values ranged between 0 1. if the value is close to unity this implies that sorption is chemical process. the more heterogenous the surface the closer 1/n value is to 0. in fact, for tested dyes the 1/n values are close to 0 (table 3). the values of r2 are generally regarded as a measure of the goodness of fit of experimental data on the isotherm models (al-asheh et al., 2000; basha et al., 2008). as can be seen from table 3 higher coefficients of determination r2 were obtained for the freundlich model (r2= 0.983 for by1, r2 = 0.976 for by2, r2 = 0.945 for bg4) compared to langmuir isotherm (r2 = 0.906 for by1, r2 = 0.947 for by2, r2 = 0.789 for bg4). 0 100 200 300 400 500 600 700 0 40 80 120 160 200 240 280 320 360 experimental data langmuir freundlich q eq [ μ m ol /g ] ceq [μmol/l] a nova biotechnologica 9-1 (2009) 59 0 200 400 600 800 1000 1200 0 50 100 150 200 250 300 350 400 450 experimental data langmuir freundlich q eq [ μ m ol /g ] ceq [μmol/l] 0 50 100 150 200 250 0 50 100 150 200 250 300 350 400 experimental data langmuir freundlich q eq [μ m ol /g ] ceq [μmol/l] fig. 2. fit of the langmuir and freundlich adsorption isotherms of by1 (a), by2 (b) and bg4 (c) biosorption by r.. squarrosus (1.0 g /l, d.w.) from single dye solutions at 25°c and initial ph 4.0. equilibrium ph 5.4. the corresponding data are shown in table 3. b c 60 remenárová l. et al. table 3. adsorption isotherm models and corresponding parameters for by1, by2 and bg4 sorption by r.. squarrosus from single dye solutions at ph 4 obtained by using non-linear regression analysis. model dye qmax [µmol/g] d.w. b [l/ µmol] k [l/g] 1/n r2 langmuir by1 310 ± 10 0.06 ± 0.01 0.906 by2 382 ± 11 0.026 ± 0.004 0.947 bg4 354 ± 33 0.04 ± 0.02 0.789 freundlich by1 99.7 ± 5.5 0.19 ± 0.01 0.983 by2 93.2 ± 7.7 0.21 ± 0.01 0.976 bg4 59.7 ± 10.5 0.32 ± 0.04 0.945 this shows that the freundlich isotherm is better fitted to the experimental data of by1, by2 and bg4 sorption by r. squarrosus in the concentration range studied and describes the process well and quantitatively. the freundlich model provides a more realistic description of dye sorption by organic matter because it accounts for sorption to heterogeneous surfaces or surfaces supporting sites of varied affinity. however, we draw attention to some published papers stressing that the application of adsorption models is not able to explain the biosorption mechanisms of complex biological systems (fraile et al., 2005; cordero et al., 2004). 4. conclusions sorption capability of biosorbent prepared from moss r. squarrosus was tested for cationic dyes bg4, by1 and by2 in batch experiments. the removal of dyes by moss biosorbent was found to be rapid at an initial stage and the equilibrium was reached within 1-2 hours. the kinetic analysis showed that biosorption process could be described well with the pseudo-n-order kinetic model. the respective reaction order for bg4, by1 and by2 biosorption was estimated as 2.3, 1.7 and 2.6. equilibrium data were analysed using langmuir and freundlich isotherm models. the freundlich isotherm was demonstrated to provide a very good correlation for the biosorption of bg4, by1 and by2 onto moss biosorbent. references al-asheh, s., banat, f., al-omari, r., duvnjak, z.: predictions of binary sorption isotherms for the sorption of heavy metals by pine bark using single isotherm data. chemosphere, 41, 2000, 659-665. aksu, z.: application of biosorption for the removal of organic pollutants: a review. process biochem., 40, 2005, 997-1026. nova biotechnologica 9-1 (2009) 61 basha, s., murthy, z.v.p., jha, b.: sorption of hg (ii) from aqueous solutions onto carica papaya: application of isotherms. chem. eng. j., 47, 2008, 980-986. cordero, b., lodeiro, p., herrero, r., sastre de vicente, m.e.: biosorption of cadmium by fucus spinalis. environ. chem., 1, 2004, 180-187. crini, g.: kinetic and equilibrium studies on the removal of cationic dyes from aqueous solution by adsorption onto cyclodextrin polymer. dyes pigments, 2008, 77, 415-426. crini, g.: non-conventional low-cost adsorbents for dye removal: a review. bioresour. technol., 97, 2006, 1061-1085. crini, g., badot, p.-m.: application of chitosan, a natural aminopolysaccharide, for dye removal from aqueous solutions by adsorption processes using batch studies: a review of recent literature. progr. polymer sci., 33, 2008, 399-447. crini, g. peindy, h.n.: adsorption of c.i. basic blue 9 on cyclodextrin based material containing carboxylic groups. dyes pigment, 70, 2006, 204–211. crini, g., peindy, h.n., gimbert, f., robert, c.: removal of c.i. basic green 4 (malachite green) from aqueous solutions by adsorption using cyclodextrin-based adsorbent: kinetic and equilibrium studies. sep. purif. technol., 53, 2007, 97-110. fraile, a., penche, s., gonzáles, f., blázquez, m.l., muñoz, j. ballester, a.: biosorption of copper, zinc, cadmium and nickel by chlorella vulgaris. chem. ecol., 21, 2005, 61-75. ho, y.s., ng, j.c.y., mckay, g.: kinetics of pollutant sorption by biosorbents: review. separ. purif. meth., 29, 2000, 189-232. inbaraj, b.s., chien, j.t., ho, g.h., yang, j., chen, b.h.: equilibrium and kinetic studies on sorption of basic dyes by a natural biopolymer poly(γ-glutamic acid). biochem. eng. j., 31, 2006, 204-215. iqbal, m.j., ashiq, m.n.: adsorption of dyes from aqueous solutions on activated charcoal. j. hazard. mat., b 139, 2007, 57-66. kumar, k.v., sivanesan, s.: isotherm parameters for basic dyes onto activated carbon: comparison of linear and nonlinear methods. j. hazard. mat., 129, 2006, 147-150. liu, y., liu, y.j.: biosorption isotherms, kinetics and thermodynamics. sep. purif. technol., 61, 2008, p. 229-242. liu, y., shen, l.: a general rate law equation for biosorption. biochem. eng. j., 38, 2008, 390-394. liu, y., wang, z.w.: uncertainity of preset-order kinetic equations in description of biosorption data. bioresour. technol., 99, 2008, 3309-3312. őzer, a.: removal of pb(ii) ions from aqueous solutions by sulphuric acid-treated wheat bran. j. hazard. mat., 141, 2007, 753-761. microsoft word šimonovičova final.doc 38 nova biotechnologica et chimica 14-1 (2015) doi 10.1515/nbec-2015-0012 © university of ss. cyril and methodius in trnava influence of fine-grained montmorillonite on microfungal pellets growth in aqueous suspensions   karol jesenák1, alexandra šimonovičová2, slavomír čerňanský3 1department of inorganic chemistry, faculty of natural sciences, comenius university in bratislava, mlynská dolina, bratislava, sk-842 15, slovak republic 2department of soil science, faculty of natural sciences, comenius university in bratislava, mlynská dolina, bratislava, sk-842 15, slovak republic 3department of environmental ecology, faculty of natural sciences, comenius university in bratislava, mlynská dolina, bratislava, sk-842 15 slovak republic abstract: the paper presents an inhibition effect of clay mineral – montmorillonite – on the growth of microscopic filamentous fungus aspergillus niger in the aqueous solution. the significant reduction in growth of the final size of spherical fungal pellets as well as total amount of produced microbial biomass was found out. within the observed range of additions of clay mineral of 1, 5, 10, 15 and 20 g in the total volume of the 80 ml suspension, this size was in indirect relation to the weight of montmorillonite. however, the most significant inhibition effect was observed at the lowest concentration of the sorbent (1 g). microscopic analysis of pellets referred to the presence of mineral particles in their pore structure and the distribution of particles in the spatial structure of fungal hyphae was variable. the experiment clearly demonstrated an inhibition effect of montmorillonite. this inhibition could be answered by the experiments focused on the detection of the influence of size and shape of inorganic sorption particles together with the influence of the physicochemical properties of its surface. it could be stated that the simultaneous application of the microscopic fungus aspergillus niger and the clay mineral montmorillonite for decontamination of waste waters should be disadvantage due to their interaction if compared with the decontamination based on bioaccumulation and sorption separately. key words: montmorillonite, aspergillus niger, microfungal pellets, radial growth 1. introduction the aspergillus niger species is an ubiquitous soil microscopic filamentous fungus (šimonovičová, 2013). it could be used in bioaccumulation of various potentially toxic elements and in bioremediation of contaminated sites (enontiemonria et al., 2012; soleimanifar et al., 2011; šimonovičová et al., 2010; žemberyová et al., 2009; wasay et al., 2010). the simultaneous occurrence of microscopic filamentous fungi and microcrystalline inorganic silicates is often in natural systems. both components frequently occur in soils and sediments and they are able to decrease the concentration of various water-soluble substances in their surrounding environments. for the transport of these substances from the surrounding environment, the presence of water in its fluid phase is required, which significantly increases surface for interaction of the sorbent, also due to decreasing of particle size to smaller particles and also it bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:54 utc nova biotechnologica et chimica 14-1 (2015) 39 allows to implement significantly more effective sorption mechanisms (for example cation exchange). bioaccumulation properties of microscopic fungi and sorption properties of microcrystalline silicates result in consideration about their potential application for decontamination of the environment (fomina and gadd, 2002; saraswathy and hallberg, 2005). thereby bioaccumulation properties of microscopic fungi as well as sorption properties of microcrystalline silicates are known, overall decontamination effect in technological processes followed by implementation of bioaccumulation and sorption units may be relatively well estimated. on the contrary, decontamination effect of the simultaneous application of microscopic fungi and silicate sorbents cannot be estimated because it is complicated by their interactions. therefore, it is necessary to estimate it experimentally. it can be supposed that this effect will be lower than sum of effects of both components. the main reason should be an inhibition of transport of the solution towards the sorbent surface due to the decreasing of the circulation of fluid phase in the surroundings of fungal pellets as well as directly inside the pellets. another reason could be an inhibition effect of the sorbent on the growth of microscopic filamentous fungi. the aim of the paper is to carry out an assay of the influence of the clay mineral montmorillonite on the growth of microscopic filamentous fungus aspergillus niger in the aqueous environment. the selection of montmorillonite for this study is because of this mineral belonging to very frequent component of various soils. it also represents inexpensive industrial products with adequate purity. the overall strategy of performed experiments was based on the model system that included microscopic filamentous fungus, standard culture medium and clay mineral with no impurities identified by x-ray diffraction method and it was also enough characterized by its particle size in aqueous suspension. 2. material and methods the influence of montmorillonit on the growth of microscopic filamentous fungus aspergillus niger was observed in 70 ml sabouraud medium composed of glucose, peptone and distilled water, with addition of 10 ml of aqueous suspension of fungal spores. the a. niger strain was originally isolated from the šobov locality (šimonovičová et al., 2013). the monomineral sample of montmorillonite in addition of 1, 5, 10, 15 and 20 g was dispersed in the suspension of the culture medium with fungal spores. the cultivation was carried out for 7 days at 25 °c in 100 ml conic flasks. the flasks were then shaken using unimax 2010 shaker (heidolph, germany) at 130 rpm. there were three replicated runs for each experiment. the fungal pellets were washed by 500 ml of distilled water at the end of the experiment. montmorillonite used in the experiment was obtained by sedimentation of 4 % aqueous suspension of bentonite from the stará kremnička – jelšový potok site. the sedimentation conditions were set up to acquire the particle size of lower than 5 μm as calculated according to stokes' equation. the distribution of size of montmorillonite particles was then determined by the particle size laser analyzer (mastersizer 2000, malvern instruments ltd., uk). this analyzer is applicable for the analysis of particle size in the range from 0.02 to 2 000 μm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:54 utc 40 jesenák, k. et al. the size of fungal pellets was determined according to the photographs of the thin layer of suspension in the petri dish, in which pellets were in mono layer with negligible overlapping of the particles. the microstructure of pellets was observed by optical microscope (jenalumar, carl zeiss jena, germany) and photographs were recorded by digital camera (camedia c-5060, olympus, japan). to increase background contrast, pellets were colored by commercial histological pigment based on methylene blue (lactophenol cotton blue, himedia laboratories pvt. ltd., india). 3. results and discussion the distribution of montmorillonite particle size, determined using particle size laser analyzer (fig. 1) shows that main content of the clay mineral is formed by particles lower than 10 μm. in regard of the mineralogical homogeneity of the montmorillonite samples, these particles also formed the main weight ratio of the sample. the result of the analysis shows significant difference against diameter of particles derived from the stokes' equation. the content of non-clay impurities in the bentonite fractions obtained from the stará kremnička – jelšový potok site with the particle size lower than 10 μm is negligible (jesenák, 2007). fig. 1 the distribution of the particle size of montmorillonite isolated from bentonite obtained from the stará kremnička – jelšový potok site using a particle size laser analyzer mastersizer 2000 (malvern instruments ltd., uk). (the particle size is expressed in micrometers.) the selection from the series of experiments focused on the determination of the influence of montmorillonite on the fungal pellet growth is shown in fig. 2. it can be seen that sizes of the microfungal pellets formed in the presence of dispersed particles of montmorillonite are markedly lower than sizes of pellets formed in culture medium without addition of montmorillonite (fig. 3). the size of the first type of pellets ranges from 2 to 6 mm with the highest amount in the range of 3-4 mm. pellets formed in the presence of different additions of montmorillonite have their size lower than 1 mm with the highest amount in the size of 0.5 mm. the experiments show that approximately 1 wt% of clay mineral in culture medium markedly reduces the pellet size (almost 10-fold), however, another increasing of montmorillonite has bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:54 utc nova biotechnologica et chimica 14-1 (2015) 41 only minimal influence on the pellet sizes. another result of the experiments is the fact that with increasing weight of clay mineral, relative amount of produced fungal biomass is decreasing. this statement could be derived from the surface of pellets on the petri dishes (fig. 2). fig. 2. microfungal pellets of the a. niger species formed after 7-day cultivation at 25 °c in 80 ml of sabouraud culture medium (upper left corner). all other petri dishes show fungal pellets of the same fungal strain formed in the presence of different amounts of the clay mineral montmorillonite under the same cultivation conditions (1 g – upper right corner, 5 g – lower left corner, 10 g – lower right corner). fungal pellets were colored with commercial histological pigment based on methylene blue (lactophenol cotton blue, himedia laboratories pvt. ltd., india). fig. 3. variability in shape and size of the microfungal a. niger pellets produced in the presence of the clay mineral montmorillonite. the size of the largest pellets is approx. 5 mm. 5 mm bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:54 utc 42 jesenák, k. et al. morphology and size of pellets under variable conditions as volume of fungal inoculum and medium, carbon and nitrogen source, initial ph and temperature or adding of mineral microparticles was studied by many other authors (feng et al., 2003; gonciarz and bizukojc 2014; lópez et al., 2004; petre et al., 2005; kim and song, 2009). reduction of pellet size after addition of bentonite and kaolinite as well as after addition of talc microparticles to aspergillus glaucus inoculum was observed by fomina and gadd (2002) and gonciarz and bizukojc (2014), respectively. according to kim and song (2009) the agitation speed is the most important control factor. at the lower agitation speed of 100 rpm the large pellets are formed and the enzymatic activity is higher than that of small pellets culture at 150 rpm. a new strategy for shaping fungal morphology by modifying polarized growth genes applied in submerged fermentation in bioreactor which could be valuable for morphology improvement of industrial filamentous fungi is documented by cai et al. (2014). fig. 4. the structure of single pellet of the a. niger fungus containing closed particles of montmorillonite (mag. 40×) (a). particles of montmorillonite (dark objects) inside the filamentous structure of the fungal pellet (mag. 78×) (b). the detail of the pellet surface (mag. 157×) (c). the detail of fungal hyphae at the pellet surface (mag. 252×) (d). (the size of the pellet is 2 mm.) fig. 4 shows several details of spherical fungal pellets formed in the culture medium with addition of clay mineral montmorillonite. it could be seen that clay particles are fixed within the filamentous structure of microscopic fungus (figs. 4a, b). however clay particles are not fixed on the pellet surface regularly. the amount of a b c d bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:54 utc nova biotechnologica et chimica 14-1 (2015) 43 clay particles decreases from the middle of the pellet to its surface. this change in frequency of the occurrence of montmorillonite particles could be well explained by decreasing in the density of hyphal net towards to the pellet surface (figs. 4c, d). however, the amount of clay mineral particles in individual volume parts of pellets is not continuous. it depends on their distance from their geometric center. considerably diffusion surface of pellets shows that the objectification of dimensional properties of pellets, for example by image analysis, is very limited by considerably subjective selection of abstract line, which serves for separation of the pellets from its surroundings. these findings could be viewed as follows. the first view is the application of microscopic filamentous fungi and clay minerals in decontamination of the environment. it seems that such application of microscopic fungi and silicate sorbents would not offer any significant advantages in decontamination technologies. however, the overall influence of the interaction of both components on the decontamination effect could be shown only by the experimental observation. the second view on the findings is connected to reasons of the inhibition influence of clay minerals on the growth of microscopic filamentous fungi. the explanation of these reasons could be shown by experiments focused on intensity of this influence. 4. conclusion the main result of the experiments was a clear confirmation of the significant reduction of the size of spherical pellets of the aspergillus niger microscopic fungus due to the inhibition of its growth by clay mineral montmorillonite. there could be various reasons of this effect such as spherical inhibition of the growth, changes resulting from physicochemical interactions of the clay mineral surface with the surface of microscopic fungi as well as from interactions of clay with the components of culture medium. one of the problems of the research focused on the explanation of the effects is that none of the listed properties could be changed independently when fine-grained particulates are used. acknowledgements: the work was supported by projects of scientific grant agency vega 1/0482/15 and slovak research and development agency apvv-0344-11 and apvv-0866-12. references cai, m., zhang, y., hu, w., shen, w., yu, z., zhou, w., jiang, t., zhou, x., zhang, y.: genetically shaping morphology of the filamnentous fungus aspergillus glaucus for production of antitumor polyketide aspergiolide a. microb. cell fact., 13, 2014, 1-10. enontiemonria, e.v., kofi, h.f., cybil, o., gbenga, t.: the effects of pseudomonas aeruginosa and aspergillis niger on the bioremediation of raw and treated crude oil polluted water. int. j. sci. technol., 2, 2012, 345-352. feng, k.-ch., rou, t.-m., liu, b.-l., tzeng, y.-m., chang, y.-n.: effects of fungal pellet size on the high yield production of destruxin b by metharhizium anisopliae. enzyme microb. technol., 34, 2004, 22-25. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:54 utc 44 jesenák, k. et al. fomina, m., gadd, g.m.: influence of clay minerals on the morphology of fungal pellets. mycol. res., 106, 2002, 107-117. gonciarz, j., bizukojc, m.: adding talc microparticles to aspergillus terreus atcc 20542 preculture decreases fungal pellet size and improves lovastin production. eng. life sci., 14, 2014, 190-200. jesenák, k.: zrnitostná charakteristika bentonitových suspenzií. zborník príspevkov z 9. ročníku konference o speciálních anorganických pigmentech a práškových materiálech, 13. 9. 2007, pardubice, fakulta chemicko-technologická, univ. pardubice, 2007, 42-43 (in slovak). kim, y.-m., song, h.-g.: effect of fungal pellet morphology on enzyme activities involved in phthalate degradation. j. microbiol., 47, 2009, 420-424. lópez, j.l.c., pérez, j.a.s., sevilla, j.m.f., porcel, e.m.r., chisti, y.: pellet morphology, culture rheology and lovastin production in cultures of aspergillus terreus. j. biotechnol., 116, 2005, 61-77. petre, m., peng, m.x., mao, l.x.: the influence of culture conditions on fungal pellet formation by submerged fermentation of cordyceps sinensis (paecilomyces hepiali) cs 4. fifth international conference on mushroom biology and mushroom products. 8.-14. april 2005, shanghai, china. 12, supplement, 99-104. saraswathy, a., hallberg, r.: mycelial pellet formation by penicillium ochrochloron species due to exposure to pyrene. microbiol. res., 160, 2005, 375383. soleimanifar, h., ardejani, f.d., marandi, r.: bio-remediation of acid mine draianage in the saecheshmeh porhyry copper mine by fungi: batch and fixed bed process. i.j.m.g.e., univ. of teheran, 45, 2011, 87-102. šimonovičová, a.: biodiverzita pôdnych mikroskopických húb v pôdach slovenska. univerzita komenského v bratislave, 2013, 100 pp. (in slovak). šimonovičová, a., barteková, j., janovová, ľ., luptáková, a.: behaviour of fe, mg and ca in acid mine drainage and experimental solutions in the presence of aspergillus niger species isolated from various environment. nova biotechnol., 10, 2010, 63-69. šimonovičová, a., hlinková, e., chovanová, k., pangallo, d.: influence of the environment on the morphological and biochemical characteristics of different aspergillus niger wild type strain. ind. j. microbiol. 53, 2013, 187193. žemberyová, m., sherman, a., šimonovičová, a., hagarová, i.: bioaccumulation of as(iii) and as(v) species from water samples by two strains of aspergillus niger using hydride generation atomic absorption spectrometry. int. j. environ. anal. chem., 2009, 569-581. wasay, s.a., barrington, s.f., tokunaga, s.: using aspergillus niger to bioremediate soils contaminated by heavy metals. bioremed. j., 2, 1998, 183-190. presented at the 3rd international conference “biotechnology and metals 2014”, september 17-19, 2014, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:54 utc microsoft word al hakeim et al., 2015.doc nova biotechnologica et chimica 14-2 (2015) 141 doi 10.1515/nbec-2015-0023 © university of ss. cyril and methodius in trnava interaction of calcium phosphate nanoparticles with human chorionic gonadotropin modifies secondary and tertiary protein structure hussein k al-hakeim1, rahman s.al-zabeba1, eric grulke2, emad a. jaffar al-mulla1 1department of chemistry, faculty of science, university of kufa, p.o. box 21, annajaf 54001, iraq (imad.almulla@uokufa.edu.iq) 2department of chemical engineering, college of engineering, kentucky university, usa abstract: calcium phosphate nanoparticles (capnp) have good biocompatibility and bioactivity inside human body. in this study, the interaction between capnp and human chorionic gonadotropin (hcg) was analyzed to determine the changes in the protein structure in the presence of capnp and the quantity of protein adsorbed on the capnp surface. the results showed a significant adsorption of hcg on the capnp nanoparticle surface. the optimal fit was achieved using the sips isotherm equation with a maximum adsorption capacity of 68.23 µg/mg. the thermodynamic parameters, including ∆h° and ∆g°, of the adsorption process are positive, whereas ∆s° is negative. the circular dichroism results of the adsorption of hcg on capnp showed the changes in its secondary structure; such changes include the decomposition of α-helix strand and the increase in β-pleated sheet and random coil percentages. fluorescence study indicated minimal changes in the tertiary structure near the microenvironment of the aromatic amino acids such as tyrosine and phenyl alanine caused by the interaction forces between the capnp and hcg protein. the desorption process showed that the quantity of the hcg desorbed significantly increases as temperature increases, which indicates the weak forces between hcg and the surface. key words: calcium phosphate nanoparticles; hcg; protein; secondary structure; and tertiary structure. abbreviations: capnp, calcium phosphate nanoparticles; hcg, human chorionic gonadotropin; cd, eircular dichroism; ftir, fourier transform infrared spectroscopy; tem, transmission electron microscopy; sem, scanning electron microscopy; np, nanoparticle; qe, quantity adsorbed at equilibrium; ce, concentration of hormone at equilibrium; qm, maximum quantity adsorbed; ks, sips constant of the energy of adsorption; t, sips constant of the system heterogeneity; and eds, energy-dispersive x-ray spectrometer. 1. introduction hormones are signaling molecules that traffic information from one cell to another and can be classified according to their chemical nature into peptides, proteins, and steroids (gardner and shoback, 2011). human chorionic gonadotropin (hcg) is a pregnancy hormone that maintains adequate levels of sex steroids synthesized by the corpus luteum until the placenta performs this function (stenman et al., 2008). furthermore, hcg acts as a gonad tropic molecule that functions on the ovaries to promote steroid synthesis (cole, 2009). this hormone comprises of α-subunit that bound with β-subunit by ionic interactions and non-covalent hydrophobic (cole, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc 142 al-hakeim, h. et al. 2009; zin et al., 2010). the interaction of specific proteins with nanoparticles (nps) significantly improves clinical diagnosis, receptor-targeted delivery, in vivo gene delivery, and medical/cancer imaging. the development of biocompatible nanomaterials to enhance or modify bio-properties is the new challenge in the biotechnology field. these applications can be classified into four main categories: biomolecular interactions, application in drug and gene delivery (zamani et al., 2013; jin et al., 2014), bioimaging (holgado et al., 2014), and biosensors design (cao et al., 2012). the use of calcium phosphate nanoparticles (capnp) as carriers of different molecules into cells depends on their stability in colloidal form by charged molecules or biopolymers (epple et al., 2010). capnp has excellent biocompatibility due to its chemical similarity to human bone tissues (epple et al., 2010). therefore it used in many biological applications including gene delivery (roy et al., 2003), drug delivery (liong et al., 2008), and as fluorescing probes (chane-ching et al., 2007). these uses involve the direct contact between capnp and body fluids. therefore, understanding the possible effect of these nps on the soluble proteins is very important for the safety of uses of capnp. nps provide a highly useful tool for studying protein-surface interactions. the high surface area and optical properties of these systems allow the ready application of circular dichroism (cd) methods (fenoglio et al., 2011). cd spectroscopy efficiently determines the folding of protein and characterizes its secondary and tertiary structures, as well as the structural family to which it belongs. cd is a valuable technique to monitor the induced conformational changes in the macromolecular structures (ranjbar and gill 2009; hoidy et al., 2010). moreover, secondary structure can be determined via cd spectroscopy at a wavelength range of 190–250 nm. in this study, a comprehensive investigation was carried out to determine the interaction between hcg hormone and capnp and the possible effects of this interaction on the protein structures. 2. materials and methods 2.1preparation of nps capnp: ca3(po4)2 with a particle size of ~25 was synthesized according to the method of liu et al. (2010). briefly, 352.8 mg of cacl2.2h2o was added to 20 ml of water and then mixed with 20ml of a solution containing 172.8 mg of nah2po4. the two solutions were stirred vigorously and heated to 85°c. subsequently, 40ml of ammonium hydroxide solution (28 %) was added. the mixture was stored at 85°c for 24 hours. the expected yield of ca3(po4)2 nps is 248 mg. 2.2 interaction of hcg hormone with capnp briefly, 10 mg/ml capnp were dispersed in 40 ml of pbs buffer to obtain a concentration of 0.25 mg/ml. the mixture was mixed and ultrasonicated for 10 min twice. the following hcg concentrations were prepared in phosphate buffer: 36, 32, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc nova biotechnologica et chimica 14-2 (2015) 143 28, 24, 20, 16, 12, and 8 µg/ml. subsequently, 500 μl of the ca3(po4)2 nanoparticle suspension was mixed with 0.5ml of the different concentrations of hcg solution supplied by sigma-aldrich co. the mixture was stirred for 30 min at different temperatures (15, 25, 35, and 45°c). the mixture was ultracentrifuged at 20 000 rpm. the supernatant was separated, and the hcg concentration in the solution was determined using a ready-to-use elisa kit supplied by monobind® co. usa. isotherms were constructed between the hcg concentration in the solution at equilibrium (ce) and the amount of the hcg adsorbed on the nps (qe). the amount of the hcg hormone adsorbed was calculated using the following equation: qe=v(co-ce)/m where qe: amount of adsorption (µg/g), co: initial concentration (µg/ml), ce: concentration at equilibrium (µg/ml), m: weight of adsorbent (mg), and v: volume of solution (ml). isotherms were analyzed to determine the optimal equation applicable for practical results. thermodynamic parameters (∆h°, ∆s°, and ∆g°) were initially calculated using the vant–hoff’s equation (jackson, 2006): ln ke= −∆h°/rt + constant where ∆h°: enthalpy in reaction, ke: maximum amount adsorbed, r: gas constant = 8.314 j/mol.k. by plotting ln ke against 1/t, a straight line should be formed with a slope of -∆h°/r. to calculate the free energy change (∆g°) of the adsorption process at a specific temperature, the equilibrium constant of the adsorption–desorption process of hcg on the capnp was calculated: ke=qe×m / ce×v where keq: equilibrium constant, qe: adsorption amount (µg/mg), m: mass of nanoparticle (mg), ce: concentration of hcg at equilibrium (µg/ml), and v: volume of solution (ml). gibbs free energy change can be calculated from the following equation (jackson 2006; zhang et al., 2008): ∆g° = –rt lnke. entropy (∆s°) can be calculated from the following formula: ∆s° = (∆g°−∆h°)/t. all experiments were repeated three times and the mean of the results were taken. 2.3 preparation of nanoparticle–hcg for tem imaging the nanoparticle–hcg sample for tem was prepared by adding 0.3 mg/ml hcg to 1 mg/ml nps to form a monolayer of the hormone on the nps. after adsorption, 5 μl of the solution was placed on a copper grid and then left to dry for 2 minutes. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc 144 al-hakeim, h. et al. subsequently, 5μl of the negative stain (2% uranyl acetate in dry weight) was added and left to dry for one hour. the images were then obtained. 2.4 dynamic light scattering (dls) method the particle size distribution of the capnp powders was determined using dls (90 plus particle size, brookhaven instruments, usa) at 25°c. 2.5 fluorescence method fluorescence spectra were scanned between 250 and 450 nm after excitation at 262 nm at room temperature. excitation and emission slits were set to 5 nm. 2.6 preparation of samples for cd study the cd spectra of the 30 μm free or immobilized hcg in 50mm phosphate buffer (ph = 7.4) were obtained using a 1-mm spectrosil quartz cuvette (starna cells) and a jasco cd spectrometer at 20°c. three scans were averaged using chirascan pro-data viewer software. thermal stability was examined by monitoring the changes in ellipticity at 220 nm as a function of temperature. cd spectra were explained by the online project called k2d2, which was developed by perez–iratxeta et al., (2008). k2d2 can be accessed at k2d2 web site (http://www.ogic.ca/projects/k2d2/). k2d2 inputs a cd spectrum outputs the estimated α-helix and β-strand content of the corresponding protein and the estimated measure of error. 2.7 desorption process the percentages of the quantities of the hcg desorbed on the surface of the capnp surface were measured, as described previously al-hakeim et al. (2014), by dividing the quantity released into 1 ml of solvent = (ce/1) on the adsorbed quantity of hcg on 0.125 mg of the capnp surfaces = (qe/0.125); thus: % desorbed = [(ce/1)/(qe/0.125)] × 100% desorption processes were carried out at different temperatures (288, 298, 308, and 318°k) by adding 1 ml of the buffer to the hcg–capnp precipitate. desorption (%) was then calculated. 3. results and discussion 3.1 preparation and identification of capnp capnp was prepared in an aqueous environment. most nps are prepared in an aqueous solution to complete the nucleation and crystal growth processes (finney bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc nova biotechnologica et chimica 14-2 (2015) 145 and finke, 2008; bonevich, 2010). the particle morphology and the microstructure of the synthesized capnp were examined via tem by using the highly diluted samples prepared by dispersing and sonicating small amounts of powder in 100% ethanol. the images of capnp were obtained using an accelerating voltage of 200-kev field-emission analytical tem equipped with an oxford energy-dispersive x-ray spectrometer (eds) and a charge-coupled camera (gatan 794ccd). the dimensions and the shape of nps were estimated using a program incorporated in the tem device. the shape of the particle was sliced with dimensions equal to 65 nm × 24 nm), as shown in fig. 1. fig. 1. tem images of the prepared capnp. fig. 2. eds of capnp. eds can be used to determine the chemical composition of the materials and to plot the elements of composition maps to provide the fundamental compositional bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc 146 al-hakeim, h. et al. information for various materials (moffet et al., 2010). a typical eds spectrum is represented as a plot of energy (in kev) versus x-ray counts. the interaction of an electron beam with nps produces various emissions, including x-rays. energy peaks correspond to the various elements in the sample. the eds results of capnp are shown in fig. 2. the data from eds indicates that the percentage of ca is equal to 72.59% ± 2.02%, whereas that of p is 27.41% ± 1.53%. this result confirms the conformation of capnp. 3.2 hcg interaction with capnp the incubation of hcg with capnp decreases the hcg concentration in the solution, which indicates that some hormones are bound to the nanoparticle surface with different surface phenomena, namely, adsorption processes. adsorption refers to the accumulation of a dissolved solute onto the surface of a solid-adsorbing material. adsorption is achieved by establishing a contact between the solution and the adsorbing material (adsorbent). the adsorbed substance is called adsorbate, whereas the surface that adsorbs this substance is called adsorbent. adsorption is classified into chemical (chemisorption) and physical (van der waals adsorption) (umoren et al., 2013). the energy distribution of the adsorption of protein on the nps shows a heterogeneous behavior. this behavior results from the surface heterogeneity and the lateral effect between the adsorbed molecules. the sips isotherm model is a combination of the freundlich and langmuir isotherm-type models and describes the heterogeneous surface more accurately (wang et al., 2012). qe = qm × ks × c t e/(1 + ks × c t e) the sips isotherm approaches the freundlich isotherm at low adsorbate concentrations, whereas approaches the langmuir isotherm at high concentrations. ks is a sips constant related to the energy of adsorption, and parameter (t) is the parameter characterizing the system heterogeneity (ahmed and dhedan, 2012; monemtabary et al., 2013). the adsorption isotherms of the different hcg concentrations from aqueous solutions on a constant weight of capnp at 25 °c and ph 7.4 are plotted in fig. 3. this figure showed the successful prediction of the hcg hormone adsorption isotherm data using the sips isotherm. the fitting of the sips equation for the adsorption isotherm of the hcg hormone adsorption on capnp is consistent with the general finding in the ability of sips equation to explain the adsorption of proteins on different nps (liu et al., 2010). other studies confirmed the ability of the sips isotherm to predict the adsorption of large molecules and proteins on the surface of nps (ahmed and dhedan 2012; kumar et al., 2011). the adsorption of protein on nps is the first step of a complex series of biophysical and biochemical processes that determine the biocompatibility of the material. adsorption is affected by many factors, including shape, energy, and functional group of the nps surface and protein bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc nova biotechnologica et chimica 14-2 (2015) 147 properties (charge distribution, net charge and hydrogen bonding) (wang et al., 2012). the present findings demonstrated the heterogeneous adsorption behavior of hcg on capnp. heterogeneity is a general feature of the surface properties caused by different unsaturation of a sorption on the sites leading to their different energetic characteristics. surface imperfection and the presence of impurities are thus important; different types of adsorption forces on different active sites resulting in the creation of clusters or assemblies of adsorption molecules on the surface (wang et al., 2012). fig. 3. sips adsorption isotherm of hcg hormone on capnp at 25 °c. the sips model for heterogeneous adsorption produces three important constants, which describe the maximum adsorption amount (qm), ks, and t. these constants can be estimated as part of the fitting exercise but can also be used to determine the parameters affected by the general shape of the molecule (toth, 2002). the three constants extracted from the adsorption isotherm of hcg on capnp are qm = 68.2 µg/mg, t = 2.4, and ks = 0.2. the sips equation is also used to model the cooperative adsorption among the adsorbed macromolecules. the t value for capnp is higher than 1 that represents a favorable adsorption condition; thus, the adsorbed hcg affects the adsorption of other hcg molecules. the magnitude of t has been linked to the factors affecting heterogeneous adsorption. at 0 < t < 1, heterogeneity is linked to the variations in the solid surface. when the adsorbed molecule has a strong affinity to other adsorbent molecules (a positive cooperative effect), t is higher than 1 (toth, 2002). this mechanism is consistent with the low hcg adsorption at low solution concentrations followed by a rapid increase. this lateral effect (positive cooperative effect) is also consistent with the energy site distribution analysis in many studies (toth, 2002; ahmed and dhedan 2012). 3.3 morphology of the immobilized hcg on capnp the morphologies of the hcg adsorbed on the surface of the capnp are presented in fig. 4. the figure shows several interesting conclusions, particularly in the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc 148 al-hakeim, h. et al. formation of the hcg layers on the surface of capnp. furthermore, capnp aggregate with each other after hcg adsorption, which indicates the cement-like action of the adsorbed layer of hcg and the cooperative interaction among the adsorbed hcg molecules. this finding indicates the high possibility of forming more the one adsorbed layer of hcg on the surface of capnp. fig. 4. tem morphology of the hcg–np conjugates in addition to the aggregation of the np-hcg flakes caused by the hcg outer layer. the interaction of the adsorbed hcg molecules with each other indicates that more adsorbed solutes results in easier accumulation of the additional amounts. this finding suggests a lateral association among the adsorbed molecules that holds them on the surface. this process is called "cooperative adsorption" (shemetov et al., 2012). the adsorption of proteins onto capnp is interesting because the interaction between proteins and capnp is fundamental to understand biomineralization. a higher concentration of protein on the surface indicates that more proteins are adsorbed on the nps surface and make the protein molecules easy to interact laterally (fei and perrett, 2009). the effective forces among the adsorbed protein molecules are stronger and more long-ranged than that in the solution; thus, the protein molecules aggregate on the surface but in the bulk at similar conditions (neogi and wang, 2011). these explanations may be applied for the adsorption process of hcg on the surfaces of capnp. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc nova biotechnologica et chimica 14-2 (2015) 149 3.4 thermodynamics of the interaction between hcg and np the thermodynamic data (changes in enthalpy (δh), entropy (δs), and free energy (δg)) obtained from all protein nps interactions can be used to detect the nature of the interaction due the fact that thermodynamics regulates most aspects of biomolecular interactions. the heat of adsorption (δh°) provides a direct measure of the strength of the binding forces between the adsorbate molecules and the surface of the adsorbent. to analyze the thermodynamics of the adsorption of hcg on capnp, adsorption processes were performed at different temperatures (288, 298, 308, and 318°k) by using 18 μg/ml hcg hormone and 0.125 mg/ml capnp. the results of adsorption at different temperatures are demonstrated in table 1. table 1. amount of the hcg hormone adsorbed on capnp at different temperatures. t (°k) c0 (µg/ml) ce (µg/ml) qe (µg/g) 288 8 12.05 47.63 298 8 11.82 49.37 308 8 11.20 54.36 318 8 10.91 56.81 the results showed that the amount of adsorption slightly increases with the increase in temperature. the plotting of vant–hoff’s equation for the adsorption process is presented in fig. 5. fig. 5. vant–hoff's lines of the adsorption of hcg on capnp at different temperatures (288, 298, 308, and 318 °k). the positive value of ∆h° (7.45 kj/mol) indicates that the adsorption process is endothermic. the adsorption of protein to a surface may induce conformational changes in the protein. the degree of conformational changes is determined with the combination of the native stability of a protein, the hydrophobicity and charges of the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc 150 al-hakeim, h. et al. protein, and the sorbent surface. protein adsorption can be driven by conformational entropy changes, particularly if the adsorption is endothermic (czeslik et al., 2002). other research found that the adsorption capacity of protein increases with increasing temperature, which indicates an endothermic process. the values of the changes in free energy and entropy are presented in table 2. table 2. thermodynamic parameters of hcg adsorption on capnp at four different temperatures. thermodynamic quantities reveal that hcg-nps complexes involve electrostatic interaction and other non-covalent forces, including hydrophobic, hydrogen bonding, and π–π interaction obtained from the surface functional groups of the nps (de, 2009). adsorption energy is consistent with the adsorption energy derived as the sum of the electrostatic and van der waals forces as seen previously (adamczyk et al., 2011). the stability of the adsorption system (hcg–np) depends on the equilibrium between the entropy and enthalpy change on adsorption. 3.5 cd spectroscopy the cd spectrum of hcg was analyzed to obtain the secondary structural topographies, such as the percentage of α-helical, β-sheet, or random coil regions. at the cd conditions the most important chromophore in the protein molecules is the peptide bond. the spectra of the hcg molecules in the far-uv regions are dominated by the transitions of the amide groups (π → π* and n/π*) and are affected by the morphology and the environment of the polypeptide skeleton which reflects the different types of secondary structures (zhang et al., 2008). fig. 6. cd spectra of the free hcg and hcg–capnp. temperature (ºk) lnke ∆hº (kj/mol) ∆gº (kj/mol) ∆sº (j/mol.k) 288 -0.71 7.45 1.69 -20.27 298 -0.65 1.61 -19.59 308 -0.50 1.28 -20.03 318 -0.43 1.13 -19.86 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc nova biotechnologica et chimica 14-2 (2015) 151 the cd signal is strong for α-helix but weak for β-sheets. cd is very useful for estimation the changes in the conformation of proteins at different environments. the cd spectra for the free hcg hormone and the hcg bound with capnp are provided in fig. 6. after the cd spectra of the free and nanoparticle–immobilized hcg were analyzed by the k2d2 server, the obtained results are listed in table 3. table 3. percentages of the secondary structures of the free and immobilized hcg obtained from the cd spectra. secondary structure free hcg (%) capnp-hcg (%) change in the structure after adsorption (%) α-helix% 84.27 67.45 -16.82 β-sheet% 1.24 3.24 +2.00 random coiled% 14.49 29.31 +14.82 the secondary structure of the free hcg consist of 84.27 % α-helix and 1.24 % βsheet; after binding to capnp, the secondary structure of the immobilized hcg consists of 67 % α-helix and 3.24 % β-sheet. these results indicate that β-sheet (+2 %) and random coils (+14.82 %) increase, whereas α-helix decreases by −16.82 %, which estimated a high interference between the hcg molecules and the surface of capnp. moreover, these changes indicate the presence of more than one interaction site between the hcg molecule and the surface; the sites interact with α domains, whereas the other site with β-domains. other research showed similar conclusion regarding the decrease in α-helix percentage, which affects the bioactivity of the adsorbed molecules (shirazi et al., 2013). the change in the protein structure after the interaction of the protein molecule with the nps surface depends mainly on the type and properties of the nps surface. a study showed that the secondary structure of glucose oxide enzyme (protein) changes after adsorption on the nanoparticle surface, whereas maintains its native structure when adsorbed on other type of np (zhaoa et al., 2013). another factor that affects the interaction between the hcg and nps is the differences in the particle curvature, which significantly affects the amount of hcg adsorption and subsequent changes in its secondary structure. as the size of the nanostructure decreases, the curvature of np increases. therefore, in the smaller nanostructures, only a small part of the approaching molecule are attached with the absorbent surface and thus results in less interaction with the protein. larger nps allow the formation of larger contact surfaces and thus cause larger changes in the secondary structure of the protein upon binding (lundqvist et al., 2004). the cd spectra in fig. 6 and table 3 is due mainly to the α-helix structure, which shows a large peak at 208 nm, whereas the β-sheet percentage of hcg is very low with unrecognized peak around the common wavelength of β-sheet (216–220 nm). the amide chromophore of the peptide bond dominates the cd spectra of the proteins at lower than 250 nm. the transition n/π* is electrically prohibited but magnetically permitted; this transition is primarily responsible for the negative bands at 222 nm, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc 152 al-hakeim, h. et al. which is the characteristic of the α-helix spectrum, and at 216–218 nm, which is the characteristics of the β-sheet spectrum. the transitions n/π* and π → π* are primarily responsible for the negative band at 208 nm and the positive band around 190 nm, which are the characteristics of the α-helix spectrum, as well as for the positive band at 198 nm, which is the characteristic of the β-sheet spectrum (khan et al., 2012). cd provides very good information about the residue fractions in the secondary structure involved in α-helix, β-sheet, or random coils (chaudhuri et al., 2011). cd also provides important additional information on the effect of temperature on the secondary structure of the proteins (corrêa and ramos, 2009). another useful function of cd is molar ellipticity, which is plotted in fig. 7. molar ellipticity is useful for comparing the results of the present study with those of different studies; thus, molarity should be considered in this case. fig. 7. molar ellipticity of the free hcg and hcg–capnp. the molar ellipticity of hcg showed the same general conclusion regarding the changes in the secondary structure of hcg when adsorbed on the surface of capnp. the affected secondary structures depend on the changes in their orientation and indicate the structural changes within a protein molecule. structural changes, in which the secondary and/or tertiary structure of the protein changes because of adsorption or desorption, have been observed. these changes could be a substantial driving force for adsorption. the three dimensional structure of hcg is well determined by lapthorn et al., (1994) and wu et al., (1994). the disulphide bonds and all covalent bonds, determined by these studies, are not changed after adsorption on the surface of nps. these results extracted from the thermodynamic values that showed relatively low free energy change and from desorption ratios. 3.6 fluorescence spectroscopy as shown in fig. 8, the intrinsic fluorescence spectrum was different between the free hcg and hcg–capnp. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc nova biotechnologica et chimica 14-2 (2015) 153 fig. 8. fluorescence spectra of the free hcg and hcg–capnp. valuable information regarding the local environment and the tertiary conformational variations can be achieved via fluorescence spectroscopy. the fluorescence emission spectra of the protein is produced from the contribution of the aromatic amino acids, including tryptophan, tyrosine, and phenylalanine, and the intrinsic fluorescence of the proteins are highly sensitive to their surrounding environment. this difference can be explained through the significant changes in the microenvironment of aromatic residues after adsorption on the surface of capnp through different forces. the hcg used in the present experiment is a tryptophan-free protein, hence the decrease in the fluorescence signal in the fluorescent spectrum is due mainly to tyrosine and phenyl alanine. in a protein containing the three fluorescent amino acids, detecting tyrosine and phenylalanine fluorescence is complicated because of the interference by tryptophan with resonance energy transfer and the weak signal of phenylalanine (corrêa and ramos, 2009). therefore, the application of tyrosine and phenylalanine fluorescence is limited to tryptophan-free proteins, such as the hcg in the present work. 3.7 desorption of protein on the surface desorption refers to the dissociation of the surface-molecule bonds, and the molecule leaves the surface to the bulk. the quantity desorbed with a suitable solvent may refer to the type of interaction between the adsorbent and adsorbate. the values of desorption (%) of hcg from capnp at four different temperatures are provided in fig. 9. fig. 9 shows that the quantity desorbed depends on temperature; thus, the quantity increases as the temperature increases. generally, when the quantity desorbed is high, the adsorption forces are weak and thus the linkage between the adsorbent and adsorbate can be easily broken. the adsorption of protein on the hydrophobic surfaces bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc 154 al-hakeim, h. et al. is mostly irreversible (miller et al., 2005). hence, when a layer of the adsorbed protein is in contact with a solvent or buffer, no (or only small) desorption can be observed over experimental time scales. this finding is a direct consequence of the large number of interactions that a protein can arrange with the functional groups of a given surface. all bonds between the adsorbed protein and the surface should be damaged to allow desorption. the activation energy for such process may be very high, rendering a constant but low desorption rate that depends strongly on the protein, surface, and buffer used (camarero et al., 2004). desorption is high when the adsorbed quantity is high because of the heterogeneous part of the adsorbent surface (the less favorable protein adsorption sites are desorbed first) (miller et al., 2005). fig. 9. desorption percentages of hcg as a function of temperature. the desorption of hcg from capnp depends on temperature, that is, the quantity of the hcg desorbed increases as temperature increases. this phenomenon may be due to the weak forces between hcg molecules and the nanoparticle surface; such forces can be broken by increasing the temperature. another phenomenon is due to the increase in the kinetic energy of the adsorbed molecule until the energy is higher than the forces of interaction with the surface of the nps. other authors showed a similar desorption behavior (mansur et al., 2001; van der veen et al., 2007). 4. conclusion the interaction between hcg and capnp is mainly caused by the adsorption phenomenon that obeyed the sips isotherm. the adsorption of hcg changes the secondary and tertiary protein structures and subsequently changes the biological activity of the protein. hence, the interaction between other nps used in vivo with different human proteins should be evaluated prior to use. references adamczyk, z., barbasz, j.: cieśla m. mechanisms of fibrinogen adsorption at solid substrates. langmuir, 27, 2011, 6868 6878. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:56 utc nova biotechnologica et chimica 14-2 (2015) 155 ahmed, m.j., dhedan, s.k.: equilibrium 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heruntergeladen 17.01.20 09:56 utc nova biotechnologica et chimica 15-1 (2016) 1 doi 10.1515/nbec-2016-0001 © university of ss. cyril and methodius in trnava in vitro regeneration potential of seven commercial soybean cultivars (glycine max l.) for use in biotechnology jana sojková1, iwona žur2, zuzana gregorová1,3, mária zimová1,3, ildikó matušíková4, daniel mihálik5,6, ján kraic5,6, jana moravčíková3* 1department of botany and genetics, faculty of natural sciences, the constantine philosopher university, nábrežie mládeže 91, sk-949 74 nitra, slovak republic 2polish academy of sciences, franciszek gorski institute of plant physiology, niezapominajek 21, pl-30-239 kraków, polland 3institute of plant genetics and biotechnology slovak academy of sciences, akademická 2, p.o. box 39a, sk-950 07 nitra, slovak republic (jana.moravcikova@savba.sk) 4department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, sk-917 01 trnava, slovak republic 5department of biotechnology, faculty of natural sciences, university of ss, cyril and methodius in trnava, nám. j. herdu 2, sk-917 01 trnava, slovakia 6research institute of plant production, national agricultural and food center, sk-921 68 piešťany, slovakia abstract: this work is aimed to evaluate in vitro regeneration potential of seven commercial soybean varieties bohemians, cardiff, gallec, merlin, moravians, naya and silensia (glycine max l.) cultivated in central europe. our results showed the half-seeds could be effectively used as an explant source for all tested cultivars. the regeneration was initiated on the media containing growth regulators 1.67 mg.l-1 bap and 0.25 mg.l-1 ga3. within the first five days culture, green chlorophyll-containing explants were observed with frequency from 18.3% to 55.9%. two weeks later, the explants responded by production of calli with the efficiency up to 83.0%. first shoots appeared after 2-3 weeks of subculture on the media. the soybean regeneration showed to be genotype-dependent with variable efficiencies from 5.7% (cv. naya) to 37.7% (cv. gallec). the cultivars cardiff, merlin and gallec appear to be the most promising candidates for further biotechnological use. application of antioxidants such as l-cysteine, dithiothreitol and sodium thiosulfate does not have effect on the explant regeneration for the first five days. key words: antioxidants, half-seeds, chlorophyll fluorescence, in vitro, soybean 1. introduction soybean (glycine max (l.) merrill) is one of the major legume crops in the world. the seeds are important source of protein and edible vegetable oil for livestock, human consumption and industrial sector. soybean is one of the few plants that contain all eight amino acids essential for human health. the consumption of soybean reduces cancer, cholesterol, osteoporosis and heart diseases (birt et al., 2004). because soybean has been growing for many centuries around the world under bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:55 utc 2 sojková, j. et al. different climatic conditions, there is a wide range of soybean varieties available. the major producers are the united states, brazil and argentina. the first genetically modified (gm) roundup ready soybeans were planted in the usa in 1996. in 2011-2012 soybeans were grown on about 30 million hectares in the usa, with roundup ready gm soy contributing 93–94% of the production (bøhn et al., 2014). the successful application of genetic engineering in plant improvement is dependent on availability of the efficient in vitro regeneration protocol. the regeneration of soybean has been reported using immature embryos (barwale et al., 1986), immature cotyledons (ishimoto et al., 2010), hypocotyls (wang and xu, 2008), mature cotyledonary nodes (liu et al., 2008) or half-seeds (paz et al., 2006). however, the regeneration efficiency was variable depending on the genotype and the explant type used (reviewed by verma et al., 2014). the half-seeds represent alternative cotyledonary explants derived from mature soybean seeds. the half-seed system has been developed as a demand for elimination of deliberate tissue wounding prior agrobacterium infection (paz et al., 2006). using the half-seeds as an explant source, the regeneration efficiency and the number of obtained transgenic plants were 1.5-fold higher (paz et al. 2006) compared to the cotyledonary node method (paz et al. 2004). many plant tissue cultures respond to the culture manipulation by oxidative browning and necrosis that typically result in poor regeneration (reviewed by dan et al. 2008). beside, tissue browning is associated with plant defence response to infection with agrobacterium (kuta and tripathi, 2005). several studies reported that adding antioxidants into the culture media can considerably improve plant regeneration (reviewed by verma et al., 2014). adding of l-cysteine and other thiol compounds sufficiently inhibited woundand agrobacterium-induced responses of soybean cotyledonary node cells and increased the number of regenerated transgenic plants (olhoft and somers, 2001; olhoft et al., 2001). therefore, thiol compounds are common part of the regeneration media in soybean cotyledonary node and half-seed transformation and regeneration systems (paz et al., 2004; paz et al., 2006; wang and xu, 2008; kim et al., 2012). in this work, seven commercial soybean varieties bohemians, cardiff, gallec, merlin, moravians, naya and silensia were studied for their ability to regenerate in vitro. the half-seeds were used as the explant source. the regeneration approach designed here led to obtaining of plants from each of tested cultivars, however, with variable efficiencies. this is (up to our knowledge) the first report on in vitro regeneration of these commercial soybean cultivars. 2. material and methods 2.1 plant material and explant preparation the soybean cultivars (glycine max (l). merrill) bohemians, cardiff, gallec, merlin, moravians, naya and silensia were obtained from the company matex s.r.o (veškovce, slovak republic). mature seeds were surface-sterilized for 20 hours bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:55 utc nova biotechnologica et chimica 15-1 (2016) 3 using chlorine gas produced by mixing 3.5 ml 12 mol.l-1 with 100 ml commercial bleach (savo) described by di et al. (1996). one day prior experiments, sterilised seeds were soaked in sterile water for about 20 hours in petri dishes covered with aluminium foil. the cotyledons were separated by longitudinal cut along hilum. the half-seed explants were obtained by the excision of the embryogenic axis. 2.2 plant regeneration the half-seed explants were cultivated on callus induction medium (cima, table 1) lined with sterile filter paper. the explants were placed adaxial side (flat side) down on the medium. following 5 days, the explants were transferred on the shoot-induction medium (sim, table 1) so the nodal end of the half-seed explant was imbedded into the media. after 14 days, the explants were transferred to the fresh sim medium. after 5 weeks of culture, the explants were transferred on the shoot elongation medium (sem, table 1). the explants were regenerated at 24°c and 16 h/ 8 h light/dark photoperiod under 50 μe m-2 s-1 light intensity. 2.3 chlorophyll fluorescence measurement chlorophyll fluorescence was measured using portable fluorometer handy fluorcam fc 1000-h (psi s.r.o. czech republic). measurement was performed on petri dishes containing half-seed explants (10 explants per petri dish). petri dishes containing explants were placed in the dark for 30 min prior to the measurement. the chlorophyll fluorescence was measured directly from the top of dishes with removed lids according to the protocol “short” kautsky effect in continuous light. we measured minimum chlorophyll fluorescence (f0), variable fluorescence (fv = fm f0) and quantum efficiency of open psii centers in a dark-adapted state (qymax = fv/fm). 2.4 histochemical staining the cell viability was determined after staining with a 0.25 % (w/v) evans blue for 30 min at room temperature and subsequent washing three times with distilled water, for 10 min each according to tamas et al. (2008). lipid peroxidation was detected using schiff´s reagent for 60 min as described previously pompella et al. (1987) 2.5 explant sampling and statistical analyses the sets of analysed explants for in vitro regeneration potential were subjected to analyses at (1) 5 th day and (2) 5th weeks culture. for chlorophyll fluorescence measurement, the explants were analysed at 5th dayculture on the callus-induction media with or without antioxidants (cima or cimb, respectively). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:55 utc 4 sojková, j. et al. the cell viability and lipid peroxidation was detected on explants immediately after separation of cotyledons and excision of embryogenic axis. data are presented as the means of three replications. statistical significance of the experimental results was evaluated by duncan’s test with help of statistica_version 7.1. table 1. composition of media used in regeneration experiments. cima 0.32 g.l-1 gambor b5 including vitamins (duchefa), 2.78 mg.l-1 feso4.7h2o, 3.72 mg.l -1 naedta, , 4.26 mg.l-1 mes, 30 g.l-1 sacharose, 1.67 mg.l-1 bap, 0.25 mg.l-1 ga3, 5 g.l -1 agar ph 5.4, 400 mg.l-1 l-cysteine, 0.154 g.l-1 dtt, 0.158 mg.l-1 sts and 5 g.l-1 agno3. cimb 0.32 g.l-1gambor b5 including vitamins (duchefa), 2.78 mg.l-1 feso4.7h2o, 3.72 mg.l -1 naedta, 4.26 mg.l-1 mes, 30 g.l-1 sacharose, 1.67 mg.l-1 bap, 0.25 mg.l-1 ga3, 5 g.l -1 agar ph 5.4 sim 3.2 g.l-1 gambor b5 including vitamins (duchefa), 278 mg.l-1 feso4.7h2o, 372 mg.l-1 naedta, , 0.64 mg.l-1 mes, 30 g.l-1 sacharose, 1.67 mg.l-1 bap, 5 g.l-1 agno3, 7 g.l -1 agar ph 5.7 sem 3.2 g.l-1 gambor b5 including vitamins (duchefa), 278 mg.l-1 feso4.7h2o, 372 mg.l-1 naedta, 0.64 mg.l-1 mes, 20 g.l-1 sacharose, 0.5 mg.l-1 ga3, 0.1 mg.l-1 iaa, 1 mg.l-1 zeatín, 50 mg.l-1 asparagine, 7 g.l-1 agar ph 5.7 cima callus induction medium, sim shoot induction medium, sem shoot elongation medium, mes 2-(n-morpholino) ethanesulfonic acid, bapbenzylaminopurine, ga3 – gibberellic acid, dtt – 1,4 dithiothreitol, sts – sodium thiosulfate, iaa – indolyl acetic acid 3. results and discussion soybean is one of the most important crops worldwide. presently, biotechnology offers soybean traits that provide healthier ingredients for our diets and the diets of farm animals, as well as increased disease and insect resistance. however, soybean is considered particularly difficult to transform due to different factors, including regeneration efficiency in vitro. here we studied in vitro regeneration potential of selected soybean cultivars, yet not tested elsewhere, using the half-seeds as an explant source (fig 1a). this type of the explant has been previously shown to be a convenient material not only for soybean regeneration itself (janani et al., 2013) but also for genetic modification via a. tumefaciens (paz et al., 2006; kim et al. 2012). the regeneration efficiency was genotype dependent and ranged from 1.4% to 8.7%. the half-seed explants were prepared by splitting of imbibed seeds and excision of embryogenic axis. such manipulation is very often accompanied with browning and necrosis of the affected tissue that can lead to poor in vitro regeneration. generally, mechanical wounding is associated with production of reactive oxygen species (ros), common components of plant defence response against various stresses (orozco-cárdenas et al., 2001; demidchik et al., 2015). however, ros at low level regulate numerous plant biological processes (del río et al., 2015). for example, soybean seed germination is regulated through ethylene production in response to ros (ishibashi et al., 2013). contrary, high level of ros promotes oxidative stress through oxidation of the cell compounds (demidchik et al., 2015) and often leads to loss of cell function and apoptosis (marnett 2000). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:55 utc nova biotechnologica et chimica 15-1 (2016) 5 fig. 1. representative photos of in vitro regeneration process of the cv. merlin. a) the half-seeds were prepared by splitting of imbibed seeds and excision of embryogenic axis and placed on the cima medium; b) the half seeds after five days culture on the cima medium; c) first shoots after two weeks subculture on the sim medium; d) shoots after five weeks subculture on the sim medium. bars 500 µm. fig. 2. detection of cell viability (a,b,c, d) and membrane lipid peroxidation (e, f, g, h) in the half-seeds after splitting and excision of the embryogenic axis. the cell death was determined using staining with evans blue. the membrane lipid peroxidation was stained using schiff´s reagent. a, c) histochemical staining with evans blue in the explants of the cvs. naya and gallec (respectively); b, d) cross sections of the explants of the cv. naya indicating cell death at the side of the embryogenic axis removing; e, g) histochemical staining with schiff´s reagent in the explants of the cvs. naya and gallec (respectively); f, h) cross sections of the explants of the cv. naya and gallec (respectively) indicating the extent of the lipid peroxidation. bars 500 µm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:55 utc 6 sojková, j. et al. in our experiments, histochemical staining proved peroxidation of membrane lipids all over the surface of the half-seeds immediately after explant preparation (fig. 2a, 2b). besides, the stain was detected in the cross section of the half-seed explants (fig. 2c, fig 2d). the sides of wounding areas of death cells were detected histochemically as a result of the separation of cotyledons (fig. 2e, 2f) and at the site of excision of embryogenic axis (fig 2g, 2h). similar histochemical patterns were observed for all cultivars. several studies reported the inclusion of antioxidants in the culture media can prevent necrosis and consequently improve plant in vitro regeneration (reviewed by dan 2008). moreover, antioxidants can reduce cell damage following agrobacterium-mediated transformation by inhibiting of woundand plant pathogen induced responses (olhoft et al., 2001, olhoft and somers, 2001). in cotyledonary node transformation of soybean, adding a mixture of thiol compounds such as l-cysteine, dithiothreitol and sodium thiosulfate into the co-cultivation medium improved regeneration potential of transformed cells (olhoft et al., 2003; paz et al., 2004; wang and xu, 2008; kim et al., 2012). however, a high level of thiol compounds might also have a negative effect on in vitro regeneration. for example, the application of l-cysteine at concentration higher than 600 mg.l-1 resulted in reduced number of transformed soybean shoots (liu et al., 2008). thus, above mentioned thiol compounds were applied at concentrations (table 1) recommended by others (olhoft et al., 2003; paz et al., 2004; kim et al., 2012). the half-seeds were cultivated on the cim medium supplemented with (cima) or without (cimb) antioxidants. after 5 days culture on the media (fig 1b), the chlorophyll fluorescence of the explants (qymax) as an indicator of regeneration was measured (fig. 3). the results showed the chlorophyll content significantly varied with genotype (at p≤0.001) (table 2). we did not observe any negative effects of these compounds on the halfseed regeneration. however, the effect of thiols as protective agents against ros produced by wounding was not statistically significant (table 2). nevertheless, considering that promising soybean cultivars are expected to be subjected to genetic modification via agrobacterium, these thiols (table 1) were also applied in our further regeneration experiments. it is well known the regeneration is essential requirement for transformation. moreover, regeneration potential of transformed cells can be dramatically decreased also for plant genotypes that are routinely regenerated. for example, regeneration efficiency of 50% was reduced to 1.3% after agrobacterium-mediated transformation of cotyledonary petiole explants of oilseed rape cultivar campino (boszoradova et al., 2011). thus, identifying genotypes with high regeneration potential represents one of the prerequisites for successful transformation experiments. table 2. variance analyses. source a f empirical b photosynthetic activity (1) 16.75 *** (2) 1.81 ns (1) × (2) 1.06 ns regeneration efficiency (1) 52.27 *** a effect of (1) genotype, (2) treatment with or without antioxidants statistical significance at ***p≤0.001; ** p≤0.01; * p≤0.05; ns – not significant bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:55 utc nova biotechnologica et chimica 15-1 (2016) 7 fig. 3. effect of antioxidants on the maximum quantum yield of psii (qymax = fv/fm). the chlorophyll fluorescence was measured in the half-seed explants after 5th days culture on the media with (cima) or without (cimb) antioxidants. bars represents means ± standard deviations of three replications. distinct letters denote statistically significant differences with duncan’s tests. the half seed explants (fig 1a) were cultured on the callus inducing (cima) media supplemented with the plant hormones 1.67 mg.l-1 bap and 0.25 mg.l-1 ga3. such combination of phytohormones has been successfully applied for regeneration of transformed cotyledonary node (olhoft et al., 2003; paz et al., 2004) and halfseed (paz et al., 2006) explants of different soybean varieties. during the first five days, the most of explants positively responded by producing of green chlorophyll. (fig. 1b). the number of green explants varied from 18.3% (cv. naya) to 55.9% (cv. gallec) (table 3). besides, direct regeneration of shoots from cotyledonary explants and/or from shoot apical meristem was observed (fig. 1b). following five days, the explants were sub cultured on the shoot-inducing (sim) medium (fig. 1c). the calli and the shoots were appeared after 2-3 weeks culture on the sim medium. the first larger shoots were excised and discarded. treatment with bap breaks down apical dominance and triggers adventitious shoot formation (wright et al., 1986). as was noticed by others (sairam et al., 2003), only the callus induced from the nodal region gave rise to shoots. healthy shoots (fig. 1d) were excised, transferred to the shoot-elongation (sem) medium. the efficiency of regeneration of cultivars was evaluated five weeks after subculture on the sim medium. it was calculated as percentage of the number of explants producing at least one shoot to the total number of explant used. data are given in fig. 4. the best regeneration responses were observed for the cultivars merlin (36.6%) and gallec (37.7%) while the lowest was found for the cultivars naya (5.7%) and silensia (8.0%). the regeneration was clearly genotype dependent (p≤0,001) (table 2). based on the data obtained (table 3), the response of the explants to the culture media for the first five days seems to define the regeneration potential. generally, low regeneration potential is associated with sensitivity and strong plant stress responses to the in vitro manipulation (benson, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:55 utc 8 sojková, j. et al. 2000). however, histochemical staining of some parameters of stress (fig. 2) did not reveal obvious differences between individual genotypes, probably because points on the surface layer of epidermal cells that are usually most damaged. we suppose that lower regeneration efficiencies achieved for some cultivars as naya and silensia might coincide rather with the composition of the regeneration media. optimizing of the exogenous hormone levels in media might lead to increased regeneration efficiencies of these genotypes, albeit this would require much loads of time and other inputs thus are not usable for routine screenings. nevertheless, the half-seed regeneration system appears to be suitable for regeneration of soybean plants from all tested genotypes. table 3. results from in vitro regeneration of soybean cultivars number of responded explants no. expl.a green b [%] c calli d [%]e calli and shoots f [%]g bohemians 103 30 29.1 69 67.0 24 34.8 cardiff 111 46 41.4 91 82.0 36 39.6 gallec 104 55 55.9 86 82.7 39 37.2 merlin 106 50 47.2 88 83.0 39 44.4 moravians 113 42 37.2 68 60.2 34 50.0 naya 109 20 18.3 52 47.7 6 11.5 silensia 136 28 20.6 68 50.0 11 16.2 a total number of explants used in experiments b the number of green chlorophyll containing explants after 5th day culture on the cima medium c the number of green chlorophyll containing explants as a percentage of the total number of explants used d the number of explants producing calli e the number of callus-producing explants as a percentage of the total number of explants used. f the number of callusand shoot -producing explants after 5th weeks culture on the media g the number of callusand shoot -producing explants after 5th weeks culture on the media as a percentage of the number of callus-producing explants. fig. 4. regeneration efficiencies of soybean cultivars. regeneration efficiency was calculated as percentage of the number of explants producing at least one shoot to the total number of explant used. bars represents means ± standard deviations of three replications. distinct letters denote statistically significant differences with duncan’s tests. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:55 utc nova biotechnologica et chimica 15-1 (2016) 9 4. conclusions in vitro regeneration potential of commercially important soybean cultivars bohemians, cardiff, gallec, merlin, moravians, naya and silensia was studied. halfseeds as an explant source were used. within five weeks subculture on the regeneration media, shoots were generated from the explants of all cultivars. adding the given antioxidants into the callus-induction medium had no significant effect on the cell regeneration. regeneration efficiency was genotype dependent and varied between 5.7% and 37.7%. we concluded that the genotypes naya and silensia are likely to appear as recalcitrant for regeneration after transformation with agrobacteria. on the other hand, the cultivars with the high regeneration potential such as cardiff (32.6%), merlin (36.6%) and gallec (37.7%) appear to be more promising for further biotechnological applications via genetic transformations. acknowledgment: this work was supported by the bilateral project sav-pav (2016-2018), by the slovak grant agency vega 1/0061/15 and by the university grant agency ukf ugaviii/35/16. references barwale, u. b., kerns, h. r., widholm, j. m.: plant-regeneration from callus-cultures of several soybean genotypes via embryogenesis and organogenesis. planta, 167, 1986, 473-481. benson, e. e.: in vitro plant recalcitrance: an introduction. in vitro cell. dev-pl., 36, 2000, 141-148. birt, d.f., hendrich, s., anthony, m., alekel, d.l.: soybeans and the prevention of chronic human disease. soybeans: improvement, production and uses. j. specht, r. boerma, eds. 3rd ed., american society of agronomy, madison, wi, 2004, 1047–1117 bohn, t., cuhra, m., traavik, t., sanden, m., fagan, j., primicerio, r.: compositional differences in soybeans on the market: glyphosate accumulates in roundup ready gm soybeans. food chem., 153, 2014, 207-215. boszoradova, e., libantova, j., matusikova, i., poloniova, z., jopcik, m., berenyi, m., moravcikova, j.: agrobacterium-mediated genetic transformation of economically important oilseed rape cultivars. plant cell tiss. org., 107, 2011, 317-323. dan, y.: biological functions of antioxidants in plant transformation. in vitro cell. dev-pl., 44, 2008, 149-161. del rio, l. a.: ros and rns in plant physiology: an overview. j. exp. bot., 66, 2015, 2827-2837. demidchik, v.: mechanisms of oxidative stress in plants: from classical chemistry to cell biology. environ. exp. bot., 109, 2015, 212-228. di, r., purcell, v., collins, g. b., ghabrial, s. a.: production of transgenic soybean lines expressing the bean pod mottle virus coat protein precursor gene. plant cell rep., 15, 1996, 746-750. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:55 utc 10 sojková, j. et al. ishibashi, y., koda, y., zheng, s.-h., yuasa, t., iwaya-inoue, m.: regulation of soybean seed germination through ethylene production in response to reactive oxygen species. ann. bot., 111, 2013, 95-102. ishimoto, m., rahman, s. m., hanafy, m. s., khalafalla, m. m., elshemy, h. a., nakamoto, y., kita, y., takanashi, k., matsuda, f., murano, y., funabashi, t., miyagawa, h.,wakasa, k.: evaluation of amino acid content and nutritional quality of transgenic soybean seeds with highlevel tryptophan accumulation. mol. breeding, 25, 2010, 313-326. janani, c., kumari, b.d.: in vitro plant regeneration from cotyledonary node and half seed explants of glycine max l. (js335). ann. biol. res., 4, 2013, 60-66. kim, m.j., kim, j. k., kim, h. j., pak, j. h., lee, j.h., kim, d.h., choi, h. k., jung, h. w., lee, j.d., chung, y.s.,ha, s.h.: genetic modification of the soybean to enhance the beta-carotene content through seed-specific expression. plos one, 7, 2012. kuta, d. d., tripathi, l.: agrobacterium-induced hypersensitive necrotic reaction in plant cells: a resistance response against agrobacterium-mediated dna transfer. afr. j. biotechnol., 4, 2005, 752-757. liu, s.j., wei, z.m., huang, j.q.: the effect of co-cultivation and selection parameters on agrobacterium-mediated transformation of chinese soybean varieties. plant cell rep., 27, 2008, 489-498. marnett, l. j.: oxyradicals and dna damage. carcinogenesis, 21, 2000, 361-370. olhoft, p. m., flagel, l. e., donovan, c. m., somers, d. a.: efficient soybean transformation using hygromycin b selection in the cotyledonary-node method. planta, 216, 2003, 723-735. olhoft, p. m., lin, k., galbraith, j., nielsen, n. c., somers, d. a.: the role of thiol compounds in increasing agrobacterium-mediated transformation of soybean cotyledonary-node cells. plant cell rep., 20, 2001, 731-737. olhoft, p. m., somers, d. a.: l-cysteine increases agrobacterium-mediated tdna delivery into soybean cotyledonary-node cells. plant cell rep., 20, 2001, 706-711. orozco-cardenas, m. l., narvaez-vasquez, j.,ryan, c. a.: hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. plant cell, 13, 2001, 179-191. paz, m. m., martinez, j. c., kalvig, a. b., fonger, t. m., wang, k.: improved cotyledonary node method using an alternative explant derived from mature seed for efficient agrobacterium-mediated soybean transformation. plant cell rep., 25, 2006, 206-213. paz, m. m., shou, h. x., guo, z. b., zhang, z. y., banerjee, a. k., wang, k.: assessment of conditions affecting agrobacterium-mediated soybean transformation using the cotyledonary node explant. euphytica, 136, 2004, 167179. pompella, a., maellaro, e., casini, a.f., comporti, m.: histochemical detection of lipid peroxidation in the liver of bromobenzenepoisoned mice. am. j. pathol., 129, 1987, 295–301. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:55 utc nova biotechnologica et chimica 15-1 (2016) 11 sairam, r. v., franklin, g., hassel, r., smith, b., meeker, k., kashikar, n., parani, m., al abed, d., ismail, s., berry, k.,goldman, s. l.: a study on the effect of genotypes, plant growth regulators and sugars in promoting plant regeneration via organogenesis from soybean cotyledonary nodal callus. plant cell tiss. org., 75, 2003, 79-85. tamas, l., dudikova, j., durcekova, k., halugkova, l.u., huttova, j., mistrik, i., olle, m.: alterations of the gene expression, lipid peroxidation, proline and thiol content along the barley root exposed to cadmium. j. plant physiol. 165, 2008, 1193–1203 verma, k., saini, r., rani, a.: recent advances in the regeneration and genetic transformation of soybean. j. innov. biol., 1, 2014, 015-026. wang, g., xu, y.: hypocotyl-based agrobacterium-mediated transformation of soybean (glycine max) and application for rna interference. plant cell rep., 27, 2008, 1177-1184. wright, m. s., koehler, s. m., hinchee, m. a., carnes, m. g.: plantregeneration by organogenesis in glycine-max. plant cell rep., 5, 1986, 150-154 received 23 march 2016 accepted 5 may 2016 . bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:55 utc microsoft word nova biotechnologica gubisova rev.doc nova biotechnologica et chimica 11-1 (2012) 57 doi 10.2478/v10296-012-0006-z ©university of ss. cyril and methodius in trnava optimization of barley mature embryo regeneration and comparison with immature embryos of local cultivars marcela gubišová1,2, daniel mihálik1, jozef gubiš1 1plant production research institute piešťany, plant production research center piešťany, bratislavská cesta 122, sk-921 68 piešťany, slovak republic (gubisova@vurv.sk) 2department of botany and genetics, faculty of natural sciences, constantine the philosopher university in nitra, nábrežie mládeže 91, sk-949 74 nitra, slovak republic abstract: regeneration ability of plant cells or tissues in explant culture is one of the key factors affecting success of genetic transformation. in experiments, the effect of explant type (whole embryo, scutellum, embryonic axis, meristematic/central zone of embryonic axis) and plant growth regulators (bap or tdz) on mature embryo regeneration was determined. explant type significantly affected regeneration efficiency. while no regenerants were observed using mature scutella, whole embryos or embryonic axes produced the highest number of regenerants. using embryonic axes with discarded apical and basal parts, regeneration efficiency dramatically decreased. no statistical differences in regeneration were observed between bap and tdz added to the regeneration medium in concentration 0.1 or 1 mg l-1. at last, regeneration ability of mature embryos of nine slovak spring barley cultivars (donaris, ezer, levan, ludan, nitran, pribina, sladar, orbit, pax) and golden promise as a model cultivar was examined and compared with regeneration ability of immature embryos which have been usually used for genetic transformation of barley. although the regeneration from mature embryos was very weak, the same cultivars golden promise, pribina and levan showed the best regeneration ability by using both, immature and mature embryos. on the other hand, cultivars ezer and pax belonged to the weakest ones in both experiments. key words: bap, barley regeneration, explant type, immature embryo, mature embryo, thidiazuron (tdz) 1. introduction regeneration ability of plant species in tissue culture is strongly affected by several factors, mainly by the species, genotype, explant type and its developmental stage and composition of culture medium. likewise, the donor plant quality and environmental conditions have also a significant impact (dahleen, 1999; klčová et al., 2004). immature scutellum has been the most widely used type of explant for genetic transformation of barley (wan and lemaux, 1994; tingay et al., 1997; bartlett et al., 2008) because of its higher regeneration potential in the comparison with other explant types. however, there are efforts to improve regeneration ability of mature embryos (ganeshan et al., 2003; sharma et al., 2004; sharma et al., 2005) for their availability throughout the year, avoiding the need for laborious growing of donor plants in controlled conditions and eliminating inter-seasonal variation. choice of properly treated explant type and modification in media composition could provide the opportunity to achieve this aim. dahleen (1995) described improvement of regeneration efficiency for barley immature embryos bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:10 utc 58 gubišová, m. et al. by increasing copper level; cho et al. (1998) confirmed the positive effect of an intermediate cultivation medium containing bap in combination with 2,4-d on induction of barley regeneration. plant hormones play a significant role in culture medium. until dicamba (3,6-dichloro-o-anisic acid) or 2,4-d (2,4dichlorphenoxyacetic acid) are successfully used for callogenesis induction, bap (6benzylaminopurine) is usually used in regeneration media for barley. several authors (shan et al., 2000; ganeshan et al., 2003; sharma et al., 2004) described positive effect of addition of tdz (thidiazuron) instead of bap on plant regeneration. in this work, the effect of genotype, explant type and media composition on in vitro regeneration of barley from mature embryos was described. 2. materials and methods mature seeds of two spring barley cultivars (hordeum vulgare l.) golden promise (gp) and orbit were used to determine the effect of explant type on in vitro regeneration ability. seeds were surface sterilized by immersion in 70% alcohol for 2 min, then in 4 % solution of sodium hypochlorite for 15 min with gentle shaking, and rinsed three times with sterile redistilled water. then the seeds were left to imbibe for two days in sterile water with the addition of 6 mg l-1 2,4-d at 4°c according to sharma et al. (2005). in the first experiment, four types of explants were aseptically prepared from this seeds: whole embryos (es), sculella (s), embryonic axes (e) and embryonic axes with discarded apical and basal parts (em, fig. 1). for callogenesis and regeneration, modified media of sharma et al. (2004, 2005) were used (tab. 1). after one week of culture, emerged shoots and roots were cut, so as not to inhibit callogenesis. callogenesis took place four weeks on j1 medium in the dark at 25°c, regeneration two weeks on j2 medium and next 3 weeks on j3 medium under the photoperiod 16 h light/8 h dark with a light intensity approximately 50 μmol m-2 s-1 at 25/20°c. in the second experiment, the effect of growth regulators bap and tdz on plant regeneration from embryonic axes of mature seeds of cultivars golden promise, orbit and levan were studied. explants were cultivated as above, but medium j1 didn’t contain bap, medium j2 was omitted and medium j3 was supplemented with 1 mg l -1 2,4-d and 0.1 or 1 mg l -1 bap or tdz. calli were left to regenerate 5 weeks on j3 medium. at last, regeneration efficiency of mature embryonic axes of 9 slovak spring barley cultivars (donaris, ezer, levan, ludan, nitran, pribina, sladar, orbit, pax) and a model cultivar golden promise was examined and compared with regeneration ability of immature embryos of the same cultivars. regeneration process for mature embryonic axes was conducted as was mentioned above in the first experiment. protocol for regeneration of immature embryos (scutella) has been described in gubišová et al. (2011). the frequency of callogenesis and regeneration (%), number of regenerants per regenerating callus and efficiency of regeneration represented by the number of regenerants per plated explant were evaluated in all experiments. experiments were repeated twice with 64 96 explants per each variant. data were processed by one-way bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:10 utc nova biotechnologica et chimica 11-1 (2012) 59 analysis of variance (anova) followed by lsd test (α = 0.05). percentage data were transformed by arcsin √ × before analysis. table 1. composition of media for barley regeneration. media were adjusted to ph 5.8 and autoclaved at 121°c for 20 min. j 1 j 2 j 3 4.3 g l-1 ms (murashige and skoog, 1962) salts (duchefa) 60 g l-1 maltose 1 g l-1 casein hydrolysate 1.25 mg l-1 cuso4.5h2o 200 mg l-1 myo-inozitol 500 mg l-1 proline 1 mg l-1 thiamine hcl 6 mg l-1 2,4-d 0.001 mg l-1 bap 3.0 g l-1 gelrite 4.3 g l-1 ms (murashige a skoog, 1962) salts (duchefa) 30 g l-1 maltose 1.25 mg l-1 cuso4.5h2o 500 mg l-1 proline 500 mg l-1 glutamine 200 mg l-1 myo-inozitol 1 mg l-1 thiamine hcl 6 mg l-1 2,4-d 0.01 mg l-1 bap 2.5 g l-1 gelrite 4.3 g l-1 ms (murashige a skoog, 1962) salts (duchefa) 30 g l-1 maltose 1.25 mg l-1 cuso4.5h2o 500 mg l-1 glutamine 200 mg l-1 myo-inozitol 1 mg l-1 thiamine hcl 1 mg l-1 2,4-d 0.1 mg l-1 bap 2.5 g l-1 gelrite 3. results and discussion comparing responses of different explant types of barley cultivars golden promise and orbit significant differences were observed for frequency of callogenesis, regeneration and the number of regenerants. mature scutella didn’t produce any regenerants and frequency of callogenesis was also lower in comparison with other explant types (fig. 2). sharma et al. (2005) observed no response of scutella also. in their experiments, scutella of cultivar gp didn’t produce any callus, while 73% of scutela were callogenic in our experiments. frequency of regeneration varied from 0 % for mature scutella up to 89.7% for whole embryos of gp. frequency of regeneration of whole embryos was higher for gp in comparison with cv. orbit, although efficiency of regeneration of embryonic axes and embryonic axes with discarded apical and basal parts was similar for both cultivars. sharma et al. (2005) described that scutella inhibited callus proliferation of whole embryos. we didn’t observe such effect and whole embryos of gp (fig. 1) produced even more regenerants than embryonic axes alone. in the next experiments, embryonic axes were only used due to lower germination on induction medium. embryonic axes with discarded apical and basal parts were used with the aim to eliminate germination of embryos in culture, but regeneration frequency of embryonic axes treated in this way dramatically decreased. the effect of bap or tdz in concentration 0.1 or 1 mg l-1 on barley mature embryonic axes regeneration was studied in the second experiment. although ganeshan et al. (2003) and sharma et al. (2004) recommend using tdz for regeneration of barley from meristematic shoot segments of mature embryos and shan et al. (2000) for immature embryos of barley, we didn’t observe significant differences between tdz and bap in the frequency of regeneration and the number of regenerants from mature embryonic axes. there were only differences comparing cv. gp (29.43 % regenerating callus; 0.56 regenerants per plated explant) with other two bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:10 utc 60 gubišová, m. et al. cultivars orbit and levan (15.33 and 16.46 % regenerating explant respectively and 0.22 regenerants for both cultivars). fig. 1. detail of barley embryonic axis with discarded apical and basal parts (left) and regeneration of cv. golden promise from whole mature embryo (right). 0 20 40 60 80 100 es s e em 0 0,2 0,4 0,6 0,8 1 gp call orbit call gp reg orbit reg fig. 2. frequency (%) of callogenic explants (call) and the number of regenerants (reg) per plated explant of four types of explants (whole embryo – es, scutellum – s, embryonic axis – e and embryonic axis with discarded apical and basal parts – em) from mature seeds of spring barley cultivars golden promise and orbit. 0 10 20 30 40 50 60 70 80 90 donaris ezer levan ludan nitran pribina sladar orbit pax gp ie me fig. 3. frequency (%) of regenerating explants of slovak spring barley cultivars and cv. golden promise using mature embryonic axes (me) in comparison with immature scutella (ie) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:10 utc nova biotechnologica et chimica 11-1 (2012) 61 statistically significant differences were observed comparing nine slovak spring barley cultivars and cv. golden promise with respect to their frequency of regeneration and the number of regenerants. the best responses were observed for cv. golden promise and pribina. cultivar pribina actually showed higher regeneration frequency and higher number of regenerants per regenerating callus than gp (fig. 3, 4). these two cultivars and cv. levan also showed the best regeneration ability in experiments with both, mature and immature embryos. on the other hand, ezer and pax were the weakest regenerating cultivars by using mature as well as immature embryos. 0,0 0,5 1,0 1,5 2,0 2,5 3,0 do na ris ez er le va n lu da n ni tra n pr ibi na sl ad ar or bit pa x gp reg./rc -i e reg./rc me reg./pe ie reg./pe me fig. 4. number of regenerants per regenerating callus (reg./rc) and per plated explant (reg./pe) of slovak spring barley cultivars and cv. golden promise using mature embryonic axes (me) in comparison with immature scutella (ie). 4. conclusions finally, we can conclude that while scutella from immature embryos have good regeneration capacity, scutella from mature seeds are not regenerable. as for mature embryos, embryonic axes or whole embryos are suitable for regeneration in explant culture. slovak cultivars of spring barley pribina and levan were chosen to be the most regenerable by using mature as well as immature embryos. because the regeneration from mature embryos is still not effective and would need further investigations, immature scutella of chosen cultivars can be recommended for genetic transformation of barley. acknowledgements: this work was supported by operational programme research and development: development of new types of genetically modified plants with farm traits (itms 26220220027) from european regional development fund. we thank mrs. klára križanová from hordeum ltd. sládkovičovo for providing seeds of slovak barley cultivars. references bartlett, j.g., alves, s.c., smedley, m., snape, j.w., harwood, w.a.: high-throughput agrobacterium-mediated barley transformation. plant methods, 4 (22), 2008, http://www.plantmethods.com/content/4/1/22. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:10 utc 62 gubišová, m. et al. cho, m.j., jiang, w., lemaux, p.g.: transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism. plant science, 138 (2), 1998, 229-244. dahleen, l.s.: improved plant regeneration from barley callus cultures by increasing copper levels. plant cell tiss.org. cult., 43, 1995, 267-269. dahleen, l.s.: donor-plant environment effects on regeneration from barley embryo-derived callus. crop sci., 39, 1999, 682-685. ganeshan, s., baga, m., harvey, b.l., rossnagel, b.g., scoles, g.j., chibbar, r.n.: production of multiple shoots from thidiazuron-treated mature embryos and leaf-base/apical meristems of barley (hordeum vulgare). plant cell tiss. org. cult., 73, 2003, 57-64. gubišová, m., mihálik, d., konôpková, ľ.: regeneration efficiency of slovak spring barley cultivars and winter wheat cultivars. agriculture, 57 (2), 2011, 76-83. klčová, l., havrlentová, m., faragó, j.: cultivar and environmental conditions affect the morphogenic ability of barley (hordeum vulgare) scutellumderived calli. biologia, bratislava, 59 (4), 2004, 501-504. murashige, t., skoog, f.: a revised medium for rapid growth and bioassay with tobacco tissue cultures. physiologia plantarum, 15 (3), 1962, 473-479. shan, x., li, d., ou, r.: thidiazuron promotes in vitro regeneration of wheat and barley. in vitro cell. dev. biol.-plant, 36, 2000, 207-210. sharma, v.k., hansch, r.,. mendel, r.r, schulze, j.: a highly efficient plant regeneration system through multiple shoot differentiation from commercial cultivars of barley (hordeum vulgare l.) using meristematic shoot segments excised from germinated mature embryos. plant cell rep., 23, 2004, 9-16. sharma, v.k., hansch, r.,. mendel, r.r, schulze, j.:mature embryo axisbased high frequency somatic embryogenesis and plant regeneration from multiple cultivars of barley (hordeum vulgare l.). j. exp. bot., 56 (417), 2005, 1913-1922. tingay, s., mcelroy, d., kalla, r., fieg, s., wang, m., thornton, s., brettell, r.: agrobacterium tumefaciens-mediated barley transformation. the plant journal, 11 (6), 1997, 1369-1376. wan, y., lemaux, p.g.: generation of large number of independently transformed fertile barley plants. plant physiology, 104 (1), 1994, 37-48. presented at the 3rd international scientific conference “applied natural sciences 2011”, october 5–7, 2011, častá papiernička, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:10 utc << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (iso coated) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.3 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /perceptual /detectblends true /detectcurves 0.1000 /colorconversionstrategy /srgb /dothumbnails true /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true 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can be opened with acrobat and adobe reader 5.0 and later.) >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [2834.650 2834.650] >> setpagedevice microsoft word durcekova nb.doc nova biotechnologica vii-1 (2007) 123 study of anaesthetical activity of alkoxyphenylcarbamic acid esters using statistical methods tatiana durčeková1, ján mocák1, 2, jozef lehotay1, 2, jozef čižmárik3 1department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (tatiana.durcekova@ucm.sk) 2institute of analytical chemistry, slovakuniversity of technology, radlinskeho 9, sk-81237 bratislava, slovak republic 3department of pharmaceutical chemistry, comenius university, odbojarov 10, sk-83232 bratislava, slovak republic abstract: anaesthetical activity of 113 morpholinoethyl-, piperidinoethyl-, piperidinopropyland azepanoethylester derivatives of alkoxyphenylcarbamic acid was characterized by several chemometrical techniques. the surface anaesthetical activity, a, and the infiltration anaesthetical activity, b, were correlated to lipophilicity, (expressed by the logarithm of the hplc retention factor, log k), the length of the side alkoxy chain (represented by the number n of carbon atoms), molar mass m as well as the ester type. principal component analysis and cluster analysis were used for predicting both types of the anaesthetic activity of the alkoxyphenylcarbamic acid esters. key words: alkoxyphenylcarbamic acid esters, anaesthetical activity, correlation analysis, cluster analysis, principal component analysis. 1. introduction the prediction of the compound property is the basic problem of the quantitative studies of the relationships structure activity. the basic assumption is that the compounds with similar structure dispose of a similar biological activity (kuchař, rejholec, 1987), as it was also stated in a similar anaesthetics study of hatrík et al., 1995). after discovery of a biologically active compound with a new structure, the next phase of the activity optimization is commonly characterized by varying the basic structure in order to achieve the maximal biological activity (hatrík et al., 1995a). the derivatives of basic ethyl and propyl esters of alkoxysubstituted phenylcarbamic acids have been prepared and studied as local anaesthetics due to their low toxicity (waisser et al., 2004). the hplc capacity factor was used for characterization of the lipophilicity of the tested compounds (hatrík et al., 1995a). in this paper an attempt is made to correlate both types of the anaesthetical activity to the chemical shifts in the 1h nmr and 13c nmr spectra of the examinated compounds. when simulated chemical shifts are used then it is possible to omit laborious chemical synthesis – at least in the first phase of such study. 124 ďurčeková, t. et al. 2. experimental the measured values of infiltration anaesthesia and surface anaesthesia were taken from the papers of hatrík et al. (1995), hatrík et al. (1995a) and hatrík et al. (1995b). the subject of the study was the data set comprising 48 compounds, for which both log a and log b were found, plus two data sets consisting of 62 and 68 compounds, for which log a and log b were acquired, respectively. all studied compounds were divided into 3 groups (categories) by increasing anaesthetical activity (the lower, middle and upper terciles). 19 variables describing molecule properties were used in our calculations: log k – the hplc retention factor, n number of carbon atoms on the side alkoxy chain, m – molar mass, c1, c2, c3, c4, c5, c6, c8, c10, c12 – the 13c nmr chemical shifts of the corresponding carbons numbered in fig. 1, hch3, h1, h2, h12 the 1h nmr chemical shifts of the terminal methyl and the numbered protons (fig. 1), respectively, qh – the quaternary nitrogen proton located in the ester ring. the mentioned nine 13c and five 1h nmr chemical shifts were simulated using the software package acd labs, ver. 7.0. the position numbering in fig. 1 was also taken from acd labs. for basic data processing the ms excel spreadsheets were used. the correlation analysis, cluster analysis and principal component analysis were performed using software packages jmp 7.0, spss 15.0 and statgraphics plus 5.1. hn o o o(ch2)nch3 r h1, c1 h2, c2 c 6 c 8 h12, c12 c 3 c 4 c 5 c 10 hch3 . hcl fig. 1: structure of the studied derivatives of alkoxyphenylcarbamic acid differing by n ( n = 0 9), and the ester part r (r = morpholin-4-yl or piperidin-1-yl or azepan-1-yl). 3. results and discussion the correlation analysis shows the measure of correlation expressed by the pair (pearson) correlation coefficients between all pairs of the studied variables. in the studied problem, the significant dependences of both log a and log b on the logarithm of the hplc retention factor, log k, and mainly on ηch3 (1h nmr methyl chemical shift) have been observed. in addition, further very significant dependences were discovered: log a – n, log a – m, and log b – qh. some further simulated nmr chemical shifts correlate significantly with anaesthetical activity in the form of log a and/or log b. more details are summarized in table 1. nova biotechnologica vii-1 (2007) 125 table 1. the pair correlation coefficients of the selected molecule properties with anaesthetical activity in the form of log a and log b, respectively. property log a log b property log a log b log k 0.4584 0.2064 c8 -0.2300 0.0201 n 0.5041 0.1199 c10 0.1562 -0.2967 m 0.5548 0.0563 c12 -0.1589 0.1929 c1 -0.1309 -0.0593 hch3 -0.6062 -0.1979 c2 0.1969 -0.1224 h1 0.1178 0.1304 c3 -0.2910 0.1266 h2 0.1166 -0.1283 c4 0.0416 -0.1298 h12 -0.1516 0.2123 c5 0.1164 0.1229 qh -0.0162 -0.3328 c6 -0.0056 -0.1230 note: rcrit = 0.2109 (n = 62, α = 0.05) for the dataset with log a; rcrit = 0.2012 (n = 68, α = 0.05) for the for the dataset with log b. cluster analysis is represented by a dendrogram showing the distances among the objects (the studied compounds in this case) or the used variables (the selected properties of the molecule). the shorter distance in the dendrogram, the more similar are the clustered items. the results of cluster analysis depicted in fig. 2 and fig. 3 demonstrate that the shortest distance (expressed on the dendrogram ordinate) to the anaesthetical activity log a and therefore the largest similarity was found for log k, then for the number of alkoxy carbon atoms n and the molar mass m both for the squared euclidean and the city-block distances. the anaesthetical activity log b is most similar to n, then to log k and c1 if the squared euclidean distances are used. when using the city-block distances, the closest distance to the log b variable exhibit log k and n; then the next shortest distance exhibits c10. dendrogram ward's method, squared euclidean d is ta nc e 0 200 400 600 800 1000 1200 c 1 c 10 c 12 c 2 c 3 c 4 c 5 c 6 c 8 h 1 h 12 h 2 h c h 3 lo ga lo gk m n qh fig. 2. cluster analysis of 18 variables, 62 compounds, anaesthetical activity is expressed by log a. software jmp 6.0. ward’s clustering method utilizing the squared-euclidean distances. the symbols used for describing the horizontal axis are introduced in experimental part. 126 ďurčeková, t. et al. it is noteworthy that the results of cluster analysis, both for log a as well as log b, are in good accordance with the results of the correlation analysis and confirm the previous results. the principal component analysis was performed in the biplot form. the biplot describes the studied problem representing the used variables (molecule properties) by the rays and the objects (the studied compounds) by the object (compound) numbers or, alternatively, by the number of the category (lower, middle and upper) created according to the log a or log b measured values. when considering the variables, the most important mutual dependence exhibit those of them, which are represented by the rays forming an angle as close as possible to 0° or 180° (in such a case an inverse proportional dependence is supposed). dendrogram ward's method, squared euclidean d is ta nc e 0 200 400 600 800 1000 1200 c 1 c 10 c 12 c 2 c 3 c 4 c 5 c 6 c 8 h 1 h 12 h 2 h c h 3 lo gb lo gk mn qh fig. 3. cluster analysis of 18 variables, 68 compounds, anaesthetical activity is expressed by log b. software jmp 6.0. ward’s clustering method utilizing the squared-euclidean distances. the symbols used for describing the horizontal axis are introduced in experimental part. the pca results were received separately for the dataset with the log a measurements and that with log b. neither log a nor log b was important with respect to the pc1 and pc2 variables but they were very important for the pc3. it was demonstrated by a long ray of the corresponding anaesthetical activity on the pc3-pc1 biplot, as it is visible in fig. 4, calculated for the dataset with log a. in this figure, log a is closely correlated to n, m and then to log k. in addition, a very strong inverse dependence on the 1h nmr chemical shift of the terminal ch3 is observed. it was also found that the most correlated variables to log b are n and m, then log k and the 13c nmr chemical shift of the carbon c1. similarly to log a, a strong inverse correlation of log b with the 1h nmr chemical shift of the terminal ch3 was also observed. it was also observable from the biplots calculated for both datasets that two groups of the studied compounds were clearly separated. by a detailed inspection of the dataset with log a it was found that one group is created mostly by ortho and para derivatives (the right group in fig. 4) and another group is formed mainly by the meta nova biotechnologica vii-1 (2007) 127 derivatives. the dataset with log b consists only from the ortho and meta derivatives, which (with some exceptions) formed two observed distinct groups. biplot pc1 (39.6%) p c 3 (1 8. 1% ) c1c10 c12 c2 c3 c4 c5 c6 c8 h1 h12 h2 hch3 loga logk mn qh -4.1 -2.1 -0.1 1.9 3.9 -5.3 -3.3 -1.3 0.7 2.7 4.7 fig. 4. biplot pca, 18 variables and 62 compounds. software statgraphics 5.1. rays denote the used variables (molecule properties) and the symbols represent the studied compounds. the pc3 axis can be assigned to anaesthetical activity – the higher pc3 the larger log a or log b. since no one of the above mentioned two groups of compounds are located at higher or lower pc3 values it can be concluded that the position of the alkoxy chain is not a deciding factor determining any of the anaesthetical activity. much more important is the length of the alkoxy chain, which is consistent with the larger molar mass and the hplc retention factor so that a larger anaesthetical activity is predicted for a higher lipophilicity. the 1h nmr chemical shift of the terminal ch3 is inversely dependent on the length of the alkoxy chain (the distance from oxygen), which is in accord with the mentioned conclusions. acknowledgement: the research contribution of individual authors of this paper was supported by the following grants: vega 1/3584/06, apvv-0057-06. references hatrík, š., lehotay, j., čižmárik, j.: possibility of anaesthetical activity prediction of n-(pyrrolidinyl)ethyl esters of alkoxyphenylcarbamic acids. collect. czech. chem. commun., 60, 1995, 1410-1414. hatrík, š., lehotay, j., čižmárik, j.: neural network method, the tool for studying biological activity of compounds. relationship between infiltration anaesthesia, coded structural information, and chromatographic properties applied in homologous series of alkoxy-substituted esters of phenylcarbamic acids. chem. papers, 49 (3), 1995a, 149-154. 128 ďurčeková, t. et al. hatrík, š., lehotay, j., čižmárik, j.: study of relationship between surface anaesthesia and chromatographic properties of alkoxyesters of phenylcarbamic acid by neural network method. part i. collect. czech. chem. commun., 60, 1995b, 960-965. kuchař, m., rejholec, v.: základy kvantitativních vztahú mezi strukturou a biologickou aktivitou. in: využití kvantitativních vztahú mezi strukturou a biologickou aktivitou (principles of the quantitative dependences between structure and biological activity. in: utilization of quantitative relationships between structure and biological activity), academia, prague, 1987, 11 18. waisser, k., dražková, k., čižmárik, j., kaustová, j.: a new group of potential antituberculotics: hydrochlorides of piperidinylalkylesters of alkoxysubsituted phenylcarbamic acids. folia microbiol., 49 (3), 2004, 265-268. nova biotechnologica et chimica 13-2 (2014) 137 doi 10.1515/nbec-2015-0004 © university of ss. cyril and methodius in trnava the response of artificial aging to sorption properties of biochar for potentially toxic heavy metals vladimír frišták1*, wolfgang friesl-hanl1, martin pipíška2; barbora richveisová micháleková2, gerhard soja1 1department of health & environment, ait austrian institute of technology gmbh, tulln, 3430, austria (vladimir.fristak@ait.ac.at) 2department of ecochemistry and radioecology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic abstract: this paper evaluates the effect of simulated conditions of artificial aging on sorption capacity of two types of biochar. these were produced by slow pyrolysis from different feedstock beech wood chips (bc a) and garden green waste residues (bc b). cadmium served as a model for potentially toxic metals. twenty freeze-thaw cycles were used to simulate physical aging. the determination of biochar physicochemical properties showed main changes in cec and sa values of aged sorbents. the maximum sorption capacities of aged bc a sorbent were higher by about 26 % and aged bc b sorbent by about 20% compared to qmax of non-aged biochar. qmax of aged bc b peaked at 9.4 mg g-1 whereas bc a sorbed significantly less cd. ft-ir analyses confirmed the changes in structural composition and content of functional groups on biochar surfaces. the artificial physical aging model was assessed as an efficient tool for investigation of natural weathering conditions. key words: biochar, artificial aging, sorption, cadmium 1. introduction soil contamination with potentially toxic heavy metals (pthm) usually cd (cadmium), cr (chromium), cu (copper), pb (lead), ni (nickel) and zn (zinc), is one of the main concerns in environmental protection due to their non-biodegradability and eco-toxicity, even at trace concentrations (o’connell et al., 2008). various technologies have been developed for removing metals from liquid wastes, such as extraction, coagulation, precipitation, reduction, separation with membrane systems, ion exchange and reverse osmosis (frišták et al., 2013; xiong et al., 2015) and for immobilizing pthm in soils, a process of adding metal-sorbing amendments is utilized (friesl et al., 2009). sorption separation as a proven and practical treatment method leads to significant metal recovery and simple process realization. for heavy metal sorption removal from aqueous waste solutions a wide range of heterogeneous biological and non-biological materials can be used such as algae (kaduková and horváthová, 2012), moss (pipíška et al., 2010), dried sewage sludge (frišták et al., 2014a), synthetic zeolites (remenárová et al., 2014), natural zeolites (chmielewska, 2013) or chitosan (mi et al., 2015). biochar represents a solid by-product from biomass pyrolysis which is the thermal decomposition of organic materials at elevated temperatures (< 1000 °c) with little or no available oxygen. theoretically, biochar could be produced from any carbon-rich biomass bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:29 utc 138 frišták et al. (lehmann and joseph, 2009). this pyrolysis product has attracted attention mainly for its potential use as a soil amendment to improve soil quality (karer et al., 2013; kloss et al., 2014). biochar can be characterized by many important physico-chemical properties, mainly for rich porosity, high exchange capacity, water holding capacity, surface area, low bulk density, which play decisive roles in a wide range of agricultural or soil remediation applications. sorption separation of toxic metals from aqueous or soil solutions provides a new field for biochar utilization as sorbent and in-situ stabilization tool. generally, sorption capacities of sorbents can be modified by physic-chemical effects. abiotic and/or biotic processes (so-called aging) affect the main characteristics and the strength of pthm sorption. biological, chemical and physical aging have been recognized to contribute to artificial aging (hale et al., 2011). however, the studies to investigate the change in biochar sorption capacity under different temperature conditions over time of storage are scarce and limited. based on these scenarios, the main aim of this paper was to simulate the conditions of physical biochar aging and to assess the influence of different biochar feedstocks and aged sorbent characteristics on the cd sorption capacity as a model of pthm. 2. material and methods 2.1 biochar production biochar samples were produced in slow pyrolysis process from two different feedstocks: beech wood chips (bc a) and garden green waste residues (bc b). both materials had maximum dimensions 2 cm x 2 cm x 2 cm and had been pyrolyzed at a highest treatment temperature of 500°c and residence time 120 min in a rotary furnace (fig.1). for ensuring inert and uniform heating conditions, nitrogen was used as flush gas. the biochars were ground and sieved to particles with size < 2 mm. sorbents were pretreated by rinsing in deionised water with conductivity < 0.4 µs cm-1 to remove the ash impurities. 2.2 biochar characterization the active and potential ph values of bc a and bc b were measured after stirring the biochars with deionized water and 1.0 mol l-1 kcl (ratio 1:2.5) for 1 h and stabilization for 1 h (inolab ph level 2p, weilheim, germany). the electrical conductivities (ec) of bc a and bc b were measured in a 1:10 deionised water extracts after 24 h mixing (inolab ph level 2p, weilheim, germany). the cation exchange capacities (cec) of biochars were determined by the bacl2 method described in our previous paper (frišták et al., 2013). surface areas (sa) were estimated by the titration method with naoh according to melichová and hromada (2013). the total c, n and h contents of biochars were measured by an elemental analyzer (chns-o ea 1108, carlo erba instruments, italy). initial concentrations of cd, zn and cu in biochar samples were determined after aqua regia digestion by icp-ms (perkin elmer, elan drce 9000). the intra-structures of bc a bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:29 utc nova biotechnologica et chimica 13-2 (2014) 139 and bc b sorbents and surface structure analysis were obtained by scanning electron microscopy (sem) using electron microscope jeol jsm7600f (japan). the analyses was performed at voltage 30kv, vacuum pressure 9.0×10-3 pa and magnification 250×. fig. 1. scheme of the pyrolysis reactor. 2.3 artificial aging for simulation of biochar artificial aging, a physical approach was used. for the sample series with freezing cycles, bc a and bc b were moved into a freezer at -18°c firstly. as a second stage, the biochar samples were then kept in an incubator at 25°c for thawing. each of 20 freeze-thawing cycles consisted of 12 h freezing and 12 h thawing. after the last cycle, sorbents were dried at 50°c. fig. 2 sem micrographs of bc a (a) and bc b (b) at magnitude 250 ×. 2.4 sorption experiments the sorption process of cd from single component system was studied using a batch equilibration method according to the oecd guideline 106 (oecd, 2001), a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:29 utc 140 frišták et al. modified by lair et al. (2006). 1 g of aged bc a or bc b sorbent was suspended in polypropylene centrifuge tubes filled with 20 ml of 0.01 mol l-1 cacl2 (ph = 5.65) and placed on a horizontal shaker for 24 h (200 rpm). the solution of cacl2 was used to ease phase separation and to keep ionic strength similar to natural soil solutions. deionised water (<0.4 μs cm-1), spiked with cd as cdcl2, was added to each tubes, resulting in a concentration range from 10 to 75 mg l-1 and ph 7.0±0.1. biocharsolution ratio was 1:30. solution ph changed from 5.65-5.50 with increasing concentration of metals. after agitation for 24 h, tubes were centrifuged (38 400 g, 15 min) and supernatant was filtered through a 0.45 µm pore size membrane filter (schleicher and schuell, germany) to remove colloids from the solution. filters were tested for retention of metals before use. concentration of cd in liquid phase was measured by atomic absorption spectrometry with flame atomization (faas, aa 400, perkin elmer, usa). all experiments were realized in triplicates. sorptions of cadmium were calculated according to eq.1: m/v)cc(q eqeq ×−= 0 (1) where: qeq is the cadmium uptake (mg g -1), c0 is the initial liquid-phase concentrations of cadmium (mg l-1), ceq is the equilibrium liquid-phase concentrations of cadmium (mg l-1), v is the volume (l) and m is the amount of biochar (g). obtained data were analysed by mathematical models with terms of langmuir, freundlich and dubinin-kaganer-radushkevich (dkr) isotherms (table 1). table 1. models of adsorption isotherms used to describe the sorption equilibrium of single metal system. adsorption model equation coefficients langmuir eq eq eq bc cbq q + = 1 max qeq – amount of sorbed metal at equilibrium b – isotherm coefficient characterizing affinity sorbent to metal ion in solution qmax – maximum metal sorption capacity at saturated sorbent binding sites ceq – metal equilibrium concentration in solution freundlich )/1( n eqeq kcq = qeq – amount of sorbed metal at equilibrium k,n – freundlich empirical constants characterizing parameters of sorption process ceq – metal equilibrium concentration in solution dkr ⎥ ⎥ ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎢ ⎢ ⎣ ⎡ − + = 22 )) 1 1ln(( exp e c rt qq eqmeq qeq – amount of sorbed metal at equilibrium qm – monolayer maximum metal sorption capacity r – gas constant t – temperature ceq – metal equilibrium concentration in solution e – sorption energy bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:29 utc nova biotechnologica et chimica 13-2 (2014) 141 parameters of adsorption isotherms were calculated by non-linear regression analysis using the program microcal origin 8.0 professional (originlab corporation, northampton, usa). mentioned program was applied also for paired t-test of selected physico-chemical properties of unaged and aged biochar. 2.5 ft-ir analysis spectral analysis of bc a and bc b in infrared region was used to determine binding characteristics, determination of functional groups of biochar and comparison of unaged and physically aged sorbent. the surface functional groups of bc a and bc b were detected by infrared spectroscope with fourier transformation (ft-ir) (nicolet nexus 470, thermo scientific, usa). the spectra were recorded from 4000 to 400 cm−1. 3. results and discussion 3.1 characterization of biochar-derived sorbents both biochar samples prepared from two different feedstocks contained more than 75% of carbon (table 2), which is among the typical reported values of other biochars produces at these pyrolysis conditions (kloss et al., 2012). the c/n ratios of biochars were 200.75 for bc a and 122.74 for bc b. lehmann and joseph (2009) identified the c/n ratio lower than 20 as a ratio of good soil amendment. although it is still unclear whether the c/n ratio criterion is directly applicable to biochars which do not decompose at the same rate as other organic amendments, applications of biochars with higher c/n ratios may lead to lower n uptake and a delay in n turnover in soil (prommer et al., 2014). comparison of physicochemical properties of bc a and bc b showed the main differences in ash content, cec and sa values. the initial concentration of cd was lower than lod of analytical measurement (<0.002 mg g-1) for both biochar samples. table 2. physico-chemical characteristics of unaged bc a and bc b. bc a bc b ph‡ (h2o) 8.78 ± 0.12 9.03 ± 0.08 ec‡ (ms cm-1) 0.54 ± 0.02 1.67 ± 0.05 ash content† (%) 15.20 19.30 density† (kg l-1) 0.36 0.34 cec‡ (mmol 100 ml-1) 9.83 ± 0.15 12.85 ± 0.12 sa‡ (m2 g-1) 27.24 ± 0.30 31.54 ± 0.27 c† (%) 80.30 79.78 h† (%) 1.60 1.59 n† (%) 0.40 0.65 c/n ratio 200.75 122.74 cd‡ (mg g-1) <0.002* <0.002* * concentration lower than lod of analytical equipment † single determination ‡ determined in triplicate (mean ± sd) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:29 utc 142 frišták et al. additionally, artificial aging caused the changes of basic physico-chemical properties (data not shown). the main differences in cec and sa values were determined. aged bc a provided an ssa of 28.21 ± 0.09 m2 g-1 and bc b 33.09 ± 0.12 m2 g-1. in comparison with cec of unaged biochars, aged bc a had 10.01 ± 0.05 mmol 100 ml-1 and bc b 13.91 ± 0.08 mmol 100 ml-1. the cec and sa values are important parameters for heavy metal sorption behaviour assessment (frišták et al., 2014b). nonetheless, paired t-test (α = 0.05) confirmed the small statistical significance of obtained data for aged and unaged biochar. lehrsch (1998) showed the main effect of freeze-thaw cycles on soil characteristics mainly in breakdown or formation of new aggregates. equally, zhao et al. (2013) proposed a conceptual model of the mechanisms involved into freeze-thaw cycles of soil particles. however, a mechanism of this process remains unclear for biochar as a potential soil amendment. 3.2 effect of artificial aging on cd sorption process the effect of cd initial concentration to sorption capacity of artificially aged bc a and bc b was determined within the range of 10-75 mg l-1. in general, the obtained data revealed that sorption capacity increased with increasing the initial cd concentration in the reaction solution. for description of equilibrium relationships, langmuir, freundlich and dkr mathematical equations of adsorption models were used. comparison of the coefficients of determination calculated by non-linear regression analyses showed that langmuir model fitted the cadmium sorption data for ba a and bc b better than the freundlich and dkr model (table 3). frequently, the langmuir model better describes adsorption on homogeneous surfaces, while the freundlich model is better for heterogeneous surfaces. table 3. langmuir (l), freundlich (f) and dubinin-kaganer-radushkevich (d) equilibrium parameters (± sd) for cd sorption by aged bc a and bc b obtained by non-linear regression analysis. sorbent model qmax [mg g-1] b [l mg-1] k [l g-1] 1/n qm [mg g-1] β [mol2 j-2] r 2 l 2.51 ± 0.12 0.08 ± 0.015 0.993 f 0.28 ± 0.012 0.64 ± 0.017 0.979 bc a d 1.39 ± 0.02 -9.58*10 -7 0.867 l 9.43 ± 0.32 0.16 ± 0.019 0.996 f 0.68 ± 0.042 0.92 ± 0.044 0.979 bc b d 2.19 ± 0.06 -4.72*10 -7 0.866 langmuir parameters demonstrated that the effect of freeze-thaw cycles caused increases of sorption capacity for both bc a and bc b. values qmax of bc a and bc bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:29 utc nova biotechnologica et chimica 13-2 (2014) 143 b were higher by about 26 % and 20% compared to qmax of non-aged biochar (table 4). hale et al. (2011) showed the crucial effect of freeze-thaw cycles on structure and also sorption properties of biochar. authors highlighted the correlation between increased sa and cec values of aged biochar samples. in the report of yao et al. (2010) a detailed simulation of geochemical weathering of biochar samples using physico-chemical methods was described. in our study, increasing sorption capacity of both aged biochar-based sorbents can be caused by several processes. freezing alters biochar properties through various mechanisms, and has direct or indirect chemical and biological consequences as well. freeze-thaw process significantly influences the amount and chemical forms of biochar water-extractable components and also possible reaction sites for cadmium sorption. additionally, wang et al. (2007) confirmed the crucial effect of temperature conditions on sorption and desorption of phosphorus by wetland soil and highlighted the role of altered micropores structures. biochar as a porous material provides a huge volume of microand mezopores, both involved in sorption processes (frišták et al., 2015). freeze-thaw process can contribute to formation or damaging of these structures and thus changing the total porosity of biochar-based sorbents in terms of different water bonding and effect of liquid-solid phase. table 4. comparison of maximum sorption capacities (qmax ± sd) of bc a and bc b for cd before and after artificial aging process obtained from langmuir adsorption isotherm. as illustrated in fig. 3 and 4, ft-ir spectra of unaged bc a and bc b indicated carboxyl and hydroxyl groups were present in abundance. similar broad peaks in ftir spectra were found in the study of štefušová et al. (2012) for rapeseed and wheat straw-derived biochar. these functional groups can serve as proton donors and hence the deprotonated hydroxyl and carboxyl groups can be involved in coordination of free cd ions (lu et al., 2012). artificial aging caused mainly the changes in absorption intensity at σ 3418 cm-1 (bc a) and σ 3415 cm-1 (bc b) that are responsible for stretching vibration of –oh functional groups of water, alcohols and phenols. the absorption bands at σ 1559 cm-1 and 1445 cm-1 for bc a and σ 1581 cm-1 and 1437 cm-1 for bc b after freeze-thaw cycles were changed by shifts and extensions of peak areas. the highlighted differences could be induced by the freezethaw processes during the artificial aging induced that caused the main changes in biochar consistency and structure. a crucial role in this process was played by the water bonded capillary forces between biochar particles. after freeze-thaw cycles the structures for water bonding could be damaged. after the sorption of cd by aged biochar , shifts and changes of absorption bands of c=o and c=c vibrations at wave numbers 1579 and 1445 cm-1 for bc a and 1581 and 1437 cm-1 for bc b were detected. for c-h bending at σ 1042 cm-1 (bc a) sorbent qmax [mg g-1] unaged aged bc a 1.99 ± 0.19 2.51 ± 0.12 bc b 7.80 ± 0.70 9.43 ± 0.32 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:29 utc 144 frišták et al. and σ 1038 cm-1 (for bc b) only small changes in the spectra after cadmium sorption process were found. 4000 3500 3000 2500 2000 1500 1000 15 20 25 30 35 40 45 t ( % ) 1/cm bc a bc a after aging process bc a after aging process and cd sorption fig. 3. representative ft-ir spectra of unaged bc a and aged bc a before and after cadmium sorption process. 4000 3500 3000 2500 2000 1500 1000 15 20 25 30 35 40 45 50 55 60 t ( % ) 1/cm bc b bc b after aging process bc b after aging process and cd sorption fig. 4. representative ft-ir spectra of unaged bc b and aged bc b before and after cadmium sorption process. 4. conclusions this paper illuminates the utilization of biochar as an efficient sorbent for cadmium removal from liquid waste and waste waters. mainly, the positive effect of artificial aging by freeze-thaw cycles on sorption capacity of these biochar-based sorbents was confirmed. the maximum sorption capacities of aged bc a sorbent were bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:29 utc nova biotechnologica et chimica 13-2 (2014) 145 higher about 26 % and aged bc b sorbent about 20% compared to qmax of non-aged biochar. the changes of temperature could lead to the structural changes (porosity) of biochar-based sorbent in terms of changes in water bonding and effect of water liquidsolid phase on biochar. however, the differences in sorption capacity of the two biochars were much larger (bc b sorbed more than three times as efficiently as bc a), demonstrating the importance of biochar feedstock material for the sorption capacity of biochar. apparently, biochar artificial aging by freeze-thaw cycles presents a promising new model for studying weathering effects assessment which should be further investigated and compared with real-world conditions of biochar aging in a soil matrix. references chmielewska, e., hodossyová, r., bujdoš, m.: kinetic and thermodynamic studies for phosphate removal using natural adsorption materials. pol. j. environ. stud., 22, 2013, 1307-1316. friesl-hanl, w., platzer, k., horak, o., gerzabek, m. h.: immobilising of cd, pb, and zn contaminated arable soils close to a former pb/zn smelter: a field study in austria over 5 years. environ. geochem. health, 31, 2009, 581-594. frišták, v., pipíška, m., horník, m., augustín, j., lesný, j.: sludge of wastewater treatment plants as co2+ ions adsorbent. chem. pap., 67, 2013, 265273. frišták, v., pipíška, m., valovčiaková, m., lesný, j., rozložník, m.: monitoring 60co activity for the characterization of the sorption process of co2+ ions in municipal activated sludge. j. radioanal. nucl. ch., 299, 2014a, 16071614. frišták, v., pipíška, m., nováková, m., lesný, j., packová, a.: sorption separation of cadmium from aqueous solutions by alginate material: kinetic and equilibrium study. desalin. water treat., 2014b, doi 10.1080/19443994.2014.935811. frišták, v., pipíška, m., lesný, j., soja, g., friesl-hanl, w., packová, a.: utilization of biochar sorbents for cd2+, zn2+, and cu2+ ions separation from aqueous solutions: comparative study. environ. monit. assess., 187, 2015, 4093. hale, s.e., hanley, k., lehmann, j., zimmerman, a.r., cornelissen, g.: effects of chemical, biological and physical aging as well as soil addition on the sorption of pyrene to activated carbon and biochar. environ. sci. technol., 45, 2011, 10445-10453. kaduková, j., horváthová, h.: biosorption of copper, zinc and nickel from multi-ion solutions. nova biotechnol. chim., 11, 2012, 125-132. karer, j., wimmer; b., zehetner, f., kloss, s., soja, g.: biochar application to temperate soils: effects on nutrient uptake and crop yield under field conditions. agr. food. sci., 22, 2013, 390-403. kloss, s., zehetner, f., dellantonio, a., hamid, r., ottner, f., liedtke, v., schawanninger m., gerzabek, m.h., soja, g.: bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:29 utc 146 frišták et al. characterization of slow pyrolysis biochars: effects of feedstocks and pyrolysis temperature on biochar properties. j. environ. qual., 41, 2012, 990-1000. kloss, s., zehetner, f., oburger, e., buecker, j., kitzler, b., wenzel, w.w., wimmer, b., soja, g.: trace element concentration in leachates and mustard plant tissue (sinapis alba l.) after biochar application to temperate soil. sci. total environ., 481, 2014, 498-508. lair, g.j., gerzabek, m.h., haberhauer, g., jakusch, m., kirchmann, h.: response of the sorption behavior of cu, cd, and zn to different soil management. j. plant nutr. soil sci., 169, 2006, 60-68. lehmann, j., joseph, s.: biochar for environmental management: science and technology, earthscan/james james, 2009. lehrsch, g.a: freeze-thaw cycles increase near-surface aggregate stability. soil. sci., 163, 1998, 63-70. lu, h., zhang, w., yang, y., huang, x., wang, s., qiu, r.: relative distribution of pb2+ sorption mechanisms by sludge-derived biochar. water res., 46, 2012, 854-862. melichová, z., hromada, l.: adsorption of pb2+ and cu2+ ions from aqueous solutions on natural bentonite. pol. j. environ. stud., 22, 2012, 457-464. mi, f.l., wu, s.j., lin, f.m.: adsorption of copper (ii) ions by chitosan-oxalate complex biosorbent. int. j. biol. macromol., 72, 2015, 136-144. o’connell, d.w., birkinshaw, c., o’dwyer, t.f.: heavy metal adsorbents prepared from the modification of cellulose: a review. bioresour. technol., 99, 2008, 6709-6724. oecd-guideline 106: oecd guideline for the testing of chemicals. adsorptiondesorption using a batch equilibrium method. organisation for economic cooperation and development (oecd), paris, 2001. pipíška, m., horník, m., remenárová, l., augustín, j., lesný, j.: biosorption of cadmium, cobalt and zinc by moss rhytidiadelphus squarrosus in the single and binary component system. acta chim. slov., 57, 2010, 163-172. prommer, j., wanek, w., hofhansl, f., trojan, d., offre, p., ulrich t., schleper, c., sassmann s., kitzler, b., soja, g., hoodnowotny, r.c.: biochar decelerates soil organic nitrogen cycling but stimulates soil nitrification in a temperate arable field trial. plos one 9, e86388, 2014, doi 10.1371/journal.pone.0086388. remenárová, l., pipíška, m., florková, e., horník, m., rozložník, m., augustín, j.: zeolites from coal fly ash as efficient sorbents for cadmium ions. clean technol. environ. policy, 16, 2014, 1551-1564. štefušová, k., lovás, m., zubrik, a., matik, m., václavíková, m.: removal of cd2+ and pb2+ from aqueous solutions using bio-char residues. nova biotechnol. chim., 11, 2012, 139-146. wang; g.p., liu; j.s., zhao, h.y., wang, j.d., yu, j.b.: phosphorus sorption by freeze-thaw treated wetland soils derived from a winter-cold zone (sanjiang plain, northeast china). geoderma, 138, 2007, 153-161. xiong, ch., li, y., wang, g., fang, l., zhou, s., yao, c., chen, q., zheng, x., qi, d., fu, y., zhu, y.: selective removal of hg (ii) with bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:29 utc nova biotechnologica et chimica 13-2 (2014) 147 polyacrylonitrile-2-amino-1,3,4-thiadiazole chelating resin: batch and column study. chem. eng. j., 259, 2015, 257-265. yao, f.x., arbestain, m.c., virgel, s., blanco, f., arostegui, j., maciá-agullo, j.a., macias, f.; simulated geochemical weathering of amineral ash-rich biochar in a modified soxhlet reactor. chemosphere, 80, 2010, 724-732. zhao, q., xing, b., tai, p., li, h., song, l., zhang, l., li, p.: effect of freezethawing cycles on soil aging behavior of individually spked phenanthrene and pyrene at different concentrations. sci. total environ., 444, 2013, 311-319. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:29 utc nova biotechnologica et chimica 15-1 (2016) 35 doi 10.1515/nbec-2016-0004 © university of ss. cyril and methodius in trnava pyrolysis products as soil fertilizers: screening of potentially hazardous aromatic compounds vladimír frišták1*, marion graser1, martin pipíška2,3, barbora micháleková-richveisová2, gerhard soja1 1energy department, ait austrian institute of technology gmbh, tulln, 3430, austria (vladimir.fristak@ait.ac.at) 2department of ecochemistry and radioecology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic 3department of chemistry, trnava university, trnava, sk-918 43, slovak republic abstract: this study investigated the concentrations of polycyclic aromatic hydrocarbons (pahs) in pyrogenic carbonaceous materials (pcm) produced from three waste materials during slow pyrolysis at 400 and 500°c. as feedstocks bone meal (bm), biogas slurry (bc) and chicken manure (cm) were used. as potentially problematic substances 1and 2methylnaphthalene were analysed as indicators for methylated hydrocarbons in pyrolysis products. the phytotoxic effect of soil amendments was evaluated by a standard cress germination test with lepidium sativum l. the analysis showed higher concentrations of the sum of 16 us-epa pahs in samples produced at lower temperature and in samples produced from biogas slurry. concentrations of 1and 2-methylnaphthalene showed similar trends with concentrations in a range of 35205% of the sum of 16 pahs. germination tests showed inhibition effects of products from biogas slurry when applied in concentrations of >10 % to standard substrate/. apparently pyrolysis of biogas slurry requires special attention to avoid accumulation of pahs and methylated naphthalenes. key words: biochar, pahs, methylated naphthalene, phytotoxicity 1. introduction in a sustainable society renewable resources should substitute non-renewable ones wherever possible. waste and by-products from various industrial processes and municipal waste are still frequently disposed in landfills whereas in the past they often have been discharged to the sewage water system or to the aquatic environment (frišták and soja, 2015). the application of untreated sewage sludges to agricultural land is regionally either completely banned or tightly controlled. depending on national regulations, mono-incineration of sludge is seen as a promising utilization strategy but the nutrient constituents may be lost if the resulting ash is landfilled. the importance of organic waste and manures recycling to maintain crop production has been recognized by farmers for thousands of years. however, repeated and excess application of these wastes to limited acreage of land has occasionally resulted in excessive nutrient accumulation in soils with subsequent runoff and leaching losses to water bodies, causing water eutrophication and quality deterioration (moreno-jimenez et al., 2016). pyrolysis as a carbonization technique that converts organic materials thermo-chemically into recalcitrant aromatic carbon compounds represents a highly effective and reliable treatment method to avoid bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:05 utc 36 frišták et al. nutrient losses and create a more stable material for fertilization (galamboš et al., 2015; lehmann and joseph, 2015; víglašová et al., 2016). the process requires an inert atmosphere with low or no oxygen and temperatures usually in the range 300-1000°c. for the production of biochar as a nutrient carrier for crops it is necessary to use residual or waste materials (livestock manures, slurries, animal residues, food processing wastes, bones) which need to be monitored for potential contaminations with organic and inorganic xenobiotics. incomplete combustion and pyrolysis also may cause the formation of polycyclic aromatic hydrocarbons (pahs). at lower pyrolysis temperature as a main condition for pah formation dehydrogenation, dealkylation, cyclization and aromatization of present polymers can be observed (hilber et al. 2012). simoneit (1998) mentioned the effect of high pyrolysis temperature on organic compounds from feedstock via cracking into small and highly reactive free radicals followed by pyrosynthesis. in this process free radicals form more stable aromatic structures through recombination reactions. however, the effect of feedstock composition is still not well understood. hale et al. (2012) described this factor as main determinant in the assessment of the aromatic compound formation potential during the carbonization processes. authors showed the general trends in pahs formation linked to higher production temperatures. as we know moisture of feedstock and oxygen level as well contribute to pahs formation during pyrolysis process. finally pyrolysis time represent important parameter which could enhances the pahs formation. zielenska and oleszczuk (2015) as well hossain et al. (2011) discussed the effect of pyrolysis temperature and different feedstock composition on the creation of pahs in sewage sludge-derived biochars. additionally, pyrolysis products can be also contaminated during pyrolysis by recondensation of pyrolysis vapours (buss and mašek, 2014). table 1. comparison of selected physico-chemical properties of naphthalene with 1and 2 methylnaphthalene. property naphthalene 1-methylnaphtalene 2-methylnaphtalen chemical structure molecular weight 128.19 142.20 142.20 melting point 80.50 °c -22°c 34.6°c boiling point 218°c 244.6°c 241°c density at 20°c 1.145 g/ml 1.0202 g/ml 1.0058 g/ml solubility in water at 25°c 31.7 mg/l 25.8 mg/l 24.6 mg/l soluble in organic agents alcohol, benzene, ether, acetone alcohol, ether, benzene alcohol, ether, benzene log kow 3.29 3.87 3.86 log koc 2.97 3.39 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:05 utc nova biotechnologica et chimica 15-1 (2016) 37 the application of biochars or pyrolysis products as soil fertilizers with critical concentrations of aromatic compounds can lead to increasing pahs concentrations fixed on soil structures. therefore the screening of hazardous aromatic compounds is relevant and much in demand. generally, in most existing biochar quality standardization protocols the 16 epa-pahs of the united states environmental protection agency (usepa, 2000) have been established as a main quality control parameter to evaluate the potential risk of aromatic compounds in soil amendments for soil contamination. however, recently the scientific discussions have shifted their focus also on methylated structures of naphthalene (table 1) which could be analyzed in char products from animal residues and bones (wu et al., 2012). these aromatics are present naturally in cigarette smoke, wood smoke, tar, and asphalt. they represent relevant structures which could be included in the assessment of pahs contamination as an indicator for clean production conditions. the main aim of this study was to quantify the content of 16 epa pahs, 1methylnaphthalene and 2-methylnaphthalene as new structures in pyrolysis products produced from bone meal, biogas slurry and chicken manure and finally evaluate the potential risk for plant seed germination. 2. material and methods 2.1 feedstock preparation and pyrolysis commercially available materials such as bone-meal (bm), biogas slurry (bs) and chicken manure (cm) were oven dried or lyophilised (table 2). pyrogenic carbonaceous materials (pcm) were produced by slow pyrolysis from all three treated feedstocks at two temperatures (400 and 500°c). thermochemical conversion was carried out at a residence time of 120 min in a modified lab-scale pyrolysis reactor with nitrogen as flush gas (frišták et al., 2014). after pyrolysis process the resulting products with production yields 30 40 % were ground and sieved to achieve a fraction <200 μm. the obtained pyrolyzed samples of bone-meal (bm-pcm), biogas slurry (bs-pcm) and chicken manure (cm-pcm) were directly stored in polypropylene boxes under dry and cool (4 °c) conditions for further characterization and experiments. table 2. feedstock characterization, pre-treatment and conditions of pyrolysis process. sample feedstock pre-treatment pyrolysis conditions bm 400 bone-meal drying 60°c/72 h 400°c / 2h / n2 bm 500 bone-meal drying 60°c/72 h 500°c / 2h / n2 bs 400 biogas slurry lyophilisation 400°c / 2h / n2 bs 500 biogas slurry lyophilisation 500°c / 2h / n2 cm 400 chicken manure lyophilisation 400°c / 2h / n2 cm 500 chicken manure lyophilisation 500°c / 2h / n2 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:05 utc 38 frišták et al. 2.2 pyrolysis products characterization the active ph values of samples were measured after mixing the samples with deionized water (ratio 1:15 m/v) for 1 h and 1 h of stabilization. electrical conductivities (ec) of samples were measured in deionized water (1:10 m/v) after 24h of mixing. the total contents of c, h, n and s in samples were determined using a chns-o elemental analyzer (chns-o ea 1108, carlo erba instruments, italy). total cd, cu and pb concentrations in materials were determined using icp-ms (perkin elmer, elan drce 9000, usa) after wet digestion with hno3 and h2o2 (enders and lehmann, 2012). 2.3 determination of pahs in pyrolysis products the total concentrations of the 16 us-epa pahs in pyrogenic carbonaceous materials were determined by soxhlet extraction (toluene, solid/liquid: 1/15, 6h, 120°c) and a modified method described by hilber et al. (2012). for comparison ethyl-acetate (solid/liquid: 1/15, 6h, 80°c) as extraction agent was used as alternative extractant. the obtained toluene and ethyl-acetate extracts were evaporated to 1 ml using a rotary vacuum concentrator. after purification and filtration, the concentrated extracts were analysed by hplc coupled with dad (λ = 310 nm) and fld (ex = 260 nm, em = 350 nm, 420 nm, 440 nm, 500 nm) detectors. 16 us-epa pahs standard solution (merck, germany) was used to prepare internal standard solutions of known concentrations. fig. 1. characteristic chromatogram of a standard with 2 mg l-1 1-methylnaphtalene (—) and 4 mg l-1 2-methylnaphtalene (—). the chromatographic apparatus consisted of a agilent 1100er series model, degasser g1379a, quaternary pump g1311a, column grace/vydac® c18 polymeric reversed-phase, 4.6 × 250 mm, 5 μm and grace/ vydac® c18 guard lu min bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:05 utc nova biotechnologica et chimica 15-1 (2016) 39 column, 4.6 × 7.5 mm, 5μm. the composition of mobile phase as a mixture of ultrapure water and acetonitrile varied during time of analysis. the determination of 1and 2-methylnaphthalene was done in the extracts of bmpcm, bspcm and cmpcm with the analogue design of hplc coupled to fld detector (ex = 260 nm, em = 350 nm). standard solutions of 1and 2-methylnaphtalene (250 mg l-1, merck, germany) were used to prepare internal mix solutions at known concentrations. the limit of detection (lod) and limit of quantification (loq) for both substances were determined as 8 μg l-1 and 20 μg l-1, respectively. good repeatability (rsd 0.4 % (1methylnaphthalene) and 1.3 % (2-methylnaphthalene) and linearity of the applied method was confirmed for the range 0.05-125 mg l-1. characteristic chromatograms of studied substances are shown in fig. 1. 2.4 germination test the potentially phytotoxic effects of pyrolysis products were assessed with a germination test according to a standardized method (önorm s2023). briefly, test materials bm-pcm 400, bm-pcm 500, bs-pcm 400, bs-pcm 500 cm-pcm 400 and cm-pcm 500 were mixed with the standard substrate (mixture of einheitserde ed63 (natural clays, peat, block peat) and sand; 9:1) in proportions of 10 and 30% and moistened to 60 % water holding capacity. the substrate samples were placed in pots according to the experimental design shown in fig. 2, followed by incorporation of 30 cress seeds (lepidium sativum l.). the germination rate was determined during the first 6 days of growth under greenhouse conditions (22°c, light period l/d 12/12h) and constant water content. fig. 2. experimental design of pot layout for cress-germination test. 3. results and discussion 3.1 elemental characteristics of pyrolysis products basic physico-chemical characterizations of pyrogenic carbonaceous materials bm-pcm 400, bm-pcm 500, bs-pcm 400, bs-pcm 500 cm-pcm 400 and cmbereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:05 utc 40 frišták et al. pcm 500 showed slight differences in ph and ec between samples prepared at 400 and 500°c. generally ph increased in the order of bm-pcm (9.9-10.0) < cm-pcm (10.1-10.2) < bs-pcm (10.4-10.5). table 3. elemental analyses of studied pyrolysis products; for sample abbreviations see table 2. sample c* (%) h* (%) n* (%) s* (%) cd mg kg-1 cu mg kg-1 pb mg kg-1 bm-pcm 400 30.3 2.5 5.2 2.3 <0.01 19.04 ± 0.51 <1 bmpcm 500 33.9 1.3 5.0 1.8 0.11 ± 0.01 21.10 ± 1.24 <1 bspcm 400 35.3 2.6 2.1 12.0 0.37 ± 0.02 80.08 ± 0.47 8.44 ± 0.47 bspcm 500 29.7 1.9 1.8 9.6 0.43 ± 0.02 81.47 ± 0.21 10.07 ± 0.45 cmpcm 400 44.8 2.6 4.4 8.7 0.63 ± 0.02 98.54 ±1.27 2.77 ± 0.02 cmpcm 500 46.3 1.6 4.1 8.4 0.64 ± 0.01 110.01 ±1.98 3.09 ± 0.01 *values represent means of 5 material measurements with sd lower than 1% determination ec values showed values 4.75 ± 0.25 ms cm-1 for bm-pcm, 4.08 ± 0.12 ms cm-1 for cm-pcm and 10.36 ± 0.58 ms cm-1 for bs-pcm. the trends in ph and ec values of pyrolysis products reflect similar trends in feedstocks characteristics (data not shown). elemental analyses (table 3) confirmed differences in c, h, n and s contents of materials prepared from different bio-waste feedstocks. as the pyrolysis temperature increased, the total carbon concentrations of bone meal and chicken manure-derived pyrogenic carbonaceous material increased, while hydrogen, nitrogen and sulphur decreased. at higher pyrolysis temperatures some amorphous carbon could have been converted into aromatic carbon (keiluweit et al., 2010; ren et al., 2016). additionally, with rising temperature an increased volatilization of proteins and decomposition of remaining lignocelluloses can be observed (tsai et al., 2012). ren et al. (2016) showed after pyrolysis of pig manure increases in total c content, dependent on pyrolysis temperature. their pig manure-derived materials pyrolyzed at 300°c contained more than 62 % total c. as we realized the chicken manure feedstock contained a variable amount of straw which could affect total carbon content in pyrolysis products. in our study pcm-products produced by thermochemical conversion of biogas slurry showed slightly increasing total c, h, n and s contents at lower temperature. these heterogeneous mixtures which are characterized by comparable compositions to sewage sludges apparently contain more volatile compounds and have potentially lower lignocellulosic content. by pyrolysis of these materials a carbon content of 28-35 % can be reached in the pyrogenic products, depending on temperature. additionally, table 3 shows the total cd, cu and pb concentrations which were 1.5 2 folds higher compared to metal contents in feedstocks due to h, n and o losses. organic wastes such as biosolids, slurries and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:05 utc nova biotechnologica et chimica 15-1 (2016) 41 sludges are generally known to contain high concentrations of potentially toxic elements (ptes) which remain in the final pyrogenic products (lu et al., 2013). cely et al. (2015) showed similar contents for comparison of different manure-based pyrolysis products and evaluation of their agronomic properties. our results revealed relatively high sulphur and lead contents in bs-pcm samples which can be one of the factors to restrict the application of these heterogeneous materials as soil amendments. elevated concentrations of ptes in soil fertilizers can affect seed germination and plant metabolism. most of the ptes concentrations in studied materials were lower as thresholds permitted by european biochar certificate ebc (2012) such as cd <1.5 mg kg-1, cu <100 mg kg-1 and pb <150 mg kg-1. singla et al. (2014) found in digested slurry-derived pyrolytic materials produced at 330-370°c concentrations of 0.4 mg cd kg-1, 76 mg cu kg-1 and 2.5 mg pb kg-1. 3.2 pah concentrations polycyclic aromatic hydrocarbons are produced during pyrolysis process of lignocellulose materials and are dependent on pyrolysis temperature (lehmann and joseph, 2015). high temperatures (>600°c) during pyrolysis of plant residues were reported to be propitious for reducing the content of pahs due to nuclear condensation into large, non-extractable sheets and strong sorptive retention by condensed phases (qiu et al., 2016). however, in case of more heterogeneous feedstocks such as slurries, sludges and manures this trend can be completely different. our analyses revealed quite variable concentrations of the sum of 16 pahs in bone meal(bmpcm), biogas slurry(bs-pcm) and chicken manure(cm-pcm) derived pyrogenic carbonaceous materials produced at 400 and 500°c (table 4). we could not confirm the direct dependence of 16 us-epa pahs concentrations on the temperature of thermochemical conversion. table 4. mean concentrations of σ16 epa pah compounds, 1and 2methylnaphthalene in toluene extracts of bone meal(bm-pcm), biogas slurry(bs-pcm) and chicken manure(cm-pcm) derived pyrogenic carbonaceous materials produced at 400 and 500°c. sample σ16 epa pahs 1-methylnaphtalene 2-methylnaphtalene bmpcm 400 4.87 ± 0.47 0.69 ± 0.03 2.51 ± 0.04 bmpcm 500 5.56 ± 0.58 1.16 ± 0.08 2.83 ± 0.08 bspcm 400 37.97 ±2.13 8.76 ±0.45 20.35 ± 0.84 bspcm 500 24.20 ± 2.45 2.96 ± 0.98 5.43 ± 1.85 cmpcm 400 6.18 ± 0.41 1.86 ± 0.08 7.34 ± 0.55 cmpcm 500 3.35 ± 0.12 1.81 ± 0.11 5.08 ± 0.13 samples produced from bone meal and chicken manure showed generally lower levels than the limits established in the european biochar certificate for basic quality but not for premium quality (ebc, 2012). on the other hand, samples bs-pcm 400 and bs-pcm 500 exceeded the basic quality thresholds for the sum of 16 us-epa pahs, with the highest concentrations for phenanthrene, fluorene, naphthalene and chrysene. naphthalene was the major contributor (>65%) to the total pahs content in bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:05 utc 42 frišták et al. samples derived from biogas slurry. the extraction efficiency tested by toluene and ethyl-acetate extraction protocols showed similar and comparable results (r2 0.87). 3.3 concentrations of 1-methylnaphthalene and 2-methylnaphthalene methylated pahs such as 1and 2methylnaphthalene provide 10-20 times lower degradation efficiency and thus are more persistent in water and aquatic ecosystems compared to non-methylated naphthalene (heitkamp and cerniglia, 1987). determination of concentrations of both chemical compounds (fig. 4) in toluene and ethyl-acetate extracts of studied samples showed similar trend as in σ16 us-epa pahs contents. generally, contents of 1-methylnaphthalene and 2-methylnaphtalene increased in order bm-pcm < cm-pcm < bs-pcm. fig. 4. characteristic chromatograms of standard 1-methylnaphtalene (—) at concentration 2 mg l-1, 2methylnaphtalene (—) at concentration 4 mg l-1 and ethyl acetate extract of bs-pcm produced 500°c (—). the highest concentrations were determined in samples produced from biogas slurry and chicken manure at 400°c. concentrations of methylated structures represented 35-205 % of the sum of 16 us-epa pahs in the analysed samples. this fact showed a potential risk to be considered in the assessment of carbonaceous materials as soil fertilizers. for comparison, shackley et al. (2012) found concentrations of 1.77 and 1.36 mg kg-1 for 1-methylnaphthalene and 2methylnaphtalene, respectively, in rice husk char produced by gasification process. however, standardized concentration thresholds for these chemical structures in potential soil fertilizers produced from heterogeneous materials such as sludges or slurries are still missing and provide a new question in environmental policy for this kind of products. 3.4 germination test of pyrolysis products germination rates of cress seeds (lepidium sativum l) were affected by all tested materials when 10 or 30% were added to the growth substrate (fig. 5). the higher lu min bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:05 utc nova biotechnologica et chimica 15-1 (2016) 43 application rate of pyrolysis materials intensified adverse effects or reduced positive effects on cress germination. in comparison with control (non-amended sample) cmpcm400 and cm-pcm500 samples in 10% application rate showed positive effects and bs-pcm400, bs-pcm500 negative effect on seeds germination. at samples bmpcm400, bm-pcm500 no significant change was observed compared to control. 30% application rate showed 10-20% reduction compared to an addition rate of 10%. positive or neutral effects on cress germination were confirmed at cm-pcm500 and cm-pcm400 treatments. on the other hand, bs-pcm400, bs-pcm500, bmpcm400, and bm-pcm500 showed significantly negative effects on germination rates. this trend is in accordance with the chemical characterizations of pah concentrations and metals (such as cd and cu) that apparently pose a risk by releasing germination inhibitors when pcms are applied in such high concentrations as in these tests. bmpcm 400 bmpcm 500 bspcm 400 bspcm 500 cmpcm 400 cmpcm 500 c 0 20 40 60 80 100 g er m in at io n (% ) 10% application rate 30% application rate fig. 5. effects of 10 and 30% application rates of bone meal-, biogas slurryand chicken manure-derived pyrogenic carbonaceous materials on germination rate (%) of cress seeds after 6 days. values represent means ± sd (n=3). a phytotoxic influence of mobile organic compounds on cress germination was extensively described by buss and mašek (2014). the authors identified specific volatile organic compounds (vapours) and pyrolysis liquids (mainly crude-oils) which could play a role in inhibition of biochemical and biological processes. as mentioned before, pyrolysis products could be contaminated by production and re-condensation of various organic compounds during the pyrolysis process. subsequently, a solid pyrolysis product can release organic structures which may become toxic for biological systems and can have an adverse effect on germination. based on these scenarios, it is required to take a closer look on product characteristics not only of the solid but also of the liquid and gaseous fraction. more volatile forms such as phenolic substances could be relevant for phytotoxic effect evaluation (lentz and ippolito, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:05 utc 44 frišták et al. 2012). due to their micro-structure and high porosity, biochars and pyrogenic carbonaceous products provide a large storage potential for such substances. the study of new structures (pahs, phenolic species) which could be relevant during the carbonization process should be incorporated into the quality control of new pyrolysis products which are intended as soil amendments. 4. conclusions re-condensation of liquid and gaseous substances during the pyrolysis process resulted in pyrogenic carbonaceous materials with partially high content of organic compounds that could have phytotoxic effect. the quantification of standardized sum of 16-usepa pahs and methylated naphthalene structures showed a potential risk in utilization of heterogeneous waste materials such as biogas slurry for fertilizer production. our study confirmed for concentrations of pahs the possibility to comply with ebc thresholds (4 or 12 mg kg-1 resp.) for materials produced from chicken manure and bone meal. these materials showed no-toxic effects in germination tests with cress seeds. when using biogas slurry as feedstock, pah accumulation in the pyrolysis product has to be carefully observed and effectively avoided by advanced management of volatiles in the reactor. acknowledgment: the authors thank the austrian ffg for financial support of the research studio ferti-mine, project nr. 844744. references buss, w., mašek, o.: mobile organic compounds in biochar – a potential source of contamination – phytotoxic effects on cress seed (lepidium sativum) germination. j. environ. manage., 137, 2014, 111-119. cely, p., gascó, g., paz-ferreiro, j., méndez, a.: agronomic properties of biochars from different manure wastes. j. anal. appl. pyrolysis., 111, 2015, 173182. enders, a., lehmann, j.: comparison of wet-digestion and dry-ashing methods for total elemental analysis of biochar. commun. soil sci. plan., 43, 2012, 10421052. frišták, v., soja, g.: effect of wood-based biochar and sewage sludge amendments for soil phosphorus availability. nova biotechnol. chim., 14, 2015, 104-104. galamboš, m., daňo, m., víglašová, e., krivosudský, l., rosskopfová, o., novák, i., berek, d., rajec, p.: effect of competing anions on pertechnetate adsorption by activated carbon. j. radioanal. nucl. chem., 304, 2015, 1219-1224. hale, s.e., lehmann, j., rutherford, d., zimmermann, a.r., bachmann, r.t., shitumbanuma, v., o`toole, a., sundqvist, k.l., arp, h.p.h., cornelissen, g.: quantifying the total and bioavailable polycyclic aromatic hydrocarbons and dioxins in biochars. environ. sci. technol., 46, 2012, 2830-2838. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:05 utc nova biotechnologica et chimica 15-1 (2016) 45 heitkamp, m.a., cerniglia, c.e.: effects of chemical structure and exposure on the microbial degradation of polycyclic aromatic hydrocarbons in fresh water and estuarine ecosystems. environ. toxicol. chem., 6, 1987, 535546. hilber, i., blum, f., leifeld, j., schmidt, h.p., bucheli, t.d.: quantitative determination of pahs in biochar: a prerequisite to ensure its quality and safe application. j. agric. food chem., 60, 2012, 3042-3050. hossain, m.k., strezov, v., yin chan, k., nelson p.f.: agronomic properties of wastewater sludge biochar and bioavailability of metals in production of cherry tomato (lycopersicon esculentum). chemosphere, 78, 2010, 1167-1171. keiluweit, m., nico, p.s., johnson, m.g., kleber, m.: dynamic molecular structure of plant biomass-derived black carbon (biochar). environ. sci. technol. 44, 2010, 1247-1253. lehmann, j., joseph, s.: biochar for environmental management: science, technology and implementation. earthscan from routledge: london, uk, 2015, 944pp. lentz, r.d., ippolito, j.a.: biochar and manure affect calcareous soil and corn silage nutrient concentrations and uptake. j. environ. qual., 41, 2012, 1033-1043. lu, h., zhang, w., wang, s., zhuang, l., yang, y., qiu, r.: characterization of sewage sludge-derived biochars from different feedstocks and pyrolysis temperatures. j. anal. appl. pyrol., 102, 2013, 137-143. moreno-jimenez, e., fernández, j.m., puschenreiter, m., williams, p. n., plaza, c.: availability and transfer to grain of as, cd, cu, ni and zn in a barley agri-system: impact of biochar, organic and mineral fertilizers. agric. ecosyst. environ., 219, 2016, 171-178. önorm s 2023: untersuchungsmethoden und güteüberwachung von komposten. front cover. österreichisches normungsinstitut, 1993 16 pages. qiu, m., sun, k., jin, j., han, l., sun, h., zhao, y., xia, x., wu, f., xing, b.: metal/metalloid elements and polycyclic aromatic hydrocarbon in various biochars: the effect of feedstock, temperature, minerals, and properties. environ. pollut., 206, 2015, 298-305. ren, x., sun, h., wang, f., cao, f.: the changes in biochar properties and sorption capacities after being cultured with wheat for 3 months. chemosphere, 144, 2016, 2257-2263. shackley, s., carter, s., knowles, t., middelink, e., haefele, s., sohi, s., cross, a., haszeline, s.: sustainable gasification-biochar systems? a case-study of rice-husk gasification in cambodia, part i: context, chemical properties, environmental and health and safety issues. energy pol., 41, 2012, 618623. simoneit, b.r.: biomarker pahs in the environment. in neilson, a.h. (ed.) pahs and related compounds. springer, berlin, 1998, pp. 175-221. singla, a., dubey, s.k., singh, a., inubushi, k.: effect of biogas digested slurry-based biochar on methane flux and methanogenic archael diversity in paddy soil. agric. ecosyst. environ. 197, 2014, 278-287. us-epa: process design manual, land application of sewage sludge and domestic septage. office of research and development, washington, dc, united states environmental protection agency, epa/625/r-95/001, 1995. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:05 utc 46 frišták et al. víglašová, e., daňo, m., galamboš, m., rosskopfová, o., rajec, p., novák, i.: column studies for the separation of 99mtc using activated carbon. j. radioanal. nucl. chem., 307, 2016, 591-597. wu, x., markham, j., sun, x., wang, d.: optimizing catalytic fast pyrolysis of biomass for hydrocarbon yield. transactions of asabe, 55, 2012, 1879-1885. zielińska, a., oleszczuk, p.: the conversion of sewage sludge into biochar reduces polycyclic aromatic hydrocarbon content and ecotoxicity but increases trace metal content. biomass bioenerg., 75, 2015, 235-244. received 23 march 2016 accepted 6 july 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:05 utc microsoft word pristas nbc 2015.doc 62 nova biotechnologica et chimica 14-1 (2015) doi 10.1515/nbec-2015-0015 © university of ss. cyril and methodius in trnava non-ferrous metal industry waste disposal sites as a source of polyextremotolerant bacteria peter pristas1, 2, zuzana stramova3, simona kvasnova4, jana judova4, zuzana perhacova5, barbora vidova6, zuzana sramkova6, andrej godany6 1institute of animal physiology, slovak academy of sciences, soltesovej 4-6, 04001 kosice, slovakia (pristas@saske.sk) 2institute of biology and ecology, faculty of science, pavol josef safarik university, srobarova 2, 04154 kosice, slovakia 3institute of chemistry, faculty of science, pavol josef safarik university, srobarova 2, 04154 kosice, slovakia 4faculty of natural science, matej bel university, tajovskeho 40, 97401 banska bystrica, slovakia 5faculty of ecology and environmental sciences, technical university in zvolen, t. g. masaryka 24, 96001 zvolen, slovakia 6department of biology, faculty of natural sciences, university of ss. cyril and methodius in trnava, j. herdu 2, trnava, sk-917 01, slovakia abstract: waste disposal sites from non-ferrous metal industry constitute environments very hostile for life due to the presence of very specialized abiotic factors (ph, salt concentration, heavy metals content). in our experiments microflora of two waste disposal sites in slovakia – brown mud disposal site from aluminium production near ziar nad hronom and nickel sludge disposal site near sered was analyzed for cultivable bacteria. isolated bacteria were characterized by a combination of classical microbiological approaches and molecular methods and the most of isolated bacteria shown a poly-extremotolerant phenotype. the most frequently halotolerant (resistant to the high level of salt concentrations) and alkalitolerant (resistant to the high ph level) bacteria belonging to the actinobacteria class were detected. the most of bacteria shown very high level of heavy metal resistance e.g. more than 500 µg/ml for zn2+ or cu2+. based on our data, waste disposal sites thus on one side represents an important environmental burden but on other side they are a source of new poly-extremotolerant bacterial strains and species possibly used in many biotechnology and bioremediation applications. key words: metal industry, waste, heavy metals, environment, extremotolerant bacteria, biotechnology 1. introduction during billions years of evolution bacteria have adapted to nearly all possible environments on earth and evolutionary changes in the prokaryotes focused on metabolic diversity and the genetic capacities to explore and eventually colonize every conceivable environment on earth, including extreme environments (horikoshi and grant, 1998). extremophile bacteria not only survive, but thrive under extreme conditions. this contrasts with an extremotolerant bacteria that may tolerate and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:07 utc nova biotechnologica et chimica 14-1 (2015) 63 survive extreme conditions, but grows optimally under less extreme conditions. extremotolerant bacteria can resist extreme temperatures, ph values, salinity, radioactivity, high concentration of heavy metals etc. which are detrimental to the majority of life on earth. there are a variety of natural extreme environments on earth that have very specialized abiotic factors. these habitats can be found in deep marine settings, earth crust interiors, highly mineralized soils, extreme radiation surroundings or extreme aquatic chemical environments e.g. hydrothermal deep-sea vents, thermal deep-sea black smokers, hot springs, soda lakes, salt pans etc. (pikuta et al., 2007). with the advent of industrial technologies a new type of extreme environments appeared with conditions unseen before. especially waste disposal sites from nonferrous metal industry constitute environments very hostile for life. mining and ore processing consists of extracting minerals and metals from surrounding earth and ore. the extracted ore is then sent for crushing, washing, and various physical or chemical separation processes, like smelting, leaching, electrolyzing etc. through the extraction and subsequent mineral processing, metals and metal compounds tend to become chemically more available, which can result in the generation of acid or alkaline drainage. in each stage of these operations the undesirable constituents are discarded as liquid or solid wastes. the residual waste frequently contains the toxic processing chemicals and/or elevated levels of metals (adriano, 2001) and it is disposed by landfill which led to the pollution of environment especially groundwater. in our experiments cultivable bacteria of two waste disposal sites in slovakia – brown mud disposal site from aluminium production near ziar nad hronom (schwarz and lalik, 2012) and nickel sludge disposal site near sered (michaeli et al., 2012) were analyzed for the resistance to the extreme conditions. 2. materials and methods 2.1 sample collection, isolation, growth, and identification of bacteria brown mud samples, samples of drainage water, and nickel sludge samples were collected from the slovalco co. disposal site near ziar nad hronom (slovakia) and nickel sludge disposal site of the nickel smelter near sereď (slovakia), respectively. solid samples were taken from at least two different locations and mixed together, the water sample was taken from a reservoir of drainage water. the samples were immediately brought to the laboratory and stored in the cold (4 ºc) until microbiological analysis. ph of the brown mud sample was determined according to the iso 10390:2005 standard. to the 0.5 g of solid sample 10 ml of sterile phosphate-buffered saline solution were added and after 20 min of intensive mixing aliquots were spread on non-selective agar medium (tsa tryptone soya agar, oxoid, usa). the samples of drainage water (50 µl per plate) were directly spreaded on tsa plates. cultivation was conducted under aerobic conditions at 22 °c for 24 72 h. the individual isolates were selected on the basis of cell and colony morphology and used for further analyses. after repeated subculturing and control for the purity, the isolates were typed and identified by the maldi tof (matrix-assisted laser desorption ionization time-offlight) mass spectrometry approach as specified recently (kopcakova et al., 2014). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:07 utc 64 pristas, p. et al. 2.2 heavy metal resistance analysis the minimal inhibitory concentrations (mic) of the metals of isolated bacteria were determined by the plate dilution method as adopted by aleem et al. (2003). the lowest concentration that prevented bacterial growth was considered the mic. solid nutrient medium 2 (oxoid, usa) was used for heavy metal resistance determination in order to minimize complexation of the heavy metal ions tested. 2.3 nacl and ph resistance analysis for determining the salt tolerance of the isolated bacteria, they were streaked on tsa agar supplemented with increasing concentration of nacl – 3 %, 5 %, and 10 % and cultivated for 24 hours at 22 °c. after the appearance of colonies the isolates were scored positive or negative for their ability to grow in different concentration of nacl. similar approach was used to test the ph tolerance of the isolated bacteria. the bacteria were streaked on buffered tsa plates (tsa medium with 100 mm trishcl, ph 7.5 or 10), and the growth was scored after 24 hours cultivation at 22 °c. 3. results and discussion the brown mud disposal site from aluminium production near ziar nad hronom and nickel sludge disposal site of the nickel smelter near sered belong to the most dangerous landfills in slovakia. brown mud is disposed as a slurry having a solid concentration in the range of 10-30 %, ph in the range of 13 and high ionic strength (schwarz and lalik, 2012). the nickel sludge contains of about 80 % of fe and high concentrations of other metals e.g. 3.5 % of cr2o3 and 0.17 % of ni (michaeli et al., 2012). despite extremely harsh conditions (extreme ph values and heavy metal content in brown mud disposal site from aluminium production or high heavy metal content in nickel sludge) relatively high numbers of bacteria were recovered (table 1) from these environments. the highest bacterial counts were detected in nickel sludge (32 000 cfu/g), the lowest in highly alkaline drainage water of brown mud landfill from aluminium production (80 cfu/ml). it seems there is a correlation between alkalinity and number of cultivable bacteria in studied ecosystems. in natural ecosystems such extreme ph values are very rare and for example in the lonar lake in india, having ph 10.5, the total number of cultivated bacteria was found to be between 102-106 cfu/ml (joshi et al., 2006). isolated bacteria were characterized by a combination of classical microbiological approaches (gram reaction, cell and colony morphology, catalase activity, spore formation, h2s production, and ability to grow on tsa plates at 22 and 37 °c) and maldi tof analysis. maldi tof analysis identified the highest variability of bacteria in the brown mud sludge (at least 16 taxa) followed by nickel sludge (at least 8 taxa) and the drainage water sample (at least 6 taxa). however, only limited number of isolates could be identified using maldi tof analysis (kopcakova et al., 2014). further microbiological analyses shown that in all tested samples grambereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:07 utc nova biotechnologica et chimica 14-1 (2015) 65 positive bacteria dominate (table 1). while the nickel sludge and drainage water was dominated by actinobacteria, the brown mud sample was dominated by firmicutes (low g+c content gram-positive bacteria). in both brown mud and nickel sludge actinomycetes were detected and identified as streptomyces variabilis and s. malaysiensis for isolates from brown mud and as nocardiopsis alba for isolate from nickel sludge respectively (data not shown). similar halo-alkaliphilic actinomycetes such as nocardiopsis sp. and streptomyces sp. were collected e.g. from the mud soil of solar salt works in india (jose and jebakumar, 2012). table 1. characteristics of studied environments and occurrence of poly-extremotolerant phenotypes in isolated bacteria. brown mud disposal site near ziar nad hronom characteristics drainage water brown mud nickel sludge disposal site near sered ph cultivable bacteria counts total number of isolates gram-positive actinobacteria actinomycetes gram-negative number of species dominant genus frequency of isolates growing at: ph=7.5 ph=10.0 3 % nacl 5 % nacl 10 % nacl 500 µg/ml zn2+ 500 µg/ml ni2+ 200 µg/ml co2+ 500 µg/ml cu2+ 13.1 80 cfu/ml 12 10 7 0 2 6 microbacterium 12/12 12/12 12/12 11/12 5/12 0/12 0/12 0/12 0/12 11.6 3 500 cfu/g 21 18 8 2 3 16 bacillus 21/21 19/21 21/21 8/21 2/21 19/21 14/21 14/21 14/21 8.1 32 000 cfu/g 24 24 21 1 0 8 arthrobacter 24/24 22/24 24/24 23/23 0/23 24/24 3/24 23/24 24/24 microflora of metal industry waste disposal sites is not well understood and in the study of microflora of bauxite residue treated by using various added nutrients hamdy and williams (2001) reported the presence of multiple bacterial genera including firmicutes and actinobacteria (e.g. bacillus, lactobacillus, leuconostoc, micrococcus, staphylococcus, pseudomonas, flavobacterium, and enterobacter). bacterial population in alkaline lonar lake was dominated by firmicutes, followed by γ-proteobacteria, and α-proteobacteria. actinobacteria were found to be the least bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:07 utc 66 pristas, p. et al. represented class of bacteria (joshi et al., 2006). in extremely alkaline (ph > 12) ground water of the lake calumet area of chicago, illinois, where historic dumping of steel slag has filled in a wetland, the presence and growth of a variety of alkaliphilic βproteobacteria, bacillus, and clostridium species was detected (roadcap et al., 2006). in this report the non-cultivation based approach was used however. most of the isolated bacteria have shown poly-extremotolerant phenotype. the most frequently halotolerant (resistant to the high level of salt concentrations) and alkalitolerant (resistant to the high ph level) bacteria belonging to the actinobacteria class were detected (table 1). the highest level of haloand alkalitolerance was observed among isolates from the drainage water. the isolates from solid wastes have shown similar degree of alkalitolerance despite dramatically different ph in these environments (11.6 in brown mud versus 8.1 in nickel sludge). surprisingly, two isolates from the brown mud (both gram-negative) were unable to grow at ph 10 under in vitro conditions. the lowest salt tolerance was observed in isolates from nickel sludge. in both waste disposal sites, relatively high concentrations of heavy metals were detected. brown mud heavy metal content is 10 ppm for hg, 220 ppm for cu, 400 ppm for cr, 700 ppm for v, 150 ppm for pb and 800 ppm for as (kopcakova et al., 2014). the average heavy metal content of nickel sludge is fe (45.9 %), cr2o3 (2.1 %), sio2 (15.0 %), al2o3 (4.1 %), cao (3.5 %), nio (0.2 %), mgo (2.3 %) (michaeli et al., 2012). heavy metals may exert an inhibitory action on microorganisms by blocking essential functional groups, displacing essential metal ions, or modifying the active sites of biological molecules (moffett et al., 2002). numerous studies have examined the heavy metal sensitivity or resistance of bacteria isolated from different habitats and many microorganisms shown adaptation to the toxic metals to which they are exposed (müller et al., 2001). accordingly, most of isolates from brown mud and nickel sludge shown very high levels of resistance against heavy metal present in disposal sites (e.g. more than 500 μg/ml for cu2+ or co2+) as well as to zn2+ used as a control. surprisingly, the nickel resistance was more frequently encountered in the brown mud compared to the nickel sludge isolates. all three actinomycetes belonged among the most heavy metal resistant isolates. heavy metal resistance is frequently occurred in actinomycetes (alvarez et al., 2013). on other hand, the isolates from the drainage water were much more sensitive to the heavy metals, probably as a consequence of extreme ph in drainage water at which most heavy metals form insoluble hydroxides. thus heavy metal concentrations in drainage water are much lower compared to the brown mud (e.g. 0.3 ppm of cu in drainage water compared to 220 ppm of cu in brown mud (unpublished data). in the last years an interest in extremotolerant bacteria increased dramatically either in biotechnology or bioremediation applications. the use of metabolic abilities of extremotolerant bacteria for degradation/removal of environmental pollutants provides an economic and safe alternative compared to other methodologies (perpetuo et al., 2011). based on our data, metal industry waste disposal sites on one side represents an important environmental burden but on other side they are a source of new poly-extremotolerant bacterial strains and species possibly used in many biotechnology and bioremediation applications. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:07 utc nova biotechnologica et chimica 14-1 (2015) 67 4. conclusions despite extremely harsh conditions (extreme ph values and heavy metal content in brown mud disposal site from aluminium production or high heavy metal content in nickel sludge) relatively high numbers of bacteria were recovered from both metal industry disposal sites. at least some of isolated bacteria are poly-extremotolerant. the most frequently halotolerant (resistant to the high level of salt concentrations) and alkalitolerant (resistant to the high ph level) bacteria belonging to the actinobacteria class were detected. the most of bacteria showed very high heavy metal resistance levels. waste disposal sites are a source of new poly-extremotolerant bacterial strains and species possibly used in many biotechnology and bioremediation applications. acknowledgment: this publication is the result of the project no. 26220120001 implementation supported by the research & development operational programme funded by the erdf. references adriano, d.c.: trace elements in terrestrial environments: biogeochemistry, bioavailability, and risks of metals. 2ed. springer verlag, new york, 2001, 867 pp. aleem, a., isar, j., malik, a.: impact of long-term application of industrial wastewater on the emergence of resistance traits in azotobacter chroococcum isolated from rhizosphere soil. bioresour. technol., 86. 2003, 7-13 alvarez, a., catalano, s.a., amoroso, m.j.: heavy metal resistant strains are widespread along streptomyces phylogeny. mol. phylogenet. evol., 66, 2013, 1083-1088. hamdy, m.k., williams, f.s.: bacterial amelioration of bauxite residue waste of industrial alumina plants. j. ind. microbiol. biot., 27, 2001, 228-233 horikoshi, k., grant, w.d.: extremophiles — microbial life in extreme environments. wiley-liss, new york, 1998, 322 pp. jose, p.a., jebakumar, s.r.d.: phylogenetic diversity of actinomycetes cultured from coastal multipond solar saltern in tuticorin, india. aquat. biosyst., 2012, 8, 23. joshi, a.a., kanekar, p.p., kelkar, a.s., shouche, y.s., vani, a.a., borgave, s.b., sarnaik, s.s.: cultivable bacterial diversity of alkaline lonar lake, india. microb. ecol., 2008, 55, 163-172. kopcakova, a., stramova, z., kvasnova, s., godany, a., perhacova, z., pristas, p.: need for database extension for reliable identification of bacteria from extreme environments using maldi tof mass spectrometry. chem. pap., 68, 2014, 1435-1442. michaeli, e., boltižiar, m., solár, v., ivanová, m.: the landfill of industrial waste – lúženec near the former nickel smelter at sereď town as an example of environmental load. životné prostredie, 46, 2012, 63-68 (in slovak). moffett, b., nicholson, a., uwakwe, c., chambers, b., harris, a. hill, j.: zinc contamination decreases the bacterial diversity of agricultural soil. fems microbiol. ecol., 1472, 2002, 1-7. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:07 utc 68 pristas, p. et al. müller, a.k., westergaard, k., christensen, s., sørensen, s.j.: the effect of long-term mercury pollution on the soil microbial community. fems microbiol. ecol., 36, 2001, 11-19. perpetuo, e.a, souza, c.b., nascimento, c.a.o.: engineering bacteria for bioremediation. in: carpi, a. (ed.) progress in molecular and environmental bioengineering – from analysis and modeling to technology applications, intech, croatia, 2011, 606-632. pikuta, e.v., hoover, r.b., tang, j.: microbial extremophiles at the limits of life. crit. rev. microbiol., 33, 2007, 183-209. schwarz, m., lalik, m. possibilities of exploitation of bauxite residue from alumina production. in: nusheh, m. (ed.) recent researches in metallurgical engineering from extraction to forming, intech, croatia, 2012, 3-22. roadcap, g.s., sanford, r.a., jin, q., pardinas, j.r., bethke, c.m.: extremely alkaline (ph > 12) ground water hosts diverse microbial community. ground water, 44, 2006, 511-517. presented at the 3rd international conference “biotechnology and metals 2014”, september 17-19, 2014, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:07 utc nova biotechnologica et chimica 11-2 (2012) 87 doi 10.2478/v10296-012-0009-9 © university of ss. cyril and methodius in trnava secondary iron minerals present in amd sediments from smolník abandoned mine zuzana dakos1, daniel kupka1, michal kovařík1, katarína jablonovská1, václav krištúfek2, marcela achimovičová1 1institute of geotechnics of the slovak academy of sciences, košice, slovak republic,(dankup@saske.sk) 2institute of soil biology of the academy of sciences of the czech republic na sadkach 7, 370 05 české budejovice, czech republic abstract: the genesis of acid mine drainage (amd) is conditioned by existence of indigenous chemolithotrophic iron and sulfur oxidizing bacteria, especially of genus acidithiobacillus. the result of the oxidizing weathering of metal sulfides is a sequential formation of ochreous precipitates in drainage systems and in the surroundings of amd seepage on the surface. the long-term monitoring of amd waters collected at the shaft pech that receives the majority of waters draining the flooded smolník mine area point out the enduring contamination risk of particular components in the environment of smolník mine area. elemental analysis, x-ray diffraction, mossbauer spectroscopy and scanning electron microscopy of the ochreous precipitates formed from smolník amd stream revealed schwertmannite as the dominant solid phase in the precipitates. the chemical analysis of amd effluents and the elemental composition of related sediments indicated considerable scavenging potential of the ochreous precipitates towards metal cations and oxyanions of arsenic and sulfate. key words: acidithiobacillus, acid mine drainage (amd), ochreous precipitates, schwertmannite 1. introduction extremely acidic environment (ph <3) occurs naturally, though they are more frequently associated with human activities, particularly the mining of coals and metal ores (johnson, 1991). the biotic component of these environments is predominantly microbiological. accordingly most research interest has focused on the iron-oxidizing bacteria (genus acidithiobacillus) since their metal mobilizing activities are exploited in the biological processing of sulphidic ores, and also because their oxidation of mineral sulfides is a primary cause of acid mine drainage formation (norris, 1990). the result of microbial oxidation of metal sulfides in oxic zones is the formation of acidic, metal laden drainage water and precipitation of secondary weathering products, primarily schwertmannite and minerals of the jarosite group. the precipitation of ferric iron as fe(iii)-hydroxysulfates is ph-dependent, and the chemical composition of amd effluents also greatly influences the mineralogical composition of forming precipitates (bigham and nordstrom, 2000). the ph range from 3.0 to 4.5 and sulfate concentrations in the range of 1000 to 3000 mg l-1 facilitate the schwertmannite formation, while lower ph values at sufficiently high [so4] 2 concentrations lead to jarosite formation. furthermore, schwertmannite formation does not require the presence of monovalent or divalent cations and removes more ferric iron and fewer sulfates from the solution phase compared to jarosite precipitation bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:43 utc 88 dakos, z. et al. (bigham et al., 1994, bigham, 1994). this work points out the correlation between aqueous geochemistry of amd and ochreous precipitates. it also presents the results of the analysis of selected physical and chemical parameters of mine drainage and associated stream sediments at the abandoned smolník deposit (east slovakia). 2. materials and methods samples of amd were collected from the sampling site at the shaft pech quarterly according to the water act no. 364/204 coll. as amended by the act no. 384/2009 coll. and related legislative documents. the measurement of temperature, ph and redox potential were carried out in the field using a ph electrode and a combined ptag/agcl redox electrode respectively. water samples for the consequent analysis were acidified with concentrated hno3. metals dissolved in amd were measured with atomic absorption spectrometry (aas). total dissolved solids (tds) were determined gravimetrically and the sulfate concentration by nephelometric method with bacl2. ochreous precipitates were collected from the bottom and walls of shaft pech receiving the majority of waters draining the flooded smolník mine area. the precipitates were washed with distilled water. the solid phase was separated by centrifugation, air dried and homogenized before the analysis. the chemical composition of solid samples was determined by aas after digestion with concentrated hno3 and hcl (1:1) in a microwave digester. xrd patterns were measured by philips pw 1820 powder diffractometer with cukα radiation. the jcpds pdf database was utilized for phase identification. mössbauer spectra were taken in transmission geometry at temperature t = 293 k. a 57co/rh γ-ray source was used. the velocity scale was calibrated relative to 57fe in rh. the recoil spectral analysis software was used for the quantitative evaluation of the mössbauer spectra. morphology of the solid precipitates was examined by cryo field-emission sem jsm 7401-f (jeol ltd., tokyo, japan). the density of the precipitates was determined by pycnometric method and specific surface area by bet method (gemini 2360, micromeritics, usa). 3. results and discussion the ph of the water effluent from the shaft pech after the end of flooding (june 1994) was 3.1 and during the following years it increased to 3.7. it does not change either after intense rains. concentrations of some alkaline earth metals, total dissolved solids, sulfates and heavy metals present in these water samples are depicted in fig. 1. the column graphs indicate the amd water quality immediately after the flooding of the smolník mine, compared to average values of the monitored parameters during the period 1995-2010. the third column indicates seasonal variation of individual parameters in connection to intensive rainfall in june 2010. fig. 2 shows ochreous precipitates from the amd. the chemical composition of the precipitate is stated in table 1 and 2. the smolník precipitate sample contained 44.89% fe and 5.53% s, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:43 utc nova biotechnologica et chimica 11-2 (2012) 89 yielding a molar fe/s ratio of 4.7. this value is within the range of naturally occurring schwertmannites (bigham et al., 1994). fe mg 0 200 400 600 800 1000 μ g l-1 after the flooding from 1995to april 2010 after intense rains m g l-1 cu mn al zn ca 0 40 80 120 160 m g l-1 as pb cr sb cd 0 10 20 30 0 4000 8000 12000 16000 tds (so4) 2m g l-1 fig. 1. chemical analysis of water samples from shaft pech fig. 2. ochreous precipitates formed at the amd flows bottoms and rims table 1. chemical analysis of major elements in the ochreous precipitate collected from shaft pech in smolník abandoned mine. the values are in weight %. fe s al ca mg k na 44.89 5.53 0.38 0.1 0.07 1.13 0.02 table 2. chemical analysis of heavy metals in the ochreous precipitate collected from shaft pech in smolník abandoned mine. the values are in ppm. as cu mn zn ni cd pb cr sb 2800 700 210 235 16,3 0.7 1074.5 23.5 102 powder xrd analysis of schwertmannite can be readily distinguished from the associated minerals if specimens are reasonably pure. in the case of mixed assemblage with other minerals it may be difficult because of its poor crystallinity. the powder diffraction pattern of schwertmannite consists of eight broad peaks for d >1.4 å (bigham and nordstrom, 2000). the strongest peak for natural sample (fig. 3) occurs at about 2.54 å (212). although it can be easily confused with the strongest peak of ferrihydrite, but its symmetrical character confirms the presence of schwertmannite as well as the reflection at 1.64 å (522), which is significantly displaced from that at 1.72 å for ferrihydrite. distinct identification of two following bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:43 utc 90 dakos, z. et al. reflections occurring at 2.27 (302) and 1.94 å (113) is difficult, because the d-spacing characteristics for schwertmannite (2.28 and 1.95 å) very close to d-values characteristic for ferrihydrite (2.21 and 1.96 å). similar situation occurs also in the case of reflections exhibited at 1.51 (004) and 1.45 å (204), but in the case of ferrihydrite, the intensity ratios are reversed. furthermore, in contrast to ferrihydrite, schwertmannite exhibits two more reflections at 3.39 and 4.86 å, from which the peak at 3.38 å (310) is detected on the xrd-pattern of natural sample. the d-values mentioned above responding to reference of schwertmannite [47-1775] were also confirmed by rietveld lebail profile matching technique (kupka et al. 2011). 10 20 30 40 50 60 70 80 0 50 100 150 200 250 20 4 00 4 52 2 11 3 30 2 21 2 31 0 in te ns ity 2θ fig. 3. x-ray diffraction analysis of the smolník precipitate gave the characteristic schwertmannite pattern with broad peaks and a relatively high background. the 57fe mössbauer spectrum of natural sample measured at room temperature (fig. 4) consists of an asymmetric doublet that was fitted with a combination of three ferric doublets with following mössbauer parameters (table 3). an average quadrupole splitting 0.69 mm/s of these doublets is characteristic of schwertmannite (murad and cashion, 2004). table 3. mössbauer parameters, isomer shift (is), quadrupole splitting (qs) and area of natural sample at 293 k. phase is [mm/s] qs [mm/s] area [%] doublet 1 0.35 0.89 69 doublet 2 0.35 0.51 16 doublet 3 0.36 0.68 15 the specific density of natural schwertmannite (precipitates settled down on the bottom of the amd effluent channel) was 2.86 ± 0.02 g.cm-3. specific surface area was 4.54 ± 0.06 m2.g-1. sem and edx analysis of schwertmannite is depicted in fig. 5 and 6. sem examination of the precipitate showed characteristic pin-cushion morphology of schwertmannite described by bigham et al. (1994). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:43 utc nova biotechnologica et chimica 11-2 (2012) 91 -4 -2 0 2 4 98,5 99,0 99,5 100,0 t ra ns m is si on [% ] velocity [mm/s] fig. 4. fitted 57fe mössbauer spectrum of the smolník precipitate. fig. 5. sem micrograph of the precipitate sample containing schwertmannite as the dominant mineral phase fig. 6. edx analysis revealed 69 % fe, 5.72 % s, 23.8 % o, 0.46 % al and 0.39 % si content in this mineral phase 4. conclusions this work deals with the correlation between the chemical composition of amd and ochreous precipitates formation in them and their recipients in the presence of indigenous iron-oxidizing bacteria. the parameters of amd effluent from the shaft pech fit well the geochemical conditions which are conducive to iron precipitation as schwertmannite. in regard to the continuous surface and groundwater contamination, the need of solving the problem of water resources contaminated with acid mine drainage effluents is urgent and is calling for immediate attention. the results acquired during this research provide information about the possibilities to use natural processes leading to formation of ochreous precipitates for attenuation of negative impact of amd effluents on the environment. the bacterial iron oxidation step promotes the formation of ferric iron hydroxysulfate minerals that bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:43 utc 92 dakos, z. et al. accommodate large quantities of iron and sulfate ions in their structure. furthermore, the precipitates possess considerable scavenging potential for arsenic and other metal species. fresh amd precipitates are metastable and readily transform to thermodynamically more stable mineral forms. these interconversions modify the mobility and environmental impact of adsorbed and co-precipitated elements (cornell and schwertmann, 2003). immediate separation of ferric sludge could prevent metals migration in both soluble and suspended form downstream from the contaminant source. acknowledgment this work was supported by the operational programme research and development through the project: centre of excellence for integrated research of the earth's geosphere (itms: 26220120064), which is cofinanced through the european regional development fund. and by the slovak grant agency vega, grant no. 2/0166/11). references bigham j.m., carlson, l., murad, e.: schwertmannite, a new iron oxyhydroxysulphate from pyhäsalmi, finland and other localities. mineral. mag., 58, 1994, 641-648. bigham j.m., nordstrom d.k.: iron and aluminum hydroxysulfates from acid sulfate waters. rev. mineral geochem., 40, 2000, 351-403. bigham, j.m.: mineralogy of ochre deposits formed by sulfide oxidation. in: blowes, d.w., lambor, j.l. (eds.), the environmental geochemistry of sulfide mine-wastes. mineralogical association of canada, waterloo, ontario, canada, 1994, p. 103-132. cornell, r.m, schwertmann, u.: the iron oxides. wiley-vch, weinheim, 2003, isbn 3-527-30274-3. 703 p. johnson, d.b.: diversity of microbial life in highly acidic, mesophilic environments. in: diversity of environmental biogechemistry (berthelin, j., ed.), pp. 225-238, 1991, elsevier, amsterdam kupka, d.: effluent water quality and the ochre deposit characteristics of the abandoned smolník mine, east slovakia, acta montanistica (in press). murad, e., cashion, j.: mössbauer spectroscopy of environmental materials and their industrial utilization. springer, boston/norwell, massachusetts, usa, 2004, p. 418 norris, p.r.: acidophilic bacteria and their activity in mineral sulphide oxidation. in: microbial mineral recovery (ehrlich, h.l. and brierley, c.l., eds.), pp. 3-27, 1990, mcgraw-hill, new york. presented at the 2nd international conference “biotechnology and metals 2011”, september 22–23, 2011, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:43 utc nova biotechnologica et chimica 11-2 (2012) 153 doi 10.2478/v10296-012-0018-8 © university of ss. cyril and methodius in trnava 137cs uptake and translocation in leafy vegetable: a study with lactuca sativa l. grown under hydroponic conditions anna šuňovská, miroslav horník, jana marešová, martin pipíška, jozef augustín department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, sk-917 01 trnava, slovak republic (hornikm@ucm.sk) abstract: a hydroponic study involving lettuce plants (lactuca sativa l.) as a leafy vegetable was conducted to evaluate the 137cs uptake and translocation in plant tissues in dependence on the presence or absence of k+ or/and nh4+ ions in cultivation media according to hoagland (hm) during 8 d plants growth under hydroponic conditions. significant increase of the 137cs+ uptake by lettuce plants and the decrease of 137cs+ translocation efficiency from roots to leaves were observed in 50 % hm deficient in k+ and nh4+ ions. speciation analysis using visual minteq program showed that at micromolar concentration of cscl (5 µmol/dm3) in 50 % hm at ph 6.0 and 25 °c, cesium was occurred practically only in the free cationic cs+ form − 98.8 %, with minor proportions of other cesium species: cscl − 0.4 %, csno3 − 0.4 %, and csso4− 0.4 %. surplus of cl-, no3and so42ions in hm causes the increase of proportions of the cesium species cscl, csno3 and csso4-, respectively at the expense of bioavailable cs+ form. radiocesium 137cs taken up via roots was removed from lettuce leaves with high efficiency by boiling in diluted nacl solution. at ambient temperature the extraction of 137cs with diluted acetic acid was concentration and time dependent process, and was succeeded by leakage of tissue components absorbing at 260 nm. these findings are important for the risk assessment of radiocesium entry into the food chain via contaminated leafy vegetable. key words: 137cs, root uptake, translocation, lactuca sativa, speciation, food chain 1. introduction contamination of the environment has become a serious problem all over the world. in contrast to heavy metals, which can present in high toxic levels in all parts of the environment, radionuclides are generally presented in low concentration, but from radiological unacceptable impact on live organisms point of view in non-negligible quantities. especially, the presence of artificial radionuclides in the environment poses a serious risk to human health through the food chain. the artificial sources of radionuclides in the environment represent: testing of nuclear weapons (gabrieli et al., 2011), nuclear waste disposal (grambow, 2008), accidents resulting from the nuclear power generation as well as manipulation with nuclear fuel (hu et al., 2010). in the case of the chernobyl accident in 1986, the radionuclides released from the reactor that caused exposure of individuals were mainly radioiodine and radiocesium (unscear, 2000). these nuclides can be transferred to humans rapidly from the air via leafy vegetables, milk and other products of the food chain (ingestion pathway) (tschiersch et al., 2009). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:15 utc 154 šuňovská, a. et al. from the radiological point of view, the releases of 134cs (τ = 2.06 y) and 137cs (τ = 30.2 y), estimated to have been 54 and 85 pbq, respectively, are the most important to consider (soudek et al., 2004). the accident of fukushima nuclear power plant (npp) on 11 march 2011 that resulted from the earthquake and tsunami contaminated large areas with radionuclides consisting primarily of 131i, 134cs, and 137cs (mext, 2012). as reported by the japanese government to the iaea on june 2011, the total amount of radioactivity released into the atmosphere might have been around 1.5 x 1017 bq of 131i and 1.5 x 1017 bq of 137cs, and the total radioactivity discharged into the sea might have been around 4.7 x 1015 bq (rjg, 2011). baeza et al. (2012) assume that released radioactivity correspond to approximately 6 % (131i) and 42 % (137cs) of the radioactivity released into the environment after the nuclear reactor accident at chernobyl. radionuclides released into the environment under normal or accidental functioning of the nuclear facilities, can contaminate plants via root uptake, direct deposition onto above-ground parts of plant or resuspension of contaminated soil particles. in the case of a nuclear accident occurring during the growing season, dry or/and wet deposition of aerosols on cultivated lands are the major and most direct steps in the food chain contamination (anspaugh et al., 2002). however, the release of new contamination still persists even after several years. also, climate/environmental changes e.g. global warming (dowdall et al., 2008) can play a possible role in distribution of radionuclides in soils as well as in soil-to-plant transfer processes. following a nuclear accident during the growing period, long-lived radionuclides, compared to the time elapsed between contamination and harvest, could constitute a potential hazard to human when present as residues in consumed agricultural products. ingestion of contaminated agricultural foodstuffs can constitute the major exposure pathway for the contribution to the total dose to human beings (madoz-escande et al., 2004). it is therefore essential to study radionuclides uptake and translocation into edible parts of plants (mainly vegetables) as well as the effect of culinary preparation on radionuclide ingestion risks (see e.g. adriano et al., 2000). contamination of the edible parts of „leafy vegetables“ (salad, for example) can comparatively occur throughout the growing period and contamination of ground vegetables, such as radishes, generally occurs through translocation from the contaminated aerial parts and root transfer from contaminated soil (madoz-escande et al., 2004). the bioassay using l. sativa l. (lettuce) studies is considered a simple, quick and sensitive way of evaluating potential environmental risk (gopalan, 1999) and can be successfully applied in soil with a high concentration of heavy metals of natural and anthropogenic origin (farré and barceló, 2003; escoto et al., 2007; bagur-gonzález et al., 2011). world production of lettuce in the year 2009 was approximately 24 million metric tons, with a cultivation area of one million hectares (usda, 2011). however, the significant production of lettuce is realized under hydroponic conditions. hydroponic cultivation has been reported not only to be associated to higher production yields but also to allow better control and standardization of the cultivation process, thus reducing overall production costs (domingues et al., 2012). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:15 utc nova biotechnologica et chimica 11-2 (2012) 155 our previous papers reported bioaccumulation and distribution of cesium (137cs), cobalt (60co), cadmium (109cd) and zinc (65zn) in tissues of tobacco (nicotiana tabacum l.) (vrtoch et al., 2007; horník et al., 2007; horník et al., 2009; guldanová et al., 2010), celery (apium graveolens l.) (horník et al., 2007) and sunflower (helianthus annuus l.) (horník et al., 2005; horník et al., 2007). also, the foliar uptake and translocation of zinc (65zn) in tobacco (n. tabacum l.) and hop (humulus lupulus l.) plants we studied (pipíška et al., 2008; marešová et al., 2012). this paper deals with 137cs uptake and translocation in lettuce (lactuca sativa l.) plants as a model of leafy vegetable contaminated with radiocesium and behaviour of radiocesium after treatment of lettuce leaves in the context of radiocesium releasing from contaminated leaf tissues. 2. materials and methods 2.1. plant material seeds of lettuce (lactuca sativa l. var. capitata l.,) obtained from seva seed, ltd., cr were germinated and grown in pots filled with commercial garden substrate (agro cs slovakia, sr) and illuminated by natural sunlight. the plants were periodically watered with tap water. after 3-4 weeks of pre-cultivation seedlings were gently removed from the substrate, roots were thoroughly washed by deionized water for removing of substrate particles and used in hydroponic experiments. 2.2. hydroponic experiments selected lettuce plants with more than 10 leaves (height approx. 10 cm, 3 g wet weight) from the pre-cultivation phase were transferred into series of 250 cm3 erlenmeyer flask containing 150 cm3 of 50 % hoagland medium (hoagland, 1920) spiked with 133cscl and 137cscl as a tracer. cultivation medium was adjusted with 1 m naoh to the value ph 6.0. the composition of the full strength (100 %) hoagland medium was (mmol/dm3): mgso4.7h2o – 1.5; kno3 – 4.0; cacl2 – 4.0; nah2po4.2h2o – 2.1; na2hpo4.12h2o – 0.13; feso4.7h2o – 6.4.10 -2; nano3 – 4.0; nh4cl – 4.0; nh4no3 – 2.0; h3bo3 – 0.14; na2moo4.2h2o – 2.5.10 -4; mnso4.5h2o – 2.1.10 -2; znso4.7h2o – 2.3.10 -3; cuso4.5h2o – 3.2.10 -3. all flasks were covered with black foil to protect plant roots against the lights and plants were cultivated under hydroponic conditions in triplicate series at artificial illumination (4 000 lx) and 16 h light / 8 dark photoperiod. in time intervals aliquot samples of cultivation media were taken, remaining 137cs radioactivity was measured by gamma-spectrometry and subsequently samples were returned back into the medium. at the same time the reduction of cultivation medium volume in flasks caused by transpiration activity of plants was recorded and the difference was compensated by glass balls addition due to keeping of roots submersion in media. at the end of experiments, lettuce plants were removed from cultivation media, roots bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:15 utc 156 šuňovská, a. et al. were carefully washed in deionized water and incorporated 137cs radioactivity in roots and aboveground parts of plants was analysed. dry weight of roots and leaves was estimated after 48 h drying at 60 °c. growth value (gv) for lettuce plants was estimated according to the equation (1): 0 0 m mm gv t − = (1) where mt or m0 are fresh weight of plants at the start or end of experiments, respectively. 2.3. extraction of 137cs from lettuce leaf tissues one-step extraction of 137cs from leaves of lettuce plants obtained from hydroponic experiments in 50 % hm spiked with 137cscl was performed by diluted acetic acid under occasional shaking at [biomass] : [extractant] ratio 1 : 50 and 23 ± 2 °c. 2.4. radiometric analysis two gamma-spectrometric scintillation detectors 54bp54/2-x and 76bp76/3 with well type crystal nai(tl) (scionix, nl) and data processing software scintivision-32 (ortec, usa) were used for determination of 137cs in cultivation medium and individual parts of lettuce plants. for energy and efficiency calibration a library of radionuclides was built by selecting characteristic γ-ray peaks (88.04 kev for 109cd, 661.66 kev for 137cs, 834.81 kev for 54mn and 1115.52 kev for 65zn). standardized solution of 137cs in the form of 137cscl (5.723 mbq/cm3, 20 mg/dm3 cscl in 3 g/dm 3 hcl) was provided from czech metrological institute (prague, cr). 2.5. speciation modelling the proportion of individual cs ionic forms in cultivation medium was predicted by speciation modelling software visual minteq (ver. 3.0) obtained from gustafsson (2010) in consideration of total salt concentration in medium, ph value, ionic strength and temperature. 2.6. statistical analysis all analytical determinations were performed in triplicate. statistical significance of differences in calculated values of 137cs distribution and translocation in lettuce plant tissues on the basis of different cultivation conditions were evaluated by multiple range test to ascertain differences between individual groups. the level of significance was p = 0.05 in all cases. origin 8.5 pro (originlab corp., usa) and statgraphics centurion ver. 15 (statpoint, inc., usa) were used for graphing and statistical analyses, respectively. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:15 utc nova biotechnologica et chimica 11-2 (2012) 157 3. results and discussion 3.1. the effect of k+ and nh4+ ions on 137cs uptake uptake and translocation of 137cs in lettuce plants (l. sativa l.) were studied under hydroponic conditions. these conditions on the one hand imitate in some details conditions of soil solution and on the other hand represent one way of commercial production of lettuce. thus, from the point of view of soil contamination with radiocesium the mentioned arrangement of experiments describes only 137cs transport in sequence soil solution − plant roots − aboveground parts of plant without characterisation of sorption-desorption processes between soil particles and soil solution. it is generally known, that 137cs shows high affinity to sorption onto clay minerals (aluminosilicates). from this reason the radiocesium binding in mentioned soil components is a limiting factor, which determines the behaviour, bioavailability and transport of cs into the environment. in generally, these processes are also affected by chemical speciation of analysed metal or radionuclide in given environment e.g. the portion of individual ionic form of metal or radionuclide in soil solution. table 1. chemical speciation of cesium in complete 50 % hoagland medium (hm), in hm deficient in k+ and nh4+ ions and in hm with surplus of cl, no3and so42+ anions. calculated by visual minteq ver. 3.0 for ph 6.0 and 25 °c and c0 = 5 μmol/dm3 cscl. molar fraction [x].100 hoagland medium cs+ cscl csno3 csso4 hm complete 98.8 0.4 0.4 0.4 hm deficient for k+ 99.0 0.4 0.2 0.4 hm deficient for nh4+ 99.0 0.3 0.3 0.4 hm deficient for k+ and nh4+ 99.1 0.2 0.2 0.5 surplus of clanions* 10-times (60.005 mmol/dm3) 100-times (600.005 mmol/dm3) 96.2 80.4 3.1 0.3 0.4 19.2 0.2 0.2 surplus of no3anions* 10-times (50.000 mmol/dm3) 100-times (500.000 mmol/dm3) 95.8 78.5 0.3 3.5 0.4 0.2 21.1 0.2 surplus of so42anions* 10-times (7.950 mmol/dm3) 100-times (79.500 mmol/dm3) 95.3 78.0 0.3 0.4 4.0 0.2 0.2 21.6 * concentration of anions in 50 % hm (mmol/dm3): cl– 6.005; no3– 5.000; so42– 0.795. in this context the speciation modelling program visual minteq for prediction of individual ionic forms of cs in hoagland media (hm) was used. we found that in 50 % hm containing 5 µmol/dm3 cscl the cesium was occurred mainly in the free cationic form cs+ (98.8 %) with minor presence of cscl (0.4 %), csno3 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:15 utc 158 šuňovská, a. et al. (0.4 %) and csso4 (0.4 %) forms at ph 6.0 and 25 °c (tab. 1). in the case cultivation media without k+ (kno3 as source), nh4 + (nh4cl and nh4no3 as source) or without both cations the slight increase of free cs+ (up to 99.1 %) was observed. on the basis of cscl, csno3 and csso4 existence in 50 % hm under given conditions, it can be concluded that in the decrease or increase of cs+ cations portion in media the concentration of cl-, no3 or so4 2anions will play important role. in tab. 1, it is evident that if we increase 10-times or 100-times the concentration of mentioned anions the cs+ portion in 50 % hm will significantly decrease. this phenomenon can be expected also in real soil conditions and soil solution. 0 1 2 3 4 5 6 7 8 9 0 200 400 600 800 1000 1200 1400 1600 u pt ak e of 13 7 c s [b q/ g] ; f re sh w t. time [day] 2mm k+; 3mm nh+ 4 2mm k+; 0mm nh+ 4 0mm k+; 3mm nh+ 4 0mm k+; 0mm nh+ 4 fig. 1. the effect of absence or presence of k+ and nh4+ ions in cultivation media on cesium uptake by root system of lettuce plants (l. sativa l.) during 8 d hydroponic cultivation in 50 % hoagland medium (hm) spiked with 5 μmol/dm3 cscl (36.4 kbq/dm3 137cscl), ph 6.0 at photoperiod 16 h light / 8 h dark (4 000 lx) and 25 ± 2 °c. error bars represent standard deviation of the mean (n = 3). the initial average fresh weight (fresh wt.) of lettuce plants was 2.68 g and the average increase of plants weight during experiments was gv = 1.26 ± 0.15 (sd; n = 12 − the mean of values obtained at all cultivation conditions). fig. 1 depicts cs uptake by root system of lettuce plants (l. sativa l.) during 8 days hydroponic cultivation in the complete 50 % hm spiked with 5 μmol/dm3 cscl (36.4 kbq/dm3 137cscl) and in 50 % hm without k+, nh4 + or without both ions. the choice of plant cultivation time length was adequate to evaluation of 137cs uptake and translocation into lettuce plant tissues. sabbarese et al. (2002) observed that in his time the maximum radionuclide 60co or 137cs transport was reached in the case of lettuce plants. we found that the significant increase of 137cs+ uptake by lettuce plants was observed in 50 % hm fully deficient in both k+ and nh4 + ions. in media deficient in k+ or nh4 + ions the significant increase of cs uptake, but in less extent, was shown for k+ ions. also, we observed that in complete media or media without k+ or nh4 + ions the cs uptake by roots of lettuce plants showed time linear dependence. however, in the case of media deficient in both k+ and nh4 + ions can be seen three phases: i) the first phase characterized by rapid cs uptake (after 24 h more than 60 % of the total cs was uptake); ii) the second equilibrium phase and iii) the third phase bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:15 utc nova biotechnologica et chimica 11-2 (2012) 159 of gradually increasing in cs uptake from solution of 50 % hm. all these differences in cs uptake at different composition of 50 % hm can not be explained on the basis of differences in chemical speciation of cs in media (see previous paragraph). results of authors soudek et al. (2004; 2006) show, that the concentrations of k+ and nh4 + ions in media play a decisive role as competitive ions (inhibitors) in cs uptake by plants. chou et al. (2005) found that the transfer of 137cs from contaminated soil to lettuce plants (l. sativa l.) was significantly diminished in the case of amended soil with 100 ppm kcl. these facts are crucial from the point of view of possible risks of 137cs transfer from contaminated environment to food chain e.g. through leafy vegetables. thus, in the case of the environment (soil or water) deficient mainly in k+ ions, there is a high risk of plants contamination with 137cs. on the other hand, the environment rich in k+ or nh4 + ions inhibit the transfer of 137cs into plants by competitive relationships between these macroelements and 137cs+ cations. in this context, in table 2 we present determined specific radioactivity 137cs (bq/g; fresh wt.) in lettuce plants at different [cs+] : [k+], [cs+] : [nh4 +] or [cs+] : [k+ + nh4 +] molar concentration ratios. table 2. content of 137cs [bq/g] (fresh wt.) in lettuce plants (l. sativa l.) after 2 and 8 d growth in 50 % hm spiked with 5 μmol/dm3 cscl (36.4 kbq/dm3 137cscl) at different [cs+] : [k+], [cs+] : [nh4+] or [cs+] : [k+ + nh4+] molar concentration ratios. c0 [mmol/dm3] molar ratio radioactivity [bq/g] k+ nh4+ [cs+] : [k+] [cs+] : [nh4+] [cs+] : [ k++ nh4+] 2 d 8 d 0.0 0.0 704.7 1032.7 0.0 3.0 1 : 600 92.7 465.4 2.0 0.0 1 : 400 65.4 311.8 2.0 3.0 1 : 1 000 60.5 301.6 it is generally known that the uptake of cs+ is operated mainly via two transport pathways on plant root cell membranes, namely k+ transporters (high-affinity mechanism) or k+ channels (low-affinity mechanism). however, the physiological role of cs+ in plant nutrition until now is no known (marschner, 1995; white and broadley, 2000). the change in 137cs uptake at an external k concentration in nutrient solution around 0.25 mmol/dm3, coincides with the concentration range at which the k uptake systems switch between a high-affinity mechanism and a lowaffinity mechanism (fu and luan, 1998; waegeneers et al., 2001). it can be expected that differences in k uptake rates in the genotype level result in different k depletion and hence different radiocesium uptake rates. waegeneers et al. (2001) found that these differences are more pronounced in soils with a low k availability and reported that high radiocesium uptake occurs (i) if the plant species has a high intrinsic cs uptake rate at low k concentrations (e.g. barley, lettuce, bentgrass), (ii) if the species have a high biomass production, resulting in a large depletion of k in the bulk soil solution (e.g. ryegrass), or (iii) if the species have a high k uptake rate per unit root surface, resulting in a high rhizospheric k depletion (e.g. lettuce). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:15 utc 160 šuňovská, a. et al. 3.2. the effect of k+ and nh4+ ions on 137cs translocation from the point of view of risks of toxic metals or radionuclides transfer into the food chain it is important that given metals or radionuclides will not be accumulated mainly in edible parts of plants. in the fig. 2 is presented the effect of k+ and nh4 + ions deficiency in cultivation media on distribution of 137cs in root and leaves of lettuce plants after 8 days hydroponic cultivation. we found that the deficiency in k+ and nh4 + ions caused a decreasing the accumulation of 137cs in leaves from the total accumulated radioactivity in lettuce plants in comparing with roots (significant differences are shown in fig. 2 at the p = 0.05 level based on multiple range test). in the complete 50 % hm (control) the portion of 137cs radioactivity in lettuce leaves represents more than 70 % of the total accumulated amount of 137cs. the absence of k+ and nh4 + ions this amount of 137cs in leaves decreased on the value less than 40 %. thus, it can be concluded that the presence of k+ and nh4 + ions in media increase the cs transport from roots to leaves of lettuce plants whereby this effect is additive. 0 20 40 60 80 100 120 2 mm k+ 0 mm nh 4 + 2 mm k+ 3 mm nh 4 + a c b 0 mm k+ 0 mm nh 4 + 0 mm k+ 3 mm nh 4 + 13 7 c s di st ri bu tio n [% ] root leaves a fig. 2. distribution of cesium in roots and leaves of lettuce plants grown 8 d at the absence or presence of k+ and nh4+ ions in hm media. for details see legend in the fig. 1. error bars represent standard deviation of the mean (n = 3). means with the same letter at columns are not significantly different at the p = 0.05 level based on multiple range test. for evaluation of 137cs mobility and translocation in conductive tissues of lettuce plants we analysed the distribution of radiocesium on the basis of the ratio of concentration in edible part of plants [cs]leaves to concentration in root system of plants [cs]root. also, as can be seen in the fig. 3, in the case of lettuce plants cultivation in 50 % hm fully deficient in both k+ and nh4 + ions was reached 4.5-times lower value of the concentration ratio [137cs]leaves : [ 137cs]root (bq/g : bq/g; dry wt.) than in control experiments with the value [137cs]leaves : [ 137cs]root = 0.73. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:15 utc nova biotechnologica et chimica 11-2 (2012) 161 our previous results (guldanová et al., 2010) obtained for tobacco plants cultivated under hydroponic conditions showed that the concentration ratio [cs]shoot : [cs]root increased with increasing hm concentration from the value 0.10 to the value 0.85, particularly at 50 % and 100 % hm, when concentration of monovalent ions (k+ and nh4 +; [k+] : [nh4 +] = 2 : 3) were 5 mmol/dm3 and 10 mm, respectively. similar effect was also observed with sunflower hydroponics, when shoot-to-root specific 137cs radioactivity ratio (bq/g : bq/g; wet wt.) increased with increasing concentration of hm from the value 0.10 to the value 0.69 (horník et al., 2005). chou et al. (2005) found that in the contaminated soil with 137cs the addition of 100 ppm kcl caused the increase of the transfer factor of 137cs for the aboveground and the decrease for the underground parts of cabbage, spinach, lettuce, radish, rape and clover plants cultured in contaminated soil during 7, 30 or 50 days. 0,0 0,2 0,4 0,6 0,8 1,0 1,2 c b a [1 37 c s] le av es /[ 13 7 c s] ro ot [b q/ g / b q/ g] ; d ry w t. a 2 mm k+ 3 mm nh 4 + 2 mm k+ 0 mm nh 4 + 0 mm k+ 3 mm nh 4 + 0 mm k+ 0 mm nh 4 + fig. 3. the effect of k+ and nh4+ ions on the [cs]leaves / [cs]root concentration ratio of lettuce plants after 8 d growth in 50 % hm. for details see legend in the fig. 1. error bars represent standard deviation of the mean (n = 3). means with the same letter at columns are not significantly different at the p = 0.05 level based on multiple range test. in the case of contaminated plants is very important to know not only the distribution of radioactivity in roots and aboveground parts, but also the distribution in individual segments of aboveground parts (old and young leaves, fruits, stems). in this term we analysed the specific radioactivity as (bq/g; dry wt.) for 137cs in individual leaves of lettuce plants (head and wrapper leaves) cultivated in complete 50 % hm or media without k+ or/and nh4 + ions. we found that the highest specific radioactivity in individual lettuce leaves was in the case of plants cultivated in 50 % hm deficient in k+ and nh4 + ions and as decreased in the order of plants cultivated in: hm deficient in k+ and nh4 + (7 950 bq/g) > hm deficient in k+ (4 160 bq/g) > hm deficient in nh4 + (3 940 bq/g) > complete hm (3 220 bq/g). also we determined that values of the specific radioactivity as in all bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:15 utc 162 šuňovská, a. et al. leaves within the analysed plant were not significantly differed. from this result, it can be concluded that the uptake of 137cs in individual leaves of lettuce plant will not be dependent on the development stage or localization of the leaf. 3.3. releasing of 137cs from lettuce leaves many papers deal with radionuclide releasing from contaminated vegetables and other foods during culinary preparation (see e.g. adriano et al., 2000) or during decontamination by washing of the plant matter after dry deposition of radionuclides (see e.g. tschiersch et al., 2009). in this part of the paper we evaluate the effect of lettuce leaves treatment on the residual concentration of radiocesium in leaves. all the 137cs radioactivity in experiments entered the plant via the roots from 137cs spiked hm and was localized within the plant tissues. 0 5 10 15 20 25 0 20 40 60 80 100 e xt ra ct ed 13 7 c s [% ] time [h] 40 g/dm3 aa 20 g/dm3 aa 5 g/dm3 aa fig. 4. one-step extraction of 137cs from leaves of lettuce plants grown in 50 % hm spiked with 137cscl. extraction with diluted acetic acid (aa) at [biomass] : [extractant] ratio 1 : 50 and 23 ± 2 °c. leaves of lettuce with specific radioactivity 370 bq/g (fresh. wt.) were obtained from hydroponic experiments described in fig. 1. radiocesium was removable from lettuce leaf biomass with high efficiency by boiling in diluted nacl solutions (data not shown). the efficiency of extraction under mild conditions such as by extraction with diluted organic acids at ambient temperature was low and time dependent. negligible amounts of 137cs were removed from the lettuce leaves after 1 h contact with diluted acetic acid and for higher efficiency of the process several hours contact times were necessary (fig. 4). the highest efficiency of 137cs removal was obtained only after 24 hour treatment with diluted acetic acid. however, at these conditions leaf bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:15 utc nova biotechnologica et chimica 11-2 (2012) 163 tissue was significantly damaged, decolorized and into the extraction media were released cell components absorbed at 260 − 280 nm region, typical for nucleic acid and proteins (fig. 5). the question of cs speciation and distribution in green plants is not completely solved. prevailing part of cesium will exists in a form of free cs+ ion and the main barrier of the cs+ ions translocation are diffuse barriers of the plant cells. in other biosystems e.g. in basidiomycetes cesium can form complexes with organic ligands such as badione or norbadione structurally related to the larger pigment family of pulvinic acids (desage-el murr et al., 2006). fig. 5. uv-vis spectrum of diluted acetic acid solutions (aa) after 24 h extraction of 137cs from lettuce leaves at 23 ± 2 °c. for details see the legend at fig. 4. 4. conclusions the results from hydroponic cultivation of lettuce plants (l. sativa l.) indicate that the uptake and translocation of 137cs in plant tissues are significantly affected by presence and concentration of k+ and nh4 + ions. we found that the significant increase of 137cs+ uptake by lettuce plants was observed in cultivation media deficient in both k+ and nh4 + ions. on the other hand, the absence of mentioned ions in media caused a decreasing of 137cs translocation from roots to leaves of lettuce plants. from the point of view of risks of toxic metals or radionuclides transfer into the food chain we also evaluate the effect of lettuce leaves treatment on the residual concentration of radiocesium in leaves. radiocesium was removable from lettuce leaf biomass with high efficiency by boiling in diluted nacl solutions and in the case of mild conditions with diluted organic acids at ambient temperature low and time dependent releasing of 137cs from contaminated lettuce leaves was observed. 1 2 4 1 – 80 g/dm3 aa 2 – 20 g/dm3 aa 3 – 5 g/dm3 aa 4 – 10 g/dm3 aa 3 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:15 utc 164 šuňovská, a. et al. acknowledgement: this work was supported by the ministry of education, science, 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phytoavailability of radiocesium are pronounced at low k intensities in soil. plant soil, 235, 2001, 11-20. white, p.j., broadley, m.r.: mechanism of caesium uptake by plants. new phytol., 147, 2000, 241-256. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:15 utc nova biotechnol chim (2023) 22(1): e1452 doi: 10.34135/nbc.1452 1 nova biotechnologica et chimica immobilization of beta-glucosidase purified from mandarin (citrus reticulata) fruit to superparamagnetic nanoparticles and its aroma quality enhancing effect mesut acar1, yusuf turan1, olcay sinan2, selma sinan2, 1department of biology, faculty of art and science, balikesir university, balikesir, turkey 2department of molecular biosciences, college of natural sciences, the university of texas at austin, austin, texas, usa  corresponding author: selmasinan@utexas.edu article info article history: received: 14th october 2021 accepted: 20th october 2022 keywords: aroma enhancement β-glucosidase immobilization magnetic nanoparticles mandarin abstract this paper reports on novel and efficient enhancement effects of fruit juice aroma using immobilized β-glucosidase, the enzyme involved in important functions in living organisms, onto superparamagnetic nanoparticles fe3o4 via carbodiimide βglucosidase was purified from mandarin (citrus reticulata) using ammonium sulfate precipitation and hydrophobic interaction chromatography. to be used in this study, superparamagnetic nanoparticles were synthesized and then the shape, size, and magnetism properties of the nanoparticles were characterized. the purified enzyme was immobilized on the nanoparticles. the optimum temperature for β-glucosidase (40 ℃) was increased by 10 ℃ after immobilization, while the optimum ph values of free and immobilized β-glucosidase were 5.5. while the km and vmax values of the free enzyme were 0.264 mm and 294 eu, immobilized enzyme’s km and vmax were 0.222 mm and 370 eu, respectively. in addition, it was determined that the storage stability of the immobilized enzyme was higher than the free enzyme. when the effect of some metal ions on the enzyme activity was examined, it was observed that fe+2 increased the enzyme activity while other metals inhibited it. according to the results obtained, the immobilized enzyme had a flavor-enhancing effect on mandarin juice. © university of ss. cyril and methodius in trnava introduction β-glucosidases (β-d-glucoside glucohydrolases, ec 3.2.1.21) are present in both prokaryotes and eukaryotes as responsible enzymes for selectively catalyzing the hydrolysis of β-glycosidic bonds either between two glycone molecules or between glycone and aglycone residues. they play various essential key roles in physiological activities and biotechnological processes, including food quality and flavor enhancement (turan 2008). it has been reported that, following the hydrolysis of glycosidic bond by using beta-glucosidase activity, the release of potential aroma constituents increases the aromatic quality of whole fruit or processed fruit products and beverages (fan et al. 2011). however, glucosidase has been reported to show mostly lower activities during food processes, and therefore exogenous glucosidase is proposed to be used for enhancing the aroma quality of fruits and their products (su et al. 2010). since the purified enzymes are essential for exogenous uses, the purification processes of the enzymes require specific techniques that result in high costs. mailto:selmasinan@utexas.edu nova biotechnol chim (2023) 22(1): e1452 2 removal of the free enzyme without activity loss is mostly impossible following catalysis in these types of industrial applications. addition of the inhibitors for the termination of the catalytic reactions causes extra contaminations of the processed foods. separation and removal of contaminants from processed food products require additional and more complicated techniques. in addition, the aroma quality of the fruit beverages may be remarkably altered by the loss of volatile aromas during long industrial processes. considering these kinds of unfavorable outcomes, immobilized enzyme systems have been established and primarily preferred for aroma enhancement applications in recent years. the use of immobilized beta-glucosidases from different sources for these applications has also been reported. shoseyov et al. (1990) used aspergillus niger endo-beta-glucosidase for immobilization on acrylic beads and corn stover cellulose particles to enrich the flavor of wine and passion fruit juices. gueguen et al. (1997) showed enhancement of the aromatic quality of muscat wine using of candida molischiana 35m5n beta-glucosidase immobilized to duolite a-568 resin. gallifuoco et al. (1999) immobilized beta-glucosidase in chitosan pellets to study the winemaking industry. yan et al. (2010) immobilized and determined the activity of betaglucosidase on different soil colloids. fan et al. (2011) reported the immobilization of orange betaglucosidase by three different methods and used it to release the bound volatile compounds in orange juice. on account of the fact that, the tea plant glucosidases show low activity under natural conditions and are destroyed to a high degree during the tea manufacturing processes of withering, rolling, and fermentation. su et al. (2010) preferred to immobilize a commercially purchased beta-glucosidase on alginate by the crosslinking–entrapment–crosslinking method and showed its aroma-increasing effect on tea beverages. pombo et al. (2011) isolated an extracellular β-glucosidase from issatchenkia terricola and immobilized it onto eupergit c for aroma enhancement of white muscat wine. magnetic nanoparticles consist of magnetic elements such as iron, cobalt, nickel, and their alloys (liu et al. 2020). magnetic nanoparticles not only have special magnetic properties such as superparamagnetism but also have unique physical properties, biocompatibility, stability (shabestari khiabani et al. 2017). fe3o4 nanoparticles are widely used in separation technology (samanta and ravoo 2014), protein immobilization (xu et al. 2009), catalysis (liu et al. 2010), medical science (lee and kang 2017), and the environment (guo et al 2015). the most important advantage of using magnetic nanoparticles is that once the enzyme is added to the reaction mixture, it is easily removed from the environment and can be used repeatedly in this way. immobilized enzyme can be selectively separated from a reaction mixture by the application of a magnetic field produced by a permanent magnet (kockar et al. 2010). this is the first time that this study presents the isolation and characterization of the betaglucosidase from mandarin (citrus reticulata) fruit to study the optimization of immobilization conditions to superparamagnetic iron oxide (fe3o4) nanoparticles, the characterization of immobilized enzyme, and its application to the release of bound aroma constituents in mandarin to determine whether this enzyme and the method performed can be utilized in industry. experimental chemicals mandarin (citrus reticulata) fruits used in this study were harvested in october from a field near balikesir, turkey. the materials, including p-npg, o-npg and carbodiimide, were obtained from sigma chem. co. iron (ii) chloride tetrahydrate (merck, ≥99 %), iron (iii) chloride hexahydrate (sigma-aldrich, ≥99 %) and ammonium hydroxide (merck, 25 % ammonium in water) were used for the synthesis. hclo4 (merck, 60 %) was used to prepare the dispersion. all chemicals were of analytical grade and used without further purification. all other chemicals were of the best available grade. enzyme assays were measured with the aid of a thermo scientific multiscango uv-visible spectrophotometer. the purification gel, which is used in this study, was synthesized at balikesir university, department of biology laboratory (sinan et al. 2006). nova biotechnol chim (2023) 22(1): e1452 3 preparation of magnetic nanoparticles the iron oxide nanoparticles (ions, fe3o4) were synthesized by co-precipitation in an n2 atmosphere at room temperature. the synthesis of ions was based on massart’s method (massart 1981) 1 m fecl3·6h2o was dissolved in 40 ml deionized water, and 2 m fecl2·4h2o was dissolved in 10 ml 2 m hcl solution. nh4oh was added to the solution under vigorous stirring for 30 min in an n2 atmosphere. after the reaction, the precipitate was washed three times with deionized water and dried in an oven overnight to remove the water. dispersion of the nanoparticles was obtained by using hclo4 as in the method of massart (massart 1981). characterizations the structural characterization of the ions was done by phillips analytical x-ray diffractometer using cukα radiation (λ=1.54056 å) between 20o<2θ<80o. fourier transform infrared spectroscopy (ftir, perkin elmer-1600 series) was also employed to investigate the structure of ions and the immobilization of β-glucosidase to ions. the shape and particle size of the nanoparticles were determined by tecnai g2 f30 model high-resolution transmission electron microscope (tem) operating at an accelerating voltage of 200 kv. the particle size of the nanoparticles (dtem) was measured from the image using the imagej program. the magnetic properties of ions and β-glucosidase bound ions were measured by a vibrating sample magnetometer (vsm, ade ev9 model) in a field range ± 20 koe (1 oe intervals) at room temperature. purification of β-glucosidase from mandarin (citrus reticulata) by hydrophobic interaction chromatography in this study, the kara et al. (2011) procedures were used for the purification of β-glucosidase. firstly, mandarin fruits were washed with distilled water three times. secondly, to prepare the crude extract, 200 g of sample tissue was cut quickly into thin slices and homogenized in a warring blender for 2 min using 100 ml of 0.1 m tris buffer (ph 8.0) including 1 m nacl, 0.02 % (w/v) nan3. after filtration of the homogenate through muslin, the filtrate was centrifuged at 15,000 g for 30 min, and the supernatant was collected. the enzyme solution was treated with solid ammonium sulfate to obtain the 40 – 80 % saturation fraction by centrifuging at 15,000 rpm for 30 min. the enzyme solution was applied to the hydrophobic column (1.0 cm diameter × 10.0 cm length), pre-equilibrated with 50 mm sodium phosphate buffer (ph 6.8) including 1.5 m (nh4)2so4. the enzyme was eluted using a linear gradient of 1.5 – 0.0 m (nh4)2so4 in the same buffer at a flow rate of 25 ml.h-1; 1.5 ml fractions were collected. the proteins containing the highest β-glucosidase activity were combined and used as immobilization studies after confirming homogeneity by gel electrophoresis. sds and native polyacrylamide gel electrophoresis sds and native polyacrylamide gel slab electrophoresis of purified enzyme was carried out according to the method of laemmli (1970). both electrophoresis were performed in a minigel system (bio-rad laboratories, usa) using 3 % stacking and 10 % separation gels. gels were fixed, stained with coomassie brilliant blue r-250 (sigma), and destained using standard methods to detect protein bands. the thermo scientific unstained protein weight marker was used to determine the size of the protein bands after electrophoresis. binding of β-glucosidase to superparamagnetic nanoparticles firstly, 0.1 g of ions were added to 2 ml of buffer a (0.003 m phosphate, ph 6, 0.1 m nacl) for each sample and blank tube. for the dispersion of ions, the mixture was incubated in a sonicator for 30 min. 0.5 ml of carbodiimide was added over this mixture. the mixture was allowed to stand in the sonicator for 15 minutes. thus, activating the surface of the nanoparticles carbodiimide was brought to the β-glucosidase enzyme, which was ready to connect. finally, 2 ml of β-glucosidase solution (0.5 – 15 mg.ml-1 in buffer a) was added, and the reaction mixture was sonicated for 30 min. the supernatant was used for the protein analysis. nova biotechnol chim (2023) 22(1): e1452 2 the precipitates were washed with buffer a, then buffer b (0.1 m tris, ph 8.0, 0.1 m nacl), and then directly used for the measurements of activity and stability. free and immobilized β-glucosidase enzyme assay during enzyme extraction and purification, free βglucosidase activity was determined using paranitrophenyl β-d-glucopyranosides (p-npg) as substrate. 70 µl of enzyme solution in 50 mm sodium acetate, ph 5.5 and 70 µl of substrate were mixed in the wells of a 96 well microliter plate in duplicate. after incubation at 37 ℃ for 30 min, the reaction was stopped by adding 70 µl of 0.5 m na2co3, and the color of the occurred pnitrophenol formation was measured at 410 nm. enzyme activity was expressed as µmol pnitrophenol formed per minute in the reaction mixture under these assay conditions. immobilized β-glucosidase enzyme assay was carried out with reference to the free enzyme activity. for this purpose, 1 ml of 2.5 mm p-npg substrate was added to tubes containing 0.1 g of βglucosidase bound ions and ions. ions were used as a blank. the mixture was incubated at 37 ℃ for 30 min with shaking (210 rpm). at the end of 30 min, nanoparticles were removed by using a magnet. each 140 µl sample and blank reaction mixture were taken, and the reaction was stopped by adding 70 µl of 0.5 m na2co3, and the color that developed because of p-nitrophenol liberation was measured at 410 nm. free and immobilized enzyme units were calculated from the p-npg graph equation. stability measurement free β-glucosidase and immobilized β-glucosidase storage stability were measured by assaying their relative activities in buffer b at 37 ℃ after being incubated in buffer b at 4 ℃ and 25 ℃ for a required period. the stabilities of immobilized βglucosidase and free β-glucosidase were investigated by measuring their relative activities up to 30 days. initial activity was assumed to be 100 and the next activity results are proportioned accordingly. determination of kinetic and biochemical parameters the kinetic parameters of the free and immobilized β-glucosidase enzyme using p-npg as a substrate were calculated by measuring their activity in buffer b with seven different substrate concentrations at ph 5.5 and 25 ℃. the concentrations of p-npg in the reaction mixture were 0.35 – 2.5 mm. a double reciprocal lineweaver–burk plot was used to calculate the parameters. the biochemical parameters, ph and temperature, of free and immobilized β-glucosidase were measured. the effect of varying the ph on enzyme activity was examined using 25 mm sodium acetate (3.0 – 5.8), citrate-phosphate (3.0 – 7.0) and phosphate (6.0 – 11.0) buffers. for optimum temperature determination, the enzyme and substrate p-npg solution mixtures were assayed in the range 20 – 60 ℃ for 30 min. to study the effect of various metals on mandarin β-glucosidase activity, enzyme activity was assayed in 1 mm final concentration of fe, cu, ni, pb, cd, zn, cr, mn, mg, and ag. the activity results of the enzyme solution, which does not contain metal ions, were statistically compared (anova) with the activity results of the solution containing metal ions. differences were considered statistically significant at p <0.05. analysis of aromatic compounds by using the immobilized β-glucosidase, glycosidic bound volatile compounds in mandarin fruit juice were isolated, and extracted with amberlite xad-2 resin, and then hydrolyzed by the immobilized β-glucosidase. the released glycosidic bound volatiles were analyzed by gcms and lc-ms. the samples extracted were analyzed with shimadzu gas chromatography gc2010 plus. compounds identified in the gc-ms (gas chromatography–mass spectrometry) analysis of the samples were determined by comparison with the wiley229 and nist27 mass spectroscopy libraries. the samples were analyzed in an electrospray ionization (electrospray ionization esi) device with the agilent lc-ms 4 nova biotechnol chim (2023) 22(1): e1452 3 (liquid chromatography–mass spectrometry) system. results and discussion purification of β-glucosidase from mandarin in this study, β-glucosidase was purified from mandarin fruits using an ammonium sulfate and sepharose 4b-l-tyrosine-1-napthylamine hydrophobic interaction column (kara et al. 2011). upon fractionation of the β-glucosidase active fractions with ammonium sulfate, 75% of the total activity was obtained in the fraction saturated with 40 – 80 % ammonium sulfate. to improve its binding efficiency, the precipitate with βglucosidase activity was dissolved and saturated with 1.5 m ammonium sulfate before applying it to the sepharose-4b-l-tyrosine-1-napthylamine column. the fractions with the highest βglucosidase activity and the relatively lower protein contents were pooled and used for enzyme assay. the fold purification and the enzyme yield (196.2 and 11 %) with the above procedure seem to be higher than with previous glucosidase purification studies from different plant sources (gerardi et al. 2001; odoux et al. 2003; li et al. 2005; yu et al. 2007). this is due to the favourable ammonium sulfate precipitation range and the specifically designed hydrophobic interaction column. fig. 1. sds-native page of purified mandarin β-glucosidase. 1 – sds-page; 2 – native-page; 1a,2s,3 – molecular weight marker (β-galactosidase, 116 kda; bovine serum albumin, 66.4 kda; egg albumin, 45 kda; lactate dehydrogenase, 35 kda; rease bsp981 (e. coli), 25 kda; β-lactoglobulin, 18.4 kda; lysozyme,14.4 kda). the purified β-glucosidase migrated as a single band during native and sds–polyacrylamide gel electrophoresis when stained with coomassie brilliant blue (fig. 1). mandarin β-glucosidase molecular weight was determined as 30 kda and 60 kda using sds and native page, respectively. therefore, it was found that the enzyme had two subunits of equal size. the estimated molecular mass of the protein is a little smaller than βglucosidases from various plant sources e.g., 64 kda from orange (citrus sinensis) fruit tissue (cameron et al. 2001), 68 kda from ripe sweet cherry (prunus avium) fruit (gerardi et al. 2001) and 62 kda from sorghum bicolor seedlings (hosel et al. 1987). optimum binding capacity of the enzyme to nanoparticles (binding efficiency) the optimum binding percentage of the enzyme to the nanoparticles in the medium and activity were measured (table 1). using the data in table 1, fig. 2 was drawn, showing the relationship between the amount of nanoparticles and the percentage binding and activity of the β-glucosidase enzyme. 5 nova biotechnol chim (2023) 22(1): e1452 3 table 1. fe3o4 nanoparticle amount, mandarin immobilized β-glucosidase percentage, and activity. nanoparticle [mg] nanoparticle [mg.ml-1] enzyme volume* [μl] protein amount [μg.ml-1] protein amount in elution immobilized enzyme [%] δa (405 nm) activity [u.ml.min-1] 25 7.69 750 (*including 294,59 μg.ml-1 protein) 67.982 26.925 60.4 1.44 1850.813 50 15.38 100 1.47 1891.463 75 23.07 1.21 1539.160 100 30.76 1.15 1457.859 125 38.46 1.08 1363.008 150 46.15 1.02 1281.707 as shown in table 1, it was found that 60.4 % of the enzyme was bound by 7.69 mg/ml fe3o4 nanoparticle in the reaction medium containing 67.982 μg.ml-1 β-glucosidase enzyme. besides the percentage of immobilized glucose, β-glucosidase increased and then remained at 100 % when the amount of nanoparticle added was above 15 15 mg.ml-1. on the other hand, as the amount of fe3o4 nanoparticles increased, the enzyme activity decreased (as is more clearly seen in fig. 2). therefore, the optimum fe3o4 nanoparticle rate in the medium was revealed to be 15 mg.ml-1. fig. 2. effect of the amount of ions added on the percentage of immobilized β-glucosidase. particle size and structure the xrd pattern of the ions is given in fig. 3. the nanoparticles have a cubic spinel structure with the characteristic (220), (311), (400), (422), (511), (440) and (622) peaks of iron oxide at around 2θ ≈ 31o, 35o, 43o, 53o, 57o, 63o and 74o respectively, according to the jcpds cards no. 019-0629 and no. 039-1346. the mean crystal size of ions, dxrd were calculated from the most intense peak (311) in the pattern using the scherrer equation (cullity 1978) and found to be 14.4 nm. tem images of the ions were given in fig. 4. the particles are mostly spherical. the physical particle size, dtem, is found to be 9.0 ± 2.3 nm. fig. 3. xrd pattern of ions. fig. 4. tem image of ions. magnetic property the magnetization curves of the ions and βglucosidase bound ions were measured at ± 20 koe and given in fig. 5. the detailed curves drawn at ± 50 oe are illustrated in the inset. ions are superparamagnetic with zero coercivity, hc. saturation magnetization, ms of the ions is 73.6 emu/g. the mean magnetic particle size, dmag (with standard deviation, σ) was calculated 6 nova biotechnol chim (2023) 22(1): e1452 2 according to the relations (morales et al. 1999; karaagac et al. 2010) and found to be 7.8 ± 2.4 nm. after the immobilization, the ms value of immobilized β-glucosidase is 56.3 emu.g-1 and the sample is superparamagnetic with a hc of 3 oe. about 24 % decrease in ms was observed after the immobilization. this could be attributed to the binding of β-glucosidase to ions, which leads to a reduction in the number of magnetic moments per weight in the volume fraction. fig. 5. magnetization curves of ions and β-glucosidase bound ions (inset shows the magnetization curves in ± 50 oe). mechanism of binding ftir analysis was demonstrated for the βglucosidase bound to magnetic nanoparticles (fig. 6). ftir spectra for the dried β-glucosidase from purified mandarin, ions, and β-glucosidase bound ions. the band around 538 cm-1 corresponds to the fe-o vibration, confirming the sample is iron oxide. ions nanoparticles were detected during a frequency peak at 538.2 cm-1. the deepest peak frequency of 619.14 cm-1 observed in pure βglucosidase shows a secondary amide group for the unique protein structure. it was evidence that the characteristic bands of protein (i.e., liao and chen 2001) at 1642 and 1631cm−1 were present in pure β-glucosidase and in the β-glucosidase-bound ions, confirming the binding of yeast alcohol dehydrogenase (yadh) to ions. the deep characteristic bands of proteins for the βglucosidase-bound ions should be due to the high enzyme loading. fig. 6. ftir analysis of nanoparticles, β-glucosidase and β-glucosidase bound nanoparticles. 7 nova biotechnol chim (2023) 22(1): e1452 3 activity and stability when the optimum fe3o4 nanoparticle rate in the medium was 15 mg.ml-1, because β-glucosidase was completely bound, the activities and stabilities of immobilized β-glucosidase were measured under this condition. as shown in fig. 7a, optimum ph values for free and immobilized β-glucosidase were the same as in previous study (su et al. 2010). however, the optimum temperature value of immobilized β-glucosidase was determined to be higher than free enzyme (50 ℃ and 40 oc respectively, fig. 7b). as a positive result, the increase in the heat resistance of the mandarin βglucosidase enzyme immobilized in the research is consistent with the results of singh et al. (2011) it seems that immobilized β-glucosidase has generally more stability than the free enzyme. fig. 7. optimal ph (a) and temperature (b) for free and immobilized β-glucosidase. fig. 8 shows the storage stabilities of immobilized β-glucosidase and free β-glucosidase at 4 and 25 ℃ in a relative activity to time plot. after an incubation time of 30 days, the relative activities of free β-glucosidase at 4 and 25 ℃ were 74 and 23 %, respectively. however, the immobilized βglucosidase retained 88 % and 47 % activity at 4 and 25 ℃ respectively, over a period of 30 days. it was observed that, the storage stability of the immobilized β-glucosidase significantly increased according to the free enzyme. interestingly, it was determined that the immobilized β-glucosidase activities at both 4 ℃ and 25 ℃ were observed to be increased compared with the free β-glucosidase activity for 5 days. similarly, many other studies (fan et al. 2011; chen et al. 2012; su et al. 2010) reported that the immobilized enzyme was more stabled than the free enzyme. fig. 8. storage stability of the immobilized and free βglucosidase at 4 ℃ and 25 ℃. km and vmax values the reaction kinetics of the immobilized and free β-glucosidase were calculated by using lineweaver–burk plots with the artificial substrate p-nitrophenyl-d-glucopyranosides (p-npg) (fig. 9). fig. 9. lineweaver–burk plots of the immobilized and free βglucosidase with p-npg. 8 nova biotechnol chim (2023) 22(1): e1452 2 according to the plots, it was found that the km and vmax values of free and immobilized enzymes were 0.264 mm and 294 eu; 0.222 mm and 370 eu, respectively. because of lower km values for immobilized enzyme, the affinity of the immobilized enzyme for p-npg was higher than for free enzyme. as a result, after the immobilization, the rate of the enzyme catalytic activity increased and consequently, the km value of the β-glucosidase enzyme decreased. in other words, the immobilization process made the enzyme work more effectively. the higher immobilized activity has also been reported in others (su et al. 2010; fan et al. 2011). the results of activity and stability are summarized in table 2. as shown in the table, it can be revealed that immobilized enzyme would be very useful in industry. table 2. activity and stability of β-glucosidase. optimum ph optimum temperature [°c] p-npg vmax [eu) p-npg km [mm) storage stabilities 4 °c 25 °c free β-glucosidase 5.5 40 294.12 0.264 44 % 23 % immobilized β-glucosidase 5.5 50 370.97 0.222 88 % 47 % the effect of various metal ions on the purified βglucosidase enzyme was examined (fig. 10). fig. 10. effect of some metal ions on β-glucosidase enzyme activity from mandarin (*p ≤0.05, nsp >0.05). of the ions tested, only fe2+ did not show an inhibitory effect on the enzyme activity, while others exhibited different levels of inhibitory effects. similar results were reported in other studies (cameron et al. 2001; kara et al. 2011). according to the result, it can be said that it is very reasonable to use fe2+ ions to immobilize the βglucosidase enzyme. results of gc and lc analyses of aromatic compounds the table below summarizes the analysis of mandarin juice hydrolyzed by immobilized βglucosidase (table 3). table 3. compounds detected from mandarin juice hydrolyzed by immobilized β-glucosidase. compound analyze type retention time [rt, min] diagnostic reference naringin lc-ms (qn) 10.699 calibrated compounds hesperidin lc-ms (qn) 11.116 calibrated compounds limonene gc-ms (ql) 16.695 wiley7 lib. benzaldehyde gc-ms (ql) 4.295 wiley229 lib. benzyl acetate gc-ms (ql) 46.840 wiley229 lib. 1,3-dimetil benzene gc-ms (ql) 8.850 wiley229 lib. ethyl benzene gc-ms (ql) 8.460 nist27 lib. benzophenone gc-ms (ql) 44.580 nist27 lib. 2-amino-1,3-propanediol gc-ms (ql) 3.235 nist27 lib. phenol-2,6-bis (1,1 dimethylethyl)-4-methyl gc-ms (ql) 39.485 wiley7 lib. abbreviations: ql qualitative; qn, quantitative 9 nova biotechnol chim (2023) 22(1): e1452 3 naringin and hesperidin were analyzed quantitatively, others qualitatively. when the chromatograms of the lc-ms analysis samples were examined, an increase was observed in the compounds that play a role in the aromatic quality of the fruit juice. the amount of hesperidin in the intact mandarin juice, which is at a level of 19.821 ppm, was determined at a level of 40.195 ppm in the sample treated with fe3o4 nanoparticles and at a level of 74.23 ppm in the sample treated with immobilized β-glucosidase. while the amount of naringin was 116.74 ppm in intact fruit juice, it was found to be 365.771 ppm and 371.060 ppm in fruit juices treated with nanoparticles and immobilized β-glucosidase, respectively. while no peak was observed in the gc-ms chromatograms of the fruit juice prepared as the control group and the juice treated with fe3o4 nanoparticles, various peaks were observed in the juice chromatograms treated with immobilized βglucosidase enzyme. these peaks were compared with the wiley229 and nist27 mass spectroscopy libraries, and limonene, benzaldehyde, benzyl acetate, 1,3-dimethyl benzene, ethyl benzene, benzophenone, 2-amino-1,3-propendiol, and phenol-2,6-bis (1,1-dimethylethyl)-4-methyl were detected. in other words, it was revealed that 8 compounds were formed by the immobilized βglucosidase. the aroma enhancing effect of βglucosidase enzyme immobilized by many researchers on orange juice (fan et al. 2011), tea (su et al. 2010), grape wine (guengen at al. 1997; pombo et al. 2011) and passion fruit juice (shoseyov et al. 1990) was reported. it is the first time to be investigated the immobilization of βglucosidase enzyme obtained from mandarin fruit on fe3o4 superparamagnetic nanoparticles and its effect on the aromatic quality of fruit juice. in conclusion, the present study has revealed the effectiveness of the purification procedure for βglucosidase mandarin (citrus reticulata). the enzyme was purified by salting it out with ammonium sulfate and using sepharose-4b-ltyrosine-1-napthylamine hydrophobic interaction chromatography. the purified enzyme obtained from mandarin was immobilized onto the nanoparticles and characterized. besides, it was determined that the immobilized enzyme had a flavor enhancing effect on mandarin juice. acknowledgement this work was some parts of msc thesis of mesut acar and supported by balikesir university grant number bap 2013/64. conflict of interests the authors declare that they have no conflict of interest. references cameron rg, manthey ja, baker ra, grohmann k (2001) purification and characterization of a β-glucosidase from citrus sinensis var. valencia fruit tissue., j. agric. food chem. 49: 4457-4462. chen l, li n, zong mh (2012) a glucose-tolerant βglucosidase from prunus domestica seeds: purification and characterization. process biochem. 47: 127-132. cullity bd (1978) elements of x-ray diffraction, addisonwesley, usa. fan g, xu y, zhang x, lei s, yang s, pan s (2011) characteristics of immobilised b-glucosidase and its effect on bound volatile compounds in orange juice. int. j. food sci. technol. 46: 2312-2320. gallifuoco a, alfani f, cantarella m, spagna g, pifferri pg (1999) immobolized β-glucosidase for winemaking industry: study of biocatalyst operational stability in laboratory-scale continuous reactors. process biochem. 35: 179-185. gerardi c, blando f, santino a, zacheo g (2001) purification and characterization of a β-glucosidase abundantly expressed in ripe sweet cherry (prunus avium l.) fruit. plant sci. 160: 795-805. gueguen y, chemardin p, pien s, arnaud a, galzy p (1997) enhancement of aromatic quality of muscat wine by the use of immobilized beta-glucosidase. j. biotechnol. 55: 151-156. guo zq, li y, pan sh, xu jz (2015) fabrication of fe3o4 cyclodextrin magnetic composite for the high-efficient removal of eu(iii). j. mol. liquids. 206: 272-277. hosel w, tober i, eklund sh, conn ee (1987) characterization of beta-glucosidases with high specificity for the cyanogenic glucoside dhurrin in sorghum bicolor (l.) moench seedlings. arch. biochem. biophys. 252: 152-162. kara h, sinan s, turan y (2011) purification of betaglucosidase from olive (olea europaea l.) fruit tissue with specifically designed hydrophobic interaction chromatography and characterization of the purified enzyme. j. chromatogr. b. 879: 1507-1512. karaagac o, kockar h, beyaz s, tanrisever t (2010) a simple way to synthesize superparamagnetic iron oxide nanoparticles in air atmosphere: iron ion concentration effect”. ieee trans. magn. 46: 3978-3983. kockar f, beyaz s, sinan s, koçkar h, demir d, eryılmaz s, tanrisever t, arslan o (2010) paraoxonase 1-bound 10 nova biotechnol chim (2023) 22(1): e1452 2 magnetic nanoparticles: preparation and characterizations. j. nanosci. nanotechnol. 10: 7554-7559. laemmli uk (1970) cleavage of structural proteins during the assembly of head of bacteriophage-t4. nature 227: 680-685. lee y-c, kang i-j (2017) optimal fabrication conditions of chitosan-fe3o 4-gold nanoshells as an anticancer drug delivery carriers. bull. kor. chem. soc. 38: 313-319. li y, jiang cj, wan xc, zhang x, li d (2005) purification and partial characterization of beta-glucosidase from fresh leaves of tea plants (camellia sinensis (l.) o. kuntze). acta biochim. biophys. sin. 37: 363-370. liao mh, chen dh (2001) immobilization of yeast alcohol dehydrogenase on magnetic nanoparticles for improving its stability. biotechnol. lett. 23: 1729-1727. liu s, chen h, lu x, deng c, zhang x, yang p (2010) facile synthesis of copper(ii) immobilized on magnetic mesoporous silica microspheres for selective enrichment of peptides for mass spectrometry analysis. angew. chem. int. ed. engl. 49: 7557-7561. liu s, yu b, wang s, shen y, cong h (2020) preparation, surface functionalization and application of fe3o4 magnetic nanoparticles. adv. colloid interface sci. jul. 281: 102165. massart r (1981) preparation of aqueous magnetic liquids in alkaline and acidic media, ieee trans. magn. mag-17: 1247-1248. morales mp, veintemillas-verdaguer s, montero mi, serna cj, roig a, casas ll, martinez b, sandiumenge f (1999) surface and internal spin canting in γ-fe2o3 nanoparticles. chem. mater. 11: 3058-3064. odoux e, chauwin a, brillouet jm (2003) purification and characterization of vanilla bean (vanilla planifolia andrews) β-glucosidase. j. agric. food chem. 51: 31683173. pombo pg, farina l, carrau f, viera fb, berna bm (2011) a novel extracellular β-glucosidase from issatchenkia terricola: isolation, immobilization and application for aroma enhancement of white muscat wine. process biochem. 46: 385-389. samanta a, ravoo bj (2014) magnetic separation of proteins by a self-assembled supramolecular ternary complex. angew. chem. int. ed. engl. 53: 2946-2950. shabestari khiabani s, farshbaf m, akbarzadeh a, davaran s (2017) magnetic nanoparticles: preparation methods, applications in cancer diagnosis and cancer therapy. artif. cells nanomed. biotechnol. 45: 6-17. shoseyov o, bravdo ba, siegel d, goldman a, cohen s, shoseyov l, kan r (1990) immobilized endo-βglucosidase enriches flavor of wine and passion fruit juice. j. agricult. food chem. 38: 1387-1390. sinan s, kockar f, arslan o (2006) novel purification strategy for human pon1 and inhibition of the activity by cephalosporin and aminoglikozide derived antibiotics. biochimie 88: 565-574. singh rk, zhang yw, nguyen np, jeya m, lee jk (2011) covalent immobilization of β-1,4-glucosidase from agaricus arvensis onto functionalized silicon oxide nanoparticles. appl. microbiol. biotechnol. 89: 337-344. su e, tao x, liping g, qianying d, zhengzhu z (2010) immobilization of beta-glucosidase and its aromaincreasing effect on tea beverage. food bioprod. proces. 88: 83-89. turan y (2008) a pseudo-β-glucosidase in arabidopsis thaliana: correction by site-directed mutagenesis, heterologous expression, purification, and characterization. biochem. (moscow) 73: 912-919. xu l, kim mj, kim kd, choa yh, kim ht (2009) surface modified fe3o4 nanoparticles as a protein delivery vehicle. colloid surface a. 350: 8-12. yan j, pan g, li l, quan g, ding c, luo a (2010) adsorption, immobilization, and activity of b-glucosidase on different soil colloids. j. colloid interface sci. 348: 565-570. yu hl, xu jh, lu wy, lin gl (2007) identification, purification and characterization of β-glucosidase from apple seed as a novel catalyst for synthesis of oglucosides. enzyme microb. technol. 40: 354-361. 11 28 nova biotechnologica et chimica 13-1 (2014) doi 10.2478/nbec-2014-0004 © university of ss. cyril and methodius in trnava the effect of culture age and initial silver concentration on biosynthesis of ag nanoparticles jana kaduková, oksana velgosová, anna mražíková, renáta marcinčáková department of material science, faculty of metallurgy, technical university of kosice, letna 9, 042 00 kosice, slovakia, (jana.kadukova@tuke.sk) abstract: many organisms or their extracts have the ability to reduce ag+ ions to ag0 and stabilize them what results in nanoparticle formation in solution. the aim of the article was to study the influence of two selected parameters – initial silver concentration and culture age, on ag nanoparticles production by green algae parachlorella kessleri. the presence of ag nanoparticles in the solution was confirmed by the uv-vis spectroscopy and tem analyses. typical curve with the peak at app. 420 nm was found for nanoparticles produced by algae. while culture age did not have any significant effect, the initial silver concentration had significant influence on nanoparticle production which influenced the rate of nanoparticle production, their amount, their size and stability, as well. key words: silver nanoparticles, parachlorella kessleri, algae 1. introduction utilisation of organisms in the field of nanoparticles production is gaining greater importance in the present time due to easy, non-toxic and environmentally friendly way of nanoparticle production (castro et al., 2010). biologically produced nanoparticles can be used the same way as nanoparticles produced by other synthetic ways in several fields such as non-linear optics, solar energy absorption (davenas et al., 2008), biosensors (dubas and pimpan, 2008), sterilisation and disinfection (yan and chen, 2003; ruparelia et al., 2008; maneerung et al., 2008), bioimaging (sanghi and verma, 2009), etc. eukaryotic organisms seem to be very promising in biological production of nanoparticles due to their ability to release great amounts of enzymes into their environment. extracellular formation in comparison with intracellular one represents easier and cheaper way of nanoparticle production (korbekandi et al., 2009). nowadays, the ability of many organisms or even their extracts to reduce metal ions to metal particles and stabilize them what often results in nanoparticle formation in solution is known and studied in broad extent (govindaraju et al., 2008; sharma et al., 2009; fayaz et al., 2011; mukunthan et al., 2011; logeswari et al., 2013; roopan et al., 2013). the disadvantage of biological systems is production of nanoparticles with different shapes and wide size distribution. however, the preparation of nanoparticles with narrow particle distribution is crucial for utilization of silver nanoparticles (agnps) in many applications as their efficiency often depends on the surface area of silver nanoparticles (lin et al., 2008). particular emphasis has recently been placed on the control of shape, because in many cases it bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:02 utc nova biotechnologica et chimica 13-1 (2014) 29 allows properties to be fine-tuned with a great versatility that gives the particles a unique nature (xiangqian et al., 2011). in comparison with plant extracts the synthesis of nanoparticles using algae as a source has been unexplored and underexploited. there are few reports about biological synthesis of noble metal nanoparticles using algae, mostly based on the utilization of extracts from algae (xie et al., 2007; jena et al., 2013) or dry algal biomass (govindaraju et al., 2008; castro et al., 2013; sharma at el., 2014). however, biosynthesis of metal nanoparticles by living algae has not been frequently reported. chakraborty et al. (2009) used living blue-green algae lyngbya majuscule, spirulina subsalsa and green alga rhizoclonium hieroglyphicum to study the biosorption and bioreduction of gold nanoparticles. they did not produced nanoparticles into the solution but desorbed them from the biomass surface. dahoumane et al. (2014) used living cells of chlamydomonas reinhardtii for production of stable bimetallic ag-au nanoparticles. the aim of the article was to study extracellular production of silver nanoparticles using alga parachlorella kessleri and the possibility to influence the nanoparticle formation process so that nanoparticles with the same shape and narrow size distribution would be produced. 2. material and methods 2.1 preparation of algal culture algae parachlorella kessleri (syn. chlorella kessleri) strain larg/1, supplied by ccala, no. 253. locality: russia, moscow, inst. general genetics of acad. sci.ussr, lab. radiation genetics were used for experiments. algae were cultivated on agar plates in petri dishes (diameter 9 cm). millieu bristol nutrient solution with 2% agar added (flake agar from imuna pharm, šarišské michaľany) was used for cultivation. algal strains were cultivated under continuous light regime, at the temperature from 20 – 25°c. 2.2 synthesis of silver nanoparticles stock silver solution was prepared by dissolution of agno3 p.a. (from mikrochem company) in deionised water with final ag+ ion concentration of 10 mm. the solution with required final concentration (in general 0.5 mm) was prepared by dilution of required volume of stock silver solution in deionised water. algae were added into erlenmeyer flasks containing silver nitrate solution. all experiments were carried out in triplicates. the silver solution with ag+ ion concentration 0.5 mm was prepared as a control sample and kept at the same conditions as experimental solutions. it was used as a reference solution for the absorbance measurement. after 24 hours (1st day) 3 ml of each solution was removed from the erlenmeyer flasks and stored in the plastic tubes in a refrigerator. 1.0 ml of that solution was used to measure the absorbance. the samples were withdrawn and absorbance measured on the 3rd, 7th, 10th and 14th days. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:02 utc 30 kaduková, j. et al. to study the effect of culture age, algal cultures cultivated for 1, 2, 3 and 4 weeks on agar plates were used. to study the effect of initial silver concentration, the solutions with ag+ ion concentrations of 0.5, 1 and 2 mm were used. for these experiments algae cultivated for 3 week were used. 2.3 uv-visible spectroscopy the silver nanoparticle formation from silver ions was monitored by measuring the uv-vis spectra of the respective solutions in 10-mm optical-path-length quartz semimicrocuvettes. the spectrum was recorded on unicam uv/vis spectrometer uv4 (from chromspec company). the absorbance in the range of wavelengths between 200 nm and 800 nm was measured using wolfram (for measurement in vis range) and deuterium (for measurement in uv range) lamps. 2.4 transmission electron microscopy (tem) the size and morphology of the nanoparticles were studied by means of a transmission electron microscope (jeol model jem-2000fx microscope operated at an accelerating voltage of 200 kv). samples for tem analyses were prepared on carbon-coated copper grids. the films on the grids were allowed to dry in air prior analyses. 3. results and discussion a study on extracellular biosynthesis of silver nanoparticles by the vital culture of parachlorella kessleri was carried out in this work. visual observation of the experimental mixture after addition of algal cells into agno3 solution showed a colour change from transparent to brown whereas no colour could be observed in control experiment without addition of cells. the appearance of brown colour of colloidal silver solution clearly indicates the formation of silver nanoparticles in the reaction mixture (pinto et al., 2010). the formation of nanoparticles was confirmed by uvvis spectroscopy. typical spectrum is the result of the radiation absorption in the visible region of the electromagnetic spectrum due to the localised surface plasmon of silver nanoparticles (dubas and pimpan, 2008; bankura et al., 2012). the surface plasmon resonance (spr) bands in absorption spectrum with the peak at app. 420 nm typical for spherical silver nanoparticles (petit et al., 1993) was found for nanoparticles produced by alga parachlorella kessleri. 3.1 influence of culture age alga parachlorella kessleri has been grown on agar plates for different duration of time such as 1, 2, 3 and 4 weeks. the influence of the culture age on nanoparticle production was studied using the uv-vis spectral analysis of silver nanoparticles produced by selected algal cultures (fig. 1a). the fact that all spectra are comprised of a single and well defined spr bands is evidence that all colloids are composed of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:02 utc nova biotechnologica et chimica 13-1 (2014) 31 small, round nanoparticles with relatively uniform size (dahoumane et al., 2014). in the uv-vis absorption spectrum, spr peaks located at 418, 420, 419 and 419 nm were observed for nanoparticles synthesized by 1, 2, 3 and 4 week-old algae, respectively. it is well documented that position of peaks, assigned to a surface plasmon, can be attributed to the nanoparticle size (pinto et al., 2010). based on the peak position of spr bands shown in fig. 1a it may be suggested that small and relatively uniform nanoparticles were produced. fig. 1. uv-vis spectrum of ag nanoparticles produced by algal cultures of different ages registered after 7 days (a), maximum measured absorbance values of nanoparticles produced by algal cultures of selected ages at different time (b). to better understand the kinetics of production of the nanoparticles by algal cells, the formation of agnps in solution was monitored for 21 days using uv-vis spectroscopy. for each sample the maximum absorbance was plotted against time. as shown in fig. 1b the same trend can be observed for all samples. almost linear relationship between the maximum absorbance and time during the first 14 days was observed. the absorbance intensity did not change significantly between the 10th and 14th day indicating that all ag+ ions were turned to nanoparticles (shaligram et al., 2009). from the 15th day the sharp decrease of maximum absorbance was recorded suggesting the aggregation and precipitation of nanoparticles. dark silver precipitates were present in the bottom of the flasks on 21st day and solution colour changed into transparent confirming the precipitation of silver. 3.2 influence of initial silver concentration the possibility of controlling the reaction rate and particle size was further investigated by changing the initial ag+ ion concentration. in order to study whether the concentration of silver ions plays an important role in the synthesis and size control of agnps, ag+ ions concentrations of 0.5, 1 and 2 mm were used. the increase in the maximum absorbance measured after 24 hours of nanoparticle biosynthesis with the increase of the initial silver ion concentration was observed (fig. 2a). the maximum synthesis of agnps occurred at 2 mm. the difference between maximum absorbance measured at concentration 1 mm and 2 mm, however, was not 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 0 5 10 15 20 25 time [day] a bs or ba nc e 1-w eek culture 2-w eek culture 3-w eek culture 4-w eek culture 0 0,2 0,4 0,6 0,8 1 1,2 300 350 400 450 500 550 600 650 700 wav elength [nm] a bs or ba nc e 1 w eek culture 2 w eek culture 3 w eek culture 4 w eek culture a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:02 utc 32 kaduková, j. et al. significant what is different from the results obtained by gurunathan et al. (2009). they found almost linear increase in the maximum absorbance up to concentration 5 mm. the agnps produced were characterised by uv-vis spectroscopy. the observation indicated that small, spherical nanoparticles were produced extracelluarly (fig. 2b). fig. 2. effect of various concentrations of silver ions on agnps biosynthesis expressed by the dependence of maximum absorbance on initial ag+ ion concentration (a) and uv-vis spectra of nanoparticles recorded after 24 hours of experiment. the kinetics of production and the stability of silver nanoparticles can be observed in more detail in fig. 3 where the maximum absorbance was plotted as a function of time. fig. 3. maximum absorbance values of nanoparticles prepared at different initial silver concentration recorded at different time. at silver ion concentration 0.5 mm linear increase of maximum absorbance with time was observed. at both higher studied concentrations (1 and 2 mm) the sharp increase of absorbance during the first 3 days was recorded then the decrease of maximum absorbance as well as formation of dark precipitates in the experimental flasks was observed suggesting the aggregation and precipitation of silver nanoparticles. 0 0,5 1 1,5 2 0 0,5 1 1,5 2 a g+ concentration [mm] m ax im um a bs or ba nc e 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 300 400 500 600 700 800 wavelength [nm] a bs or ba nc e 0.5 mm 1 mm 2 mm a b 0 0 ,5 1 1 ,5 2 2 ,5 0 5 10 15 t i m e [d ay] a bs or ba n ce 0 .5 m m 1 m m 2 m m bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:02 utc nova biotechnologica et chimica 13-1 (2014) 33 fig. 4 shows that the sizes of agnps increase with increasing ag+ ion concentrations. this indicates that the size of agnps can be modulated by the concentration of agno3. the reason of the increase in particle size with increasing agno3 is not clear yet, however, it is obvious that silver ions, by their dispersive action, have a role in controlling the growth of agnps. on the contrary to our observations, gurunathan et al. (2009) and song and kim (2009) found that nanoparticle size decreased with the increase of ag+ ion concentration in the range of 1 – 5 mm. they speculated that agno3 forms a coat on growing nanoparticles preventing their aggregation and resulting in formation of nanosized particles. in both studies mentioned above extracts from biomass were used in contrast to our experiments where algal cells were present in the experimental medium, thus, agno3 could be adsorbed on the algal surface allowing broader aggregation of silver resulting in larger nanoparticles formation. surface plasmon resonance bands were observed at 414, 431 and 455 nm for the silver nanoparticles prepared at silver ion concentrations 0.5, 1 and 2 mm, respectively. according to literature the shift of λmax to higher wavelength values is associated with an increase in size of agnps (fritsche et al., 1998), thus, this observation also confirms the increase of the nanoparticle size with increasing the initial silver ion concentration. this fact was finally confirmed by tem analyses. the average nanoparticle size was found to be 9, 14 and 18 nm at initial silver concentration 0.5, 1 and 2 mm, respectively (fig. 4). fig. 4. average size of nanoparticles formed at different initial silver concentration. the representative tem images of silver nanoparticles that were synthesised at different initial silver ion concentrations are shown in fig. 5. mono dispersed silver nanoparticles with uniform size and shape can be observed in left illustrations in fig. 5. the silver particles´ size histograms (right illustrations in fig. 5) show that the very small particles with average 9 nm were produced at silver ion concentration 0.5 mm. very narrow size distribution was found in this case in comparison with the both studied higher concentrations. more than 50% of nanoparticles were in the range of 6 – 10 nm. the presence of spherical silver nanoparticles relatively uniform in diameter in all three studied systems was confirmed by tem images. 0 5 10 15 20 25 50 100 200 initial silver c oncentration [mg/l] n an op ar tic le s iz e [n m ] bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:02 utc 34 kaduková, j. et al. fig. 5. tem images and corresponding size distribution histograms built from their analyses for agnps synthesized at initial silver concentration 0.5 mm (a), 1 mm (b) and 2 mm (c). 4. conclusions size control during synthesis of particles is an important criterion in the area of silver nanoparticle biosynthesis. depending on the size of nanoparticles, their application branch out. it has been demonstrated that the algae parachlorella kessleri are capable of producing of silver nanoparticles extracellularly and the size and 0 10 20 30 40 50 60 0-5 6-10 11-15 16-20 21-25 26-30 31-35 siz e [nm] f re qu en cy [% ] 20 nm 0 10 20 30 40 50 60 0-5 6-10 11-15 16-20 21-25 26-30 31-35 siz e [nm] f re qu en cy [% ] 50 nm 0 10 20 30 40 50 60 0-5 6-10 11-15 16-20 21-25 26-30 31-35 siz e [nm] f re qu en cy [% ] 50 nm a b c bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:02 utc nova biotechnologica et chimica 13-1 (2014) 35 stability of this nanoparticles can be controlled by the preparation conditions and nanoparticles with desirable characteristics can be produced. although culture age did not affect the nanoparticle production, the initial silver concentration has influenced the process in great extent. the controlling particles size at different initial silver ion concentrations would play an important role during optimization of a process. acknowledgement: the work was supported by slovak grant agency (vega), project no. 1/0235/12. references bankura, k.p., maity, d., mollick, m.m.r., mondal, d., bhowmick, b., bain, m.k., chakraborty, a., sarkar, j., acharya, k., chattopadhyay, d.: synthesis, characterization and antimicrobial activity of dextran stabilized silver nanoparticles in aqueous medium. 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pimpan, v.: green synthesis of silver nanoparticles for ammonia sensing. talanta, 76, 2008, 29-33. fayaz, a. m., girilal, m., rahman, m., venkatesan, r., kalaichelvan, p.t.: biosynthesis of silver and gold nanoparticles using thermophilic bacterium geobacillus stearothermophilus. process biochem., 46, 2011, 1958–1962 fritsche, w., porwol, h., wiegand, a.: in-situ formation of ag containing nanoparticles in thin polymer films. nanostruct. mater., 10, 1, 1998, 71–89 govindaraju, k., basha, s.k., kumar, v.g.., singaravelu, g.: silver, gold and bimetallic nanoparticles production using single-cell protein (spirulina platensis) geitler. j. mater. sci., 43, 2008, 5115-5122. gurunathan, s., kalishwaralal, k., vaidyanathan, r., deepak, v., pandian, s.r.k., muniyandi, j. hariharan, n., eom, s.h.: biosynthesis, purification and characterization of silver nanoparticles using escherichia coli, coll. surf. b: biointerfaces, 74, 2009, 328-335. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:02 utc 36 kaduková, j. et al. jena, j., pradhan, n., dash, b.p., sukla, l.b., panda, p.k.: biosynthesis and characterization of silver nanoparticles using microalga chlorococcum humicola and its antibacterial activity. int. j. nanomat. biostruct., 3, 1, 2013, 1-8. korbekandi, h., iravani, s., abbasi, s.: production of nanoparticles using organisms. crit. rev. biotechnol. 29, 4, 2009, 279-306. li, x., xu, h., chen, z., chen, g.: biosynthesis of nanoparticles by microorganisms and their applications. j. nanomater., 2011, art. id 270974, 16 pp. doi:10.1155/2011/270974 lin h-w., hwu w-h., ger m-d.: the dispersion of silver nanoparticles with physical dispersal procedures. j. mater. process technol. 206, 2008, 56–61. logeswari, p., silambarasan, s., abraham, j.: synthesis of silver nanoparticles using plants extract and analysis of their antimicrobial property. j. saudi chem. soc. 2013, (in press) maneerung, t., tokura, s., rujiravanit, r.: impregnation of silver nanoparticles into bacterial cellulose for antimicrobial wound dressing. carbohydr. polym. 72, 1, 2008, 43–51. mukunthan, k.s., elumalai, e.k., patel, t.n., murty, v.r.: catharanthus roseus: a natural source for the synthesis of silver nanoparticles. asian pac. j. trop. biomed., 2011, 270-274 petit, c., lixon, p., pileni, m-p.: in situ synthesis of silver nanocluster in aot reverse micelles. j. pys. chem., 97, 1993, 12974-12983. pinto, v.v., ferreira, m.j., silva, r., santos, h.a., silva, f., pereira, c.m.: long time efect on the stability of silver nanoparticles in aqueous medium: effect of the synthesis and storage conditions. coll. surf. a: physicochemical and engineering aspects, 364, 2010, 19-25. roopan, s.m., madhumitha, r.g., rahuman, a.a., kamaraj, c., bharathia, a., surendra, t.v.: low-cost and eco-friendly phyto-synthesis of silver nanoparticles using cocos nucifera coir extract and its larvicidal activity. ind. crops prod., 43, 2013, 631– 635. ruparelia j.p., chatterjee, a.k., duttagupta, s.p., mukherji, s.: strain specificity in antimicrobial activity of silver and copper nanoparticles. acta biomater., 4, 2008, 707-716 sanghi, r., verma, p.: biomimetic synthesis and characterization of protein capped silver nanoparticles. bioresour. technol., 100, 2009, 501-504. shaligram, n.s., bule, m., bhambure, r., singhal, r.s., singh, s.k., szakacs, g., pandey, a.: biosynthesis of silver nanoparticles using aqueous extract from the compactin producing fungal strain. process biochem., 44, 2009, 939–943 sharma, b., purkayastha, d.d., hazra, s., gogoi, l., bhattacharjee, ch. r., ghosh, n.n., rout, j.: biosynthesis of gold nanoparticles using freshwater green alga, prasiola crispa. mat. lett., 116, 2014, 94-97. sharma, k.v., yngard, r.a., lin, y.: silver nanoparticles: green synthesis and their antimicrobial activities. adv. colloid. interface sci., 145, 2009, 83-96. song, j.y., kim, b.s.: rapid biological synthesis of silver nanoparticles using plant leaf extracts. bioprocess biosyst. eng., 32, 2009, 79-84. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:02 utc nova biotechnologica et chimica 13-1 (2014) 37 xie, j., lee, j.y., wang, d.i.c., ting, y.p.: silver nanoplates: from biological to biomimetic synthesis. acs nano, 1, 5, 2007, 429-439. yan, j., chen, j.: colloidal nanosilver solution and method for making the same. us patent us 2003/0185889. 2003, 6pp. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:02 utc microsoft word jencarova final.doc nova biotechnologica et chimica 14-1 (2015) 87 doi 10.1515/nbec-2015-0018 © university of ss. cyril and methodius in trnava the metal and sulphate removal from mine drainage waters by biological-chemical ways jana jenčárová, alena luptáková institute of geotechnics, slovak academy of sciences, watsonova 45, 040 01 košice, sk-040 01, slovakia (jencarova@saske.sk) abstract: mine drainage waters are often characterized by high concentrations of sulphates and metals as a consequence of the mining industry of sulphide minerals. the aims of this work are to prove some biological-chemical processes utilization for the mine drainage water treatment. the studied principles of contamination elimination from these waters include sulphate reduction and metal bioprecipitation by the application of sulphate-reducing bacteria (srb). other studied process was metal sorption by prepared biogenic sorbent. mine drainage waters from slovak localities banská štiavnica and smolník were used to the pollution removal examination. in banská štiavnica water, sulphates decreased below the legislative limit. the elimination of zinc by sorption experiments achieved 84 % and 65 %, respectively. key words: metals, mine drainage water, sulphate-reducing bacteria 1. introduction industrial wastewater streams containing heavy metals such as copper, zinc, lead, chromium, nickel, cadmium, manganese, aluminium, cobalt, arsenic, silver, etc. are produced from different industries and from a variety of applications. mining influenced water (miw) is generated from chemical and biological processes in which sulphidic minerals, pyrite being the most common, are oxidized to sulphates and metallic hydroxides. the amount and toxicity of the generated water depends on several factors such as mineralogy of the rock material, surface area, crystallography, temperature, oxygen concentration, and the amount of water contacting the material (pinto et al., 2011). miw which is also defined as any water whose chemical composition has been affected by mining or mineral processing (wildeman and schmiermund, 2004) is often characterized by low ph (acid mine drainage) and high dissolved metal concentrations discharging to surface waters (miller et al., 2011). acidic drainage from abandoned metal mines is a widespread and persistent form of aquatic pollution (mayes et al., 2011). the conventional techniques for removing heavy metals from water include many processes such as chemical precipitation, flotation, ion exchange, electrochemical deposition, and adsorption. the adsorbents may be of mineral, organic or biological origin, zeolites, industrial byproducts, agricultural wastes, biomass, and polymeric materials (barakat, 2011; herkova et al., 2013; schütz et al., 2013). a possible alternative to the chemical treatment of these effluents is bioremediation using anaerobic sulphate-reducing bacteria (srb), taking advantage of the fact that these microorganisms grow in mining environments (duarte et al., 2008). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:16 utc 88 jenčárová, j. and luptáková, a. sulphate-reducing bacteria are important members of microbial communities with economic, environmental and biotechnological interest. they can exist in a variety of environments such as soils, sediments and domestic, industrial and mining wastewaters. srb are included in a group of chemoorganotrophic and strictly anaerobic bacteria, which contains representatives of the genera desulfovibrio, desulfomicrobium, desulfobacter and desulfotomaculum, among others (luptáková and kušnierová, 2005; martins et al., 2009). srb are useful to amd remediation due to two reasons. at first, because of their ability to reduce sulphates to hydrogen sulphide that additionally reacts with certain metals dissolved in the contaminated waters. on the other hand, the system acidity is reduced by their own action of sulphate reduction and by the carbon metabolism of the bacteria (garcia et al., 2001). anaerobic processes based on the use of sulphate reducing bacteria are able to use sulphate as an electron acceptor and to form hydrogen sulphide that leads to an increase in the ph of the water and the precipitation of heavy metals, forming insoluble sulphides. in addition, srb also produce bicarbonate as a by-product of the sulphate reduction, which also contributes to an increase in the ph (jimenezrodriguez et al., 2009). srb require a substrate composed of simple organic compounds using sulphate as a terminal electron acceptor. so4 2converts into h2s and hco3 −, according to the following equation. so4 2− + nutrients + h2o → h2s + hco3 − (1) sulphide precipitation is the desired mechanism of contaminant removal because metal sulphides are highly insoluble and less bio-available compared with other metal species. this process is particularly effective for removing heavy metals such as cadmium, copper, lead, mercury, zinc and iron to low concentration (kuyucak, 2002). metals can also be removed by co-precipitation with (or adsorption onto) fe and mn oxides and bacterially produced metal sulphides (jong and parry, 2003). iron sulphide solids may be beneficial for a variety of biogeochemical applications (zhou et al., 2014). it was found that iron sulphides produced by sulphate-reducing bacteria, is an excellent adsorbent for a wide range of heavy metals and had a very high specific uptake from solution for metal ions (jong and parry, 2004). the aim of this work was to examine a suitability of sulphate-reducing bacteria use to treat the mine drainage waters polluted with high concentration of sulphates and heavy metals from 2 slovak places (banská štiavnica, smolník). the next step was to evaluate the success rate of studied processes – sulphates elimination, metals precipitation and sorption. 2. material and methods 2.1 sulphate-reducing bacteria a mixed culture of sulphate-reducing bacteria (with predominant genus desulfovibrio) was isolated from mineral water collected at gajdovka spring (košice, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:16 utc nova biotechnologica et chimica 14-1 (2015) 89 slovakia) using medium postgate c (postgate, 1984). it is water with ph 7.5, h2s odour and with natural content of srb. bacteria were grown for 10 days at 30 °c in glass reaction flasks in anaerobic conditions that had been generated by introducing an inert gas (n2) and chemically with sodium thioglycollate. 2.2 mine drainage water samples of the mine drainage water were collected in slovak localities banská štiavnica and smolník. first sample from banská štiavnica (marked bš) is the outflow from dump of “new shaft” (where was the pb-zn ore deposit). second sample (marked sm) is from the shaft pech (smolník), which is acid mine drainage from the enclosed and flooded sulphide deposit (mainly fes2). therefore, waters outflows from dumps can contain increased concentrations of metals (such as fe, cu, zn, pb). they were analyzed by atomic absorption spectrometry – aas (spectrometer varian). 2.3 sulphates and metals removal process of sulphate reduction was monitored in both mine drainage water samples. 250 ml bottles were filled with modified postgate medium c (without sulphates usually present in standard medium), with mine drainage water samples (bš, sm) and inoculated with a mixture of srb (inoculum 10 %) taken from cultivation flasks. after inoculation, 1 ml of liquid phase from each sample was taken out. solutions were transferred to plastic tubes, centrifuged at 10 000 rpm for 10 minutes to small solid particles separation and then were extracts diluted and prepared for analysis. sampling was realized every second day during 2 weeks. “abiotic controls” were prepared by filling the flasks with modified postgate medium c and mine drainage water, without srb inoculation. sampling was the same. bottles were all the time stored in thermostat at 30 °c and enclosed to avoid an oxygen entry. the concentration of the sulphates in the solutions was determined by the dionex ics-5000 ion chromatograph. moreover, at the beginning and at the end of experiments mentioned above, 5 ml of each sample were taken out, filtrated, stabilized by hno3, diluted and prepared for the measurement of selected metal ions concentration by aas. there was metal bioprecipitation by the srb application and concentration decrease expected at the end. 2.4 biogenic sorbent preparation and utilization the creation of biogenic sorbent in the form of iron sulphides was realized in a modified growth medium for srb cultivation postgate c. the modification in this case consist of an addition of fe ions in form of sulphates (feso4.7h2o, fe2(so4)3.9h2o) and double dose of sodium lactate as carbon and energy source. the production was performed in glass bottles containing growth medium with ph around 7.5 % and 10 % of bacteria inoculum, at 30°c, under anaerobic conditions. the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:16 utc 90 jenčárová, j. and luptáková, a. samples composition was evaluated by using x-ray powder diffraction measurement on bruker-axs d8 advance device. sorption tests were conducted by mixing 0.1 g of sorbent samples with 100 ml of mine drainage water samples at room temperature in plastic erlenmeyer flasks for 24 hours. the mixture was during experiment continuously stirred using mechanical laboratory shaker at 250 oscillations per minute. 2 ml of liquid phase were taken out in predetermined intervals, filtered and prepared for analysis. the concentrations of the metal ions in the filtrates were determined by aas. 3. results and discussion the results of mine drainage waters analysis from banská štiavnica and smolník are summarized in table 1. table 1. the analysis of mine drainage water (selected parameters). sulphates fe zn cu sample ph mg/l banská štiavnica (bš) 6.1 950 <0.05 5.6 0.2 smolník (sm) 3.8 1660 205.1 6.9 1.3 in the past ore minerals such a sphalerite, pyrite, chalcopyrite, marcasite were mined in banská štiavnica. in smolník it was mainly pyrite and chalcopyrite. therefore, higher concentrations of fe, zn and cu were found in sampled waters. water from banská štiavnica contained higher amount of zinc. very high concentration of iron besides increased zinc presence was confirmed in water from smolník. next research was focused on metals (iron, zinc) and sulphates elimination from mine drainage waters by biological-chemical processes caused by srb performance and h2s presence. the ph of nutrient medium before inoculation and mine drainage water addition was adjusted to 7.5, the value suitable for srb growth. all three components were then mixed in the bottles and sealed with butyl rubber stoppers. the ph conditions very probably caused immediate precipitation of some amount of metals dissolved in mine drainage water samples. remaining metals were possibly eliminated by following srb activity hydrogen sulphide production and bioprecipitation or adsorption onto bacterially produced sulphides. the specific responsible mechanism was not examined. song et al. (2001) state once sulphate-reducing conditions are established, sulphide precipitation becomes the predominant mechanism of metal removal from acid mine drainage. watson and ellwood (1994) performing industrial effluents treatment found out that many of the removed metals do not form insoluble sulphides, but were removed when iron sulphide was produced. they came to the conclusion that the bacterially created iron sulphide was acting as an adsorbent for a wide range of heavy metals including many not normally precipitated as sulphides. next study bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:16 utc nova biotechnologica et chimica 14-1 (2015) 91 revealed that metal sulphide solid phases produced by sulphate-reducing bacteria can remove co, cr, cu, hg, pb, zn, mn, fe, as and al. the metal ion concentration was reduced from 10 mg/l to a few μg/l (watson et al., 1995). the batch adsorption experiments realized by marius et al. (2005) shown very successful removal of cadmium. the changes in metal concentrations in solutions with srb are summarized in table 2. iron and zinc elimination in sample with water from banská štiavnica was not very significant. but iron concentration decrease in smolník water was evident, from 64 to final value 0.7 mg/l. in blank (abiotic) samples were no or very little concentration changes and therefore are not listed. acid mine drainage treatment by srb by costa et al. (2008) showed almost complete precipitation of the main metals (fe, cu, zn). metals removal was attributed to the combined result of precipitation as metal sulphides, (oxy)hydroxides, coprecipitation and sorption onto the matrix materials surface. no enough sulphate reduction efficiency was obtained, which corresponds to less than 50 % of removal. machemer and wildeman (1992) pointed out to similar finding, where most or all of the fe, cu and zn were removed. manganese elimination was significantly lesser, about 30-40 %. the efficiency of sulphate decrease did not reach 25 %. table 2. iron and zinc concentrations in solutions. fe zn mg/l sample initial concentration (after mixing) final concentration initial concentration (after mixing) final concentration medium+srb+bš 2.1 1.4 0.4 0.3 medium+ srb+sm 64.3 0.7 0.3 <0.03 the elimination of sulphates from mine drainage water samples (bš, sm) by sulphate-reducing bacteria is shown in fig. 1. metabolic processes of bacterial culture isolated from gajdovka spring were in both experiments as expected and the required sulphate-reduction was running in created conditions. almost all sulphates in sample with banská štiavnica mine water were removed within the experiment duration. the concentration in water from smolník did not decrease below 250 mg/l, which is the limit value for sulphates in water in legislative. there is the need of supplementary nutrients for bacterial activity to lower the sulphates under the limit. it is also evident that in “abiotic controls” (ac-bš, ac-sm) were no changes in sulphates concentration noticed. the other step of investigation was aimed at biogenic sorbent composition study. as it has already been mentioned, hydrogen sulphide generated as a consequence of srb metabolic activities reacts in medium with soluble metal (iron) ions. final products are strong dependent on solution chemistry, temperature and last but not least, on length of creation. previous examinations on biogenic iron sulphide formation in natural conditions have identified poorly crystalline mackinawite and greigite as bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:16 utc 92 jenčárová, j. and luptáková, a. major solid phases (schoonen, 2004). in addition, many intermediate iron sulphides may exist between disordered mackinawite and well crystallized pyrite in anaerobic environments, including greigite, marcasite, smythite and pyrrhotite (mokone et al., 2010). 0 100 200 300 400 500 600 700 800 900 1000 0 50 100 150 200 time [hours] s u lp h at es c o n ce n tr at io n [ m g /l] bš sm ac-bš ac-sm fig. 1. decreasing of sulphates concentration during bacterial sulphate-reduction in mine drainage waters. the result of x-ray analysis of sorbent sample prepared by srb cultivation is shown in fig. 2. it revealed three compounds, identified as mackinawite, greigite, and sulphur alpha. this corresponds to gramp et al. (2010), where major peaks for biologically produced precipitates have been identified as iron sulphide minerals mackinawite and greigite. the presence of sulphur alpha may be explained by incomplete sulphate reduction or sulphides oxidation. fig. 2. xrd patterns of biogenic sorbent. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:16 utc nova biotechnologica et chimica 14-1 (2015) 93 figure 3 illustrates the sorption experiments results carried out by mixing the biogenic sorbent with mine drainage water samples (bš and sm) to verify sorption properties of created sorbent. because of low initial copper concentration in waters, only zinc removal was spotted. 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 0 20 40 60 80 100 120 140 160 180 200 220 240 tim e [m in] q [ m g /g ] zinc smolník zinc banská štiavnica fig. 3. zinc sorption from mine drainage water samples by biogenic sorbent. it is visible, that sorption from both samples became more stabilized after 120 minutes. the total zinc elimination after 4 hours in sample sm achieved 65 %, in sample bš it was about 84 %. the removal percentage value for zinc obtained by jong and parry (2004), which studied zinc sorption by bacterially produced metal sulphides from synthetic solution (ph = 7), was 93.3 %. the progress of each experiment and total removal could be influenced by different factors, such as an initial zinc concentration, ph of water, presence and amount of other metal ions in solution, etc. any other mechanism such as sorption was not in this case assumed and examined. 4. conclusion this work was oriented on the sulphate-reducing bacteria application for heavy metals and sulphates elimination from mine drainage waters. the results confirmed that srb from gajdovka spring were utilizable and under appropriate conditions (nutrient, ph, temperature), able to reduce high concentrations of sulphates in water samples to minimum and precipitate dissolved metals to insoluble form. 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poľnohospodárska knižnica | heruntergeladen 16.01.20 17:16 utc nova biotechnologica et chimica 11-2 (2012) 139 doi 10.2478/v10296-012-0016-x © university of ss. cyril and methodius in trnava removal of cd2+ and pb2+ from aqueous solutions using bio-char residues katarína štefušová, michal lovás, anton zubrik, marek matik, miroslava václavíková institute of geotechnics of slovak academy of sciences, watsonova 45, košice, sk043 53, slovak republic (stefusova@saske.sk) abstract: in this paper, wheat straw and rapeseed residues before and after microwave pyrolysis during biooil production were studied as potential sorbents of heavy metals. the sorbents were characterized by elemental analysis and ftir spectroscopy. sorption properties of the materials were investigated using batch adsorption-equilibrium experiments and the effect of initial cd and pb concentration was studied. the experimental data fit langmuir adsorption isotherm. the maximum sorption affinity of studied materials was observed in the case of rapeseed and its sorption capacity was 31.6 and 83.5 mg/g for cd and pb, respectively. key words: biomass, microwave pyrolysis, char, cadmium, lead, sorption 1. introduction water pollution is one of the most important environmental problems. among the aquatic pollutants, heavy metals have gained relatively more significance in view of their persistence, bio-magnification and toxicity. the main sources of heavy metal contamination are e.g. metal plating, mining operations, tanneries, radiator manufacturing, smelting, alloy industries and storage batteries manufactures (kadirvelu et al., 2001). treatment processes for heavy metals removal from wastewaters include precipitation, membrane filtration, ion exchange, adsorption and co-precipitation/adsorption. cost-effective alternative technologies for the treatment of metal-containing wastewaters are needed. adsorption has evolved as one of the most used treatment techniques for removing metal ions to lowers from water which cannot be removed by other techniques (mohan et al., 2007). in recent years, considerable attention has been focused on the removal of heavy metals from aqueous solution using adsorbents derived from low-cost materials, such as agricultural waste and by-products. natural materials that are available in large quantities, or certain waste products from agricultural operations, may have potential as inexpensive adsorbents. due to their low cost, after these materials have been expended, they can be disposed of without regeneration. in general, adsorbents can be assumed as low cost if they require little processing, are abundant in nature, or are a by-product or waste material from another industry (bailey et al., 1998). several methods have been studied over the years, for the production of low-cost adsorbents. pyrolysis is one of the methods that can be used for production of low-cost adsorbents (chen et al., 2011; liu et al., 2011; liu et al., 2012; mohan et al., 2007) pyrolysis can be described as the direct thermal decomposition of the organic matrix in the absence of oxygen to obtain an array of solid, liquid and gas products. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:05 utc 140 štefušová, k. et al. microwave heating technology has been shown to be more energy efficient than conventional methods in many applications. microwave irradiation is rapid and volumetric with the whole material heated simultaneously. in contrast, conventional heating is slow and the heat is introduced into the sample from the surface. microwave pyrolysis has gained momentous attraction because of the merits mentioned above. existing research works on microwave pyrolysis of biomass includes pyrolysis of wheat straw (budarin et al., 2009), corn stover (yu et al., 2007), rice straw (huang et al., 2008), biomass (wan et al., 2009), coffee hulls (domínguez et al., 2007), and pine wood sawdust (chen et al., 2008). the main purpose of this study was the evaluation of pb and cd adsorption from aqueous solutions onto straw and rapeseed char. sorption properties of the materials were studied before and after their conversion into biochars through microwave pyrolysis treatment. 2. materials and methods 2.1 sorbent preparation samples of the wheat straw and rapeseed (palma sečovce, s.r.o) were crushed by (air grinder mc – fdv), sieved to granulometric fraction under 1 mm and washed with deionized water and dried at temperature of 100°c. the material were press to briquettes and used for pyrolysis. after pyrolysis samples were crushed, sieved and dried again. microwave pyrolysis experiments were carried out at nitrogen atmosphere and power 900 w. samples were characterized by elemental analysis (chns elementar vario macro cube) and ftir spectroscopy (ft-ir bruker tensor 27). 2.1 adsorption experiments the sorption properties of raw materials and biomass char residues were tested under batch conditions. the initial cd and pb concentration was 10-200 and 10250 mg/l, respectively. model solutions of cd and pb were prepared by dissolving cd(no3)2.4h2o and pb(no3)2 in deionized water, respectively. the sorbent concentration was 2 g/l. the ph of the solutions was adjusted to value of 5.0 with 0.01 and 0.1 m naoh and hno3. the experiments were performed at constant temperature of 25°c in a rotary shaker set at 30 rpm and equilibrium time 24 hours. the metal quantity in solutions was determined by aas (varian 240 rs/240 z, australia) before and after the sorption experiments. the amount of the metal sorbed (mg) per unit mass of sorbents (g) qeq, was calculated using the eq. (1) s eq eq c cc q − = 0 (1) where c0 and ceq are initial and equilibrium metal concentration (mg/l), respectively and cs is the sorbent concentration in solution (g/l). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:05 utc nova biotechnologica et chimica 11-2 (2012) 141 3. results and discussion 3.1 characterization of sorbents the results of the elemental analysis of tested materials are given in table 1 and 2. during the conversion of biomass to biochar the original carbon is retained in the biochar, which offers a significant opportunity for creating a carbon sink. as a consequence of the pyrolysis process, the carbon content of the carbonized residue increased with regard to the original biomass, along with a deoxygenation as a consequence of the loss of functional groups during the process. there was also a noticeable decrease in the hydrogen content, probably due to the great proportion of hydrogen compounds in the volatile matter. table 1. characterization of biomass samples (a – ash, v – volatile matter, w – moisture; superscripts: d – dry basis, a – analytical specimen, od – by difference according to 100 (ad + cd + hd + nd+ sd). sample ad wa v nd[%] cd[%] hd[%] sd[%] od[%] rapeseed 7.0 4.2 74.6 5.44 46.07 6.59 0.79 34.11 wheat straw 7.3 4.7 74.8 1.7 39.67 5.96 0.24 45.13 table 2. characterization of biomass samples after microwave pyrolysis. sample recovery [%] time of pyrolysis [min] n[%] c[%] h[%] s[%] rapeseed 27 25 4.26 59.83 1.237 0.414 wheat straw 24 30 1.56 63.92 1.635 0.315 ftir spectroscopy was used to characterize the functional groups responsible for heavy metals adsorption. the ftir spectra of rapeseed and wheat straw before and after pyrolysis are given in fig. 1 and 2, respectively. it is important to note that visible vibrations presented in the spectrum represent not only one structure, but there are several group/bond vibration, which are often overlapped. the broad peak at maximum of 3287 cm-1 represents hydroxyl groups (vibration frequencies of water, alcohols, and phenols) and n–h stretching vibrations (abdel-nasser and elhendawy, 2006). the peaks at 2924 and 2854 cm-1 can be assigned to ch2 (asymmetric) and ch3 (symmetric) stretch of aliphatic structures. vibration frequency at 1743 cm-1 can be assigned to esters. theoretical vibration frequencies c=o functional group of aldehydes and ketones are shifted to lower values (around 1725 1700 cm-1), even though these compounds also contribute to this absorbance. next peak at 1634 cm-1 can be associated with weaker h-o-h bending vibration of water. moreover, in this region, there are also c=c bond of alkenes and aromatic compounds and c=o stretching vibration of carbonyl group. the aromatic skeletal c=c bonds are visible in the region at 1517 cm-1 and 1443 cm-1, respectively. c–o stretching bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:05 utc 142 štefušová, k. et al. vibration can be also presented in this region (around 1423 cm-1) (barka et al., 2010). asymmetric stretching vibration of c-o-c in esters and/or organic acids is presented at 1234 cm-1. absorption bands at 1132 cm-1 and 1023 cm-1 are assigned to deformation vibrations of c-h bonds in aromatic ring and deformation vibrations of c-o bonds in alcohols and carboxylic acids (tuzen et al., 2009). 0 0,02 0,04 0,06 0,08 5001000150020002500300035004000 wavenumber [cm-1] a bs or ba nc e r s 3287 17432855 2924 1634 i 1234 i 1518 1023 1132 i 1421 i fig. 1. ft-ir spectroscopy of wheat straw and rapeseed. degradation of the organic phase was observed in both samples after process of pyrolysis (fig. 2). decreasing in the band intensities is visible in all regions and only some peaks are presented in the ft-ir spectrum. for example, a visible intensity was recorded at 1010 cm-1 in the case of wheat straw residue. 3.2 adsorption the effect of initial metal concentration on sorption by tested materials is shown in fig. 3-6. isotherm studies provide information on the capacity of sorbent. adsorption isotherms are characterized by certain constants and describe the mathematical relationship between the quantity of adsorbate and concentration of adsorbate remaining in the solution at equilibrium. the experimental data were evaluated using langmiur isotherm (eq. 2). eq eq eq bc1 bc qq + = max (2) where qeq (mg/g) is the weight adsorbed per unit mass of adsorbent at equilibrium, qmax (mg/g) is the theoretical monolayer adsorption capacity, b (l/mg) is the langmuir constant related to the energy of adsorption and ceq (mg/l) is the equilibrium concentration. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:05 utc nova biotechnologica et chimica 11-2 (2012) 143 0 0,02 0,04 0,06 5001000150020002500300035004000 wavenumber [cm-1] a bs or ba nc e r (mp) s (mp) fig. 2. ft-ir spectra of char residues (wheat straw and rapeseed) after microwave pyrolysis. the effect of initial cd and pb concentration on sorption capacity of tested sorbents is shown in fig. 3-6. it was observed that the increase of initial metal concentration caused increasing of the metal amount adsorbed per mass unit. the maximum obtained sorption capacity of wheat straw was 5.5 and 14.0 mg/g for cd and pb, respectively. microwave pyrolysis of straw caused a considerable increase of sorption capacity of material. the maximum sorption capacity of wheat straw after pyrolysis was 10.5 and 33.5 mg/g for cd and pb, respectively. the sorption capacity of rapeseed was 31.6 and 83.5 mg/g for cd and pb, respectively. microwave pyrolysis of rapeseed caused a decrease of sorption affinity of material toward cd and pb ions. the sorption capacity of rapeseed after microwave pyrolysis was 4.8 and 26.2 mg/g for cd and pb, respectively. experimental data were in good agreement with langmuir isotherms. langmuir adsorption parameters are given in table 3. table 3. langmuir adsorption parameters. cd pb qmax[mg/g] b[l/mg] r 2 qmax[mg/g] b[l/mg] r 2 s 6.71 0.02 0.98 14.24 0.12 0.99 smp 11.99 0.04 0.99 23.91 0.72 0.97 r 49.36 0.02 0.98 88.53 0.24 0.99 rmp 5.60 0.08 0.98 26.89 0.41 0.97 the sorption of cd and pb by biomass char residues has been studied by several authors, as well. mohan et al. (2007) studied cd and pb sorption by chars produced from pyrolysis of pine wood, pine bark, oak wood and oak bark during bio-oil production. the most efficient sorbent was oak bark char with sorption capacity bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:05 utc 144 štefušová, k. et al. 2.9 and 11.3 mg/g for cd and pb (c0=10 mg/l), respectively. tangjuank et al. (2009) studied removal of cd and pb by activated carbon prepared from cashew nut shells. the maximum sorption capacity of sorbent was 14.3 and 28.9 mg/g for cd and pb (c0=40 mg/l), respectively. 0 20 40 60 80 100 120 140 160 180 200 0 2 4 6 8 10 12 s s mp q e q[m g/ g] c eq [mg/l] fig. 3. cd adsorption on wheat straw (s) and wheat straw char residues (smp). 0 20 40 60 80 100 120 140 160 0 5 10 15 20 25 30 35 r r mp q e q[m g/ g] c eq [mg/l] fig. 4. cd adsorption on rapeseed (r) and rapeseed char residues (rmp). 0 20 40 60 80 100 120 140 0 5 10 15 20 25 30 35 s s mp q e q[m g/ g] c eq [mg/l] fig. 5. pb adsorption on wheat straw (s) and wheat straw char residues (smp). 0 20 40 60 80 100 120 140 160 180 200 0 10 20 30 40 50 60 70 80 90 r r mp q e q[m g/ g] c eq [mg/l] fig. 6. pb adsorption on rapeseed (r) and rapeseed char residues (rmp). 4. conclusions in this work straw and rapeseed before and after microwave pyrolysis were studied as pb and cd sorbents. the chars produced from microwave pyrolysis of straw and rapeseed during bio-oil production were characterized and used successfully without activation for the removal of cd and pb from water. microwave pyrolysis caused significant changes in sorption properties of materials. after pyrolysis the sorption capacity of straw increased while in the case of rapeseed it decreased. the cd sorption on the sorbents followed the order: r>>smp>s~rmp. the pb sorption on the sorbents followed the order: r>>smp>rmp>s. the sample of untreated rapeseed has shown the highest sorption affinity toward cd and pb ions. the maximum sorption capacity of the untreated rapeseed was 31.6 mg and 83.5 mg/g for cd and pb, respectively. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:05 utc nova biotechnologica et chimica 11-2 (2012) 145 acknowledgements: this work has been supported by science and scientific grant agency vega, project no. 0141 and the slovak research and development agency under the contract no. apvv-0252-10. references bailey, s.e., olin, t.j., brika, r.m., adrian d.a.: a review of potentially low-cost sorbent for heavy metals. water res., 33, 1998, 2469-2479. barka, n., abdennouri, m., boussaoud, a., el makhfouk, m.: biosorption characteristics of cadmium(ii) onto scolymus hispanicus l. as lowcost natural biosorbent. desalination, 258, 2010, 66–71. budarin, v.l., clark, j.h., lanigan, b.a., shuttleworth, p., breeden, s.w., wilson, a.j., macquarrie, d.j., milkowski, k., jones, j., bridgeman, t., ross, a.: the preparation of high-grade bio-oils through the controlled, low temperature microwave activation of wheat straw. bioresour. technol., 100, 2009, 6064-6068. chen, m.q., wang, j., zhang, m.x., chen, m.g., zhu, x.f., min, f.f., tan, z.c.: catalytic effects of eight inorganics additives on pyrolysis of pine wood sawdust by microwave heating. j. anal. appl. pyrolysis, 82, 2008, 145-150. chen, x., chen, g., chen, l., chen, y., lehmann, j., mcbride m.b., hay, a.j:: adsorption of copper and zinc by biochars produced from pyrolysis of hardwood and corn straw in aqueous solution. bioresour. technol., 102, 2011, 8877-8884. domínguez, a., menéndez, j.a., fernández, y., pis, j.j., valente nabais, j.m., carrott, p.j.m., ribeiro carrott, m.m.l.: conventional and microwave induced pyrolysis of coffee hulls for the production of a hydrogen rich fuel gas. j. anal. appl. pyrolysis, 79, 2007, 128-135. el-hendawy, a.n.a.: variation in the ftir spectra of a biomass under impregnation, carbonization and oxidation conditions. j. anal. appl. pyrolysis, 75, 2006, 159–166. huang, y.f., kuan, w.h., lo, s.l., lin, c.f.: total recovery of resources and energy from rice straw using microwave-induced pyrolysis. bioresour. technol., 99, 2008, 8252-8258. kadirvelu, k., thamaraiselvi, k., namasivayam, c.: removal of heavy metals from industrial wastewaters by adsorption onto activated carbon prepared from an agricultural solid waste. bioresour. technol., 76, 2001, 63-65. liu w.j., zeng, f.x., jiang, h., zhang, x:s.: preparation of high adsorption capacity bio-chars from waste biomass. bioresour. technol., 102, 2011, 82478252. liu, y., zhao, x., li, j., ma, d., han, r.: characterization of bio-char from pyrolysis of wheat straw and its evaluation on methylene blue adsorption. desalin. water treat., 46, 2012, 115-123. mohan, d., pittman, c.u., bricka, m., smith, f., yancey, b., mahammad, j., steele, p.h., alexandre-franco, m.f., gómezserrano, v., gong, h.: sorption of arsenic, cadmium, and lead by chars bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:05 utc 146 štefušová, k. et al. produced from fast pyrolysis of wood and bark during bio-oil production. j. colloid interface sci., 310, 2007, 57-73. tangjuank, s., insuk, n., tontrakoon, j., udeye, v.: adsorption of lead(ii) and cadmium(ii) ions from aqueous solutions by adsorption on activated carbon prepared from cashew nut shells. world academy of science, engineering and technology, 52, 2009, 110-116. tuzen, m., sari, a., mendil, d., uluozlu o.d.,, soylak, m., dogan, m.: characterization of biosorption process of as(iii) on green algae ulothrix cylindricum. j. hazard. mater., 165, 2009, 566-572. wan, y., chen, p., zhang, b., yang, c., liu, y., lin, x., ruan, r.: microwave-assisted pyrolysis of biomass: catalyst to improve product selectivity. j. anal. appl. pyrolysis, 86, 2009, 161-167. yu, f., deng, s., chen, p., liu, y., wan, y., olson, a., kittelson, d., ruan, r.: physical and chemical properties of bio-oils from microwave pyrolysis of corn stover. appl. biochem. biotechnol., 136-140, 2007, 957-970. presented at the 2nd international conference “biotechnology and metals 2011”, september 22–23, 2011, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:05 utc microsoft word nagy-szabo a et al 2012.doc nova biotechnologica et chimica 11-1 (2012) 27 doi 10.2478/v10296-012-0003-2 ©university of ss. cyril and methodius in trnava monitoring of polycyclic aromatic hydrocarbons (pahs) in surface water of the hungarian upper section of the danube river andrea szabó nagy1, gábor simon1, istván vass2 1department of physics and chemistry, széchenyi university, h-9026 győr, egyetem square 1. hungary (nszaboa@sze.hu) 2laboratory of the inspectorate for environment, nature and water of the north transdanubian region, h-9028 győr, ignác török road 68. hungary (vass@edktvf.kvvm.hu) abstract: the aim of this paper is to investigate the concentrations of polycyclic aromatic hydrocarbons (pahs) in surface water of the hungarian upper section of the danube river in the period of 2007-2010. a total of 77 water samples were collected from the sampling sites located at rajka, medve and komárom (1848, 1806 and 1766 river km) under the authority of the inspectorate for environment, nature and water of the north transdanubian region designated by the hungarian national monitoring programme. sixteen pahs identified by the us environmental protection agency (usepa) as priority pollutants were monitored. concentrations of total 16 pahs (∑pahs) ranged from 25 to 357 ng⋅l-1 with the mean value of 98.27 ± 58.48 ng⋅l-1. the low and medium molecular weight pahs (2-3 and 4 ring) ranged from below method detection limit (<1) to 136 ng⋅l-1 while high molecular weight pahs (5-6 ring) were present at much lower concentrations (<1-25 ng⋅l-1). the 2-3-ring pahs contributed to about 64% while 4-6-ring pahs accounted for 36% of the ∑pahs. the dominant species are naphthalene and phenanthrene in the surface water. concentration ratios of specific pah compounds including anthracene/(anthracene+phenanthrene) and fluoranthene/(fluoranthene+pyrene) were calculated to evaluate the possible sources of pah contamination. the levels of ∑pahs determined in our study were compared with other sections of the danube and other regions of the world. keywords: polycyclic aromatic hydrocarbons, pah, danube, monitoring, surface water 1. introduction polycyclic aromatic hydrocarbons (pahs) are a large group of organic compounds with two or more fused aromatic rings. the us environmental protection agency (us epa) has identified 16 unsubstituted pahs as priority pollutants for measurement in environmental samples, some of which are considered to be possible or probable human carcinogens, and hence their distribution in the environment and potential risks to human health have been the focus of much attention. pahs originate from both the natural as well as anthropogenic sources that mainly include thermal combustion processes, vehicular emissions, oil contaminations and biomass burning. combination of their physicochemical properties, such as low aqueous solubility, moderate vapour pressure, high octanol-water partition coefficient and persistence in environment make them capable of long range transport (manoli and samara, 1999; malik et al., 2011). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:57 utc 28 szabó nagy, a. et al. atmospheric deposition is considered to be an important input of pahs to surface waters. low molecular weight pahs (twoand three-ring) occur in the atmosphere in the vapour phase whereas multi-ringed pahs (five-ring) are bound to particles. intermediate molecular weight pahs (four-ring) are partitioned between the vapour and particulate phases, depending on atmospheric temperature (srogi, 2007). beside the atmospheric fallout includes wet and dry deposition of particles the pahs enter surface waters especially via urban run-off, municipal effluents, industrial effluents and oil spillage or leakage (baker and eisenreich, 1990; manoli and samara, 1999). they have a relatively low solubility in water, but are highly lipophilic. similarly to the process observed in the atmosphere pahs associate easily with particulate matter and are finally deposited in the sediment. therefore their concentration in water is low (ko and baker, 1995; yunker et al., 2002). the concentrations of pahs in river sediments are generally much higher than in the surrounding water body (malik et al., 2011). the aim of this paper is to investigate the concentrations of pahs in surface water of the hungarian upper section of the danube river in the period of 2007-2010. the water samples were collected from the sampling sites located at rajka, medve and komárom (1848, 1806 and 1766 river km) under the authority of the inspectorate for environment, nature and water of the north transdanubian region designated by the hungarian national monitoring programme. the concentrations of 16 us epa pahs were studied. concentration ratios of specific pah compounds including anthracene/(anthracene+phenanthrene) and fluoranthene/(fluoranthene+pyrene) were calculated to evaluate the possible sources of pah contamination. the levels of total 16 pahs (∑pahs) determined in our study were compared with other sections of the danube and other regions of the world. the results of this study add new data to the international database of the danube river producing comparable and reliable information on water quality. 2. materials and methods 2.1 study area and sampling the length of the hungarian section of the danube is 417 km (1433-1850 river km) from which the length along the hungarian and slovakian border is 142 km. the danube has been diverted to the bypass canal of the gabcikovo hydroelectric power plant and the slovakian side provides water into the old riverbed from the hrusov reservoir. its water quality is characterized at the rajka sampling site (1848 rkm). above the medve sampling site (1806 rkm) the divided water of the river is reunited again. the water quality at the komárom sampling site (1766 rkm) is significantly influenced by the moson danube flowing into the danube. the sampling sites are shown in fig. 1. a total of 77 surface water samples were collected from the three sampling sites mentioned above and analysed for the 16 us epa pahs in the period of 2007-2010. the number of sampling was not consistent in the examined years or even at the sampling sites depending on the prevalent national monitoring programmes. at one bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:57 utc nova biotechnologica et chimica 11-1 (2012) 29 sampling site, a number between 1-12 samples were collected in a year at different months. the sampling location in profile was in the middle of the river (∼30 cm below the surface) on the bridge at the sampling sites of medve and komárom. the samples were collected on the right side of the danube river at the sampling site of rajka. the water samples were directly collected from the river using 2 l brown glass bottles and transferred to the laboratory directly. the samples were stored at 4 °c. rajka medve komárom fig. 1. the sampling locations in the danube river. 2.2 analysis the pah analysis was conducted in accordance with the hungarian standard method procedure (msz 1484-6:2003). 1 l of the water sample was acidified to ph = 2 with 5 mol⋅l-1 h2so4. 100 ng of a sv internal standard mix (restek) was added to the sample. liquid-liquid extraction with 30 ml n-hexane was applied in three times. the extract was dried using anhydrous sodium sulphate and concentrated to 1 ml by a rotary evaporator. pahs in the concentrated extract were fractionated by a silica gel column (200 mm long, 4 mm internal diameter). the column was first eluted with 30 ml of n-hexane and the eluate was discarded. further elution was carried out with 30 ml of dichloromethane to obtain pahs fraction. the pahs containing fraction was concentrated to 1 ml by using a rotary evaporator, then 100 ng of a wa eph aromatic hydrocarbon standard (restek) was added to the sample for further chromatographic analysis. a gas chromatography-mass spectrometry (gc-ms) system, consisting of a gas chromatograph (agilent 6890) with a gc column (rtx-5ms-integra 30 m long, 0.25 mm internal diameter, 0.25 μm coating) and a mass spectrometer (agilent 5973), was used for the determination. selected ion monitoring (sim) mode was employed for the identification and quantification of 16 individual pah compounds. by this method, the 16 pah compounds were determined with the limit of detection of about 1 ng⋅l-1. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:57 utc 30 szabó nagy, a. et al. 3. results and discussion figure 2 illustrates the spatial and time distributions of annual average concentration, standard deviation and median of the ∑pahs in surface water of the danube river at the different sampling sites in the period of 2007-2010. table 1 summarises the concentration ranges and the mean values of the individual pah compounds in all of water samples for the examined period. 0 20 40 60 80 100 120 140 160 180 200 220 n = 6 n = 7 n = 12 n = 25 2007 2008 2010 all samples ∑ p a h s ng ⋅l -1 average median rajka 0 20 40 60 80 100 120 140 160 180 200 220 n = 7 n = 12 n = 1 n = 20 2007 2008 2009 all samples ∑ p a h s ng ⋅l -1 average median komárom 0 20 40 60 80 100 120 140 160 180 200 220 n = 6 n = 10 n = 4 n = 12 n = 32 2007 2008 2009 2010 all samples ∑ p a h s ng ⋅l -1 average median medve fig. 2. mean concentrations, standard deviations and medians of total pahs in surface water of the danube river at the sampling sites (n: number of collected samples). during the study period the concentrations of ∑pahs in the river water of the hungarian upper section of the danube ranged from 25 to 357 ng⋅l-1 with the mean value of 98.27 ± 58.48 ng⋅l-1. for individual pahs, two-, threeand four-ring pahs are the most abundant, the concentrations of them ranged from below method detection limit (<1) to 136 ng⋅l-1. the concentrations of fiveand six-ring pahs were much lower, ranged from <1 to 25 ng⋅l-1. dibenz[ah]anthracene was not detected in the water samples. the naphthalene and phenanthrene were the most dominant pah compounds with the average distribution of 21 and 26% of the total pahs in the river water, respectively. the 2-3-ring and 4-ring pahs contributed to about 64 and 28% while 5-6-ring pahs accounted for 8% of the total pahs. it is remarkable that fluoranthene and pyrene had also been significantly high average contribution to the ∑pahs (each about 12%). a lot of previous studies have been reported about pahs levels measured for decades in other rivers of the world (maldonado et al., 1999; doong and lin, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:57 utc nova biotechnologica et chimica 11-1 (2012) 31 2004; nagy et al., 2007; luo et al., 2008; malik et al., 2011, and references therein), although a direct comparison of literature data is difficult due to the difference in the phase analyzed (dissolved, particulate or both), the analytical methods used, and the compounds considered in each study. comparison of the ∑pahs concentrations in our study with other data of the danube river at various sampling sites and some different rivers of the world is presented in table 2. table 1. the concentrations of individual pahs in all of water samples (n = 77) from danube river (collected at 1848, 1806 and 1766 river km) in the period of 2007-2010. concentration (ng⋅l-1) pah compounds total rings range mean ± sd naphthalene 2 1−120 21.09 ± 19 acenaphthene 3 <1−15 3.62 ± 3.20 acenaphthylene 3 <1−20 3.51 ± 3.77 fluorene 3 <1−18 6.16 ± 3.96 anthracene 3 <1−15 2.25 ± 2.28 phenanthrene 3 4−136 25.79 ± 22.55 fluoranthene 4 <1−130 11.35 ± 17.72 pyrene 4 1−90 12.49 ± 14.08 benz[a]anthracene 4 <1−13 1.90 ± 1.99 chrysene 4 <1−9 2.14 ± 1.76 benzo[b]fluoranthene 5 <1−25 2.23 ± 3.86 benzo[k]fluoranthene 5 <1−25 1.95 ± 3.12 benzo[a]pyrene 5 <1−13 1.60 ± 1.90 dibenz[ah]anthracene 5 <1 <1 indeno[123-cd]pyrene 6 <1−4 1.06 ± 0.37 benzo[ghi]perylene 6 <1−5 1.13 ± 0.59 ∑pahs 25−357 98.27 ± 58.48 for instance, pah data were collected during the aquaterra danube survey (2004) undertaken in the environmental integrated project of the 6th eu framework programme. numerous samplings were carried out along the longitudinal stretch of the river danube from the upper section (germany) to the black sea and in major tributaries. the pah concentrations in water samples at different sampling stations along the whole danube showed wide variations. it has to be noted that concentrations of pahs in our study were lower than in the surface waters of the ráckevei-soroksári danube (the longest side-branch of the hungarian danube with its 57 km length is situated south from the capital city budapest). the ∑pahs concentrations were in the range of 129-1745 ng⋅l-1 with the mean value of 523 ± 348 ng⋅l-1 in the water samples of the ráckevei-soroksári danube (nagy, 2007). the higher levels of pahs can be explained by urban area of budapest. furthermore, comparison our results with other sections of the danube or even different rivers of the world (see table 2 and the data in the mentioned references above) reveals that the pah concentrations are relatively low at the hungarian upper section. fig. 3 shows the distributions of monthly average concentration, standard deviation and median of ∑pahs in surface water of the hungarian upper section of the danube. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:57 utc 32 szabó nagy, a. et al. the average monthly concentration of total pahs (average of the three sampling sites in the examined four years) was highest in april and lowest in july. the median values were nearly consistent in the range of 65-93 ng⋅l-1 except the value of july with 25 ng⋅l-1, respectively. table 2. comparative levels of total pahs in water samples collected from different locations. concentration (ng⋅l-1) water year of sampling n n range mean ± sd reference danube river, hungarian upper section 2007-2010 77 16 25-357 98.27 ± 58.48 this study ráckevei-soroksári danube, hungary 2002-2004 240 15 129-1745 523 ± 348 nagy, 2007 danube river, germany-black sea 2004 31 16 28-1163 432 ± 336 nagy, 2007∗ elbe river, hamburg, germany 1992-1993 20 22 60.9-381.6 145.5 götz et al., 1998 york river, us 1998-1999 23 20 2.09-123 13.1 ± 24.51 countway et al., 2003 mississipi river and gulf of mexico, us 1999 6 18 0.06-433.9 50.8 ± 164.6 mitra and bianchi, 2003 gao-ping river, taiwan 1999-2000 48 16 10-9400 430 doong and lin, 2004 tonghui river, beijing, china 2002 16 16 192.5-2651 762.3 ± 777.4 zhang et al., 2004 pearl river estuary, china 2002-2003 21 15 12.9-182.4 na luo et al., 2008 gomti river, india 2004-2006 48 16 60-84210 10330 ± 19940 malik et al., 2011 n: number of collected samples, n: number of compounds of ∑pahs, na: not applicable ∗ the data were based on the aquaterra danube survey (2004) programme 0 50 100 150 200 250 300 ja nu ar y f eb ru ar y m ar ch a pr il m ay ju ne ju ly a ug us t s ep te m be r o ct ob er n ov em be r d ec em be r ∑ p a h s ng ⋅l -1 average median fig. 3. temporal variation of monthly average concentration of total pahs in surface water of the danube river in the period of 2007-2010. significantly higher value of monthly concentrations of ∑pahs was also found in april at the ráckevei-soroksári danube branch (nagy, 2007). however, it cannot be bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:57 utc nova biotechnologica et chimica 11-1 (2012) 33 explained by the more intense atmospheric fallout whereas the heating season startup can increase the amount of pahs in autumn. probably, more fine-grain bound or colloidal pahs existed in dissolved phase in april. witt (1995) associated that the lowest concentrations of pahs also measured in summer is presumably due to the seasonal changes in degradation intensities. possible explanation could be the increased degree of photodegradation in july due to the high temperature and light intensity in summer (fasnacht and blough, 2002). according to the formation mechanisms, pahs can be classified as pyrogenic and petrogenic pahs. pyrogenic pahs are formed as a consequence of incomplete combustion whereas petrogenic pahs are mainly derived from crude oil and its refined products. several concentration ratios of pah compositions are used to infer the possible sources (yunker et al., 2002; doong and lin, 2004). in this study, the anthracene/(anthracene+phenanthrene) and fluoranthene/(fluoranthene+pyrene) ratios were calculated from the data of the water samples to try identifying the possible pah origins (see fig. 4). 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 ant/(ant+phe) f l t /( f l t +p y r ) fig. 4. cross plot for the concentration ratio of anthracene/(anthracene+phenanthrene) versus fluoranthene/(fluoranthene+pyrene). the ant/(ant+phe) ratio < 0.1 indicates origin of pahs from petrogenic sources, whereas the pyrogenic/combustion origin to ratio > 0.1. the concentration ratio of flt/(flt+pyr) < 0.4 suggests origin of pahs from petrogenic sources, whereas value of flt/(flt+pyr) ratio 0.4-0.5 indicates pahs combustion of liquid fuels and ratio > 0.5 infers pahs combustion of solid fuels. the fig. 4 illustrates that pyrogenic as well as petrogenic sources are responsible for the pahs inputs in the surface water of the danube. a previous study (janák, 2007) reported that major part of the oil pollutions was originated from watercrafts, especially attributed to bilge oil drained into the water by the foreign ships in the hungarian upper section. although concentration ratios of specific pah compounds are often used for characterizing possible pollution sources, their ratios are influenced by several processes such as photooxidation, chemical oxidation and biodegradation. in addition, seasonal changes can be found in degradation intensities as it is mentioned above. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:57 utc 34 szabó nagy, a. et al. 4. conclusions concentrations of polycyclic aromatic hydrocarbons in surface water of the hungarian upper section of the danube river were studied in the period of 2007-2010. the total concentrations of the 16 usepa pahs ranged from 25 to 357 ng⋅l-1 with the mean value of 98.27 ± 58.48 ng⋅l-1. the low and medium molecular weight pahs (2-3 and 4 ring) ranged from <1 to 136 ng⋅l-1 while high molecular weight pahs (5-6 ring) were present at much lower concentrations (<1-25 ng⋅l-1). of the total pahs, the 2-3-ring pahs contributed to about 64% while 4-6-ring pahs accounted for 36%. naphthalene and phenanthrene were found as the most dominant compounds with average distribution of 21 and 26% of the total pahs in the river water. evaluating the possible sources of pahs showed that the pahs were from both the pyrogenic and petrogenic origin in the danube. comparison of the ∑pahs concentrations determined in our study with other section of the danube and different rivers of the world revealed that the pah concentrations are relatively low in the hungarian upper section. references baker, j.e., eisenreich, s.j.e.: concentrations and fluxes of polycyclic aromatic hydrocarbons and polychlorinated biphenyls across the air-water interface of lake superior. environ. sci. technol., 24, 1990, 342-352. countway, r.e., dickhut, r.m., canuel, e.a.: polycyclic aromatic hydrocarbon (pah) distributions and associations with organic matter in surface waters of the york river, va estuary. organic geochem., 34, 2003, 209-224. doong, r., lin, y.: characterization and distribution of polycyclic aromatic hydrocarbon contaminations in surface sediment and water from gao-ping river, taiwan. water res., 38, 2004, 1733-1744. fasnacht, m.p., blough, n.v.: aqueous photodegradation of polycyclic aromatic hydrocarbons. environ. sci. technol., 36, 2002, 4364-4369. götz, r., bauer, o.h., frissel, p., rock, k.: organic trace compounds in water of the river elbe near hamburg. chemosphere, 36, 1998, 2103-2118. janák, e.: important water management issues. report, inspectorate for environment, nature and water of the north transdanubian region, győr, hungary, 2007 (in hungarian) ko, f.c., baker, j.e.: partitioning of hydrophobic organic contaminants to resuspended sediments and plankton in the mesohaline chesapeake bay. mar. chem., 49, 1995, 171-188. luo, x.j., mai, b.x., yang, q.s., chen, s.j., zeng, e.y.: distribution and partition of polycyclic aromatic hydrocarbon in surface water of the pearl river estuary, south china. environ. monit. assess., 145, 2008, 427-436. maldonado, c., bayona, j.m., bodineau, l.: sources, distribution and water column processes of aliphatic and polycyclic aromatic hydrocarbons in the northwestern black sea water. environ. sci. technol., 33, 1999, 2693-2702. malik, a., verma, p., singh, a.k., singh, k.p.: distribution of polycyclic aromatic hydrocarbons in water and bed sediments. environ. monit. assess., 172, 2011, 529-545. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:57 utc nova biotechnologica et chimica 11-1 (2012) 35 manoli, e., samara, c.: polycyclic aromatic hydrocarbons in natural waters: sources, occurrence and analysis. trends anal. chem., 18, 1999, 417-428. mitra, s., bianchi, t.s.: a preliminary assessment of polycyclic aromatic hydrocarbon distributions in the lower mississippi river and gulf of mexico. mar. chem., 82, 2003, 273-288. msz 1484-6:2003: testing of waters. determination of polycyclic aromatic hydrocarbons content by gas chromatographic-mass spectrometry (in hungarian) nagy, p.g.: determination of concentration and sources of polycyclic aromatic hydrocarbons and alkylphenols in danube water and sediment. ph.d. dissertation, university of technology and economics, budapest, hungary, 2007 (in hungarian) nagy, p., fekete; j., sharma, v.k.: polycyclic aromatic hydrocarbons (pahs) in surface waters of ráckevei-soroksári danube branch, hungary. j. environ. sci. heal a, 42, 2007, 231-240. srogi, k.: monitoring of environmental exposure to polycyclic aromatic hydrocarbons: a review. environ. chem. lett., 5, 2007, 169-195. witt g.: polycyclic aromatic hydrocarbons in water and sediment of the baltic sea. mar. pollut. bull., 31, 1995, 237-48. yunker, m.b., macdonald, r.w., vingarzan, r., mitchell, h.r., goyette, d., sylvestre, s.: pahs in the fraser river basin: a critical appraisal of pah ratios as indicators of pah source and composition. organic geochem., 33, 2002, 489-515. zhang, z., huang, j., yu, g., hong, h.: occurrence of pahs, pcbs and organochlorine pesticides in the tonghui river of beijing, china. environ. pollut., 130, 2004, 249-261. presented at the 3rd international scientific conference “applied natural sciences 2011”, october 5–7, 2011, častá papiernička, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:57 utc << /ascii85encodepages false 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can be opened with acrobat and adobe reader 5.0 and later.) >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [2834.650 2834.650] >> setpagedevice microsoft word styriakova nb.doc nova biotechnologica vii-i (2007) 11 factors affecting bioleaching in the processing of non-metallics iveta štyriaková department of biotechnology, institute of geotechnics of the slovak academy of sciences, watsonova 45, košice, sk-043 53, slovak republic (bacil@saske.sk) abstract: biotechnological treatment of non-metallics is based on bacterial leaching of raw material and dissolution of fe. bacterial iron dissolution ability is dependent on various physicochemical factors as temperature, acidity of solutions, redox potential, rapidity of water circulation and presence of organic sources. the fe content in the quartz sands and feldspar samples by the biological leaching decreased as much as 60% and by subsequent using of electromagnetic separation of feldspars, the decrease of fe content in 74% was achieved. however, the application of magnetic separation of quartz sands after bioleaching resulted in total iron removal of 93% and in such combined way prepared product contained 0.024 % of fe2o3. achieved results on iron removal point to the fact that combination of leaching and magnetic separation enables to obtain product usable in glass and ceramic industry. key words: bioleaching, non-metallics, iron, bacillus sp. 1. introduction non-metallic raw materials such as silicates, carbonates and oxides contain no energy source for the microorganisms to utilize. such materials may be leached by using heterotrophic bacteria and fungi, which require an organic carbon source as a source of energy and carbon for their growth. bioleaching of non-metallic minerals may be used for the recovery of metals from low-grade ores and minerals as well as for the beneficiation of mineral raw materials, recovery of metals from sediments, and heavy metal detoxification of soils and solid residues (jain, 2004). heterotrophic bacteria and fungi have the potential for producing acidic metabolites that are able to solubilize oxide, silicate, carbonate and hydroxide minerals by reduction, acidolysis and complexation mechanisms (jain, 2004). the use of fungi presents a problem, however. the material to be leached is often adsorbed to or enclosed by the fungal mycelium. this is especially undesirable in the benefication of coal, bauxite and other materials. in this case the process must either be performed in two separated steps: (1) metabolite production and (2) leaching by the metabolite (groudev and groudeva, 1986; vachon et al., 1994; brand et al., 1999) bioleaching processes using heterotrophic bacteria have received little attention although heterotrophs in the genera bacillus and pseudomonas have been found effective in the bioleaching of non-sulfidic minerals (karavaiko et al., 1980). naturally occurring silicates contain oxidic iron minerals as coatings on grains or impregnations in the matrix. the extent of iron removal from industrial silicate minerals depends on the mineralogy and distribution of iron in silicate rocks. for this reason, bioleaching studies with industrial minerals for their beneficiation have 12 štyriaková, i. examined the kinetics of iron dissolution from the siliceous matrix (veglio, 1997). in the present works, iron reduction was monitored as a measure of bacterial activity in the bioleaching of non-metallic raw materials. the aim of this was to remit on bacterial kind of bacillus spp. can be used for bioleaching and the effect of factors on the extent of fe removal from non-metallics. 2. materials and methods the iron – bearing minerals (goethite, mica, smectite) decrease the quality of nonmetallic material. bioleaching medium containing nah2po4 – 0.5g/l, mgso4 . 7h2o 0.5g/l, (nh4)2 so4 – 1.0g/l, nacl – 0.2g/l. however, medium was changed six times during bioleaching at 17 days intervals under aseptic conditions. the carbon sources were glucose, sucrose, galactose, technical-grade sucrose and molasses (all at 20 g/l). the samples were inoculated with a mixture of both bacillus cereus, bacillus mycoides and bacillus pumilus strains. the flasks were incubated statically for 3 or 4 months at 28oc. the abiotic controls were cultivated under the same conditions. after incubation, the culture solutions were separated from the biomass by means of membrane filtration. quantitative changes in the solid phase were measured with a model 30 varian atomic absorption spectrometer (varian, inc., melbourne, vic., australia). dissolved fe2+ and fe3+ were measured spectrophotometrically using the phenantroline method (herrera et al., 1989; stucky and anderson, 1981). headspace gas was analyzed for co2 concentration using a gas chromatograph chrom 5 (l.p. praha, prague, czech republic) equipped with a tc detector. bacterial oxidation of sucrose and glucose was analyzed as described by dubois et al. (1956). morphological and chemical changes in the surfaces of individual mineral grains were investigated by scanning electron microscopy (sem) and energy-dispersion microanalysis (eds), respectively. mineral samples were coated with carbon and examined under a tesla bs 340 (tesla elmi, brno, czech republic) scanning electron microscope. solid residues were analyzed by x-ray diffraction using a philips x'pert sw–binary diffractometer with cukα radiation (40 kv, 50 ma), equipped with an automatic divergence slit, sample spinner, and a graphite secondary monochromator. data were collected for 2-60 °2θ with a step width of 0.05° and a counting time of 30 sec per 0.05°. dry electromagnetic separation was carried out using a laboratory high gradient magnetic separator with the induction of magnetic field at 1.3 t. 3. results and discussion the iron-bearing minerals can be easily removed by magnetic separation, and ultra-fine iron particles are difficult to treat by conventional mineral processing methods, biochemical leaching appears to be the alternative for the effective removal of iron minerals. biotechnological treatment is based on bacterial leaching of raw nova biotechnologica vii-i (2007) 13 material and dissolution of fe. these processes are in last decades often observed in situ at weathering of silicate minerals in soil. in order to optimize a leaching process, it is necessary to understand the nature of the biotic reactions. factors that influence these reactions include the kind of microbial population as well as the mineralogical properties of the raw materials to be leached and also physicochemical parameters: 3.1 microbial population heterotrophic bacteria of bacillus strains are ubiquitous in non-metallic deposit (štyriaková and štyriak, 2002), they are non-pathogenic and are also facultative anaerobic bacteria enabling an easy manipulation during non-metallic treatment, increased rapidly in the numbers of cell during non-metallic treatment and targently produced organic acid in silicate structures. the leaching effect, observed by measurement of fe2+ and fe3+ concentration in solution, showed higher activities bacterial kind of bacillus spp. isolated from bajkal lake and also using of yeast saccharomyces sp. during bioleaching of quartz sands. however, every kinds of bacillus spp. isolated from slovak deposit and from bajkal lake were very active in iron reduction and dissolution during bioleaching of feldspar raw material. the results of experiments confirmed the ability of bacteria of bacillus genus to corrode silicate minerals in feldspar raw materials and quartz sands with subsequent extraction of intergranularly bound iron and smectite as unrequested admixture in nonmetallic raw materials (štyriaková et al., 2003b, štyriaková et al., 2007a). 3. 2 mineralogical properties the effect of biological leaching is dependent from mineralogical composition of certain raw material and the form of iron binding. bioleaching was effective for the removal of surface layers comprising fine iron minerals, fine-grained mica and iron smectite fraction from non-metallics. quartz sands containing mineral impurities in form siderite and mica were successfully treated with bioleaching and elutriation. in the process, poorly crystalline fe-oxides that sealed siderite nodules were released due to the bacterial action from intergranular space of quartz sands. these fe-oxides formed a finegrained fraction with fe-bearing minerals and fine mica fraction which were subsequently removed by elutriation process (štyriaková et al., 2003c). other quartz sands showed the presence of iron smectite coatings in quartz grains. the bioleaching treatment removed these surface coatings, presumably through the action of organic acid produced by the bacteria, and released fine clay particles from quartz grains. independent iron minerals were removed by magnetic separation (štyriaková et al., 2007a). magnetic separation removed independent particles of iron bearing minerals such as biotite and ilmenite. the extent of iron removal moved approximately 60 74% in combination of bioleaching and magnetic separation of feldspar treatments (štyriaková et al., 2006). the application of magnetic separation of quartz sands after bioleaching resulted in total iron removal of 93 % (štyriaková et al., 2007a). 14 štyriaková, i. 2.3 temperature at slovakia, there is often colder weather. that is why active bacteria with cold shock proteins in their genomes should be suitable for the use at leaching of nonmetallic raw materials under such climatic conditions. in view of the low temperature of water at the bottom of baikal lake, it can be assumed that cold–adapted microorganisms are found in the sediments. psychrophilic and psychrotrophic microorganisms are able to grow at temperatures around 0 °c. psychrophilic microorganisms have an optimum growth temperature ≤ 15 °c and do not grow above 20 °c; psychrotrophic (psychrotolerant) microorganisms have their optimum growth above 15 °c (margesin et al., 2003). mesophilic bacteria prefer temperatures between 20 and 40 °c, but some mesophiles can slowly grow at low temperatures. exposure of bacterial cells to a cold shock leads to the synthesis of cold-shock proteins (csp) (francis et al., 1997). psychrotrophic and mesophilic strains can be differentiated using pcr by the primers bcff2, bcapf1, and bcapr1 for the amplification of mesophilic/psychrotrophic cspf and psychrotrophic cspa genes (francis et al., 1998). in the case of mesophilic strains, one 284 bp pcr product was amplified, in the case of psychrotrophic strains, two 160 bp and 284 bp pcr products were obtained. thus, as a second step for the verification of the quality of dna isolated, the pcr identification of this cold-shock protein was chosen. from the results it follows that the dna from psychrotrophic microorganisms was amplified. as the psychrotrophic bacillus mycoides was isolated from baikal lake sediments, the dnas from psychrotrophic bacillus cereus ccm145 and mesophilic bacillus thuringiensis ccm19t were used as the control in pcr, too (španová, 2006). 2.4 eh and ph of solutions heterotrophic bacteria formed anaerobic conditions by microbial respiration after each replacement of medium after 1 day. the creation of eh gradually decreased from –110 mv in solution to –380 mv in sample bed (štyriaková et al., 2006). the bacteria of bacillus spp. were inoculated into media with sample at the beginning of each batch test, it was assumed that gases were produced and ph was lowered from 7 to 4 due to the fermentation of an organic source during 5 days. cumulative measurement of co2 was made only for the first four days of incubation in the glucose-sucrose medium. the results showed the formation of 27 mg co2 from 2 g sucrose and 4 mg co2 from 2 g glucose. these values are extremely low and may reflect a high production of organic acids (štyriaková et al., 2007b). 2.5 water circulation experimental studies of the effect of microorganisms on iron removal were performed in either closed (batch) or open (percolated columns) reaction vessels. the availability of carbon source and its concentration in the medium is found to be crucial for the growth of the heterotrophic organisms and metabolite production (castro et nova biotechnologica vii-i (2007) 15 al., 2000). our results from chemical analyses of leaches and solid phases showed that iron dissolution from non-metallic raw materials via aqueous – phase transport in the batch experiment was increased by the sufficient consumption of organic source, thereby allowing a higher increase in the extent of fe removal from natural sample materials. in contrast to the batch bioleaching, a lower fe dissolution was observed by biological leaching in percolator columns (štyriaková et al., 2006). 3.6 organic materials the effect of carbon source was tested in media inoculated with b. cereus by the addition of glucose, sucrose, galactose, technical-grade sucrose and molasses. the results showed that the extent of iron dissolution was higher in the presence technicalgrade sucrose and molasses after 6 days of bioleaching of non-metallics. molasses can be used as a relatively inexpensive organic carbon source for the heterotrophs, leading to the formation of metabolites, most notably organic acids acting as leaching agents (štyriaková et al., 2007b). 4. conclusion the results were presented in the many publications may serve as a starting point for development of flexible biotechnology for non-metallic's beneficiation, where either bacterial fe3+ dissolution and fe2+ reduction would be managed, depending on the many physicochemical factors. acknowledgements: this work was supported by the slovak science and technology assistance agency (contract apvt-51-006304) and slovak academy of science (vega 2/5033/5). references castro, i.m., fietto, j.l.r., vieira, r.x., tropia, m.j.m., campos, l.m.m., paniago, e.b., brandao, r.l.: bioleaching of zinc and nickel from silicate using aspergillus niger culture. hydrometallurgy, 57, 2000, 39-49. dubois, m., gilles, k.a., hamilton, j.k., rebers, p.a., smith, f.: colorimetric method for determination of sugars and related substances. anal. chem., 28, 1956, 350-356. francis, k. p., stewart, g. s. a. b.: detection and speciation of bacteria through pcr using universal major cold-shock protein primer oligomers. j. ind. microbiol. biotechnol., 19, 1997, 286-293. francis, k. p., mayer, r., von stetten, f., stewart, g. s. a. b., scherer, s.: discrimination of psychrotrophic and mesophilic strains of the bacillus cereus group by pcr targeting of major cold shock protein genes. appl. environ. microbiol., 64, 1998, 3525-3529. groudev,s.n., groudeva,v.i.: biological leaching of aluminium from clays. biotechnol. bioeng. symp., 16, 1986, 91 – 99. 16 štyriaková, i. herrera, l., ruiz, p., aguillon, j.c., fehrman, a.: a new spectrometric method for the determination of ferrous iron in the presence of ferric iron. j. chem. technol. biotechnol., 89, 1989, 171-181. margesin, r., labbé, d., schinner, f., greer, c. w., whyte, l. g.: characterization of hydrocarbon-degrading microbial populations in contaminated and pristine alpine soils. appl. environ. microbiol., 69, 2003, 3085-3092. stucky, j.w., anderson, w.l.: the quantitative assay of minerals for fe2+ and fe3+ using 1,10-phenantroline: i. sources of variability. soil sci. soc. am. j. 45, 1981, 633-637. španová, a., rittich, b., štyriak, i., štyriaková, i., horák, d.: isolation of polymerase chain reaction-ready bacterial dna from lake baikal sediments by caroxyl-functionalised magnetic polymer microspheres, j. chromatogr. a, 1130, 2006, 115 – 121. štyriaková, i., štyriak, i.: heterotrófne baktérie v aplikovanej mineralógii a v biotechnológiach spracovania nerudných surovín, mineralia slovaca, 34, 2002, 99-104. štyriaková, i., štyriak, i., kraus, i., hradil, d., grygar, t., bezdička, p.: biodestruction and deferritization of quartz sands by bacillus species. miner. eng., 16, 2003a, 709-713. štyriaková, i., štyriak, i., galko, i., hradil, d., bezdička, p.: the release of iron-bearing minerals and dissolution of feldspars by heterotrophic bacteria of bacillus species. ceramics-silikáty, 47, 2003b, 20-26. štyriaková, i., štyriak, i., nandakumar, m.p., mattiasson, b.: bacterial destructon of mica during bioleaching of kaolin and quartz sands by bacillus cereus. world j. microbiol. biotechnol., 19, 2003c, 583 – 590. štyriaková, i., bhatti, t.m., bigham, j.m., štyriak, i., vuorinen, a., tuovinen, o.h.: weathering of phlogopite by bacillus cereus and acidithiobacillus ferrooxidans. can. j. microbiol., 50, 2004, 213-219. štyriaková, i., štyriak, i., malachovský, p., lovás, m.: biological, chemical and electromagnetic treatment of three types of feldspar raw materials. miner. eng., 19, 2006, 348-354. štyriaková, i., štyriak, i., malachovský, p., večera, z., koloušek, d.: bacterial clay release and iron dissolution during the quality improvement of quartz sands. hydrometallurgy, 89, 2007a, 99-106. štyriaková, i., štyriak, i., malachovský, p.: nutrients enhancing the bacterial iron dissolution in the processing of feldspar raw materials. ceramicssilikáty, 2007b (in press). nova biotechnologica et chimica 15-1 (2016) 65 doi 10.1515/nbec-2016-0007 © university of ss. cyril and methodius in trnava improved multiplex polymerase chain reaction for rapid staphylococcus aureus detection in meat and milk matrices zuzana šramková1,2, barbora vidová1,2, andrej godány1,2 1department of biology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (zuzana.sramkova@ucm.sk) 2institute of molecular biology, slovak academy of sciences, dúbravská cesta 21, bratislava, sk-845 51, slovak republic abstract: staphylococcal food poisoning represents one of the most frequently occurring intoxications, caused by staphylococcal enterotoxins (se-s) and staphylococcal enterotoxin-like proteins (sel-s). therefore, there is a need for rapid, sensitive and specific detection method for this human pathogen and its toxin genes in food matrices. the present work is focused on staphylococcus aureus detection by a nonaplex polymerase chain reaction, which targets the 23s rrna gene for identification of s. aureus at the species level, genes for classical se-s (sea, sec, sed), new se-s (seh, sei), sel-s (sek, sel) and tsst-1 gene (toxic shock syndrome toxin). primers were properly designed to avoid undesirable interactions and to create a reliably identifiable profile of amplicons when visualized in agarose gel. according to obtained results, this approach is able to reach the detection sensitivity of 12 colony forming units from milk and meat matrices without prior culturing and dna extraction. key words: staphylococcus aureus, enterotoxin, multiplex pcr, rapid detection 1. introduction foodborne pathogens causing toxins-associated diseases are important health problems throughout the world in both developed and developing countries (gustafson et al., 2014). s. aureus is one of the major pathogens causing foodborne intoxications. staphylococcal food poisoning (sfp) is an intoxication that results from the consumption of foods containing sufficient amount of one or more types of enterotoxin. clinical characteristics of sfp comprise acute and rapid onset of symptoms (from 30 min to 8 hours after the consumption of contaminated food), with the most common nausea, vomiting, prostration, sometimes diarrhea and lowered blood pressure (le loir et al., 2003). to date, the known repertoire of s. aureus enterotoxins comprises of the classical enterotoxins (ses) sea – see and the new ses with demonstrated emetic activity in a primate model (seg sej, ser set) and staphylococcal enterotoxin-like (sels) proteins, whose emetic properties remain unconfirmed (selk-selq, selu-selx). tsst-1, the toxic shock-syndrome staphylococcal toxin, initially designated as sef, lacks emetic activity (argudín et al., 2010; grumann et al., 2014). all of these toxins possess superantigenic activity and are encoded by mobile genetic elements, including plasmids, prophages, pathogenicity islands, genomic islands, or by genes located next to the staphylococcal cassette chromosome implicated in methicillin resistance (argudín et al., 2010; grumann et al., 2014; otto, 2014). most bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:12 utc 66 šramková, z. et al. commonly, s. aureus strains carry not only one se/sel gene and according to the results of epidemiology studies, the frequency of se types and their combinations vary considerably (rosec and gigaud, 2002; zschöck et al., 2005; bania et al., 2006; hwang et al., 2007; chiang et al., 2008; rall et al., 2008; aydin et al., 2011; cremonesi et al., 2015; zeinhom et al., 2015; shin et al., 2016; rola et al., 2016). unless heat processes are applied, staphylococci are expected to exist in any and all foods that are handled directly by humans or are of animal origin, as animals and humans are considered to be the main reservoir of s. aureus (leroy et al., 2016; ortega morente et al., 2016). once produced, staphylococcal enterotoxins survive heat processing during food manufacture in biologically active form because of their heat stability. foods frequently implicated in staphylococcal food poisoning include meat and meat products; poultry and egg products; salads, such as egg, tuna, chicken, potato, and macaroni; bakery products, such as cream-filled pastries, cream pies, and chocolate éclairs; sandwich fillings; and milk and dairy products (reed, 1993). strict application of good hygienic practices and good manufacturing practices are the key factors, which can prevent products being contaminated by s. aureus and its toxins. according to regulations of european union (commision regulation (ec) no. 1441/2007), milk and diary products must be tested for enterotoxins if coagulasepositive staphylococci are detected at levels higher than 105 cfu/g (http://eurlex.europa.eu/eli/reg/2007/1441/oj). since the incidence of staphylococcal enterotoxicosis is associated with food safety and the conventional methods used to detect foodborne pathogen are time consuming and laborious, the effort of many research teams worldwide put their mind to development of methods that would allow food manufacturers quick, efficient and highly specific detection of pathogenic and potentially pathogenic microorganisms directly from samples either in the raw materials used in manufacturing or in final products. rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods (law et al., 2015). pcr (polymerase chain reaction) and in particular multiplex pcr (mpcr) were proven as one of the most suitable way of approaching the issue of bacteria detection (sun et al., 2011; brizzio et al., 2013; hummerjohann et al., 2014; cremonesi et al., 2015; zeinhom et al., 2015; shawish et al., 2016). the pcr assay can be made within hours with high sensitivity and method accuracy, allowing for the detection of very low amounts of microorganisms. the aim of this study was to develop a rapid and highly specific method allowing routine proof of s. aureus and its toxigenic strains in meat and milk based on improved mpcr protocol, without prior culturing and dna extraction. 2. materials and methods 2.1 bacterial strains and media staphylococcus aureus subsp. aureus ccm885, donated from el spol. s.r.o. company, slovakia, with all genes tested in this work was used for multiplex pcr of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:12 utc nova biotechnologica et chimica 15-1 (2016) 67 s. aureus toxin genes. the number of colony-forming units (cfu) needed for detection of pcr product was determined by cultivation. lysogeny broth (lb medium, 1 ml) was inoculated with a single colony and incubated overnight at 37°c, and serial 10–fold dilutions in lb were prepared, giving counts in the range of 10–1010 cfu/ml. viable counts were obtained by culturing each dilution (50 µl) on lb-agar plates with overnight incubation at 37°c. 2.2 primers all primers used in the study are shown in table 1. primers were designed using the primer 3 program for reliable use in nonaplex pcr, to yield a reliably identifiable profile of amplification products with defined molecular weight when visualized in agarose gel. table 1. oligonucleotide primers used in this study. product size (bp) primer sequence (5'-3') target gene genbank accession number 1300 sau1 (f) sau2 (r) ggacgacattagacgaatca cgggcacctattttctatct gene for 23s rrna cp009681.1 856 sea1(f) sea2 (r) agcgagaaaagcgaagaaat ccataggcaccacctcctta sea l22566.1 (borst and betley, 1993) 603 sec1 (f) sec2 (r) agaccctacgccagatgagt cctggtgcaggcatcata sec kf729631.1 (hunt et al., 2014) 530 sed1 (f) sed2 (r) tcgggaaaatcacccttaac gcagataaaaatccaataataggaga sed dq630751.1 (nema et al., 2007) 463 seh1 (f) seh2 (r) tcacatcatatgcgaaagca tcggacaatatttttctgatcttt seh aj937548.1 (jørgensen et al., 2005) 385 sei1 (f) sei2 (r) aaactggatatttttggcattg caggcagtccatctcctgta sei ab060537.1 (omoe et al., 2002) 271 sek1 (f) sek2 (r) ctaataatgccagcgctcaa gtagctgtgactccaccata sek gq358928.1 322 sel1 (f) sel2 (r) gcgatgtaggtccaggaaat actgtttgatgcttgccatt sel jn689383.1 196 sats1 (f)sats2 r) gcgacaatcgctacaggttt tgatgctgccatctgtgttt tsst-1 ef531614.1 (monecke et al., 2007) specificity of oligonucleotide sequences of primers for s. aureus was analysed by primer-blast program (http://www.ncbi.nlm.nih.gov/tools/primer-blast). all oligonucleotide primers were synthesized by microsynth ag (switzerland). primer mix containing all primers at equimolar concentrations was prepared for easy and reproducible handling of the numerous primers used in multiplex pcr. all primer stock solutions were normalized to a concentration of 100 µm using pcr water, thus the resulting 10× primer mix contained each primer at 2 µm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:12 utc 68 šramková, z. et al. 2.3 cell suspensions for pcr the tested s. aureus overnight culture prepared from single colony grown on lb medium was treated in two ways: by boiling and with naclo solution. in case of boiling preparation, 1 ml of culture was centrifuged and cell pellet was suspended in 500 µl of sterile distilled water in a tube. the tube was vortexed and incubated at 96°c for 10, 20, 30 and 60 min, respectively, then chilled on ice for 5 min, and centrifuged at 10,000 × g for 2 min to remove the cell debris. the supernatant (5 µl) was added directly to the pcr mixture. in the case of naclo samples, prepared according to our previous study (vidová et al., 2011), the 1 ml of the s. aureus overnight culture was treated with naclo solution in resulting concentration of 5 × 10−4 % for 10 min. in both cases, the viability of the cells was verified by counting the number of cfu on lb-agar plate after overnight incubation at 37°c. with the aim to prepare the sample for pcr assay with milk matrix, the naclo-treated cell pellet was first resuspended in 1 ml of sterile uht-treated cow’s milk, incubated at room temperature for 30 min, and then washed with 1 ml of water and phosphate buffered saline (pbs ph 8). simultaneously, with the aim to prepare the sample with meat matrix, the washed cell sample was first mixed and incubated at room temperature for 30 min with 1 g of pork meat sample, and then washed with water and pbs. cell suspension (5 µl) was used as a template in a final volume of 20 µl of pcr mixture containing: 1× optimized pcr buffer 10× (with 1.5mm mgcl2) (applichem), dntp mix (10 mm) (applichem), taq dna polymerase (dna-free, 5u/μl) (applichem) and primer mix. 2.4 pcr conditions pcr assay was carried out directly from s. aureus cells prepared according to procedure described previously by vidová et al. (2011). the amplification programme consisted of denaturation at 95°c for 5 min, 30 cycles of denaturation at 95°c for 60s, annealing at 55°c for 90s and extension at 72°c for 2 min, followed by a final extension at 72°c for 8 min. if monoplex pcr was used, the concentration of each primer in pcr reaction was 0.625 µm and quantity of taq dna polymerase was 0.2 u. the whole pcr reactions were analyzed by electrophoresis in 0.8% agarose gel stained with 0.5 µg/ml ethidium bromide, which was carried out in 1× bbe buffer (0.65 m boric acid, 0.029 m sodium tetraborate, 0.25 m edta, ph 7.8). uv transillumination of the bands on the agarose gel ought to show different sizes of nine desired amplification products. dna molecular size marker was included in each gel (100 bp dna ladder; invitrogentm). all obtained pcr products were sequenced by microsynth ag (switzerland) to verify their identity. 3. results 3.1 exclusion of dna extraction by direct use of s. aureus cell suspensions as template for pcr extraction of chromosomal dna suitable for pcr is often the most limiting step, in particular of time consuming, dna quality, purity and integrity, which lead to bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:12 utc nova biotechnologica et chimica 15-1 (2016) 69 nonspecific results and reduction of the sensitivity of the method. therefore, in this work was dna extraction step excluded and replaced by direct use of washed bacterial cells into polymerase chain reactions. according to our previous experience (chotár et al., 2006; vidová et al., 2011), bacterial cells prepared by washing method with naclo pretreatment were used. briefly, two ways to prepare cell suspensions from overnight culture before washing steps were used; and samples (5 µl) after washing with sterile water were used directly to pcr. the rest of cell preparations were subsequently stored at 4◦c for a week. both preparations, freshly prepared and stored ones, were tested for viability. in the case of boiling preparation, the 10 min of incubation at 96◦c was enough to achieve pcr products, but only with fresh preparation. there were no pcr products observed after one week of storage, although boiling treatment was enough to kill all viable cells in sample as well. the naclo treated samples exhibited satisfactory results with both freshly prepared and stored ones, with the same results observed after more than three months of storage. therefore, the naclo samples of overnight culture were used in further experiments. the additional advantage of this treatment is that there was no need to prepare other overnight cultures, which reduce the handling with infective living cells. therefore, laboratory testing of such bacterial pathogen samples is more safety, and further testing proceeded with one standardized batch of cell suspension. in addition, naclo samples of characterized bacterial cell suspensions are able to be used as positive controls due to the possibility of their long term storage without affecting pcr sensitivity. bypassing of dna extraction still remains beneficial in such cases as well, resulting in time and labor-savings. 3.2 monoplex pcrs all primer pairs designed for identification of s. aureus toxin genes generated single pcr products of expected sizes when washed cell suspension of this pathogen was directly used in pcr reactions (fig. 1). no pcr products were generated with these primers by using negative control strain. amplification of 1300 bp pcr product was observed when species-specific primer pair (sau1 and sau2; chotár et al., 2006) was used. detection limits, i.e. number of cells needed to obtain a visible pcr product, for every single primer pair were determined by cultivation. the amount of 4 cells was enough for positive amplification of appropriate pcr products with primer pairs sea1/sea2, sec1/sec2 and sed1/sed2. two cells were enough for the amplification of pcr products with primer pairs seh1/seh2 and sei1/sei2, and even one single cell with primer pairs sek1/sek2, sel1/sel2 and sats1/sats2. the observation of detection limit for species-specific sau primer pair was the same as stated previously by chotár et al. (2006). 3.3 multiplex pcrs optimization of multiplex pcr conditions revealed crucial steps that should not be neglected. the first one is the length of initial denaturation step, which is required to be at least 5 minutes. this step is important due to presence of whole bacterial cells in reactions and allowed adequate destruction of cells, dna release and denaturation of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:12 utc 70 šramková, z. et al. contaminating cell contents that could have an inhibitory effect on taq-polymerase during pcr. the second important step is using the equal concentrations (0.2 µm) of all primers prepared in one mix. and finally, keeping the annealing time for 90 seconds showed to be essential as well. testing of multiplex pcr in this work comprised three experimental models depending on the type of matrix, in which the presence of s. aureus cells was tested. the prepared cells suspensions were then directly used in the multiplex pcr. in the first model overnight culture was treated for 10 min with naclo solution (resulting concentration of 5 × 10 −4 %), washed with water and pbs, and then directly used as template in pcr. in the second model the overnight culture was treated with naclo mixed with a sample of uht-treated cow’s milk, followed by washing with water and pbs, and then directly used as template in pcr. finally, in the third model the naclo-treated overnight culture was mixed with a sample of meat, followed by washing with water and pbs, and then directly used as template in pcr. because the sensitivity of multiplex pcr is linked with visibility of all amplicons, the results of testing suitability of cell suspension prepared only from the overnight culture for multiplex pcr were comparable with that of monoplex pcr. the visibility of the upper pcr product for 23s rrna gene was detectable for at least 8 cells in the multiplex pcr. the presence of milk background and meat matrix decreased the sensitivity by 0.5-times (to 12 cells). fig. 1. amplicons of targeted genes visualized in agarose gel (monoplex and multiplex pcrs). a: sec gene (603 bp), b: sek gene (271 bp), c: sei gene (385 bp), d: tsst-1gene (196 bp), e: sel gene (322 bp), f: seh gene (463 bp), g: nonaplex pcr with 23s rrna gene (1500 bp) h: sea gene (856 bp), i: sed gene (530 bp); mwm: standard of molecular weight (100 bp dna ladder). 4. discussion in our work, we initially designed primer pairs for 23s rrna gene and for se/sel genes sea, sec, sed, seh, sei, sek, sel and tsst-1. monoplex pcrs for individual genes bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:12 utc nova biotechnologica et chimica 15-1 (2016) 71 targeted were standardized to determine the optimum amplification conditions on collection strain of s. aureus. after successful optimization of pcr conditions, amplified products were sequenced and the obtained data confirmed identity with expected sequences (according to the genbank). we used intact cells of s. aureus as a template for pcr. in our laboratory, this advanced procedure without prior dna extraction from bacterial cells proved to be more sensitive in comparison to protocol with extracted dna. we were able to reach the detection sensitivity of 12 cfu in milk (ml-1) and meat (g-1). nagaraj et al. (2014) reported much lower detection sensitivity of 106 cfu/ml or cfu/g in food when using extracted dna as a template for mpcr assay. dna extraction is the commonly used step in pcr protocols published in articles dealing with similar issue (aydin et al., 2011; quigley et al., 2011; ali et al., 2014; lu et al., 2015). there are some studies about optimization of dna extraction as an essential step for successful pcr (ramesh et al., 2002; cremonesi et al., 2006; binet et al., 2014). however, we found out that this step can be completely dropped, thus shortening the time and reducing the costs for analysis. tilsala-timisjarvi and alatossava (2004) used a membranebased method for dna preparation from diary samples, avoiding the contact with volatile solvents or other toxic reagents used during conventional dna isolation but this approach was i) more expensive, ii) required more time than procedure described in our work and iii) the sensitivity was comparable with conventional method. subsequently, the conditions for multiplex pcr were optimized. primer pairs generated a set of amplicons with molecular sizes expected from the gene sequences (fig. 1): 1300 bp (sequence coding 23s rrna), 856 bp (sea), 603 bp (sec), 530 bp (sed), 463 bp (seh), 385 bp (sei), 271 bp (sek), 322 bp (sel), 196 bp (tsst-1). these products were reliably separated in 0.8 % agarose gel. the resulting profile represents a s. aureus reference dna marker. we chose this set of primers for multiplex pcr because it covers i) the most frequently occurring classical enterotoxins (a, c, d), ii) some new types of se (h, i), iii) se-like superantigens (k, l) and iv) toxic shock syndrome toxin 1 (tsst-1). the superantigenic toxin tsst-1 is found to be associated with enterotoxins (johler et al., 2013). there are reports regarding the production of tsst-1 from s. aureus strains isolated from food handlers (sospedra et al., 2012). analysis of 147 isolates of s. aureus from patients associated with staphylococcal food poisoning outbreaks in taiwan confirmed 91.8 % of strains to be positive for at least one se or tsst-1 (chiang et al., 2008). tsst-1 was found also in raw bulk tank milk samples from switzerland (scherrer et al., 2004). distribution of classical and new se and sags is variable and depends on country of origin and sample used for isolation of s. aureus (becker et al., 2004; omoe et al., 2005; morandi et al., 2007; trnčíková et al., 2010). classical enterotoxins are known as common cause of the food poisoning outbreaks. type a is the mostly occurring one in various food samples (chen et al., 2004; hwang et al., 2007; rall et al., 2008; medveďová et al., 2014; rall et al., 2014), while type e is not frequently detected. however, it was identified and quantified in the cheese responsible for food poisoning, which was the first evidence of food poisoning in france caused by this enterotoxin (ostyn et al., 2010). as there are still many cases with unknown cause, the newly described ses and sel proteins are also of importance bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:12 utc 72 šramková, z. et al. because they can be the reason explaining some of these enterotoxicosis with unknown causative agent. 5. conclusions highly specific methodology utilizing pcr and multiplex pcr for routine detection of isolates of s. aureus in dairy and meat products has been developed. method described in this study enables to identify the presence of coagulase-positive s. aureus and selected staphylococcal toxins using specifically designed primers directly in one sample at a time without prior culturing and dna extraction, avoiding the contamination of people and the environment. this methodology gives a clear overview of the presence of pathogenic s. aureus and various toxic genes in food matrices. it is potentially applicable in the agricultural and food industry in the control of animal health and food quality, which enables fast 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encoding for enterotoxins. j. food nutr. res., 49, 2010, 215-220. vidová, b., tóthová, e., blahut, l., horváthová, v., godány, a.: multiplex pcr for detection of escherichia coli o157:h7 in foods. biologia, 66, 2011, 401–405. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:12 utc 76 šramková, z. et al. zeinhom, m.m., abdel-latef, g.k., jordan, k.: the use of multiplex pcr to determine the prevalence of enterotoxigenic staphylococcus aureus isolated from raw milk, feta cheese, and hand swabs. j. food sci., 80, 2015, m2932-6. zschöck, m., kloppert, b., wolter, w., hamann, h.p., lämmler, ch.: pattern of enterotoxin genes seg, seh, sei and sej positive staphylococcus aureus isolated from bovine mastitis. vet. microbiol., 108, 2005, 243-249. received 18 may 2016 accepted 7 july 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:12 utc nova biotechnologica et chimica 11-2 (2012) 117 doi 10.2478/v10296-012-0013-0 © university of ss. cyril and methodius in trnava study of ni and cd bioleaching from spent ni-cd batteries oksana velgosová, jana kaduková, renáta marcinčáková technical university in košice, faculty of metallurgy, department of material science, letná 9, košice 04200, slovak republic (oksana.velgosova@tuke.sk) abstract: in the present work the ni and cd extraction from the electrode material of spent ni-cd batteries by bioleaching using the bacteria acidithiobacillus ferrooxidans was examined. the possibility of the use of the bacteria for both the ni and cd recovery was analyzed. the anode of ni-cd batteries contains ni0, ni(oh)2, cd0 and cd(oh)2. the cathode is covered by nickel hydroxides and nickel oxy-hydroxides. the ph values and content of ni and cd in the solution were monitored throughout the experiment (the initial ph was 1.5, the experiment took 28 days). the bioleaching efficiency of ni and cd from anode powder reached 5.5% and 98%, respectively. during the cathode powder bioleaching ni and cd efficiency reached 45% and 100%, respectively. throughout the bioleaching process mainly the dissolution of hydroxides occurred meanwhile ni0 leaching was not observed. the aas analysis was used to analyze the amount of ni and cd in the solutions. the amount of ni and cd present in the solid samples before and after bioleaching was examined using x-ray analysis. keywords: ni-cd batteries, bioleaching, acidithiobacillus ferrooxidans, heavy metals 1. introduction since ni-cd batteries are still being used in various areas and the amount of spent ni-cd batteries increases, it is necessary to find eco-friendly way how to recycle and treat them and subsequently to recover valuable metals. more than 55 % of the battery weight forms the material of electrodes. nickel, cadmium and their hydroxides are main compounds of electrode material, which make ni-cd spent batteries an attractive material for heavy metals obtaining (nogueira and margarido, 2007). pyrometallurgical and hydrometallurgical processes or combination both of them are traditional techniques for the spent ni-cd batteries treatment (bartolozzi et al. 1995; cox and fray, 1999; espinosa et al. 2004; freitas and rosalem, 2005). hydrometallurgical processes are more acceptable than pyrometallurgical ones due to the elimination of toxic air emissions and a high purity of the final metal product. the hydrometallurgical processes of ni-cd batteries treatment deal with ni and cd solubilisation in the different solutions and under the different conditions. hydrometallurgical processes offer many possibilities of changing the process conditions so that the required results may be achieved. different leaching solutions, ph, temperature, treatment of solid stage (granularity, morphology, chemical treatment) or using bacteria are several variables that can influence the efficiency and duration of hydrometallurgical processes. nowadays, biohydrometallurgy comes to the forefront as an alternative method of metal obtaining. the bioleaching processes, in comparison to commonly used bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:56 utc 118 velgosová, o. et al. processes, are less expensive and therefore they can be used as an option for the treatment of ni-cd batteries. the acidophilic bacteria are the most widely used microorganisms. the bacteria acidithiobacillus ferrooxidans gain energy from the oxidation of ferrous ions or elemental sulphur to meet their growth need and consequently produce sulfuric acid and ferric ions that play important role as leaching agents in the bioleaching process of sulfide ores. many authors apply bioleaching mechanism of sulfide ores that have been described well for bioleaching of non-sulfidic material (e.g. spent secondary batteries) (chan et al. 2003; chen and lin, 2004; eccles, 1999; meruane et al. 2003; rudnik and nikiel, 2007; zhao et al. 2008; zhu et al. 2003), however, we should take into account the fact that they contain no sulfides but in the case of the spent batteries especially elemental metals, metal oxides and metal hydroxides are present therefore the bioleaching mechanisms might not be necessary the same. the bioleaching processes depend on many factors; therefore the explanation of their influences under certain conditions is necessary for the understanding of the bioleaching process. by the studying of the particular leaching factors we tried to shed light on the mechanisms involved and responsible for the waste bioleaching processes. the purpose of this work was to study the bioleaching process of electrode powder from spent ni-cd batteries using acidithiobacillus ferrooxidans, to analyze the possibility of using bacteria for ni and cd recovery, and to analyze the efficiency of ni and cd bioleaching processes. 2. materials and methods in this research spent rechargeable ni-cd batteries were used as the experimental material. the batteries were weighted and manually cut up into different portions. the electrode material forms about 55 % of the battery weight. the active electrode material, cathode and anode powders were physically removed from the metal grid, ground and sieved to obtain a mesh size of less than 40 μm. all remaining components of the battery were metal parts and separator with plastic parts. for the experiments cathode and anode electrode materials were used separately. for the bioleaching the adapted bacteria acidithiobacillus ferrooxidans for the electrode powder material were used. the pure culture of acidithiobacillus ferrooxidans was obtained from the institute of geotechnics of slovak academy of science, košice. the microorganisms were cultured in erlenmeyer flasks containing 200 ml of 9 k medium at the initial ph of 1.5 in the incubator at 30°c. then 2 g of cathode or anode powder was added to these cultured bacteria. after 3 weeks, 5 ml of adapted bacteria were poured into the erlenmeyer flask containing 295 ml of basic 9k medium and were cultivated for another 4 days. after that 3 g of electrode powder was added. the experiments were carried out in the incubator at 30°c, the solid / liquid (w/v) ratio was 1/100 in all experiments. the samples were withdrawn at these days: 1, 3, 7, 10, 13, 21, and 28. all experiments were conducted in triplicates. the leachate (5 ml) was periodically taken and filtrated. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:56 utc nova biotechnologica et chimica 11-2 (2012) 119 atomic absorption spectrometry (aas) (spectraa 20 plus varian) was used to determine the ni and cd concentrations in the solutions. the ph of each experiment was measured throughout the experimental period by the ph meter gryf 208 l using a combine electrode. x-ray analysis was used to analyze the input of the solid samples and the solid residues. 3. results and discussion the chemical composition of the electrode powder obtained from the ni-cd spent batteries was determined by x-ray analyses. the anode consists of cadmium and cadmium hydroxide in combination with a small amount of nickel and nickel hydroxide (fig. 1). the cathode is covered by nickel hydroxide and nickel oxyhydroxide, (fig. 2). the small amount of cobalt was present in both samples. it is apparent that the anode contains a high amount of cd and a small amount of ni. this fact was also confirmed by the aas analysis. the results of aas analysis of cathode and anode input samples showed that ni content in the battery was 47.1 % and 22.2 % for cathode and anode, respectively. cd content was 6.3 % and 37.5 % for cathode and anode, respectively in the electrode powder bioleaching process the solution ph did not change significantly (fig. 3). the ph increased from the value of 1.5 to 2.8 up to 21th day of the experiment, after that the ph value remained constant. the ph increase was probably caused by the neutralization when, at least part, cadmium and nickel hydroxides dissolved. fig. 3. dependence of ph on leaching time. fig. 1. x-ray of the initial anode material of spent ni-cd battery. fig. 2. x-ray of the initial cathode material of spent ni-cd battery. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:56 utc 120 velgosová, o. et al. ni and cd bioleaching efficiency of the anode powder was 5% and 98%, respectively (fig. 4) and the bioleaching efficiency of ni and cd from the cathode powder reached 45% and 100% respectively (fig. 5). the results exhibited evident difference in cd and ni dissolution behavior during the bioleaching process. these findings can be explained by the fact that cadmium as a less noble element is oxidized easier than metallic nickel and cd hydroxides are dissolved faster than hydroxides of nickel, too. the solubility of metal ni is significantly affected by its resistance to corrosion, from thermodynamic point of view the dissolution of ni is possible at ph bellow 6.3. many authors studied the passivation of nickel in different acidic solutions. it was reported that the passive film formed on nickel surface in low concentrations of sulfuric acid is formed by niooh or nio and ni2o3. on the other hand, βniso4.6h2o was suggested as the passive compound formed in very high concentrations of sulfuric acid (abdallah et al. 2003; ohtsuka et al. 1979; kovtun et al. 2005). but ni solubilization can be reached by modification of leaching conditions e.g. by increasing of the leaching potential or temperature (nogueira et al. 2004; pietrelli et al. 2005; goyal et al. 2003). during the bioleaching process the microbial oxidation of ferrous to ferric ions occurs as follows: ( ) ohsofesohofeso ferrooxa 2342 .. 4222 1 42 +⎯⎯⎯ →⎯++ (1) the consumption of protons also occurs and this is together with the dissolution of ni and cd hydroxides probably responsible for the increase of the leaching solution ph value up to 2.8 (fig. 3). but despite the total dissolution of hydroxides just a very small increase of the solution ph was observed. it might be due to jarosite formation. this process is expressed as follows: ( ) ( )3242342 2366 ohfesohohsofe ++→+ −+ (2) the dissolution of ferric sulphate and its subsequent hydrolysis causes the release of protons to the solution. it is supposed that ferric hydroxides and parts of the fig. 4. the efficiency of ni and cd bioleaching from anode powder. fig. 5. the efficiency of ni and cd bioleaching from cathode powder. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:56 utc nova biotechnologica et chimica 11-2 (2012) 121 sulphate ions formed during this reaction (2) participate in the jarosite formation. potassium, which is a part of the jarosite molecule, is getting to the process from an electrolyte. this process can be described as follows: ( ) ( ) +−+−+ +→++++ hohsokfeohsofeohk 62432 2 4 3 235 (3) consequently, during this reaction equilibrium shifts to the right, and the release of protons results in the decrease of ph values. this process helps to maintain the low leaching solution ph values which lead to the further dissolution of hydroxides up to their total elimination. x-ray analysis confirmed jarosite formation and the removal of hydroxides from the solution (fig.6 and fig.7). the x-ray analysis of solid residuum obtained after the anode powder bioleaching showed the negligible amount of cd hydroxides, metallic ni and jarosite. after the cathode powder bioleaching only metallic ni remained. the large majority of jarosite was present in the solid residuum in the both samples. one of the factors that can influence the process rate inhibition and causes lower efficiency of ni leaching is the presence of cadmium, which is to a certain extent toxic to bacteria. however acidithiobacillus ferrooxidans have good resistibility to toxic elements their high concentration can kill the bacteria. according to cabrera et al. 2005, it is possible to establish the order of metal toxicity to acidithiobacillus ferrooxidans bacteria: cr(iii)>cu(ii)>cd(ii)>ni(ii)>zn(ii). for nickel, the tolerance up to 40 g ni/l was found. 4. conclusions series of experiments were carried out to contribute to understanding of the mechanisms of non-sulfidic material bioleaching processes. the results of the bioleaching experiments can be summarized as follows: 1. under the studied conditions, all ni hydroxides are leached to the solution but the leaching of metallic ni is negligible. 2. the better leachability was observed in the case of cd bioleaching. the efficiency of cd bioleaching from anode and cathode powders reached almost 100%. 3. the jarosite formation was confirmed by x-ray analysis. fig. 6. x-ray of bioleached anode material. fig. 7. x-ray of bioleached cathode material. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:56 utc 122 velgosová, o. et al. it would be of value to carry out these bioleaching experiments under other conditions to extract ni, for example, to change the chemical composition of the bioleaching media since not only the presence of iron ions but also its amount in the media can significantly influence the bioleaching process rate, to use mixed acidophilic bacteria, two-step leaching system or to use continuous bioleaching and bioleaching with retention time in bioreactors. acknowledgement: the work was fully supported by a grant from the slovak national grant agency under the vega project 1/0235/12. references abdallah, m., el-etre, a.y.: corrosion inhibition of nickel in sulfuric acid using tween surfactants, portug. electrochimica acta, 21, 2003, 315-326. bartolozzi, m., braccini, g., bonvini, s., marconi, p.f.: hydrometallurgical recovery process for nickel–cadmium spent batteries. j. power sources, 55, 1995, 247–250. cabrera, g., gómez, j.m., cantero, d.: influence of heavy metals on growth and ferrous sulphate oxidation by acidithiobacillus ferrooxidans in pure and mixed cultures, process biochem., 40, 2005, 2683-2687. chan, l.c., gu, x.y., wong, j.w.c.: comparison of bioleaching of heavy metals from sewage sludge using ironand sulfur-oxidizing bacteria. adv. in environ. res. 7, 2003, 603–607. chen, s.y., lin, j-g..: bioleaching of heavy metals from livestock sludge by indigenous sulfur-oxidizing bacteria: effects of sludge solids concentration. chemosphere, 54, 2004, 283–289. cox, a., fray, d.j.: recycling of cadmium from domestic sealed nicd battery waste by use of chlorination. trans. instn. min. metall. – c: mineral process. extr. metall. 108, 1999, 153–158. eccles, h.: treatment of metal-contaminated wastes: why select a biological process? trends in biotechnology, 17, 1999, 462-465. espinosa, d.c.r., bernandes, a.m., tenorio, j.a.s.: an overview on the current processes for the recycling of batteries. j. power sources, 135, 2004, 311319. freitas, m.b.j.g., rosalém, s.f.: electrochemical recovery of cadmium from spent ni–cd batteries. j. power sources, 139, 2005, 366–370. goyal, n., jain, s.c., banerjee, u.c.: comparative studies on the microbial adsorption of heavy metals. adv. environ. res., 7, 2003, 311-319. kovtun, v.n., bolotin, a.v.: dynamic behavior of ni-h2so4 system at high anodic potentials and different electrolysis conditions, russ. j. electrochem., 41, 2005, 111-115. meruane, g., vargas, t.: bacterial oxidation of ferrous iron by acidithiobacillus ferrooxidans in the ph range 2.5-7.0. hydrometallurgy, 71, 2003, 149-158. nogueira, c.a., margarido, f.: leaching behaviour of electrode materials of spent nickel-cadmium batteries in sulphuric acid media. hydrometallurgy, 72, 2004, 111-118. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:56 utc nova biotechnologica et chimica 11-2 (2012) 123 nogueira, c.a., margarido, f.: chemical and physical characterization of electrode materials of spent sealed ni–cd batteries. waste manage., 27, 2007, 1570–1579. ohtsuka, t., heusler, k.e.: potential-dependent properties of the passivating film on nickel in sulphuric acid solution from multiple-angle-of-incidence reflectivity measurements, j. electroanal. chem., 102, 1979, 175-187. pietrelli, l., bellomo, b., fontana, d., montereali, m.: characterization and leaching of nicd and nimh spent batteries for the recovery of metals, waste managem., 25, 2005, 221-226 rudnik, e., nikiel, m.: hydrometallurgical recovery of cadmium and nickel from spent ni–cd batteries. hydrometallurgy, 89, 2007. 61–71. zhao, l., yang, d., zhu, n-w.: bioleaching of spent ni–cd batteries by continuous flow system: effect of hydraulic retention time and process load. j. hazard. mater., 160, 2008, 648–654. zhu, n., zhang, l., li, c., cai, c.: recycling of spent nickel–cadmium batteries based on bioleaching process. waste manage., 23, 2003, 703–708. presented at the 2nd international conference “biotechnology and metals 2011”, september 22–23, 2011, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:56 utc microsoft word mrazikova final.doc nova biotechnologica et chimica 14-1 (2015) 45 doi 10.1515/nbec-2015-0013 © university of ss. cyril and methodius in trnava influence of used bacterial culture on zinc and aluminium bioleaching from printed circuit boards anna mrazikova1, renata marcincakova1, jana kadukova1, oksana velgosova1, magdalena balintova2 1technical university in košice, faculty of metallurgy, letná 9, 042 00 košice, slovak republic 2technical university in košice, faculty of civil engineering, institute of environmental engineering, vysokoskolska 4, 042 00 kosice, slovak republic abstract: bioleaching processes were used to solubilize metals (cu, ni, zn and al) from printed circuit boards (pcbs). in this study, a pcbs-adapted pure culture of acidithiobacillus ferrooxidans, pure culture of acidithiobacillus thiooxidans and pcbs-adapted mixed culture of a. ferrooxidans and a. thiooxidans were used for recovery of the metals. the study showed that the mixed bacterial culture has the greatest potential to dissolve metals. the maximum metal bioleaching efficiencies were found to be 100, 92, 89 and 20% of cu, ni, zn and al, respectively. the mixed culture revealed higher bacterial stability. the main factor responsible for high metal recovery was the ability of the mixed culture to maintain the low ph during the whole process. the pure culture of a. thiooxidans had no significant effect on metal bioleaching from pcbs. keywords: bioleaching, electronic waste, zinc, aluminium, acidithiobacillus ferrooxidans, acidithiobacillus thiooxidans 1. introduction waste from printed circuit boards (pcbs) belongs to the typical waste of electric and electronic equipments and their recycling has attracted a great attention not only from the perspective of waste treatment but also of valuable metal recovery. to date, pcbs waste recycling was still limited due to heterogeneity and complexity in their components. the content of most components present in pcbs is: major metals 28 % 30 %, plastics 19 %, bromine 4 %, glass and ceramics 49 %. the content of metals can be expressed as follows: copper: 10-20 %, lead: 1-5 %, nickel: 1-3 % and precious metals like silver, platinum and gold are also present in the electronic scrap to a total of 0.3-0.4 % (ilyas et al., 2013; liang et al., 2013). the quantity of metals turns the electronic scrap into an interesting raw material according to the economic point of view. if pcbs are not recycled or treated appropriately and consequently they are made to be dispersed to environment, the heavy metals such as copper, lead and nickel and halogenated burn – resisting materials being a part of them can lead to serious environment problems (wang et al., 2009). the use of microorganisms for metal recovery from waste electronic equipment could be an economical alternative to such processes as pyrometallurgy, hydrometallurgy or mechanical processes, since using biological techniques for bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:58 utc 46 mrazikova, a. et al. recycling pcbs, the recovery efficiency could be increased whereas thermal or physico-chemical methods alone are less successful as shown in copper and gold mining where low-grade ores are biologically treated to obtain metal values (wang et al., 2009; xiang et al., 2010). acidithiobacillus ferrooxidans is a major participant in consortia of microorganisms used for industrial recovery of copper (bioleaching or biomining) (valdés et al., 2008; bálintová and luptáková, 2012). initially, an acid environment was established by acidithiobacillus thiooxidans through producing h2so4 to dissolve pcbs fine powder (eq. 1). then, a. ferrooxidans oxidized fe2+ into fe3+ (eq. 2) which can oxidize metals into corresponding ionic forms (eq. 3). afterwards, fe2+ will be oxidized into fe3+ to begin the next cycle reaction. a. ferrooxidans and a. thiooxidans can grow and utilize ferrous iron or elemental sulphur and thus produce ferric ions or sulphuric acid that are important as leaching agents of metals (zhao et al., 2008; lee and pandey, 2011; liang et al., 2010; liang, et al., 2013). s0+ 1,5o2 + h2o → so4 2 + 2h+ (1) 2fe2+ + 0,5o2 + 2h + →2fe3+ + h2o (2) m (zn, al) + fe2(so4)3 → mso4 +2feso4 (3) high amounts of different metals present in pcbs have a toxic effect on bacteria; however, as it was reported by brandl et al. (2001) the bacterial strains of a. ferrooxidans and a. thiooxidans can to a certain extent tolerate high metal concentrations. it is known, that metals bioleaching from pcbs closely relate to bacterial growth. the aim of this study was to evaluate the influence of the pure cultures of the acidophilic bacteria and their mixture on copper, nickel, zinc and aluminium recovery from pcbs. additionally, the contribution of particular bacterial strains to bioleaching mechanisms was examined. 2. materials and methods the isolates used in the experiments were recovered from the acid mine drainage water in smolník, slovakia. the pure culture of a. ferrooxidans and a. thiooxidans and their mixture, as well, were cultured and acclimated in presence of pcbs for two weeks and consequently used as the bioleaching bacteria for zinc and aluminium recovery from pcbs waste. the pure culture of a. ferrooxidans and a. thiooxidans and mixed culture of a. ferrooxidans and a. thiooxidans are designed as main influential factors in examination of percentage of zinc and aluminium solubilisation from the pcbs. the chemical composition of nutrient media needed for bacterial growths, which were also used as the bioleaching media, are shown in table 1. the ph values of the bioleaching media were adjusted to 1.5 with 10 m h2so4. the unwashed scraps of actual pcbs, which were used in our experiments, were crushed to particle size of 1 1 000 mm. analysis of the electronic scrap before bioleaching revealed the presence bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:58 utc nova biotechnologica et chimica 14-1 (2015) 47 of zn (1.2 %) and al (1.7 %). metal ion concentration in the leach liquor was measured by an atomic absorption spectrophotometer (perkin elmer 3100). table 1. chemical composition of nutrient and bioleaching media, as well (g.l-1). composition waksman and joffe medium (a. thiooxidans) 9k medium (a. ferrooxidans) mixed medium (a. ferrooxidans and a. thiooxidans) kcl 0.1 0.1 (nh4)2so4 0.2 3.0 2.0 k2hpo4 3.0 0.5 0.25 mgso4.7h2o 0.5 0.5 0.25 ca(no3)2.4h2o 0.014 cacl2.6h2o 0.25 feso4.7h2o trace amount 44.2 44.2 h2so4 1-5 ml 1-5 ml 1-5 ml sulphur powder 10 5 bioleaching experiments were carried out in 250 ml erlenmeyer flasks containing 190 ml of the nutrient media, depicted in table 1, to which 2 g pcbs were added. each flask was inoculated with 10 ml pcbs-adapted pure bacterial culture of a. ferrooxidans, pcbs-adapted pure bacterial culture of a. thiooxidans or mixture of a. ferrooxidans and a. thiooxidans. 3. results and discussion the overall cu, ni, zn and al bioleaching efficiencies using the pure culture of a. nferrooxidans, a. thiooxidans, and the mixed culture of a. ferrooxidans and a. thiooxidans are shown in fig. 1. as it can be seen the pure culture of a. ferrooxidans was the most effective in zn and cu dissolution, when 100 % and 85 %, respectively, were dissolved. the percentages of ni and al dissolved were 35 % and 33 %, respectively. the effectiveness of the pure culture of a. thiooxidans was the lowest. during the whole bioleaching process only 1 %, 3 % and 17 % of cu, zn and al were leached out. however, the bioleaching efficiency of ni was 50 % that was higher than by the pure culture of a. ferrooxidans. contrary to the results obtained by the pure bacterial cultures, shown in fig. 1, it is obvious that mixed bacterial culture was the most effective. the percentages of cu, ni, zn and al bioleaching after 28 days were 100 %, 92 %, 89 % and 20%, respectively. compared to the results obtained during the bioleaching by the pure culture of a. ferrooxidans zn and al bioleaching efficiency was slightly lower. positive influence of the mixed bacterial culture on metal dissolution was observed by several authors. ilyas et al. (2007) observed maximum bioleachability of metals from pcbs using mixed consortium of metal – adapted culture of acidophilic heterotrophs (sulfobacillus thermosulfidooxidans) and acidophilic autotrophs (a. ferrooxidans). in their study 81, 79 % and 83 % of ni, al and zn, respectively, were leached out. one of the main advantages of using heterotrophs in above mentioned mixed culture was the crossbereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:58 utc 48 mrazikova, a. et al. feeding. the heterotrophs use extracellular metabolites from autotrophs as a carbon source enhancing the stability of the mixed bacterial culture (fournier et al., 1998). fig. 1. percentages of copper, nickel, zinc and aluminium solubilized from pcbs by the pure culture of a. ferrooxidans, a. thiooxidans and mixed culture of a. ferrooxidans and a. thiooxidans. the presence of a. thiooxidans in mixed culture obviously enhanced the bioleaching process. although the main leaching bacteria (especially in the case of cu and zn recovery) were probably a. ferrooxidans the positive effect of a. thiooxidans was in the maintaining of the low ph values in media. many authors agreed that acid environment and strong oxidants are needed for dissolution of metals present in pcbs (liang et al., 2010). thus, the maximal metal recovery could be achieved if these two strains were cultured together to generate h2so4 and fe 3+ simultaneously. in the presence of a. ferrooxidans ferrous ions are oxidized into ferric ions (eq. 2) which as the strong oxidation agents consequently oxidized metals from m0 into m2+ (3+) form (eq. 3). the oxidation of fe2+ into fe3+ and metals dissolution resulted in a rapid increase of ph from the initial ph = 1.5 up to value of 2.2 observed in the first ten days of the bioleaching processes (fig. 2). on the following days the ph remained stable until the end of the experimental period. based on the fact that the ph remained stable since day 10 up to the end of the bioleaching processes, it might be assumed that there was a balance between the simultaneous consumption of protons (eq. 2) and release of protons during the, bacterial sulphur oxidation (eq. 1), hydrolysis of fe3+ ions (eq. 4, 5, 6) and jarosite formation (eq. 7). fe3+ + h2o ↔ feoh 2+ + h+ (4) fe3+ + 2h2o ↔ fe(oh)2 + h+ (5) fe3+ + 2h2o ↔ fe(oh)2 + h+ (6) 3fe3+ + 6h2o + m + + 2hso4 → mfe3(so4)2(oh)6 + 8h + (7) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:58 utc nova biotechnologica et chimica 14-1 (2015) 49 as it was reported by daoud and karamanev (2006) the presence of jarosite has negative effect on the metal dissolution due to the kinetic barrier formation which inhibits the diffusion of reactants and products through the precipitation zone. fig. 2. changes in ph during bioleaching studies. the presence of jarosite was confirmed by the x-ray diffraction analysis in the studied system (fig. 3).the maintenance of low ph values by the mixed bacterial culture delayed the jarosite formation significantly resulting in the higher recovery of metals in comparison with the pure a. ferrooxidans culture utilization. fig. 3. x – ray diffraction of pcbs residuum after bioleaching process. the maintenance of low ph values by the mixed bacterial culture delayed the jarosite formation significantly resulting in the higher recovery of metals in comparison with the pure a. ferrooxidans culture utilization. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:58 utc 50 mrazikova, a. et al. 4. conclusion the main purpose of this work was to investigate the influence of the three kinds of acidophilic bacterial cultures: the pure culture of a. ferrooxidans, of a. thiooxidans and mixed culture of a. ferrooxidans and a. thiooxidans on copper, nickel, zinc and aluminium solubilisation from pcbs. the results from this study demonstrate that mixed culture of a. ferrooxidans and a. thiooxidans had the highest metals bioleaching capacity. the mixed consortium was able to dissolve 100 %, 92 %, 89 % and 20 % of cu, ni, zn and al, respectively. the main advantage of the mixed bacterial culture was the ability of the bacteria to maintain the low ph values resulting in higher metal bioleaching efficiencies. the experimental results also showed that the pure culture of a. ferrooxidans was the most effective in zn (100 %) and cu (85 %) dissolution. the pure culture of a. thiooxidans was not efficient. acknowledgements: the work was fully supported by a grant from the slovak national grant agency under the vega project 1/0235/12 and 1/0197/15. references bálintová, m., luptáková, a.: úprava kyslých banských vôd. košice, stavebná fakulta, technická univerzita, 2012, 131 pp. (in slovak). brandl, h., bosshard, r., wegmann, m.: computer-munching microbes: metal leaching from electronic scrap by bacteria and fungi. hydrometallurgy, 59, 200, 319-326. daoud, j., karamanev, d.: formation of jarosite during fe2+ oxidation by acidithiobacillus ferrooxidans. minerals eng., 19, 2006, 960-967. fournier, d., lemieux, r., couillard, d.: essential interactions between thiobacillus ferrooxidans and heterotrophic microorganisms during wastewater sludge bioleaching process. environ. pollut., 101, 1988, 303-309. ilyas, s., anwar, m. a., niazi, s. b., ghauri, m. a.: bioleaching of metals from electronic scrap by moderately thermophilic bacteria. hydrometallurgy, 88, 2007, 180-188. ilyas, s., lee, j., chi, r.: bioleaching of metals from electronic scrap and its potential for commercial exploitation. hydrometallurgy, 131-132, 2013, 138 – 143. lee, j., pandey, b.d.: bio – processing of solid wastes and secondary resources for metal extraction – a review. waste manage., 32, 2012, 3-18. liang, g., tang, j., liu, w., zhou, q.: optimizing mixed culture of two acidophiles to improve copper recovery from printed circuit boards (pcbs). j. hazard. mater., 250-251, 2013, 238-245. liang, g., yiwei, m., zhou, q.: novel strategies of bioleaching metals from printed circuit boards (pcbs) in mixed cultivation of two acidophiles. enzyme microb. technol., 47, 2010, 322-326. valdés, j., pedroso, i., quatrini, r., dodson, r.j., tettelin, h., blake ii, r., eisen, j. a., holmes, d.s.: acidithiobacillus ferrooxidans metabolism: from genome sequence to industrial applications. bmc genomics, 9, 2008, 597. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:58 utc nova biotechnologica et chimica 14-1 (2015) 51 wang, j., bai, j., xu, j., liang, b.: bioleaching of metals from printed wire boards by acidithiobacillus ferrooxidans and acidithiobacillus thiooxidans and their mixture. j. hazard. mater., 172, 2009, 1100-1105. xiang, y., wu, p., zhu, n., zhang, t., l, w., w., j. and li , p: bioleaching of copper from waste printed circuit boards by bacterial consortium enriched from acid mine drainage. j. hazard. mater., 184, 2010, 812-818. zhao, l., zhu, n.w., wang, x.h.: comparison of bio-dissolution of spent ni-cd batteries by sewage sludge using ferrous ions and elemental sulfur as subrate. chemosphere, 70, 2008, 974-981. presented at the 3rd international conference “biotechnology and metals 2014”, september 17-19, 2014, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:58 utc microsoft word 6_janairo et al nova biotechnologica et chimica 15-2 (2016) 156 doi 10.1515/nbec-2016-0016 © university of ss. cyril and methodius in trnava in silico site-directed mutagenesis of the anopheles gambiae odorant binding protein 20 jose isagani b. janairo1,*, patricia isabel k. bravo2, ninna louise g. morano2, derrick ethelbhert c. yu2 1 biology department, de la salle university, 2401 taft avenue, manila, 0922, philippines (*jose.isagani.janairo@dlsu.ed) 2 chemistry department, de la salle university, 2401 taft avenue, manila, 0922, philippines abstract: the anopheles gambiae is a highly anthropophilic mosquito which is the leading vector for malaria. this disease has affected more than 500 million people worldwide. the anopheles gambiae targets its hosts through the odors of the human skin and sweat where odorant molecules radiate. these odors elicit specific responses from the insect through the odorant – binding proteins (obp). recently, a specific type of obp has been characterized which is known as the anopheles gambiae odorant – binding protein 20 (agamobp20). this obp is highly expressed in the female mosquito antennae during the peak of its host – seeking behavior and thus may play a role in olfactory perception. the binding site of the agamobp20 is composed primarily of hydrophobic residues wherein the importance of each residue is herein analysed to further understand the properties of agamobp20. this was carried out through computer – aided site – directed mutagenesis coupled with homology modelling and docking simulations wherein each residue in the binding site was changed to alanine and serine. probable key amino acid residues were identified as leu106, leu107, and met53 which are hypothesized to play a significant role in the protein – ligand interaction. these residues had the greatest impact in the binding free energy when mutated with alanine and serine. the presented results suggest that steric hindrance and hydrophobic interaction are crucial factors to consider on the manner in which the ligand binds with agamobp20. the molecular features and parameters obtained may be utilized for the development of new pesticides and repellents that are able to block the function of agamobp20 and may result to the disarray of the host – seeking behavior of the anopheles gambiae. keywords: odorant binding proteins, anopheles gambiae, site-directed mutagenesis 1. introduction malaria is a disease acquired from the female anopheles mosquitoes. it is caused by parasites that belong to the genus plasmodium. previous studies indicated that the parasite is introduced to the female mosquito vector through its accumulation of blood from infected humans. the parasite develops and reproduces inside the host where infection may spread once the mosquito bites more humans. the common symptoms of malaria include fever and a flu-like illness, which will lead to death if not treated. according to the center for disease control (2015), about 3.2 billion people are at great risk of acquiring malaria. a total of 97 countries were calculated to have been experiencing malaria transmission within their domain in the year of 2015. there have been approximately 198 million cases of this disease bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:56 utc 157 janairo, j.i.b. et al. around the world and about 500,000 people have died due to malaria. the anopheles gambiae mosquitoes are believed to able to locate their targets through olfactory sensing facilitated by odorant binding proteins (obp) (zhou, 2010; pelosi, 1994; pelosi and maida, 1990). the hypothesized role of obps in odor perception is that they are odorant molecule carriers. they are able to bind with odorant molecules and carry them across the mucus barrier to the olfactory receptor, triggering the mosquito’s mechanism of olfaction (pelosi, 1994; pelosi and maida, 1990; pelosi and tirindelli, 1989; pevsner et al., 1988). obps are generally hydrophobic in nature and thus, aid hydrophobic compounds to traverse across the aqueous mucus layer. one of the newly discovered obps in the a. gambiae mosquito is the obp20. a recent study using dna microarray analysis on the a. gambiae under light/dark cycles showed that the gene expression of the agamobp20 is regulated by light/dark cycles, the period where its host seeking behavior is at its peak, suggesting that this obp has a vital role in the olfactory responses (ziemba et al., 2013; rund et al., 2011). evidently, using microarray-based survey on mrna levels of the a. gambiae, showed that the agamobp20 is one of the obps that are expressed highest in the female antennae exhibiting around 3-6 fold elevated expression levels (biessmann et al., 2005). the crystal structure of the agamobp20 has a specific route for the arrival and the departure of the odorant molecules from humans into a certain binding pocket. the binding pocket in the agamobp20 is lined up with mostly hydrophobic residues which are: met6, met7, gly10, ile13, met50, met53, ile70, ile73, met82, leu86, leu106, leu107, leu110, phe117, ile118, phe119 and pro120. the polar residues thr55 and ser66 are quite close on one edge of the protein and are strategically positioned so that they will be able to hydrogen bond with the polar groups of the ligands. these residues greatly contribute to the activity of the agamobp20 (ziemba et al., 2013). the manner of the conformation of the agamobp20 is associated with the binding free energy of the ligand. currently, insufficient information is present on the odorant preference and the manner of which this obp adapts to its odorants. however, a study on the docking simulations performed on certain carboxylic acids found in sweat and limburger cheese with agamobp20 showed that oleic acid could be a potential ligand since it exhibited the most favourable binding free energy value when docked with agamobp20. (janairo et al., 2015). evidently, oleic acid is a component of limburger cheese, having an odor similar to the human skin and is one of the main attractant for the female anopheles gambiae, further supporting the role of oleic acid as a potential ligand (knols et al., 1997). site – directed mutagenesis using in silico methods was employed by mutating each residue, within the binding site, with alanine and serine in order to identify the key amino acid residue that had the greatest effect on the protein – ligand interaction and to analyse the dominant molecular interactions between the agamobp20 and its potential ligand, oleic acid. alanine and serine were used in order to determine the effect of polarity and steric hindrances. the binding behaviour of the mutated agamobp20 with the ligand was assessed through docking simulations. results of the docking simulations were then analysed using the binding bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:56 utc nova biotechnologica et chimica 15-2 (2016) 158 free energy values that were calculated in order to determine the prevailing trend. structure – based method was also employed in order to observe the changes in the conformation of the ligand and the surrounding residues. results may be of use for the development of repellents and pesticides that are able to inhibit the function of the agamobp20. 2. materials and methods 2.1. mutagenesis of the agamobp20 the sequence of the agamobp20 was obtained and used as a template for site-directed mutagenesis that was employed for each of the residues on the binding site. the sequence was obtained from the research collaboratory for structural bioinformatics protein databank (rcsb pdb), an online portal that feature free and a readily available storehouse of biomolecular 3d structures (berman et al., 2000). the sequence was obtained as a fasta sequence using its protein id 3vb1 with a resolution of 2.00 angstroms (ziemba et al., 2013). each residue was replaced with alanine and serine according to their localization on the binding site. the mutated sequences for both alanine and serine were collected and were used to generate homolog model of the agamobp20. 2.2. homology modelling homolog models of the agamobp20 mutants were produced using swiss model server, a fully automated server for protein homology – modelling (biasini et al., 2014; bordoli et al., 2009; bordoli et al., 2006). the templates selected were based on their identity match with the target sequence, global model quality estimation, and the quality mean. the global model quality estimation (gmqe) is a quality estimation that combines properties from the target – template alignment. the quality mean (qmean) is a composite scoring function that estimates the global and local model quality. each residue is assigned a reliability score between 0 to 1 for both gmqe and qmean, which indicates the expected similarity with the target structure. the template with the highest value of the identity match, gmqe and qmean were chosen. 2.3. ligand selection the ligand selected for docking with the agamobp20 was oleic acid since it obtained the most favourable binding free energy value out of the major components in sweat and limburger cheese that were also docked with the agamobp20 (janairo et al., 2015). the ligand structure was optimized using spartan ’14 (wavefunction, inc.) software at the dft b3lyp level of theory. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:56 utc 159 janairo, j.i.b. et al. 2.4. docking simulations docking simulation was then performed in order to closely observe the difference in the binding orientation of oleic acid for each of the mutated agamobp20 as well as the wild type, and to energetically determine the interactions involved. the homologs generated from the mutated sequences with alanine and serine were docked with oleic acid using arguslab (planaria software), a publicly available software that is reliable for virtual screening, docking simulations, molecular modelling, ligand pose construction and ligand pose selection (janairo and janairo 2012; oda et al., 2009). the simulation was confined only within 15 angstroms of the x, y, and z axes of the binding site with a 0.4 angstroms of grid resolution, having a regular precision and a maximum of 150 poses. binding interaction was assessed in thermodynamic parameters using the binding free energy of the best ligand pose. the binding free energies obtained from the mutated sequence were compared with the binding free energy of the wild type. structure-based observations were also employed to determine the changes in the conformation of the ligand and the surrounding residues. 3. results and discussion the binding site of the agamobp20 is composed of hydrophobic residues namely m6, m7, g10, i13, m50, m53, i70, i73, m82, l86, l106, l107, l110, f117, i118, f119, and p120. site – directed mutagenesis was conducted through homology modeling by replacing each residue with alanine and serine. docking simulation was then employed for the mutated obp20 and the wild type with oleic acid used as the ligand. docking was performed in order to observe the difference in the binding of the ligand for each mutation in contrast to the binding of the ligand to the wild type. the key residues that are said to play an important role in the binding of the agamobp20 with its ligand were selected based on the greatest difference in the value of the calculated binding free energy of the alanine and serine mutations compared with the wild type. in order to maintain consistency, mutant obp20 with its corresponding mutation will be denoted as mobp20-mutation (i.e. mobp20leu107a etc.) the obp20 with the mutated residues and the wild type are listed in table 1 with their corresponding binding free energy after it has been docked with the potential ligand, oleic acid. from the residues altered with alanine and serine, the following can be observed. the mobp20-leu107a and mobp20 leu106s garnered the lowest value of binding free energy, indicating enhanced spontaneity. taking into account the binding free energy of the wild type, which is -11.9752 kcal/mol, mobp20leu107a and mobp20-leu106s resulted into a more favorable binding free energy. the percentage difference of the binding free energy of the extremes with respect to the binding free energy of the wild type is shown in table 2. the alteration of leucine with both alanine and serine resulted into a binding free energy that is more favorable with the percentage difference of 12.68 % and 6.38 % from the wild type. since both alanine and leucine are hydrophobic amino acids, it is difficult to conclude bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:56 utc nova biotechnologica et chimica 15-2 (2016) 160 that the enhanced spontaneity brought about by alanine substitution is absolutely due to hydrophobicity. table 1. binding free energies of the mutated obp20 and the wild type using oleic acid as the ligand. mutated obp20 alanine mutation binding free energy (kcal/mol) mutated obp20 serine mutation binding free energy (kcal/mol) wild type binding free energy (kcal/mol) l107a -13.49 l106s -12.74 obp20 -11.98 p120a -12.71 p120s -12.55 i13a -12.65 l107s -12.55 m7a -12.47 m50s -12.39 i70a -12.20 i118s -12.36 m82a -12.07 i73s -12.19 f117a -12.04 g10s -12.00 f119a -12.01 f117s -11.97 l110a -12.01 m7s -11.95 g10a -11.98 i13s -11.94 i73a -11.98 f119s -11.85 m50a -11.92 l86s -11.83 m6a -11.73 m6s -11.59 l106a -11.64 m82s -11.40 i118a -11.59 l110s -11.38 l86a -11.54 i70s -10.82 m53a -11.14 m53s -10.26 this is further supported with the observations wherein serine mutations at l106, p120, l107, i118 and i73 resulted into a more favorable binding. this suggests that the ligand preferred a residue that is less bulky since both alanine and serine are less bulky than leucine. steric interactions within the protein binding site have strong influence on the manner of which the ligand binds (snyder et al., 2011; hlavacek et al., 1999). however, the role of hydrophobicity in ligand binding cannot be discounted and still remains a crucial factor. this was observed when the values generated for alanine and serine mutations were compared. table 2. percentage difference of the binding free energy values of the extremes with the wild type. mutated obp20 alanine mutation percentage difference (%) mutated obp20 serine mutation percentage difference (%) l107a 12.68 l106s 6.38 m53a -6.95 m53s -14.32 it is evident that the binding free energy values generated from the mutated residues with alanine obtained lower values than that of the serine mutants indicating that the ligand preferred nonpolar residues to interact with. when m53 was altered with both alanine and serine, it resulted to a higher value of binding free energy. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:56 utc 161 janairo, j.i.b. et al. this indicates that the affinity of the protein with the ligand did not become favorable. however, upon observing the values of other methionine residues such as m7, m82, m50 and m6, it was not consistent with the trend of m53 so it is safe to conclude that m53 garnered the highest value of binding free energy when replaced with alanine and serine due to its position with respect to the orientation of the ligand. fig. 1. comparison of the binding of the ligand oleic acid (yellow) with the wild type (a) and the mutated agamobp20 (b,c,d,e) in ribbon model after docking. fig. 1b shows the mutated obp20-l107a after docking. fig. 1c shows mutated obp20-m53a after docking. changes in the orientation of the residues as well as the ligand orientation in contrast to fig. 1a is notable. fig. 1d shows mobp20-l106s after it has been docked with oleic acid. fig. 1e shows mobp20-m53s after docking. notable variations in the ligand orientation are observed as well as with the specific residues the ligand interacts with. these conformational differences can be attributed to the mutations conducted. differences in ligand orientation among the mutants were thereafter assessed. the images of the mutated agamobp20 (l107a and l106s) were compared with the docked wild type (fig. 1). fig. 1a shows the wild type obp20 (ribbon model) interacting with the oleic acid (yellow). the figure shows that most of the residues that are close to the ligand were the nonpolar residues such as v49, m50, and l61. however, there are also presence of polar residues such as n62 and t55. the obp20 mutated with l107a is depicted in fig. 1b. it can be deduced from fig. 1b that the conformation of oleic acid is bent wherein the terminal atoms are observed to potentially express a hydrophobic interaction with f117 while the median portion of the oleic acid are shown to potentially express hydrophobic interaction a. b. c. d. e. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:56 utc nova biotechnologica et chimica 15-2 (2016) 162 with nonpolar residues such as a107 and m50. it can also be depicted that the manner of which the ligand is bent may be due to the orientation of residues such as f117, bulky in nature, that are oriented towards the ligand. the obp20 mutated with l106s is shown in fig. 1d. it can be observed from fig. 1d that the alkyl chain of the oleic acid is bent and is surrounded with nonpolar residues such as l61, l86, a93, m50 and f117. these residues are oriented towards the ligand that may have been the reason why the ligand bent itself in that manner. presence of polar residues can also be noted such as s106. fig. 2. ribbon model of the wild type (a) and the mutated obp20 with the extremes l107a (b), m53a (c), l106s (d), m53s in (e). overall, variations in ligand orientation and localization within the binding pocket which can be attributed to the mutations are notable. to observe the ligand orientation with respect to the conformation of the agamobp20, docked images of the wild type and the mutated obp20 containing the extremes were collected (fig. 2). the ribbon model of the wild type agamobp20 after docking with oleic acid is presented in fig. 2a. it can be observed that the ligand is elongated in such a way that all parts of the chain are located on the outer most portion of the protein. it is evident that the carboxylic side is in close proximity with helices 2 and 3 where the upper middle portion of the alkyl chain is bent to such degree that it is relatively close to the lower portion of helix 3. likewise, the terminal chain is bent in such a way that it is relatively close to the uppermost portion of helix 4. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:56 utc 163 janairo, j.i.b. et al. the ribbon model of the obp20 with the extremes l107a and m53a after it has been docked with oleic acid is presented in fig. 2b and 2c, respectively. in fig. 2b, it is clearly evident that there was a change in the conformation of oleic acid in contrast to that shown in fig. 2c however, the ligand is still located on the outermost core of the protein. in here, the carboxylic side is now in close proximity with the upper portion of the helix 4. the alkyl chain is within the perimeter of helix 3. in fig. 2c, where the obp20 containing m53a was docked with oleic acid, the carboxylic side is now located near helix 3 and is elongated in such a way that the terminal side is near the proximity of helix 4. the ribbon model of the obp20 with the extremes l106s and m53s is depicted in fig. 2d and 2e, respectively. it is clearly evident that the ligand is now located onto the inner core of the protein. for obp20 with the mutation l106s, the carboxylic side of the ligand is near the proximity of the helix 6. the ligand is bent to such degree that the middle portion of the alkyl chain is near the c terminal tail of the protein. the terminal tail of the ligand exists in the median core of the protein. for the obp20 containing m53s, depicted in fig. 2e, the ligand is also located within the inner portion of the protein where the carboxylic side is also near helix 6 however, the alkyl chain is more bent to such degree that the median portion of the alkyl chain is the located on the median core of the protein. the terminal portion of the alkyl chain exits near the c terminal tail of the protein. 4. conclusions in summary, the results of the calculations and modelling provided insights regarding the binding of oleic acid with agamobp20. from these calculations, key amino acids residues were identified which are hypothesized to play key roles in the binding of the ligand. these residues are l106, l107 and m53. these residues had the greatest impact in the binding free energy when these were mutated with alanine and serine. the significant change in the binding free energy of these mutants from the wild type is attributed to a decrease in steric hindrance within the binding site. these results suggest that both hydrophobic interaction and steric hindrance are crucial factors to consider in the binding of the ligand with this odorant – binding protein. the molecular features and parameters obtained may be a potential target for the new drugs and pesticides that are able to inhibit the function of agamobp20 and may result to the blocking of the olfactory mechanism of the anopheles gambiae, the mosquito responsible for transmitting malaria in humans, leading to the disarray of its host seeking behaviour. references arnold, k., bordoli, l., kopp, j., schwede, t.: the swiss-model workspace: a web-based environment for protein structure homology modelling. bioinformatics, 22(2), 2005, 195-201. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:56 utc nova biotechnologica et chimica 15-2 (2016) 164 berman, h.m., westbrook, j., 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naturally occurring pancreatic glucokinase mutants. philipp. sci. lett., 5(1), 2012, 1-6. janairo, j.i.b., carandang, j., vi, amalin, d.: docking simulations and regression analysis on the binding of several carboxylic acids with the odorant binding protein 20 of anopheles gambiae. rom. j. biochem., 52(1), 2015, 61-65. knols, b.g.j., van loon, j.j.a., cork, a., robinson, r.d., adam, w., meijerink, j., de jong, r., takken, w.: behavioural and electrophysiological responses of the female malaria mosquito anopheles gambiae (diptera: culicidae) to limburger cheese volatiles, bull. entomol. res., 87, 1997, 151-159. oda, a., takahashi, o.: validation of arguslab efficiencies for binding free energy calculations. chem. bio. informat. j., 9, 2009, 52-61. pelosi, p., maida, r.: odorant binding proteins in vertebrates and insects: similarities and possible common function. chem. senses., 15, 1990, 205215. pelosi, p.: odorant-binding proteins. crit. rev. biochem. mol. biol., 29(3), 1994, 199-228. pelosi, p., tirindelli, r.: structure/activity studies and characterization of an odorant-binding protein. receptor events and transduction in taste and olfaction. chem. senses, 1, 1989, 207-226. pevsner, j., reed, r.r., feinstein, p.g., snyder, s.h.: molecular cloning of odorant-binding protein: member of a ligand carrier family. science, 241, 1988, 336-339. rund, s.s.c., hou, t.y., ward, s.m., collins, f.h., duffield, g.e.: genome-wide profiling of diel and circadian gene expression in the malaria vector anopheles gambiae. proc. natl. acad. sci. usa, 108, 2001, e421-e430. schwede, t., kopp, j., guex, n., peitsch, m.c.: swiss-model: an automated protein homologymodeling server. nuc. acids res., 31(13), 2003, 3381-3385. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:56 utc 165 janairo, j.i.b. et al. snyder, p.w., mecinovic, j., moustakas, d.t., thomas, s.w., harder, m., mack, e.t., lockett, m.r., héroux, a., sherman, w., whitesides, g.m.: mechanism of the hydrophobic effect in the biomolecular recognition of arylsulfonamides by carbonic anhydrase. proc. natl. acad. sci. usa, 108(44), 2011, 17889-17894. zhou, j.: odorant-binding proteins in insects. vitam. horm., 83, 2010, 241-272. ziemba, b.p., murphy, e.j., edlin, h.t., jones, d.n.m.: a novel mechanism of ligand binding and release in the odorant binding protein 20 from the malaria mosquito anopheles gambiae, protein sci., 22, 2013, 11-21. received 18 october 2016 accepted 7 december 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:56 utc microsoft word 9_miklovic et al. nova biotechnologica et chimica 15-2 (2016) 190 doi 10.1515/nbec-2016-0019 © university of ss. cyril and methodius in trnava synthesis, crystal structure and spectral properties of copper(ii) 2-chloronicotinato complexes with n-heterocyclic ligands jozef miklovič1, dušan valigura1, ingrid svoboda2, ján moncol3, milan mazúr4 1 department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic (jozef.miklovic@ucm.sk) 2 material sciences, darmstadt university of technology, darmstadt, 64287, germany 3 institute of inorganic technology, fchpt, slovak university of technology, bratislava, sk-812 37, slovak republic 4 institute of physical chemistry and chemical physic, fchpt, slovak university of technology, bratislava, sk-812 37, slovak republic abstract: the synthesis and characterization of nine new copper(ii) complexes [cu(2-clnic)2l2] (where 2-clnic is 2-chloronicotinate anion, l is imidazole – im, benzimidazole – bim, furo[3,2-c]pyridine – fp, 2-methylfuro[3,2-c]pyridine – mfp, or [1]benzofuro[3,2-c]pyridine – bfp), [cu(2-clnic)2(ina)] (where ina is isonicotinamide), [cu(2-clnic)2(4-py)]·h2o (where 4-py is 4-methylpyridine) and [cu2(2-clnic)4(iq)2] (where iq is isoquinoline) are reported. the characterizations were based on elemental analysis, infrared, electronic and epr spectra. the dimeric character of [cu2(2-clnic)4(iq)2] is assumed on the epr spectrum and the other spectral methods. the crystal structure of the [cu(2-clnic)2(bim)2] and [cu(2-clnic)2(fp)2] complexes have been determined by x-ray crystal structure analysis. both complexes exhibit the hexacoordination coordination polyhedra around copper atom that lies in the crystallographic center of symmetry. the distorted tetragonal-bipyramidal (4+2) arrangement is in good agreement with spectral data that have suggested an asymmetric chelate coordination of the carboxylic group. key words: complex, copper(ii), crystal structure, carboxylate, ir, electronic and epr spectra 1. introduction the metal carboxylates are interesting from a chemical point of view as the carboxylate ion can coordinate to metals in number of ways: as a unidentate ligand, as a chelating ligand, as a bridging ligand, or as a monoatomic bridging ligand. this causes the existence of a rich family of compounds with various structures (deacon and phillips 1980, rao et al., 2004). moreover pyridinecarboxylates due to presence of pyridine nitrogen atom can act as n-donor ligands in addition to their carboxylate o-donor ability. some crystal structures of copper(ii) 2-chloronicotinate complexes have been published (jin et al., 2012; 2014; 2015; moncol et al., 2002; 2006; 2007). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:05 utc 191 miklovič, j. et al. in this paper, we described synthesis, spectral properties and crystal structure of 2-chloronicotinate copper(ii) complexes with n-heterocyclic ligands: [cu(2clnic)2(l)2] (l = im, bim, fp, mfp, bfp); [cu2(2-clnic)4(iq)2]; [cu(2-clnic)2(4-py)2]·h2o; [cu(2-clnic)2(ina)]; [cu(2-clnic)2(3,5-py)(meoh)]. the crystal and molecular structure of the complexes under study [cu(2-clnic)2(bim)2] and [cu(2-clnic)2(fp)2] has also been studied by x-ray structure analyses. sketch and abbreviations of 2-chloronicotinate and heterocyclic ligands used in assembling new cu(ii) complexes are presented in fig. 1. n n ch3 n conh2 n h3c ch3 n h n n h n o n o n o n h3c n cl coo furo[3,2-c]pyridine fp 2-metylfuro[3,2-c]pyridine mfp [1]benzofuro[3,2-c]pyridine bfp iso quinoline iq imidazole im benzimidazole bim 4-methylpyridine 4-py 3,5-dimetylpyridine 3,5-pyiso nicotinamide ina 2-chloronicotinate 2-clnic fig. 1. sketch and abbreviations of 2-chloronicotinate and n-heterocyclic ligands. 2. material and methods 2.1. chemical reagents, analysis and physical measurements all used chemicals were of reagent grade and used without further purifications. derivatives of furopyridine (fp, mfp and bfp) have been prepared using eloyderyckere procedure (eloy and deryckere, 1971). the complex [cu2(2-clnic)4(h2o)2] was prepared by procedure described in (moncol et al., 2006). carbon, hydrogen, nitrogen and sulfur were determined by microanalytical methods (thermo electron flash ea 1112). analytical data for the complexes are given in table 1. electronic spectra (9 000 – 50 000 cm-1) of the powdered samples were recorded on a specord 200 (karl-zeiss). ir spectra were recorded on ft-ir spectrometer (nicolet 5700, thermo scientific) with a smartorbittm diamond atr accessory in range of 4 000 – 400 cm-1 at room temperature (r.t.). epr spectra of powdered samples were measured a bruker 200d src x-band (9.4 ghz) at room bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:05 utc nova biotechnologica et chimica 15-2 (2016) 192 temperature. the simulations of the epr spectra were performed using the commercially available program simfonia (bruker). table 1. analytical data for the cu(ii) complexes. compound empirical formula formula weight (g mol-1) calc / found (%) c h n [cu2(2-clnic)4(iq)2] 1 c42h26cl4cu2n6o8 1011.60 49.87 50.12 2.59 2.46 8.31 8.30 [cu(2-clnic)2(4-py)2]·h2o 2 c24h22cl2cun4o5 580.91 49.62 49.14 3.82 3.51 9.64 9.54 [cu(2-clnic)2(ina)] 3 c18h12cl2cun4o5 498.77 43.34 43.28 2.42 2.43 11.23 10.91 [cu(2-clnic)2(3,5-py)(meoh)] 4 c20h19cl2cun3o5 515.83 46.57 46.91 3.71 3.88 8.15 8.62 [cu(2-clnic)2(im)2] 5 c18h14cl2cun6o4 512.80 42.16 42.07 2.75 2.64 16.39 16.12 [cu(2-clnic)2(bim)2] 6 c26h18cl2cun6o4 612.92 50.95 51.30 2.96 2.87 13.71 13.68 [cu(2-clnic)2(fp)2] 7 c26h16cl2cun4o6 614.88 50.79 50.56 2.62 2.52 9.11 9.07 [cu(2-clnic)2(mfp)2] 8 c28h20cl2cun4o6 642.94 52.31 52.40 3.13 3.11 8.71 8.54 [cu(2-clnic)2(bfp)2] 9 c34h20cl2cun4o6 715.00 57.11 57.63 2.82 2.86 7.83 8.00 2.2. crystallography data collection and cell refinement of 1 and 2 were carried out using a κ-axis diffractometer xcalibur s ccd (oxford diffraction) with graphite monochromated mokα radiation. the diffraction intensities were corrected for lorentz and polarization factors. the structures were solved using program shelxt (sheldrick, 2015a) or olex2.solve (bourhis et al., 2015) and refined by the full-matrix least-squares procedure with shelxl (version 2016/4) (sheldrick, 2015b). geometrical analyses were performed with shelxl. the structures were drawn using the olex2 package (dolomanov et al., 2009). crystal data and conditions of data collection and refinement are reported in table 2. 2.3. preparation of the complexes [cu2(2-clnic)4(iq)2] 1; [cu(2-clnic)2(4-py)2]·h2o 2; [cu(2-clnic)2(ina)] 3; [cu(2-clnic)2(3,5-py)(meoh)] 4: complex [cu2(2-clnic)4(h2o)2] (0.5 mmol; 0.395 g) was suspended in methanol (20 cm3) and ligand 2 mmol (iq = 0.258 g; 4-py = 0.186 g; ina = 0.244 g; 3,5-py = 0.214 g) in methanol (10 cm3) was added. solution was then heated to reflux for 15 minutes and then filtered off. mixture then evaporated at r. t. and subsequent crystals were separated, washed with methanol bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:05 utc 193 miklovič, j. et al. and dried at r. t.. yield: complex 1 – 0.3 g (59 %, green); 2 – 0.17 g (29 %, blue); 3 – 0.39 g (78 %, blue green); 4 – 0.32 g (62 %, blue). table 2. crystallographic data for the reported compounds. compound 6 7 [cu(2-clnic)2(bim)2] [cu(2-clnic)2(fp)2] empirical formula c26h18cun6o4 c26h16cun4o6 formula weight 612.90 614.87 temperature (k) 293(1) 293(1) wavelength (å) 0.71073 0.71073 crystal system monoclinic monoclinic space group p21/c p21/c a (å) 7.6684(3) 12.9680(4) b (å) 22.9040(10) 13.2636(4) c (å) 7.6341(5) 7.3937(2) α (°) 90 90 β (°) 107.504(6) 99.284(3) γ (°) 90 90 v (å3) 1278.74(12) 1255.08(6) z, ρcalc g.cm-3 2, 1.592 2, 1.627 radiation type mo-kα mo-kα μ (mm-1) 1.109 1.134 f (000) 622 622 crystal size (mm) 0.20x0.36x0.40 0.16x0.50x0.50 2θ ranges (°) 5.57 to 55.35 8.30 to 52.73 final r indices [i > 2σ(i)] r1= 0.0353 r1= 0.0493 wr2= 0.0823 wr2= 0.1203 r indices (all data) r1= 0.0418 r1= 0.0616 wr2= 0.0865 wr2= 0.1297 s 1.099 1.038 ccdc number 1511127 1511128 [cu(2-clnic)2(im)2] 5; [cu(2-clnic)2(bim)2] 6: complex [cu2(2-clnic)4(h2o)2] (0.5 mmol; 0.395 g) was suspended in methanol (20 cm3) and ligand 2 mmol (im = 0.136 g; bim = 0.236 g) in methanol (10 cm3) was added. solution was then heated to reflux for 15 minutes and then filtered off. mixture was then evaporated at r. t.. solid was recrystallized from ethanol and dried at r .t.. yield: complex 5 – 0.15 g (29 %, blue); 6 – 0.21 g (34 %, blue). [cu(2-clnic)2(fp)2] 7; [cu(2-clnic)2(mfp)2] 8; [cu(2-clnic)2(bfp)2] 9: copper(ii) acetate monhydrate (0.5 mmol, 0.1 g) was dissolved in mixture of methanol (10 cm3) and water (1 cm3). to this solution was added ligand (fp = 0.26 g; mfp = 0.29 g; bfp = 0.37 g) in methanol (5 cm3). 2-chloronicotinic acid (1 mmol, 0.157 g) in methanol (10 cm3) was then added to the solution. reaction mixture was stirred for 1 hour and filtered of. mixture was then evaporated at r. t. and crystals were formed and separated. yield: 7 – 0.2 g (65 %, blue); 8 – 0.15 g (47 %, blue); 9 – 0.3 g (84 %, blue). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:05 utc nova biotechnologica et chimica 15-2 (2016) 194 3. results and discussion 3.1. description of crystal structure of [cu(2-clnic)2(bim)2](6) and [cu(2-clnic)2(fp)2](7) molecular structure of complexes 6 and 7 are given in fig. 2. both cases crystallize in monoclinic system with space group p21/c. the molecular structures show the both compound have mononuclear units with a trans square planar configuration, in which the copper(ii) atoms are coordinated by carboxylate oxygen atoms of two 2-chloronicotinate anions and two nitrogen atoms of benzimidazole (6) or furo[3,2-c]pyridine (7) ligands. the distances of cu–o1 are 1.961(1) and 2.160(3) å, respectively, and cu–n1 are 1.920(2) and 1.998(3) å, respectively. the remaining carboxylate oxygen atoms of both complexes, which are weakly (6) or more strongly (7) bonded to the copper atom [cu2–o2 = 2.890(2) å or 2.281(4) å, respectively] in the direction of the cu1–o2 bonds, lie at 50.14(6) and 58.59(11)°, respectively, from the normal to the cuo2n2 plane and complete a tetragonal-bipyramidal (4+2) coordination. the complexes 6 and 7 represent two opposite examples of variability of the tetragonal-bipyramidal coordination of copper(ii) carboxylate complexes (moncol et al., 2004). fig. 2. the molecular structures of [cu(2-clnic)2(bim)2] (left) (6) and [cu(2-clnic)2(fp)2] (right) (7). the complex molecules of 6 are linked through n–h···o hydrogen bonds between imidazole nitrogen atoms (n2) and carboxylate oxygen atoms (o2) of neighboring complex molecules [n2–h2···o2 (-x, 1-y, 2-z) with n2···o distance of 2.757(2) å and n2–h2···o angle of 160°] into 1d supramolecular chains (fig. 3). the crystal structure of 6 contains also π-π stacking interactions (janiak, 2000) between imidazole rings of benzimidazole ligands [angle between two planes of π-π stacking interactions of 0.0°, the centroid-centroid distance of 3.88å with shift distance of 1.66å], and between pyridine rings of 2-chloronicotinate ligands [angles between two planes of π-π stacking interactions of 5.4°, the centroid-centroid distances of 3.86 and 3.87å with shift distances of 1.10 and 1.28å] (fig. 4). the π-π stacking bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:05 utc 195 miklovič, j. et al. interaction (janiak, 2000) have been also observed in crystal structure of 7 between pyridine rings of furo[3,2-c]pyridine ligands [angles between two planes of π-π stacking interactions of 12.4°, the centroid-centroid distances of 3.88å with shift distances of 1.93 and 1.38å] (fig. 4). fig. 3. supramolecular chain formed from connecting complex molecules of 6 through n–h···o hydrogen bonds. the hydrogen atoms are omitted for clarity. fig. 4. the π-π stacking interactions in crystal structures of 6 (top) and 7 (bottom). the hydrogen atoms are omitted for clarity. table 3. selected bond lengths and angles of 6 and 7. compound 6 7 [cu(2-clnic)2(bim)2] [cu(2-clnic)2(fp)2] cu1–o1* 1.961(1) 2.160(3) cu1–o2* 2.890(2) 2.281(4) cu1–n1* 1.982(2) 1.998(3) o1–cu1–o2 50.14(6) 58.59(11) n1–cu1–o1 92.28(6) 89.59(10) n1–cu1–o2 81.41(6) 89.34(10) * also for cu1–d* symmetry codes: 1-x, 1-y, 2-z (for 6), 1-x, 1-y, 1-z (for 7). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:05 utc nova biotechnologica et chimica 15-2 (2016) 196 3.2. ir, electronic and epr data all the typical features of ir spectra are clearly compatible with the structural characteristics of the complexes under study. some characteristic ir bands of the sodium salt 2-clnic na·h2o as well as of cu(ii) complexes are given in table 4. the ir spectrum of complex 2 shows absorption bands in the region from 3 200 to 3 500 cm-1. these bands correspond to the antisymmetric and symmetric oh stretch and confirm the presence of water. ir spectrum of the complex 4 has sharp band at 3 492 cm-1. this is stretching vibration of oh and confirms presence of methanol in complex structure that is in good agreement with elemental analyses. table 4. spectroscopic dataa (in cm-1) of cu(ii) complexes. compound infrared data electronic data carboxyl group νas(coo-) νs(coo-) δb band i band ii 2-clnicna 1577s 1383vs 194 [cu2(2-clnic)4(iq)2] 1 1574s 1377vs 197 13 200 25 100sh [cu(2-clnic)2(4-py)2]·h2o 2 1576vs 1389vs 187 16 700 [cu(2-clnic)2(ina)] 3 1573vs 1376vs 197 14 400 [cu(2-clnic)2(3,5-py)(meoh)] 4 1573s 1374vs 199 16 400 [cu(2-clnic)2(im)2] 5 1572vs 1390vs 182 17 300 14 600sh [cu(2-clnic)2(bim)2] 6 1593vs 1365vs 228 18 100 14 400sh 28 600 [cu(2-clnic)2(fp)2] 7 1575vs 1374vs 201 16 800 [cu(2-clnic)2(mfp)2] 8 1590s 1382vs 208 13 100 [cu(2-clnic)2(bfp)2] 9 1574vs 1382vs 192 16 800 a vs – very strong; s – strong; m – medium; br – broad; sh – shoulder, bδ = νas(coo-) νs(coo-). the difference between the antisymmetric stretch and symmetric stretch (δ) gives information on carboxylic bonding mode for the complexes after comparison with δ of compounds with ionic carboxylic groups (nakamoto, 1977). the difference between the antisymmetric stretch and symmetric stretch for 2-clnic complexes could not be determined accurately due to an overlap of νas(coo -) with the stretching vibration of c=n of the pyridine ring. for sodium 2-chloronicotinate, the δ value is 194 cm-1. similar δ value for the complex 1 (197 cm-1) and 3 suggest bridging carboxylic group. for complex 1 it is confirm from electronic spectra. the greater δ value for the complexes 4 (199 cm-1), 6 (228 cm-1), 7 (201 cm-1) and 8 (208 cm-1) suggest, that carboxylic group is probably coordinated in an asymmetric chelating manner. in this case, the δ values are comparable to those of unidentate complexes (nakamoto, 1977). the lower δ value for the complexes 2 (187 cm-1) and 5 (182 cm-1) suggest, that carboxylic group is probably coordinated in chelating manner. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:05 utc 197 miklovič, j. et al. the positions of bands which correspond to pyridine ring deformation of neutral ligands are shifted to higher wavenumbers [iq (601 → 641 cm-1), fp (602 → 648 cm-1), mfp (601 → 648 cm-1) and bfp (596 → 648 cm-1)] show, that these ligands are coordinated through the nitrogen atom of the pyridine ring (nakamoto, 1977). the positions of band to the skeletal vibrations for 4-py (802 and 995 cm-1) and 3,5-py (858 and 1064 cm-1) for free ligands are shifted to higher wavenumbers 847, 1033 cm-1; complex 2, and 867, 1064 cm-1, complex 4 respectively. this shift suggest coordination these ligand via nitrogen atom of pyridine ring (milata et al., 2008). the band of stretching vibration amide group c=o is at 1 626 cm-1 for free ligand, and is shifted to 1 686 cm-1 in the complex 3. this suggests that ligand ina is in the complex 3 as bridging. table 5. the epr data of monomeric cu(ii) complexes. compound epr data g׀׀ gi g⊥ gav * g* [cu(2-clnic)2(4-py)2]·h2o 2 2.28 2.07 2.14 4 [cu(2-clnic)2(ina)] 3 2.16 [cu(2-clnic)2(3,5-py)(meoh)] 4 2.37 2.07 2.17 5.3 [cu(2-clnic)2(im)2] 5 2.25 2.08 2.14 3 [cu(2-clnic)2(bim)2] 6 2.26 2.07 2.13 3.8 [cu(2-clnic)2(fp)2] 7 2.41 2.09 2.2 4.5 [cu(2-clnic)2(mfp)2] 8 2.39 2.10 2.2 3.9 [cu(2-clnic)2(bfp)2] 9 2.39 2.09 2.19 4.3 * gav = 1/3(2g⊥ + g׀׀); g = (g2 – ׀׀)/(g⊥ – 2). the solid state electronic spectra of complex 1 show a broad absorption band (band i) v visible region with maximum at 13 200 cm-1 (table 4), which is assigned to a dxy,yz → dx 2 -y 2 transition (kato and muto, 1988). moreover, the spectrum of complex 1 displays a shoulder at about 25 100 cm-1 (band ii). band ii has been assigned to charge transfer absorption and is believed to be indicate of dimeric complex. finally, complex 1 displays band i and ii in the usual range for cu(ii) compounds in square-pyramidal cuo4n environment. electronic spectra of all other copper(ii) complexes under study exhibit a asymmetrical broad ligand field band with a maximum at from 13 100 cm-1 to 18 100 cm−1. this type of d–d spectra for complexes 2 – 9 is typical for tetragonally distorted octahedral copper(ii) complexes (lever, 1984). in the complexes 5 and 6 it is possible watch little evolve shoulders near 14 600 and 14 400 cm-1 by jahn-teller effect. in others complexes is splitting d-d transition very little of shining and it is not possible determination of position individual bands. the solid state epr spectra of complexes 2, 4, 5, 6, 7, 8 and 9 are of monomeric type, exhibiting allowed transitions (δms=1) characteristic of species with s=1/2. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:05 utc nova biotechnologica et chimica 15-2 (2016) 198 the epr spectra exhibit axial symmetry pattern giving g-tensor values listed in table 5. the axial character with g|| > g⊥ g values close to four are in agreement with the elongated pseudooctahedral geometry having a dx 2-y 2 ground state. acknowledgements: this work was financially supported by the grants apvv-14-0073, vega 1/0534/16 of the slovak grant agency for science. references bourhis, l.j., dolomanov, o.v., gildea, r.j., howard, j.a.k., puschmann, h.: the anatomy of a comprehensive constrained, restrained refinement program for the modern computing environment olex2 dissected. acta crystallogr. sect. a, 71, 2015, 59-75. deacon, g.b., 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copper acetate, 3,5-dimethylpyrazole and carboxylates. polyhedron, 81, 2014, 382-395. jin, s.w, liu, h., chen, g.q., an, z.y., lou, y.l., huang, k., wang, d.q.: syntheses and crystal structures of copper(ii), zinc(ii) and cadmium(ii) complexes containing pyridine, quinoline and 2-methylquinoline. polyhedron, 95, 2015, 91107. kato, m., muto, y.: factors affecting the magnetic properties of dimeric copper(ii) complexes. coord. chem. rev., 92, 1988, 45-83. lever, a.b.p.: inorganic electronic spectroscopy, 2nd ed., elsevier, amsterdam, 1984, 480 p. milata, v., segľa, p., gatial, a., kováčik, v., miglierini, m., stankovský, š., šíma, j.: aplikovaná molekulová spektroskopia, vydavateľstvo stu, 2008; 600 p. moncol, j., palicová, m., segľa, p., koman, m., melník, m., valko, m., glowiak, t.: crystal and spectral study of copper(ii) pyridinecarboxylates: crystal and molecular structure of trans-diaqua-bis(n,n-diethylnicotinamiden)bis(2-chloronicotinate-o)copper(ii). polyhedron, 21, 2002, 365-370. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:05 utc 199 miklovič, j. et al. moncol, j., múdra, m., , lonnecke, p., koman, m., melník, m.: copper(ii) halogenopropionates: low-temperature crystal and molecular structure of bis(2,2-dichloropropionato)-di(methyl-3-pyridylcarbamate)copper(ii) and bis(2-bromopropionato)-di(2-pyridylmethanol)copper(ii). j. coord. chem., 57, 2004, 1065-1078. moncol, j., segľa, p., , mikloš, d., mazúr, m., melník, m., glowiak, t., valko, m., koman, m.: structural diversity of coordination polymers with bridging 3-pyridylmethanol ligand: new type of coordination polymer with different stereochemistry of copper(ii) atom. polyhedron, 25, 2006, 15611566. moncol, j., korabik, m., segľa, p., koman, m., mikloš, d., jašková, j., glowiak, t., melník, m., mrozinski, j., sundberg, m.r.: preparation, structure, and magnetic properties of copper(ii) halogenonicotinates: crystal and molecular structure of tetrakis(μ-2-chloronicotinato-o,o')diaquadicopper(ii). z. anorg. allg. chem., 633, 2007, 298-305. nakamoto, k.: infrared and raman spectra of inorg. and coord. compounds, part b, 5th edition, new york, 1997, 384-387. rao, c.n.r., natarajan, r., vaidhyanathan, r.: metal carboxylates with open architectures. angew. chem., int. ed., 43, 2004, 1466-1496. sheldrick, g.m.: shelxt integrated space-group and crystal-structure determination. acta crystallogr. sect. a, 71, 2015a, 3-8. sheldrick, g.m.: crystal structure refinement with shelxl. acta crystallogr. sect. c, 71, 2015b, 3-8. received 16 november 2016 accepted 5 december 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:05 utc microsoft word 3_moravčíková et al. nova biotechnologica et chimica 15-2 (2016) 122 doi 10.1515/nbec-2016-0013 © university of ss. cyril and methodius in trnava beta-1,3-glucanase activities in wheat and relative species jana moravčíková1, denisa margetínyová2, zdenka gálová2, iwona žur3, zuzana gregorová1, mária zimová1,4, eva boszorádová1, ildikó matušíková5 1 institute of plant genetics and biotechnology slovak academy of sciences, akademická 2, p.o. box 39a, nitra, sk-950 07, slovak republic (jana.moravcikova@savba.sk) 2 slovak university of agriculture, faculty of biotechnology and food science, tr. a. hlinku 2, nitra, sk949 76, slovak republic 3 polish academy of sciences the franciszek gorski institute of plant physiology, niezapominajek 21, kraków, pl-30-239, poland 4 department of botany and genetics, faculty of natural sciences, the constantine philosopher university, nábrežie mládeže 91, nitra, sk-949 74, slovak republic 5 department of ecochemistry and radioecology, university of cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic abstract: the (1,3)-β-d-glucan also referred to as callose is a main component of cell walls of higher plants. many physiological processes are associated with the changes in callose deposition. callose is synthesised by the callose synthase complex while its degradation is regulated by the hydrolytic enzymes β-1,3-glucanases. the latter one specifically degrade (1,3)-β-d-glucans. this work is aimed to study β-1,3-glucanase activities in the leaves of plants at two leaf stage in two diploids (agilops tauschii, triticum monococcum l.), four tetraploids (ae. cylindrica, ae. triuncialis, t. araraticum, t. dicoccum) and two hexaploids (t. aestivum l, t. spelta l.). the leaves were subjected to qualitative and quantitative β-1,3-glucanase activity assays. our results showed that the total β-1,3-glucanase activities were variable and genotype dependent. no significant correlation between β-1,3-glucanase activities and ploidy level was observed. the gel activity assays revealed a single fraction of ~52 kda glu1 that was found in all genotypes. the glu1 fraction corresponds to a single or two acidic glu isoforms in dependence on genotype. however, none of the acidic glu fractions can be assigned as a specific for di-, tetraor hexaploid genotypes. a single basic gluf isoform was detected and found as present in all genotypes. key words: (1,3)-β-d-glucans, β-1,3-glucanases, callose, laminarin, ploidy, wheat 1. introduction β-d-glucans are non-starch polysaccharides that are main component of the cell walls of most plants, fungi or microorganisms. certain β-d-glucans of different origin were studied mainly for their health-promoting effect. they were demonstrated to have hypocholesterolemic and anticoagulant activities; and chemoprotective effect as well. they can also be a potential source of long chain probiotics. not surprisingly that organisms rich of β-glucans are of economic interest. the biological activities and application potential of β-d-glucans are mainly determined by their chemical structure. β-d-glucans consist of d-glucose monomers bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:47 utc 123 moravčíková, j. et al. linked by (1,3)-β, (1,4)-β or (1,6)-β glycosidic bonds. in higher plants, β-d-glucans are synthesized as linear (1,3)-β-d-glucans, (1,4)-β-d-glucans and mixed (1,3)(1,4)-β-dglucans. cellulose or (1,4)-β-d-glucan is the major component of plant cell walls (minic et al., 2008) while the mixed (1,3)(1,4)-β-d-glucan is present mainly in the cell walls of starchy endosperm (havrlentova and kraic, 2006). the (1,3)-β-d-glucans are commonly referred to as callose. during plant growth development it can be found in the cell plate during cell division. callose deposition is important during pollen development, microsporogenesis or seed germination (pirselova and matusikova, 2013). besides, callose is synthesized between the cell wall and the plasma membrane after exposure of plants to various (a)biotic stresses (zavaliev et al., 2011). callose degradation and thus various plant physiological processes are regulated by the β-glucan endohydrolases namely β-1,3-glucanases (ec 3.2.1.39) (pirselova and matusikova, 2013). the β-1,3-glucanases (gh 17) catalyse the hydrolysis of (1,3)-β-d-glucosidic linkages in (1,3)-β-d-glucans but not in (1,3)(1,4)-β-d-glucans (høj and fincher, 1995). the β-1,3-glucanases have widely been studied for their potential to inhibit fungal pathogen (moravcikova et al., 2004), while recently their importance in plant defence against abiotic stresses has also been proven e.g. low temperature (romero et al., 2008), drought (gregorova et al., 2015) or heavy metals (pirselova et al., 2011). plant β-1,3-glucanases are referred to as “pathogenesis-related proteins” (pr2). they are grouped into the classes i-iv. the class i are vacuolar basic proteins that are accumulated in mature leaves and roots upon pathogen infection. the classes ii and iii are acidic extracellular proteins. the class iv is similar to the class ii; however, β-1,3-glucanases of the class iv are not inducible upon pathogen attack (leubnermetzgner, 2003; minic, 2008). wheat is a plant with complicated genetic information. due to the evolutionary hybridisation, domestication and/or selection steps; the wheat genome was evolved to diploid, tetraploid and hexaploid genomes. during polyploidization, many duplicated genes were retained and the redundancy conferred by duplicated genes may lead to their novel or divergent functions (chen et al., 2007). in this work, we aimed to study the activities of β-1,3-glucanases in two wheat and six crop relatives species at early growth stages. we studied the plants of two diploids (aegilops tauschii, triticum monococcum l.), four tetraploids (ae. cylindrica, ae. triuncialis, t. araraticum, t. dicoccum) and two hexaploids (t. aestivum l, t. spelta l.) they were grown to two leaf stage and the leaves were subjected to qualitative and quantitative β-1,3-glucanase activity assays. 2. material and methods 2.1. plant material and growth conditions seeds of wheat and crop relative species (table 1) were obtained from the gene bank of the slovak republic (national agricultural and food centre, pieštany). the seeds were germinated on the watered sterile filter paper in dark at room bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:47 utc nova biotechnologica et chimica 15-2 (2016) 124 temperature for 3 days. then, germinated seeds were transferred to the pots with the commercial substrate bora and cultivated at 22 oc and 16 h/8 h light/dark photoperiod under 50 µe/m2.s1 light intensity for 3 weeks. the leaves of the plants (10 plants/genotype) at two leaf stage (zadoks stage 12) were collected and used for further analyses. table 1. wheat and crop relatives species used in experiments. gene banka ploidy genome triticum monococcum l. azesvk2009-84 diploid aa m aegilops tauschii armen06-40 diploid dd aegilops cylindrical armen06-02 tetraploid ccdd aegilops triuncialis armen06-06 tetraploid uucc triticum araraticum azesvk2009-47 tetraploid ggaa u triticum dicoccum azesvk2009-78 tetraploid bbaa u triticum aestivum, l. astella hexaploid bbaa u dd triticum spelta, l. brun 5/9 hexaploid bbaa u dd a accession number in the gene bank of the slovak republic 2.2. protein extraction crude protein extracts were isolated from the leaves using an extraction buffer that contained 0.1 mol/dm3 sodium acetate (ph 5.0) and 0.02 % (v/v) β-mercaptoethanol according to the protocol described previously (žur et al., 2013). protein concentration was determined spectrophotometrically. 2.3. detection of β-l,3-glucanase activities in the gel protein extracts (30 µg) were separated on 12.5 % (w/v) sds-containing polyacrylamide slab gels (laemmli, 1970) with 2.5 % (w/v) laminarin from laminaria digitata (sigma l-9634), as an enzyme substrate. the gels were run at 8 oc at a constant voltage of 120 v for 2 h. after electrophoresis, proteins were re-naturated by shaking the gel in 50 mmol/dm3 sodium acetate buffer (ph 5.0), 1 % (v/v) triton x-100 for 1 hour. separation of proteins under native conditions (for acidic/neutral or basic/neutral proteins, separately) was performed according to konotop et al. (2012) using 11 % (w/v) acrylamide gels with 2.5 % (w/v) laminarin. the β-l,3-glucanase activity was visualised by boiling the gel for 10 min in 1 mol/dm3 naoh containing 0.1 % (w/v) 2,3,5-triphenyltetrazolium chloride according to the method of pan et al. (1991). after enzyme activity detection, the gels were stained with coomassie brilliant blue r 250. 2.4. β-l,3-glucanase quantitative assays β-l,3-glucanase activities were assayed spectrophotometrically with laminarin as a substrate (sigma l-9634) using dinitrosalicylic acid (dns) method (miller, 1959). the absorbance was measured at 500 nm (ultra spect 100, pharmacia biotech). the enzyme activity was expressed in nmol as an amount of released bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:47 utc 125 moravčíková, j. et al. reducing sugar (d-glucose) per hour per milligram of soluble proteins. all data are the means of three replications. statistical significance of the experimental results was evaluated by anova/manova and duncan’s tests, using statistica® ver. 7.1. 3. results and discussion plant β-l,3-glucanases are abundant and highly regulated enzymes that are commonly found throughout the plant kingdom (leubner-metzgner, 2003). so far, these enzymes were studied mainly for their anti-pathogenic functions (minic, 2008) and for their role in reversible deposition of (1,3) β-d-glucans found in the cellwall sheath surrounding plasmodesmata orifices during plant response to stress (levy et al., 2007). their role in plant growth and developmental processes has been also proven (morohashi and matshushima, 2000; leubner-metzgner, 2003; ruan et al., 2004). however, they were mainly studied in dicot plants such as arabidopsis (doxey et al., 2007), tobacco (leubner-metzgner, 2003), soybean (jin et al., 1999) or tomato (morohashi and matshushima, 2000). relatively little is known about β-1,3-glucanases in wheat. so far, the uniprot/ncbi databases contain up to 11 characterised sequences concerning β-1,3-glucanases only in the hexaploid t. aestivum (table 2). moreover, β-1,3-glucanases were studied mainly for their induction upon fungal infection. in this work we aimed to study the activities of β-1,3-glucanases in wheat and crop relatives species with different genome background (table 1). the seedlings of two wheat and six crop relatives species were grown under controlled conditions up to two leaf stage. subsequently, the leaves were collected and subjected to the qualitative and quantitative activity assays of the β-l,3-glucanases. the activities were evaluated based on the ability of plant β-l,3-glucanases to hydrolyse laminarin, a linear (1,3)-β-d-glucan with a low degree of glucosyl substitution at 0-6. even though, there are available other (1,3)-β-d-glucans such as curdlan or pachyman (hrmova and fincher, 1993), laminarin was found to be the most suitable substrate for plant β-l,3-glucanases (pan et al., 1991; hrmova and fincher, 1993; pirselova et al., 2011; žur et al., 2013; gregorova et al., 2015). the total β-l,3-glucanase activity detected was variable. data are summarised in fig. 1. the highest enzyme activity was detected for genotypes ae. tauschii (4.47 nmol d-glucose/h/mg) and t. spelta (4.11 nmol d-glucose/h/mg) while the lowest activity was detected for ae. cylindrica (1.82 nmol d-glucose/h/mg). the β-l,3-glucanase activity was shown to be genotype dependent (at p ≤ 0.001) (fig. 1a, table 2). however, no significant differences (at p ≤ 0.05) between di-, tetra and hexaploid genotypes were observed (fig. 1b). the enzyme activities may be related to diversification of wheat due to domestication, natural hybridization and allopolyploid speciation. for example, the genome of hexaploid t. aestivum is composed of three closely-related and independently maintained genomes as a result of hybridization of tetraploid t. turgidum dicoccum or emmer wheat (genome aabb) with ae. tauschii (genome dd) (matsuoka, 2011). this supports also the fact that even within the group of tetraploid cultivars significant differences (at p ≤ 0.01) in β-l,3-glucanase activities were detected (table 2). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:47 utc nova biotechnologica et chimica 15-2 (2016) 126 t ab le 2 . β1, 3gl uc an as es in t . a es tiv um l . a nd th ei r c ha ra ct er is at io n ba se d on th e d at a in th e u n ip r o t a n c b i d at ab as es (j ul y 20 16 ). u n ip r o t / n am e a a m w d n a d es cr ip ti on /f un ct io n l it er at ur e n c b i [ kd a] [b p] p5 24 09 /   β -1 ,3 -g lu ca na se (g lc 1) 46 1 48 .9 2 07 2 a bi ot ic s tr es s (a l3 + ) c r u z -o r t e g a e t a l., 1 99 7 u 30 32 3 q 1e r f8 / e nd oβ1, 3gl uc an as e 3 32 3 5. 0 1 15 0 a bu nd an t i n he al th y le av es h ig a -n is h iy a m a e t a l., 2 00 6 a b 24 46 41 (t ag lb 2e ) q 1e r f9 / e nd oβ1, 3gl uc an as e 3 36 3 5. 4 1 09 3 a bu nd an t i n he al th y sp ik es h ig a -n is h iy a m a e t a l., 2 00 6 a b 24 46 40 (t ag lb 2d ) q 1e r g 0/ e nd oβ1, 3gl uc an as e 3 36 3 5. 5 1 08 9 a bu nd an t i n he al th y sp ik es h ig a -n is h iy a m a e t a l., 2 00 6 a b 24 46 39 (t ag lb 2c ) q 1e r f7 / e nd oβ1, 3gl uc an as e 3 42 3 6. 1 1 18 1 a bu nd an t i n le av es h ig a -n is h iy a m a e t a l., 2 00 6 a b 24 46 42 (t ag lb 2f ) q 1e r g 1/ e nd oβ1, 3gl uc an as e 3 40 3 5. 9 1 43 8 b io tic s tr es s h ig a -n is h iy a m a e t a l., 2 00 6 a b 24 46 38 (t ag lb 2b ) ( e ry si ph e gr am in is , f us ar iu m g ra m in ea ru m ) b 5a 7b 8/ β1, 3gl uc an as e (g lc 2) 3 99 44 .2 2 15 2 – – e u 81 69 11 q 9x e n 7/ β -1 ,3 -g lu ca na se (g lb 3) 3 34 3 4. 7 1 43 9 b io tic s tr es s l i e t a l., 2 00 1 a f1 12 96 7 ( f us ar iu m g ra m in ea ru m ) q 9x e n 5/ β -1 ,3 -g lu ca na se (g lb 3) 3 34 3 4. 9 1 26 9 b io tic s tr es s l i e t a l., 2 00 1 a f1 12 96 5 (f us ar iu m g ra m in ea ru m ) q 4j h 28 / β -1 ,3 -g lu ca na se 33 4 35 .4 1 33 8 b io tic s tr es s – d q 09 09 46 (p uc ci ni a st ri ifo rm is ) q 4j k 90 / β -1 ,3 -g lu ca na se 33 4 3 5. 4 1 33 7 b io tic s tr es s – d q 07 82 55 ( p uc ci ni a st ri ifo rm is ) a a – a m in o ac id s; m w – m ol ec ul ar w ei gh t bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:47 utc 127 moravčíková, j. et al. fig. 1. total β-1,3-glucanase activities in wheat and crop relatives species (a) and in dependence on ploidy level (b). the enzyme activities were assayed spectrophotometrically using laminarin as a substrate. the activity was expressed in nmol of d-glucose released per hour per mg of soluble proteins. bars represent means ± standard deviations of three replications. distinct letters denote statistically significant differences with duncan’s test at p ≤ 0.001, ns – not significant at p ≤ 0.05. the total β-l,3-glucanase activities measured can comprise several glucanase isoforms (fig. 2, table 3). we identified a single glucanase fraction of a ~52 kda in all analysed genotypes (fig. 2b). in literature, wheat β-l,3-glucanases were studied mainly in the context of a/biotic stresses (li et al. 2001; higa-nishiyama et al., 2006; pirselova et al., 2011; gregorova et al., 2015). thus, only data from non-stressed wheat plants might be used for comparison. besides, these experiments were performed mainly on bread wheat t. aestivum, moreover, at different stage of growth. for example, gregorova et al. (2015) observed up to four glucanase isoforms of sizes ranging from ~30 kda to ~150 kda in the breeding line sk-196 at growth stage after tillering. similarly, western blot analyses performed on the control plants of the cv. bobwhite (t. aestivum l.) revealed three glucanases bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:47 utc nova biotechnologica et chimica 15-2 (2016) 128 of molecular weight ranging from ~30 kda to ~60 kda (jayaraj et al., 2004). a higher number of the glucanase isoforms in latter stages of wheat plant might coincide with reversible callose deposition and degradation growth (jayaraj et al., 2004; gregorova et al., 2015). ruan et al. (2004) showed the expression of the fibre-specific β-1,3-glucanase gene ghgluc1 was undetectable when callose was deposited but became evident at the time of callose degradation. they suggested that callose deposition and degradation might correlate with timing of plasmodesmata closure and reopening that is important for division, growth and differentiation of plant cells. table 2. variance analyses. β-1,3-glucanases vs. f empiricala genotype 10.51*** ploidy level 1.01 ns teptraploid species 14.07** a statistical significance at *** p ≤ 0.001; ** p ≤ 0.01, * p ≤ 0.05 fig. 2. detection of β-1,3-glucanase activities after separation of crude protein extracts in sds-page (b); and under native conditions in page for acidic/neutral (c); and basic/neutral proteins (d). separated proteins were visualised with coomassie brilliant blue r 250 (a). numbers on the left refer to the molecular mass marker. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:47 utc 129 moravčíková, j. et al. the glucanase fraction of a ~52 kda can comprise up to five acidic (glua-glue) (fig. 2c) and a single basic (gluf) isoforms (fig. 2d) (table 3). neither of the analysed genotypes contained the all five acidic isoforms. in most of them, a combination of two acidic isoforms was found out. however, none of the acidic isoforms can be associated with ploidy level. a single basic gluf was detected in the all analysed genotypes (table 3). based on the classification by stintzi et al. (1993) the acidic isoforms are considered as extracellular while basic as vacuolar. however, several authors pointed out that such classification cannot be fully applied for wheat. analyses performed on apoplastic fluid of wheat leaves revealed accumulation of not only acidic but also basic glucanase fractions (van der westhuizen et al., 1998). our results (fig. 1, fig. 2) proved that β-1,3-glucanases are accumulated in the leaves of all studied genotypes. the genotype has significant effect on β-1,3-glucanase activities. however, no significant differences between ditetra and hexaploid genotypes were observed. since, the β-1,3-glucanases might be involved in defence against pathogens, the role of individual β-1,3-glucanase isoforms during biotic stress might be in future evaluated for its use in stress marker assistance breeding or biotechnology programmes. table 3. overview of the β-1,3-glucanase activities in wheat and crop relatives species. β-1,3-glucanase isoform totala acidic/neutralb basic/neutralc glu1 [~52kda] glua glub gluc glud glue gluf ae. tauschii + + + + t. monococcum l. + + + + t. araraticum + + + t. dicoccum + + + ae. cylindrica + + + ae. triuncialis + + + + t. aestivum l. + + + + t. spelta l. + + + + a size of the fraction detected in the gel after re-naturation of separated proteins in the sds-page (fig. 2b) b fractions detected in the gel after separation of the proteins in the page under conditions for acidic/neutral proteins (fig. 2c) c fractions detected in the gel after separation of the proteins in the page under conditions for acidic/neutral proteins (fig. 2d) +/presence/absence of fractions with β-1,3-glucanase activities 4. conclusions the activities of β-1,3-glucanases in the leaves of two diploids (ae.s tauschii, t. monococcum l.), four tetraploids (ae. cylindrica, ae. triuncialis, t. araraticum, t. dicoccum) and two hexaploids (t. aestivum l., t. spelta l.) were studied. the total β-1,3-glucanase activities were variable and genotype dependent. we did not observe significant correlation between β-1,3-glucanase activities and ploidy level. the gel activity assays revealed a single ~52 kda glu1 fraction that was found in all bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:47 utc nova biotechnologica et chimica 15-2 (2016) 130 genotypes. the glu1 isoform comprised a single or a combination of two acidic glu isoforms in dependence on genotypes. neither of the acidic glu isoforms could be assigned as specific for di-, tetraor hexaploid genotypes. the basic gluf isoform was present in all genotypes. a large variability in enzyme activities might be a result of genome evolution. due to the involvement of β-1,3-glucanases in plant defence, the profile of the activities of these enzymes might be indicative for plant performance in various environmental conditions, thus it deserves further investigations for their use in biotechnology and breeding programmes. acknowledgment: this work was supported by the bilateral project apvv sk-pl-2015-044, by the project apvv-15-0051 and by the university grant agency ukf uga viii/35/16. references chen, z.j: genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids. annu. review plant biol., 58, 2007, 377-406. doxey, a.c., yaish, m.w.f., moffatt, b.a., griffith, m., mcconkey, b.j.: functional divergence in the arabidopsis beta-1,3-glucanase gene family inferred by phylogenetic reconstruction of expression states. mol. biol. evol., 24, 2007, 1045-1055. gregorova, z., kovacik, j., klejdus, b. , maglovski, m., kuna, r., hauptvogel, p., matusikova, i.: drought-induced responses of physiology, metabolites, and pr proteins in triticum aestivum. j. agr. food chem., 63, 2015, 8125-8133. havrlentova, m., kraic, j.: content of beta-d-glucan in cereal grains. j. food nutr. res., 45, 2006, 97-103. higa-nishiyama, a., ohsato, s., banno, s., woo, s. h., fujimura, m., yamaguchi, i., kimura, m.: 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role in defense against pathogens. biochimie, 75, 1993, 687-706. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:47 utc nova biotechnologica et chimica 15-2 (2016) 132 van der westhuizen, a.j., qian, x.m., botha, a.m.: beta-1,3-glucanases in wheat and resistance to the russian wheat aphid. physiol. plant., 103, 1998, 125-131. zavaliev, r., ueki, s., epel, b.l., citovsky, v.: biology of callose (beta1,3-glucan) turnover at plasmodesmata. protoplasma, 248, 2011, 117-130. žur, i., golębiowska, g., dubas, e., golemiec, a., matusikova, i., libantova, j., moravcikova, j.: β-1,3-glucanase and chitinase activities in winter triticales during cold hardening and subsequent infection by microdochium nivale. biologia, 68(2), 2013, 241-248. received 12 november 2016 accepted 2 december 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:47 utc 182 nova biotechnologica et chimica 13-2 (2014) doi 10.1515/nbec-2015-0008  university of ss. cyril and methodius in trnava prediction of wine sensorial quality by routinely measured chemical properties adriána bednárová1, roman kranvogl2, darinka brodnjak-vončina2, tjaša jug3 1department of chemistry, faculty of natural sciences, university of ss cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic (adriana.bednarova@ucm.sk) 2 faculty of chemistry and chemical engineering, university of maribor, smetanova 17, 2000 maribor, slovenia 3 chamber of agriculture and forestry of slovenia, institute for agriculture and forestry, pri hrastu 18, 5000 nova gorica, slovenia abstract: the determination of the sensorial quality of wines is of great interest for wine consumers and producers since it declares the quality in most of the cases. the sensorial assays carried out by a group of experts are time-consuming and expensive especially when dealing with large batches of wines. therefore, an attempt was made to assess the possibility of estimating the wine sensorial quality with using routinely measured chemical descriptors as predictors. for this purpose, 131 slovenian red wine samples of different varieties and years of production were analysed and correlation and principal component analysis were applied to find inter-relations between the studied oenological descriptors. the method of artificial neural networks (anns) was utilised as the prediction tool for estimating overall sensorial quality of red wines. each model was rigorously validated and sensitivity analysis was applied as a method for selecting the most important predictors. consequently, acceptable results were obtained, when data representing only one year of production were included in the analysis. in this case, the coefficient of determination (r2) associated with training data was 0.95 and that for validation data was 0.90. when estimating sensorial quality in categorical form, 94 % and 85 % of correctly classified samples were achieved for training and validation subset, respectively. key words: overall sensorial quality, prediction, slovenian wine, artificial neural networks, multivariate data analysis 1. introduction wine is a complex mixture of organic as well as inorganic compounds determining the final organoleptic properties of wine. the factors influencing the quality of wine are related to winemaking environment including ground, climate and the variety of the employed oenological practice (pohl, 2007). there are numerous studies showing the connection of chemical compounds, especially phenolic and volatile as well as some non-volatile compounds with human perception (sáenz-navajas et al., 2012; lorrain et al., 2013). sensory analysis is the most common method of assessing the biological characteristics of foodstuffs and therefore its quality. typical tests to estimate wine quality rely on sensorial assays carried out by a group of experts. although these methods are probably the best option for evaluating a small number of samples, they are time-consuming and expensive since an extensive training of panellists is necessary for reproducible results and even the trained panel can perform only a limited number of analyses per day due to fatigue or environmental bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc nova biotechnologica et chimica 13-2 (2014) 183 interferences. successful application of conventional analytical techniques to the evaluation of sensory properties of foodstuffs is very difficult, primarily because the relationship between chemical composition and flavour is not properly known and also due to high complexity and variability of food composition (legin et al., 2003). though, it was reported that the global signal provided by sophisticated analytical methods can be considered the fingerprint of sample flavour and used as the analytical signal for characterising the commodity, especially, when chemometrics is used to obtain the most detailed information from the sample (lópez-feria et al., 2008). in addition, using equipment such as electronic nose and electronic tongue connected with appropriate predictive modelling techniques could improve the performance of estimating sensory properties (legin et al., 2003; buratti et al., 2007). methods of multivariate data analysis (mva) are frequently used for investigation of relations and interactions inside a large data table. for wine classification the mva methods are especially useful when different attributes of wine authenticity are predicted utilising various types of analytes (arvanitoyannis et al., 1999; cozzolino et al., 2009; saurina, 2010; kruzlicova et al., 2013). however, since institutions controlling wine quality or wineries do not commonly possess modern analytical equipment, it would be interesting to assess the feasibility of estimating the sensorial quality of wine only by simple chemical properties such as alcoholic grade, density, ph, content of extract or so2, that are routinely measured at the mentioned institutions or wineries. thus, the goal of present study was to examine the possibility of predicting the overall sensorial quality of slovenian red wines based on the results of routinely chemical analyses performed in the control institution. for this purpose, artificial neural networks (anns), specifically multilayer perceptron, was utilised. in addition, the interrelations among the studied oenological descriptors were discovered in detail. 2. materials and methods 2.1 wine samples and analytical procedures altogether 131 red wine samples originated in primorska winegrowing region in slovenia were analysed during five years of production (2002 – 2006). more specifically, the analysed wine samples represented varieties such as cabernet sauvignon (51 samples), merlot (46 samples), and a blend of several red wines varieties (34 samples). sampling was made by the chamber of agriculture and forestry of slovenia in nova gorica. the following wine descriptors were determined: relative density at 20 °c, contents of total extract (g/dm3), reducing sugars (g/dm3), ash (g/dm3), free so2 (mg/dm 3), ethanol (%), total acidity (g/dm3), volatile acidity (g/dm3), and ph. in addition, the sensorial quality was obtained by group of experts evaluating the wine properties (colour, aroma, taste and harmony) using a twenty point scale in total. all analytical methods were accomplished according to official gazette republic of slovenia no. 43/01, and the sensorial analysis was performed according to official gazette republic of slovenia no. 32/00. sensory evaluation was performed in a group of 5 experts, representatives of consumers, producers and wine experts. they bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc 184 bednárová, a. et al. were allowed to evaluate up to 40 samples per day. in every evaluation they give up to 2 points for clarity, 2 for colour, up to 4 for odour, up to 6 for taste and 6 for harmony. the winescan ft 120 instrument utilising ftir was employed for simultaneous determination of mentioned wine descriptors. the analytical procedures are described in detail in (bednárová et al., 2013). 2.2 statistical analyses in addition to the mentioned oenological characteristics, i.e. relative density (denoted as density), total extract (extract), reducing sugars (redsug), ash (ash), free so2 (so2free), ethanol (ethanol), total acidity (totacid), volatile acidity (volac), and ph (ph), further descriptors, namely non-volatile acidity (g/dm3) and reduced extract (g/dm3) were calculated as follows: non-volatile acidity (nvolac) = totacid – volac reduced extract (sfe) = extract – (redsug – 1) furthermore, ratios volac/totacid (va_ta) and nvolac/totacid (nva_ta) were added to the list of analysed variables. after performing exploratory analysis of the available data, i.e. checking normality and presence of outliers, the interrelations among all oenological descriptors were discovered by correlation analysis (ca) and principal component analysis (pca). however, the main regard was taken on the significant correlations of the target variable – overall sensorial quality (denoted as quality) with the studied oenological descriptors. secondly, the target variable quality was transformed into categorical form and nonparametric tests were applied to perform detailed insight to the connection of sensorial quality with wine chemical descriptors. the main objective of this work was to discover the possibility of estimating the wine sensorial quality by routinely measured chemical properties. for this purpose, two approaches were used: (1) classification of samples into three created groups of quality_cat by artificial neural networks (anns) and linear discriminant analysis (lda) and (2) prediction of the continual target variable quality by anns. in all model building procedures, a method for input variables selection was performed as well as proper validation of the created model. to assess the predictive ability (predictivity) of the model, i.e. the measure of how well the model can predict values of new data, the data not utilised in model calculations were employed. external validation, when performed judiciously, is generally regarded as the most rigorous assessment of predictivity, since predictions are made for samples not used in the model development in comparison to e.g. cross-validation methods. the results of the predictions were evaluated by the coefficient of determination (r2) between the actual and predicted values of quality and root mean square error (rmse) as a measure of the difference between values predicted by a model and the values actually observed. the rmse of a model prediction with respect to the estimated variable xmodel is defined as the square root of the mean squared error: n )x(x =rmse imodel,iobs,  2 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc nova biotechnologica et chimica 13-2 (2014) 185 where xobs is set of observed values and xmodel is set of modelled values at time/place i. regarding the classification of wine samples according to the quality_cat, the success of classification was evaluated by the percentage of correctly classified objects into the categories. 2.3 ann model optimisation learning the input/output relationship through the training process is the key feature of artificial neural networks (zupan and gasteiger, 1993; bishop, 1995; haykin, 1999). a prescribed set of well-defined rules for the solution of a learning problem is called a learning algorithm. in our case, the bfgs (broydenfletcher-goldfarb-shanno) algorithm was applied as one of the most recommended techniques in neural networks. ann models have been built by employing the automated network search included in the statistica software (statsoft, tulsa, usa) where different network architectures were automatically tested and the best alternatives were determined. to verify the generalisation (prediction) of a model, the validation data (subset of samples not used in model calculation) were applied to demonstrate whether the input-output relationship computed by network utilising training data was correct. the complexity of the model in terms of the selection of the input (predictor) variables was examined with the aid of so-called sensitivity analysis indicating the importance of input variables by particular neural network (hunter et al., 2000). thus, the results of sensitivity analysis were used for informative purposes, but also to perform input pruning. also, the number of neurons in the hidden layer was optimised due to avoiding the over-training the network. therefore, a third subset of data was selected for computing prediction error periodically during training to avoid over-training. consequently, together three subsets of data were created by random dividing the whole data set into three parts: (1) training subset, (2) test subset used to determine if over-training has occurred, and (3) external validation subset for evaluation of the predictivity of the model. 3. results and discussion 3.1. correlation analysis the direct inter-relations between the studied descriptors and the target overall sensorial quality were analysed by the correlation analysis. moreover, the information about the correlations among all descriptors was important in the further model building part, especially in the step of selection of input variables subset. all data with exception of two outliers were included (n = 129) in the correlation analysis, containing all varieties and all vintages. since departures from normal distribution were identified by shapiro-wilk test (p < 0.05) in case of several studied variables, the spearman correlation analysis was employed as a non-parametric alternative to pearson correlation (miller and miller, 2010). another detail of importance due to a large size of data (129 samples) also relatively small values of the correlation coefficient meant a statistically significant correlation. for the sake of differentiation, a blue colour is used for the highest significant correlations (p < 10-6) in table 1. the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc 186 bednárová, a. et al. less important correlations are marked in black. as expected, the target variable quality correlated highly to ethanol (rs = 0.42) as shown in table 1. in addition, positive correlations of quality with sfe, extract, ph as well as volac and negative correlations of the target variable with totacid and nvolac were found. this means, that high content of non-volatile acids could affect decreasing the overall sensorial quality of wine, which is in agreement with oenological practice: wines with higher acidity are usually rougher. on the other hand, great level of volatile acids and especially of ethanol indicates high wine quality. table 1. correlation table with spearman correlation coefficients (n = 129). statistically significant (p < 0.05) correlation coefficients are bold; highly significant (p < 10-6) correlation coefficients are denoted by blue colour. d en si ty e th an ol e xt ra ct to ta ci d n v ol a c v ol a c a sh so 2f re e r ed su g sf e ph ethanol -0.38 extract 0.71 0.31 totacid 0.32 -0.21 0.13 nvolac 0.40 -0.30 0.14 0.94 volac -0.32 0.41 -0.04 -0.25 -0.50 ash 0.04 0.07 0.16 -0.43 -0.47 0.15 so2free 0.25 -0.10 0.17 -0.22 -0.17 -0.13 0.13 redsug 0.43 0.21 0.59 0.01 0.03 0.11 -0.03 0.13 sfe 0.68 0.29 0.95 0.18 0.18 -0.06 0.18 0.14 0.38 ph -0.17 0.19 0.00 -0.71 -0.71 0.22 0.67 0.06 -0.19 0.03 quality -0.03 0.42 0.28 -0.31 -0.30 0.22 0.10 0.00 0.12 0.28 0.22 the highly significant correlations between variables totacid and nvolac (0.94) as well as extract and sfe (0.95) corresponded with fact, that variables nvolac and sfe were calculated from measured descriptors totacid and extract, respectively. thus, correlations of totacid and nvolac as well as other linearly dependent variables to other descriptors are analogous. further significant correlations were proved; for instance, the correlation of ph with totacid and nvolac (-0.71) was logical. however, the significant positive correlation of ph with ash (0.67) was not expected, although higher mineral content means more cations ant therefore higher ph. additionally, ethanol negatively correlated with density (-0.38), nvolac (-0.30), totacid (-0.21) and positively with volac (0.41), ph (0.19) and a group of mutually positively correlated variables extract, sfe and redsug. in the study (sáenz-navajas et al., 2012), similar conventional oenological descriptors of red wines were determined and their correlations between the attributes of in-mouth sensory perception evaluated. the authors found out that the titratable acidity positively correlated with the perceived acidity of wine, and the ethanol content was an active contributor to the astringency perception. interestingly, the content of reducing sugars was not significantly correlated with sensory sweetness. however, the correlations of total sensorial quality were not included in the mentioned study. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc nova biotechnologica et chimica 13-2 (2014) 187 3.2. nonparametric tests an important issue in this research was also the determination of factors affecting the variability of data. it is well known, that different wine varieties could be distinguished by their different chemical properties (beltrán et al., 2006; câmara et al., 2006; kruzlicova et al., 2009). similarly, the vintage, i.e. the year of production as a factor covering especially the climatic conditions of wine production, contributes significantly to the differentiation of the wine chemical composition and hence also sensory characteristics. indeed, according to the kruskalwallis test, statistically significant differences (p < 0.01) among categories of vintage used as a factor were found in case of descriptors ethanol, volac, sfe, extract and more importantly, also in case of target variable quality. when testing variety as a factor, statistically significant differences between varieties were proved only by ash and ph. however, the aim of this paper was to build a model capable to predict the overall sensorial quality with minimum limitation of domain of application, i.e. including data originated from various wine varieties and vintages. furthermore, non-parametric tests were applied to see the differences in oenological descriptors when three categories of wine samples were built according to their quality value: (1) premium quality wines with quality value greater than 18.0 (n = 45); (2) quality wines with quality greater than 17.0 but less than or equal to 18.0 (n = 45) and (3) quality and country wines with quality less than or equal to 17.0 (n = 39). the results are summarised in table 2. to examine statistically significant differences between particular categories, mann-whitney test was performed. table 2. summary of investigated oenological descriptors with the median values for each category of quality_cat. the statistically significant results of kruskal-wallis test are denoted bold. category of quality_cat / category code descriptor p-value 1/ a 2/ b 3/ c ethanol 5.10-07 12.8 b,c 12.1 a 12.2 a density 0.665 0.994 0.994 0.994 totacid 0.001 5.47 c 5.67 c 6.18 a,b nvolac 0.003 4.75 c 5.05 c 5.48 a,b volac 0.044 0.590 b 0.470 a 0.530 ph 0.086 3.49 c 3.49 3.46 a ash 0.011 2.65 2.79 c 2.61b extract 0.012 27.6 b,c 25.8 a 26.1a redsug 0.527 1.90 1.90 1.70 sfe 0.006 26.6 b,c 24.6 a 25.6 a so2free 0.79 24.0 26.0 26.0 nva_ta 0.015 0.863 b,c 0.890 a 0.893 a va_ta 0.017 0.110 b,c 0.089 a 0.085 a descriptor units are introduced in materials and methods. a,b,c the median value of the given category is significantly different from the mean value of the coded category (tested by mann-whitney test). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc 188 bednárová, a. et al. our results differed from the study (šnuderl et al., 2009) where analysis of variance was used for discovering significant differences between three groups of sensorial quality. statistically significant differences were proved only in case of ash content and density in the mentioned study. however, the disagreement with our study was obvious since different varieties were considered. 3.2. principal component analysis principal component analysis (pca) is the most widely used multivariate data analysis method for transforming the original measurement variables into new, uncorrelated variables called principal components (varmuza and filzmoser, 2009). using this procedure, a set of orthogonal axes that represents the direction of greatest variance in the data is found (gemperline, 2006). usually, only two or three principal components are necessary to explain a significant fraction of the information present in multivariate data when the original (measurement) variables are inter-correlated. in addition, pca graphical output provides displaying inter-relations among original variables in the space of newly calculated pcs together with detecting possible natural grouping of samples. the first two pcs calculated from the studied descriptors accounted for 56.61 % of the total data variability. as it was obvious in the biplot (fig. 1), the target variable quality was closely located to descriptors ash, ethanol, ph and volac representing their positive correlation. fig. 1. biplot for all red wine samples labelled by numbers according to their variety (1 – cabernet sauvignon, 2 – mixture of varieties, 3 – merlot) and by colour according to their year of production (yellow – 2002, red – 2003, blue – 2004, black – 2005, green – 2006). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc nova biotechnologica et chimica 13-2 (2014) 189 on the contrary, quality was oppositely situated to adjacent descriptors nvolac and totacid, i.e. quality was in negative and significant correlation to them. the mutual position of the studied descriptors was in good agreement with the results of the correlation analysis. in addition, the objects (wine samples) labelled according to their variety and vintage were mixed in the plane of pc 2 vs. pc 1 (fig. 1). this means that no natural grouping of samples according to their variety and vintage occurred. though, it is worth noting that the right half of graph (related to the highest positive values of pc 1) contained mostly objects representing only one vintage (2005). on the contrary, objects representing all other vintages were located mostly by negative values of pc 1. this is important due to the distribution of samples in relation to the axis pc 1 representing the original descriptors nvolac, totacid, ph, ash, ethanol and quality and volac following their loadings. accordingly, wine samples with high values of nvolac and totacid and lower values of ph, ash, ethanol and quality originated mostly from vintage 2005. this agreed with results of kruskal-wallis test proving the statistically significant differences in vintage categories for variable quality. 3.3. prediction of sensorial quality using multilayer perceptron artificial neural networks (anns) do not assume any initial mathematical relationship between the input and output variables, so they are particularly useful when the underlying mathematical model is unknown or uncertain (miller and miller, 2010). finding the proper network design (number of layers and neurons) is usually a trial and error procedure (zupan and gasteiger, 1993). nevertheless, the key decision on the number of hidden layers and neurons was accomplished by automated network search (ans) involved in software statistica, which was helpful for creating a variety of different network architectures and for choosing the network with the best performance. it was found in all cases that the best network type was the three-layer perceptron (3-mlp), i.e. the architecture with one hidden layer. the optimal number of hidden neurons was sought by examining several types of the 3-mlp with regard to the performance of the network. to avoid over-training of the network, a subset of data selected for monitoring the error periodically during the training was used (so-called test subset). thus, the whole data set was randomly divided into three subsets: (1) training (65 % of data), (2) test (10 %) and (3) validation data (25 %) which were not included in the training and utilised to assess the predictive (generalisation) ability of developed model. according to the sensitivity analysis, the predictors (input variables) with repeatedly low value of sensitivity were sequentially removed from the set of inputs. firstly, all data were included in analyses (n =129) and only oenological descriptors were utilised as continual input variables. after the selection of predictors, the optimum architecture for networks utilising eleven inputs (sfe, extract, totacid, nvolac, ph, va_ta, so2free, ethanol, ash, volac and nva_ta) contained six hidden units. the predictive ability decreased gradually when more neurons were added, i.e. with higher number of hidden neurons the network became over-trained. on the other hand, when less hidden neurons were included in the model calculation, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc 190 bednárová, a. et al. the performance of model decreased considerably. the criterion used to select the adequate ann model consisted of selecting the number of neurons, which gave a minimum final error in a minimal number of iterations during the training phase. the best performance was obtained when a network utilised the bfgs algorithm for 63 epochs and when the hyperbolic tangent in the hidden layer and logistic function in the output layer were used as activation functions. however, after optimisation of number of input and hidden neurons, the final performances of networks achieved only unsatisfactory results – the maximum value of coefficient of determination (r2) related to training data was 0.64 and that for validation data was 0.42. table 3. the results of comparing predicted vs. actual values of quality for selected 3-mlp models in case of all samples (n = 129) and samples representing only one vintage (n = 66) with using different inputs. r2 rmse number of objects input variables t ra in in g t es t v al id at io n t ra in in g t es t v al id at io n 129 sfe, extract, totacid, nvolac, ph, va_ta, so2free, ethanol, ash, volac, nva_ta 0.637 0.514 0.421 0.430 0.582 0.686 129 vintage, variety, totacid, nvolac, sfe, nva_ta, volac, ph, ethanol, ash, va_ta, so2free, extract, density 0.830 0.671 0.616 0.294 0.475 0.561 66 nvolac, totacid, ash, so2free, ethanol, redsug, sfe, ph, nva_ta, density, volac 0.947 0.936 0.901 0.186 0.274 0.229 note: the input variables are ordered by decreasing importance according to the sensitivity analysis to improve the performance of neural networks, factors vintage and variety were included in analyses as categorical input variables. this led to decreasing error in computations and consequently to improving results (table 3). the bfgs algorithm for 37 epochs was selected as optimum with using hyperbolic tangent and logistic function as activation functions in hidden and output layer, respectively. six hidden neurons were selected to get acceptable training and testing error. the resulting sensitivity values indicated the importance of predictors in following order: vintage, variety, totacid, nvolac, sfe, nva_ta, volac, ph, ethanol, ash, va_ta, so2free, extract and density. this means that factors vintage and variety significantly influenced the recognition and extraction of data information for quality prediction. the results for comparing predicted vs. actual target descriptor quality are summarised in table 3 showing increased values of r2 and decreasing rmse values in all data subsets. furthermore, as it was found by the kruskal-wallis test (section 3.2.), there were proved statistically significant differences between categories of factor vintage regarding few oenological descriptors as well as the target descriptor quality. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc nova biotechnologica et chimica 13-2 (2014) 191 accordingly, the next step was to discover the possibility of predicting the overall sensorial quality when only samples originated from one year of production were involved in the analysis. since the highest number of samples was obtained in vintage 2005 (n = 66), further model building was accomplished with data characterising only wine samples originated from 2005. in this case, the random distribution of objects into training, test and validation subsets was following: (1) 70 % of data used as training subset, (2) 10 % as test subset and (3) 20 % of data for validation. the results markedly improved as is shown in table 3. the best 3-mlp contained 6 hidden neurons and again, hyperbolic tangent in hidden and logistic function in output layer as activation functions, and bfgs algorithm for 57 epochs were employed. the performance of the best ann model is presented in fig. 2. fig. 2. correlation between the predicted and actual values of quality for the 3-mlp achieving the best performance. the objects characterising wine samples originating only from vintage 2005 are divided into three subsets: (1) 47 objects used as training subset, labelled by blue squares (□), (2) 6 samples (○) used as test subset and (3) 13 samples (∆) used for external validation. accordingly, the routinely measured chemical descriptors are strongly connected with the year of wine production and adding data representing more vintages makes the predictions more demanding. to compare the predictive performance of the developed ann models, other studies dealing with estimating sensorial characteristics are briefly discussed here. for instance, the overall sensory quality of 15 wine samples was estimated by means of measurements based on electronic nose, amperometric electronic tongue and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc 192 bednárová, a. et al. spectrophotometric determinations in the study of (buratti et al., 2007) where genetic algorithms were utilised for variables selection. consequently, the response of carbon electrode, contents of total flavonoids, non-anthocyanin flavonoids and total anthocyanins turned out to be the most important and resulted in predictive power of 66 % determined by bootstrap procedure. furthermore, different parameters of taste and flavour of wine were predicted by back-propagation neural network in the work of (legin et al., 2003) on the basis of measurements by electronic tongue. the mean relative error was within 4 – 27 % for external validation data. in the study of (sáenz-navajas et al., 2012), the sensory astringency was predicted by partial least squares models for 34 red wine samples. among different non-volatile compounds as well as other analytes, only few were selected the most important, such as contents of ethanol, protocatechuic acid ethyl ester, protein-precipitable proanthocyanidins, trans-p-coumaric, cis-aconitic, and cis-caftaric acids. it is noteworthy that the ethanol content was found the most important contributor to the astringency estimation. resulting r2 associated with validation of model was 0.629 and the rmse was 0.441. thus, we could conclude that the predictive ability of routinely measured oenological descriptors for sensorial quality estimation tested in our study was comparable with studies mentioned previously. 3.4. classification according to the sensorial quality furthermore, an alternative approach was applied to find a proper model for prediction of overall wine sensorial quality with using routinely measured oenological descriptors. in this case, the categorical form of target variable – quality_cat was employed as a categorical dependent variable. for this purpose, the wine samples were categorised into three groups according to their quality value: (1) premium quality wines with quality value greater than 18.0 (n = 45); (2) quality wines with quality value greater than 17.0 but less than or equal to 18.0 (n = 45) and (3) quality and country wines with quality less than or equal to 17.0 (n = 39). regarding the distribution of the objects into training, test and validation subsets, similar way was used as in previous analyses when all vintages were included. each mlp was trained using the bfgs (broyden-fletcher-goldfarb-shanno) algorithm for a maximum of 200 training cycles. the networks were optimised against the cross-entropy error function (bishop, 1995) and again, the sensitivity analysis was utilised to eliminate predictors with poor discrimination ability. several configurations (one hidden layer with four to seven neurons) of the ann model network have been tried out to establish the optimum network architecture for the best prediction performance. it was observed that the network predictive ability (performance for the validation subset) decreased with adding more than six neurons of hidden layer due to over-training. the perceptron with best performance achieved considerable results (table 4) when six neurons and hyperbolic tangent as activation function were employed in the hidden layer. similarly as in section 3.3, factors vintage and variety involved as predictors improved the results of classification. in this case, six hidden neurons and logistic activation function in hidden layer were appropriate. in training subset, 79 from 84 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc nova biotechnologica et chimica 13-2 (2014) 193 objects were correctly classified. regarding validation data, 8 objects from 32 were misclassified. table 4. the results of predicting categorical quality_cat for selected 3-mlp models in case of all samples (n = 129) and samples representing only one vintage (n = 66) with using different inputs. correctly classified objects (%) number of objects input variables training test validation 129 sfe, totacid, nvolac, ethanol, density, ash, ph, volac, nva_ta, extract, so2free 86.9 66.7 71.9 129 vintage, variety, nva_ta, ph, nvolac, totacid, volac, sfe, extract, ethanol, ash, so2free 94.1 75.0 75.0 66 nvolac, totacid, ph, density, sfe, va_ta, ethanol, volac, ash, redsug, so2free 93.6 66.6 84.6 note: the input variables are ordered by decreasing importance according to the sensitivity analysis finally, the prediction of quality_cat was performed including only data characterising vintage 2005 (n = 66) in the next step. consequently, significantly higher ann performances were achieved regarding classification results (table 4). in this case, lower number of samples was incorporated and the optimum number of hidden units was 5 with logistic activation function in hidden layer. briefly, 70 % of data were selected as training subset (n = 47), 10 % as test subset (n = 6) and 20 % of data were used for validation (n = 13). the classification success achieved 94 % for training set, i.e. 3 objects were misclassified; and the prediction ability evaluated by classification success for validation subset was 85 %, this means 11 objects from total 13 were correctly classified (table 4). the predictors were selected by sensitivity analysis according to their importance in following order: nvolac, totacid, ph, density, sfe, va_ta, ethanol, volac, ash, redsug and so2free. in addition, linear discriminant analysis (lda) was accomplished to compare the performance of ann with other classification technique. in lda, the highest percentage of correctly classified objects for training set was 71.2 % and 65.2 % for leave-one-out validation. in this classification model, the stepwise selection procedure was applied to optimise the set of input variables and as a result, only four descriptors sufficed, concretely density, ash, ph and totacid. still, the classification success of lda was markedly lower in comparison to results of ann. it should be stressed that the number of papers dealing with the classification of wines according to their overall sensorial quality is limited. nevertheless, in (šnuderl et al., 2009), lda was applied to classify the wine samples into three categories of their sensorial quality. the best classification success (78.3 %) was obtained with nine selected input variables, concretely contents of free and total so2, total extract, reducing sugars, ash, polyphenols, and lactic, tartaric and citric acids. to our knowledge, the application of ann for classification of wine samples according to the sensorial quality was not yet reported. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc 194 bednárová, a. et al. 4. conclusions since the sophisticated and modern analytical equipment is often not available to the most wine producers, the utilisation of routinely determined chemical properties as predictors of wine quality would be of high importance for practical applications. this work showed the possibility of estimating the overall sensorial quality of slovenian red wines by simple oenological descriptors. artificial neural networks (anns), specifically three-layer perceptrons (3-mlps) with the bfgs (broyden-fletchergoldfarb-shanno) algorithm, were employed as prediction tool for achieving considerable results. the exploratory data analysis was accomplished to discover the relations among the studied descriptors and the target sensorial quality using correlation analysis, principal component analysis as well as non-parametric tests. numerous statistically significant correlations were proved and a good agreement amongst the applied computational methods has been observed. the highest positive correlation of sensorial quality was found with content of ethanol and the highest negative correlation with total acidity. the importance of vintage as a factor was also obvious in the prediction performances of developed ann models since considerably higher performances of ann models were reached when only data representing one vintage were included in predictions. two approaches of sensorial quality prediction were performed in the terms of using target variable in continual as well as categorical form. in both, the set of input variables was optimised and the resulting models were tested by external validation subset of data. consequently, the predictive ability of the best 3-mlp model for estimation of sensorial quality in continual form achieved r2 value of 0.62 in case of including all data, and 0.90 when wine samples originated only from one vintage were selected. in addition, the predictive power of 3-mlp models used in classification of wine samples into three categories was 75 % for all data and 85 % for data representing only vintage 2005. accordingly, markedly better results were achieved in case of predictions including only data representing one vintage in comparison of predictions covering data of all vintages. hence, involvement of more data related to wines of different origins in predictions demands more complex characterisation of samples in terms of incorporating various types of analytes in analyses. acknowledgement: the support of slovak grant agency vega 1/0233/12 is highly acknowledged. references arvanitoyannis, i.s., katsota, m.n., psarra, e.p., soufleros, e.h., kallithrakay, s.: application of quality control methods for assessing wine authenticity: use of multivariate analysis (chemometrics). trends food sci. tech., 10, 1999, 321-336. bednárová, a., kranvogl, r., brodnjak-vončina, d., jug, t., beinrohr, e.: characterization of slovenian wines using multidimensional bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc nova biotechnologica et chimica 13-2 (2014) 195 data analysis from simple enological descriptors. acta chim. slov., 60, 2013, 274-286. beltrán, n.h., duarte-mermoud, m.a., bustos, m.a., salah, s.a., loyola, e.a., peña-neira, a.i., jalocha, j.w.: feature extraction and classification of chilean wines. j. food eng., 75, 2006, 1-10. buratti, s., ballabio, d., benedetti, s., cosio, m.s.: prediction of italian red wine sensorial descriptors from electronic nose, electronic tongue and spectrophotometric measurements by means of genetic algorithm regression models. food chem., 100, 2007, 211-218. bishop, c.m.: neural networks for pattern recognition, clarendon press, oxford, 1995, 482 pp. câmara, j.s., alves, m.a., marques, j.c.: multivariate analysis for the classification and differentiation of madeira wines according to main grape varieties. talanta, 68, 2006, 1512-1521. cozzolino, d., cynkar, w.u., shah n., dambergs r.g., smith p.a.: a brief introduction to multivariate methods in grape and wine analysis. int. j. wine res., 1, 2009, 123-130. gemperline, p.: practical guide to chemometrics, crc press, boca raton, 2006, 520 pp. haykin, s.: neural networks: a comprehensive foundation, pearson education, dehli, 1999, 823 pp. hunter, a., kennedy, l., henry, j., ferguson, i.: application of neural networks and sensitivity analysis to improved prediction of trauma survival. comput. meth. prog. bio., 62, 2000, 11-19. kruzlicova, d., mocak, j., balla, b., petka, j., farkova, m., havel, j.: classification of slovak white wines using artificial neural networks and discriminant analysis. food chem., 112, 2009, 1046-1052. kruzlicova, d., fiket, ž., kniewald, g.: classification of croatian wine varieties using multivariate analysis of data obtained by high resolution icp-ms analysis. food res. int., 54, 2013, 621-626. legin, a., rudnitskaya, a., lvova, l., vlasov, y., di natale, c., d’amico, a.: evaluation of italian wine by the electronic tongue recognition, quantitative analysis and correlation with human sensory perception. anal. chim. acta, 484, 2003, 33-44. lópez-feria, s., cárdenas, s., valcárcel, m.: simplifying chromatographic analysis of the volatile fraction of foods. trends anal. chem., 27, 2008, 794-803. lorrain, b., tempere, s., iturmendi, n., moine, v., de revel, g., teissedre, p.-l.: influence of phenolic compounds on the sensorial perception and volatility of red wine esters in model solution: an insight at the molecular level. food chem., 140, 2013, 76-82. miller, j.n., miller, j.c.: statistics and chemometrics for analytical chemistry, pearson education limited, harlow, 2010, 278 pp. sáenz-navajas, m.-p., avizcuri, j.-m., ferreira, v., fernándezzurbano, p.: insights on the chemical basis of the astringency of spanish red wines. food chem., 134, 2012, 1484-1493. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc 196 bednárová, a. et al. pohl, p.: what do metals tell us about wine? trends anal. chem., 26, 2007, 941949. saurina, j.: characterization of wines using compositional profiles and chemometrics. trends anal. chem., 29, 2010, 234-245. šnuderl, k., mocak, j., brodnjak-vončina, d., sedláčková, b.: classification of white varietal wines using chemical analysis and sensorial evaluations. acta chim. slov., 56, 2009, 765-772. varmuza, k., filzmoser, p.: introduction to multivariate statistical analysis in chemometrics, crc press, boca raton, 2009, 321 pp. zupan, j., gasteiger, j.: neural networks for chemists: an introduction, vch, new york, 1993, 305 pp. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:39 utc 12 nova biotechnologica et chimica 12-1 (2013) doi 10.2478/nbec-2013-0002 © university of ss. cyril and methodius in trnava sorption of synthetic dyes onto river sediments: a laboratory study miroslava bachratá, anna šuňovská, miroslav horník, martin pipíška, jozef augustín department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, sk-917 01 trnava, slovak republic (hornik@ucm.sk) abstract: this work deals with sorption of cationic synthetic dye thioflavine t (tht) onto the river sediment obtained from the váh river under conditions of batch and column system using spectrophotometric methods. we found that sorption of tht onto river sediment was a rapid process with reaching of concentration equilibrium within 2 h of interaction. the values of distribution coefficient (dc) defined as concentration ratio [tht]sediment : [tht]solution were linearly increased with increasing concentration of river sediment in solution within the range csediment 1.25 – 10.0 g/dm3 and minimally changed in the range of initial ph values 2 – 6. the increasing concentration of tht in model solution caused exponentially decrease in the value of dc. the sorption processes characterized by dependence between equilibrium specific sorption qeq and tht concentration ceq in solution were better described by adsorption isotherm according to freundlich (r2 = 0.979) than according to langmuir adsorption isotherm (r2 = 0.914). from evaluation of tht sorption onto river sediment in column system containing of 5 cm sediment layer with 30 cm of water column on the basis of tht concentration changes in infiltrated water we found that these processes were significantly dependent on the rate of infiltrated water flow through the sediment layer riw as well as on qualitative and quantitative composition of water. the highest tht desorption from the sediment layer was found in seepage of 50 % (v/v) ethanol (etoh) solution through the sediment and efficiency of tht desorption decreased in the order: 50 % (v/v) etoh > 0.1 mol/dm3 hcl > deionized water. obtained data from the point of view of physico-chemical characteristics of the river sediment, such as ph, phzpc (potentiometric titration), cation-exchange capacity (cec) and elemental composition (x-ray fluorescence spectrometry), was also discussed. key words: thioflavine t, synthetic dyes, river sediments, sorption, desorption, column system 1. introduction water pollution by organic or inorganic contaminants is one of the most common problems and has been of worldwide concern for many years. besides organic compounds such as polychlorinated biphenyls, pesticides, polycyclic aromatic hydrocarbons, and halogenated aliphatic hydrocarbons in terms of environmental protection and remediation of contaminated localities the attention also focuses on synthetic dyes. synthetic dyes are widely used in various types of industry such as the textile, rubber, paper, plastic, leather, cosmetic, pharmaceuticals and food productions. having a complex molecular structure and synthetic origin, making them biologically not degradable and resistant to environmental conditions, dyes are stable and difficult to treat (forgacs et al., 2004; rai et al., 2005). the use of dyes in expanding industries is accompanied by increasing volume of dyeing effluents. according to bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc nova biotechnologica et chimica 12-1 (2013) 13 incomplete statistics, there are more than 10 000 types of dyes in commercial circulation (altinişik et al., 2010; senturk et al., 2010). about 7.105 tons of dyes are manufactured worldwide each year, and 10 – 15 % is discharged into water bodies as effluents that seriously pollute the environment (türgay et al., 2011; moussavi and khosravi, 2011). they significantly affect equally the human health and the aquatic ecosystem. even in very small quantities dyes lead to changes in salinity and visible coloration of the water, reducing sunlight penetration and thus hinder photosynthetic activity of aquatic organisms, while some of them are moreover toxic and carcinogenic (asgher and bhatti, 2012; fan et al., 2012; mahmoud et al., 2012). synthetic dyes are classified according to their physico-chemical characteristics and their behaviour in solutions as follows: anionic – direct, acid and reactive dyes; cationic – basic dyes; non-ionic – disperse dyes (fu and viraraghavan, 2001). according to epa and oecd, basic dyes are considered one of the most toxic substances and the amounts of non-fixed basic dyes that may be discharged into the effluents were 1 % and 2 – 3 %, respectively (hessel et al., 2007; wawrzkiewicz, 2013). basic (cationic) dyes are quaternary salts whose cations have the positive charge most often on the atom n (ammonium salts), rarely on c (carbonium), o (oxonium) and s (sulfone). in the fate of organic pollutants in all compartments of the environment sorption processes play a decisive role. there are two main factors controlling sorption of organic pollutants: the content and the chemical structure of the soil or sedimentary organic mater (chefetz et al., 2004). however, sorption processes consist of at least two steps: forward (sorption) and reverse (desorption) processes. thus, desorption processes have also significant environmental effects during the remediation of contaminated rivers, where the rate and level of sorbed pollutants which can be released into the water phase are determined mainly by these processes. therefore, understanding the mechanisms that cause sorption-irreversibility is of interest from both a scientific and technological view-point (oren and chefetz, 2005). numerous papers have investigated dye sorption (affinity) to textiles and fibers (e.g. saleem et al., 2005; saleem et al., 2007) as well as attended to finding or evaluating new, inexpensive sorbents derived mainly from the waste biomass (e.g. asgher and bhatti, 2012; piccin et al., 2012), but few examined the affinity of dyes for natural components that are likely to impact the fate of dyes in the environment. our previous papers deal with sorption of radionuclides 137cs and 60co in components of freshwater system (horník et al., 2007a) and bioaccumulation of mentioned radionuclides in freshwater plants (horník et al., 2007b; horník et al., 2008). in this paper we characterize sorption and desorption processes of thioflavine t (tht) binding, as model of benzothiazole cationic dye, onto river sediment obtained from the váh river under batch conditions as well as conditions of laboratory column system in short-time experiments by using spectrophotometry. also, in this work physico-chemical characterization of obtained river sediment as well as the study of environmental conditions effect on sorption processes were realized. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc 14 bachratá, m. et al. 2. materials and methods 2.1 river sediment samples of river bank sediment were taken from the váh river − water reservoir kráľová near the city of šaľa (slovak republic; n 48°12'35"; e 17°48'02") in spring period. the sampling depth was 0 – 10 cm from the surface of the sediment at least 1 m away from the riverbank. subsequently, the river sediment was dried at laboratory temperature (23±2 °c), mixed thoroughly and homogenized by sieved through a 0.31 mm mesh. for the comparison treated river sediments by 3-times washing in deionized water or 0.1 mol/dm3 hcl solution and laboratory chemically treated sea sand (fraction < 0.31 mm) as a model material were also used. the process of the sediment washing was carried out on rotary incubator shaker (250 min-1) with concentration of the sediment 200 g/dm3 within 2 h and at 25 ºc. 2.2 physico-chemical characterization of river sediment potentiometric titration to characterize the potential functional groups involved in the cationic dyes binding onto the surface of river sediment the potentiometric titration as described by the authors zhang et al. (2010) was used. the sediment (0.30 g) was transferred into erlenmeyer flask containing a mixture of 100 cm3 of 0.1 mol/dm3 hcl and 0.1 mol/dm3 nacl, stirred on orbital incubator shaker (250 min-1, 25 ºc) and after 2 h the sediment was centrifugated (3 500 rpm for 5 min). subsequently, the sediment was transferred into 100 cm3 of 0.1 mol/dm3 nacl solution and left to stir under identical conditions during 2 h. potentiometric titration was carried out in erlenmeyer flask by the successive addition of mixture 0.1 mol/dm3 naoh and 0.1 mol/dm3 nacl. after each addition of mentioned mixture and stabilization of the system the ph was measured using a glass electrode (three-point calibration with solution – ph 4.0; 7.0 and 10.0) in the range of ph values from 2.0 to 11.0 at 25 ºc. the relationship between the volume of added titrant 0.1 m naoh (cm3) and the measured ph values was analyzed by modelling program protofit ver. 2.1 (turner and fein, 2006) with the aim to determine pka values for individual functional groups and binding sites concentration can. cation-exchange capacity (cec) the determination of cation-exchange capacity (cec) for studied river and treated sediment was carried out according to iso standard method no. 11260. the sediment (0.25 g) was transferred into 3 cm3 of 0.1 mol/dm3 bacl2 solution and the suspension was allowed to shake on orbital incubator shaker (150 min-1) for 1 h at 25 ºc. subsequently, the sediment was centrifugated (3 500 rpm for 5 min) and the supernatant was decanted. the procedure of bacl2 solution addition, shaking, sediment centrifugation and decanting of supernatant was repeated two more times. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc nova biotechnologica et chimica 12-1 (2013) 15 the solid sediment phase was then saturated by addition of 3 cm3 0.025 mol/dm3 bacl2 solution and the suspension was allowed to shake (150 min -1) during 19 hours at 25 ºc. after this time, the sediment was centrifugated (3 500 rpm for 5 min) and the supernatant was decanted. subsequently, 3 cm3 of 0.02 mol/dm3 mgso4 solution was added into the centrifugated sediment and the suspension was allowed to shake (150 min-1) for the next 19 hours at 25 ºc. after the centrifugation, the concentration of mg was determined in the decanted supernatant by chelatometric titration using 0.02 mol/dm3 edta-na2 (standardization with caco3 solution), eriochrome black t indicator and ammonia buffer. ph the determination of the river and treated sediment ph value was carried out according to iso standard method no. 10390 for the routine determination of ph using glass electrode in the soil suspension. the ph was measured in sediment suspension with distilled water, 1 mol/dm3 kcl or 0.01 mol/dm3 cacl2 at volume ratio v(sediment) : v(solution) = 1 : 5. prepared suspension was stirred on orbital incubator shaker (150 min-1) for 1 h at 25 ºc. subsequently, the suspension was allowed to settle for at least 1 h and the ph was measured directly in the suspension at 25 º c. x-ray fluorescence spectrometry the elemental analysis of river and treated sediment was performed by x-ray fluorescence spectrometry using the high performance x-ray fluorescence spectrometer x-lab 2000, spectro (germany). the content of as, ba, bi, br, ca, cd, ce, cr, cs, cu, fe, ga, ge, la, mg, mn, mo, nb, ni, pb, rb, sb, se, sn, sr, th, u, v, w, y, zn and zr was analyzed in sediment samples treated to granularity < 0.063 mm. 2.3 model of synthetic dye and synthetic freshwater stock solution of benzothiazole cationic dye thioflavine t (tht; purity 75 %, cas 2390-54-7; c.i. 49 005; mr = 318.86; fluka, usa) with the final concentration 200 mg/dm3 was prepared in deionized water or synthetic freshwater. for experimental purposes, the stock solution was diluted with given type of solution for obtaining the desired concentrations. chemical structure of tht is shown in fig. 1. fig. 1. chemical structure of thioflavine t dye. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc 16 bachratá, m. et al. the preparation of synthetic freshwater was carried out according to work smith et al. (2002). table 1 shows the composition of stock solutions for the preparation of synthetic model freshwater imitating the composition of water in lake rostherne mere (lancashire) in the uk. table 1. composition of stock solutions for preparation of synthetic freshwater imitating the water of lake rostherne mere (lancashire) in the uk according to work smith et al. (2002). model of freshwater solution composition concentration [mg/dm3] required volume* h1 cacl2 ca(no3)2.4h2o 7.49 1.18 10 cm3 h2 caco3 0.092 910 cm3 h3 khco3 kh2po4 0.751 0.408 10 cm3 h4 nahco3 na2so4 2.27 2.81 10 cm3 rostherne mere (ph = 7.0; χ = 111 µs/cm) h5 mgso4.7h2o 10.0 10 cm3 * required volume of individual stock solutions for the preparation of 1 dm3of synthetic freshwater. 2.4 spectrophotometric analysis for the determination of the thioflavine t (tht) amount in the analyzed samples of solution the uv-vis spectrophotometer varian, cary 50 (aus) was used. calibration curve for determination of tht was obtained by measuring the absorbance of standard tht solutions at a wavelength λmax = 412 nm. 2.5 sorption of dye in batch system into a series of 100 cm3 erlenmeyer flasks containing 20 cm3 of solution with known concentration of thioflavine t (tht) and ph the defined amount of river sediment was added. exposition was carried out on rotary incubator shaker (250 min-1) at 25 ºc. the changes in concentration of tht caused by evaporating of water were avoided by covering of flasks with parafilm. in time periods (10; 20; 40; 60; 120; 240; 360 and 1 440 minute) or in the end of experiments aliquot samples of solution were taken from flasks for spectrophotometric analysis at a wavelength λmax = 412 nm and calculation of tht dye concentration. the amount of tht sorbed onto river sediment was calculated by the following equation (1): s v ccq t )( 0 −= (1) where: q – the amount of dye sorbed onto river sediment (mg/g; dry weight); bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc nova biotechnologica et chimica 12-1 (2013) 17 c0 and ct – the initial concentration of dye in solution at the time t = 0 min and at the time of solution sampling (mg/dm3); v and s – the volume of dye solution (dm3) and the amount of river sediment in the experiment (g). for characterization of environment effect on sorption processes at reaching of concentration equilibrium [tht]solution : [tht]sediment distribution coefficient (dc) was calculated according to the following equation (2): eqc q dc = (2) where: dc – the distribution coefficient (cm3/g); ceq – the concentration of dye in solution at equilibrium reaching (mg/dm 3). 2.6 sorption and desorption of dye in column system sorption of thioflavine t (tht) onto river sediments was also realized in column system imitating conditions nearby shallow river bank using a glass chromatography column with inside diameter 0.8 cm and height 40 cm. in the bottom of the column the river sediment bed was arranged to height 5 cm (5.0±0.1 g; dry weight) and over sediment the model solution containing tht was maintained with height 30 cm by periodic addition of fresh solution and infiltrated water samples taking. the changes in height of model solution column represented ± 5 % (± 1.5 cm). samples of infiltrated water from the bottom of the column system were obtained in aliquot volumes (2 cm3) and were analyzed in the term of tht concentration in solution or ph values. desorption of tht from river sediment was carried out by replacing of model solution containing tht with deionized water, 0.1 mol/dm3 hcl or 50 % (v/v) ethanol solutions. sampling of infiltrated solution and determination of tht concentration were realized identically as in the case of sorption experiments, except of the case of tht determination in 0.1 mol/dm3 hcl or 50 % (v/v) ethanol solutions, when changes in value of molar extinction coefficient ε were taken into account. 3. results and discussion 3.1 characterization of river sediment it is generally known that xenobiotic binding into abiotic and biotic components of water bodies or water systems is affected by several primary factors mainly defined by physico-chemical characteristics of components themselves, xenobiotics and waters. this work is focused on characterization of cationic synthetic dye thioflavine t (tht) sorption onto the river bank sediment obtained from the váh river under laboratory conditions. therefore in first step we carried out physico-chemical bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc 18 bachratá, m. et al. characterization of studied river sediment. in these analyses treated river sediments by washing in deionized water or in 0.1 mol/dm3 hcl solution as well as laboratory chemically treated sea sand were included. from the view of cationic dyes sorption, as well as xenobiotics molecules show cationic charge in solutions, the qualitative and quantitative occurrence of negatively charged functional groups on sorbent play an important role. for qualitative analysis of functional groups on different sorts of sorbents is wide and commonly used ft – ir analysis. however, in this work for qualitative characterization of functional groups as well as quantitative determination of binding sites concentration can for each predicted functional group the potentiometric titration with application of modelling program protofit ver. 2.1 was used. 3 4 5 6 7 8 9 10 11 0,0 0,5 1,0 1,5 2,0 2,5 3,0 a dd iti on o f 0 .1 m ol /d m 3 n ao h [c m 3 ] ph non-treated sediment treated sediment in deion. h 2 o treated sediment in 0.1 mol/dm3 hcl sea sand fig. 2. potentiometric titration curves for non-treated river sediment obtained from the váh river, treated river sediment in deionized water or 0.1 mol/dm3 hcl solution and chemically treated sea sand (3.0 g/dm3). the titration was carried out with addition of 0.1 mol/dm3 naoh in the presence of background electrolyte 0.1 mol/dm3 nacl and at 25 ºc. the fig. 2 shows potentiometric titration curves describing the dependence between added volume of 0.1 mol/dm3 naoh solution and measured value of ph in solution containing protonated non-treated or treated river sediment in hcl solution or deionized water as well as laboratory chemically treated sea sand. in generally, it can be expected that in sorption of cationic xenobiotics mainly negatively charged functional groups such as carboxyl (–cooh), hydroxyl (–oh), phosphate (–po3h2) or amino (–nh2) groups play an important role. in the case of river sediments content of organic matrix bond or involved on the surface of inorganic particles of sediments (mainly silica sands) can play a decisive role in this term. we found that obtained data showed best-fit to the non-electrostatic model characterizing four binding sites. in the case of river sediment the qualitative analysis confirmed the presence of carboxyl (–cooh), hydroxyl (–oh), phosphate (–po3h2) as well as amino (–nh2) group, whereby hydroxyl and amino groups were presented bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc nova biotechnologica et chimica 12-1 (2013) 19 in higher concentration can. in comparison, the laboratory sea sand did not showed the presence of carboxyl group (data not shown). the abovementioned modelling program also allowed to determine the phzpc value (ph value of zero point of charge), which represents the ph value of environment when matrix shows zero surface charge. in the case, if ph < phzpc then matrix has positively charged surface and if ph > phzpc matrix shows negatively charged surface. among other important parameters, which characterize the ability of matrix to bind negatively charged organic compounds belong the ph value and cation-exchange capacity (cec). table 2 presents obtained values of cec, phzpc as well as ph of studied river sediments and laboratory sea sand. table 2. comparison of obtained values of ph (analyzed in distilled water − phh2o, 0.01 mol/dm3 cacl2 − phcacl2, 1 mol/dm3 kcl − phkcl) determined according to iso standard method no. 10390, cation-exchange capacity (cec) determined according to iso standard method no. 112 60 and phzpc predicted by the modelling program protofit ver. 2.1 from potentiometric titration data according to authors zhang et al. (2010). data characterize non-treated or treated river sediment obtained from the váh river and chemically treated sea sand. sediment treatment phh2o phcacl2 phkcl cec [meq/100g] phzpc non-treated 7.91 7.55 8.56 12.6 9.78 deionized h2o 8.06 7.38 8.44 7.60 10.2 0.1 mol/dm3 hcl 7.24 7.10 7.37 4.00 10.8 sea sand 7.33 7.39 8.60 2.60 10.8 these values are routinely determined in the case of soil samples in terms of evaluation of bond and mobility of metal ions. in general, ph value suggests which type of substances or ions will significantly bind on analyzed matrix, respectively. from this it can be expected that if value of ph << 7 then bond of negatively charge ions will be dominant and if ph >> 7 then bond of positively charged ions will be dominant. cec quantitatively indicates the ability of matrix to bind cations. in the case of clay or organic soils the cec assumes values up to 20 meq/100 g. on the other hand, sandy soils with low ability to bind ions show values only 2 − 3 meq/100 g (pansu and gautheyrou, 2003). as additional analysis, the determination of selected element content in studied river sediments by x-ray fluorescence spectrometry was realized (table 3). united states environmental pollution agency (us epa) developed parameters the effects range low (erl) and effects range median (erm) as predictive tools for characterizing contamination in sediments. both parameters have been widely applied and found to be effective predictive tools (zaharescu et al., 2009; yin et al., 2011). the erl represents the tenth percentile of the effects database, below which harmful effects on aquatic biota are rarely observed. the erm represents the fiftieth percentile of the effects data and is indicative of concentrations above which harmful effects are often observed (us epa, 2002). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc 20 bachratá, m. et al. table 3. the presence of selected elements in river sediments obtained from the váh river determined by x-ray fluorescence spectrometry. a. – non-treated sediment; b. – sediment treated in deionized water; c. – sediment treated in 0.1 mol/dm3 hcl. element sample as ba ca cd cr cu fe mg a −* 305 ppm 6.0 % −* 37 ppm 9 ppm 0.9 % 1.6 % b −* 283 ppm 6.3 % −* 38 ppm 7 ppm 0.9 % 1.6 % c −* 301 ppm 4.1 % −* 36 ppm 9 ppm 0.9 % 1.4 % mn ni pb rb sr v zn zr a 0.03 % 14 ppm 13 ppm 41 ppm 200 ppm 31 ppm 29 ppm 134 ppm b 0.03 % 14 ppm 16 ppm 39 ppm 198 ppm 27 ppm 49 ppm 132 ppm c 0.02 % 15 ppm 13 ppm 43 ppm 165 ppm 26 ppm 29 ppm 151 ppm * value below detection limits of x-ray fluorescence spectrometry (for as and cd < 2 ppm). we found that in obtained river sediment from the váh river abovementioned limits were not exceed and determined values of metal content were minimally 2-times lower than erl values. the influence of river sediment treatment by washing with deionized water or 0.1 mol/dm3 hcl on metals presence in matrix was significantly observed only in the case of diluted solution of hcl and metals of alkaline earths (mg, ca and sr). this fact indicates that metals weakly bound into sediment can be released at slightly acidic conditions. in the case of cationic synthetic dyes, it can be expected a similar effect, however more remarkable in the case of less polar solutions conditions (e.g. diluted solutions of alcohols or organic solvents). 3.2 sorption of dye in batch system in this part of the work we quantitatively characterize sorption of thioflavine t (tht) onto river sediment obtained from the váh river under batch non-equilibrium and equilibrium conditions. experiments were realized in deionized water without addition of macroand microelements or source of carbon, which could support the growth of microorganisms presented in sediments. to some extent batch experiments imitated a possible processes occurring during the movement of particles from upper layers of sediment caused by benthic organisms activity and bioturbation in rivers or lakes. it is generally known that these processes represent dominant mechanisms of sediment particles movement (see e.g. bentley et al., 2006). on fig. 3a is depict the kinetic of tht binding onto river sediment at initial concentration of tht c0 = 40 mg/dm 3 and ph 7.0. we found that in tht binding bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc nova biotechnologica et chimica 12-1 (2013) 21 a rapid sorption processes play a decisive role when in exposition time 10 min nearly 40 % of tht was bound onto sediment. after 2 h of interaction the concentration equilibrium [tht]solution : [tht]sediment with maximal specific sorption q = 4.5 mg/g was observed. also, we found that kinetic of this process is characterized by two phases: first, rapid phase of dye molecules sorption from solution onto surface of sediment particles, and second, slow phase with concentration equilibrium reaching. baughman (1995) in the work focused on adsorption of 9 acid and 5 direct dyes in sediments obtained from oconee river (greece) found that initial rapid dye removing from solution was followed by slow dye removing. from this result, he concluded that rapid sorption process was followed by intraparticle diffusion processes. 0 200 400 600 800 1000 1200 1400 1600 0 2 4 6 8 10 t ht s or pt io n [m g/ g] time [min] 0 2 4 6 8 10 12 14 0 20 40 60 80 100 d c [c m 3 / g] c sediment [g/dm3] linear regression: y = a + b*x parameter value s.e. a 26.12 0.76 b 3.53 0.13 r2 0.996 fig. 3 a. kinetic of tht sorption onto river sediment obtained from the váh river (5 g/dm3) during 24 h of interaction at initial concentration of tht c0 = 40 mg/dm3 and ph 4.0, on rotary incubator shaker (250 min-1) and at 25 °c. the value of ph after 24 h phf = 8.1. b. effect of river sediment concentration on distribution coefficient (dc) for tht sorption onto sediment (5 g/dm3) after 2 h of interaction on rotary incubator shaker (250 min-1) and at 25 °c. initial tht concentration c0 = 100 mg/dm3 and ph 4.0. final ph value at the end of experiments: 1.25 g/dm3 – 7.0; 2.5 g/dm3 – 7.5; 5.0 g/dm3 – 7.7; 10.0 g/dm3 – 8.0; 12.5 g/dm3 – 8.0. data represents arithmetical mean from two independent experiments. also, the results suggest that many acid and direct dyes will be stable in aquatic systems for long periods of time unless other transformation pathways (e.g. photochemical) are rate determining. varlikli et al. (2009) observed the concentration equilibrium reaching for dyes sorption onto sand obtained from sahara at the time 10 min and found that sorption capacity of sand was higher in the case of cationic dyes (75 % of dye removed from solution) than in the case of anionic dyes (only 21 % of dye removed from solution). on the other hand, lazaridis and keenan (2010) determined the concentration equilibrium reaching after 5 h of model soil composed from sand, sediment and clays (ratio 85:10:5) interaction with solution containing azo dye reactive black 5. from the point of view of chemical composition of sediments, it may be stated that the portion of organic and inorganic components of sediments will significantly affect their sorption capacity as well as the rate of these processes. for example liu et al. (2001) found the increasing of cationic dyes sorption onto sediment obtained a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc 22 bachratá, m. et al. from qinghe river (china) after organic carbon (oc) removing and in the case of anionic dyes the decreasing of sorption after oc removing was observed. in the real conditions, it can be expected that microorganisms, especially in the form of biofilms, in interaction of toxic organic compounds with components of river or lake sediments will also play a decisive role from the point of view of their accumulation, biodegradation and sorption abilities. in the next experiments we studied the effect of river sediment concentration on tht sorption in the range csediment 1.25 – 12.5 g/dm 3, in time of concentration equilibrium reaching (2 h) and at initial tht concentration c0 = 100 mg/dm 3. the dye sorption onto sediment was evaluated by distribution coefficient (dc) defined as concentration ratio [tht]sediment : [tht]solution. we found that the value of dc was increased with increasing sediment concentration and this increase showed linear dependence (r2 = 0.996) within sediment concentration csediment 1.25 – 10.0 g/dm 3 (fig. 3b). similar result also obtained by varlikli et al. (2009) at methylene blue sorption in suspension of sahara sand. however, it can be expected that sorption ability of river or lake sediments will significantly depend on the size or specific surface of sediment particles as well as aggregates formed between abiological and biological components of sediments, respectively. 2 3 4 5 6 7 8 0 40 80 120 160 200 treated sediment in deioniz. h 2 o initial ph 0 final ph f initial ph 0 final ph f d c [c m 3 / g] ph 0 ph f non-treated sediment 0 40 80 120 160 200 0 200 400 600 800 1000 d c [c m 3 / g] c 0 [mg/dm3] fig. 4. a. effect of ph value (initial ph0; final phf at the end of experiment) on distribution coefficient (dc) for tht sorption onto non-treated and treated (3-times washed in deionized water) river sediment obtained from the váh river (5 g/dm3) after 2 h of interaction on rotary incubator shaker (250 min-1) at initial concentration of tht c0 = 40 mg/dm3 and 25 °c. b. effect of initial concentration of tht on dc for tht sorption onto sediment (5 g/dm3) after 2 h of interaction on rotary incubator shaker (250 min-1) at initial ph 4.0 and 25 °c. the final ph value at the end of experiment: 20 mg/dm3 – 8.1; 40 mg/dm3 – 8.1; 80 mg/dm3 – 7.6; 100 mg/dm3 – 7.6; 200 mg/dm3 – 7.6. data represents arithmetical mean from two independent experiments. it is generally known that ph value of the environment strongly affects the behaviour of toxic compounds as well as their binding in the environment. this effect is not only in terms of changes in physico-chemical characteristics of compounds but also from the point of view of functional groups dissociation on the surface of sediment compartments responsible for sorption of these substances. on the fig. 4a the dependence between dc value and initial ph value within a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc nova biotechnologica et chimica 12-1 (2013) 23 the range 2 – 6 or final, equilibrium ph value, respectively are depicted. we found that at all experiments the ph value was increased from the initial value to the final, equilibrium value ph 8, which correspond with value determined according to iso method no. 10390 (table 2). this effect was also observed in the case of treated river sediment by 3-times washing in deionized water, however treated sediment showed practically 3-times lower dc values. from the fig. 4a, it is also clear that dc value was increased minimally with increasing initial as well as equilibrium ph value. similar result was observed by liu et al. (2001) at sorption of cationic dyes basic yellow x-5gl and basic red 13 onto sediment from qinghe river within the range of initial ph values 2 – 10. another important parameter in the evaluation of toxic organic substances behaviour as well as the risks associated with their input into aquatic systems is their concentration. from this reason, we realized a series of experiments within the concentration range of tht 20 − 200 mg/dm3 in deionized water at ph 7.0. from obtained results, it can be concluded that dc value exponentially decreased with increasing initial concentration of tht in solution (fig. 4b). obtained equilibrium data characterizing the sorption of tht onto river sediment also allow to describe sorption processes by equilibrium adsorption isotherms. among the most commonly used mathematical models describing sorption processes include adsorption isotherms according to langmuir and freundlich. langmuir adsorption isotherm describes mono-layer sorption on the surface of sorbent with a limited number of identical sites. in equation includes the parameter qmax defining the maximum specific sorption, which permits to evaluate a various sorbents in terms of their sorption properties as well as the effect of physico-chemical changes in environment on compounds sorption. in contrast, the freundlich adsorption isotherm calculates with non-homogeneous binding sites thus describes the sorption on heterogeneous surface containing binding sites with different affinity for sorbate. -20 0 20 40 60 80 100 120 140 160 180 3,2 3,6 4,0 4,4 4,8 5,2 langmuir freundlich model: freundlich r^2 = 0.979 a 2.99 ±0.10 b 0.094±0.008 model: langmuir r^2 = 0.914 a 4.61 ±0.12 b 0.657±0.153 q eq [m g/ g] c eq [mg/dm3] fig. 5. description of tht sorption onto river sediment obtained from the váh river according to langmuir and freundlich adsorption isotherms. details of experimental conditions see in fig. 4b. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc 24 bachratá, m. et al. from description of obtained data as well as from the comparison of coefficients of determination (r2), it can be concluded that tht sorption onto river sediment was better described by adsorption isotherm according to freundlich (r2 = 0.979) than by langmuir adsorption isotherm (r2 = 0.914), which determines the value of qmax parameter 4.61±0.12 mg/g (±sd) (fig. 5). obtained parameters from both mathematical equations of adsorption isotherms are presented in table 4. also, liu et al. (2001) found that the sorption of five cationic and anionic dyes onto sediment obtained from qinghe river (china) was better described by freundlich adsorption isotherm with coefficients of determination in the range r2 = 0.941 − 0.985. in the case of cationic dyes basic yellow x-5gl and basic red 13 determined values of freundlich constants kf 7.04 and 7.01, respectively, as well as 1/n 0.386 and 0.687, respectively. similarly, a better description of the sorption of anionic dyes by suspension of sandy and loamy soils with freundlich adsorption isotherms was observed by morris et al. (2008) and qu et al. (2008). also, the freundlich adsorption isotherm was an appropriate model for description of cationic surfactant cetyltrimethylammonium bromide (ctab) sorption on marine sediment (cao et al., 2011). table 4. obtained parameters from adsorption isotherms according to langmuir and freundlich describing tht sorption onto river sediment obtained from the váh river. details of experimental conditions see in fig. 4b. langmuir isotherm eq eq eq cb cbq q *1 **max + = freundlich isotherm neqfeq ckq 1 *= qmax b r2 kf 1/n n r2 4.61±0.12 0.657±0.153 0.914 2.99±0.10 0.094±0.008 10.6 0.979 qmax – langmuir constant represents the maximal sorption capacity (mg/g); b – langmuir constant represents affinity between sorbate and sorbent (dm3/mg); r2 – coefficient of determination; kf – freundlich constant relating with sorption capacity (dm3/g of sediment); n – freundlich constant relating with sorption intensity (non-dimensional coefficient). 3.3 sorption and desorption of dye in column system in this type of experiments a glass column with inside diameter 0.8 cm and height 40 cm was used. in bottom of the column the river sediment obtained from the váh river was arranged with sediment bed height 5 cm and over sediment the model solution containing thioflavine t (tht) was maintained with height 30 cm by periodic addition of fresh solution and infiltrated water samples taking. samples of infiltrated water in aliquot volumes (2 cm3) were collected and spectrophotometrically analyzed for residual concentration of tht. the aim of these experiments was to imitate the conditions occurring near banks of shallow rivers, where vertical transport processes (convection) governed bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc nova biotechnologica et chimica 12-1 (2013) 25 by gravitation and bank infiltration play a decisive role. on the other hand, in the case of bottom and sediments of the river distant from the bank the horizontal transport processes (advection) take place. 0 40 80 120 160 200 240 0 20 40 60 80 100 c iw [m g/ dm 3 ] v iw [cm3] r iw = 0.71 cm3/min r iw = 0.56 cm3/min fig. 6. concentration of tht in infiltrated water from experimental column system containing river sediment obtained from the váh river. experiment was realized in glass column (diameter 0.8 cm and height 40 cm) with 5 cm of sediment bed and 30±1.5 cm level of model solution with initial concentration of tht c0 = 100 mg/dm3 in deionized water, ph 4.0 and at 22±2 °c. the average flow rate of infiltration water: -■-■riw = 0.71±0.08 cm3/min (±sd); -o-oriw = 0.56±0.06 cm3/min (±sd). fig. 6 depicts results from the determination of tht concentration in infiltrated water that passes through the 5 cm layer of river sediment. we found that in the first tens of millilitres of infiltrated water the concentration of tht was negligible, and subsequently, the tht concentration in infiltrated water exponentially increased up to the value of tht concentration in water column above the sediment layer (c0 = 100 mg/dm 3). it means that in this phase the river sediment was saturated by tht dye to such an extent that tht passed through the sediment without significant interaction. also, we found that at a slower flow rate of infiltrated water riv = 0.56 cm3/min tht concentration in infiltrated water began to increase in 2-times greater volume of solution (120 cm3) which passed through the sediment bed in comparison with faster flow rate riv = 0.71 cm 3/min of solution under identical conditions of column system. in the next experiment we studied the effect of water composition containing tht on dye transport through the layer of river sediment. from fig. 7, it is evident that in the case of synthetic freshwater imitating the composition of water in lake rostherne mere (lancashire) in the uk and containing tht with initial concentration c0 = 100 mg/dm 3 we found enhanced transport of tht through the sediment in comparison with deionized water. this fact can be explained by competitive effect between monovalent or bivalent metal cations occurring in synthetic freshwater and cationic dye tht as well as saturation of sediment no only with tht, but also with metal ions contained in the freshwater. it means that the qualitative and quantitative composition of the water of lakes or rivers will be significantly affect bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc 26 bachratá, m. et al. the transport of toxic organic compounds, such as cationic or anionic synthetic dyes through sediments. 0 40 80 120 160 200 0 20 40 60 80 100 c iw [m g/ dm 3 ] v iw [cm3] deionized water synthetic freshwater fig. 7. concentration of tht in infiltrated water from experimental column system containing river sediment obtained from the váh river. experiment was realized in glass column (diameter 0.8 cm and height 40 cm) with 5 cm of sediment bed and 30±1.5 cm level of model solution with initial concentration of tht c0 = 100 mg/dm3 in deionized water or synthetic freshwater according to smith et al. (2002), ph 4.0 and at 22±2 °c. the average flow rate of infiltration water: -■-■riw = 0.71±0.08 cm3/min (±sd); -o-oriw = 0.62±0.06 cm3/min (±sd). 0 40 80 120 160 200 0 20 40 60 80 100 c iw [m g/ dm 3 ] v iw [cm3] 0 10 20 30 40 50 60 70 0 20 40 60 80 100 50 % (v/v) ethanol c iw [m g/ dm 3 ] v iw [cm3] deioniz. h 2 o 0.1 mol/dm3 hcl fig. 8 a. concentration of tht in infiltrated water from experimental column system containing river sediment obtained from the váh river. experiment was realized in glass column (diameter 0.8 cm and height 40 cm) with 5 cm of sediment bed and 30±1.5 cm level of solution with tht concentration c0 = 100 mg/dm3 in deionized water, ph 4.0 and at 22±2 °c. the average flow rate of infiltration water riw = 0.71±0.08 cm3/min (±sd). b. concentration of tht in infiltrated solution from experimental column system containing river sediment after sorption experiment (fig. 8a). experiment was carried out identically as sorption experiment with difference that model solution of tht was replaced with deionized water (10 fraction with 2 cm3 infiltrated water), 0.1 mol/dm3 hcl (14 fraction) and 50 % (v/v) ethanol solution (9 fraction). from the point of view of synthetic dyes transport and behaviour in the environment their chemical stability plays an important role. for this reason it is necessary to study the remobilization of dyes from sediments into the water of rivers a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc nova biotechnologica et chimica 12-1 (2013) 27 or lakes, as well as the gradual release of these substances from lower sediment layers up to groundwater. in terms of this request the desorption of tht from river sediment by gradually replacement of model dye solution with deionized water, 0.1 mol/dm3 hcl or 50 % (v/v) ethanol (etoh) solution we carried out. we found that sorbed amount of tht in sediment layer was not released by seepage of deionized water and only minimal desorption of tht in the case of 0.1 mol/dm3 hcl solution was observed (fig. 8). the highest desorption of tht from the sediment layer was found in application of 50 % (v/v) etoh solution into the column system, and the efficiency of tht desorption from 5 cm of sediment layer decreased in the order: 50 % etoh > 0.1 mol/dm3 hcl > deionized water. from obtained results it can be concluded that the binding of tht, and possibly other cationic synthetic dyes, onto river sediments under natural conditions will be practically irreversible. 4. conclusions obtained data suggest that sorption of benzothiazole cationic dye thioflavine t (tht), and possibly other cationic synthetic dyes, onto river sediments under natural conditions will be a rapid and practically irreversible process as well as the further fate of these substances after their input into the aquatic systems and sediments will be probably determined by degradation processes carried out mainly by autochthonous microorganisms as well as photochemical reactions. acknowledgement: this work was supported by project of the operational program research and development and co-financed with european regional development fund (erdf) with the grant number itms 26220220106. also, authors want to thank dr. daniela mackových for x-ray fluorescence spectrometric analyses. references altinişik, a., gür, e., seki, y.: a natural sorbent, luffa cylindrica for the removal of a model basic dye. j. hazard. mater., 179, 2010, 658-664. asgher, m., bhatti, h.n.: evaluation of thermodynamics and effect of chemical treatments on sorption potential of citrus waste biomass for removal of anionic dyes from aqueous solutions. ecol. eng., 38, 2012, 79-85. baughman, g.l.: fate of dyes in aquatic systems. part 3: the role of suspended sediments in adsorption and reaction of acid and direct dyes. dyes pigments, 27, 1995, 197-210. bentley, s., thibodeaux, l., adriaens, p., li, m.y., romerogonzález, m., banwart, s.a., filip, l., demnerova, k., reible, 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risk assessment of heavy metals in surface sediments from lake taihu, china. environ. res. lett., 6, 2011, 1-11. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:21 utc nova biotechnol chim (2017) 16(1): 12-19 doi: 10.1515/nbec-2017-0002  corresponding author: jana.moravcikova@savba.sk nova biotechnologica et chimica perception of biotech trees by slovak university students – a comparative survey jana moravčíkováa, , ildikó matušíkováb, peter nemečekc, alžbeta blehovád, želmíra balážováe, zdenka gálováe, patrik mészárosf and ján kraicg,h a institute of plant genetics and biotechnology, centre of biology and biodiversity sas, akademická 2, p.o. box 39a, nitra, sk-950 07, slovak republic b department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic c department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic d department of plant physiology, faculty of natural sciences, comenius university, mlynská dolina b2, bratislava, sk-842 15, slovak republic e faculty of biotechnology and food science, slovak university of agriculture, tr. a. hlinku 2, nitra, sk-949 76, slovak republic f department of botany and genetics, faculty of natural sciences, constantine the philosopher university, nábrežie mládeže 91, nitra, sk-949 74, slovak republic g department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic h research institute of plant production, national agricultural and food center, piešťany sk-921 68, slovak republic article info article history: received: 8th january 2017 accepted: 27th february 2017 keywords: biotechnology forest trees genetic modification gmo public attitude survey transgenic university students abstract acceptance of genetically modified plants is restricted in eu by legislation, while the attitude of public is not favourable as well. surveys show that knowledge about gm plants is getting increased. newly developed strategies on gm safety for environment can be a crucial aspect for the (partial) acceptance in future. gm trees as non-edible plants might appear as more admissible, however, are relatively rarely discussed. we performed a comparative survey on knowledge and perception of gm forest trees among students at four slovak universities. we also compared their responses between as well as with the outcome of similar cross-country survey in frames of the cost action fp0905. the results point to very similar attitude of slovak students when compared with students from other countries, no significant difference between responses of males and females, but also influence of age as well as orientation of their study (natural sciences vs. economy) on view of gm tree safety and placing on the market.  university of ss. cyril and methodius in trnava introduction it is some 20 years since biotech or genetically modified (gm) crops were commercialised and largely adopted by farmers mainly in usa, brazil, argentina or canada. in 2015, biotech crops were planted on 179.7 hectares in 28 countries (clive 2015), while the total area of biotech plants has increased more than one hundred fold since 1996. hitherto, the international service for the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:28 utc mailto:jana.moravcikova@savba.sk nova biotechnol chim (2017) 16(1): 12-19 13 table 1. overview of gm woody plants with the regulatory approval according to the isaaa gm approval database (january 2017). authorisation crop trade name event name trait country type of approval a malus x domestica arctic™ "golden delicious" apple gd743 non-browning phenotype canada usa a (2015), b (2015), c (2015) a (2015), b (2015), c (2015) malus x domestica arctic™ gs784 non-browning phenotype canada usa a (2015), b (2015), c (2015) a (2015), b (2015), c (2015) malus x domestica arctic™ fuji apple nf872 non-browning phenotype usa a (2016), b (2016), c (2016) carica papaya rainbow, sunup 55-1 viral disease resistance canada japan usa a (2003) a (2011), c (2011) a (1997), b (1997), c (1996) carica papaya not available 63-1 viral disease resistance usa c (1996) carica papaya huanong no. 1 huanong no. 1 viral disease resistance china c (2006) carica papaya not available x17-2 viral disease resistance usa a (2008), b (2008), c (2009) eucalyptus sp. gm eucalyptus h421 volumetric wood increase brazil a (2015), b (2015), c (2015) prunus domestica not available c-5 viral disease resistance usa a (2009), b (2009), c (2008) populus sp. bt poplar, poplar 12 (populus nigra) not available lepidopteran insect resistance china c (1998) popupus sp. hybrid poplar clone 741 not available lepidopteran insect resistance china c (2001) a a – food, direct use or processing; b – feed direct use or processing; c – cultivation domestic or non-domestic use. in the brackets the years of approval are given. of agri-biotech applications (isaaa) gm approval database contains more than four hundred entries concerning biotech/gm events with regulatory evaluations and approvals (isaaa 2017). the most common genetically engineered crops are soybean, maize, canola and cotton with traits ensuring tolerance to herbicides and/or insect resistance. in european union, deliberate release of gm plants for research purpose and placing on the market are directed by eu directive 2001/18/ec (ec 2001) that was implemented into national legislations. the directive was amended by eu 2015/412 (ec 2015) as regards the possibility for the member states to restrict and prohibit gm organisms in their territory after they have been authorised to be placed on the union market. the eu legislation requires upfront evaluation of direct, indirect, immediate and delayed effects as well as the cumulative long term effects on human health and the environment. comprehensive and strict legal regulations allow using biotech plants with eu authorisation only for food/feed purposes (isaaa 2017; eu register of authorised gmos 2017). the first gm (poplar) tree was reported by filatti et al. (1987) five years later as the first ever gm (tobacco) plant was generated. gm trees were developed and studied in greenhouse or field conditions for improved woody quality, faster growth, herbicide tolerance, insect and disease resistance or abiotic stress tolerance. of nearly 800 gm field trials approved worldwide, however, fewer than 50 were in europe, mainly for research purposes (haggman et al. 2013) at strictly controlled dissemination and sexual maturity (pilate et al. 2016). technical limitation, biosafety concerns and existing legislation hinder research progress and commercial application of gm tree technology in europe. so far, only small number of gm woody plants has been successfully commercialised (table 1) and include woody plants such as papaya, eucalyptus, apple and plum trees with authorisation in canada, usa, japan, brazil bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:28 utc nova biotechnol chim (2017) 16(1): 12-19 14 or china. among forest trees, only gm poplar is commercialised in china (table 1). in spite of obvious economical benefits from commercial plantation of gm trees, the majority of public discussion is focused on their unintended effect on environment. however, the key arguments are associated with ethical consideration and moral imperatives. most of studies on public attitude of gmos are referred to gm crops (lucht et al. 2015) but only few gm trees (nonić et al. 2015; kazana et al. 2016). here, students of four slovak universities with different field of study were asked to give anonymously their opinion on gm tree plantation. we deliberately focused on students aged from 18 to 25, future experts that should not be blinkered from gmos. we aimed to estimate their i) knowledge concerning gm forest trees, ii) agreement with gm trees commercialisation and iii) perception of gm trees (adoption) safety. the survey extends the screening carried out within the frame of european cost action fp0905 “biosafety of forest transgenic trees and eu police directives” (vettori et al. 2016). table 2. socio-demographic profile of respondents. university faculty field of study no. students gender male/female average age comenius university in bratislava (cu) faculty of natural sciences (fns) plant physiology, genetics 24 7/17 22 university of ss. cyril and methodius in trnava (ucm) faculty of natural sciences (fns) biology 22 7/15 22 slovak university of agriculture in nitra (sua) faculty of biotechnology and food sciences (fbfs) biotechnology 20 6/14 24 faculty of economics and managements (fem) accounting 23 9/14 22 horticulture and landscape engineering faculty (hlef) horticulture 20 10/10 21 constantine the philosopher university in nitra (ukf) faculty of natural sciences (fns) biology 21 5/16 20 total 130 44/86 x̄ = 22 experimental a survey was conducted among students of comenius university in bratislava, university of ss. cyril and methodius in trnava, slovak university of agriculture in nitra and constantine the philosopher university in nitra. target groups were students of disciplines related to natural sciences and economics. socio-demographic profile of respondents is given in table 2. the questionnaire contained nine questions (q1–q9) and was organised into four sections: i) socio-demographic information, ii) knowledge about gm forest trees (q1–q3), iii) acceptance of cultivation of gm forest trees (q4–q7) and iv) perception of gm trees (adoption) safety (q8–q9). the questions were as follows: q1 – do you know what a genetically modified forest tree (transgenic forest tree) is? q2 – do you know if transgenic forest plantations are grown commercially? q3 – do you know if final products of transgenic forest plantations (wood, biofuel, pulp, paper) are being sold in the market (stores, supermarkets etc.)? bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:28 utc nova biotechnol chim (2017) 16(1): 12-19 15 q4 – would you agree with forest transgenic crops to be approved for commercial planting? q5 – would you purchase the final products (wood products, pulp, paper etc.) produced from transgenic forest plantations? q6 – would you agree with the final products produced from transgenic forest plantations to be labelled to indicate that they originate from genetically modified trees? if yes, would you agree with the labelling of such products to be legally mandatory? (q6.a) q7 – which of the following benefits resulting through adoption of transgenic forest crops do you think may be important in your country?: use of less chemicals and energy to process cellulose (q7.a), harvesting of a smaller number of trees for consumption (q7.b) use of less pesticides in forest plantations (q7.c), less herbicide treatments of forest plantations (q7.d), restoration of contaminated soils (q7.e), less old growth logging (q7.f), better timber quality/higher value product (q7.g), higher pulping efficiency (q7.h), more efficient biofuel production from gm forest trees (q7.i), stronger timber construction materials (q7.j), higher tree productivity (q7.k). q8 – which of the following issues concerns you the most regarding adoption of transgenic forest crops? which of the following do you think if it occurs when adopting a transgenic forest crop may constitute a hazard?: forest trees less fit (q8.a), forest trees more vulnerable to viral diseases (q8.b), higher rates of soil decomposition (q8.c), more pesticide resistant forest species (q8.d), more use of broad spectrum herbicides (q8.e), loss of biodiversity (q8.f), adverse effects on bio-trophic processes of host ecosystems (q8.g), increased cost of controlling pest outbreaks. (q8.h), cultural adaptation to changing biodiversity (q8.i), transgene genes become inactive (q8.j). table 3. comparison of responses to selected questions (q) among individual groups of respondents using fisher's lsd test. scorea dependent variableb mean difference (i-j) std. error significance 95 % confidence interval lower bound upper bound qa sua cu 0.1859* 0.0808 0.023 0.026 0.346 fns fem -0.2501* 0.0806 0.002 -0.410 -0.091 qb ukf ucm -0.2123* 0.1024 0.040 -0.415 -0.010 sua ucm -0.1830* 0.0831 0.029 -0.347 -0.019 ukf ucm -0.2576* 0.0990 0.010 -0.454 -0.062 fbfs fem -0.2782* 0.0923 0.003 -0.461 -0.096 q8 fns hlef -1.549* 0.669 0.022 -2.87 -0.23 a group qa includes questions q1, q2 and q3; group qb includes questions q4, q5, q6 and q6a. b comparisons between universities and between faculties. * the mean difference is significant at the 0.05 level. q9 – which of the following items do you think may constitute a hazard when adopting a transgenic forest crop?: forest trees less fit (q9.a), forest trees more vulnerable to viral diseases (q9.b), higher rates of soil decomposition (q9.c), more pesticide resistant forest species (q9.d), more use of broad spectrum herbicides (q9.e), loss of biodiversity (q9.f), adverse effects on bio-trophic processes of host ecosystems (q9.g), increased cost of controlling pest outbreaks. (q9.h), cultural adaptation to changing biodiversity conditions (q9.i). the questions q1–q6 were type of yes/no, while the question q7 was evaluated using four-level rating scale: very important (4), slightly important (3), not important (2) and i do not know (1). in the question q8, students had to select only one safety issue. the question q9 was evaluated using scale: serious hazard (4), slight hazard (3), no hazard (2) and i do not know (1). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:28 utc nova biotechnol chim (2017) 16(1): 12-19 16 for data processing, fisher's lsd test was applied to compute the pooled standard deviation from groups qa (q1, q2, q3), qb (q4, q5, q6) and q8 using the statistical program ibm spss 22. results and discussion the questionnaire was submitted to a total of 130 students of four slovak universities (table 2). respondents were aged from 18 to 25 with average age of 22. a total of 66 % students were women (table 2). fig. 1. respondents’ positive attitude in percentage concerning knowledge on gm forest trees (q1), their commercialisation (q2) and placing on the market (q3). the first section of the questionnaire qa (q1–q3) was focused on knowledge of respondents about gm trees. data are summarised in fig. 1. in average, more than 57 % students of environmental disciplines indicated that they knew meaning of forest gm trees (q1). the highest percentage was recorded in students of cu-fns (100 %) and sua-fbfs (95 %). the lowest number of positive responses (39 %) was recorded in students of disciplines related to economics. despite that high number of respondents indicated a positive answer on the question q1, abundance of positive responses on remaining two questions (q2 and q3) was significantly lower. less than 40 % respondents knew if gm forest trees are grown commercially (q2) and less than 41 % knew if final products of gm trees are placed on the market (q3). similar non-uniform pattern was observed in the cross-country survey focused on public attitude towards the use of gm trees performed by kazana et al. (2016) or in the survey conducted in serbia by nonić et al. (2015). for example, 82.5 % of students of university of belgrade declared that they know meaning of gm tree; however, only 51.5 % knew if gm trees are grown commercially. it may coincide with the fact that until now only few gm trees were authorised, moreover outside of the eu (table 1). overall, 26 out of 130 (20 %) slovak respondents answered positively on all three questions (q1, q2 and q3). these respondents can be considered as well informed. to compare answers between universities and faculties, all responses were statistically analysed. at university level the differences among answers to questions q1, q2 and q3 (score qa) of respondents were in general at the border of significance (p=0.057), while respondents from cu and sua differed in their knowledge on gm trees most obviously (at p≤0.05) (table 3). however, significant differences (at p≤0.05) were observed between individual faculties oriented towards natural sciences (fns) or biotechnologies (fbfs) exerted significantly different knowledge comparing to those studying economy (fem) (at p≤0.05). fig. 2. respondents’ positive attitude in percentage concerning acceptance of gm trees for commercial use (q4), purchase of the final product from gm trees (q5) and labelling of such products (q6, q6a). respondents´ attitudes towards benefits resulting from adoption of gm trees (q7) were evaluated using four-step scale: very important (4), slightly bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:28 utc nova biotechnol chim (2017) 16(1): 12-19 17 fig. 3. the most important safety issues of gm trees (q8) evaluated according to the respondents. table 4. opinions of students about potential benefits from gm forest tree adoption (questions q7). individual sub questions q7a-k are described in experimental. cu-fns [%]* ukf-fns [%]* ucm-fns [%]* 1 2 3 4 1 2 3 4 1 2 3 4 q7.a 0 4.2 29.2 70.8 9.5 0.0 19.0 71.4 0 0 40.9 59.1 q7.b 0 4.2 12.5 83.3 0.0 0.0 9.5 90.5 0 0 4.5 95.5 q7.c 0 8.3 33.3 50.0 4.8 14.3 19.0 61.9 4.5 0 31.8 63.6 q7.d 0 12.5 37.5 50.0 4.8 9.5 42.9 42.9 9.1 18.2 27.3 45.5 q7.e 4.2 0 45.8 50.0 0 19.0 19.0 61.9 4.5 0 4.5 90.9 q7.f 0 8.7 21.7 69.6 4.8 9.5 19.0 66.7 0 4.5 27.3 68.2 q7.g 8.7 17.4 65.2 8.7 19.0 9.5 42.9 28.6 22.7 9.1 54.5 13.6 q7.h 0 0 56.2 43.5 4.8 4.8 23.8 66.7 0 4.5 50.0 45.5 q7.i 8.7 21.7 43.5 26.1 14.3 4.8 14.3 66.7 9.1 9.1 13.6 68.2 q7.j 4.3 13.0 65.2 17.4 9.5 9.5 38.1 38.1 0.0 22.7 45.5 31.8 q7.k 0 0 43.5 56.5 14.3 4.8 28.6 52.4 4.5 13.6 40.9 40.9 sua-fbfs [%]* sua-hlef [%]* sua-fem [%]* 1 2 3 4 1 2 3 4 1 2 3 4 q7.a 0 5.0 45.0 50.0 0 5.3 42.1 52.6 0.0 4.3 34.8 60.9 q7.b 0 0 25.0 75.0 0 0 5.3 94.7 4.3 0.0 17.4 78.3 q7.c 5 5.0 30.0 60.0 0 5.3 42.1 52.6 4.3 8.7 30.4 34.8 q7.d 0 10.0 30.0 60.0 5.3 0.0 26.3 68.4 17.4 13.0 65.2 30.4 q7.e 5 10.0 20.0 65.0 21.1 5.3 10.5 63.2 4.3 8.7 26.1 60.9 q7.f 0 10.0 35.0 60.0 5.3 0 21.1 73.7 8.7 8.7 26.1 56.5 q7.g 10.0 10.0 70.0 10.0 47.4 15.8 26.3 10.5 26.1 8.7 52.2 13.0 q7.h 0 10.0 40.0 50.0 26.3 0 21.1 52.6 4.3 4.3 60.9 30.4 q7.i 0 25.0 25.0 50.0 21.1 10.5 15.8 52.6 13.0 8.7 47.8 30.4 q7.j 5.0 20.0 30.0 45.0 0 26.3 26.3 47.4 8.7 17.4 56.5 17.4 q7.k 5.0 5.0 45.0 45.0 0 5.3 31.6 63.2 8.7 4.3 43.5 43.5 * percentage of students considering the given benefit as (4) very important, (3) slightly important, (2) not important, (1) i do not know. important (3), not important (2) and i do not know (1). majority of students indicated as very important the use of less chemicals (q7.a), harvesting of smaller number of trees (q7.b), restoration of poison contaminated soil (q7.e) and less old growth logging (q7.f). slightly lower importance was attributed by students to the use of less bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:28 utc nova biotechnol chim (2017) 16(1): 12-19 18 insecticides/pesticides/herbicides (q7.c/q7.d), higher pulping efficiency (q7.g), better timber quality q7.h), more efficient biofuel production (q7.i), stronger timber construction (q7.j) or higher tree productivity (q7.k). data are summarised in table 4. table 5. respondents’ attitudes about potential hazards related to the gm tree plantations (q9). individual sub questions (q9a-i are described in experimental. cu-fns [%]* ukf-fns [%]* ucm-fns [%]* 1 2 3 4 1 2 3 4 1 2 3 4 q9.a 8.3 12.5 54.2 25 4.8 19.0 57.1 19.0 18.2 4.5 50.0 27.3 q9.b 4.2 4.2 41.7 50 14.3 14.3 23.8 47.6 22.7 4.5 40.9 31.8 q9.c 25 4.2 54.2 16.7 19.0 9.5 19.0 52.4 13.6 9.1 40.9 36.4 q9.d 8.3 4.2 45.8 41.7 4.8 14.3 52.4 28.6 9.1 27.3 45.5 18.2 q9.e 4.2 4.2 29.2 62.5 4.8 4.8 38.1 52.4 18.2 13.6 18.2 50.0 q9.f 0 0 25.0 75.0 0 0 28.6 71.4 13.6 9.1 18.2 59.1 q9.g 4.2 12.5 29.2 54.2 9.5 9.5 42.9 38.1 9.1 9.1 36.4 45.5 q9.h 4.2 16.7 16.7 62.5 4.8 9.5 42.9 42.9 13.6 13.6 27.3 45.5 q9.i 4.2 16.7 33.3 45.8 14.3 9.5 52.4 23.8 13.6 18.2 45.5 22.7 sua-fbfs [%]* sua-hlef [%]* sua-fem [%]* 1 2 3 4 1 2 3 4 1 2 3 4 q9.a 0 26.3 52.6 21.1 15.8 5.3 47.4 31.6 4.3 8.7 56.5 30.4 q9.b 5.3 10.5 36.8 47.4 5.0 10.0 35.0 50.0 13.0 4.3 56.5 26.1 q9.c 21.1 10.5 52.6 15.8 20.0 15.0 35.0 30.0 0 26.1 43.5 30.4 q9.d 0 10.0 35.0 55.0 5.0 5.0 50.0 40.0 34.8 13.0 43.5 8.7 q9.e 0 0 63.2 36.8 15.0 20.0 15.0 50.0 30.4 26.1 30.4 13.0 q9.f 0 15.0 20.0 65.0 10.0 5.0 20.0 65.0 13.0 0 30.4 56.5 q9.g 5.3 10.5 63.2 21.1 15.0 5.0 20.0 60.0 8.7 4.3 30.4 56.5 q9.h 5.3 15.8 57.9 21.1 0 15.0 50.0 35.0 0 17.4 69.6 13.0 q9.i 10.5 21.1 36.8 31.6 5.0 15.0 35.0 45.0 8.7 4.3 30.4 56.5 * percentage of students considering the potential hazard of gm trees as (4) serious hazard, (3) slight hazard, (2) no hazard (1) i do not know. conclusions our survey shows that in a relatively small country like slovakia the students of four selected universities share similar opinion on gm trees, though differences related to study orientation (economy vs. science-related) were identified. remarkable coincidence can be found between responses of males and females, in contrast different age categories face the issue of gm tree differently. nevertheless, the attitude of slovak students coincides with those described in other european countries. based on our survey we suggest that knowledge of students about gms should be extended since was lower compared to other published european surveys. moreover, better knowledge on gms is a prerequisite towards acceptance of this potentially powerful tool to solve several serious issues of humankind, despite of current restriction in eu countries. acknowledgements this work was supported by the project vega 2/0035/17. references clive j (2015) global status of commercialized biotech/gm crops. isaaa brief no. 51. isaaa: ithaca, ny. ec (2001) directive 2001/18/ec of the european parliament and of the council of 12 march 2001 on the deliberate release into the environment of genetically modified organisms and repealing council directive 90/220/eec ec (2015) directive (eu) 2015/412 of the european parliament and of the council of 11 march 2015 amending directive 2001/18/ec as regards the possibility for the member states to restrict or prohibit the cultivation of genetically modified organisms (gmos) in their territory. eu register of authorised gmos (2017) european commission gmos register (regulation ec 1829/2003) and the products subject to ec decisions on withdrawal from the market. fillatti jj, sellmer j, mccown b, haissig b, comai l (1987) agrobacterium mediated transformation bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:28 utc nova biotechnol chim (2017) 16(1): 12-19 19 and regeneration of populus. mol. gen. genet. 206: 192199. haggman h, raybould a, borem a, fox t, handley l, hertzberg m, lu mz, macdonald p, oguchi t, pasquali g, pearson l, peter g, quemada h, seguin a,tattersall k, ulian e, walter c, mclean m (2013) genetically engineered trees for plantation forests: key considerations for environmental risk assessment. plant biotech. j. 11: 785-798. isaaa (2017) international service for the acquisition of agri-biotech applications gm approval database, 54 p. kazana v, tsourgiannis l, iakovoglou v, stamatiou c, alexandrov a, araujo s, bogdan s, bozic g, brus r, bossinger g, boutsimea a, celepirovic n, cvrčková h, fladung m, ivankovic m, kazaklis a, koutsona p, luthar z, máchová p, malá j, mara k, mataruga m, moravčíková j, paffetti d, paiva j ap, raptis d, sanchez c, sharry s, salaj t, šijačič-nikolič m, telzur n, tsvetkov i, vettori c, vidal n (2015) public attitudes towards the use of transgenic forest trees: a cross-country pilot survey. iforest 9: 344-353. lucht jm (2015) public acceptance of plant biotechnology and gm crops. viruses 7: 4254-4281. nonic m, radojevic u, milovanovic j, perovic m, sijacicnikolic m (2015) comparative analysis of students' attitudes toward implementation of genetically modified trees in serbia. iforest 8: 714-718. pilate g, allona i, boerjan w, déjardin a, fladung m, gallardo, f, häggman h, jansson r, acker v, halpin c (2016) lessons from 25 years of gm tree field trials in europe and prospects for the future. in biosafety of forest transgenic trees: improving the scientific basis for safe development and implementation of eu policy directives. springer netherlands, 67-101. vettori c, gallargo f, häggman h, kazana v, migliacci f, pilate g, fladung m (2016) biosafety of forest transgenic trees: improving the scientific basis for safe development and implementation of eu policy directives. springer netherlands, 329 p. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:28 utc http://apps.webofknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&colname=wos&sid=2a7usr6nn8pmekklbez&author_name=pearson,%20l&dais_id=26708946&excludeeventconfig=excludeiffromfullrecpage http://apps.webofknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&colname=wos&sid=2a7usr6nn8pmekklbez&author_name=peter,%20g&dais_id=26977196&excludeeventconfig=excludeiffromfullrecpage 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http://apps.webofknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&colname=wos&sid=2a7usr6nn8pmekklbez&author_name=ulian,%20e&dais_id=81648630&excludeeventconfig=excludeiffromfullrecpage http://apps.webofknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&colname=wos&sid=2a7usr6nn8pmekklbez&author_name=walter,%20c&dais_id=18179062&excludeeventconfig=excludeiffromfullrecpage http://apps.webofknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&colname=wos&sid=2a7usr6nn8pmekklbez&author_name=mclean,%20m&dais_id=22243171&excludeeventconfig=excludeiffromfullrecpage nova biotechnologica et chimica 15-1 (2016) 47 doi 10.1515/nbec-2016-0005 © university of ss. cyril and methodius in trnava in vitro investigation of dand l enantiomer synergistic efects of some amino acids zuzana bystrická1, jozef lehotay2 1institute of medical chemistry, biochemistry and clinical biochemistry, faculty of medicine, comenius university, sasinkova 2, bratislava, sk-811 08, slovak republic (zuzana.bystricka@centrum.sk) 2department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic abstract: d-amino acids can arise from endogenous microbial flora, from ingestion with the diet or from spontaneous racemization of l-amino acids during ageing. in this work, the behavior of methionine, homocysteine and cysteine enantiomers was investigated in human serum in vitro during 0-72 h at incubation temperature 37 °c. the separation of enantiomers was realized in two dimensional on-line system (the connection of an achiral column purospher rp-18 endcapped and a chiral column chirobiotic tag). this system allowed simultaneous monitoring all tested amino acids and their enantiomers. the possible effect d-enantiomer on the behavior of its l-enantiomer (the synergistic effect) was evaluated during incubation time. the first results have showed that no synergistic effect of d-enantiomer on its lisomer has been observed in our experimental conditions in vitro. key words: amino acid; enantiomers; serum; synergism 1. introduction fuchs et al. (2005) reported that d-amino acids can arise from endogenous microbial flora, from ingestion with the diet or from spontaneous racemization of lamino acids incorporated in polypeptides during ageing. from all mentioned origin of d-amino acids, food is the most significant source. there are formed d-isomers from common l-amino acids either in the production processes or as a consequence of changes in the microbiological quality of the diet (friedman, 1999). some animal experiments have demonstrated that d-methionine (met) acts an otoprotective agent and its oral administration can be useful in mitigating hearing loss from platinum based chemotherapy, aminoglycosides and excessive noise (campell et al., 2007). on the other hand, other studies have showed that d-met is poorly utilized in humans (efron et al., 1969; kies et al., 1975; zezulka and calloway, 1976; printen et al., 1979; friedman and gumbmann, 1984). the conversion of d-met into l-met in rats was also indicated using a stable isotope methodology in the work of hasegawa et al. (2005). their results showed that almost all (more than 90 %) of administered d-met has been converted into lenantiomer in vivo. the conversion of d-met can be possible through oxidative deamination by d-amino acid oxidase to form α-keto-γ-methiolbutyric acid and after that α-keto-γ-methiolbutyric acid is stereospecifically reaminated by transaminases to form l-met. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:08 utc 48 bystrická, z. and lehotay, j. d-cysteine (cys) has significantly depressed the growth of mice and therefore it can be considered nutritionally antagonistic or toxic. after oral administration of dcys, the concentration of sulfate in rat urine was shown to increase (about 55 % of the dose), but the concentration of taurine in the serum did not increase indicating that it is probably not or only slowly converted to taurine. the administration of l-cys caused an increase the concentration of sulfate in the urine (about 33 % of the dose) as well as the concentration of taurine in the serum (krijgsheld et al., 1981; friedman and gumbmann, 1984). other studies have showed that d-cys has beneficial effects in acute alcohol intoxication and in cancer therapeutics and it shows no mutagenicity for humans (glatt and oesch, 1985; tsukamoto et al., 1990; roberts, 1995; oancea and formaggio, 2008). this work originated from the effort to simulate processes in vivo by using in vitro. we focused on investigation of the behavior of selected amino acid enantiomers in human serum and for these purposes two dimensional on-line system was used. this system allowed simultaneous monitoring all tested amino acids as well as their enantiomers. 2. materials and methods 2.1 chemicals acetonitrile, sodium phosphate monobasic monohydrate, perchloric acid, sodium borohydride, d-met, dl-homocysteine (hcy) and d-cys were purchased from sigma-aldrich (usa). ortho-phosphoric acid and 1-octanesulfonic acid sodium salt were purchased from fluka biochemika (switzerland). sodium hydroxide and methanol were obtained from merck (germany). d-hcy was not available and its racemate was used for measurements. 2.2 preparation of working solutions all standard solutions were prepared in water. doubly deionized water (≥ 18 mω cm) was produced on the apparatus rodem 6 (rodem water s.r.o., slovakia). working solutions of these amino acids were obtained by mixing of stock solutions. all solutions were stored at -80 °c until use. 2.3 calibration curves to determine molar concentrations, regression equations y = 159.5x + 737 (r2 = 0.999) for met, y = 381.7x – 1681 (r2 = 0.995) for hcy and y = 327.7x 7746 (r2 = 0.984) for cys were used. calibration curves were constructed in the range of 5-200 µmol/l for met, 2.5-30 µmol/l for hcy and 25-500 µmol/l for cys. 2.4 blood collection and sample pretreatment the blood was obtained from two healthy female volunteers (age 28 and 29). venous blood samples were collected after 12 h overnight fast. within 1 h of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:08 utc nova biotechnologica et chimica 15-1 (2016) 49 collection, the blood was centrifuged at 2200 g for 15 min. at 4 °c. obtained serum was storaged at -80 °c until the analysis. serum samples were pre-treated according to garaiova et al. (2013). shortly, 40 µl of sodium borohydride (1 mol/l in 0.1 mol/l naoh) was added to 100 μl of the serum, mixed and incubated at 50 °c for 30 min. 100 μl of perchloric acid (0.6 mol/l in water) was added and samples centrifuged at 14 000 g for 15 min. non-diluted supernatant (20 μl) was injected into the chromatographic system. 2.5 instrumentation and chromatographic conditions the chromatographic system consisted of an isocratic pump (deltachrom sds 030, watrex, praha, czech republic) and an electrochemical detector (coulochem ii, esa, chelmsford, uk). the detector was composed of guard cell (model 5020) and analytical cell (model 5010a, esa, chelmsford, uk) with porous graphite electrodes. analytical cells had potential +0.7 v (e1) and +0.9 v (e2), and the guard cell potential was set to +1.4 v. achiral separations were achieved on purospher rp-18 endcapped 250-4 mm (5 µm) (merck, darmstadt, germany) used together with a pre-column purospher star rp-18e (5 µm) (merck, darmstadt, germany). chiral separations were performed on chirobiotic tag 250-4.6 mm (5 µm) column (astec, usa) in an on-line system. the mobile phase contained 25 mmol/l phosphate buffer, 1 mmol/l octanesulfonic acid (ph 2.7), acetonitrile and methanol with ratio 94 : 3 : 3 (v/v/v). the separation system was proposed in our previous work (deáková et al., 2015). the flow rate was 0.4 ml/min. a nylon filter (47 mm in diameter) was used for mobile phase filtering. both columns were thermostated with jet stream ii plus hplc column thermostat (wo industrial electronics, austria) at 20 °c. sample incubations were realized by using two thermostats thermostat 1 (bibby stuart scientific block heater, uk) set to 50 °c for the sample treatment and thermostat 2 (eppendorf thermomixer 5437, germany) set to 37 °c for sample incubation. 2.6 experiment design the serum sample was divided into 2 aliquots (2 × 500 µl) and incubated at 37 °c during 0, 24, 48 and 72 h for total time of the analysis (150 min.). in first aliquot, only l-amino acids were present (without d-amino acids addition). in second aliquot, standards of d-enantiomes were artificially added to the serum (10 µl of denantiomers to 500 µl of the serum). d-enantiomers were added so that their final concentrations were at equimolar (or two times higher) concentrations of their corresponding l-enantiomers. in given time, 100 µl of the serum was picked up and pre-treated as described above. measurements of both aliquots were realized on the same day and concentration changes were evaluated in dependence on incubation time. the synergistic effect was estimated via differences in the behavior of l-enantiomer with and without denantiomer presence. the significance was determined by student´s t-test that was chosen according to fisher f-test results. statistically significant results were obtained bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:08 utc 50 bystrická, z. and lehotay, j. when p-values were lower than 0.05. measurements were realized on two independent samples in two replicates. 3. results and discussion gibson and wiseman (1951) reported that l-forms of amino acids disappeared from intestine of rats more rapidly than their corresponding d-enantiomers and therefore they regarded existence of a stereochemically specific mechanism for active absorption of l-amino acids. later results (viseman, 1953; agar et al., 1953; matthews and smyth, 1954) showed that transport of l-amino acids involves an active process and it was assumed that this did not apply to d-amino acids. on the other hand, jervis and smyth (1959) showed that competition between some dhistidine and l-met was possible using in vivo as well as in vitro techniques (wiseman, 1955). in the later work, jervis and smyth (1960) suggested that the transfer mechanism for l-met has not been absolutely stereochemically specific and that d-met is able to utilize this mechanism in vitro. utilization of d-met during parenteral nutrition in adult rats was investigated by cho and stegink (1979). infusion of the solution containing dl-met significantly increased plasma met levels during protein sparing therapy with accumulation of the d-isomer. there were also urinary met losses small as well as losses of methionine sulfoxide and alpha-keto-gamma-methiolbutyrate. in contrast to humans, adult rats utilized more than 99% of parenterally administered dor l-met. data obtained from the study focusing on the feeding of young infants by the formulas containing dl-met showed that substantial concentrations of d-met circulated in the plasma (stegink et al., 1971). these findings have indicated that the nutritional utilization of different d-amino acids varies widely in animals and humans. some d-amino acids may be both beneficial and deleterious. thus, whereas d-met is largely utilized as a nutritional source of its l-isomer, d-serine induces histological changes in the rat kidney. because d-amino acids are consumed by animals as well as humans as part of their normal diets, there exists a need for understanding of their roles in nutrition, food safety, microbiology, physiology and medicine (friedman, 1999). in the present study, l-amino acid concentrations of met, hcy and cys were estimated in the serum and concentration changes of l-enantiomers were evaluated in dependence on incubation time. the same procedure was realized in the presence of d-enantiomers which have equimolar concentrations of corresponding l-amino acids. differences in the behavior of l-enantiomers in the presence and absence of denantiomers were evaluated statistically. results showed that d-enantiomers had not significant influence on the behavior of their l-enantiomers (p = 0.087 for met, p = 0.177 for hcy and p = 0.173 for cys). for confirmation of these results, two times higher concentrations of d-enantiomers were added to the serum. we have assumed that higher concentrations of d-enantiomers could have had larger effect on their l-enantiomers. however, results did not confirm our assumptions (p = 0.319 for met, p = 0.453 for hcy and p = 0.731 for cys). more detailed results are shown in table 1. representative chromatograms of human serum after addition of d-amino acids at time 0 h and 24 h are shown in fig. 1. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:08 utc nova biotechnologica et chimica 15-1 (2016) 51 fig. 1. chromatograms of human serum after addition of d-amino acids at time 0 h and 24 h. d-amino acids were added to the serum at equimolar concentrations of their corresponding l-enantiomers. cysteine enantiomers were determined at electrode cell potential +0.7 v and homocysteine as well as methionine enantiomers were determined at +0.9 v. obtained data were also used for the comparison of the behavior of land denantiomers in human serum in vitro. there were used values of l-enantiomers and also d-enantiomers that were added to the serum so that they had equimolar concentrations to their corresponding l-enantiomers. the behavior of land disomers of same amino acid was evaluated at chosen incubation time. however, the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:08 utc 52 bystrická, z. and lehotay, j. standard of d-hcy was not available and its racemate was used for measurements, in the order to compare the behavior of these amino acid enantiomers, we calculated concentrations of l-hcy when racemate was used as total concentrations of l-hcy minus concentrations of d-hcy. results of t-test have showed no significant differences in the behavior of land d-enantiomers (p = 0.285 for met, p = 0.067 for hcy and p = 0.071 for cys) and therefore probably no stereospecific behavior of enzymes was observed in these experimental conditions. table 1. amino acid level changes with and without d-amino acids addition into serum. incubation time amino acid unit 0 h 24 h 48 h 72 h l-met µmol/l 22.7 21.9 15.1 15.2 l-met (d) µmol/l 21.6 16.5 12.1 10.8 l-hcy µmol/l 7.5 6.6 5.6 5.4 l-hcy (d) µmol/l 7.8 7.2 6.4 5.8 l-cys µmol/l 269.7 225.6 213.2 213.5 l-cys (d) µmol/l 270.6 211.9 206.4 203.1 d-amino acids were added to the serum at two times higher concentrations as their corresponding lenantiomers had. data are present as average values of two measurements (n=2, n-number of volunteers) abbreviations: l-met is a nomenclature for l-met without the presence of d-met and l-met (d) is a nomenclature for l-met in the presence of d-met. basing on obtained results, we can only hypothesize that necessary enzymes inclusive in the methionine metabolism should be not active in our experimental conditions or however, d-enantiomers had not the influence on the behavior of their l-isomers, the presence of l-enantiomers might have inhibitory effect on denantiomers as it published jervis and smyth (1960). 4. conclusions obtained results can provide the first view for us on in vitro behavior of amino acid enantiomers in human serum. no effect of d-enantiomers on the behavior of their l-enantiomers (no synergistic effect) was observed during chosen incubation time. also, no conversion of selected d-amino acids to their l-forms and no differences in the behavior of dand l-enantiomers were observed. taking into consideration knowledge of methionine metabolism, it is hard to simulate processes in the living organism by using in vitro system because the behavior of amino acids in vivo and in vitro is apparently different. moreover, there probably exists the difference between the utilization (behavior) of amino acids in humans and animals and therefore it is hard to generalize our conclusions. acknowledgements: the authors would like to thank all the volunteers who took part in this study. special thanks go to mrs. m. piatková for blood taking and prof. d.w. armstrong for chiral column lending. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:08 utc nova biotechnologica et chimica 15-1 (2016) 53 references agar, w.t., hird, f.j.r., sidhfu, g.s.: the active absorption of amino-acids by the intestine. j. physiol., 121, 1953, 255-263. campell, k.c.m., meech, r.p., klemens, j.j., gerberi, m.t., dyrstad, s.s.w., larsen, d.l., mitchell, d.l., el-azizi, m., verhuls, s.j.t., hughes, l.f.: prevention of noiseand drug-induced hearing loss with dmethionine. hearing res., 226, 2007, 92-103. deáková, z., ďuračková, z., armstrong, d.w., lehotay, j.: twodimensional high performance liquid chromatography for determination of homocysteine, methionine and cysteine enantiomers in human serum. j. chromatogr a, 1408, 2015, 118-124. efron, m.l., mcpherson, t.c., shih, v.e., welsh, c.f., mac creadz, r.a.: d-methioninuria due to dl-methionine ingestion. am. j. dis. chid., 117, 1969, 104-107. friedman, m.: chemistry, nutrition, and microbiology of d-amino acids. j. agric. food chem., 47, 1999, 3457-3479. friedman, m., gumbmann, m.r.: the utilization and safety of isomeric sulfurcontaining amino acids in mice. j. nutr., 114, 1984, 2301-2310. fuchs, s.a., berger, r., klomp, l.w.j, de koning t.j.: d-amino acids in the central nervous system in health and disease: minireview. mol. genet. metab., 85, 2005, 168-180. garaiova, i., muchova, j., nagyova, z., mislanova, c., oravec, s., dukat, a., wang, d., plummer, s.f., durackova, z.: effect of a plant sterol, fish oil and b vitamin combination on cardiovascular risk factors in hypercholesterolemic children and adolescents: a pilot study. nutr. j., 12, 2013, 18. gibson, q.h., wiseman, g.: selective absorption of stereo-isomers of aminoacids from loops of the small intestine of the rat. biochem. j., 48, 1951, 426-429. glatt, h., oesch, f.: mutagenicity of cysteine and penicillamine and its enantiomeric selectivity. biochem. pharmacol., 34, 1985, 3725-3728. hasegawa, h., shinohara, y., akahane, k., hashimoto, t.: direct detection and evaluation of conversion of d-methionine into l-methionine in rats by stable isotope methodology. j. nutr., 35, 2005, 2001-2005. cho, e.s., stegink, l.d.: d-methionine utilization during parenteral nutrition in adult rats. j. nutr., 109, 1979, 1086-1093. jervis, e.l., smyth, d. h.: competition between enantiomorphs of amino acids during intestinal absorption. j. physiol., 145, 1959, 57-65. jervis, e.l., smyth, d. h.: the active transfer of d-methionine bz the rat intestine in vitro. j. physiol., 151, 1960, 51-58. kies, c., fox, h., aprahamian, s.: comparative value of l-, dland dmethionine supplementation of an oat-based diet for humans. j. nutr., 105, 1975, 809-814. krijgsheld, k.r., glazenburg, e.j., scholtens, e., mulder, g.j.: the oxidation of land d-cysteine to inorganic sulfate ad taurine in the rat. biochim. biophys. acta, 18, 1981, 7-12. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:08 utc 54 bystrická, z. and lehotay, j. matthews, d.m., smyth, d.h.: the intestinal absorption of amino acid enantiomorphs. j. physiol., 126, 1954, 96-100. oancea, s., formaggio, f.: biological role of d-α-amino acids and their occurrence in foodstuffs – review. acta universitatis cibiniensis series e: food technology, 12, 2008, 3-18. printen, k.j., brummel, m.c., ericson, m.s., stegink, l.d.: utilization of d-methionine during total parenteral nutrition in post-surgica patients. am. j. clin. nutr., 32, 1979, 1200-1205. roberts, j.c.: stereoisomers of cysteine and its analogs potential effects on chemo and radioprotection strategies. amino acids, 8, 1995, 113-124. stegink, l.d., schmitt, j.l., meyer, p.d., kain, p.h.: effect of diets fortified with dl-methionine on urinary and plasma methionine levels in young infants. j. pediatr., 79, 1971, 648-655. tsukamoto, s., kanegae, t., nagoya, t., shimamura, m., mieda, y., nomura, m., hojo, k., okubo, h.: effects of amino acids on acute alcohol intoxication in mice-concentrations of ethanol, acetaldehyde, acetate and acetone in blood and tissues. arukoru kenkyuto yakubutsu ison (japan), 25, 1990, 429440. wiseman, g.: absorption of amino-acids using an in vitro technique. j. physiol., 120, 1953, 63-72. wiseman, g.: preferential transference of amino acids from amino-acid mixtures by sacs of everted small intestine of the golden hamster (memocricetus auratus). j. physiol., 127, 1955, 414-422. zezulka, a.y., calloway, d.h.: nitrogen retention in men fed isolated soybean supplemented with l-methionine, d-methionine, n-acetyl-l-methionine or inorganic sulfate. j. nutr., 106, 1976, 1286-1291. received 29 march 2016 accepted 6 july 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:08 utc nova biotechnologica et chimica 11-2 (2012) 93 doi 10.2478/v10296-012-0010-3 © university of ss. cyril and methodius in trnava determination of the functional groups in algae parachlorella kessleri by potentiometric titrations dana ivánová1, jana kaduková2, jana kavuličová1, hedviga horváthová2 1technical university in košice, faculty of metallurgy, department of chemistry, letná 9, košice, 042 00, slovak republic (dana.ivanova@tuke.sk) 2technical university in košice, faculty of metallurgy, department of material science, letná 9, košice, 042 00, slovak republic (jana.kadukova@tuke.sk) abstract: the acidic functional groups of the cell wall of native algae parachlorella kessleri were evaluated by potentiometric titrations. the gran´s method was applied to determination of the total, strong, weak and very weak acidities. the total organic acidity obtained for biomass was 3.93 mmol g-1, the largest content belonged to the strong acidic groups (2.13 mmol g-1) together with the weak acidic carboxylic groups (1.28 mmol g-1). very weak acidities represented by the amine groups (0.52 mmol g-1) did not exceed 14% and they formed the lowest numerous part of all acidic functional groups. key words: potentiometric titrations, gran´s method, acidic functional groups, algae parachlorella kessleri 1. introduction it is essential to understand the complex chemistry and mechanisms involved in metal biosorption in order to exploit algal biomass on an industrial scale for water, wastewater and effluent treatment (malik, 1999). metal biosorption by biomass mainly depends on the components of the cell, especially through cell surface and the spatial structure of the cell wall. various polysaccharides, proteins and lipids existing in algal cell walls have been proved to play a very important role in metal binding. some functional groups have been found to bind metal ions, especially carboxyl groups. there is some evidence confirming that the o-, n-, s-, or p-containing groups participate directly in binding of certain metals (wang and chen, 2009). green algae consist of mainly cellulose and high percentage of the cell wall is proteins (10-70%) bonded to polysaccharides forming glycoproteins (romera et al., 2007). proteins can contribute significantly to metal binding, offering the functional groups of amino acids (hydroxyl, carboxyl, sulfhydryl, amine, amide, imidazole). the cell walls of brown algae generally contain cellulose, alginic acids and sulphated polysaccharides. the predominant active groups of mentioned compounds are carboxyl and sulphate groups (romera et al., 2007). alginate and the sulphated matrix polysaccharide fucoidan constitute about 10-40% of the brown algal dry weight, 5-20% respectively. in brown algae the protein content is less than 30%. alginate and fucoidan are known for their metal binding properties whereby ion exchange between metal ions occurs (schiewer and wong, 2000; davis et al., 2003). in brown algae, the carboxyl groups of alginate are more abundant than either bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:47 utc 94 ivánová, d. et al. carboxyl or amine groups of the proteins and are therefore likely to be the main binding sites. sulphate groups appear to be of secondary importance (schiewer and wong, 2000; volesky, 2003; davis et al., 2003). hydroxyl groups are present in all polysaccharides but they only become negatively charged at ph > 10, at lower ph hydroxyl groups play a secondary role, too (volesky, 2003; davis et al., 2003). the applications of titration techniques become a powerful tool for the characterization of heterogeneous materials involved in biosorption and bioremediation processes. the acid-base titration can yield valuable specific data for further quantitative sorption work (naja et al., 2005). the aim of this study is to evaluate native biomass of parachlorella kessleri and to attempt to quantify the acidic functional groups in the cell wall of algae by potentiometric titrations. the gran´s method is applied to determine the total acidity of the functional groups, the quantity of the three types of acidities-strong, weak or very weak. 2. materials and methods 2.1 potentiometric titrations for titration, 0.25 g of dried untreated algal biomass – parachlorella kessleri was dispersed in 50 cm3 distilled water. an initial ph value was adjusted to 1.2 by addition with a known quantity of hcl. the suspension was titrated with 0.1 m naoh to ph 11.75. similar titration was performed on a blank solution (control) without algae. 2.2 gran´s method gran´s method (gran, 1952; rossotti and rossotti, 1965) consists on transforming a titration curve into two linear functions g1 versus the volume of added titrant before the end-point and g2 versus the volume of added titrant after the endpoint titration. the intersections of the linear portions with the volume axis correspond to the equivalence points va and vb. the definition of the gran´s function is as follows: g1 = (v0+v).10 -ph at ph < 7 (1) g2 = (v0+v).10 (ph-14) at ph > 7 (2) where v0 – the initial sample volume (cm 3) v – the volume of added titrant (cm3) the total organic acidity (ato) of a sample can be divided into three different chemical acidic groups depending on their apparent ionization constants (naja et al., 2005): strong acidities at ph < 4 0 ana1 s n.m )vv( )meqg(a − =− (3) weak acidities at 4 < ph < 7 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:47 utc nova biotechnologica et chimica 11-2 (2012) 95 0 anenae1 w n.m )vv()vv( )meqg(a −−− =− (4) very weak acidities at ph > 7 0 bnnbenbneb1 vw n.m )]vv()vv)][(vv()vv[( )meqg(a −−−−−− =− (5) vwwsto aaaa ++= (6) where n0 – the normality of the base used, v and vn – the total volume of the base added after titration of the sample and control, respectively, ve– the volume of base in the end-point titration, m – sample weight (g). the suffix n corresponds to the control. 3. results and discussion the potentiometric titration curves of the untreated algae parachlorella kessleri and the control, curves fitted by boltzmann function are shown in figure 1a) and gran´s plot of the sample and the control and their linearization are shown in figure 1b). four equivalence points were extrapolated according to the gran´s method. van a va are the equivalence volumes determined by the acidic slopes (before the equivalence point) of the gran´s function of the control and the sample. vbn a vb are the equivalence volumes determined by the basic slopes (after the equivalence point) of the gran´s function of the control and the sample. in addition to these equivalence points, other points indicating the endpoint of the control ven, the first and second endpoints of the sample ve1 and ve2 were determined by boltzmann function minimizing the mean square deviations between model and experimental data. van is the naoh volume necessary to titrate the excess of hcl over the sample total acidity, va – van is the titrant volume necessary to reaction with strong acidic groups, such as phosphoric or sulfonate groups, as well as carboxylic groups linked to aromatic functions at ph < 4, ve1 – va and ve2 – ve1 are attributed to the ionization of carboxylic and some proteic groups at 4 < ph < 7, vb – ve2 is attributed to phenolic and amine groups of proteins at ph > 7 (brunelot et al., 1989; naja et al., 2005). the strong, weak, very weak and total acidities calculated according to eqs. (3)– (6) as well as the quantity of acidic groups for other green and brown algal cells are indicated in table 1. the total number of acidic groups was found 3.93 mmol g-1. it may be observed from the data in table 1 that approximately 54.2% of all binding sites in the cell wall of biomass consist of strong acidic groups (phosphoric, sulfonate and carboxylic groups linked to aromatic compounds). weak acidic sites attributed to the carboxylic groups present the third of total acidity, whereas very weak acidities of amine groups on the biomass surface represent 13.2% of all acidic groups. it could be noted that the weak acidity attributed to carboxylic and the very weak acidity to amine functions of proteins formed nearly the half of total organic acidity. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:47 utc 96 ivánová, d. et al. 0 1 2 3 4 5 6 7 8 9 10 11 12 4 6 8 10 12 14 16 18 20 22 24 v n aoh [ml] ph algae algae-calculated control control-calculated v a v e1 vb v e2 v an v en v bn0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 4 6 8 10 12 14 16 18 20 22 24 vn aoh [ml] g algae control fig. 1. a) titration curves of a sample (untreated algae parachlorella kessleri) and a control (without algae) and curves fitted by boltzmann function, b) gran´s function (g) of the sample ( ) and of the control ( ) and their linearization. a) b) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:47 utc nova biotechnologica et chimica 11-2 (2012) 97 it appears that only content of the strong acidic sites is slightly higher in comparison with other green and brown algae whereas values of weak and very weak acidities correspond to these obtained in other studies (table 1). the carboxylic groups are generally the most abundant acidic functional groups in the brown algae and they constitute the highest content of total acidic sites (aw is greater than 50%). table 1. types and quantity of acidic sites for untreated algae parachlorella kessleri and comparison with other green, brown algae. as aw avw ato algal type (mmol g-1) references parachlorella kessleri 2.13 1.28 0.52 3.93 present work chaetophora elegans 0.85 2.99 3.83 (andrade et al., 2005) caulerpa scalpelliformis 2.89 (aravindhan et al., 2007) ulva lactuca 0.19 1.62 1.81 (murphy et al., 2007) ulva spp. 0.44 1.50 1.94 (murphy et al., 2007) gr ee n chlorella vulgaris 0.1 0.11 0.22 (hadjoudja et al.., 2010) fucus serratus 1.28 1.93 0.36 3.56 (ahmady-asbchin et al., 2008) sargassum vulgare 0.5 1.5 0.75 2.75 (davis et al., 2000) sargassum fluitans 0.3 1.5 0.75 2.55 (davis et al., 2000) sargassum filipendula 0.3 1.6 0.80 2.70 (davis et al., 2000) b ro w n fucus vesiculosus 2.46 (grimm et al., 2008) differences in the metal biosorption capacity of mentioned green and brown algae may be attributed to differences in cell wall compounds, providing different functional groups responsible for metal biosorption (davis et al., 2003). the maximum sorption capacity for copper uptake by green and brown algae is showed as comparable parameter in table 2. table 2. a comparison of copper biosorption capacities for various biosorbents. qmax algal type (mg g-1) references parachlorella kessleri 43 (kaduková and horváthová, 2011) codium vermilara 16.9 (romera et al., 2007) spirogyra insignis 21.3 (romera et al., 2007) ulva lactuca 43.8 (murphy et al., 2009) g re en ulva spp. 20.7 (murphy et al., 2007) fucus serratus 101.6 (ahmady-asbchin et al., 2008) sargassum vulgare 59.1 (davis et al., 2000) sargassum fluitans 50.8 (davis et al., 2000) sargassum filipendula 56.5 (davis et al., 2000) b ro w n fucus vesiculosus 23.4 (grimm et al., 2008) green algae showed lower sorption capacity than brown algae. the carboxyl or amine groups of the proteins are likely to participate as main metal-binding sites in the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:47 utc 98 ivánová, d. et al. green algae. on the other hand, the metal adsorption capacities of the brown algae are directly related to the presence of carboxylic groups on the alginate polymer, which counts as a significant component of the dried seaweed biomass (ahmadyasbchin et al., 2008; grimm et al., 2008). according to the data presented in literature (davis et al., 2003, murphy et al., 2007, grimm et al., 2008) it is obvious that metal uptake by both, green and brown algae, is proportional to the amount of acidic sites. 4. conclusions polysaccharides and proteins of biomass provide various possibilities for metal binding. titration method is subservient to indication of functional acidic groups on the surface of green algae. the total organic acidity associated with strong, weak and very weak acidic active sites was established. in green algae parachlorella kessleri, the quantity of strong acidic groups represents 54.2% of all binding sites in the cell wall of biomass. weak acidic sites attributed to the carboxylic groups present 32.6% of total acidity. very weak acidities on the biomass surface represent 13.2% of all acidic groups. in general, in scientific literature results of biosorption or potentiometric titrations are published separately. but to understand the role of functional groups the interconnection would be very helpful. carboxylic and amine groups of proteins could be the key functional groups of the algal cell wall responsible for metal binding within biosorption. further research is required to understand the role of binding sites on the surface of biomass in biosorption. acknowledgement: this work was supported by the slovak grant agency vega, grant no. 1/0235/12. references ahmady-asbchin, s., andres, y., gérente, c., le cloirec, p.: biosorption of cu(ii) from aqueous solution by fucus serratus: surface characterization and sorption mechanisms. bioresour. technol., 99, 2008, 61506155. aravindhan, r., rao, j.r., nair, b.u.: removal of basic yellow dye from aqueous by sorption on green alga caulerpa scalpelliformis. j. hazard. mater., 142, 2007, 68-76. brunelot, g., adrian, p., rouiller, j., guillet, b., andreux, f.: determination of dissociable acid groups of organic compounds extracted from soils, using automated potentiometric titration. chemosphere, 19, 1989, 14131419. davis, t.a., volesky, b., vieira, r.h.s.f.: sargassum seaweed as biosorbent for heavy metals. water res., 34, 2000, 4270-4278. davis, t.a., volesky, b., mucci, a.: a review of the biochemistry of heavy metal biosorption by brown algae. water res., 37, 2003, 4311-4330. gran, g.: determination of the equivalence point in potentiometric titrations. part ii, analyst, 77, 1952, 661-671. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:47 utc nova biotechnologica et chimica 11-2 (2012) 99 grimm, a., zanzi, r., björnbom, e., cukierman, a.l.: comparison of different types of biomasses for copper biosorption. bioresour. technol., 99, 2008, 2559-2565. hadjoudja, s., deluchat, v., baudu, m.: cell surface characterization of microcystic aeruginosa and chlorella vulgaris. j. colloid interface sci., 342, 2010, 293-299. kaduková, j., horváthová, h.: biosorption of copper, zinc and nickel from multi-ion solutions. biotechnology&metals, 2nd international conference, košice, 2011, 45-48. malik, d.j., streat, j., greig, j.: characterization and evaluation of seaweedbased sorbents for treating toxic metal-bearing solutions. process saf. environ. protect., 77, 1999, 227-233. murphy, v., hughes, h., mcloughlin, p.: cu(ii) binding dried biomass of red, green and brown macroalgae. water res., 41, 2007, 731-740. murphy, v., hughes, h., mcloughlin, p.: enhancement strategies for cu(ii), cr(iii) and cr(vi) remediation by variety of seaweed species. j. hazard. mater., 166, 2009, 318-326. naja, g., mustin, ch., volesky, b., berthelin, j.: a high-resolution titrator: a new approach to studying binding sites of microbial biosorbents. water res., 39, 2005, 579-588. romera, e., gonzález, f., ballester, a., blázquez, m.l., muñoz, j.a.: comparative study of biosorption of heavy metals using different types of algae. bioresour. technol., 98, 2007, 3344-3353. rossotti, f.j.c., rossotti, h.: potentiometric titrations using gran plots. j. chem. educ., 42, no. 7, 1965, 375-378. schiewer, s., wong, m.h.: ionic strength effects in biosorption of metals by marine algae. chemosphere, 41, 2000, 271-282. volesky, b.: sorption and biosorption. bv sorbex, inc., montreal, 2003, 316 pp. wang, j., chen, c.: biosorbents for heavy metals removal and their future. biotechnol. adv., 27, 2009, 195-226. presented at the 2nd international conference “biotechnology and metals 2011”, september 22–23, 2011, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:47 utc 12 nova biotechnologica et chimica 15-1 (2016) doi 10.1515/nbec-2016-0002 © university of ss. cyril and methodius in trnava hplc enantioseparation of phenylcarbamic acid derivatives by using macrocyclic chiral stationary phases ( katarína hroboňová1, jozef lehotay2, jozef čižmárik3 1institute of analytical chemistry, faculty of chemical and food technology, slovak university of technology, radlinského 9, sk-812 37 bratislava, slovak republic (katarina.hrobonova@stuba.sk) 2faculty of natural sciences, university of ss.cyril and methodius, j. herdu 2, sk-917 01 trnava, slovak republic 3comenius university, faculty of pharmacy, department of pharmaceutical chemistry, odbojárov 10, sk-832 32 bratislava, slovak republic abstract: the hplc by using chiral stationary phases based on macrocyclic antibiotics, dimethylphenyl carbamate cyklofructan 7 and β-cyclodextrin in terms of polar-organic separation mode (mobile phase methanol/acetonitrile/acetic acid/triethylamine) were used for enantioseparation of alkoxy derivatives of phenylcarbamic acid. the effect of the analyte structures on the efficiency of enantioseparation was investigated. the most suitable stationary phase was teicoplanin aglycone, where the separations of the enantiomers were obtained (the resolution value from 0.65 to 2.90, depending on the structure of the analyte). significant effect on the resolution of the enantiomers has position of alkoxy substituent in the hydrophobic part of the molecule. the enantiorecognition was achieved for 3-alkoxysubstituted derivatives. key words: hplc, chiral stationary phase, structure of the analyte, esters of phenylcarbamic acid 1. introduction chiral separation is based on the ability of the chiral selector to form diastereoisomeric complexes preferably with one of the enantiomer of a chiral analyte. the higher affinity differences of selectors to enantiomers lead to greater efficiency of the chiral recognition. the formation of stable complexes between chiral selector and enantiomer, and their structural complementarity are the main requirement for selection of suitable chiral selector (zhang et al., 1979). chromatographic techniques, particularly high performance liquid chromatography (hplc) have become the most used in the separation of enantiomers due to the availability of a large number of stationary phases containing different chiral selectors, high reproducibility, and often universality. the most challenging aspect in the development of chiral hplc method is to select the chiral stationary phase (csp). the many of available csps provides a more "degrees of freedom" for the selection of a suitable stationary phase. on the other hand, a testing of all available csp for the analysis of all chiral molecules is time-consuming. the most commonly used are csps based on saccharides and macrocyclic antibiotic chiral selectors due to their multifunctionality and the ability to use a several separation modes (reversed-phase, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:59 utc nova biotechnologica et chimica 15-1 (2016) 13 normal-phase, and polar-organic phase mode). the main requirement for newly developed csp is particularly high enantioseparation efficiency for a wide range of structurally different compounds, or an increase in separation efficiency compared with existing csp (beesley, 2011). the racemic compounds used in this study are derivatives of alkoxysubstituented esters of phenylcarbamic acid which are potential local anesthetics (fig. 1). chemically, they are weak bases which molecules contain lipophilic and hydrophilic part interlinked with connecting chain. the various csps can be used for hplc enantioseparation of local anesthetics of phenylcarbamic acid type. normal-phase separation mode (np) was suitable for enantioseparations on polysaccharide (zhang et al., 2005) and cyclofructan csps (sun et al., 2009). reversed-phase mode (rp) was especially suitable for cyclodextrin-based (hroboňová et al., 2003) and polysaccharide-based csps (peng et al., 2010). polar-organic (po) mode was mostly used for separations on chiral stationary phases based on macrocyclic antibiotics (rojkovičová et al., 2003; 2005). the selection of separation mode related with the solubility of analytes and type of matrix. the goal of the work was to separate the enantiomers of alkoxyphenylcarbamic acid derivatives by hplc using various types of chiral stationary phases in terms of polar-organic separation mode. chiral stationary phases tested were based on i) the macrocyclic antibiotics (teicoplanin and teicoplanin aglycone); ii) β-cyclodextrin and iii) cyclofructan (3,5-dimethylphenyl carbamate of cyclofructan 7). all types of tested columns are multimodal in relation to the chromatographic conditions (np, rp, po separation mode). the influence of the structure of analytes on the efficiency of enantioseparation on different types of csps was studied as a contribution to the systematic approach to selection of suitable stationary phase. * r1 r 2r 3 o o n h 2 3 nr. r1 r2 r3 nr. r1 r2 r3 1 2-oc5h11 15 2-oc4h9 2 och3 3-oc5h11 16 och3 3-oc4h9 3 2-oc4h9 17 2-oc4h9 4 2-oc5h11 18 2-oc5h11 5 2-oc6h13 19 2-oc6h13 6 3-oc4h9 20 3-oc4h9 7 3-oc5h11 21 3-oc5h11 8 oc2h5 3-oc6h13 22 oc2h5 3-oc6h13 9 2-oc4h9 23 2-oc4h9 10 2-oc5h11 24 2-oc6h13 11 2-oc6h13 25 3-oc4h9 12 3-oc4h9 26 3-oc6h13 13 3-oc5h11 14 n oc3h7 3-oc6h13 n oc3h7 fig. 1. chemical structures of alkoxysubstituted derivatives of phenylcarbamic acid under study. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:59 utc 14 hroboňová, k. et al. 2. material and methods 2.1 materials the racemates of alkoxyphenylcarbamic acid derivatives were synthesized according the literature (búčiová et al., 1987; búčiová et al., 1991; búčiová et al., 1992). the stock solutions were prepared by dissolving the substance in the mobile phase (concentration of 1.0 mg⋅ml-1, storage at 4 °c). methanol and acetonitrile (hplc grade, jt baker), triethylamine (for synthesis, merck) and glacial acetic acid (100 %, merck) were used for the preparation of the mobile phase. 2.2 hplc instrumentation and conditions the liquid chromatograph agilent 1100 series, consisting of a binary high pressure pump, injection valve rheodyne, column thermostat, and diode array detector was used. separation of the enantiomers were performed on chirobiotic t (astec), chirobiotic tag (astec), 3,5-dimethylphenylcarbamate cyclofructan 7 (astec), βcyclodextrin chiradex (merck) (4×250 mm i.d., 5 μm) chiral chromatographic columns. the mixture of acetonitrile/methanol/acetic acid/triethylamine (20/80/0.3/0.2 v/v/v/v) was used as mobile phase. the flow rate was 0.8 min⋅ml-1, the injection volume 20 μl. the column temperature was maintained at 22 °c. the chromatograms were recorded at wavelength 240 nm. all measurements were done in three replicates. the retention factor (k) values of first and second eluted enantiomer were calculated as the ratio of differences between retention time of enantiomer (tr) and dead time (tm) to dead time. the selectivity factor were calculated as the ratio of retention factors of second and first eluted enantiomer (α = k2/k1). the resolution values (rs) of enantiomeric forms were calculated by ratio of difference between retention times of enantiomers (tr1, tr2) to sum of peak widths at half peak height (w05,1, w05,2) as follow: rs = 1.18 * (tr2 tr1) / (w05,1 + w05,2). 3. results and discussion 3.1 chromatographic conditions in the previous work (hroboňová et al., 2003), the β-cyclodextrin csp in reversed-phase separation mode was used for separation of selected compounds. the mobile phase was a mixture of acetonitrile and 0.1 mol⋅l-1 sodium acetate, ph=5.2 (88/12, v/v). the enatioseparation only 2-alkoxysubstituted derivatives was achieved (the referred chromatographic conditions were not suitable for enantioseparation of 3alkoxysubstituted derivatives, rs ∼ 0). the resolution values (rs) ranged from 0.6 to 1.2, depending on the length of the alkoxysubstituent (c4 – c7). the highest resolution values were obtained for compounds with hexyloxy substituent, which, in terms of biological activity, have the highest local anesthetic activity. dominant types of interactions responsible for the enantiorecognition were steric effects influenced by bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:59 utc nova biotechnologica et chimica 15-1 (2016) 15 the length and position of the alkoxy chain. (hroboňová et al., 2003) the mechanism of enantiomeric recognition on β-cyclodextrin csp in rp mode was based on the formation of inclusion complexes driven by the hydrophobic interactions between the analytes and the cyclodextrin. the hydrophobic part of the analyte penetrated into the cyclodextrin cavity and displaced the molecules of solvent. inside the cavity the complex is stabilized by van der waals interactions optionally by other hydrophilic interactions (hydrogen bonding, dipole-dipole interactions) with the hydroxyl groups of the cyclodextrin skeleton. the π-π interactions can be dominant for aromatic compounds. the inclusion into the cavity is related to the size of the molecule. (lämmerhofer, 2010). kopecky and coworkers in the micellization study of the local anesthetic drug carbisocaine present the inclusion of the carbisocaine c7 alkyl chain into the cyclodextrin cavity and the role of the hydrophobic interaction in complexation with β-cyclodextrin in aqueous solution (kopecky et al., 2002). the present work was orientated on separation of the enantiomers of the whole group of substances (2-positioned and 3-positioned alkoxyderivatives), and therefore other types of stationary and mobile phases were tested. the polar-organic separation mode is most commonly used for csp based on macrocycles. the mobile phase consists of a mixture of polar organic solvents (methanol, acetonitrile) with the addition of ionic modifiers (acids and bases). methanol molecules are able to participate in the formation of hydrogen bonds, acetonitrile contributes to dipol interactions, which influenced the enantioseparation (value rs). the acid/base ratio in the mobile phase influenced also the ionization of groups (amino, hydroxyl, carboxyl) in stationary phases and thus to affect the ion interaction with ionizable groups of alkoxyphenylcarbamic acid derivatives under study (quaternary nitrogen in piperidine or pyrrolidine heterocycle of phenylcarbamic acid derivative molecule) (meričko et al., 2008). the separation of the enantiomers of selected analytes was realized by using a methanol/acetonitrile/acetic acid/triethylamine 20/80/0.3/0.2 (v/v/v/v) as mobile phase at 22 °c. the ratio of the concentrations of ionic modifiers in the mobile phase, which is most effective for achieving the separation was 3:2 (glacial acetic acid:triethylamine). the acidic nature of the mobile phase supports the ionization of amino groups in the molecules of separated compounds and the stationary phase. it can be assumed that the charge interactions had a positive influence on enantioseparation. 3.2 comparison of enantioseparation on different csps the stationary phases with different types of chiral selectors were used for enantioseparations of alkoxyphenylcarbamic acids derivatives. the aim was to compare the selectivity and efficiency. in the case po mode is the inside of the β-cyclodextrin cavity blocked with the molecules of solvent of mobile phase, and therefore it is not available for the formation of inclusion complexes with molecules of the analyte. the hydrophilic interactions between the molecules of analyte and the polar surface of the βcyclodextrin skeleton are supported. the enantioselectivity can be influenced by the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:59 utc 16 hroboňová, k. et al. strength of polar interactions (hydrogen bonding, dipole-dipole interactions). (lämmerhofer, 2010). the variety of stereoselective interactions (hydrogen bonding, π-π, electrostatic interactions, hydrophobic, steric or repulsive interactions, formation of inclusion complexes) of macrocyclic antibiotic (teicoplanin, teicoplanin aglycone) csps related to the presence of many stereogenic centers, the heterogenity of functional groups and structural specificity of different types of macrocyclic antibiotic molecules. this group of csp is predominantly used for the separation of enantiomers containing ionic groups at/or near the stereogenic center (wang, 2000). cyclofructan 7 (cf7) is macrocyclic oligosaccharide consisting of seven β-linked fructofuranose units. the fructofuranose units are distributed internally or externally around the "crown" ether skeleton. the cf connected hydrophilic and hydrophobic groups. in particular, derivatized (aliphatic or aromatic) cyclofructan csps are effective for enantiomer separation of compounds with amine functional groups. aromatically derivatized cf csps provide effective separation based on the use of hydrogen, π-π and dipole-dipole interactions supplemented by steric effects (sun et al., 2009). table 1 summarizes the retention factor, resolution, and selectivity factor for the separation of selected phenylcarbamic acid derivatives by using the four types of csps. teicoplanin and teicoplanin aglycone csps in po separation mode were appropriate for separation of enantiomers of 3-alkoxysubstituted derivatives while the 2-alkoxysubstituted derivatives were not separated. highest selectivity factors were achieved generally by using the csp without saccharide units in the molecule (teicoplanin aglycone). the retention factors of the enantiomers ranged from1.29 − 6.30 and the resolution value from 1.09 to 2.90. the values of retention factors achieved on teicoplanin csp were lower (1.03 − 4.31) in comparison with teicoplanin aglycone csp. resolution values ranged from 1.72 to 2.75. the most effective enantioseparation (rs = 2.90 for teicoplanin aglycone csp, rs = 2.75 for teicoplanin csp) was achieved for compound (2) (r1 = pyrrolidine, r2 = -oc3h7, r3 = oc6h13) as is documented in fig. 2. the most effective enantioseparation (rs = 2.90 for teicoplanin aglycone csp, rs = 2.75 for teicoplanin csp) was achieved for compound (2) (r1 = pyrrolidine, r2 = -oc3h7, r3 = -oc6h13). it can be assumed that saccharide units of the native teicoplanin molecule may have negative influence on chiral recognition process of enantiomeric forms. it is probably the effect of steric hindrance (berthod et al., 2000). by using β-cyclodextrin csp only the partial separation (rs = 0.36 and 0.44) of the compounds (2) and (16) (r2 = -och3) was achieved. compounds with longer r2 (ethoxy-, propoxy-) substituents near the stereogenic center were not resolved. this is probably due to steric hindrance and shading of stereogenic center. the values of retention factor obtained on β-cyclodextrin csp were the lowest (k1 = 0.08 − 0.38) in comparison with other csp tested. the reason of poor enantioseparation was probably the unavailability inclusion of analyte molecules into the β-cyclodextrin cavity in the po separation mode. the cyclofructan csp (dmp-cf7) in po separation mode was not suitable for resolution of phenylcarbamic acid derivatives under study. the retention of analytes bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:59 utc nova biotechnologica et chimica 15-1 (2016) 17 was higher in comparison to cyclodextrin csp (about two times), but any separation of enantiomers was observed. table 1. chromatographic results (retention factor (k1), resolution (rs) and selectivity factor (α)) for alkoxysubstituted derivatives of phenylcarbamic acid obtained on four chiral stationary phases in the polarorganic separation mode. csp teicoplanin teicoplanin aglycone β-cyclodextrin dmp -cf7 nr. k1 α rs k1 α rs k1 α rs k1 1 2.42 3.37 0.17 0.80 2 3.96 1.14 2.75 5.74 1.25 2.90 0.32 1.01 0.36 0.85 3 2.33 3.69 0.13 0.81 4 2.22 3.20 0.15 0.80 5 2.08 3.14 0.15 0.75 6 3.83 1.14 2.34 6.30 1.20 2.46 0.28 0.80 7 3.77 1.14 2.66 5.52 1.21 2.66 0.29 0.82 8 3.57 1.13 2.69 5.47 1.21 2.87 0.28 0.78 9 2.65 3.43 0.13 0.75 10 2.46 2.90 0.13 0.74 11 2.36 2.76 0.14 0.72 12 4.31 1.13 1.95 5.72 1.22 2.55 0.38 0.74 13 4.17 1.13 1.99 5.36 1.21 2.44 0.38 0.73 14 4.06 1.13 2.17 5.21 1.22 2.52 0.37 0.72 15 1.03 1.57 0.11 0.47 16 2.02 1.12 2.13 3.70 1.16 1.65 0.25 1.03 0.44 0.49 17 1.84 1.75 0.08 0.45 18 0.95 1.61 0.09 0.44 19 0.93 1.53 0.09 0.43 20 1.93 1.10 1.81 3.58 1.14 1.25 0.20 0.47 21 1.67 1.11 1.84 3.47 1.14 1.21 0.21 0.46 22 1.52 1.11 1.97 3.35 1.13 1.20 0.21 0.46 23 1.19 1.42 0.11 0.44 24 1.07 1.29 0.11 0.44 25 1.81 1.11 1.72 0.24 0.42 rsd ≤ 3 %, n = 3; chromatographic conditions: mobile phase: acetonitrile/methanol/acetic acid/triethylamine (80/20/0.3/0.2 v/v/v/v); column temperature: 22 °c; flow rate: 0.8 min⋅ml-1; detection: uv 240 nm; tm determined as the retention time of acetonitrile; tm(teicoplanin) = 3,81 min; tm(teicoplanin aglycone) = 3.85 min; tm(dmp-cf7) = 3.93 min; tm(β-cyclodextrin) = 3.13 min. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:59 utc 18 hroboňová, k. et al. a b fig. 2. hplc chromatograms of compound (2) separated on teicoplanin (a) and teicoplanin aglycone (b) csps. chromatographic conditions: as in table 1. 3.3 the effect of structure of the analyte on the separation of enantiomers the chemical structure of analyte has a dominant influence on the enantiomeric recognition. this may affect the availability to the chiral centers and the functional groups of the chiral selector, and the type of interactions responsible for chiral recognition. the work was focused on the investigation of the effect of structure alkoxyphenylcarbamic acid derivatives on efficiency of enantioseparation using macrocyclic antibiotics (teicoplanin, teicoplanin aglycone) csps. from the chiral separation point of view the influence of i) the position, ii) length of alkoxy substituent (r3) in the lipophilic part of the molecule, and iii) the effect of alkoxy substituent (r2) in the connecting chain of the molecule were evaluated. β-cyclodextrin csp in the rp mode was suitable for the enantioseparation of 2alkoxysubstituted derivatives, while the derivatives with 3-alkoxysubstitution provided no enantioseparation. the position of alkoxy substituent near stereogenic center was preferred from the reason of molecule compatibility for inclusion into the cyclodextrin cavity and separations results. (hroboňová et al., 2003) 4-alkoxysubstituted derivatives exhibit very low local-anesthetic activity and they were not synthesized. table 1 has documented that the compounds which have a substituent r3 in the 3position on the aromatic ring are separated (rs = 0.96 to 2.90) on glycoprotein (teicoplanin, teicoplanin aglycone) csps. the substituent near the stereogenic center (2-positioned derivatives) of the separated compound may intervene in the chiral recognition process by steric hindrance, where the substituent shades stereogenic center and blocking possible interaction sites. the values of the retention factors of 3alkoxy derivatives were about two times higher in comparison with 2-alkoxy derivatives. the separation of enantiomers is slightly affected by non-polar interactions. increase of lipophilicity of separated compounds (r3: c4-c6) caused decrease of retention factors and resolution values of the enantiomers on both stationary phases. the hydrophilic part of the molecule contains the nitrogen heterocycle (r1: piperidino, pyrrolidino). higher values of resolution and retention factors were achieved for pyrrolidino derivatives on both tested columns. the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:59 utc nova biotechnologica et chimica 15-1 (2016) 19 piperidino substituent is probably steric hindrance in chiral recognition process. the values of the retention factor and resolution decreased with increase of carbon atoms (c1 – c3) in the r2 substituent. 3.4 separation of the local anaesthetics diperodon (3-(1-piperidinyl)-1,2-propanediol bis(phenylcarbamate)) and carbisocaine ((2-(heptyloxy)phenyl)-2-(diethylamino)-1-methylethyl ester of carbamic acid) are a local anesthetic drugs used as the hydrochloride salt; applied to the skin for abrasions, irritations, and pruritus and intrarectally for relief of pain from hemorrhoids.(cohen et al., 1977) table 2 summarised the chromatographic parameters for carbizocaine and diperodon enantioseparation on a teicoplanin, teicoplanin aglycone, β-cyclodextrin, and 3,5-dimethylphenyl carbamate cyclofructan 7 csps by using the same mobile phase. even though the retention of carbisocaine was significantly higher on macrocyclic antibiotic csps in po separation mode, the any enantiorecognition was observed. carbizocaine is the the derivative of phenylcarbamic acid with heptyloxysubstitution in the 2-position on the aromatic ring (table 2). csps and mobile phase used are not suitable for separate the enantiomers of compounds with substitution near the stereogenic center, in comparison to 3positioned derivatives. diperodon enantiomers were separated on csps based on macrocyclic antibiotics, where the highest value of resolution (rs = 1.79) was achieved using teicoplanin aglycone column. obtained results were compatible with thus obtained previously (part 3.3). figure 3 shows the chromatogram of the enantioselective separation of diperodon. table 2. chromatographic results for carbisocaine and diperodon obtained on four chiral stationary phases in the polar-organic separation mode. csp k1 α rs teicoplanin 0.99 1.14 1.01 teicoplanin aglycone 2.86 1.29 1.79 β-cyclodextrin 0.85 1.0 diperodon n . hcl o o o n h n h o * dmp-cf7 0.11 1.0 teicoplanin 2.11 1.0 teicoplanin aglycone 2.32 1.0 β-cyclodextrin 0.16 1.0 carbizocaine *o o n h n o . hcl dmp-cf7 0.76 1.0 rsd ≤ 3%; n = 3, chromatographic conditions as in table 1. fig. 3. hplc separation of diperodon enantiomers on teicoplanin aglycone csp. chromatographic conditions: as in table 1. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:59 utc 20 hroboňová, k. et al. 4. conclusions hplc enantioseparation of potential local anesthetics of alkoxysubstituted phenylcarbamic acid type was achieved in terms of a polar-organic separation mode using macrocyclic antibiotic csp (teicoplanin, teicoplanin aglycone), in contrast to the β-cyclodextrin and 3,5-dimethylphenylcarbamate cyclofructan 7 based chiral stationary phases. comparison of the results shows that while the position of the alkoxy substituent near stereogenic center significantly affected retention and recognition of enantiomers, the length of the substituent has only slightly effect. enantiomeric separation was achieved for the 3-alkoxy substituted derivatives. alkoxy substituent in the connecting chain of molecule had not significant impact on the separation of the enantiomers. increasing of nitrogen heterocycle (piperidine, pyrrolidine) caused decrease of resolution value. macrocyclic antibiotic columns in polar-organic separation mode show enantioselectivity for local anaesthetic drug diperodone. these obtained results are complementary to study on β-cyclodextrin csp in reversed-phase separation mode, where was reached the enanantioseparation only for 2-alkoxysubstituted derivatives (hroboňová et al., 2003; hroboňová et al., 2002b). acknowledgments: support of this work by vega grant no. 1/0499/14 of the scientific grant agency of the ministry of education, science, research, and sport of the slovak republic and the slovak academy of sciences. the authors are grateful to d.w. armstrong for providing of macrocyclic and cyclofructan hplc columns. references beesley, t.e.: review of chiral stationary phase development and chiral applications. lc/gc europe, 24, 2011, 270-276. búčiová, ľ., borovanský, a., čižmárik, j., csöllei, j., švec, p., kozlovský, j., račanská, e., beneš, l.: štúdium lokálnych anestetík. lxxxix. štúdium vplyvu obmien v spojovacom reťazci na biologickú aktivitu v skupine bázických fenylkarbamátov. českoslov. farm., 36, 1987, 339-344. búčiová, ľ., csőllei, j., borovanský, a., čižmárik, j., račanská, e.: štúdium lokálnych anestetík. xcvii. príprava a účinnosť 1-etoxymetyl-2-/1pyrolidinyl/, 2-piperidinoa 2-/1-perhydroazepinyl/-etylesterov kyseliny oa malkoxyfenylkarbámovej. českoslov. farm., 40, 1991, 102-105. bučiová, l., csölei, j., račanská, e., švec, p.: investigations on local anaesthetics, ic1: syntheses and local anaesthetic properties of alkoxyphenylcarbamates. arch. pharm. (weinheim), 325, 1992, 393-396. cohen, j.l.: profiles drug substances (london) 6, 1977. čižmárik, j., lehotay, j., hromuľáková, k., pokorná, m., lacuška, m.: hplc separation of enantiomers of carbisocaine. pharmazie, 52, 1997, 402403. hermansson, j., grahn, a.: optimization of the separation of the enantiomers of basic drugs. retention mechanism and dinamic modification of the chiral bonding properties on an αacid glicoprotein column. j. chromatogr. a, 694, 1995, 57-69. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:59 utc nova biotechnologica et chimica 15-1 (2016) 21 hroboňová, k., lehotay, j., čižmárik, j., armstrong, d.w.: in vitro study of enzymatic hydrolysis of diperodon enantiomers in blood serum by twodimensional lc. j. pharm. biomed. anal., 30, 2002a, 875-880. hroboňová, k., lehotay, j., čižmárik, j., renčová, m., armstrong, d.w.: study of mechanism of enantioseparation ii. hplc chiral analysis of alkoxysubstututed esters of 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2008, 167-176. peng, l., jayapalan, s., chankvetadze, b., farkas, t.: reversed-phase chiral hplc and lc/ms analysis with tris(chloromethylphenylcarbamate) derivatives of cellulose and amylose as chiral stationary phases. j. chromatogr. a, 1217, 2010, 6942–6955. rojkovičová, t., lehotay, j., čižmárik j.: vplyv sacharidových častí v teikoplanínovej stacionárnej fáze na separáciu niektorých enantiomérov fenylkarbámového typu metódou hplc, čes. slov. farm., 52, 2003, 97-101. rojkovičová, t., lehotay, j., čižmárik, j.: hplc separácia racemátov bázických esterov alkoxyfenylkarbámovej kyseliny použitím dvoch teikoplanínových chirálnych stacionárnych fáz, čes. slov. farm., 54, 2005, 173– 177. rustichelli, c., ferioli, v., gamberini, g., stancanelli, r.: enantiomeric separation of local anaesthetic drug by hplc on chiral stationary phases. chromatographia, 54, 2001, 731–736. sun, p., wang, c., breitbach, z.s., zhang, y., armstrong, d.w.: development of new hplc chiral stationary phases based on native and derivatized cyclofructans. anal. chem., 81/24, 2009, 10215-10226. zang, t., kientzy, c., franco, p., ohnishi, a., kagahimara, y., kurosava, h.: solvent versatility of immobilized 3,5dimethylphenylcarbamate of amylose in enantiomeric separations by hplc. j. chromatogr. a, 1075, 2005, 65-75. zhang, x.x., bradshaw, j.s., izatt, r.m.: enantiomeric recognition of amine compounds by chiral macrocyclic receptors. chem. rev., 97, 1997, 3313-3361. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:59 utc 22 hroboňová, k. et al. wang, a.x., lee, j.t., beesley, t.e.: coupling chiral stationary phases as a fast screening approach for hplc method development. lc-gc, 18, 2000, 626-639. received 21 january 2016 accepted 19 april 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:59 utc nova biotechnol chim (2023) 22(1): e1414 doi: 10.34135/nbc.1414 1 nova biotechnologica et chimica effects of dihydroquercetin, 1-aryltetrahydroisoquinoline, and conjugate on the functional condition mitochondrial membrane of the rat liver zafarjon m. ernazarov1,, mamurjon k. pozilov2, muzaffar i. asrarov1, sherzod n. zhurakulov 2,3 1institute of biophysics and biochemistry at the national university of uzbekistan named after mirzo ulugbek, almazar district, student town, 174, tashkent 100174, uzbekistan 2 national university of uzbekistan named after mirzo ulugbek, university str. 4, tashkent 100174, uzbekistan 3acad. s. yunusov institute of the chemistry of plant substances academy of sciences of uzbekistan, m. ulugbek st. 77, tashkent 100170, uzbekistan  corresponding author: zafarbek1985@gmail.com article info article history: received: 14th october 2021 accepted: 20th october 2022 keywords: dhq-11 conjugate f-18 isoquinoline alkaloid fe2+/citrate lipid peroxidation mitochondria abstract in the current research paper, the flavonoid dihydroquercetin, 1-(2´-bromine-4´,5´dimethoxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (f-18) and 2-(3,4dihydroxyphenyl)-6-(1-(2´-bromine-4´, 5´-dimethoxyphenyl)-6,7-dimethoxy-3,4dihydroisoquinoline-2 (1n)-il) methyl-3. the effects of 5,7-trigidroxychroman-4-on (dhq-11) conjugate on rat liver mitochondrial calcium megachannels and on lipid peroxidation (lpo) induced using fe2+/citrate were investigated in vitro experiments. white male rats weighing 180-200 grams were used in the experiments. it was found that the dhq-11 conjugate was identified to have an inhibitory effect on rat liver mitochondria to calcium megachannels and peroxidation of lipids induced by fe2+/citrate. the inhibitory properties of dhq-11 conjugate on hepatic mitochondrial calcium megachannels and mitochondrial membrane lipid peroxidation were identified as active against dihydroquercetin and the f-18 isoquinoline alkaloid. © university of ss. cyril and methodius in trnava introduction these days, biologically active substances that are being extracted from plants are widely utilised in medicine for the purpose of treatment and prevention of diseases. this is because substances that are derived from plants are distinguished from synthetic drugs by their non-toxic effects on cells and their high biological activity in small concentrations. cell mitochondria serve as molecular targets for a number of biologically active compounds. being located in the outer and inner membrane of the mitochondria, the number of ion transport channels modulated by specific activators and inhibitors (drahota et al. 2009). the calcium megachannel (mitochondrial permeability transition pore-mptp) can modify its physiological conformation due to increased loading of ca2+ ions in the matrix (paul et al. 2008). currently, there are many tools that could increase mptp permeabilization. however, pharmaceuticals that inhibit mptp permeability are encountered quite mailto:zafarbek1985@gmail.com nova biotechnol chim (2023) 22(1): e1414 2 rarely (shengrong et al. 2002). many flavonoid compounds extracted from plants are considered to physiologically regulate mptp permeability by inhibiting mitochondrial swelling. one of such biologically active substances, the flavonoid dihydroquercetin, distinguishes itself from other polyphenolic compounds by its biological activity. dihydroquercetin is estimated to be the main flavonoid compound isolated from the larix sibirica tree. currently, the bioactive properties of the dihydroquercetin flavonoid are being investigated in a number of scientific laboratories around the world. dihydroquercetin stabilizes cell membranes by inhibiting the formation of free radicals formed as a result of lipid peroxidation (lpo) in biomembranes. it has been found that it inhibits the development of dystrophic and sclerotic changes in tissues as well as normalizes capillary vascular permeability (babkin et al. 2003). the effects of dihydroquercetin on antioxidant activity in blood cells, platelet aggregation, and erythrocyte hemolysis have been identified (chen et al. 2009). dihydroquercetin has been clinically evaluated and clinical trials have revealed that it has strong natural antioxidant properties (babkin et al. 2004). among 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydro isoquinolines, sedative-anxiolytic (mirzaev et al. 2017), cytotoxic (terentyeva et al. 2019), antiarrhythmic, anti-inflammatory (rakhmanova et al. 2022), atypical neuroleptic (mirzaev et al. 2020) activities, and regulating the volume of thymocytes (terentyeva et al. 2016) substances have been found within their active characteristics. some authors have explored the inotoropy and antiarrhythmic effects of isoquinoline alkaloids on heart muscle tissue (zhumaev et al. 2020), as well as the mechanisms of action on cardiomyocyte ca2+ transport systems (zhumaev et al. 2019). taking for experimental purposes 1-(2´-bromine4´,5´-dimethoxyphenyl)-6,7-dimethoxy-1,2,3,4-te trahydroisoquinoline (f-18), 3,4-dimitoxy phynilethylamine, and 2-bromine are considered as compounds synthesized on the basis of 4,5dimethoxybenzaldehyde, which is characterized by its high physiological effect on biomembranes. among the chemical compounds with heterocyclic structure, isoquinoline alkaloids possess a range of physiological effects. isoquinoline alkaloids have been investigated for their cardiovascular, antispasmodic, analgesic, and anti-inflammatory quality. the effect of the f-18 isoquinoline alkaloid on papillary muscle contraction in rats has been studied in vitro (rustamov et al. 2021). nowadays, intensive scientific research is being conducted on the effects of existing compounds on membrane ultrastructures. dihydroquercetin flavonoid, 1-(2´bromine-4´,5´-dimethoxyphenyl)-6,7-dimethoxy-1, 2,3,4-tetrahydroisoquinoline (f-18) synthesized on the basis of these compounds 2-(3,4dihydroxyphenyl)-6-(1-(2´-bromine-4´, 5´-di methoxy-phenyl)-6,7-dimethoxy-3,4-dihydroiso quinoline-2 (1n)-il) methyl-3,5. the effect of 7trigidroxychroman-4-on (dhq-11) conjugate on biomembranes due to structural activity can reduce membrane lpo intensity and effectively affect the functional activity of the cell’s energy organoid mitochondria. however, since the mechanisms of action of dihydroquercetin flavonoid and f-18 isoquinoline alkaloid and their conjugate (dhq-11) on the liver mitochondrial membrane have not been studied, we aimed to study the effect of these bioactive substances on the lpo process and calcium megachannel in mitochondria. experimental experimental chemistry part 1h nmr spectrum was registered on unity-400 spectrometers (solvent сdcl 3, internal standardhmds). the rf values were determined by tlc on silica gel ls 5/40 plates utilizing a chloroform: methanol 6 :1 elution system. the temperature of substance melting point 3 was determined by a ‘boetius’ microtable. dihydroquercetin (dhq), chemical name: 3,5,7,3',4'-penta-hydroxyflavanonone obtained from larix sibirica (babkin et al. 2004) (cas no. 480-18-2), 1-(2´-bromo-4´,5´-dimethoxyphenyl), 7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (2) was synthesized according to the previously described method (zhurakulov et al. 2013), the physicochemical characteristics corresponded to the literature data. synthesis of 2-(3,4-dihydroxyphenyl)-6-(1-(2´bromo-4´,5´-dimethoxyphenyl)-6,7-dimethoxy-3,4dihydroisoquinolin-2 (1h)-yl) methyl-3,5,7-tri hydroxy-chroman-4-one (3), dhq-11. nova biotechnol chim (2023) 22(1): e1414 3 a solution of dihydroquercetin (1) (0.15 g, 0.49 mmol) in 5 ml of isopropyl alcohol with stirring for 10 min. the reaction mixture was stirred for 30 min at 30 °c, then 0.05 ml (0.54 mmol) of 30 % formalin solution (d = 1.092) was added dropwise. precipitation began immediately. the reaction mixture was stirred for another 4 h at 30 °c and left at room temperature for 10 h. the progress of the reaction was monitored by tlc. then the precipitate was filtered off, washed with isopropyl alcohol. the residue was crystallized from ethanol. yield 0.31 g (89 %), mp. 169-171 °c (from ethyl alcohol), rf 0.67 (chloroform : methanol, 6:1). 1h nmr spectrum (400 mhz, сdcl 3, , m.d., j/hz): dihydroquercetin fragment: 3.70 (2h, s, nch2), 4.33 (1h, d, j = 11.4, h-3), 4.66 (1h, br. signal, н-2), 5.81 (1н, s, н-8), 6.72, 6.78 (each 1н, d, j = 7.7, н-5´, 6´), 6.88 (1н, s, н-2´); 1-(2´bromo-4´,5´-dimethoxyphenyl)-6,7-dimethoxy-1, 2,3,4-tetrahydroisoquinoline fragment: 2.70, 3.03, 3.21 (4h, all m, h-3, 4 ), 3.59 (3h, s, 7-och3), 3.64 (3h, s, 6-och3), 3.81 (6h, s, 4´, 5'-och3), 5.13 (1h, s, h-1), 6.10 (1h, s, h-8), 6.56 (1h, s, h6´), 6.73 (1h, s h-5), 6.99 (1h, s, 3´). compound 2-(3,4-dihydroxyphenyl)-6-(1-(2´-bromo-4´,5´-dimethoxyphenyl)-6,7-dimethoxy-3,4-dihydroisoquinolin-2 (1н)-yl) methyl-3,5,7trihydroxychroman-4-one (3) was obtained by the approach according to (zhurakulov et al. 2015). the structural formulas of alkaloids were drawn by the chemoffice 2002, chemdraw ultra 7.0 software (fig. 1). isoquinoline dihydroquercetine 1 3 5 7 1 1 3 1 5 1 1 35 7 1 1 3 1 5 1 2 3 1 o oh ho oh oh o oh + h3co h3co nh och3 och3 br ch2o h3co h3co n o oh ho oh oh o oh och3 och3 br fig. 1. scheme for the preparation of 2-(3,4-dihydroxyphenyl)-6-(1-(2'-bromo-4',5'-dimethoxyphenyl)-6,7-dimethoxy-3,4dihydroisoquinoline-2 (1h)-yl) methyl-3,5,7-trihydroxychroman-4-oh. dihydroquercetin (1), f-18 isoquinoline alkaloid (2), dhq-11 (3). animal treatment in the experiments on white male rats weighing 180 – 220 g, the international declaration of helsinki, the council of international organizations of medical sciences (cioms; 1985) and the institute of biophysics and biochemistry at the national university of uzbekistan named after mirzo ulugbek "in scientific research was used in accordance with the regulation on the bioethics of the use of laboratory animals" (report dated 22.02.2019). rats are immobilized under light ether anaesthesia and decapitated under standard laboratory conditions (20 – 24 °c; natural sunlight; 65 % humidity, food, and water available). isolation of mitochondria mitochondria were isolated from livers by conventional differential centrifugation described by schneider and hageboom (1951). rat liver was homogenized in a medium containing 250 mm sucrose, 10 mm tris (hydroxymethyl) aminomethane hydrochloride (tris-hcl), 1 mm ethylenediamine tetraacetic acid na2-salt (edta), ph 7.4 centrifuged at 1,500  g for 7 min (-2 °c, -4 °c). mitochondria were sedimented by centrifugation of the supernatant at 6,000  g for 15 min (-2° c, -4°c). the final mitochondrial pellet was suspended in a small volume of medium containing 250 mm sucrose, 10 mm tris-hcl, was kept on ice prior to experiments. the mitochondrial protein content was determined by the lowry method modified by peterson (1977). measurement of mitochondrial swelling the state of mptp was assessed by the rate of ca2+ -dependent swelling of mitochondria, recording the light scattering of the suspension of mitochondria nova biotechnol chim (2023) 22(1): e1414 2 at 540 nm (he and lemasters 2003). experimental conditions (mm): sucrose 200, kh2po4 1, succinate 5, ca2+ egta buffer – 0.02, hepes 20, tris-hcl 20, rotenone – 0.002 μg.ml-1, oligomycin 1 μg.ml-1, ph = 7.2; additives: cacl2 10 μm, csa 10 μm. incubation medium (mm); under these conditions (in the presence of ca2+ egta buffer), swelling of mitochondria is considered the opening of mptp. lipid peroxidation as measured by fe2+/citrate the induction of fe2+/citrate-dependent swelling of mitochondria was induced by the addition of 50 μm feso4 and 2 mm citrate mitochondrial suspension and recorded on a v-5000 spectrophotometer at 540 nm. the incubation medium contained (mm): sucrose-250, tris chloride-10, edta-1; ph 7.4. in this case, the amount of mitochondrial protein was 0.5 mg per 1 ml of the incubation medium. the previously established linear correlation relationship between the intensity of lipid peroxidation (lpo) processes induced by the fe2+/citrate (and fe2+/ascorbate) system and the rate of high amplitude swelling of mitochondria makes it possible to judge the state of lpo reactions by the intensity of swelling and use this model as a test system for studying the antioxidant properties of various biologically active substances. despite the fact that the results obtained by the mitochondrial swelling method used by the authors are indirect, the method itself is distinguished by a short analysis time, relative simplicity, and information content sufficient to establish/confirm the desired properties of the tested compound. drugs and chemicals the following chemical reagents were used: edta (sandoz, switzerland), egta, tris-hcl (serva, germany), sucrose, feso4, citrate, kh2po4, succinate, cacl2 (chemreaktivsnab, russia), hepes, rotenone, oligomycin, and cyclosporin-a (selleck, usa). all reagents were p.a. grade. data analysis the results were analysed statistically using origin pro 8.6 (microsoft, usa). the data were evaluated using a parametric student’s t-test and are expressed as m ± m. the results that were deemed significant are indicated as follows: * p <0.05 and ** p <0.01. results conducted experiments revealed that the effect of substances on the ptp conduction of rat liver mitochondria was investigated in vitro. the classic inhibitor cyclosporin-a (csa) was applied to inhibit mptp conduction. ca2+-bound swelling of the liver mitochondria was induced using 30 μm cacl2. mitochondrial swelling caused by ca 2+ ions was taken as a 100% control. in subsequent experiments, the conjugate (dhq-11) synthesized on the basis of mitochondrial swelling of dihydroquercetin flavonoid and isoquinoline alkaloid (f-18) in the mitochondria of rat liver was studied at a semi-maximal ic50 = 0.5 μm concentration of this mptp classic inhibitor csa. in the first experiment, the effect of the dihydroquercetin flavonoid on the liver mptp was studied. according to accumulated results, the mptp permeability of dihydroquercetin flavonoid at 10, 25, 50, 75, and 100 μm concentrations formed 9.9 ± 3.4 %, 30.2 ± 2.5 %, 56.7 ± 4.6 %, respectively, compared to the control 73.2 ± 3.2 % and 82.2 ± 3.1 % inhibition were determined (fig. 2). in the process of exposing dihydroquercetin concentrations of 10, 25, 50, 75, and 100 μm with csa (ic50 = 0.5 μm) in an incubation medium, mitochondrial swelling was 44.9 ± 4.6 %, 59.2 ± 3.9 %, respectively, compared to the control. inhibition of 76.1 ± 2.5 %, 83.9 ± 2.6 % and 95.7 ± 4.1 % was observed (fig. 2). experimental results indicate that the dihydroquercetin flavonoid inhibited hepatic mptp permeability, while a pore inhibitor in combination with csa demonstrated a stronger inhibition of permeability. in our following experiment, the effect of the f-18 isoquinoline alkaloid on the mptp permeability of rat liver was studied. concentrations of the f-18 isoquinoline alkaloid 10, 30, 50, 70, 90, 120 μm in the hepatic mitochondria were 18.2 ± 3.5 %, 38 ± 2.3 %, 59.8 ± 4.04 %, respectively, relative to the control, which inhibited 72.7 ± 2.3 %, 82.3 ± 2.03 % and 88.8 ± 2.2 % of the mptp (fig. 3). 4 nova biotechnol chim (2023) 22(1): e1414 3 0 25 50 75 100 0 25 50 75 100 * ** ** * * ** ** * * ic 50 =43,5 mkm mkm dihydroquercetin dihydroquercetin+csa (0,5 mkm) ** δ а 5 4 0 /m in ., % fig. 2. effect of dihydroquercetin flavonoid on rat liver mitochondrial swelling. (*p <0.05; **p <0.01; n = 6). the next experiments, the half-maximum inhibitory concentration of csa and the f-18 isoquinoline alkaloid at concentrations of 10, 30, 50, 70, 90, and 120 μm were affected by 55.2 ± 3.7 %, 68.1 ± 2.1 %, 80.7 ± 3.4 %, 86.1 ± 3.5 %, 89.8 ± 2.6 % and 94.3 ± 2.9 % respectively, when exposed to mptp conductivity (fig. 3). more active inhibition was detected at, respectively. 0 30 60 90 120 0 25 50 75 100 * ** * * ** * ** ** ** * ** ic 50 =40,4 mkm mkm f-18 f-18+csa (0,5 mkm)* δ а 5 4 0 /m in ., % fig. 3. effect of f-18 isoquinoline alkaloid on rat liver mitochondrial swelling. (*p <0.05; **p <0.01; n = 6). in our next experiment, we studied the effect of dihydroquercetin on the conjugate (dhq-11) that shaped a chemical bond between the 6-position carbon atom in ring a and the f-18 isoquinoline alkaloid through the ch2 bridge with the 2-position nitrogen atom in ring b on rat liver mptp. according to the results accumulated, the dhq-11 conjugate at 10, 20, 30, 40, 50 μm compared to the control mptp, 19.6±3.7%, 44.2 ± 3.2%, 74.7 ± 2.3%, 82.7±2.4%, and 85.7±2.6% inhibitions were noted (fig. 4). 0 10 20 30 40 50 0 25 50 75 100 ** ** ** * ** ** * * * ic 50 =22 mkm mkm dhq-11 dhq-11+csa (0,5 mkm)* δ а 5 4 0 /m in ., % fig. 4. effect of dhq-11 conjugate on rat liver mitochondrial swelling. (*p <0.05; **p <0.01; n = 6). proceeding the experiment, the incidence of mitochondrial edema was 54.98 ± 4.1 %, 70.8 ± 2.6 %, and 70.8 ± 2.6 %, respectively, when the dhq11 conjugate was exposed to concentrations of 10, 20, 30, 40, 50 μm in the incubation medium in accordance with the semi-maximum inhibitory concentration of csa, 94 ± 4.6 %, 90.9 ± 2.1 %, and 92.8 ± 2.8 % more active inhibition, respectively (fig. 4). in our next experiment, the effect of mitochondrial membrane lipids of compounds on lpo induced by fe2+/citrate was studied in in vitro conditions. the fe2+/citrate system leads to a sharp increase in the mitochondrial membrane lpo process, which was taken as 100% in the control score. under the influence of fe2+/citrate, the barrier function of mitochondrial membranes is impaired, and their volume increases relative to control (almeida et al. 2006). in experiments, it was defined that a concentration of dihydroquercetin 10 μm inhibited mitochondrial lpo process control by 30.0 ± 2.4 % (fig. 6a). as the concentration of dihydroquercetin in the incubation medium increased, its inhibitory effect on membrane lpo became more pronounced. at the same time, the concentrations of dihydroquercetin 40, 60, and 100 μm lpo were 67.0 ± 5.2 %, respectively, relative to the control, 5 nova biotechnol chim (2023) 22(1): e1414 2 decreased by 76.0 ± 4.8 % and 90.0 ± 6.6 %, respectively. experiments have shown that the semi-maximal inhibitory concentration (ic50) of dihydroquercetin in the rat liver mitochondrial membrane was 22.2 μm (fig. 5a). in our next experiment, 1-(2´-bromine-4´,5´dimethoxyphenyl)-6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline (f-18) (10 – 100 μm) rat liver mitochondrial membrane the effect on lpo induced by fe2+/citrate was studied (fig. 5b). 0 25 50 75 100 * ** * * 1005040302010 * 0 25 50 75 100 ** * ** * ** 1008060402010 * mkm δ а 5 4 0 /m in ., % δ а 5 4 0 /m in ., % а b mkm fig. 5. the effects of dihydroquercetin (a) and f-18 isoquinoline alkaloid (b) on fe2+/ citrate-induced lpo process of rats' liver mitochondria. (*p <0.05; **p <0.01; n = 6). the results revealed that concentrations of 10, 20, and 30 μm of the isoquinoline alkaloid (f-18) in the incubation medium were 34.8 ± 3.2 %, respectively, of the control of hepatic mitochondrial lpo; inhibition contributed 56.9 ± 3.6 % and 62.1 ± 4.1 %, respectively. their inhibitory effect on mitochondrial lpo was more pronounced when the concentrations of the isoquinoline alkaloid in the incubation medium were increased to 40, 50, and 100 μm. at the same time, 40, 50, and 100 μm levels of isoquinoline were 70.2 ± 3.4 %, respectively, of lpo induced by hepatic mitochondria fe2+/citrate; decreases were found to be 73.0 ± 4.8 % and 89.1 ± 5.6 %, respectively. experiments have demonstrated that the semi-maximal inhibitory concentration (ic50) of the liver mitochondrial membrane of the f-18 isoquinoline alkaloid was 17.6 μm (fig. 5b). thus, no significant changes in the effect of dihydroquercetin and isoquinoline alkaloids (f-18) on rat liver mitochondrial lpo intensity were seen. however, the fact that the semi-maximal inhibitory concentration of these compounds on lpo induced by fe2+/citrate in the mitochondrial membrane is slightly lowered at the isoquinoline alkaloid (f-18) indicates that it is slightly more active than dihydroquercetin. normally, in an lpo system, antioxidants are balanced and work on the feedback principle. increased activity of antioxidant compounds such as dihydroquercetin flavonoids leads to a decrease in the generation of free radicals, which in turn alters the properties of lipids located in the membrane. in subsequent experiments, the conjugate (dhq11) synthesized on the basis of dihydroquercetin and isoquinoline alkaloid (f-18). effects on lpo induced by fe2+/citrate in mitochondrial membranes isolated from rat liver at concentrations of 10–100 μm were performed in in vitro experiments (fig. 6). the results present that 10 and 20 μm of dhq-11 conjugate inhibited hepatic mitochondrial membrane lpo by 47.1 ± 4.1 % and 55.9 ± 4.0 %, respectively, relative to control. when the concentration of dhq-11 conjugate in the incubation medium was enhanced to 30 and 40 μm, it was clear that its lpo intensity decreased by 64.0 ± 4.4 % and 72.0 ± 3.3 %, respectively, relative to control. when the concentration of dhq-11 conjugate was increased to 100 μm, the 6 nova biotechnol chim (2023) 22(1): e1414 3 membrane lpo intensity decreased by 88.2 ± 6.7 % relative to control (fig. 6). 0 25 50 75 100 ** * * * * 1005040302010 * δ а 5 4 0 /m in ., % mkm fig. 6. effect of dhq-11 conjugate on rat lpo process in rat liver mitochondria with fe2+/ citrate. (*p <0.05; **p <0.01; n = 6). it was obvious from the experiments that the semimaximal inhibitory concentration (ic50) of the dhq-11 conjugate to the mitochondrial membrane of the rat was 14.4 μm. hence, the dhq-11 conjugate (10–100 μm) has an inhibitory effect on lpo intensity in rat liver mitochondria. in this case, the inhibitory property of the dhq-11 conjugate with fe2+/citrate-induced mitochondrial membrane lpo was active against the dihydroquercetin and isoquinoline alkaloids (f-18). discussion an increased load of ca2+ ions in the incubation medium leads to mitochondria swelling. mitochondrial swelling caused by ca2+ ions indicates that its ptp has led to a highly conductive state. exceeding the maximum limit of mitochondrial swelling leads to the release of water and water-soluble molecules from its matrix towards the cytosol. the mptp blocker was reported to inhibit mitochondrial contraction by cyclosporin a (basso et al. 2008). it has been detected that the flavonoid dihydroquercetin obtained for the experiment, has an activating effect on the mitochondrial atp-dependent potassium channel (gayibov et al. 2021). concentrations of 10–120 μm isoquinoline alkaloid f-18 have been considered to inhibit mitochondrial edema. at the same time, it was established that a high concentration of the isoquinoline alkaloid f18, 50 – 120 μm is effectively inhibited by the control of mitochondrial edema caused by ca2+ ions. in this condition, the isoquinoline alkaloid f18 was synthesized on the basis of 3,4dimitoxyphyleneethylamine and 2-bromo-4,5dimethoxybenzaldehyde, the presence of bromine in the structure might enhance its inhibitory properties. in the dhq-11 conjugate, dihydroquercetin is chemically bonded to the carbon atom in position 6 of ring a, and the isoquinoline alkaloid f-18 is chemically bonded to the nitrogen atom in position 2 of ring b through the ch2 bridge. membrane lipid peroxidation increases in the highly permeable state of liver mitochondria. during the experiments, lipid peroxidation was induced with fe2+/citrate. thus, there were no significant changes were observed in the effect of dihydroquercetin and isoquinoline alkaloids (f-18) on the intensity of lpo in rat liver mitochondria. however, the fact that the halfmaximal inhibitory concentration of these compounds against lpo induced by fe2+/citrate in the mitochondrial membrane is lower for the isoquinoline alkaloid (f-18) indicates that it is somewhat more active than dihydroquercetin. antioxidants in the lipid peroxidation system work according to a balanced feedback principle under normal conditions. the accelerated activity of antioxidant compounds, such as dihydroquercetin flavonoids, leads to a decrease in the formation of free radicals, which, in turn, changes the properties of lipids located in the membrane. conclusions according to the results achieved, it can be summed up that inhibition of hepatic mitochondrial swelling has been determined. inhibition of the sharp contraction of mitochondria induced by the dhq-11 conjugate with fe2+/citrate might depend on their antioxidant activity, the number of hydroxyl groups in the structure, and their mutual arrangement. moreover, the investigated compounds may have high antioxidant and antiradical properties and may not destroy biomembranes. for further clarification of the 7 nova biotechnol chim (2023) 22(1): e1414 2 antioxidant properties of the dhq-11 conjugate, their effect on the activity of antioxidant enzymes is being investigated. conflict of interest the authors declare that they have no conflict of interest. references almeida am, bertoncini cr (2006) mitochondrial dna damage associated with lipid peroxidation of the mitochondrial membrane induced by fe2+-citrate. an. acad. bras. cienc. 78: 505-514. babkin va, ostroukhova la, ivanova sz (2004) products of deep chemical processing of larch biomass. rus. chem. j. m. xlviii, 3: 62 (in russian language). babkin va, ostroukhova la, malkov ya 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10.2478/v10296-012-0007-y ©university of ss. cyril and methodius in trnava solid-phase extraction for photometric determination of rosmarinic acid in lemon balm (melissa officinalis) extracts miroslav ondrejovič1,2, tibor maliar1, hana benkovičová1, jana kubincová2 1department of biotechnology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (miroslav.ondrejovic@ucm.sk) 2department biocentrum, food research institute, kostolná 5, modra, sk-900 01, slovak republic abstract: the aim of this study was evaluation of the solid-phase extraction for elimination of interference compounds from lemon balm extracts aimed for photometric determination of rosmarinic acid. in experiments, evaluated conditions were as follows: composition and volume of mobile phase, ratio between volume of sample and mass of stationary phase and flow rate of mobile phase during separation. the results indicated that interfered compounds were eliminated. the lemon balm extracts should be pretreated by adsorption on normal stationary phase (silica gel) in ratio sample volume to silica gel weight 1:1 (v/w), elution by mobile phase – diethyl ether: acetic acid (9:1; v/v) – volume 40 times of crude extract volume – with flow rate 5 ml/min. after selection of spe conditions, the method was validated with comparison to hplc analysis. the results suggest that this method may be useable for determination of rosmarinic acid by photometric measurement based on the complexation of fe2+ ions with rosmarinic acid. key words: rosmarinic acid, determination, lemon balm, hplc, spe, photometry 1. introduction rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid (pereira et al., 2005; petersen, 1997). it is commonly found in species of the boraginaceae and lamiaceae. one of these plants is lemon balm (melissa officinalis) that express broad spectrum of biological activities. m. officinalis was used in traditional medicine dating back as far back as ancient greek and roman times for treating disorders of the nervous system and melancholy (adinee et al., 2008). today, balm is very useful for nervous agitation, and for promoting sleep, and ameliorates functional gastrointestinal dysfunction (bousbia et al., 2009). it is recommended as a plant juice, cream or tea infusion for nervous complaints, lower abdominal disorders, gastric complaints, hysteria and melancholia, chronic bronchial catarrh, migraine, nervous debility, toothache, earache, headache and high blood pressure and externally, for rheumatism, nerve pains and stiff necks (compress) (dastmalchi et al., 2007). rosmarinic acid is responsible for many published activities, especially antimicrobial (bais et al., 2002), antiallergic (ito et al., 1998; matsuno et al., 2002), antiviral and anti-inflammatory activity (georgiev et al., 2006; huang et al., 2006; kamatou et al., 2005). for analysis of its content during the extraction process in technological scale, the photometric methods were developed (lopez-arnaldos et al., 1995; öztürk et bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:13 utc 64 ondrejovič, m. et al. al., 2010). these methods are based on the increasing of uv-vis absorbance by the forming of complex between rosmarinic acid with fe2+ (lopez-arnaldos et al., 1995) and zr4+ ions (öztürk et al., 2010). photometric methods in comparison with high performance liquid chromatography (hplc) or capillary electrophoresis are rapid, cheep and sufficiently accurate. disadvantage of these methods is small selectivity of complexation reactions. increasing of absorbance at studied wavelengths by the reaction of fe2+ and zr4+ with another organic acid can introduce mistakes to the analysis. in present work, the application of solid-phase extraction aimed for removal of interference compounds by the determination of rosmarinic acid by photometric analysis was studied. 2. material and methods 2.1 chemicals rosmarinic acid 95% (sigma), methanol, ethanol, propan-2-ol, ethyl acetate, dichloromethane, chloroform, diethyl ether and acetic acid all p.a. grade (mikrochem), ferrous sulphate p.a. (chemapol), tris-(hydroxymethyl)-aminomethane p.a. (sigma) and silica gel (macherey-nagel). 2.2 plant material dry leaves of lemon balm (melissa officinalis) were obtained from local area diviaky, district banska bystrica, slovak republic. 2.3 preparation of crude extracts from lemon balm dried leaves of lemon balm (1 g) was extracted by 20 ml of following solvents: distilled water, methanol, ethanol, propan-2-ol and by their aqueous solutions (25, 50 and 75 %) at 40 °c for 1 h. prepared extracts were decanted into the test tubes. the final volume was measured and then the concentration of rosmarinic acid was determined by hplc and photometric method. 2.4 analytical determination 2.4.1 photometric determination of rosmarinic acid for photometric analysis of rosmarinic acid in the crude extracts from lemon balm, method based on the complex reaction with fe2+ ions was used. 40 µl sample and 4 ml of tris/acetate buffer (ph 6.0) was mixed. the buffer contained feso4 in the concentration of 0.5 g/l. the mixture was incubated for 1 h at room temperature in the darkness. the absorbance of the reaction medium was read at 572 nm. calibration was carried out with standard of rosmarinic acid and control without rosmarinic acid (lopez-arnaldos et al., 1995). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:13 utc nova biotechnologica et chimica 11-1 (2012) 65 2.4.2 hplc analysis of rosmarinic acid the rosmarinic acid content in prepared extracts was determined by hplc method using purospher star rp18e, 250 × 4 mm, 5 µm, gradient elution of 0.1 % trifluoracetic acid in water (a) and acetonitrile (b), at flow rate 1 ml/min: 0 min 15 % b, 18 min 30 % b, 25 min 80 % b, 27 min 80 % b with detection at 325 nm (ondrejovič et al., 2009). 2.4.3 tlc analysis of crude extracts from lemon balm the plant leaf extracts were additionally analyzed on silica gel plates by tlc. 10 µl of each extracts were applied and the plates were developed in different organic solvents and their mixtures (acetic acid: methanol: dichlormethane 4:15:35; acetic acid: methanol: dichloromethane 4:2:36; acetic acid: diethyl ether 1:9; acetic acid: chloroform 1:9; acetic acid: ethylacetate 1:9; acetic acid: butanol 1:9). dried plates were sprayed by reagent (tris/acetate buffer ph 6.0 with feso4) used for photometric analysis of rosmarinic acid. positive reaction of rosmarinic acid with reagent can be detected as dark blue spot on yellow background. 2.5 solid phase extraction for solid phase extraction, silica gel was used as stationary phase for sample (lemon balm crude extracts) adsorption. the ratio between stationary phase and crude extract was 1:1, 1:2, 1:4 (w/v). samples were dried in the oven 25 min at 105 °c and cooled in the desiccators. silica gel with adsorbed extracts were put into the extraction column and washed by eluent (acetic acid: diethyl ether 1:9; v/v). the first addition of eluent was 500 µl and other four additions were 250 µl of eluent. every fraction were collected into the test tubes and evaporated by rotary evaporator. obtained fractions were concentrated by 2 ml methanol. in all samples was determined concentration of rosmarinic acid by photometric method and hplc analysis. 3. results and discussion 3.1 detection of rosmarinic acid by photometric method rosmarinic acid can be determinated by two photometric methods. both are based upon the complexation of rosmarinic acid with fe2+ and zr4+ ions which caused increasing of sample absorbance at 572 and 362 nm respectively. by comparison of these methods for determination of rosmarinic acid comparable results were obtained (öztürk et al., 2010). however, determination of rosmarinic acid with fe2+ is cheaper and environmental acceptable as using of zr4+ ions. moreover, interferences caused by absorbance of non-complexated compounds in tested extracts are higher at 362 nm (zr4+) than 572 nm (fe2+). therefore, the application of photometric method of rosmarinic acid determination by fe2+ complexation for control of rosmarinic acid extraction process in preparative scale was studied. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:13 utc 66 ondrejovič, m. et al. studying of extraction process starts by selection of extraction solvent. for rosmarinic acid extraction, spectrum of organic aliphatic alcohols (such as methanol, ethanol, propan-2-ol) and their water solutions were used. rosmarinic acid content was determined by photometric and hplc analysis. the results (fig. 1) indicated disproportion between concentration of rosmarinic acid determined by photometric and hplc methods. all concentration of rosmarinic acid determined by photometric analysis is higher as concentration of rosmarinic acid determined by hplc. this finding refers to presence of other compounds which interference in photometric analysis. the result differences between both analytical methods are higher in samples prepared with water solution of used organic aliphatic alcohols. therefore interference compounds may be belonging to polar organic compounds. fig. 1. comparison of two methods of rosmarinic acid determination (ra); a dependence of ra concentration determined by photometric method and hplc; b – concentrations of ra determined by hplc and photometric method in extracts from lemon balm prepared by extraction to various extraction solvents:1 – distilled water; 2 – 25 % methanol; 3 – 25 % ethanol; 4 – 25 % propan-2-ol; 5 50 % methanol; 6 50 % ethanol; 7 – 50 % propan-2-ol; 8 – 75 % methanol; 9 – 75 % ethanol; 10 – 75 % propan-2-ol; 11 – methanol; 12 – ethanol; 13 – propan-2-ol. for determination of compounds interfered at photometric analysis of crude extracts from lemon balm, the tlc (thin-layer chromatography) analysis on the normal stationary phase by method of multiple elution was used. this method allows analyzing hydrophobic and hydrophilic compounds together. after visualization of separated compounds by spraying of reagent containing of fe2+ ions on tlc layer was observed several blue spots (see rf factors in table 1). it is evident that prepared extracts contain more compounds with positive reaction with fe2+ ions and higher differences between concentrations of rosmarinic acid determined by photometric and hplc analysis correlate with number of positive spots determined by tlc analysis. 3.2 spe purification of plant extracts 3.2.1 choice of eluent from the tlc analysis, it is possible to roughly estimate the polarity of ballasts interfered at determination of rosmarinic acid by photometric analysis. because the 0 1 2 3 4 5 6 7 0 0,5 1 1,5 2 2,5 3 concentration of ra (hplc) [g/l] c o n ce n tr at io n o f r a [ g /l ] photometric analysis hplc analysis 0 0.5 1 1.5 2 2.5 3 1 2 3 4 5 6 7 8 9 10 11 12 13 extractants c o n ce n tr at io n o f r a (h p l c ) [g /l ] 0 2 4 6 8 10 12 c o n ce n tr at io n o f r a (p h o to m et ri c) [ g /l ] hplc photometric b a bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:13 utc nova biotechnologica et chimica 11-1 (2012) 67 spot of rosmarinic acid was upper than another spots, the solid-phase extraction can be used for pretreatment of sample before the photometric determination of rosmarinic acid. in the literature, solid-phase extraction of rosmarinic acid was realized on the reverse stationary phase such as c18 (exarchou et al., 2003; stevens et al., 2007), but also normal stationary phase is suitable for separation of rosmarinic acid from lemon balm extracts and it is cheaper than reverse stationary phase. table 1. concentration of interfering compounds (ballasts) and retention factors of positive spots visualized on tlc plates after spraying with detection reagent (fe2+) in various extracts form lemon balm prepared by extraction with various extraction solvents. extraction solvents concentration of ballasts [g/l] retention factors of positive spots water 2.46 0 0.04 0.13 0.27 0.39 0.45 0.69 25% methanol 3.03 0 0.04 0.13 0.27 0.39 0.45 0.69 25% ethanol 3.34 0 0.04 0.13 0.27 0.39 0.45 0.69 25% propan-2-ol 3.94 0 0.04 0.13 0.27 0.39 0.45 0.69 50% methanol 2.47 0 0.04 0.13 0.27 0.39 0.45 0.69 50% ethanol 1.96 0 0.04 0.13 0.27 0.39 0.45 0.69 50% propan-2-ol 3.18 0 0.04 0.13 0.27 0.39 0.45 0.69 75% methanol 1.25 0.04 0.13 0.39 0.69 75% ethanol 2.75 0.04 0.13 0.27 0.39 0.45 0.69 75% propan-2-ol 2.40 0.04 0.13 0.27 0.39 0.45 0.69 methanol 1.83 0.04 0.13 0.39 0.69 ethanol 0.61 0.45 0.69 propan-2-ol 0.19 0.45 0.69 from the point of view of method application during technological production of rosmarinic acid, pretreatment of lemon balm extracts before photometric rosmarinic acid determination on normal stationary phase is more suitable. in order to choose a suitable eluent for the elution of rosmarinic acid, crude extract from lemon balm was adsorbed on the stationary phase (silica gel) and organic solvent was evaporated. thereafter the stationary phase with adsorbed crude extract without possible interference caused by extraction solvents was eluted by various organic solvents such as ethyl acetate, dichloromethane, chloroform, diethyl ether. the rosmarinic acid is found in the plant material as its salts (häusler et al., 1993). since, extracts prepared by extraction solvent without acids contain rosmarinic acid salts. therefore, all mobile phases contain also acetic acid in the ratio between organic solvent/acetic acid 9:1 (v/v). prepared fractions were analysed by tlc after spraying by detection reagent with fe2+ ions. rosmarinic acid was found in all of selected eluents, but the spot of rosmarinic acid without all interference compounds was only in the diethyl ether fractions. therefore, diethyl ether was selected as suitable component of mobile phase and it was used in another experiments. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:13 utc 68 ondrejovič, m. et al. 3.2.2 crude extract – stationary phase amount ratio in order to determinate the suitable ratio between crude extract and amount of stationary phase, various crude extracts from lemon balm was adsorbed by evaporation of extraction solvent in the presence of silica gel with subsequent elution by the evaporated solvent (fig. 2). the results indicated that the best ratio between crude extracts from lemon balm and amount of stationary phase was 1:1 (v/w). this ratio was used in next experiments. 25 35 45 55 65 75 85 95 105 5:1 2:1 1:1 1:2 1:5 sorbent:extract ratio [g/ml] y ie ld o f r a fr om e xt ra ct (% ) fig. 2. yield of rosmarinic acid eluted by mixture diethyl ether: acetic acid (9:1; v/v) from silica gel with adsorbed crude extract of lemon balm by various ratio between mass of silica gel and volume of extract. 3.2.3. effect of volume of eluent in order to investigate the eluent volume (diethyl ether: acetic acid; 9:1; v/v) for quantitative extraction of rosmarinic acid from stationary phase, rosmarinic acid in concentration varied from 0.03 to 3.0 mg/g of stationary phase was adsorbed on the silica gel by addition 0.25 ml of standard solution of rosmarinic acid to 0.25 g of silica gel. used concentration range of rosmarinic acid can be expected in lemon balm crude extracts. thereafter, rosmarinic acid from stationary phase by 1 ml of selected eluent was eluted. in first ten fractions, the rosmarinic acid was presented in detectable values and their elution yield was varied from 98 to 99 % of adsorbed rosmarinic acid. from results verified on lemon balm crude extract pretreatment, the lemon balm extracts for determination rosmarinic acid by photometric methods could be eluted by selected eluent from stationary phase minimally 40 times of volumes of adsorbed crude extract. 3.2.4. effect of flow rate the influence of flow rate was studied on the base of analysis of the retention and recovery of rosmarinic acid in the range of 0.5 – 5 ml/min. the retention of the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:13 utc nova biotechnologica et chimica 11-1 (2012) 69 rosmarinic acid on the stationary phase was not considerably affected by the sample solution flow rate. the recovery of rosmarinic acid by the elution was dependent just on the volume of selected eluent. therefore in another experiments the flow rate of 5 ml/min was used. 3.3 validation of method for validation of suggested method, concentration of rosmarinic acid in crude extract from lemon balm was determined by photometric analysis with spe sample pretreatment and hplc. in the fig. 4, concentrations determined by photometric analysis and hplc method are correlated and it is evident that differences between results achieved by both methods are minimal. y = 37,561ln(x) + 4,3121 r2 = 0,9738 5 25 45 65 85 105 0 2 4 6 8 10 12 14 kinetic of elution from sorbent [ml] y ie ld o f r a fr om e xt ra ct [% ] fig. 3. yield of rosmarinic acid obtained from silica gel with adsorbed crude extract of lemon balm. y = 1.0047x + 0.0503 r2 = 0.9979 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 rosmarinic acid [g/l] (photometric analysis) r os m ar ni c ac id [g /l ] ( h p lc ) fig. 4. comparison of rosmarinic acid concentrations determined by photometric method with spe sample pretreatment and hplc in crude extracts form lemon balm prepared by various extraction solvents. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:13 utc 70 ondrejovič, m. et al. 4. conclusion in this study, the solid-phase extraction was demonstrated to be very efficient for elimination of interfering compounds for precise photometric determination of rosmarinic acid from lemon balm extracts. the lemon balm extracts should be pretreated by adsorption on normal stationary phase (silica gel) in ratio sample volume to silica gel weight 1:1 (v/w), elution by mobile phase – diethyl ether: acetic acid (9:1; v/v) – volume 40 times of initial crude extract volume – with flow rate 5 ml/min. after selection of spe conditions, the method was validated by comparison to hplc analysis. the results indicated that this method may be useable for determination of rosmarinic acid by photometric method based on the complexation of fe2+ ions with rosmarinic acid. references adinee, j., piri, k., karami, o.: essential oil component in flower of lemon balm (melissa officinalis l.). am. j. biochem. biotechnol., 4, 2008, 277-278. bais, h. p., walker, t. s., schweizer, h. p., vivanco, j. m.: root specific elicitation and antimicrobial activity of rosmarinic acid in hairy root cultures of ocimum basilicum. plant physiol. biochem., 40, 2002, 983 – 995. bousbia, n., vian, m.a., ferhat, m.a., petitcolas, e., meklati, b.y., chemat, f.: comparison of two isolation methods for essential oil from rosemary leaves: hydrodistillation and microwave hydrodiffusion and gravity. food chem., 114, 2009, 355-362. dastmalchi, k., dorman, h.j.d., oinonen, p.p., darwis, y., laakso, i., hiltunen, r.: chemical composition and in vitro antioxidative activity of a lemon balm (melissa officinalis l.) extract. lwt, 41, 2008, 391-400. exarchou, v., godejohann, m., van beek, t.a., gerothanassis, i.p., vervoort, j.: lc-uv-solid-phase extraction-nmr-ms combined with a cryogenic flow probe and its application to the identification of compounds present in greek oregano. anal. chem., 75, 2003, 6288-6294. georgiev, m., kovatcheva, e., marcheva, n., ilieva, m.: purification rosmarinic acid extracts from lavandula vera mm cell biomass. food chem., 94, 2006, 111-114. häusler, e., petersen, m., alfermann, a. w.: isolation of protoplasts and vacuoles from cell suspension cultures of coleus blumei benth. plant cell rep., 12, 1993, 510-512. huang, s., zheng, r.: rosmarinic acid inhibits angiogenesis and its mechanism of action in vitro. cancer lett., 239, 2006, 271-280. ito, h., miyazaki, t., ono, m., sakurai, h.: antiallergic activities of rabdosiin and its related compounds: chemical and biochemical evaluations. bioorg. med. chem., 6, 1998, 1051-1056. kamatou, g.p.p., viljoen, a.m., gono-bwalya, a.b., zyl, r.l., vuuren, s.f., lourens, a.c.u., baser, k.h.c., demirci, b., lindsey, k.l., staden, j., steenkamp, p.: the in vitro pharmacological activities and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:13 utc nova biotechnologica et chimica 11-1 (2012) 71 chemical investigation of three south african salvia species. j. ethnopharmacol., 102, 2005, 382-390. lópez-arnaldos, t., lópez-serrano, m., ros baceló, a., calderón, a.a., zapata, j.m.: spectrophotometric determination of rosmarinic acid in plant cell cultures by complexation with fe2+ ions. anal. chem., 351, 1995, 311314. matsuno, m., nagatsu, a., ogihara, y., ellis, b.e., mizukami, h.: cyp98a6 from lithospermum erythrozin encodes 4-coumaroyl-4´hydroxphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis. febs lett., 514, 2002, 219 – 224. ondrejovič, m., benkovičová, h., šilhár, s.: optimization of rosmarinic acid extraction from lemon balm (melissa officinalis). nova biotechnol., 9-2, 2009, 175 – 182. öztürk, m., duru, m. e., ince, b., harmandar, m., topcu, g.: a new rapid spectrophotometric method to determine the rosmarinic acid level in plant extracts. food chem., 123, 2010, 1352-1356. pereira, p., tysca, d., oliveira, p., silva brum, l., picada, j.n., ardenghi, p.: neurobehavioral and genotic aspects of rosmarinic acid. pharmacol. res., 52, 2005, 199-203. petersen, m.: cytochrome p450-dependent hydroxylation in the biosynthesis of rosmarinic acid in coleus. phytochemistry, 45, 1997, 1165 – 1166. stevens, j., crawford, m., robinson, g., roenneburg, l.: automated post-collection concentration for purified preparative fractions via solid phase extraction. j. chromatogr. a, 1142, 2007, 81-83. presented at the 3rd international scientific conference “applied natural sciences 2011”, october 5–7, 2011, častá papiernička, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:13 utc microsoft word jamrichova a kol nbc.doc nova biotechnologica et chimica 14-2 (2015) 201 doi 10.1515/nbec-2015-0027 © university of ss. cyril and methodius in trnava optimization of expression conditions of the acetylesterase ce16 from hypocrea jecorina encoded by a synthetic gene and expressed in escherichia coli cells daniela jamrichová1, andrej godány1,2, ľubica urbániková2 1department of biology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic 2institute of molecular biology, slovak academy of sciences, dúbravská cesta 21, bratislava, sk-845 51, slovak republic abstract: acetylesterase ce16 was identified as a part of the enzymatic cocktail secreted by fungus hypocrea jecorina (anamorph: trichoderma reesei) during its growth on cellulose. later it was classified as the first member of a newly organized carbohydrate esterase family ce16. further studies showed that acetylesterase is crucial for complete deacetylation of naturally acetylated xylans enabling their saccharification by xylanases. to study the relationship between structure and function of acetylesterase, highly purified recombinant enzyme produced by trichoderma reesei rut c-30 was prepared. the enzyme was composed of 348 amino acid residues from which the 1 19 formed a secretion signal peptide. determined molecular mass of purified recombinant acetylesterase (aes1) was 45 kda which was more than molecular mass calculated from amino acid sequence. as it has been proved later, the difference was caused by the enzyme glycosylation. glycosylation of proteins increases their stability, but it can also be a source of heterogeneity, which might be a problem during crystallization. to make the future x-ray study of the enzyme easier, recombinant non-glycosylated enzyme needed to be prepared. for these purposes, a synthetic gene optimized for protein expression in escherichia coli was designed and synthetized. the first nonglycosylated acetylesterase obtained by the expression of its synthetic gene in e. coli cells was mostly insoluble or aggregated. conditions of cell cultivation, induction of gene expression and cells disruption were necessary to optimize. presently, after optimization of all mentioned steps, the non-glycosylated recombinant ce16 acetylesterase was prepared in the soluble and active form, ready for further downstream procedures, involving protein purification and crystallization. key words: acetylesterase ce16, hypocrea jecorina, trichoderma reesei, synthetic gene, gene expression, escherichia coli 1. introduction filamentous fungus hypocrea jecorina (anamorph: trichoderma reesei) is a representative organism today for commercial scale production of different cellulases and hemicellulases, which are currently used for production of biofuels, textile materials, detergents, food, feed and have negligible use in brewing industry. genus hypocrea jecorina has become a subject of intense studies from 1980 until now. with the increasing need to replace fossil fuels by cheaper and greener sources of energy, such as constantly forming plant biomass, scientific community has drawn attention to the enzymes produced by this fungus during its growth on cellulose and lactose, to the so called secretome (adav et al., 2011). plant cell wall has a complex structure and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:23 utc 202 jamrichová, d. et al. composition, and therefore its decomposition requires a complex interaction of a range of enzymes cleaving specific types of chemical bonds (caffall and mohnen, 2009). xylan, a constituent of the cell walls, is a polysaccharide made from xylose units which are usually decorated by side substituents, mostly arabinose, d-glucuronic acids or acetyl groups which influences its properties and protects plant cell walls towards hydrolysis by glycoside hydrolases, i.e. acetylation slows down its subsequent digesting (carpita and gibeau, 1993). the degree of acetylation influences the enzymatic hydrolysis, since esterified polysaccharides are less soluble and less accessible to the enzymatic hydrolysis. one of the enzymes that are able to deacetylate oligosaccharides in the plant cell wall is acetylesterase (trce16). acetylesterase (trce16) was identified for the first time in 1985 as a part of the cellulolytic enzyme cocktail produced by h. jecorina during its growth on cellulose (biely et al., 1985). according its substrate specificity, the enzyme was classified as an acetic-ester acetyl hydrolase (ec 3.1.1.6) (poutanen and sundberg, 1988). mentioned acetylesterase is regioselective enzyme. moreover, it is unique by its ability to deacetylate terminally acetylated xylooligosaccharides, predominantly at position 3 and 4, from their non-reducing ends and less effectively at position 2 (kremnický and biely, 2005; biely et al., 2011, biely et al., 2014). further studies showed that acetylesterase efficiently catalyzes transacetylation in aqueous medium saturated with vinyl acetate to the position 3 of non-reducing residues of xylooligosaccharides; and it is a crucial enzyme for complete deacetylation of naturally acetylated xylans enabling their saccharification by xylanases (kremnický and mastihuba, 2004; kremnický and biely, 2005). because of the need of large quantity of acetylesterase, an acetylesterase gene from wild-type trichoderma reesei qm 6a was cloned and transformed into hypercellulolytic mutant trichoderma reesei rut c-30 as a production strain. based on amino acid sequence of recombinant acetylesterase (aes1), a new family of carbohydrate esterases, family ce16 (cazy classification, lombard et al., 2014), was established in 2008 (li et al., 2008). up to date (10 november 2015), the family ce16 comprises 190 members. unfortunately, there is no tertiary structure information about any member yet. moreover, only three members are biochemically characterized: acetylesterases from t. reesei (li et al., 2008), myceliopthora thermophila (koutaniemi et al., 2013) and from podospora anserina (puchart et al., 2015). experimentally determined molecular mass of the purified recombinant acetylesterase (aes1) was 45 kda (li et al., 2008), what matched with the molecular mass of purified nonrecombinant acetylesterase trce16 (poutanen and sundberg, 1988), but was lower than molecular mass calculated from amino acid sequence. this difference is caused by the enzyme glycosylation. the fact that natural and recombinant acetylesterases can be n-glycosylated was confirmed by enzymatic cleavage by endo-β-n-acetylglucosaminidase, resulting in the production of lower-mass polypeptide. glycosylation of proteins increases their stability, but it can be also a source of the heterogeneity, which might cause problems during protein crystallization. besides this, the transformed strain of t. reesei produces several other acetylesterases similar to our target enzyme, which may cause problems at purification of our target acetylesterase. the problems could be minimized by the production of non-glycosylated recombinant aes1 protein in escherichia coli cells. for these purposes, the synthetic bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:23 utc nova biotechnologica et chimica 14-2 (2015) 203 gene encoding acetylesterase was designed (urbániková, unpublished data) and prepared (genscript, piscataway, usa). considering further studies on the relationship between structure and function of acetylesterase, it would seem necessary to prepare a non-glycosylated recombinant enzyme, making easier its crystallization and tertiary structure determination. the advantages of the e. coli expression system are as following: e.g. high overexpression, relatively easy production of proteins and ability to produce proteins with his6-tag sequence, which is helpful in protein purification by imac (immobilized metal affinity column), and, finally, the fact that e. coli does not possess the glycosylation machinery. heterologous expression is not easy to be performed. high yield and solubility of final protein are influenced by the properties of given enzyme (bashiri et al., 2015). in this paper, the expression conditions of the synthetic gene are optimized, with the aim to reach the maximal amount of a soluble active protein. 2. materials and methods 2.1 bacterial strains and plasmid synthetic gene (genscript, piscataway, usa) was prepared pursuant to gene aes1 encoding ce16 acetylesterase (aes1), but without the part coding signal peptide. codon usage of the synthetic gene was optimized for e. coli cells using the program jcat (grote et al., 2005). optimized synthetic gene tae1 was inserted into pet21a (novagen, usa) expression vector. the ndei and xhoi restriction enzymes were used to digest the sequence of the synthetic gene and pet21a vector and the recombinant pet21a-tae1 molecule was prepared by subsequent ligation. the pet21a-tae1 plasmid was transformed by heat-shock method to e. coli competent cells (froger and hall, 2007). four types of competent cells were used for testing of the gene expression: strain bl21(de3), nico21(de3), arcticexpress and bl21-gold(de3) (agilent technologies, usa). 2.2 media and culture conditions for cells cultivation, lb-miller medium (maniatis et al., 1982) was used, containing 1 % (w/v) bacto-tryptone (serva, germany), 0.5 % (w/v), yeast extract (difco laboratories, usa), and 1 % (w/v) nacl, ph 7.5. in order to enhance the production of soluble recombinant protein lb medium containing 1 m nacl was prepared (nagata et al., 2005, sasaki et al., 2006, sasaki et al., 2012). all cultivation media were supplemented with appropriate antibiotics. the transformed cells were grown at 37 °c in 500 ml shake flasks on the shaker at 250 rpm until the od600 = 0.6-1.0 was reached (about 2.5 h). then, the temperature in the shaker was lowered to 20° c for 30 minutes and the gene expression was induced by the addition of iptg (isopropyl–thio–β–d–1–galactopyranoside) to the final concentration of 1 mm (serva, germany). afterwards, several different incubation temperatures and length of cultivation were set and tested: (i) 16 °c for 24 h, (ii) 30 °c for 6 h, and (iii) 11 °c for 24 h when arcticexpress competent cells were used. as a control of the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:23 utc 204 jamrichová, d. et al. level of protein expression, samples with no iptg added were prepared at all conditions. in order to improve the production of soluble and well folded recombinant protein, a heat shock was tested, i. e. bacterial culture was exposed to 47 °c for 30 min followed by cooling to 20° c before the iptg addition (oganesyan et al., 2007., burgess and murray, 2009). the protein expression was analyzed in the induced cultures and non-induced controls in the adjusted time intervals (6, 20, 24 h during cultivation) by sodium dodecyl sulfate polyacrylamide gel electrophoresis (12 % sdspage, laemmli, 1970). proteins in the gel were stained with coomassie brilliant blue (serva, germany). after incubation, the cells were harvested by centrifugation at 10,600 g for 15 min at 4 °c and stored at -20 °c for further procedures. 2.3 cell disruption the cells were disrupted by two methods. at first, (i) chemical lysis by bugbuster master mix (5 ml / 1 g of pelleted cells) (merck millipore, usa) was performed. however, this type of cell disruption led to formation of soluble aggregates not able to bind to imac column. therefore, (ii) mechanical method of disruption cell walls by sonicator (soniprep 150, united kingdom) was used for complete cell lysis. the cells were thawed on ice and then resuspended in a sonication buffer composed of 20 mm sodium phosphate buffer, ph 7.5, supplemented with nacl and glucose to a final concentration of 200 mm and 25 mm. for the protein solubility improvement various detergents and additives in sonication buffer were tested. as a result, a detergent cocamidopropyl betaine, (tego betain f-50, evonik goldschmidt gmbh, germany) and reducing agent tcep (tris (2-carboxyethyl) phosphine) (sigma-aldrich, germany) were added to the sonication buffer to the final concentration of 1 % and 10 mm, respectively. the resuspended pellet was sonicated using 15 cycles of 30 s pulses at amplitude 14 with 1 min breaks and kept on ice during the whole sonication to avoid protein denaturation. optimal conditions of sonication were determined by time course analysis of sonication by sampling. the samples for analysis were removed during the sonication and then centrifuged for 5 min at 10,600 g at 4 °c. protein concentration was determined in supernatant by spectrophotometric analysis at the wavelength of 280 nm (nanodrop 2000c, thermo scientific, usa). after sonication, the lysate was clarified by centrifugation at 10,600 g for 15 min at 4 °c. the samples taken from both, soluble and insoluble fractions, were analyzed by 12 % sds-page (laemmli, 1970) with spectratm multicolor broad protein ladder (10-260 kda) (sm1841, thermo scientific, usa) used as a protein molecular weight control. the proteins bands were visualized by coomassie brilliant blue staining protocol. 3. results and discussion in this work, a synthetic gene encoding trichoderma reesei acetylesterase ce16 (tae1) was prepared, according to the sequence of aes1 (genbank dq866149), without a part encoding the signal peptide (fig. 2). codon usage was optimized for the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:23 utc nova biotechnologica et chimica 14-2 (2015) 205 expression in e. coli cells in order to overcome problems originating from the differences between the eukaryotic gene and the prokaryotic host organism (jonasson et al, 2002). the prepared gene was inserted into the pet21a vector with c-terminal his6-tag sequence and a gene responsible for ampicillin resistance (fig. 1). in general, this vector enables the expression under the control of a strong t7 promotor and also provides multi cloning site (mcs) with restriction enzymes ndei and xhoi used for the insertion of the synthetic gene tae1 (fig. 1). fig. 1. the construct of a pet21a expression vector with inserted synthetic gene tae1. inserted synthetic gene is marked by red color. nucleotide sequence of the synthetic gene tae1 with modified nucleotides in red is placed in the red box. tae1 ------------------mfpkphddfkylitfgdsytdngrlgyygshqahgpppgvmp aes1 mrsilvipsfvavlnafslfpkphddfkylitfgdsytdngrlgyygshqahgpppgvmp :***************************************** tae1 peanvtasgglqwpqyveastgatlydyaiagatcdnnnverwaafmnanypsiitdeip aes1 peanvtasgglqwpqyveastgatlydyaiagatcdnnnverwaafmnanypsiitdeip ************************************************************ tae1 sfkadrktklyrgvtsantvyalwigtndlsytgilsdsqvkgtnittyidclwnvfdai aes1 sfkadrktklyrgvtsantvyalwigtndlsytgilsdsqvkgtnittyidclwnvfdai ************************************************************ tae1 haaggrrfvilnnnalqltglyrplsdggagdnqfwqnktlynqteyaqkmleyttssnt aes1 haaggrrfvilnnnalqltglyrplsdggagdnqfwqnktlynqteyaqkmleyttssnt ************************************************************ tae1 midygvpfhllvknrwpgskvavydihslimdiynqpsrylepphnvvgyykhcdvngtn aes1 midygvpfhllvknrwpgskvavydihslimdiynqpsrylepphnvvgyykhcdvngtn ************************************************************ tae1 clygpgrldsylwydelhpsniiasyiareflnvvsgrskygtywehwlehhhhhh aes1 clygpgrldsylwydelhpsniiasyiareflnvvsgrskygtywehw------- ************************************************ fig. 2. alignment of amino acid sequences of the two recombinant acetylesterases ce16. tae1 = nonglycosylated acetylesterase produced by e.coli, aes1 = glycosylated acetylesterase produced by trichoderma reesei rutc-30, for the alignment clustal omega online tool was used (sievers and higgins, 2011). in the tae1 sequence, amino acid residues added due to the cloning strategy are in red and his6-tag is in green color. in aes1 sequence, amino acid residues corresponding to the signal peptide are in blue. n-glycosylation sites are in light blue color in both sequences. accgtttacgctctgtggatcggtaccaacgacctgtcttacaccggtatcctgtctgactctcaggttaaaggtaccaacatcaccacc tacatcgactgcctgtggaacgttttcgacgctatccacgctgctggtggtcgtcgtttcgttatcctgaacaacaacgctctgcagctg accggtctgtaccgtccgctgtctgacggtggtgctggtgacaaccagttctggcagaacaaaaccctgtacaaccagaccgaatacgct cagaaaatgctggaatacaccacctcttctaacaccatgatcgactacggtgttccgttccacctgctggttaaaaaccgttggccgggt tctaaagttgctgtttacgacatccactctctgatcatggacatctacaaccagccgtctcgttacctggaaccgccgcacaacgttgtt ggttactacaaacactgcgacgttaacggtaccaactgcctgtacggtccgggtcgtctggactcttacctgtggtacgacgaactgcac ccgtctaacatcatcgcttcttacatcgctcgtgaattcctgaacgttgtttctggtcgttctaaatacggtacctactgggaacactgg atgttcccgaaaccgcacgacgacttcaaatacctgatcaccttcggtgactcttacaccgacaacggtcgtctgggttactacggttct caccaggctcacggtccgccgccgggtgttatgccgccggaagctaacgttaccgcttctggtggtctgcagtggccgcagtacgttgaa gcttctaccggtgctaccctgtacgactacgctatcgctggtgctacctgcgacaacaacaacgttgaacgttgggctgctttcatgaac gctaactacccgtctatcatcaccgacgaaatcccgtctttcaaagctgaccgtaaaaccaaactgtaccgtggtgttacctctgctaac bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:23 utc 206 jamrichová, d. et al. fig. 3. analysis of protein production by different e.coli strains. sds page with samples taken at different time of the protein production in selected e. coli cells: a bl21(de3)-pet21a-tae1; b nico21(de3)-pet21a-tae1, c bl21-gold-(de3)-pet21a-tae1; d arcticexpress cells-pet21a-tae1. ladder: spectratm multicolor broad protein ladder (10-260 kda) (thermo scientific, usa). control = cultivation without induction. in the first experiments with a production of the non-glycosylated acetylesterase obtained by the expression of its synthetic gene in e. coli cells, the problems with protein solubility were found. production of deserved protein was satisfying, but after cell homogenization, the protein was mostly insoluble or aggregated and not able to bind to imac column. these problems suggested incorrect folding of the protein 1. 2. 3. 4. 5. 6. 7. 8. 1. 2. 3. 4. 5. 6. 7. 8. 40 kda 35 kda tae1 38.29 kda 40 kda 35 kda 1. 2. 3. 4. 5. 6. 7. 8. tae1 38.29 kda legend: 1. cells before induction 2. protein ladder 3. 6 h control 4. 6 hiptg induction 5. 20 h control 6. 20 hiptg induction 7. 24 h control 8. 24 hiptg induction legend: 1. cells before induction 2. 6 h control 3. 6 hiptg induction 4. 20 h control 5. 20 hiptg induction 6. 24 h control 7. 24 hiptg induction 8. protein ladder 40 kda tae1 38.29 kda d a legend: 1. cells before induction 2. 6 h control 3. 6 hiptg induction 4. 20 h control 5. 20 hiptg induction 6. 24 h control 7. 24 hiptg induction 8. protein ladder legend: 1. protein ladder 2. cells before induction 3. 6 h control 4. 6 hiptg induction 5. 20 h control 6. 20 hiptg induction 7. 24 h control 8. 24 hiptg induction 35 kda 1. 2. 3. 4. 5. 6. 7. 8. b 1. 2. 3. 4. 5. 6. 7. 8. 35 kd 40 kda tae1 38.29 kda b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:23 utc nova biotechnologica et chimica 14-2 (2015) 207 (handrick and hartl, 1995). with the aim to improve the recombinant protein folding and solubility, at first, different e. coli expression strains were tested, and the choice of given strains for the gene expression was closely tied to the properties of the selected expression vector. different types of expression cells were used to study the gene expression level. bl21(de3) and nico21(de3) were used as standard types of competent cells. arcticexpress competent cells were used due to their adjustment of low-temperature cultivation in order to increase the recovery of the soluble protein (schein, 1989). bl21-gold(de3) competent cells are designed for easy induction and high-level protein expression. the pet21a-tae1 plasmid was transformed into e. coli cells followed by the cultivation in a standard manner by described method. gene expression was monitored by sds-page analysis. the obtained results did not show any significant differences in the synthetic gene expression among the tested bl21(de3) (fig.3; a), nico21(de3) (fig.3, b) and bl21-gold(de3) (fig.3; c) expression cells. to emphasize, in each step of protein production and protein extraction from the cells, the total protein content was indirectly confirmed spectrophotometrically by measuring the absorbance at 280 nm. the cells were disrupted by bugbuster, but the recombinant protein was found either in the insoluble part or formed soluble aggregates and was not able to bind to imac column. for this reason, mechanical disruption by sonication was tested, but the majority of protein also stood in the insoluble part. the total amount of protein produced by arcticexpress competent e.coli cells (fig.3, d) was lower than in other three cases; and after homogenization, the protein remained mostly in the insoluble fraction. therefore, these competent cells were not used in further procedures. fig. 4. sds page with samples taken in different time of the protein production in bl21-gold-(de3) in lb medium supplemented by 1 m nacl and with a heat-shock before induction. ladder: spectratm multicolor broad protein ladder (10-260 kda) (thermo scientific, usa). control = cultivation without induction. in order to improve protein solubility and reach a high level of protein production, conditions of cultivation and cell lysis were optimized. many recombinant proteins cannot be expressed well and in a correct and soluble form in e. coli cells because of the absence of the proper folding environment or the absence of the post translational modification (sorensen et al; 2005). presently, various defense mechanisms of bacteria are known to protect proteins from aggregation and incorrect folding by 1. 2. 3. 4. 5. 6. 7. 8. 40 kda 35 kda tae1 38.29 kda legend: 1. cells before induction 2. 6 h-control 3. 6 hiptg induction 4. 20 h control 5. 20 hiptg induction 6. 24 h control 7. 24 hiptg induction 8. protein ladder bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:23 utc 208 jamrichová, d. et al. osmotic or heat stress. defense mechanisms include intracellular accumulation of osmolytes or synthesis of heat shock proteins in the cells (kempf et al; 1998; bukau et al; 1998). therefore, several cultivation conditions using the high concentration of nacl (1 m) in lb medium and a heat shock (47 °c, 30 min) before induction were applied. in the view of increased temperature during heat shock and the length of cultivation, ampicillin was replaced by its stable equivalent carbenicillin. from the protein solubility point of view, the most soluble protein was obtained after 20 h cultivation at 16 °c in salted lb medium with heat shock prior to induction (fig. 4). the conditions for improved protein solubility were adjusted by adding tego betain f50 and tcep, both before sonication. tcep is a reducing agent that selectively reduces disulphide bonds and that is nonreactive toward other functional groups commonly present in proteins (kirley et al; 1989). moreover tcep is compatible with imac column containing ni2+ ions. tego betain f50 is a zwitterionic detergent containing a quaternary ammonium cation and a carboxylate groups, shown aid in protein stability and folding (nitsch et al; 2005). the disadvantage of tego betain is high foaming, which may cause protein denaturation. to avoid denaturation and obtain the highest possible yield of the soluble protein, the concentration of tego betain and tcep in sonication buffer, as well as a volume of sonication buffer per gram of cells were optimized. the number of sonication cycles (fig. 5) was also optimized in all sonicated samples to avoid the protein denaturation; the temperature during the whole period of sonication was controlled to prevent well-known damages caused by ultrasound radiation in biological system (wood and loomis, 1927). the best result of both, well-expressed and most soluble protein was achieved by protein production using e. coli bl21-gold(de3) cells as a host, mechanical cell disruption by sonication using the buffer supplemented with tego betain f-50 and tcep to the final concentration of 1 % and 10 mm, respectively (fig. 6) and subjecting the cells to 15 cycles of sonication (15 x 15 s) with one minute breaks for better cooling of the sample. above-mentioned conditions were evaluated as the best conditions for acetylesterase ce16 production in terms of the protein yield and solubility. fig. 5. determination of optimal conditions of cell disruption by sonication. samples were collected after each three cycles of sonication. ladder: spectratm multicolor broad protein ladder (10260 kda) (thermo scientific, usa). legend: 1. tcp (total cell fraction) 2. 3 x 15 s 3. 6 x 15 s 4. 9 x 15 s 5. 12 x 15 s 6. 15 x 15 s 7. 18 x 15 s 8. 19 x 15 s 9. protein ladder 1. 2. 3. 4. 5. 6. 7. 8. 9. 40 kda 35 kda tae1 38.29 kda bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:23 utc nova biotechnologica et chimica 14-2 (2015) 209 fig. 6. influence of sonication buffer composition on the yield of soluble protein. the comparison of the sonication results analyzed on sds-page. a sonication buffer without tcep and tego betain; b sonication buffer containing tcep and tego betain. ladder: spectratm multicolor broad protein ladder (10-260 kda) (thermo scientific, usa). 4. conclusions in the present work, conditions of recombinant molecule pet21-tae1 expression and soluble recombinant acetylesterase production, i.e. conditions of cell cultivation, induction of protein expression and sonication of cell pellet were optimized. e. coli bl21gold(de3) strain was chosen for its property to maximally express the synthetic gene encoding acetylesterase tae1 and produce the protein in soluble form. the cultivation of e. coli cells bl21gold(de3) containing recombinant pet21a-tae1 construct proceeded after heat shock, until exponential phase was reached. then the expression of the gene encoding ce16 acetylesterase was inducted by adding iptg to the final concentration of 1 mm, in lb medium containing 1 m nacl for 20 h at 16 °c. collected e. coli cells (after storing at -20 °c) were sonicated; and solubilized protein was obtained by sonication in buffer supplemented with tcep reducing agent and tego betain f50, zwitterionic detergent. the best results were achieved after 15 cycles of 30 s pulses at amplitude 14. at this stage, a nonglycosylated recombinant ce16 acetylesterase was prepared as a soluble protein fraction after sonication under specific conditions, and is stored for further downstream procedures, involving especially protein isolation by immobilized metal affinity column (imac), purification by size exclusion chromatography using fplc system and crystallization for the tertiary structure determination. acknowledgement: the work was supported by the slovak research and development agency grant apvv-0602-12, by the slovak grant agency vega grant no. 2/0190/14 and by the faculty of natural sciences of ucm under the contract no. fppv-27-2015. the authors thank to lenka tišáková for the language checking. references adav, s.s., ravindran, a., chao, l.t., tan, l., singh, s., sze, s.k.: proteomic analysis of ph and strains dependent protein secretion of trichoderma reesei. j. proteome res., 10, 2011, 4579 4596. a tae1 38.29 kda 40 kda 35 kda legend: 1. tcp (total cell fraction) 2. soluble fraction 3. insoluble fraction 4. protein ladder 1. 2. 3. 4. tae1 38.29 kda 35 kda 40 kda legend: 1. tcp (total cell fraction) 2. soluble fraction 3. insoluble fraction 4. protein ladder 1. 2. 3. 4. b b a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:23 utc 210 jamrichová, d. et al. bashiri, s., vikstrom, d., 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mokrani1, amar zellagui1,, widad hadjab1, mehmet öztürk2, ozgur ceylen2 and chawki bensouici3 1laboratory of biomolecules and plant breeding, life science and nature, faculty of exact science and life science and nature,university of larbi ben mhidi oum el bouaghi, algeria 2departments of chemistry, faculty of science, mugla sitki kocman university, mugla, turkey 3research center in biotechnology, ali mendjli uv 03, constantine 25000, algeria  corresponding author: zellaguia@yahoo.com article info article history: received: 14th october 2021 accepted: 20th october 2022 keywords: antioxidant antibiofilm cv 026 e. faecalis hplc-dad propolis abstract this research aims to analyse the richness of propolis ethanolic extract from kherrata (eepkh) in phenolic and flavonoid compounds and its antioxidant effects using different methods. further investigations were conducted to evaluate the antibacterial and antibiofilm potential of propolis against enterococcus faecalis strains originated from oral diseases. the antiquorum-sensing ability against chromobacterium violaceum cv 026 was also investigated. the results revealed that ethanolic extract contains a high content of phenolic and flavonoid compounds, with an amount of 734.3 9± 11.54µg gae.mg-1 of extract and 224.30 ± 0µg qe.mg-1of extract respectively. caffeic acid (23.79 mg.g-1), hesperetin (15.42 mg.g-1), cynarin (7.59 mg.g-1), apigenin (5.91 mg.g-1), naringenin (4.90 mg.g-1), and kaempferol (3.43 mg.g-1) were identified as the major compounds by the hplc-dad analysis. the antioxidant activity showed good scavenging and reducing abilities. furthermore, eepkh demonstrated high antibacterial potency against e. faecalis strain 2 at concentration 20 mg.ml-1 with an inhibition diameter of 20.33 ± 0.57 mm. the mic and mbc values were found to range between 0.625 and 10 mg.ml-1. biofilm formation by e. faecalis strains was inhibited at mic with a percentage ranging from 65.93 ± 1.11 to 51.54 ± 0.81%. quorum sensing mechanisms in cv 026 was inhibited by eepkh, with diameter zone of 11.16 ± 0.29 mm at mic. this study indicated that propolis extract is considered as a new source of natural medication with therapeutic potential against oral pathology caused by free radicals, e. faecalis, biofilm formation and quorum-sensing. © university of ss. cyril and methodius in trnava introduction apitherapy is a branch of traditional and alternative medicine that uses products harvested and transformed by bees for therapeutic purposes (kolayli and keskin 2020). bee glue is one of the most important products of the hive collected by honey bees from a diversity of botanical sources mailto:zellaguia@yahoo.com nova biotechnol chim (2023) 22(1): e1599 2 (zulhendri et al. 2021). it has been widely recognized for a long time for its numerous medicinal benefits including antimicrobial, antioxidants, and anti-inflammatory properties (boulechfar et al. 2022; dos santos et al. 2022). the medicinal efficacy of propolis is linked to its various chemical compounds, including over 300 biologically active components such as polyphenols, essential oils, amino acids, and waxes (castaldo and capasso 2002; piccinelli et al. 2011). extraction is a critical step that requires a proper protocol to obtain the desired propolis components. maceration is the most common and traditional method to extract the active constituents of propolis (bankova et al. 2021). polyphenolic compounds in propolis increased with increasing concentration of ethanol in the solvent. this is probably because the polyphenolic compounds in propolis could be dissolved in ethanol better than in water. the optimum concentration of ethanol in water was found to be 70 – 95 % alcohol, most often 70 – 80 % (dent et al. 2013; bankova et al. 2021). oral infections are among the most common diseases that affect humans. the majority of these infectious diseases are induced by microorganisms living in biofilms (colombo et al. 2015).oral biofilm plays a crucial role in the development of several oral diseases, including periodontal disease and dental caries. bacteria present in the biofilm can spread to other organs or tissues of the human body through bacteremia and cause systemic diseases (larsen and fiehn 2017; mosaddad et al. 2019). biofilms are among the well-known virulence factors adopted by bacteria. this phenomenon confers to bacteria a great resistance capacity toward multiple antibiotics, thus increasing mortality rates; biofilm eradication has become an important research topic in recent years (ramachandran et al. 2023). e. faecalis is an antibiotic-resistant microorganism, gram-positive cocci, a facultative anaerobe, and an important opportunistic bacterium linked to a number of human diseases such as urinary tract infection, endocarditis, and oral infections (kouidhi et al. 2011; najafi et al. 2020). the pathogenicity of e. faecalis, as well as its resistance to the host immune system and antimicrobial medications, has been related to virulence factors such as extracellular surface protein, gelatinase, aggregation substance, collagen adhesion, and their ability to form biofilm, which are controlled by a quorum-sensing (qs) phenomenon (ali et al. 2017; bhardwaj et al. 2017; najafi et al. 2020). the qs system is a chemical communication process between bacteria that involves gene regulation in response to cell density, which influences a variety of functions such as virulence, acid tolerance, and biofilm formation (basavaraju et al. 2016).the mechanism of qs differs between gram-negative and gram-positive bacteria. in gram-negative bacteria, quorum sensing is effected by luxi/luxr, which uses acyl-homoserine lactones (ahls) as signalling molecules, whereas in gram-positive bacteria, quorum sensing is effected by oligopeptides, which use small peptides as signalling molecules (yada et al. 2015). for instance, chromobacterium violaceumis a pathogenic, gram-negative mutant bacterium widely used as a model for quorum-sensing studies. this bacterium communicates by quorum sensing via the c6-homoserine lactone signal (c6-hsl) (de oca-mejía et al. 2015). antioxidant mechanisms are related to the homeostasis of the organism, an imbalance between both leads to oxidative stress, which is considered a significant factor in the pathogenesis of oral diseases (dental caries, lichen planus, oral cancer, chronic periodontitis) (casas-grajales and muriel 2017; kumar et al. 2017). the goal of this research was to investigate the phenolic and flavonoid contents in propolis extract as well as its chemical constitution, antibacterial, antibiofilm, and anti-qs properties. experimental harvesting and extraction of propolis propolis was collected in october 2018 in kherrata district, situated in the bejaia region (algeria). the sample was extracted with 80 % ethanol in water for 24 h before being filtered, evaporated, and concentrated at 45 °c. phytochemical analysis the content of total phenols (tpc) in the extract was evaluated according to boulechfar et al. nova biotechnol chim (2023) 22(1): e1599 3 (2022). briefly, 20 µl of the prepared extract was combined with 100 µl of folin ciocalteu reagent, then 75 µl of na2co3 (7.5 %) was added. after incubation for 2 h in the dark, the absorbance was read at 765 nm. the result was expressed in terms μg of gallic acid equivalents mg-1 of extract (μg gae.mg-1 e). the total flavonoid content (tfc) was estimated according to bensouici et al. (2020). 50 µl of diluted extract was added to 130 μl of methanol, 10 μl of potassium acetate (1m) and 10 μl of aluminium nitrate (10 %). the absorbance was read at 415 nm after incubation for 40 min. the result was expressed as μg quercetin equivalents per mg of extract (μg qe.mg-1 e). the chemical components of propolis extract were identified by hplc-dad. the system is composed of shimadzu reverse-phase high-performance liquid chromatography (shimadzu cooperation, japan) including a shimadzu model lc-20at solvent delivery unit and the shimadzu model spd-m20a diode array detection system and controlled by lc-solution software (cbm-20a system controller shimadzu). the column temperature was adjusted to 35 c and the injected volume was 20 μl. the separation was attained using an inertsil ods-3 column (4 m, 4.0 mm × 150 mm) and an inertsil ods-3 guard column, with the mobile phase consisting of aqueous acetic acid 0.1% (a) and methanol (b). a sample stock solution was made in methanol at 8 mg.ml-1 and filtered through an agilent 0.45 m filter. the diode array detector (dad) was used for detection at a wavelength of 254 nm. the identification of eepkh compounds was revealed by comparing the retention time of each detected compound with the retention time of different employed standards (fumaric acid, gallic acid, p-benzoquinone, protocatechuic acid, theobromine, theophylline, catechin, 4-hydroxybenzoic acid, 6,7dihydroxycoumarin, methyl-1,4 benzoquinone, vanillic acid, caffeic acid, vanillin, chlorogenic acid, p-coumaric acid, ferulic acid, cynarin, coumarin, prophylgallate, rutin, trans-cinnamic acid, ellagic acid, myricetin, fisetin, quercetin, trans-cinnamic acid, luteolin, rosmarinic acid, kaempferol, apigenin, chrysin, 4hydroxylresorcinol, 1,4-dichlorobenzene, pyrocatechol, 4-hydroxy benzaldehyde, epicatechin, 2,4-dihydroxybenzaldehide, hesperidin, oleuropein, naringenin, hesperetin, genistein, curcumin). the results were presented as mg.g-1 of crude propolis (boutellaa et al. 2019). antioxidant activity the 2,2-diphenyl-1-picrylhydrazyl method (dpph) was determined as described by mazouz et al. (2020), using butylated hydroxyanisole (bha) as a standard antioxidant. briefly, 40 µl of eepkh at different concentrations was added to 160 µl of dpph solution. the absorbance was measured at 517 nm after incubation for 30 min in the dark. the percentage of dpph scavenging effect was estimated using the formula (eq. 1): (1) where, ac – absorbance of control, as – absorbance of sample. the results were presented as a 50 % inhibition concentration (ic50). the ability to scavenge 2,2′-azino-bis-(3ethylbenzothiazoline-6-sulfonic acid (abts) was performed using the method of mebrek et al. (2018). the reaction was generated by reacting 7 mm of abts with 2.45 mm of potassium persulfate (k2s2o8), then kipping in the dark for 12 h before diluting it with distilled water to an absorbance of 0.700 ± 0.020 at 734 nm. then, 160 μl of abts solution were added to 40 μl of sample solution prepared in methanol at various concentrations. after 10 min the absorbance at 734 nm was calculated. the following formula (eq. 2) was used to determine the inhibition percentage: (2) where: ac – absorbance of control, as – absorbance of sample. butylated hydroxytoluene (bht) and bha were used as standard antioxidants. cuprac assay (cupric reducing power) was estimated using the method described by lekouaghet et al. (2020). extract solution (40 μl) nova biotechnol chim (2023) 22(1): e1599 2 was combined with copper (ii) chloride solution (50 µl), neocuproine ethanolic solution (50 µl) and 60 µl of ch3coonh4 (1 m). the absorbance was read at 450 nm after one hour of incubation. the reduction capacity of the extracts was compared with bha and bht. the results were given as a0.5 value (μg.ml -1) corresponding to the concentration, indicating 0.50 absorbance. the galvinoxyl radical scavenging (gor) assay was determined using the procedure of barzegar and moosavi-movahedi (2011). briefly, 40 µl of sample at different concentrations was mixed with 160 µl of a methanolic solution of galvinoxyl (0.1 mm); the mixture was then incubated for 120 min in the dark at room temperature. absorbance was measured at 428 nm. bht and bha were used as standard antioxidants. the reducing power assay (rp) of the extract at various concentrations was determined using the method of elkolli et al. (2022). 10 μl sample + 40 μl phosphate buffer (ph 6.6) + 50 μl potassium ferricyanide (1 %) k3fe (cn)6 ((1 g k3fe (cn)6 in 100 ml h2o)) were mixed and incubated at 50 °c for 20 min. the absorbance was measured at 700 nm after adding 50 μl of trichloroacetic acid (10 %) (1 g of tca in 10 ml h2o), 40 μl of distilled water, and 10 μl of ferric chloride solution (0.1 %) (0.1 g of fecl3 in 100 ml h2o). as antioxidant standards, ascorbic acid, tannic acid, and α-tocopherol were used. phenanthroline-reducing activity was determined using the protocol of aissaoui et al. (2020). 10 µl of extract solution was mixed with 50 µl ferric chloride (0.2 %) +30 µl phenanthroline (0.5 %) + 110 µl meoh. the mixture was incubated at 30 °c for 20 min before measuring the absorbance at 510 nm. the results were given as a0.5 value, which corresponds to the concentration giving a 0.5 absorbance. bacterial strains and culture conditions four oral clinical isolates of e. faecalis were collected in the department of biology, university of oum el bouaghi (algeria). isolates e. faecalis aücc 29212 and cv026 were obtained from the department of microbiology, mugla university (tukey). strains were purified by growing them on bile esculin agar for 48 h at 37 °c. antibacterial activity the disc diffusion test was used to detect the antibacterial ability of the extract (parija 2012). a volume of 100 μl of suspensions (adjusted to 0.5 mcfarland) was spread on mueller-hinton agar. the sterile filter disks, 6mm in diameter, were placed on the surface of the inoculated medium and impregnated with 20µl of the extract in concentrations 20 – 0.625 mg.ml-1. plates were maintained at 4 °c for 1 h to allow the extract to diffuse into the agar. the inhibition zones around the discs were measured in mm after 24 h of incubation at 37 °c. as a negative control, dimethyl sulfoxide (dmso) was used. microdilution method the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) were determined as described by snoussi et al.(2016). in the presence of propolis extract at different concentrations (20 – 0.625 mg.ml-1), 10 µl of diluted bacterial suspension (0.5 mcfarland) was inserted into each well of sterile 96 well plates containing 170 µl mueller-hinton broth .the mic was determined as the lowest concentration that inhibited bacterial growth. mbc was determined by taking 10 µl of liquid culture from each well that showed no growth, sub-cultured on muellerhinton agar, and incubated at 37 °c for 24 h. mbc was the lowest concentration, with no visible growth on mha. antibiofilm assay antibiofilm capacity of propolis extract (mic to mic/16) was conducted according to ceylan and ugur (2015) method. 10 μl of diluted bacteria (5 × 105 cfu.ml-1) were introduced into wells in the presence of 170 μl of medium and 20 μl of extract. the negative control contained only tryptose-soy broth and bacterial cells. after incubation at 37 °c for 48 h, water was used to remove planktonic bacteria from the walls, which were then stained for 10 min at ambient temperature with a 0.1 % crystal violet solution. after that, the wells were washed once more to remove the crystal violet solution. a 200 µl 4 nova biotechnol chim (2023) 22(1): e1599 3 volume of glacial acetic acid (33 %) was added to the walls to dissolve the biofilm stains. finally, at 550 nm, the optical density (od) was calculated, and the proportion of inhibition of the tested extract was estimated using the equation (eq. 3): (3) where: odc – od control; ods – sample. anti-qs activity five millilitres of warm molten soft top agar were seeded with 100 µl of an overnight cv026 culture and 20 µl of 100 µg.ml-1 c6hsl was added as exogenous ahl source. this was mixed and immediately poured onto the surface of luriabertani agar. wells of 5 mm in diameter were made on each plate after the overlay had solidified. each well was filled with 50 µl of different concentrations (mic to mic/16) of sample. a white or cream-colored halo around this well against a purple lawn of activated cv026 bacteria was an indication of qsi. after three days of overnight incubation at 30 °c, the inhibition zone was measured in millimetres (koh and tham 2011). statistical analysis all operations were realized in triplicate. ic50 and a0.5 were determined by linear regression analysis. data were analysed by one-way anova and student t-test (for dpph) using graph pad prism software (version 8.0.2). p-values >0.05 indicated no significant differences, whereas p-values <0.05 were considered as significant. results amounts of polyphenols the results of the extraction yield of phenolic and flavonoid contents are presented in table 1. the eepkh shows a high phenolic and flavonoid contents estimated to 734.39 ± 11.54 µg gae.mg-1 and 224.30 ± 0 µg qe.mg-1, respectively. on the other hand, the extraction yield showed a remarkable result with a percentage of 58.91 %. table 1. extraction yield, tpcs, and tfcs in propolis ethanolic extract. extract extraction yield [%] tpc [µg gae.mg-1] tfc [µg qe.mg-1] eepkh 58.91 734.39 ± 11.54 224.30 ± 0 the findings are presented as means standard deviations of three parallel measurements. hplc-dad analysis the identified compounds in the ethanolic extract of propolis were determined as mg.g-1 extract (table 2, fig.1). caffeic acid (23.79 mg.g-1), hesperetin (15.42 mg.g-1), cynarin (7.59 mg.g-1), apigenin (5.91 mg.g-1), naringenin (4.90 mg.g-1), kaempferol (3.43 mg.g-1) were detected as the main compounds. luteolin, quercetin, p-coumaric acid, ferulic acid, hesperidin, protocatechuic acid, and oleuropein were identified in trace amounts. however, the presence of cynarin was recorded for the first time in algerian propolis extract. table 2. hplc-dad analysis of propolis ethanolic extract. antioxidant activity the antioxidant capacities are given in table 3. the results revealed that propolis extract has high compound retention time amount of compound [mg.g-1] protocatechuic acid 22.39 0.12 4-oh-benzoic acid 31.69 0.07 caffeic acid 35.19 23.79 p-coumaric acid 40.81 1.89 ferulic acid 42.92 0.57 cynarin 43.85 7.59 quercetin 55.42 1.15 luteolin 57.87 2.25 kaempferol 62.48 3.43 apigenin 64.07 5.91 1.4-diclorobenzene 73.81 87.74 hesperidin 47.38 0.49 oleuropein 49.54 0.11 naringenin 55.51 4.90 hesperetin 57.47 15.42 5 nova biotechnol chim (2023) 22(1): e1599 2 scavenging activity for abts and dpph but less than bha and bht. on the other hand, eepkh demonstrated similar gor activity to bha and bht (p >0.05). for cuprac, phenanthroline, and reducing power, eepkh showed stronger antioxidant activity than the antioxidant standards. datafile nam e:pkh_004.lcd sample nam e:pkh 20,0 25,0 30,0 35,0 40,0 45,0 50,0 55,0 60,0 65,0 70,0 75,0 min 0 25 50 75 100 125 150 175 200 225 250 275 300 mau pkh_004.lcd 254nm,4nm p ro to c a te c h ic a c id 4 -o h -b e n z o ic a c id 6 ,7 -d io h c o u m a ri n c a ff e ic a c id v a n ill in p -c o u m a ri c a c id f e rr u lic a c id c y n a ri n h e s p e re d in o le u ro p e in k e rs e ti n n a ri n g e n in h e s p e re ti n l u te o lin k a e m fe ro l a p ig e n in 1 ,4 -d ic lo ro b e n z e n e fig.1. hplc chromatogram of eepkh. table 3. antioxidant capacity of propolis ethanolic extract. samples abts dpph gor cuprac phenan throline reducing power ic50 [µg.ml-1] a0.5 [µg.ml-1] eepkh 5.37±0.07a 10.33±0.04a 4.82±1.07ab 0.96±0.69a 0 .51±0.19a 1.19±0.66a bht 1.59±0.03b nd 3.32±0.18b 9.62±0.87b 0.93±0.07ab na bha 1.03±0.00c 5.73±0.41b 5.38 ±0,06a 3.64±0.19c 2.24±0.17b na ascorbic acid na na na na na 6.77±1.15b tannic acid na na na na na 5.39±0.91b α-tocopherol na na na na na 34.93±2.38c results are shown as ic50 and a0.5 mean standard errors (n = 3). values with different superscripts (a, b, or c) in the same columns are significantly different (p <0.05). na – not applicable. antibacterial activity the diameter of inhibition zones, mics, mbcs, and mbc/mic ratios of the ethanolic extract of propolis are summarized in table 4 and table 5. this is the first study to show that algerian propolis has an antibacterial action against e. faecalis in oral diseases. eepkh exerted high antibacterial activity against all e. faecalis strains at 20 mg.ml-1 compared to the negative control. the highest activity was observed versus strain 2 with an inhibition diameter of 20.33±0.57 mm. mic and mbc values were found between 0.625 and 10 mg.ml-1. 6 nova biotechnol chim (2023) 22(1): e1599 3 table 4. anti-bacterial activity of propolis ethanolic extracts. the results are given as the means and standard deviations of three parallel measurements. the values with different superscripts (a, b) in the same columns are significantly different (p < 0.05). (-) – no activity. table 5. mic, mbc, and mbc/mic ratios of propolis ethanolic extract. antibiofilm activity the biofilm inhibition potential was examined at different concentrations (mic to mic/16) as shown in table 6. the ethanolic extract of propolis exhibited significant (p <0.05) inhibition effects on the biofilm formed by clinical strains. the greatest reduction in biofilm was observed at mic concentrations against all strains, with a percentage of inhibition ranging from 65.93 ± 1.11 % to 51.54 ± 0.81 %. this inhibition decreased as concentration decrease. anti-qs activity the mic values of eepkh against mutant strain cv 026 were found to be 20 mg.ml-1 and the antiqs properties of propolis extract are presented in (fig. 2). the inhibition zone at mic was found to be 11.16 ± 0.29 mm with no inhibition detected at mic/4, mic/8, and mic/16 for extract and dmso 10 %. table 6. effects of different concentrations of the propolis ethanolic extracton e. faecalis biofilmformation. results are expressed as means ± sd of three parallel measurements. the values with different superscripts (a, b, c or d) in the same columns are significantly different (p <0.05). (-) – no activity. e. faecalis strains zone of inhibition [mm] concentrations [mg.ml-1] dmso 20 10 5 2.5 1.25 0.625 / 1 16±0b 14.67±2.5ba 13±1.73bc 12.33±1.52b 11±0b 10±0b 2 20.33±0.57a 18.67±1.52a 17.67±2.30a 16±1.73a 15.33±0.57a 14±1a 3 17±1ba 16.33±0,57ba 15.67±0.57ba 15±0a 14±1a 10±0b 4 16±1.73b 14.33±0.57b 12.33±1.15c atcc 29212 16.67±2.82ba 14.67±1.52ba 13±2bc 12±1b 11±1b e. faecalis strains mic mbc mbc/mic 1 2.5 5 2 2 0.625 1.25 2 3 5 10 2 4 5 10 2 atcc 29212 1.25 2.5 2 e. faecalis strains concentration [mg.ml-1] eepkh [%] of biofilm inhibition 1 mic 55.52±1.19c mic/2 48.57±1.28a mic/4 38.91±1.6b mic/8 21.73±2.01b mic/16 12.95±2.29b 2 mic 51.93±1.2d mic/2 38.21±1.60c mic/4 35.02±0.68c mic/8 mic/16 3 mic 60.19±0.69b mic/2 52.03±1.89ab mic/4 43.23±2.28a mic/8 22.29±1.91b mic/16 18.27±1.89a 4 mic 65.93±1.11a mic/2 55.19±0.49b mic/4 35.77±0.49c mic/8 32.09±0.60a mic/16 atcc 29212 mic 51.54±0.81d mic/2 38.20±1.19c mic/4 mic/8 mic/16 7 nova biotechnol chim (2023) 22(1): e1599 2 m ic m ic /2 m ic /4 m ic /8 m ic /1 6 d m so 1 0% 0 5 10 15 i n h ib it io n z o n e s (m m ) concentrations (mg.ml -1 ) fig. 2. anti-quorum sensing activity of different concentration of propolis extract in mm on cv026. the data is shown as mean sd (n = 3). discussion propolis has a rich phenolic profile with numerous pharmacological effects. in this regard, much research seeks to investigate the main compounds responsible for these powerful activities (aliyazıcıoglu et al. 2013). extraction is a critical step in the isolation and purification of biocompounds from plants (jha and sit 2021). in the present study, the percentage yield of eepkh (58.91%) obtained by ethanolic maceration was higher than the yields of propolis extract obtained by ethanolic maceration for the cities of skardu (31%), islamabad (32%) situated in pakistan, and minas gerais (36.8%) localized in brazil (shabbir et al. 2016; saito et al. 2021). eepkh contains a higher amount of polyphenols (tpc: 734.39 ± 11.54 µg gae.mg-1e, tfc: 224.30 ± 0 µg qe.mg1 e) compared to the amount of polyphenols in propolis from other localities in algeria (oum el bouaghi (tpc: 270.62 ± 1.91 μg gae.mg-1 e, tfc: 54.53 ± 0.20 μg qe.mg-1 e), skikda (tpc: 524.95 ± 2.54 µg gae.mg-1 e, tfc: 47.31 ± 2.54 µg qe.mg-1 e)) (boulechfar et al. 2022). furthermore, segueni et al. (2020) discovered a lower content of total phenolic and flavonoid compounds (tpc: 219.66 ± 1.23 mg gae.g-1, 61.04 ± 0.45mg gae.g-1, 148.73 ± 0.93 mg gae.g-1, 56.98 ± 0.22 mg gae.g-1, tfc: 41.80 ± 0.84 mg qe.g-1, 17.00 ± 0.97 mg qe.g-1, 21.03 ± 0.04 mg qe.g-1, 10.21 ± 0.0mg qe.g-1) in propolis extracts from four locations in turkey (ankara, bursa, bilecik, istanbul). the chemical profiles of eepkh after hplc analysis revealed that the most abundant phenolic and flavonoid compounds identified in propolis extract were caffeic acid, hesperetin, cynarin, apigenin, naringenin, and kaempferol, with quantitative differences between the compounds. another study discovered that the main compounds in ethanolic extracts of propolis from oumtboul, ouadsabt, and ferdjiwa in algeria were caffeic and ferulic acids (daikh et al. 2020). the most common compounds found in turkish propolis samples were caffeic acid phenyl ester, caffeic acid and cinnamic acid, chrysin, and pinocembrin (guler et al. 2021).these quantitative and qualitative differences in phenolic and flavonoid compounds are influenced by various factors such as geographical origin, botanical origin, honeybee's genetics, and season (mountford-mcauley et al. 2021). polyphenolic compounds are among the most abundant secondary metabolites in the plant kingdom and have attracted much attention, mainly because of their broad-spectrum applicability in the prevention of human diseases (bié et al. 2023). propolis was identified as an important source of natural antioxidants such as caffeic acid, hesperidin, and cynarin (wilmsen et al. 2005; gülçin 2006; topal et al. 2016). the results of the current study showed that eepkh has strong antiradical activity against free radicals dpph, 8 nova biotechnol chim (2023) 22(1): e1599 3 abts, gor, cuprac, reducing power, and phenanthroline, which can be attributed to its high concentration of phenolic and flavonoid compounds. according to boulechfar et al. (2022) ethanolic extracts of propolis from different areas of algeria had higher free radical-scavenging activity for dpph and abts radicals. moreover, propolis from the region of souk-ahras, algeria demonstrated a powerful antioxidant inhibitory activity against dpph, abts, gor, and cuprac radicals (ouahab et al. 2023). previous research by miguel et al. (2014) demonstrates that propolis from different locations in morocco exhibited high antiradical activity regarding both abts and reducing power assays. on the other hand, ozdal et al. (2018) reported that turkish propolis demonstrated high scavenging capacity against the cuprac radical. the phenolic and flavonoid compounds in propolis were found to correlate with its free radical scavenging activity (kumazawa et al. 2010; segueni et al. 2020).the emergence and progression of oral pathologies are linked to an increase in bacterial biofilm resistance to antibiotics; due to the increasing bacterial resistance to currently used antibiotics, great importance is given to natural compounds for the prevention of oral bacterial growth, adhesion, and colonization (kouidhi et al. 2015). among them, propolis is well known for its powerful antimicrobial and antibiofilm effects against oral anaerobic bacteria, which can be attributed to its various chemical constituents (uzel et al. 2005; de sá assis et al. 2022). in this context, eepkh exerted antibacterial and antibiofilm action towards all e. faecalis strains, which can be attributed to their high concentration of phenolic and flavonoid compounds. it has been reported that apigenin, rutin, luteolin, naringin, morin, caffeic acid, and its esters effectively inhibit bacterial growth (kartal et al. 2003; gutiérrez-venegas et al. 2019). the results of this study confirm the findings of other researchers who found that propolis extract had the highest efficacy against e. faecalis planktonic and biofilm forms (wahjuningrum and subijanto 2014; carbajal mejía 2014). according to krishnan et al. (2010), the mbc/mic ratios of propolis extract were equal to 2, suggesting that the extract has a bactericide effect against all e. faecalis strains. propolis acts directly on the bacteria by damaging the cell wall, inhibiting bacterial dna-dependant rna polymerase and cell division (bhandari et al. 2014). it also affects biofilm formation by damaging the biofilm membrane, decreasing the polysaccharide content in the biofilm, which then releases its cellular content (wahjuningrum and subijanto 2014). bacteria regulate their biofilm formation and virulence factors through the qs system, which is controlled by chemical signalling molecules known as autoinductors (paluch et al. 2020). qs can be blocked by stopping the production of the signal molecule, destroying the signal molecule, and preventing the signal molecule from binding to its receptor (yada et al. 2015). the use of qs inhibitory agents to reduce, or even completely repress, biofilm formation by pathogenic bacteria appears to be a promising approach for the control of bacterial infections (zhou et al. 2020). this study revealed that eepkh has antiquorum sensing ability against cv026, which appeared as a cream-colored halo around the well against a purple lawn of activated cv026 bacteria. a previous study by savka et al. (2015) reported that propolis samples from different regions of the united states disrupt qs autoinducer signalling in cv026. ceylan and alıç (2020) showed that propolis ethanolic extracts originated from possess antiquorum-sensing activity toward cv026. the ability of propolis to inhibit and turkey disrupt qs, biofilm formation in pathogenic bacteria may be attributed to chemical components such as caffeic acid (kasote et al. 2015). conclusion the present study describes the chemical profile of polyphenol-rich propolis collected in the algerian region of kherrata, as well as its effect on free radicals, the pathogen e. faecalis in both forms (planktonic and biofilm), as well as the qs activity against cv026.the extract was found to be rich in phenolic compounds, in which a new flavonoid compound, cynarin, was identified for the first time in algerian propolis. the results also show that the extract showed significant inhibitory potential against free radicals, e. faecalis, and cv026, suggesting the potential health benefits of propolis as a natural drug for future therapy of oral infections. further research is needed to isolate and 9 nova biotechnol chim (2023) 22(1): e1599 2 identify active chemical compounds that can be used to study their mechanisms of action. acknowledgements the authors would like to thank prof. öztürk mehmet and prof. ozgur ceylan for their assistance in this research. the authors would like to acknowledge the algerian ministry of higher education and scientific research for their support. we also want to thank all the members of the biotechnology research centre in constantine (algeria), particularly dr. chawki bensouici, for their help and advice. funding this research was funded by the algerian ministry of higher education and scientific research, as well as the dgrsdt. conflict of interest the authors declare that they have no conflict of interest. references aissaoui m, rahmoun nm, barek s, bensouici c, el haci ia (2020) structural 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supercritical extraction of phenolic compounds from brazilian red and green propolis. sep. 11 https://ascidatabase.com/author.php?author=v.&mid=&last=murugaiyah https://ascidatabase.com/author.php?author=s.m.&mid=&last=mansor nova biotechnol chim (2023) 22(1): e1599 2 sci. technol. 56: 3119-3126. savka ma, dailey l, popova m, mihaylova r, merritt r, masek m, le p, mat nor sr, ahmad m, hudson ao, bankova v (2015) chemical composition and disruption of quorum sensing signaling in geographically diverse united states propolis. evid. based complement. alternat. med. 2015: 472593. segueni n, keskin ş, kadour b, kolaylı s, salah a (2020) comparison between phenolic content, antioxidant, and antibacterial activity of algerian and turkish propolis. comb. chem. high throughput screen. 24: 1679-1687. shabbir a, rashid m, tipu hn (2016) propolis, a hope for the future in treating resistant periodontal pathogens. cureus 8: e682. snoussi m, dehmani a, noumi e, flamini g, papetti a (2016) chemical composition and antibiofilm activity of petroselinum crispum and ocimum basilicum essential oils against vibrio spp. strains. microbial. pathogen. 90: 13-21. topal m, gocer h, topal f, kalin p, köse lp, gulçin i, çakmak kc, küçük m, durmaz l, gören ac, alwasel sh (2016) antioxidant, antiradical, and anticholinergic properties of cynarin purified from the illyrian thistle (onopordum illyricum l.). j. enzyme inhib. med. chem. 31: 266-275. uzel a, önçağ ö, çoğulu d, gençay ö (2005) chemical compositions and antimicrobial activities of four different anatolian propolis samples. microbiol. res. 160: 189-195. wahjuningrum da, subijanto a (2014) the antibiofilm activity of extract propolis against biofilm enterococcus faecalis as herbal medicine potential in root canal treatment. esp endodontic society of the philippines 8: 15-18. wilmsen pk, spada ds, salvador m (2005) antioxidant activity of the flavonoid hesperidin in chemical and biological systems. j. agric. food chem.53: 4757-4761. yada s, kamalesh b, sonwane s, guptha i, swetha rk (2015) quorum sensing inhibition, relevance to periodontics. j. int. oral health. 7: 67-69 zhou l, zhang y, ge y, zhu x and pan j (2020) regulatory mechanisms and promising applications of quorum sensing-inhibiting agents in control of bacterial biofilm formation. front. microbiol. 11:589640 . zulhendri f, felitti r, fearnley j, ravalia m (2021) the use of propolis in dentistry, oral health, and medicine: a review. j. oral biosci. 63: 23-34. 12 nova biotechnologica et chimica 13-2 (2014) 87 doi 10.1515/nbec-2015-0001  university of ss. cyril and methodius in trnava the effects of combinatorial chemistry and technologies on drug discovery and biotechnology – a mini review pierfausto seneci 1,2* , giorgio fassina 3 , vladimir frecer 4,5 , stanislav miertus 5,6* 1 dipartimento di chimica, università degli studi di milano, viale golgi 19, i-20133 milan, italy (pierfausto.seneci@unimi.it) 2 cisi scrl, via fantoli 16/15, i-20138 milan, italy 3 xeptagen s.p.a., via delle industrie 9, marghera (ve) italy 4 department of physical chemistry of drugs, faculty of pharmacy, comenius university, odbojarov 10, sk-83232 bratislava, slovakia 5 international centre for applied research and sustainable technology (icarst), jamnickeho 18, sk-84104 bratislava, slovakia 6 faculty of natural sciences, university of ss. cyril & methodius, nam. j. herdu 2, sk-91701 trnava, slovakia (stanislav.miertus@ucm.sk) abstract: the review will focus on the aspects of combinatorial chemistry and technologies that are more relevant in the modern pharmaceutical process. an historical, critical introduction is followed by three chapters, dealing with the use of combinatorial chemistry/high throughput synthesis in medicinal chemistry; the rational design of combinatorial libraries using computer-assisted combinatorial drug design; and the use of combinatorial technologies in biotechnology. the impact of “combinatorial thinking” in drug discovery in general, and in the examples reported in details, is critically discussed. finally, an expert opinion on current and future trends in combinatorial chemistry and combinatorial technologies is provided. key words: combinatorial chemistry, combinatorial technologies, compound libraries, computer-assisted combinatorial drug design, virtual screening, display libraries, chemical tools, chemical biology, target identification, fragment-based drug design, combinatorial proteomics 1. introduction the expression “combinatorial chemistry” (“combichem”) became extremely popular in the late ‘90s-early ‘00s, according to its recurrence in publications. a scifinder search in early november 2013 shows that “combinatorial chemistry” is referenced significantly (>50 citations per year) since 1995 (n=60); then, it climbs steadily through 100 (1996, n=126), 500 (1999, n=592), 750 (2000, n=787) and 1000 citations (2002 and 2003, respectively n=1199 and 1203). during 2004 the decrease starts (n=995), gently continues to 800 (2010, n=791) and more steeply to ≈500 (2012, n=529). partial data for 2013 (n=246, jan to oct) confirm the tendency. this decrease mirrors the rise and the fall for several key technologies in the past: the breakthrough reports in the late ‘80s (the seminal – and often forgotten – papers from furka et al. (1988; 1991), the first combinatorial libraries to fulfil hts expectations in terms of chemical diversity by geysen et al. (1984; 1985), houghten (houghten, 1985; houghten et al., 1991), lam et al. (1991), ellman (bunin et al., 1994) and pirrung (fodor et al., 1991), its fast spreading into the pharma community bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc mailto:pierfausto.seneci@unimi.it 88 seneci, p. et al. around mid ‘90s (synthesis/biosynthesis and characterization of large libraries, new automated instrumentation, new library formats), the first concerns about quality and diversity representation in the early ‘00s (large peptide libraries, mix-and-split formats, false positives and negatives in screening), the “extraction” of useful bits and pieces from combichem around mid-late ‘00s (high throughput synthesis and purification of discrete libraries, solid-supported scavengers/reagents/purifying agents, computer-assisted diversity selection, virtual hts, display libraries). combinatorial chemistry fell from the early breakthrough technology status to the current not-sopopular/useful tool for drug discovery. no one denies that the stumbling progress during the early combichem years led to excessive expectations in the scientific and industrial community. no one, though, should neglect how even routine operations today are available through discoveries made and technologies developed by combi-chemists. we will provide here a sampling of examples of today’s combichem usefulness and applications. this contribution is divided into three sections, according to the main applications for combinatorial chemistry in synthetic/medicinal chemistry, computer-assisted combichem and biotechnology. 2. combichem in synthetic/medicinal chemistry two recent reviews provide an articulate description of what combichem in medicinal chemistry was, is and will be. the former (kodadek, 2011) highlights how key questions (library size and format, hts screening technologies, “developability” of hits from combichem libraries) should be addressed to take advantage of combinatorial chemistry without improperly making it the focus of drug discovery projects. the latter (merritt, 2012) is a detailed account of high throughput chemistry in drug discovery that describes combichem-derived techniques, strategies and synthetic methodologies in use in many industrial drug discovery labs today. they provide a balanced view of the usefulness and the opportunities provided by combichem in pharmaceutical research. let’s examine here two examples of important achievements attained through the use of a combinatorial chemistry-biased approach in a drug discovery project. 3. target identification and validation histone deacetylases (hdacs) are zinc hydrolases that modulate gene expression through deacetylation of the n-acetyl lysine residues of histone proteins. eleven hdac isoforms belong to four sub-classes (grozinger and schreiber, 2002). they are involved in many cellular pathways, and it is crucial to link their effects with specific isoforms. in 2001, naturally occurring unselective hdac inhibitors trichostatin a (tsuji et al., 1976) and trapoxin (kijima et al., 1993) were known, but isoform-specific hdac inhibitors were unavailable. the x-ray structure of the complex between tsa and an hdac ortholog (houghten, 1985) suggested the variation of the the dimethylaminophenyl radical in tsa to identify isoform-specific molecular interactions. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc nova biotechnologica et chimica 13-2 (2014) 89 schreiber performed the sp synthesis of a 7,392 membered pool library of zincbinding carboxylates, hydroxamates and o-aminoanilides analogues (respectively l1l3, fig. 1) (sternson et al., 2001). the three zinc-binding groups are connected to a linker region made by a c3-c6 alkyl chain ending with an amide bond. a bulky cap region, indeed capping each library individual, is composed of a disubstituted chiral 1,3-dioxane core anchored onto the linker via an o-, mor p-phenyl group. chemical diversity is introduced in one of the chiral 1,3-dioxane substitutions, while the other substituent is determined by final resin cleavage (sternson et al., 2001). the library was prepared using chemical encoding on polystyrene macrobeads, taking advantage of a sophisticated library synthesis/quality control/hts platform (blackwell et al., 2001; clemons et al., 2001). m = 0,1 n = 2-5 l1 zbp = oh l2 zbp = nhoh l3 zbp = l1 l3 fig. 1. structure of carboxylate (l1), hydroxamate (l2) and o-aminoanilide (l3) libraries. the library was conceived to identify selective inhibitors of histone deacetylases (hdacs), and to eventually validate one or more hdacs as therapeutical targets. the library was tested on hts cellular assays (haggarty et al., 2003), determining a) the inhibition of histone (hdac1-like) and α-tubulin (hdac6-like) deacetylation; b) the selectivity of an inhibitor for either deacetylation reaction; and c) a preliminary sar. 617 library individuals, i.e. ≈8% of the library, inhibit hdac deacetylation (point a). 475 compounds inhibit α-tubulin and 344 inhibit histone deacetylation (202 being dual deacetylase inhibitors); according to the zinc-binding motifs (zbms), 18 o-aminoanilides, up to 80 carboxylates, and up to 519 hydroxamates (points b and c) inhibit one or both enzymatic activities. further profiling of inhibitors identified tubacin (tubulin acetylation inducer, 1, fig. 2), the first hdac6-selective small molecule inhibitor (haggarty et al., 2003). its use allowed the dissection of hdac6-selective physiological and pathological events in cells, and in models of cancer (aldana-masangkay et al., 2011), neurodegeneration (d´ydewalle et al., 2011) and inflammation (de zoeten et al., 2011). it also allowed the rationalization of hdac6-selective structural features (estiu et al., 2008), the design and structural optimization of second generation, drug-like hdac6 inhibitors (wong et al., 2003). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc 90 seneci, p. et al. libraries l1-l3 are a strongly biased chemical diversity effort (few zbm-linker variations, one substituent on a position of the 1,3-dioxane ring, wide substituent sampling on the other). nevertheless, the exploitation via combinatorial chemistry of synthetically accessible modifications of scaffolds inspired by the structure of biologically active natural products allowed the characterization of hdac isoforms, and the subsequent identification of drug-like, selective hdac6 inhibitors (kalin and bergman, 2013). similar examples are available in literature; others could stem from a mixed “serendipitous/rational” approach, inspired by schreiber’s efforts. 1 fig. 2 structures of hdac6-selective tubacin (1). 4. chemical tools the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene nucleus (bodipy, 2, fig. 3) is a fluorescent probe for biological applications (loudet and burgess, 2007). it conjugates excellent photophysical properties with lack of toxicity, and the decoration of its nucleus provides specificity for single molecular targets. bodipy-based probes were reported in the past (boens et al., 2012), but a thorough exploration of the chemical space around 2 required a combinatorial chemistry approach. 2 fig. 3. the bodipy scaffold (2). chang reported the library l4 of 317 mono-styryl bodipy analogues, obtained via microwave-assisted knoevenagel condensation on the asymmetric scaffold 3 (fig. 4) (lee et al., 2009; lee et al., 2011). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc nova biotechnologica et chimica 13-2 (2014) 91 5 4 l4 fig. 4. structure of the 317-membered mono-styryl bodipy library l4, of a bsa-selective (4) and a dopamine-selective (5) bodipy-based fluorescent probe from l4. the library was tested on 94 biomolecules in intracellular-like conditions, observing the fluorescence variations at four concentrations (317 compounds, 94 biomolecules, 4 concentrations = 117,192 data points). a sar map was obtained, determining the highest response for proteins; the high fluorescence changes at low ph for p-benzylamineor heterocycle-decorated library members; and their fluorescence decrease in presence of negatively charged biomolecules (dna, rna) (lee et al., 2011). two compounds showed excellent turn on (4, fig. 4, binding to bovine serum albumin/bsa with a 212-fold fluorescence increase) and turn off properties (5, fig. 4, binding to dopamine with a 10-fold fluorescence emission decrease), with good correlations between fluorescence modulation and concentration of the binding partner (lee et al., 2011). l7 8, r1 = h, r2 = ph l6 7, r1 = p-ph-b(oh)2 l5 6, r1 = p-ch=ch-ph fig. 5. structures of the bodipy libraries l5-l7, of a igg-selective (6), a fructose-selective (7), and a human serum albumin-selective (8) bodipy-based fluorescent probe. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc 92 seneci, p. et al. other library/active pairs (fig. 5) include l5/6 (vendrell et al., 2011) (160 library members, solid-phase synthesis, 6 showing a 75-fold fluorescence increase with immunoglobulin igg); l6/7 (zhai et al., 2012) (160 library members, 7 showing a 24-fold fluorescence increase with fructose); and l7/8 (er et al., 2013) (120 library members, 8 showing a 220-fold fluorescence increase with human serum albumin). bodipy sensors 4-8 show excellent selectivity for their target biomolecule, and similar targets (i.e., albumins from animals for 4 and 8; saccharides for 7) do not cause comparable fluorescence changes. 9 10 11 14 1312 fig. 6. structures of the isocyanate 9, and of bodipy-based adducts resulting from the passerini (10), bienaymè-blackburn-groebke (11), 4-mcr (12) and 3-mcr ugi (13,14) reactions. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc nova biotechnologica et chimica 13-2 (2014) 93 modern combinatorial chemistry shifts its attention towards diversity, rather than numerosity. a small number of bodipy analogues are prepared from diversitymultiplying multicomponent reactions (mcrs) on the isocyanate intermediate 9 (vazquez-romero et al., 2013) (fig. 6). 3-mcr passerini (banfi and riva, 2005), 3-mcr bienaymè-blackburn-groebcke (bienaymé and bouzid, 1998), and 3and 4-mcr ugi (ugi et al., 1996) protocols were used to prepare 10-14 (fig. 6). each compound could lead to a combinatorial library of analogues. compound 14 (phagogreen) is a cell-permeable, non-toxic, ph-sensitive fluorescent probe to image phagosomal acidification in macrophages with sensitivity and specificity (vazquezromero et al., 2013). 5. computer-assisted combinatorial drug design combinatorial chemistry has revolutionized the drug discovery process in both academic and industrial settings. in the early years of the combinatorial boom drug design relied mainly on the screening of large diversity libraries. however, the initial screening results have been disappointing in terms of the achieved hit rates. in addition, the screening often produced hits with undesirable molecular properties, which were not suitable as lead compounds (gillet, 2002; leach and hann, 2000). although the number of compounds synthesised and screened has increased by several orders of magnitude, the quantity of new chemical entities per year remained virtually unchanged. it was soon recognized that the diversity libraries are more appropriate for initial screening of compounds against a range of biological targets to identify biological activities pertinent to the portion of chemistry space covered by the library. an increased rate of identification of drug-like hits was observed when the libraries were rationally designed and focused by applying compound filtering and selection methods. application of the lipinski’s rule-of five for the identification of analogues, to avoid insufficient oral bioavailability, was a first step in this direction. training of neural networks with drugs and chemicals, performed in industrial research laboratories resulted in the design of combinatorial libraries with a higher content of biologically active compounds (kubinyi, 2002). it became apparent that incorporation of relevant information on the targeted biomolecule or knowledge about active compounds affecting the target is beneficial for the library design. thus focused (targeted) libraries were designed to cover restricted regions of the chemistry space with boundaries defined by the available information on the biological target. additional restraints dictated by computed ligand-receptor interaction energies, structures of known active molecules and admet (absorption, distribution, metabolism, excretion and toxicity) properties shifted the emphasize from drug-like libraries to more biologically focused lead-like libraries (rose and stevens, 2003; oprea, 2002). moreover, it has turned out to be more cost effective to design and in silico screen virtual libraries of molecular models to identify subsets of the chemistry space that contain promising molecules, before going to the synthesis of compounds and to their high-throughput screening. computational modelling led to an increased hit rate of drug-like molecules in combinatorial libraries, and focused the synthetic efforts onto those synthetically accessible molecules that are most likely to bind to the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc 94 seneci, p. et al. target, as predicted by a computational algorithm. later on, computer-assisted drug design methods such as generation of virtual libraries, analogue docking and in silico screening against a three-dimensional model of a targeted biomolecule or a pharmacophore derived from active analogues, became the standard procedure routinely used in medicinal chemistry. this blend of combinatorial chemistry and structure-based design (also referred to as combinatorial docking) (böhm and stahl, 2000) has emerged as a new and promising approach to drug discovery. today, similarity searching, clustering, nonlinear mapping and other established virtual screening tools are used to aid in the selection of the most interesting library subsets. a new approach to generation of drug-like libraries was introduced by the combigen program (wolber and langer, 2001). this approach combined several hundreds of ‘privileged’ drug-specific fragments to produce structurally diverse virtual combinatorial libraries with an elevated content of drug-like molecules. if the 3d structure of the biological target is known, methods of flexible docking, which simultaneously consider the flexibility of the ligand and the binding site, are applied for compounds selection. another successful strategy is the combinatorial design of ligands within the receptor binding site (böhm et al., 1999; erlanson, 2006; villar et al., 2004; blundell and patel, 2004). instead of docking thousands of individual analogues from a combinatorial library to a receptor model, this approach constructs the whole library directly within the receptor binding site. in an attempt to increase the hit rates, the emphasis in computational and medicinal chemistry has shifted toward the rational design of small focused libraries that are biased toward one specific biological target. a greater understanding of new biochemical targets through genomics and chemical biology has also increased the number of novel drug targets for which biological screens are being developed. libraries are now being focused, through the use of computer-assisted design strategies intended to hit a single specific therapeutic target. results from docking and in silico screening of a virtual library and/or predictions of activity from a qsar (quantitative structure-activity relationships) analysis or 3d pharmacophore models are now routinely used for selection of combinatorial subsets of targeted libraries. focus on biological targets represents one aspect of a multi-objective optimisation design process, which considers also synthetic feasibility, availability and cost of reagents, diversity, drugor lead-likeness, admet properties and other commercial factors (gillet et al., 2002; agrafiotis, 2002). from a methodological point of view, structure-based combinatorial library design is an extension of traditional structure-based drug design, with the design and screening applied to virtual libraries of analogues instead of individual compounds (beavers and chen, 2002). consequently, the major computational strategies that have been developed for structure-based library design originate from the field of small molecule docking and scoring (böhm et al., 1999), or alternatively from library searching for analogues that match a pharmacophore model (rollinger et al., 2008; griffith et al., 2005). the frequently used focussing strategy is straightforward. all possible members of a virtual library are first enumerated, according to the available reagents and established synthetic scheme. individual members are then separately docked into the binding site of a receptor and finally, a bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc nova biotechnologica et chimica 13-2 (2014) 95 library subset is selected for synthesis, based on the ranking of their docking scores and/or predicted admet properties (kitchen et al., 2004). alternatively only the substituents (fragments) may be scored by attaching them to the common scaffold docked to the receptor binding site (beavers and chen, 2002; zhou, 2008). docking combined with the use of a scoring function is a fast method for ranking compounds in terms of binding potency, or complementarity to a biological target which can be used for relatively large libraries. during the docking, ligand poses are generated by fitting conformers of the ligand into three-dimensional model of the binding pocket of the macromolecular receptor. then, poses are evaluated based on a scoring function, which yields estimates of binding affinity to the receptor. docking algorithms may perform systematic or random searches for ligand conformations and then map them to the three-dimensional structure of the receptor. many scoring functions have been developed, and they may score using a force field-based, empirically based, knowledgebased, or consensus-based algorithms. there are two key requirements of all computational algorithms for combinatorial docking: first, the ability to correctly predict the conformation of the docked ligand; and second, the ability to correctly predict the binding affinity of a putative ligand. it is clear that getting the geometry correct is a prerequisite for being able to predict binding affinities (böhm and stahl, 2000; kitchen et al., 2004; zhou, 2008; rupasinghe and spaller, 2006; duffy et al., 2012; mcinnes, 2007). often only a single rigid receptor conformation is used to dock and score a ligand for the sake of computational efficiency. however, there are ways in which docking programs can account for the receptor flexibility. protein flexibility namely plays a major role in biomolecular recognition. structural changes associated with ligand binding can be significant, with deviations in overall backbone structure of the receptor, or they can be more subtle such as side-chain rotations. either way, the algorithms that predict the affinity of biomolecular binding require relatively accurate predictions of the bound structure to give an accurate assessment of the energy involved in ligand-receptor association. more accurate algorithms were subsequently developed, that accommodate the receptor flexibility during ligand docking algorithms and also explore enhanced sampling techniques. the understanding and allowance for receptor flexibility are helping to make the predictions of ligand protein binding more accurate (sinko et al., 2013; heikamp and bajorath, 2013; dawis et al., 2009). two theories have been proposed to describe the receptor configurational change upon ligand interaction. the first is conformational selection, in which all conformations are present in the unbound receptor, but the populations of each configuration change in the bound form (ma et al., 1999). the second theory is the induced fit, in which a single receptor configuration is forced into another one by the ligand binding event (sherman et al., 2006). besides receptor flexibility, other tools are available to improve the estimates of ligand binding affinities of virtual libraries. in our laboratories, we tested the performance of implicit solvent effect (frecer et al., 1998) and implementation of target-specific scoring functions. these are generic scoring functions adjusted to predict binding free energies to a specific protein receptor, by assuming a linear relationship between the computed score and predicted inhibition constants, with bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc 96 seneci, p. et al. coefficients of this linear dependence derived by linear regression from a qsar analysis. the qsar model is typically obtained for a training set of homologous molecules, for which binding affinities to the targeted macromolecule were experimentally determined. this approach was used to design peptidomimetic and cyclic urea inhibitors of hiv-1 protease, non-peptidic inhibitors of enoyl-acyl carrier protein of p. falciparum, of neuraminidase from influenza h5n1 virus, as well as peptidomimetic inhibitors of ns2b-ns3 protease from dengue virus (frecer et al., 2005; frecer et al., 2009; rungrotmongkol et al., 2009; frecer and miertus, 2010; frecer et al., 2011). another popular and powerful tool for lead discovery and optimization is the fragment-based drug design (erlanson, 2006; villar et al., 2004; blundell and patel, 2004; zhou, 2008; hajduk and greer, 2007; hubbard et al., 2007). in this approach, libraries of small fragments are screened experimentally by either nmr spectroscopy, crystallography or other biophysical techniques to find low affinity hits (small molecules, fragments). when the 3d structure of the target is known, screening of libraries for active fragment hits can also be done computationally. in the next step, for the hits that occupy different subpockets of the receptor binding, site proper linkers connecting these hits while maintaining their relative positions in the subpockets are designed. high affinity leads can be found by linking two to three suitable fragments or alternatively, individual fragment hits can be grown into leads by step-by-step functionalization (mortier et al., 2012; sancineto et al., 2013). 6. combinatorial approaches in biotechnology combinatorial chemistry significantly accelerated the development of a whole set of combinatorial tools comprising efficient synthetic methods, reagents for solid phase synthesis, linkers, addressing strategies, screening methods, etc. this is a clear indication of the importance of the combinatorial approach in chemistry especially medicinal chemistry -, but “combinatorial thinking” quickly spread to many other branches of pure and applied science. for example, combinatorial technologies are now primary tools in very different and apparently unrelated fields of research such as catalyst development (woo, 2007; dominguez, 2005) and material science (maier et al., 2007). the growing interest for combinatorial technologies and their efficacy in producing valuable results, especially when coupled to molecular modelling, makes it difficult to foresee the real number of their possible applications. biotechnology is among the main impacted areas, as we will see below. 7. biological libraries the generation of molecular diversity can take advantage of the biochemical mechanisms evolved by biological systems. the immune system is an outstanding example of combinatorial chemistry embodied in living organisms, and antibody libraries can be considered naturally evolved combinatorial libraries. the libraries of antibodies generated as the result of antigenic challenges to the immune system bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc nova biotechnologica et chimica 13-2 (2014) 97 invariably contain high affinity binders or receptors. this feature of the immune response was also exploited to evolve antibodies against transition state analogues, thus generating antibodies endowed with catalytic activity (tanaka, 2002; kochetkov, 1998). another way to exploit the biology of living organisms to generate combinatorial libraries is the biological display technology, which allows the preparation of random peptides fused to proteins normally expressed on the surface of microorganisms. biological display of peptides requires the introduction of the genetic information (dna) that codes for the peptides into a microorganism. typically, chosen organisms are bacteriophages (smith and petrenko, 1997; smith, 1985; stemberg and hoess, 1995) or bacteria (jose, 2006; levin and weiss, 2006). once inside the microorganism, the dna is transcribed and translated into proteins, according to the genetic code. the dna fragment coding for the peptides is inserted into specific dna molecules called vectors. in phage display the vector is constituted by the viral genome, while in bacterial display an extra-chromosomal circular dna, called plasmid, is used. vectors are usually genetically modified to allow the insertion of dna fragments at specific sites and, sometimes, genes or regulatory regions are deleted or inserted. phage display is nowadays a mature technology, and pre-made libraries and cloning vectors are available from standard suppliers. peptides of different length and topology, spanning between linear 7-12mers and cyclic peptides comprising a disulfide bond, can be readily expressed. antibodies can also be expressed on the surface of filamentous bacteriophages. in peptide phage display, the randomized peptide sequences are expressed at the n-terminus of the minor coat protein piii which is located on the top of the filamentous phage. from a general point of view, a peptide displaying phage could be considered a “bio-bead” with peptides bound to its surface and carrying a tag, the dna, coding for them. the success of this approach is related to the efficacy of the method used for the selection of phages displaying the peptide with the highest affinity for the target molecules (panning). the pool of phages displaying the random peptide library is allowed to interact with a solid support coated with the target molecules. the unbound phages are removed by washing the support, and the specifically bound phages are eluted in more stringent conditions. the eluted phages are amplified by propagation in e.coli, and the new pool is allowed to interact again with the target molecule immobilised on solid support. after each cycle of binding/amplification, the pool of phages is enriched with clones displaying peptides with highest affinity for the target molecule. usually after 3-4 rounds, individual clones are characterized by dna sequencing and immunochemical assays. of course, this approach allows the preparation of peptides made of proteinogenic aminoacids, even though the introduction of unnatural amino acids was reported (tiam et al., 2004). phage display libraries have been used for many applications, such as epitope mapping or identification of peptide ligands for several receptors. usually both purified target molecules or intact cells expressing the target molecule on the cell surface can be used in the panning procedure (tinoco et al., 2002; stratmann et al., 2002; gazouli et al., 2002). rasmussen and co-workers reported a successful phage display approach for tumour cell-targeting, in particular, the authors identified phage clones able to selectively bind to colorectal widr tumour cell lines (rasmussen et al., 2002). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc 98 seneci, p. et al. one of most exciting potential of phage displayed combinatorial peptide libraries is to obtain small peptide molecules that mimic an antigen, at least with respect to a particular epitope. in addition to their interest as research tools, such mimotopes could in principle be useful as diagnostic tools or for eliciting antibodies to a predefined epitope. however, the reduction of the phage insert sequence to a short peptide that can compete with the antigenic and in particular with the immunogenic properties of the natural antigen faces considerable difficulties. the difficulties to use mimotopes to induce antibodies that bind to the natural antigen (crossreactive immunogenicity) and the considerable discrepancy between antigenicity and immunogenicity of phagederived peptides have been bypassed by peptides selected with antibodies from phage displayed random peptide libraries. such peptides may represent low molecular weight substitutes of the natural antigen, not only for proteins, but also carbohydrates and nucleic acids, and thus are now being developed as vaccines for some pathological situations including cancer (ashok et al., 2003). combinatorial phage peptide libraries are also useful for identification of the specific substrates of various proteases. a substrate phage library has a random peptide sequence at the n-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. when these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. this provides a novel approach to define substrate specificity (nixon, 2002). the delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined. last but not least, combinatorial approaches, based on many different biological methods, have been used to increase enzyme stability or to refine enzyme specificity (cobb et al., 2013) but the most challenging application in biotechnology is the selection of enzymes with novel catalytic properties (acevedo-rocha et al., 2014). 8. combinatorial proteomictm the naturally generated diversity of antibody libraries has been used to develop an innovative platform technology named combinatorial proteomictm, which allows to profile the expression and function of protein families in complex proteomes. the use of antibodies allows the detection of iper and ipo-expressed proteins even at picomolar concentrations, overcoming one of the main limitations of proteomics: the difficulty in detecting low abundance proteins (corthals et al., 2000; lopez, 2000). combinatorial proteomictm shares with “classical” proteomics the goals of developing and applying technologies for the global analysis of protein expression and function. its key advantage lies in the comparison of cells from normal tissue with those representing a disease state. such comparisons enable the identification of disease-specific biomarkers that could be used for diagnostic tests, or to target proteins that have the potential for drug intervention. the striking feature of combinatorial bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=11883502&dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed?term=acevedo-rocha%20cg%5bauthor%5d&cauthor=true&cauthor_uid=25055773 http://www.ncbi.nlm.nih.gov/pubmed?term=acevedo-rocha%20cg%5bauthor%5d&cauthor=true&cauthor_uid=25055773 nova biotechnologica et chimica 13-2 (2014) 99 proteomictm is to exploit the diversity of antibody libraries in order to select specific antibodies with high avidity for selected protein families. a single individual may produce a population of antibody specificities, an antibody repertoire, which is a reflection of all the b cell clones (lymphocyte repertoire) capable of immunoglobulin (ig) synthesis and secretion in response to antigenic stimulation, but also in absence of exposure to environmental pathogens (casali and schettino, 1996). natural antibodies, in fact, are germ line-encoded molecules produced by a distinct population of peritoneal b cells bearing the cell surface marker cd5 and are present in the sera and interstitial fluids of healthy individuals (kasaian et al., 1992; greenberg, 1985; kasaian and casali, 1993). the majority of natural antibodies are polyvalent immunoglobulin m (igm) isotypes with varying – usually low – affinities. they nevertheless bind very tightly to multivalent antigens, because many low-affinity interactions can produce a single high-avidity interaction. such a feature of igm makes it the most suitable molecule to be used in the combinatorial proteomictm technology. combinatorial proteomictm comprises the following steps: 1. in silico analysis of human proteome using the pattern matcher patscan and prosite database; 2. combinatorial synthesis of selected signature libraries by standard fmoc chemistry and mix-split methods; 3. immobilization on solid support of selected signature libraries; 4. purification of signature-specific igm antibodies by affinity chromatography experiments; 5. differential analysis of cellular protein expression levels. comparative analysis of protein and peptide levels can be conducted by a differential elisa assay on biological samples from affected and healthy individuals. theoretically, any disease could be studied by using molecular profiling. specific clinical goals derived from such studies include screening tests for early disease detection, improved diagnostic markers, improved prognostic markers, new therapeutic targets, markers to evaluate therapeutic efficacy and new approaches and technologies for less invasive screening and diagnosis. 9. ligands for bio-macromolecules monoclonal antibodies for therapeutic use are one of the main products deriving from biotechnology. in the quest for a synthetic ligand able to bind to monoclonal antibodies, and suitable for affinity-chromatographic applications at the industrial level, the synthesis and screening of combinatorial libraries of peptide dendrimers (fassina et al., 1996) has been exploited. previous studies have shown that peptide ligand multimerisation enhances retention of recognition properties after immobilization on solid supports for the preparation of affinity columns (fassina, 1992; fournier et al., 1992; butz et al., 1994). based on these considerations, a tetrameric peptide library has been designed, where four identical peptide chains are assembled starting from a tetradentate lysine core. the process is similar to that used for the production of multimeric antigenic peptides (tam, 1988). the multimeric bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc 100 seneci, p. et al. library, composed of 5832 (183) randomized tripeptide tetramers, has been realized by solid-phase peptide synthesis, following a simple manual procedure (ruvo et al., 1994). screening of the activity of the multimeric library in terms of antibody recognition has been carried out by measuring the ability to interfere with the interaction between protein a and biotinylated immunoglobulins, monitored on a solid phase by elisa. the screening cycles allowed the final identification of the most active multimer as (arg–thr–tyr)4–k2–k–g, named pam (protein a mimetic ligand). pam can be synthesized by solution or solid-phase methods in high yield and at limited costs. its affinity constant for igg, as determined by optical biosensor determinations, is close to 0.3 µm. the tetrameric ligand pam can be easily immobilized on pre-activated solid supports, since the presence of the symmetric central core and the four peptide chains allow an oriented immobilization and the support-bound chain acts as a built-in spacer. the peptide was found to interact with other immunoglobulins such as ige (palombo et al., 1998) and igy (verdoliva et al., 2000). a partial inverso analogue of the pam ligand was found to impair the interaction between igg and the fcγ receptor (marino et al., 2000). following a similar approach, a cyclic peptide able to bind to immunoglobulins was discovered as well. linear peptides derived from the screening of combinatorial libraries, often do not display enough structural rigidity to provide recognition surfaces sufficiently selective for biotechnological or pharmaceutical applications. cyclic peptides, on the other hand, show increased resistance to enzymatic degradation and constrained flexibility compared with the linear form. by screening dimeric tripeptide libraries, produced by starting from a bifunctional lysine residue at the c-terminus, and structurally constrained by the presence of a disulfide bond formed by two cysteine residues at the n-terminus (fassina et al., 1995), a ligand for mouse igg purification has been identified. the screening assay has been performed by immobilizing the library, as a set of sublibraries on microtiter plates for elisa determination by noncovalent adsorption and then treating the microtiter plate wells with a fixed concentration of mouse monoclonal antibody. the presence of bound antibody was detected by a subsequent treatment with a goat anti-mouse igg, labeled with peroxidase, followed by chromogenic reaction with abts. this screening strategy led to the identification of peptide h, a cyclic dimeric peptide of formula (c–f–h–h)2k-g, where the two cysteine residues at the n-terminus are covalently linked by a disulfide bridge. when tested in affinity chromatography experiments, this ligand proved useful for mouse and rat igg purification (marino et al., 1999). the use of combinatorial libraries to identify synthetic ligands to be used in affinity chromatography for the purification of antibodies (naik et al., 2011; menegatti et al., 2013) or proteins (noppe et al., 2006; jacobsen et al., 2007) remains today one of the most promising applications in biotechnology. 9. conclusions how many drugs, or at least clinical candidates, have benefited from combichem? that’s usually the question causing endless debates. if you try to find hard evidence from the literature, you won’t find any: no one is compelled to say if at any point of a bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:19 utc nova biotechnologica et chimica 13-2 (2014) 101 drug discovery project combichem was employed. if one thinks, though, of how many papers now mention that a first hit series originated either from “wet” or virtual hts, there’s your answer: compound libraries usually are made with a heavy combichem contribution, while computer-assisted computational drug design is of a great assistance to assemble meaningful virtual collections for in silico hts. several small-medium enterprises (smes) have today a strong commitment in combinatorial technologies applied to catalysis, biotechnology, material sciences, biomedical devices and pharmaceutical research. the robustness and relative affordability of combinatorial methodologies that survived the disposal of less-thanperfect approaches is now widely accepted, although rarely recognized. combinatorial libraries composed of inorganics, metallo-organics, peptides, oligonucleotides, or small molecules represent different tools for innovative research, all of them either as the products of combinatorial technologies or as part of a proprietary technology. some sme devise, and will continue to devise their own combinatorial technologies as platforms for research and product development. one should not see “combinatorial” and “rational” approaches as being juxtaposed concepts. for example, pharmaceutical companies now often work on small, targetfocused solution-phase combinatorial libraries, where good computational design has significant impact to design the “best” library members, and is essential to increase the chances of a successful outcome – hits to be progressed to leads and candidates. “combinatorial thinking” represents a tool and a mindset, which – when applied to a project where rational design drives the planning and execution of activities – allows to increase productivity, to explore the meaningful diversity space, and consequently to maximize the probability to extract useful information (and industrially applicable products) from the project itself. references acevedo-rocha, cg., hoebenreich, s., reetz, mt.: iterative saturation mutagenesis: a powerful 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(2015) 69 doi 10.1515/nbec-2015-0016 © university of ss. cyril and methodius in trnava assessment of biologically synthesized ag nanoparticles toxicity against e. coli, staphylococcus aureus, parachlorella kessleri and sinapis alba jana kaduková1, oksana velgosová1, anna mražíková1, renáta marcinčáková1, eva tkáčová2 1technical university in košice, faculty of metallurgy, department of material science, park komenského 11, košice, sk042 00, slovak republic 2regional office of the public health, ipeľská 1, košice, sk-040 11, detached office department of environmental microbiology, roosveltova 8, košice, sk040 01, slovak republic abstract: in general, ag+ ions and agnps are considered to be the most toxic for bacterial cells and less toxic for higher organisms. in the present work inhibitory effects of biologically prepared silver nanoparticles on the growth of bacteria e. coli ccm 3954 and staphylococcus aureus ccm 3953, green microscopic alga parachlorella kessleri larg/1 and seed germination and root growth of plant sinapis alba seeds were investigated. surprisingly, silver nanoparticles showed much stronger inhibitory effects on plant seed germination and root growth than on the bacterial growth. at concentration of 75 mg/l agnps both seed germination and root growth of sinapis alba was inhibited whereas inhibition of the growth of e. coli and s. aureus was observed at >195 mg/l. growth inhibition of alga parachlorella kessleri was recorded at 300 mg/l agnps concentration. the inhibitory effect of silver ions was much higher compared to silver nanoparticles. even 20 mg/l concentration of ag+ ions inhibited the root growth and concentration > 45 mg/l inhibited germination of sinapis alba seeds. inhibition zones in both studied bacteria were found at concentration > 140 mg/l. keywords: silver nanoparticles, growth inhibition, germination tests, sinapis alba, parachlorella kessleri, e. coli, staphylococcus aureus 1. introduction engineered nanoparticles have received a lot of attention recently due to their rapidly increasing applications. rapid developments in the manufacture and use of engineered nanoparticles have also led to an urgent need for assessing their possible risk to humans and the environment, but environmental risk assessment significantly lags behind invention and today’s global consumption volumes (wang et al., 2014; moos et al., 2014). according to the woodrow wilson database there were more than 1300 nanotechnological consumer products on the market by march 2011 and 313 of them contained nanosilver. currently, nanosilver is perhaps the most preferred antimicrobial nanomaterial (ivask et al., 2014). however, release of biocidal nps from consumer and household products into the waste streams and further into the environment may pose treat to non-target organism (wang et al., 2014). antimicrobial properties of silver nanoparticles are mostly studied using common tests with bacteria, rarely by tests involving algae or higher plants. but there is bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:10 utc 70 kaduková, j. et al. significant difference among toxic concentrations found by different scientists, while some of them declare as toxic concentrations in nanomoles others found that millimoles are effective against particular organisms (sharma et al., 2009). so there is still need to find what influence the toxicity of nanoparticles. therefore, better knowledge of agnps toxicity mechanisms is required in order to evaluate the environmental risk of their toxicity (oukarroum et al., 2012). as there are some assumptions that biologically produced nanoparticles are less toxic against organisms the aim of the article was to focus on the toxic effects of biologically produced agnps on gand g+ bacteria, microscopic algae and higher plants (seeds of sinapis alba). 2. materials and methods 2.1 nanoparticle characterisation synthesis of agnps by the algae parachlorella kessleri (syn. chlorella kessleri) strain larg/1, supplied by institute of botany, slovak academy of sciences was carried out according to the method described previously (kadukova et al., 2014). algae were added into erlenmeyer flasks containing 0.5 mm silver nitrate solution. spherical silver nanoparticles in the 12 nm average size range were used in the testing. prepared nanoparticles were characterised by uv-vis spectroscopy (unicam uv/vis spectrometer uv4, chromspec company) and tem microscopy (jeol model jem-2000fx microscope operated at an accelerating voltage of 200 kv). 2.2 determination of the bacterial growth inhibition bacterial strains staphylococcus aureus ccm 3953 and escherichia coli ccm 3954 from the czech collection of microorganisms, faculty of science, masaryk university, brno, czech republic were used. the supplied disk with bacteria was impaled on the sterile needle and transferred onto the agar slope in the tube. it was incubated for 1 – 4 hours at 37±2 °c. after that the culture was left to flow on the whole agar slope surface by the slow movement of the tube and it was consequently incubated for 24 – 48 hours. pre-cultivated culture was inoculated on the sterile blood agar (m 144 a columbia blood agar base 1 % from himedia company with the following composition: peptone 23 g/l, corn starch 1 g/l, nacl 5 g/l, agar 10 g/l, final ph 7.3) in petri dishes. it was cultivated at 37±2 °c for 24 hours. the 25 μl of nanoparticle suspension was dropped in the centre of both upper and lower part of the inoculated agar plate. the silver ion and silver nanoparticle concentration of 20, 45, 75 and 300 mg/l were tested. the agar plates were incubated at 36±2 °c for 22±4 hours. the growth inhibition zone around the tested drop was measured in mm. if no inhibition zone was observed, the agar plates were incubated next 24 hours. 2.3 determination of the algal growth inhibition algal strain parachlorella kessleri, (syn. chlorella kessleri) strain larg/1 supplied by institute of botany, slovak academy of sciences was used for the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:10 utc nova biotechnologica et chimica 14-1 (2015) 71 experiments. algae were inoculated on milieu bristol agar plates. the composition of millieu bristol solution is: 750 mg/l nano3, 25 mg/l cacl2.2h2o, 75 mg/l mgso4.7h2o, 20 mg/l fe-edta, 75 mg/l k2hpo4, 175 mg/l kh2po4, 20 mg nacl, 2,86 mg/l h3bo3, 0,22 mg/l znso4.7h2o, 1,81 mg/l mncl2.4h2o, 0,0306 mg/l moo3, 0,08 mg/l cuso4.5h2o, 0,09 mg/l coso4.7h2o. the ph of the solution was adjusted to 7 before sterilization. for the study of the agnps and ag+ ions effect on algal culture green alga p. kessleri was inoculated into 50 ml of sterile solutions containing silver nanoparticles or agno3. silver concentration in agnps solutions and ag + solutions were 20, 45, 75 and 300 mg/l. the culture were incubated under continuous lighting (four 36 w, cool white fluorescent tubes) with average light intensity 3 n720 lx at 20 – 24 °c for 14 days. the 4 drops (25 μl) of tested nanoparticle suspension were placed on the plate surface. the procedure was repeated for each selected silver nanoparticle concentration (20, 45, 75 and 300 mg/l). the same procedure was also carried out with the silver ion solutions. the plates were incubated under continuous lighting (four 36 w, cool white fluorescent tubes) with average light intensity 3 720 lx at 20 – 24 °c for 7 days. inhibition zone was measured in mm. all experiments were done in triplicates. 2.4 determination of the germination inhibition inhibition of germination was performed using the seeds of sinapis alba according to the standard procedure stn 83 8303. the seeds were kept in a dry and dark place at room temperature before use. before the experiments the seeds were surface sterilized in a 10 % sodium hypochlorite solution for 20 min followed by rinsing thrice with deionised water. the tested samples of 10 ml agnps or agno3 solutions at concentrations of ag 20, 45, 75 and 300 mg/l were added into 90×10 mm petri dishes. the bottom of the used petri dishes was covered with two pieces of filter paper. after that the seeds were transferred onto the filter paper, with 30 seeds per dish at 1 cm or larger distance between each seed. deionised water was used in control samples. petri dishes were covered and placed in the dark place at room temperature. germination and root length were evaluated every day during 3 days. all studies were done in triplicates. root growth inhibition expressed by ic50 was calculated as follows: 100. k vk i l ll i − = (1) where ii is a growth inhibition [%], lk is an average root length in control [cm], lv is an average root length at tested concentration [cm]. from the graphical plot for ii as a function of log ci the value ic50 was calculated. 3. results and discussion 3.1 bacterial growth inhibition no growth inhibition of bacteria e. coli and staphylococcus aureus were observed at agnps concentrations 20 mg/l after 24 and 48 hours, as well. the 12 mm inhibition bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:10 utc 72 kaduková, j. et al. zones were observed in e. coli culture as well as s. aureus culture at ag nanoparticles concentration of 195 mg/l. at concentration 75 mg/l growth inhibition in the location of the drop for s. aureus and partial growth inhibition for e. coli (visibly less colonies in the drop location) but no inhibition zone was observed. some authors (saravanakumar et al., 2015) reported stronger inhibition effects on gbacteria e. coli in comparison with g+ s. aureus but we did not observed it. results showed that silver ions caused stronger growth inhibition of the bacteria than silver nanoparticles. the silver ions concentration at which the 10 mm inhibition zone was observed at both studied bacteria was 140 mg/l. at 300 mg/l ag+ ion concentration 11 mm inhibition zone was present. according to choi et al. (2008) ic50 of silver nanoparticles for e. coli was found to be 4 μm (0.43 mg/l). growth inhibition of 55 % was found at the ag nanoparticle concentration of app. 4.2 μm (corresponding to 0.5 mg/l). at the same silver ion (ag+) concentration the growth inhibition of e. coli culture was 100 %. independently of silver form no growth was observed, when 1 mg/l of ag (9.3 μm ag) was added (choi et al., 2008). according to panacek et al. (2006) and vimala et al. (2009) with the increase of the nanoparticle sizes their bactericidal effects decrease. s. aureus was more sensitive than e. coli. toxic silver nanoparticle concentrations with the size of 44 nm for e. coli and s. aureus were 27 and 6.75 mg/l, respectively, and the nanoparticles at the size of 50 nm were toxic for s. aureus at the concentration of 54 mg/l. silver nanoparticle and silver ion concentrations causing growth inhibition in selected bacteria are listed in the table 1. on the other hand some authors (sharma et al., 2009; miao et al., 2009) claim that silver nanoparticle toxicity is significantly higher than silver ion toxicity and whereas the toxic concentration of silver nanoparticles is at the range of nanomoles the silver ions is at micromoles. rai et al. (2008) found that the silver nanoparticles have 1.4 – 1.9 stronger antibacterial effects than silver ions. it was supposed that the shape of nanoparticles is also important for silver nanoparticle toxicity the most toxic are triangular nanoparticles in comparison with the spherical ones and the least toxic are rod shaped (pal et al., 2007). morones et al. (2005) found that the toxic concentration of cubooctahedral, icosahedral, decahedral silver nanoparticles with the size from 1 to 100 nm for e. coli was 75 mg/l. 3.2 algal growth inhibition during microscopic observation of algal culture no significant changes in the cell morphology were observed at the concentrations of 20, 45 and 75 mg/l. however, the colour of cells cultivated at the concentration of 300 mg/l turned from green to brown and the formation of aggregates occurred. the large aggregate formation was also observed by oukarroum et al. (2012), however, at much lower nanoparticle concentration (10 mg/l). the authors suggested that aggregate formations were caused by the presence of large nanoparticles (over 50 nm) which cannot directly enter the cell but can act as binding agents between algal cells resulting in algal cells growth inhibition. however, the size of our nanoparticles was up to 18 nm. authors mentioned above studied the effects of chemically prepared ag nanoparticles on the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:10 utc nova biotechnologica et chimica 14-1 (2015) 73 chlorella vulgaris cells and found that nanoparticles at concentration of 1 mg/l and 10 mg/l reduced the presence of viable cells by 33 and 88 %, respectively. table 1. inhibitory concentration of silver nanoparticles and silver ions for the growth of selected bacteria. bacteria inhibitory concentration of silver [mg/l] form of ag/size of particle [nm] source 3 nano ag sharma et al. (2009) 14.4 nano ag1/9 guzman et al. (2012) 28.8 nano ag2/14 guzman et al. (2012) 258.9 nano ag3/24 guzman et al. (2012) staphylococcus aureus mrsa 215.7 nano ag4/30 guzman et al. (2012) staphylococcus aureus ccm3953 5 nano ag sharma et al. (2009) 6.75 nano ag/25 panacek et al. (2006) 6.75 ag+ panacek et al. (2006) 3.125 nano ag saravanakumar et al. (2015) 14.4 nano ag1/9 guzman et al. (2012) 28.8 nano ag2/14 guzman et al. (2012) 258.9 nano ag3/24 guzman et al. (2012) 215.7 nano ag4/30 guzman et al. (2012) 195 nano ag/12 this work staphylococcus aureus 140 ag+ this work 3.38 nano ag/25 panacek et al. (2006) 1.69 ag+ panacek et al. (2006) 1.8 nano ag sharma et al. (2009) 14.4 nano ag1/9 guzman et al. (2012) 28.8 nano ag2/14 guzman et al. (2012) 258.9 nano ag3/24 guzman et al. (2012) 215.7 nano ag4/30 guzman et al. (2012) 10 nanotriangles ag pal et al. (2007) 125 nanospheres ag pal et al. (2007) 1 nano ag choi et al. (2008) 0.5 ag+ choi et al. (2008) 3.125 nano ag saravanakumar et al. (2015) 195 nano ag/12 this work e. coli 140 ag+ this work 1prepared by sodium dodecyl sulphate + hydrazine; 2prepared by sodium dodecyl sulphate + hydrazine + sodium citrate; 3prepared by hydrazine + 1 mm sodium citrate; 4prepared by hydrazine + 2 mm sodium citrate the inhibition of algal growth on the agar plates was clearly visible only if solution with 300 mg/l nanoparticle concentration was dropped on the plate surface. the inhibition zone of 6.6 mm was observed. at lower nanoparticle concentration bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:10 utc 74 kaduková, j. et al. (75 mg/l) the inhibition zone was not observed but in the drop location slower cell growth was observed within first 6 days. after 6 days algal cells grew on the whole agar surface. at the lowest studied nanoparticle concentration (45 mg/l) no differences were observed on the agar surface. 3.3 determination of the germination inhibition germination assay is a basic procedure to determine toxic effects on plants. seed germination and the early seedling growth are more sensitive to metal ions toxicity in comparison with mature plants because some of the defense mechanisms have not been developed yet (xiong and wang, 2005). dependence of inhibition of sinapis alba seed germination on silver nanoparticle or ag+ ions concentration is shown in fig. 1. at concentrations of 20 and 45 mg/l slight inhibition effects were observed. the concentration of 300 mg/l almost entirely inhibited the germination. at concentrations of 75 and 300 mg/l dark zones surrounding the seeds were observed. the ag+ ions showed significant inhibitory effect on seed germination compared to the silver nanoparticles. the germination inhibition was 65 % at concentration of 45 mg/l and at the 300 mg/l the inhibition was 100 %. but the concentration of 20 mg/l did not have such detrimental effects. although, it caused slowing of the germination processes, the inhibition of germination was only 20 %. fig. 1 inhibition of seed germination by silver nanoparticles and ag+ ions at concentration of 20, 45, 75 and 300 mg/l ag nps and ag+ ions in the form of agno3. barrena et al. (2009) and el-temsah and joner (2012) reported that silver nanoparticles (size 29 nm) exerted visible inhibitory effects on the germination of cucumber and lettuce seeds, nanoparticles with the size < 100 nm inhibited germination of ryegrass and barley, however, no other toxicity effects were observed. regarding the root growth the similar inhibitory influence of nanoparticles as that in germination was observed, as well (fig. 2). significant difference of the inhibitory effect between the concentration of 20 and 45 mg/l was not found. however, a further increase of the concentrations up to 300 mg/l led to the significant increase of root bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:10 utc nova biotechnologica et chimica 14-1 (2015) 75 growth inhibition. the inhibition of root growth by ag+ ions was even stronger. at concentration > 45 mg/l it was above 90 %. the colour of seeds treated with the 75 and 300 mg/l of agnps solutions turned to dark brown. however, no root colour changes as was mentioned by lee et al. (2012) were observed in our experiments. interesting was the formation of darker zones around the seeds (at the highest concentration almost black). accumulation of ag nps could cause the zone formation which could be a part of detoxifying mechanisms. fig. 2 inhibition of root elongation by silver nanoparticles and ag+ ions at concentration of 20, 45, 75 and 300 mg/l ag nps and ag+ ions in the form of agno3. lee et al. (2012) observed that the growths of phaseolus radiatus seedlings and sorghum bicolor seedlings growing on agar plates were adversely affected by increasing the agnps exposure concentrations from 5 to 40 mg/l. the growth reduction was from 35 – 80 % and 40 – 53 % for p. radiatus and s. bicolour, respectively. the authors found the ec50s of the agnps of p. radiatus and s. bicolor were 13 and 26 mg/l, respectively. brown tips and necrosis were detected in the exposed roots of both plants. in their study the citrate-coated nanoparticles (size 5 – 25 nm) were used. in the case of silver ion solution, brown colours of seeds or brown spots on their surfaces were more often present in comparison with agnps. on the other hand, zones around seeds had lighter colour. not only root elongation but also development of seed leaves was strongly affected by silver ion presence as seed leaves were observed only at ag+ ions concentrations of 20 and 45 mg/l. in both studied parameters (germination and root length) significant inhibition was observed in the case of silver ions. according to the standard procedure stn 83 8303 the preliminary ic50s 5.2 mg/l and 42.8 mg/l for ag + ions and silver nanoparticles, respectively, were calculated. 4. conclusion in general, ag+ ions and agnps are considered to be the most toxic for bacterial cells and less toxic for higher organisms. however, our results revealed that agnps bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:10 utc 76 kaduková, j. et al. expressed much stronger inhibitory effects on plants than bacteria. at concentration of 75 mg/l agnps both seed germination and root growth of sinapis alba was inhibited whereas inhibition of the growth of e. coli and s. aureus was observed at >195 mg/l. silver ions had stronger inhibitory effects on bacterial growth as well as sinapis alba seeds germination and root growth than agnps. in the case of the green alga parachlorella kessleri slight algal growth inhibition on agar plates was observed at agnps concentration of 75 mg/l and visible inhibition zones were reported at 300 mg/l. acknowledgements: this work was supported by the slovak grant agency vega, grant no. 1/0197/15. references barrena, r., casals, e., colón, j., font, x., sánchez, a., puntes, v.: evaluation of the ecotoxicity of model nanoparticles. chemosphere, 75, 2009, 850–857. choi, o., deng, k.k., kim, n-j., ross, l., surampalli, r.y., hu, z.: the inhibitory effects of silver nanoparticles, silver ions and silver chloride colloids on microbial growth. water res., 42, 2008, 3066-3074. el-temsah, y.s., joner, e.j.: impact of fe and ag nanoparticles on seed germination and differences in bioavailability during exposure in aqueous suspension and soil. environ. toxicol., 2012, 27, 1, 42-49. guzman, m., dille, j., godet, s.: synthesis and antibacterial activity of silver nanoparticles against gram-positive and gram-negative bacteria. nanomedicine: nbm, 8, 2012, 37-45. ivask, a., juganson, k., bondarenko, o., mortimer, m., aruoja, v., kasemets, k., blinova, i., heinlaan, m., slaveykova, v., kahru, a.: mechanisms of toxic action of ag, zno and cuo nanoparticles to selected ecotoxicological test organisms and mammalian cells in vitro: a comparative review. nanotoxicology, 8, s1, 2014, 57-71 kaduková, j., velgosová, o., mražíková, a., marcinčáková, r.: the effect of culture age and initial silver concentration on biosynthesis of ag nanoparticles. nova biotechnol. chim., 13, 1, 2014, 16-25. lee, w.m., kwak, j.i., an, y-j..: effect of silver nanoparticles in crop plants phaseolus radiatus and sorghum bicolor: media effect on phytotoxicity. chemosphere, 83, 2012, s. 491–499. miao, a-j., schwehr, k.a., xu, ch., zhang, s-j., luo, z., quigg, a., santschi, p.h.: the algal toxicity of silver engineered nanoparticles and detoxification by exopolymeric substances. environ. pollut., 157, 2009, 30343041. moos von, n., bowen, p., slaveykova, v.i.: bioavailability of inorganic nanoparticles to planktonic bacteria and aquatic microalgae in freshwater. enviro. sci. nano, 1, 2014, 214-232. morones, j.r., elechiguerra, j.l., camacho, a., ramirez, j.t.: the bactericidal effect of silver nanoparticles. nanotechnology, 16, 2005, 2346-2353. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:10 utc nova biotechnologica et chimica 14-1 (2015) 77 oukarroum, a., bras, s., perreault, f., popovic, r.: inhibitory effects of silver nanoaprticles in two green algae, chlorella vulgaris and dunaliella tertiolecta. ecotoxic. enviro. safe, 78, 2012, 80-85. pal, s., tak, y.k., song, j.m.: does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticles? a study of the gramnegative bacterium escherichia coli. app. environ. microbiol., 27, 6, 2007, 17121720. panacek, a., kvitek, l., prucek, r., kolar, m., vecerova, r,, pizurova, n,, sharma, v.k., nevecna, t., zboril, r.: silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity. j. phys. chem. b, 110, 2006, 16248-16253. rai, m., yadav, a., gade, a.: silver nanoparticles as a new generation of antimicrobials. biotechn. adv., 27, 2009, 76-83. saravanakumar, a., ganesh, m., jayaprakash, j., jang, h.t.: biosynthesis of silver nanoparticles using cassia tora leaf extract and its antioxidant and antibacterial activities. j. ind. eng. chem., 2015 (article in press). sharma, k.v., yngard, r.a., lin, y.: silver nanoparticles: green synthesis and their antimicrobial activities. adv. coll. interface sci., 145, 2009, 83-96. stn 83 8303 testing of dangerous properties of wastes. ecotoxicity. acute toxicity tests on aquatic organisms and growth inhibition tests of algae and higher cultivated plants. vimala, k., sivudu, k.s., mohan, y.m., sreedhar, b., raju, k.m.: controlled silver nanoparticles synthesis in semi-hydrogel networks of poly(acrylamide) and carbohydrates: a rational methodology for antibacterial application. carbohyd. polym., 75, 2009, 463-471. wang, x.z., yang, y., li, r., mcguinnes, c., adamson, j., megson i.l., donaldson, k.: principal component and causal analysis of structural and acute in vitro toxicity data for nanoparticles. nanotoxicology, 8, 5, 2014, 465-476. xiong, z. t., wang, h.: copper toxicity and bioaccumulation in chinese cabbage (brassica pekinensis rupr.). environ. toxicol., 20, 2005, 188-194. presented at the 3rd international conference “biotechnology and metals 2014”, september 17-19, 2014, košice, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:10 utc 90 nova biotechnologica et chimica 15-1 (2016) doi 10.1515/nbec-2016-0010 © university of ss. cyril and methodius in trnava degradation of synthetic dyes by laccases – a mini-review barbora legerská, daniela chmelová, miroslav ondrejovič department of biotechnologies, faculty of natural sciences, university of ss. cyril and methodius, nam.j. herdu 2, trnava, sk-917 01, slovak republic (daniela.chmelova@ucm.sk) abstract: laccases provide a promising future as a tool to be used in the field of biodegradation of synthetic dyes with different chemical structures. these enzymes are able to oxidize a wide range of phenolic substrates without the presence of additional co-factors. laccases have been confirmed for their potential of synthetic dye degradation from wastewater and degradation products of these enzymatic reactions become less toxic than selected dyes. this study discusses the potential of laccase enzymes as agents for laccase-catalyzed degradation in terms of biodegradation efficiency of synthetic dyes, specifically: azo dyes, triphenylmethane, indigo and anthraquinone dyes. review also summarizes the laccase-catalyzed degradation mechanisms of the selected synthetic dyes, as well as the degradation products and the toxicity of the dyes and their degradation products. key words: laccase, synthetic dye, biodegradation, toxicity, degradation products. 1. introduction synthetic dyes have a nearly exhaustible range of application in various types of industry including food, pharmaceutical, textile, printing, paper or chemical. it has been estimated that approximately 5-10 % of dyes used in the industrial sector could remain persistent in wastewater (dafale et al., 2008). untreated dyeing effluents are a serious environmental problem in the twenty first century. the release of dyes into the environment is harmful due to toxicity, carcinogenic and/or mutagenic effects on living organisms. the presence of synthetic dyes in wastewater can cause an increase of bod (biochemical oxygen demand) and cod (chemical oxygen demand) levels. moreover, the chromophoric groups strongly absorb sunlight and therefore, photosynthetic activity of organisms is inhibited (da silva et al., 2010; kagalkar et al., 2010). the negative impact of synthetic dyeshas been observed in the oestrous cycle and reproductive system in rats (nath et al., 2015), in biochemical markers of vital organs, such as the liver and kidney (amin et al., 2010) and to foetal growth (wan et al., 2011; gopinathan et al., 2015). a correlation between the presence of synthetic dyes and high clastogenic activity in bone marrow cells has been observed in rats (rajaguru et al., 1999). additional studies have shown that a correlation exists between synthetics dyes and mitotic abnormalities (azmi et al., 1998), as well as cirrhosis from chronic consumption of selected dyes (axon et al., 2012). because of the aforementioned reasons, wastewater treatment containing synthetic dyes is a global problem requiring an immediate yet cost-effective solution. degradation of synthetic dyes using biological methods is a promising, environmentalfriendly process and these methods are presented as a cheaper alternative to the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc nova biotechnologica et chimica 15-1 (2016) 91 expensive physicochemical methods producing large amount of sludge, which must be degraded by other processes. using biological methods, the degradation of synthetic dyes can occur in a cost efficient, eco-friendly format with certain advantages. not only are the products of enzyme-catalyzed reaction less toxic than synthetic dyes (campos et al., 2001; telke et al., 2011; adnan et al., 2015), but the degradation products can be utilized by various natural organisms. these biological methods are founded on enzymes, which are produced by organisms. in the case of enzymatic degradation, azoreductases, peroxidases and phenol oxidases have a potential for dye biodegradation, but in certain aspects each of these enzyme falters in their biodegradation efficiency. azoreductases (ec 1.7.1.6) require additional co-factors such as nadh2, nadph2 and fadh2 for the activation of the catalyst. additionally, azoreductases can degrade only azo dyes via reductive cleavage of azo bonds (pandey et al., 2007). one of the main disadvantages of this enzyme-catalyzed degradation processes the formation of toxic products (platzek et al., 1999). it is known that azoreductases are intracellular enzymes and their use in the pure form without production organisms is problematic due to issues of stability and necessary regeneration of co-factors. furthermore, the direct application of microorganism produced azoreductases can result in additional problems from the inability of dye diffusion through cell membranes, due to the molecular weight of azo dyes (robinson et al., 2001). peroxidases (ec 1.11.1.x), such as horseradish peroxidase, chloroperoxidases, lignin peroxidases and manganese peroxidases, are other enzymes that could be useful for synthetic dye biodegradation but have a factor that limit their benefits in synthetic dye degradation. all of these enzymes belong to hemoproteins which catalyse the chemical reactions in the presence of hydrogen peroxide (duran et al., 2002). these reactions require specialized attention from because the presence of hydrogen peroxide at too high of a concentration can cause the inactivation of peroxidases (aitken et al., 1994). the last enzyme group described in the literature as useful for synthetic dye decolorization includes phenoloxidases. these enzymes belong to oxidases which catalyse the oxidation of phenolic compounds in the presence of oxygen without additional co-factors. one of these enzymes, laccase (ec 1.10.3.2), is a member of the multicopper oxidase family (telke et al., 2011). laccases catalyse the removal of a hydrogen atom from the hydroxyl group via electron oxidation (bollag, 1992), which generates non-toxic products during synthetic dye biodegradation (campos et al., 2001; telke et al., 2010; adnan et al., 2015). although the biodegradation potential of laccases is mostly evaluated by the change of a absorption spectrum of dye (suzuki et al., 2001; bibi et al., 2011; chmelová and ondrejovič, 2015), the destruction of chromophores can led to dye degradation (chen and ting, 2015). in this mini-review, the synthetic dye degradation of laccase-catalyze reaction has been observed with regards to their degradation products and their potential toxicity. 2. laccases laccases (ec 1.10.3.2, p-diphenol: dioxygen oxidoreductases) are classified as phenol oxidases which are able to catalyse one-electron oxidation of the substrate associated with the simultaneous reduction of oxygen in water. these enzymes are bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc 92 legerská et al. produced by a wide spectrum of organisms such as bacteria, filamentous fungi or plants (rodríguez-couto and herrera, 2006; bibi et al., 2011; zhou and xiang, 2013). laccases are monomeric, dimeric or tetrameric glycoproteins with four copper atoms per monomer located in catalytic sites. type 1 copper (cu t1, ligated by at least one cys and two his) is paramagnetic and responsible for the characteristic blue colour and the oxidation of the substrate. type 2 copper (cu t2, ligated by two his) and two copper atoms of type 3 (cu t3, each ligated by three his) conforming trinuclear cluster play a key role in the reduction of molecular oxygen to two molecules of water (davies and ducros, 2006; mot and silaghidumitrescu, 2012). laccase can oxidize different organic compounds such as monoand di-phenols or their derivatives with hydroxy-, carboxy-, methoxy-, aminoor sulphofunctional groups by radical mechanism. phenols are typical substrates for laccases due to their low redox potential which allows for electron abstraction by the cu t1. therefore, the ability for laccase enzymes to oxidize molecules is determined by the redox potential of cu t1 (e0 cu t1). e0 values have been determined using potentiometric titrations for various laccases (table 1). according to e0 cu t1, the laccases can be classified into three categories: low-, mediumand high-redox potential laccases. this redox potential is a result of combined factors such as copper-ligand interactions, the effect of desolvation around the t1 site, the intermolecular electrostatic interactions, and the restrictions in protein folding (li et al., 2004). bacterial and plant laccases have lowredox potential (below +460 mv vs. nhe – normal hydrogen electrode) while fungal laccases belong to mediumand high-redox potential laccases (mate and alcalde, 2015). laccases with medium-redox potential (+460 to +710 mv vs. nhe) are produced mainly by ascomycetes and basidiomycetes. laccases with highredox potential (+730 to +790 mv vs. nhe) are commonly found in white-rot fungi (basidiomycetes). for industrial application, laccases with a high-redox potential are the most relevant and most useful. the efficiency of synthetic dye oxidation by laccases depends on differences between the redox potential of dye and potential of enzyme (yang et al., 2015). for phenolic substrate, the reaction starts with the deprotonation of the phenolic hydroxyl group. this results in the formulation of unstable phenoxy radicals which lead to quinone formation. this process combines the oxidative transformation without the coupling of forming products, where oxidation cleavage leads to a reduction of product molecular mass and oxidative decarboxylation of different products (polak and jarosz-wilkolazka, 2012). for diversification of substrate specificity, laccases need the presence of lowmolecular weight compounds (redox mediators) because they usually are not able to oxidize non-phenolic compounds or molecules with high redox potential (giardina et al., 2010). these compounds act as intermediate substrates for laccases. the initial step is the oxidation of a redox mediator to forming a radical (1) (fabbrini et al., 2002). the substrate is attacked the radical thereby giving rise to product and producing mediator back. med-oh laccase med-oh med-o (1) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc nova biotechnologica et chimica 15-1 (2016) 93 table 1. the values of redox potential e0 (cu t1) for laccases isolated from various organisms. organism mw (kda) ph e 0 (cu t1) (mv vs. nhe) reference rhus vernicifera 5.5-8.5 0.410 johnson et al., 2003 trametes ochracea 64±2 3.74.9 0.79±0.1 shleev et al., 2004; shleev et al., 2005 trametes hirsuta 70±2 3.5-4.5 0.78±0.1 shleev et al., 2004; shleev et al., 2005 coprinus cinereus 58 5.5 0.55 schneider et al., 1999 cerrena maxima 67±4 4.0-6.0 0.75±0.05 shleev et al., 2004; shleev et al., 2005 coriolopsis fulvocinerea 65±2 3.9-5.2 0.78±0.1 shleev et al., 2004; shleev et al., 2005 ganoderma sp. 62 3.0-5.0 0.63 sharma et al., 2013 marasmius guercophilus c30 65 5.7 0.73 klonowska et al., 2002 melanocarpus alomyces 8.0 0.46±0.01 kruus et al., 2003 pleurotus ostreatus 3.0 0.588 dai et al., 2016 pycnoporus sanguineus 67 4.5 0.747 zimbardi et al., 2016 redox mediators can be natural or synthetic.3-hydroxyanthranilic acid, 4-hydroxybenzoic acid, 4-hydroxybenzyl alcohol, syringaldehyde, sinapinic acid, acetovanillon, ferulic acid, vanillin, p-coumaric acid (moreira et al., 2014) belong to the group of natural mediators. generally these compounds are phenolic substrates produced by white-rot fungi. synthetic redox mediators are 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), 1-hydroxybenzotriazole, n-hydroxyphthalimide, violuric acid or n-hydroxyacetanilide. the application of artificial mediators has several disadvantages such as high cost and toxicity (canas and camarero, 2010). laccases are able to degrade various organic pollutants with different chemical structures via direct oxidation (bollag, 1992). this ability depends on the enzyme as well as dye structures. the presence and the location of different functional groups in dye structure can affect degradation of selected dyes. for most laccases, the presence of ortoand metasubstituents in dye structure is preferable for laccase oxidation as these in para-position (baldrian, 2006; tauber et al., 2008). electron-donating substituents (-oh, -ch3, -nh2, -n(ch3)2) contribute to increased biodegradability while electron-withdrawing substituents (-cooh, -so3h, -no2, -cl, -br) make a ring less susceptible to biological oxidation (suzuki et al., 2001). for example chivukula and renganathan (1995) found that laccase from pyricularia oryzae oxidizes only azo dyes with methylor methoxysubstituents in azo structure while non-substituted 4-(4´-sulfophenylazo)-phenol and its 2-chloroand 2-nitro-analogs were not oxidized. the negative effect of halogen groups (-cl, -br) in the structure of triphenylmethane dyes such as bromophenol blue and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc 94 legerská et al. a b c d bromochlorophenol blue to their decolorization by laccase was confirmed also in our recent work (chmelová and ondrejovič, 2015). 3. synthetic dyes synthetic dyes are complex aromatic compounds which are produced via chemical synthesis which provide a wide range of colours. the most industrially used synthetic dyes can be classified as azo dyes, anthraquinone, triphenylmethane and indigo dyes (fig. 1) according to the main structure which often includes chromophores (-c=c-, -c=o, -c=n-, -no2, -n=nand quinonoid rings) responsible for absorption of visible light. n n nh2 nh2 o o oh oh nn ch3 h3c ch3 ch3 h n n h o o fig. 1. the basic structure of selected synthetic dyes. a – azo dye (chrysoidine), b – anthraquinone (alizarine), c – triphenylmethane (malachite green) a d – indigo dye (indigo). diversion of compounds belonging to individual synthetic dye groups is caused mainly by the auxochrome groups e.g. (ch3)2n-, -nh2, -oh, -och3, ch3co-, ch3-, no2, -so3h, -cor, -co2h, halogens (forgacs et al., 2004). 3.1 mechanisms of azo dye degradation azo dyes belong to the group of aromatic compounds which contain one or more azo bonds (-n=n-). azo dyes are produced by the copulation of diazonium salts with amines or phenols or naphtol, respectively. azo bonds are substituted with benzene or naphthalene which can contain different functional groups such as -cl, -ch3, -no2, -nh2, -oh and -co. in the enzymatic degradation pathway, azo dyes can be cleaved symmetrically or asymmetrically (telke et al., 2009) through a highly non-specific free radical mechanism, forming phenolic products. the biggest problem with enzymatic cleavage of azo dyes is the formation of toxic products, mainly amines. therefore, it is important to identify and evaluate toxicity and/or mutagenicity of degradation products (chhabra et al., 2009). azo dye degradation by laccases starts by asymmetric cleavage of the azo bond followed by oxidative cleavage, desulfonation, deamination, demethylation and dihydroxylation, depending on dye structure (telke et al., 2009; telke, et al., 2011; adnan et al., 2015; yang et al., 2015; zheng et al., 2016). some authors describe degradation of azo dyes without the cleavage of the azo bond (chen, 2006; pereira et al., 2009). this mechanism includes the formation of phenolic type compounds resulting from highly non-specific free radical mechanism (chen, 2006). the inability of laccase to cleave azo bond in the structure of dyes may be related to bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc nova biotechnologica et chimica 15-1 (2016) 95 the redox potential of laccase. pereira et al. (2009) suggested the proposed mechanism for biotransformation of the mono azo dye sudan orange g by laccase from bacillus subtilis. the enzymatic oxidation of sudan orange g results in the production of oligomers, and possibly polymers through radical coupling reactions without the cleavage of azo bond. laccase from b. subtilis has a low-redox potential (455 mv vs. nhe) (durao et al., 2006), so it belongs to the group of low-potential laccases. on the other hand, laccases from fungal organisms are more applicable for degradation of azo dyes because they belong to the group of high-redox potential laccases (mate and alcalde, 2015). an example can be found in the work of telke et al. (2010) which describe the degradation of azo dye (methyl orange) by laccases from aspergillus ochraceus ncim-1146 (fig. 2). n nho3s n(ch3)2 asymetric cleavage decolorization hn nho3s n(ch3)2 p-hydroxybenzene sulfonic acid p-n,n'-dimethylamine phenyldiazine fig. 2. degradation mechanism of mono azo methyl orange by laccase from aspergillus ochraceus (telke et al., 2010). the first step of mono azo dye decolorization by laccases is carbocation, the formation of an electron-deficient reaction centre and therefore highly reactive intermediates. these can be subject to the nucleophilic attack by -so3, -oh or halogen nucleophiles resulting in asymmetric cleavage of azo bond (telke et al., 2010). degradation products formed by laccase from methyl orange in this way are p-n,n'-dimethylamine phenyldiazine and p-hydroxybenzene sulfonic acid (fig. 2). although many authors marked these compounds as toxic (wang et al., 2008), du et al. (2015) concluded that degradation products (amine and amide derivatives) formed by biological treatment of methyl orange in water solution by aeromonas sp. strain dh-6 have lower phytotoxicity levels tested on chinese cabbage. degradation of bis azo dyes is a more complicated process. in the same way, azo bonds are cleaved asymmetrically by laccases (si et al., 2013; adnan et al., 2015; zheng et al., 2016) but this reaction requires electrons for reduction (nam and renganathan, 2010). laccases can transfer electron to azo dye because laccases contain four histidine-rich copper binding domains in the catalytic centre (zheng et al., 2016). biodegradation of the bis azo dye congo red by high-redox potential laccases from trametes pubescens (e0 cu t1 = 738 – 745 ± 5 mv vs. nhe; shleev et al., 2007) led to the formation of naphthalene amine (fig. 3) but phytotoxic test have shown that the purified laccase can detoxify azo dye congo red (si et al., 2013). this means that congo red degradation does not finish in this phase but probably continues forming other non-toxic degradation products. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc 96 legerská et al. similarly, biodegradation of the bis azo dye reactive black 5 by laccase from trichoderma artroviride f03 was initiated by the cleavage of the bis azo bond and followed by deamination, hydroxylation and sulphonation. the genus trichoderma is characterized by production of laccases with medium-redox potential (sadhasivam et al., 2008). the biodegradation mechanism continued with the aromatic ring fission of naphthalene-1,2,8-triol, where its oxygenated ring at c1 and c2 position was cleaved to 2-(2-carboxy-ethyl)-6-hydroxy-benzoic acid via 8-hydroxy-[1,2]-naphthoquinone. 2-(2-carboxyethyl)-6-hydroxy-benzoic acid can be degraded via two possible pathways (i) it undergoes decarboxylation and methylation to form 2,4-ditertbutylphenol (detected at 7th day) and (ii) it is transformed to benzoic acid by decarboxylation mechanism (adnan et al., 2015). moreover, adnan et al. (2015) found out that laccase from t. artroviride d03 did not generate toxic aromatic amines. similarly, zheng et al. (2016) found that acid black 172 biodegradation by laccase does not produce toxic amines. h n h n h n h n nh2 nh2 so3na so3na asymetric cleavage decolorization h nh2n h nnh2 o nh2 nh2 so3na so3na h2nh2n h n nh2 so3na desulfonation deamination desulfonation deamination oxidative cleavage nh2 hn n deamination naphtalene amine biphenyl napthalene diazonium fig. 3. degradation mechanism of congo red by purified laccases from trametes pubescens (si et al., 2013). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc nova biotechnologica et chimica 15-1 (2016) 97 3.2 mechanisms of indigo dye degradation indigo dyes are organic compounds with a characteristic blue colour obtained from plant material such as indigo feratinctoria l., indigofera suffructicosa mill., isatis tinctoria l. and polygonum tinctorium. at present these dyes are produced by chemical synthesis via baeyer-drewson reaction. indigo dyes are non-soluble in water, alcohols and ether because these dyes contain intraand inter-molecular hydrogen bonds. for the highest solubility of indigo dyes in water it is necessary to solubilize the dye in water with a base such as sodium hydroxide. indigo and its derivatives such as indigo carmine play a key role in various sectors such as the food, textile or cosmetic industries and medicine because of their colour but they are highly toxic and contact with skin or eyes may cause irritation. the oxidation of indigo dyes by laccases is observed as the sequential taking of four electrons from the molecule of indigo. the first step in indigo and indigo derivatives degradation is electrochemical oxidation to dehydroindigo (beggiato et al., 1993) followed by an attack of nucleophile (e.g. water) which lead to the incorporation of o-atoms into the degradation products (campos et al., 2001). the proposed pathway of indigo degradation by laccase from trametes hirsuta is shown in fig. 4-a (campos et al., 2001). laccases are able to degrade indigo via the formation of isatine (indol-2,3-dion). next isatine is degraded to anthranilic acid (2-aminobenzoic acid) spontaneously via decarboxylation of isatic acid, which is intermediate formed hydrolytically after isatine degradation. n n o h h o n + n o h o n o o h 2 nh2 oh o isatin anthranilic acid n n o h h o nao3s so3na nh2 oh o anthranilic acid n h o so3na o isatin-5-sulfonic acid a b desulfonation fig. 4. degradation mechanism of indigo (a) and indigo carmine (b) by laccase from trametes hirsuta (campos et al., 2001; singh et al., 2007). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc 98 legerská et al. younes and sayadi (2013) found that enzymatic degradation of indigo carmine by laccase from the fungus scytalidium thermophillum reached 98 % decolorization after 24 hours. toxicity level of degradation products was lower toward bacteria escherichia coli and bacillus megaterium than indigo carmine itself. the bacterial strain γ-proteobacterium jb and the white-rot fungus t. hirsuta were able to effectively degrade indigo carmine to anthranilic acid and isatin-5-sulfonic acid (singh et al., 2007), but higher amount of anthranilic acid was measured in the reaction system catalyzed by fungal laccase (98.3 %) than bacterial laccase (56.4 %) (fig. 4-b). additionally, laccase from t. hirsuta had the higher rate of reaction. these results were confirmed by campos et al. (2001) which found that degradation of indigo is faster in the presence of laccase from the fungal strain t. hirsuta than laccase from the bacterial strain sclerotium rolfsii, but the same degradation products (anthranilic acid and isatin) were created after biodegradation by both laccases. this difference in the reaction rate was caused by the different redox potential of selected laccases because fungal laccases have higher e0 cu t1 potential than bacterial laccases (mate and alcalde, 2015). 3.3 mechanisms of triphenylmethane dye degradation triphenylmethane (tpm) dyes belong to the group of older dyes with intense colour to be used in the textile, paper, food, cosmetic and leather industries and medicine. the basic structure of these dyes is triphenylmethane. tpm dyes are known as resistant to enzymatic decolorization and the process requires more time for dye degradation (forootanfar et al., 2012). although srivastava et al. (2004) did not confirm mutagenicity of tpm dyes such as basic green 4 and acid violet 17 (srivastava et al., 2004), malachite green (mg) is a typical tpm dye used in farmed fish for controlling of protozoan and fungal infection. degradation of mg by laccase as well as toxicity of mg and its degradation products has been extensively studied by many authors (kumar et al., 2012; zhuo et al., 2015; yang et al., 2015). compared with phytotoxicity level of mg expressed by inhibition of seed germination, phytotoxicity levels of its degradation products were decreased. similarly, decrease toxicity of tpm dyes after laccase treatment was observed in other studies (kalyani et al., 2008; sathishkumar et al., 2013; yang et al., 2015). laccases can oxidize the methyl carbon attached in tpm dye structure, giving stable products which are affected by p-substituted phenyl. bibi et al. (2011) suggested that n-demethylation was the key factor of tpm degradation. casas et al. (2009) found that laccases are able to degrade tpm dyes, but non-substituted tpm dyes were not completely degraded by laccases. laccase from cyathus bulleri is able to degrade basic green 4 via demethylation. yang et al. (2015) proposed two parallel degradation pathways of mg by laccase of cerrena sp. (fig. 5). the first pathway starts with demethylation of mg followed by degradation or polymerization of mg for chromophore destruction. in the second pathway, mg is firstly hydroxylated to its carbinol form (fisher et al., 2011) which is quickly broken down. the presence of both pathways contributes to fast and efficient degradation of mg by laccases. the pathway is probably depended on laccase type and reaction conditions. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc nova biotechnologica et chimica 15-1 (2016) 99 kumar et al. (2012) confirmed the formation of four different non-identified degradation products of mg after treatment by laccase from pleurotus ostreatus. on the other hand, after degradation of brilliant green 1 closing related to mg by laccase from trametes versicolor (e0 cu t1 = 785 mv vs. nhe; reinhammar et al., 1972), the formation of only two compounds (benzoic acid and diethylamine) were observed (casas et al., 2009). nn ch3 ch3 h3c h3c nn ch3 h h3c h3c nn ch3 ch3 h3c h3c i ii hydroxylationdemethylation oh oxidation demethylation demethylation n h3c ch3 o n h h h demethylation demethylation demethylation nn h h h h tetradesmethyl mg (amino phenyl)-phenyl-methanonen,n-dimetylanilin fig. 5. pathways (i, ii) for malachite green (mg) degradation by laccase of cerrena sp. (yang et al., 2015). 3.4 mechanisms of anthraquinone dye degradation anthraquinone dyes are the second most important class of textile dyes (baughman and weber, 1994). they provide a wide range of colours including violet, blue and green and show excellent long-term colour stability (meng et al., 2003). the oldest dye from this group is alizarin which was extracted from the root of durable plant rubia tinctorum for the first time. the π-conjugated electron system is delocalized in the core and in the substituents. the important anthraquinone dyes bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc 100 legerská et al. commonly used in textile production process are acid blue 129 and remazol brilliant blue r (rbbr). these dyes were used as reactive dyes for polymeric dye production and represent the class of often toxic and recalcitrant pollutants. there are several experimental works (yang et al., 2009; zeng et al., 2012; afreen et al., 2016) concerning the study of an anthraquinone dye decolorization by laccases. laccases have been shown to decolorize anthraquinone dyes more efficiently than other classes of dyes (zeng et al., 2011). additionally zeng et al. (2012) described anthraquinone dyes acting as redox mediators for azo dye decolorization catalyzed by laccases. during the degradation of anthraquinone dyes by laccase, the chromophore of dye can be broken down forming smaller molecules with potential lower toxicity levels. osma et al. (2010) observed biodegradation of rbbr by laccase from t. pubescens and suggested the reaction pathway including the identified intermediates and degradation products (fig. 6-a). during rbbr degradation by laccase was observed reduction, hydroxylation, deamination, and oxidation reactions. in the later work of hadibarata et al. (2011) confirmed the effective decomposition of rbbr dye by laccase from polyporus sp. s133 (fig. 6-b) (hadibarata et al, 2011). the analogous deamination reaction is not observed in rbbr biodegradation by laccase from polyporus sp. s133 resulting in products which contain an amino group in their structure. o o hn nh2 so3na s o o o so3na o so3na o s o o o o so3na o nh2 h2n s o o o so3na oh a b o fig. 6. pathway for remazol brilliant blue r degradation by the laccase of trametes pubescens (a) (osma et al., 2010) and laccase of polyporus sp. s133 (hadibarata et al, 2011). although hadibarata et al. (2011) found the presence of low-molecular weight products after laccase-catalyzed reaction but toxicity levels of these products were not lower. on the other hand, osma et al. (2010) evaluated phytotoxicity of rbbr and its degradation products after laccase treatment using ryegrass as a representative of grasslands species. degradation products of rbbr were still toxic for ryegrass (germination index – gi of 69 %) but less than original dye (gi of 26 %). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc nova biotechnologica et chimica 15-1 (2016) 101 4. conclusion it has been demonstrated that laccases are able to degrade synthetic dyes with different chemical structures. the efficiency of biodegradation is primarily affected by type of laccase-producing organism which determines the redox potential of laccases, different substrate specificities and, last but not least, rates of dye biodegradation. while bacterial and selected plant species produce laccases with a low-redox potential, filamentous fungi produce laccases with mediumor high-redox potential. the whiterot fungi group is particularly interesting in this 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govindwar, s.p.: influence of organic and inorganic compounds on oxidoreductive decolorization of sulfonated azo dye c.i. reactive orange 16. j. hazard. mater., 172, 2009, 298-309. wan, h., weng, s., liang, s., lu, q., he, j.: evaluation of the developmental toxicity of leucomalachite green administered orally to rats. food chem. toxicol., 49, 2011, 3031-3037. wang, x., cheng, x., sun, d., qi, h.: biodecolorization and partial mineralization of reactive black 5 by a strain of rhodopseudomonas palustris. j. environ. sci., 20, 2008, 1218-1225. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc 106 legerská et al. yang, j., yang, x., lin, y., ng, t.b., lin, j., ye, x.: laccase-catalyzed decolorization of malachite green: performance optimization and degradation mechanism. plos one, 28, 2015, 1-14. yang, x.q., zhao, x.x., liu, c.y., zheng, y., qian, s.j.: decolorization of azo, triphenylmethane and anthraquinone dyes by a newly isolated trametes sp. 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sci., 18, 2013, 540-546. zhuo, r., he, f., zhang, x., yang, y.: characterization of a yeast recombinant laccase rlac-en3-1 and its application in decolorizing synthetic dye with the coexistence of metal ions and organic solvents. biochem. eng. j., 93, 2015, 63-72. zimbardi, a.l.r.l., camargo, r.f., carli, s., neto, s.a., meleiro, l.p., rosa, j.c., de andrade, a.r., jorge, j.a., furriel, r.p.m.: a high redox potential laccase from pycnoporus sanguineus rp15: potential application for dye decolorization. int. j. mol. sci., 17, 2016, 1-42. received 19 june 2016 accepted 15 july 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:37 utc nova biotechnol chim (2017) 16(1): 54-60 doi: 10.1515/nbec-2017-0008  corresponding author: josef.trogl@ujep.cz nova biotechnologica et chimica comparison of bioindicator eukaryotes of activated sludge biocenoses on two water-treatment plants: a case study farida y. achmadulinaa, rustem k. zakirova, elena s. balymovaa, vera denisovaa, taťjána brovdyováb,c, josef tröglb, and martin nerudab a faculty of food technology, kazan national research technological university, karl-marx-str. 8, 420 015 kazan, russian federation b faculty of environment, jan evangelista purkyně university in ústí nad labem, králova výšina 3132/7, ústí nad labem, 400 96, czech republic c faculty of production technologies and management, jan evangelista purkyně university in ústí nad labem, pasteurova 3334/7, ústí nad labem, 400 96, czech republic article info article history: received: 16th february 2017 accepted: 28th june 2017 keywords: activated sludge sludge microbial communities waste-water treatment plant biodiversity index abstract activated sludge biocenoses were compared on waste-water treatment plants in the city of kazan, russian federation and the city of teplice, czech republic. based on palia-kovnatski index, acanthamoeba in kazan, epistylis in teplice, and acanthamoeba and centropyxis were dominant genera in both plants. the major subdominant generas identified were arcella, opercularia and aspidisca. this indicates high nitrification ability, high water purification potential and matured activated sludge. chemical composition of the waste-water was identified as the main factor determining the sludge biocenoses diversity. higher sludge biodiversity (shannon, margalef, and sorensen indexes) was found in kazan corresponding to more concentrated inflow water.  university of ss. cyril and methodius in trnava introduction currently, an assessment of the quality of wastewater incoming to and outgoing from a waste-water treatment plant (wwtp) is carried out mainly on the basis of chemical analyses. this brings accurate data with low detection limits however in longer time period required for analyses, without evaluation of the biological effect and providing limited information in conditions of multicomponent industrial effluents. the solution of the problem is in the complex use of chemical and biological monitoring of the treated waters and biocenoses of active sludge, since biological control methods allow an integral assessment of the conditions for the functioning of the bioagent (activated sludge) at any point in the biological treatment of wastewater. nevertheless since their establishment the methods of bioindication have not developed significantly (zhmur et al. 1997; wanner et. al 2000). the influence of the biodiversity of a mixed population of microorganisms in water treatment processes on the composition of treated effluent is confirmed by earlier findings as well as by experience from operating wastewater treatment plants (wanner et. al 2000; ghanizadeh and sarrafpour 2001; seviour and nielsen 2010; zhmur 2003). however, the question remains open bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:37 utc mailto:josef.trogl@ujep.cz nova biotechnol chim (2017) 16(1): 54-60 55 whether the differences in the biological purification process can affect qualitative composition of the activated sludge biocenosis in wastewater. recent works show the importance of knowledge of the composition and changes in the physiological state of microorganisms as indicator for assessing and predicting the effectiveness of biological oxidation process (akhmadullina et al. 2016; babko et al. 2014; hreiz et al. 2015; shchegolkova et al. 2016). since this issue is not only of theoretical but also practical significance, we decided to carry out this case study and compare the activated sludge biocenoses of two wastewater-treatment plants (wwtp). the two localities chosen included the wwtp of the city of kazan, russian federation, that is the industrial heart of autonomous tatarstan republic with ~1.2 mil. equivalent inhabitants. the second one, the city of teplice, czech republic, is aspa city with ~155 000 equivalent inhabitants. both plant types show similarities in incoming water and sludge but differ in technology pattern. a comparison of the indicator organism profiles from two treatment plants by bio-indication (miller et al. 2009) was carried out. the aim was the identification of dominant bioindicator microorganisms and evaluation of biodiversity (on the genus level) and similarities of sludge microbial communities based on diversity indices, especially palia-kovnatski. table 1. composition of wastewater in studied waste-water treatment plants. 1purified water abbreviations: bod biochemical oxygen demand; cod chemical oxygen demand; wwtp waste-water treatment plant experimental sludge samples formed from similar urban wastewaters (for composition see table 1) originated from two waste-water treatment plants. the first was localized in kazan, russian federation, with traditional configuration of activation process (mechanical and biological wastewater treatment in aeration tanks that foresee the regeneration zone and aeration) (fig. 1a). the second plant was by the spa city with low industry teplice, czech republic, with nitrification-denitrification configuration (fig. 1b). samples were collected from the regeneration phase of activation process. bioindication was carried out according to standards of russian federation (bolotina 1987) using microscopes micmed-5 (lomo, russia) and lambda dn45 th59f (lambda praha, czech republic). a small drop of liquid sludge on a microscopy glass slide was covered by cover glass and observed first at low magnification (eyepiece 10 or 15, lens 8), and then at large magnification (eyepiece 10 or 15 lens 40). microscopic examination was carried out without removing the hand from the micrometer screw in order to be able to change focal lengths and consider the entire surface of the field. identification of genera was carried out according to atlas of water and sludge microorganisms (sládeček and sládečková 1996, 1997). a genus-level of the sludge microbial community was investigated. previous experiences of wastewater treatment aiming to control the activation process on-line showed that this level is sufficient for such purpose (akhmadullina et al. 2016; zakirov et al. 2013). all analyses were carried out in triplicate and evaluated using microsoft excel. components (mg/l) wwtp kazan wwtp teplice influent (mg/l) efluent1 (mg/l) purification rate (%) influent (mg/l) efluent1 (mg/l) purification rate (%) bod5 148.9 9.6 93.6 157.4 12.1 92.4 cod 576 68 88.2 335.1 50.2 85.1 suspended solids 231.4 10.03 95.7 169.2 15.1 91.1 total nitrogen 31.94 18.6 41.8 35.3 14.3 60.3 total phosphorus 3.1 1.9 38.7 4.8 1.5 68.6 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:37 utc nova biotechnol chim (2017) 16(1): 54-60 56 fig. 1. scheme of biological treatment processes and sludge sampling sites: a) waste water treatment plant kazan; b) waste water treatment plant teplice. as – activated sludge, w-w – waste water. table 2. classification of determined indicator organisms. type subtype class genus at the locality kazan teplice sarcomastigophora mastigophora phymomastigophorea astasia – zoomastigophorea bodo bodo sarcodina lobosea acanthamoeba arcella centropyxis acanthamoeba arcella centropyxis filosea trinema euglypha trinema euglypha ciliophora ciliata oligohymenophora paramecium – kinetophragminophora holophrya chilodonella – holophrya chilodonella colpidium peritricha epistylis opercularia vorticella thuricola epistylis opercularia – – polyhymenophora aspidisca aspidisca suctoria suctorida podophrya tokophrya – tokophrya nemathelminthes – rotifera lecane cephalodella rotaria gastrotricha callidina gastrotricha – – lecane cephalodella rotaria – – – encentrum philodina nematodes tobrilus nematoda tobrilus nematoda annelida – oligochaeta aelosoma aelosoma bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:37 utc nova biotechnol chim (2017) 16(1): 54-60 57 table 3. indexes of biodiversity and similarities (shitikov et al. 2003). wwtp indexes shannon margalef sorensen kazan 3.88 3.42 0.77 teplice 2.94 2.84 results and discussion results of hydrobiological analyses are summarized in table 2. these visual observations and calculated indexes of biodiversity and similarity (table 3) show a highly similar profile of indicator organisms of the two studied sludge samples. further they confirmed that composition of the waste water is the main determinant of the qualitative composition of microbial communities. the more rich genetic diversity of activated sludge treatment facilities in kazan indicates to a more complex composition of treated wastewater. this was expected considering a half million inhabitants of the metropolis and industrial infrastructure development compared to teplice with almost ten times less inhabitants. systematization of data taking into account the specific conditions of existence of the identified biomarkers allowed us to classify active sludge of both wwtp as low-loaded, nitrifying, and performing the complete oxidation of pollutants. however, a comparison of the dominant aquatic biocenosis based on palia-kovnatsky index (d) (table 4) confirmed the expected effect of arrangement of the biological treatment process on the composition of a mixed population of microorganisms (table 4). table 4. palia-kovnatsky indexes (d) of dominance in the plants at the two localities studied. class genus palia-kovnatsky index (d) at the locality kazan teplice phymomastigophorea astasia 1.6 – zoomastigophorea bodo 0.096 0.748 lobosea acanthamoeba 14.56 4.988 arcella 3.2 4.239 centropyxis 14.88 18.703 filosea trinema 3.072 0.935 euglypha 14.24 17.706 oligohymenophora paramecium 1.632 – kinetofragminophora holophryа 1.152 0.623 colpidium – 0.062 chilodonella 0.32 0.623 peritricha epistylis 3.552 18.516 vorticella 5.12 – opercularia 4.416 8.354 thuricola 0.096 – polyhymenophora aspidisca 7.168 2.431 suctoria podophrya 0.032 – rotifera gastrotricha 1.408 – rotaria 0.064 0.249 lecane 0.672 0.374 cephalodella 1.28 0.249 encentrum – 0.249 nematodes tobrilus 0.032 0.249 nematoda 0.064 0.249 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:37 utc http://medbiol.ru/medbiol/dog/000dadae.htm nova biotechnol chim (2017) 16(1): 54-60 58 fig. 2. dominant genera of the waste water treatment plant of the city of kazan: а1, а2) acanthamoeba; b) centropyxis; c) euglypha. amorphous genera acanthamoeba, centropyxis, and euglypha were dominant for the wwtp of the city of kazan (d ≥10) (fig. 2). subdominant basic bio-indicators of water treatment process (d<10) were mainly ciliates – i.e. g. aspidisca (fig. 3). these are characteristic for nitrifying sludge and exhibit a wide range of tolerance to a variety of environmental factors. the presence of large ciliates of the genera vorticella and opercularia, also subdominant bioindicators, and high turbidity of the sludge liquid provide the evidence of deep regeneration of the activated sludge accompanied by its deflocculation (fig. 3). in such conditions amorphous amoebea become competitive since they are capable of both saprozoic and holozoic type of nutrition (zhmur 1997). dominant bioindicator organisms found in wwtp teplice, as expected, were the amorhous genera centropyxis and euglypha, typical for deep nitrifying sludge. as a confirmation of the latter fact is the dominance of peritrichous ciliates of the genus epistylis, testifying to the high quality and good cleaning properties of nitrifying sludge. among subdominants amorphous genera acanthamoeba, arcella, and ciliates opercularia and aspidisca prevail, characterizing ripe sludge. in this experimental case study, the relationships between the main technological factors (modification of the process) determining bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:37 utc nova biotechnol chim (2017) 16(1): 54-60 59 the functioning of the biological unit system and the biocenosis of the activated sludge were first investigated. the main impact of the results aims on the praxis of wwtp control (especially in the czech republic and russian federation, however, applicable worldwide) and its possible changes. currently, an assessment of the quality of incoming and treated wastewater on wwtp is carried out, predominantly on the basis of chemical analysis data. in this regard, the informative nature of the chemical control of the treatment facilities is drastically reduced, causing an unrealistic reflection of the conditions for the functioning of the biocenosis of the activated sludge. this can result in low quality of the biologically treated effluents (wanner et. al 2000; balymova et al. 2011). also toxic and poorly oxidizable components of industrial effluents might act in synergy on a mixed population of microorganisms and thus negatively affect the biological oxidation process. the complex use of chemical and biological monitoring of the treated waters and biocenosis of active sludge is possible way to resolve this problem, since biological control methods allow an integral assessment of the conditions for the functioning of the bioagent (activated sludge) at any point in the biological treatment of wastewater. in addition they are fast and they do not require sophisticated pricy equipment (except for optical microscope). fig. 3. deflocculating fibers of activated sludge (wwtp kazan). conclusions this study compared the qualitative composition of microbiota of two waste-water treatment plants. composition of the waste-water was determined as the main factor determining composition of the sludge biocenoses. higher sludge biodiversity (based on shannon, margalef, and sorensen indexes) was found in kazan corresponding to more concentrated inflow water. acknowledgements the authors acknowledge the assistance provided by the research infrastructure nanoenvicz, supported by the ministry of education, youth and sports of the czech republic under project no. lm2015073. references akhmadullina fy, zakirov r, balymova e (2012) biomathematical approach to rapid assessment of activated sludge biocenosis in extended aeration processes of petrochemical complex wastewater. voda: khimiya i ekologiya 2: 50-56. babko r, kuzmina t, jaromin-glen k, bieganowski a (2014) bioindication assessment of activated sludge adaptation in a lab-scale experiment. ecological chemistry and engineering s-chemia i inzynieria ekologiczna s 21: 605-616. balymova ys (2011) realizatsiya biomatematicheskogo podkhoda dlya ekspress-otsenkisostoyaniya biosenoza aktivnogo ila v protsessakh prodlennoy aeratsii stochnykh vod neftekhimicheskogo kompleksa. voda: khimiya i ekologiya 11: 52-57. bolotina ot (1987) metodicheskoye rukovodstvo po gidrobiologicheskomu kontrolyu za rabotoy sooruzheniy biologicheskoy ochistki stochnykh vod: minvodkhoz sssr.– m. ghanizadeh g, sarrafpour r (2001) the effects of temperature and ph on settlability of activated sludge flocs. iran. j. pub. health, 30: 139-142. hreiz r, latifi ma, roche n (2015) optimal design and operation of activated sludge processes: state-ofthe-art. chem. eng. j. 281: 900-920. miller fp, vandome af, mcbrewster j (2009) water pollution: bioindicator, sewage treatment, industrial wastewatertreatment, agricultural wastewater treatment, urbanrunoff, clean water act, united states environmental protection agency. alphascript publishing. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:37 utc nova biotechnol chim (2017) 16(1): 54-60 60 seviour rj, nielsen ph (2010) microbial ecology of activated sludge. iwa publishing. shchegolkova nm, krasnov gs, belova aa, dmitriev aa, kharitonov sl, klimina km, kudryavtseva av (2016) microbial community structure of activated sludge in treatment plants with different wastewater compositions. front. microbiol. 7: 90. shitikov v, rosenberg g, zinchenko t (2003) quantitative hydroecology: methods of system identification. tolyatti: ievb ran: 463. sládeček v, sládečková a (1996) atlas vodních organismů se zřetelem na vodárenství, povrchové vody a čistírny odpadních vod: destruenti a producenti. díl. 1: česká vědeckotechnická vodohospodářská společnost. sládeček v, sládečková a (1997) atlas vodních organismů se zřetelem na vodárenství, povrchové vody a čistírny odpadních vod: destruenti a producenti. díl. 2: česká vědeckotechnická vodohospodářská společnost. wanner j, růžičková i, krhůtková o, přibyl m (2000) activated sludge population dynamics and wastewater treatment plant design and operation. wat. sci. tech. 41: 217-225. zakirov r, balymova y, sidorov o, akhmadullina f (2013) izucheniye vozmozhnosti ispol'zovaniya izbytochnogo aktivnogo ila v kachestve istochnika biogennykh elementov. vestnik kazanskogo tekhnologicheskogo universiteta 22: 219-221. zhmur n (2003) tekhnologicheskiye i biokhimicheskiye protsessy ochistki stochnykh vod na sooruzheniyakh s aerotenkami. moscow: akvaros, 512. zhmur n (1997) upravlenie protsessom i kontrol'rezul’tata ochistki stochnykh vod na sooruzheniyakh s aerotenkami: moscow: luch. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:37 utc nova biotechnologica et chimica 11-2 (2012) 167 doi 10.2478/v10296-012-0019-7 © university of ss. cyril and methodius in trnava simultaneous and sequential extraction protocols as tools for determination of zinc bioavailability in dried anaerobic sludge vladimír frišták, michaela valovčiaková, martin pipíška, jozef augustín department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, sk-917 01 trnava, slovak republic (vladimir.fristak@ucm.sk) abstract: production of unsettleable sewage sludge with high water content is one of the problems of intensification of industrial activities and environmental protection. sewage sludge with low toxic metals concentrations can be utilized as fertilizer and soil conditioner in agriculture. for determination of metal bioavailability, a wide range of extraction protocols and fractionation analyses can be used. we studied the distribution and quantified the leaching and bioavailability of zinc from dried anaerobic sludge by simultaneous, multi-step and three different sequential extraction protocols. for determination of zinc, the galvanostatic stripping chronopotentiometry (scp) and electrothermal atomic absorption spectrometry (etaas) were used. the distribution of zn in sequential extraction protocols was determined using a fivestep chemical fractionation procedures (bcr, tessier and van hullebusch protocols). the potential bioavailability (0.9% nacl, 0.1 mol/dm3 hcl, 0.1 mol/dm3 hno3, 0.1 mol/dm3 ch3cooh, 0.1 mol/dm3 na2edta, 0.1 mol/dm3 cacl2, 0.1 mol/dm3 mgcl2, 0.1 mol/dm3 (nh4)2c2o4.h2o extraction) and pseudo total (aqua regia extraction and etaas analyses) content of zn in sludge was determined. the amount of aqua regia extractable zinc in sludge samples was 650 ± 12 mg/kg (d.w.). we found out that the zinc was extractable from anaerobic sludge in first hour of contact time for all tested agents. zinc was extracted with highest efficiency by 0.1 mol/dm3 (nh4)2c2o4.h2o, 0.1 mol/dm3 hcl and 0.1 mol/dm3 na2edta. sequential extraction protocols showed that the maximum extractable amount of zinc 126.3 ± 2.6 mg zn / kg d.w. was bound to organic matter and sulfides. high concentrations of zinc in residual fractions were leachable under extraction conditions of strong acids only. key words: anaerobic sludge, zn, bioavailability, extraction procedures, galvanostatic stripping chronopotentiometry 1. introduction the amount of sewage sludge from wastewater treatment plants and a number of technological processes in the world are constantly increasing. production of unsettleable sewage sludge with high water content is one of the problems of intensification of industrial activities and environmental protection. sewage sludge with low toxic metals concentration can be utilized as fertilizer and soil conditioner in agriculture. information about metal binding into sorption sites of sludges are limited for application of sludges into the soil. the ecological and toxicological effect of potentially toxic metals from sewage sludge on agro-ecosystem is directly related to their partitioning between mobile and complexed species in the highly heterogeneous soil system (merrington et al., 1997). many factors influence this partitioning of toxic metals in soil amended with sludge including the soil and sludge characteristics. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:19 utc 168 frišták, v. et al. thus, it is necessary to realize that for a safe use of sewage sludge as soil conditioner, the undesirable elements have to be removed, so reducing environmental contamination to a minimum. for extraction of heavy metals from sewage sludges, a variety of single and sequential extraction techniques can be used. these techniques were recently reviewed by babel and dacera (2006). however, owing to the wide range of extractants used in these extraction techniques, the results obtained were not comparable. in 1987, the community bureau of reference (bcr) started a programme to harmonize the methodology used in sequential extraction schemes for determination of metals in soils and sediments (ure et al., 1993). the methodology was successfully applied to different types of sewage sludges (fuentes et al., 2004). the type of applied sequential extraction scheme can lead to the anomalies in the results, as sequential extraction schemes are greatly influenced by the type of extractants used, the operating conditions (ph, contact time and temperature) and the sequence in which the extractions steps are applied. van hullebusch et al. (2005) compared the sequential extraction procedure by tessier et al. (1979), the procedure according to stover et al. (1976), the bcr protocol for fractionation of co, ni, cu, zn, mn and fe. the main aim of our paper was to describe distribution, leaching and bioavailability of zinc from dried anaerobic sludge by simultaneous, multi-step and sequential extraction protocols. owing to the scarcity of data associated with the characterization and fractionation of heavy metals in anaerobic sludge from municipal wastewater treatment plants, this paper produces a useful data for an area disposal for this type of waste as soil conditioner. 2. materials and method 2.1 sample anaerobic digested sludge was obtained from mechanical-biological waste water treatment plant (wwtp) in zeleneč (slovak republic). after washing in deionised water, sludge biomass was oven dried at 105°c for 48 h, ground, sieved and stored in closed bottles at laboratory temperature. for extraction experiments fraction < 2 mm was used. 2.2 total (aqua regia extractable) zinc fraction the pseudototal concentration of zinc in sludge samples was estimated by aqua regia extraction in triplicate. extracts were analysed by atomic absorption spectrometry with electrothermal atomization (etaas) device shimadzu aa-6300 (usa) with electrothermal atomizers shimadzu gfa-ex7i using an automatic dispenser shimadzu asc 6100 and background correction method of smith-hieftje, after microwave digestion of the samples by the multiwave system mw 3000 (anton paar gmbh, aus). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:19 utc nova biotechnologica et chimica 11-2 (2012) 169 2.3 single-step extractable zinc leachable and bioavailable fractions of zinc from anaerobic sludge were obtained after simultaneous extractions with variable set of extractants. extraction experiments were carried out by suspending of dried anaerobic sludge (10 g/dm3 d.w.) in 10 cm3 of reagents: deionized water, rain water (ph 5.34; conductivity 45.6 µs/cm), 0.9% nacl, 0.1 mol/dm3 hcl, 0.1 mol/dm3 hno3, 0.1 mol/dm 3 ch3cooh, 0.1 mol/dm 3 na2edta, 0.1 mol/dm 3 cacl2, 0.1 mol/dm 3 mgcl2, 0.1 mol/dm 3 (nh4)2c2o4.h2o. samples of the sludge were extracted in five extraction steps (1 h, 150 min-1, 22 °c). the supernatant after centrifugation (5 min, 4500 min-1) was isolated from the sediment after each extraction step. sediment was used in the next extraction step. all experiments were carried out in triplicates. 2.4 sequential extractable zinc for investigation of the distribution of zinc among different fractions of anaerobic sludge, the following extraction protocol was used: 1. modified extraction protocol by tessier (1979) for analysis of soil and sediments (tab. 1), 2. extraction protocol for sewage sludge according to van hullebusch et al. (2005) (tab. 2), 3. bcr extraction protocol (tab. 3). in all cases, 1g of dried anaerobic sludge sample was used. table 1. extraction conditions of modified sequential extraction protocol of dried anaerobic sludge (tessier, 1979). extraction conditions fraction extracting agent reaction time temperature exchangeable 8 ml mgcl2 (1 mol/dm3, ph 7.0) 1 h 20 °c carbonates 8 ml naoac (1 mol/dm3, ph 7.0) 5 h 20 °c iron and manganese oxides 8 ml nh2oh.hcl (0,1 mol/dm3, ph 3.0) 16 h 35 °c organic matter and sulphides 8ml h2o2 (30%) 5 h 35 °c residual 10 ml hcl/hno3 (3:1) 26 min 180 °c * *microwave conditions (p 20 bar, tmax 180°c) 2.4 treatment of extracts and galvanostatic stripping chronopotentiometry zinc in extracts was determined by galvanostatic stripping chronopotentiometry on macroporous electrode e104 l using electrochemical analyser ecaflow model glp 150 (istran, ltd., bratislava, slovakia). 5 ml of extracts was added to flask with 20-30 ml 0.1 mol/dm3 hcl and 0.5 ml 0.01 mol/dm3 kmno4. the solution was heated to 80bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:19 utc 170 frišták, v. et al. 95 °c for 5-10 min. oxalic acid was added to reduce the excess of permanganate. the volume of samples was adjusted to 50 ml with 0.1 mol/dm3 hcl. solid particles were removed by filtering, centrifugation or sedimentation. table 2. extraction conditions of modified sequential extraction protocol of dried anaerobic sludge (van hullebusch et al., 2005). extraction conditions fraction extracting agent reaction time temperature exchangeable 10 ml ch3coonh4 (1 mol/dm3, ph 7.0) 1 h 20 °c carbonates 10 ml ch3cooh (1 mol/dm3, ph 5.5) 1 h 20 °c organic matter and sulphides 5 ml h2o2 (30%, ph 2.0) 3 h 35 °c residual 10 ml hcl/hno3 (3:1) 26 min 180 °c* *microwave conditions (p 20 bar, tmax 180°c) table 3. extraction conditions of bcr modified extraction procedure of dried anaerobic sludge (van hullebusch et al., 2005). extraction conditions fraction extracting agent reaction time temperature exchangeable 40 ml ch3cooh (0,11 mol/dm3, ph 7.0) 16 h 20 °c iron and manganese oxides 40 ml nh2oh.hcl (0,5 mol/dm3, ph 1.5) 16 h 20 °c organic matter and sulphides 20 ml h2o2 (30 %, ph 2.0) 50 ml ch3coonh4 (1 mol/dm3, ph 2.0) 16 h 20 °c residual 10 ml hcl/hno3 (3:1) 26 min 180 °c *microwave conditions (p 20 bar, tmax 180°c) 3. results and discussion 3.1 pseudototal zinc content concentration of mobile zinc in the sewage sludge is important for using the sludge as soil conditioner and fertilizer. in our paper, pseudototal zinc content in anaerobic sewage sludge refers to the fraction of aqua regia soluble. the amount of zinc in sludge samples was 649.5 ± 12 mg/kg (d.w.). this digestion is considered adequate for analysing total-recoverable heavy metals in soils (meers et al., 2007). residual elements that are not released by aqua regia digestion are mostly bound to bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:19 utc nova biotechnologica et chimica 11-2 (2012) 171 silicate minerals and are considered unimportant for estimating the mobility and behaviour of metals (niskavaara et al., 1997). concentration of zinc determined by etaas was comparable with concentration determined by stripping chronopotentiometry. 3.2 single-step and multi-step extraction protocols it has been reported that the addition of different amendments to soil changes soil reaction and consequently the mobility and bioavailability of potentially toxic metals (hettiarachchi and pierzynski, 2004). it is widely recognized that not all chemical forms of potentially chemical metals interact with living organisms in the same manner and that their potential bioavailability has to be considered when assesing soil pollution. however, no single method is recognized universally (tica et al., 2011). simultaneous and multi-step extractions are one of possible techniques for quantification of leachable elements from soil, sediments and sludge. 1 2 3 4 5 0 5 10 15 20 25 30 35 ([ z n] so lu te / [z n] to ta l) .1 00 [% ] extraction step fig. 1. efficiency of five-step extraction protocols of zinc (%) from the anaerobic sludge (100 g/dm3). extraction conditions: t1.step = 1 h, 22 °c, 150 min-1, extractants: deionized water ( ),rain water ( ),0.9 % nacl ( ),0.1 mol/dm3 hcl ( ),0.1 mol/dm3 hno3 ( ),0.1 mol/dm3 ch3cooh ( ),0.1 mol/dm3 na2edta ( ),0.1 mol/dm3 cacl2 ( ),0.1 mol/dm3 mgcl2 ( ),0.1 mol/dm3 (nh4)2c2o4.h2o ( ). all experiments were realised in triplicates. error bars represent standard deviation of the mean (±sd) as extracting agents in extraction procedures, strong acid and their mixtures (hno3, h2so4, hcl, aqua regia), neutral solution of salts (cacl2, mgcl2, nacl), pending puffer and complex-forming agents (na2edta) can be used (meers et al., 2007). the extraction protocols used in our paper were initially developed for the assessment of zinc bioavailability but they are also used for the assessment of potentially toxic metals released from the sludge enriched soils and their consequent potential availability to all soil organisms (mclaughlin et al., 2000). in our paper, 10 extracting agents for determination of mobile fraction of zinc in anaerobic digested sludge were used. we found out that zinc is extractable from anaerobic sludge in the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:19 utc 172 frišták, v. et al. first hour of contact time. zinc was extracted with highest efficiency by 0.1 mol/dm3 (nh4)2c2o4.h2o, 0.1 mol/dm 3 hcl and 0.1 mol/dm3 na2edta. sediments of all extraction protocols were extracted subsequently 5 times. the efficiency of agents increases to the third step of extraction process (fig. 1). extraction efficiency of used extracting agents decreases in order: 0.1 mol/dm3 hcl (408.82 ± 9.23 mg/kg) > 0.1 mol/dm3 na2edta (383.12 ± 7.14 mg/kg) > 0.1 mol/dm 3 hno3 (81.72 ± 3.54 mg/kg) > 0.1 mol/dm 3 (nh4)2c2o4.h2o (77.87 ± 3.27 mg/kg)> 0.1 mol/dm 3 cacl2 (36.54 ± 1.88 mg/kg) > 0.1 mol/dm3 ch3cooh (36.12 ± 1.87 mg/kg) > deionized water (26.22 ± 1.41 mg/kg) > rain water (24.54 ± 1.71 mg/kg) > 0.9% nacl (16.89 ± 1.03 mg/kg) > 0.1 mol/dm3 mgcl2 (14.32 ± 0.98 mg/kg). nonextractable zinc fraction was electrochemically quantified in residual sediment after the fifth step of extraction protocol and aqua regia digestion. the sum of total extractable zinc and nonextractable zinc corresponded to the concentration detected by etaas in the initial characterization studies (as can be seen in chapter 3.1). 3.3 fraction analyses of zinc by sequential extraction protocols results of the three extraction protocols for the zinc concentration are presented in tab. 5 and fig. 2. for each extraction, three samples were simultaneously analysed to determine the precision of the measurements. tab. 4 shows that all extraction protocols provide a satisfactory recovery of zinc from anaerobic sludge. the sum of four steps and of the residue expressed as percentages of the total concentration varies from 99 to 101 %. the reproducibility of the tessier and bcr protocols is higher than the reproducibility of the stover extraction scheme (rudd et al., 1988). table 4. comparation of zn content in dried sludge estimated by tessier, van hullebush and bcr extraction protocols (mean ± % sd, n= 3). tessier protocol van hullebusch protocol bcr protocol σ [mg/kg] 656.6 (±1.5%) 650.1 (±1.1%) 645.9 (±0.7%) mt [mg/kg] 649.5 (±1.9%) 649.5 (±1.9%) 649.5 (±1.9%) recovery [%] 101 100 99 σ the sum of all extraction steps; mt the pseudo total zinc concentration. neutral salt extraction efficiency depends on the type of cation (different affinity to surface binding sites) and decreases in the order h+ > ca2+ > mg2+ > na+= nh4+ (vojteková and krakovská, 2006). in our paper, extraction with ion exchangeable agents (mgcl2, ch3coonh4, ch3cooh) confirmed the sorption characteristics of zn in concentrations 2.2 ± 0.3 mg/kg (tessier protocol), 15.8 ± 0.6 mg/kg (van hullebusch protocol) and 21.5 ± 0.5 mg/kg (bcr protocol). the comparison of leaching data between the exchangeable/ carbonates fraction from the bcr procedure, the sum of exchangeable and carbonates fractions from the modified tessier protocol and the sum of exchangeable and carbonates fractions from the van hullebusch protocol shows some interesting trends. the amount of zinc that was bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:19 utc nova biotechnologica et chimica 11-2 (2012) 173 extracted in these fractions is in the same order of magnitude between each extraction protocol. extraction of sludge with mgcl2 has a lower efficiency of zinc leaching attributed to lower solubilisation of organic components on sludge surface (ure et al., 1995). solution of ch3coonh4 dissolves a considerable amount of caco3, mgco3, baco3, caso4 and can act on oxide-metal (mn, fe) layers on the sludge surface (vojteková and krakovská, 2006). reducible fraction represents the content of zinc bound to fe/mn oxides that are released if matrix of sludge is subjected to reductive conditions. iron and manganese oxides are relatively scarce in this organicrich sludge (van hullebusch et al., 2005). leachable zinc into acetic acid and its salts can be regarded as almost quantitatively, without dissolving the organic fraction, oxides of mn, fe and aluminum silicate minerals (wilber and hunter, 1979). 0 100 200 300 400 500 600 edcb [z n] so lu te [m g/ kg ] a fig. 2. zinc partitioning profile of anaerobic sludge for exchangeable fraction (a), fraction of carbonates (b), fraction of fe and mn oxides (c) , fraction of organic matter and sulphides (d), residual fraction (e) according to tessier extraction protocol ( ), van hullebusch extraction protocol ( ), and bcr procedure ( ) (mean ± sd, n=3, lod = 0.15 µg/dm3). sequential extraction by tessier confirmed the presence of zn bound to this fraction, respectively. concentration of extracted zinc from sludge according to the van hullebusch protocol was below the limit of detection of equipments used. we found that the maximum extractable concentration of zinc 126.3 ± 2.6 mg zn / kg d.w. was bound to organic matter and sulfides (fig. 2). compared to the other two protocols, we found out that the fraction of zinc bound to organic matter and sulphides is major bioavailable fraction of zinc from anaerobic sludge. high concentrations of zinc in residual fractions are leachable under radical extraction conditions of strong acids only. the contents of zn in the residual fractions obtained from the three sequential extraction protocols after exposure and microwave digestion are comparable to each other. nemati et al. (2009) determined reducible fraction of zinc to 39.9 ± 3.5 mg zn / kg sewage sludge, oxidizable fraction of zinc bound to organic matter and sulfides in the amount of 58.1 ± 0.2 mg/kg of sludge. rudd et al. (1988) showed that zinc sulfides precipitates are relatively difficult to extract in the sulfides bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:19 utc 174 frišták, v. et al. fraction of extraction protocols. in this respect, rudd et al. (1988) suggested that it can be more appropriate to classify the sulfides and residual fractions as a single phase. 4. conclusions in our paper, we found out that amount of aqua regia extractable zinc in sludge samples was 649.5 ± 12 mg/kg (d.w.). zinc was extractable from anaerobic sludge in first hour of contact time in extraction by 0.9% nacl, 0.1 mol/dm3 hcl, 0.1 mol/dm3 hno3, 0.1 mol/dm 3 ch3cooh, 0.1 mol/dm 3 na2edta, 0.1 mol/dm 3 cacl2, 0.1 mol/dm3 mgcl2 and 0.1 mol/dm 3 (nh4)2c2o4.h2o. zinc was extracted with highest efficiency by 0.1 mol/dm3 hcl (408.82 ± 9.23 mg/kg), 0.1 mol/dm3 na2edta (383.12 ± 7.14 mg/kg) and 0.1 mol/dm3 hno3 (81.72 ± 3.54 mg/kg). sequential extraction protocols showed that the maximum extractable concentration of zinc 126.3 ± 2.6 mg zn / kg d.w. was bound to organic matter and sulphides. high concentrations of zinc in residual fractions were leachable under extraction conditions by strong acids only. the contents of zn in the residual fractions obtained from the bcr, tessiers and van hullebuschs sequential extraction protocols after exposure and microwave digestion were comparable to each other. data of characterization and fractionation of zinc in anaerobic sludge from municipal wastewater treatment plants are useful for assessment of zinc mobility in sewage sludge as soil amendment. references babel, s., dacera, d.d.m.: heavy metal removal from contaminated sludge for land application. j. waste manag., 26, 2006, 988-1004. fuentes, a., llorens, m., saez, j., soler, a., aguilar, m., ortuno, f.: phytotoxicity and heavy metals speciation of stabilized sewage sludges. j. hazard. mater., 108, 2004, 161–169. hettiarachchi, r.m., pierzynski, r.m.: soil lead bioavailability and in situ remediation of lead-contaminated soils: a review. environ. prog., 23, 2004, 78-93. mclaughlin,m.j., hamon, r.e., mclaren, r.g., speir, t.w., rogers, s.l.: a bioavailability-based rationale for controlling metal and metalloid contamination of agricultural land in australia and new zealand. aust. j. soil res., 38, 2000, 1037-1086. meers, e., laing, d, d., unamuno, v., ruttens, a., vangronsveld, j., tack, f.m.g., verloo, m.g.: comparison of cadmium extractability from soils by commonly used single extraction protocols. geoderma, 141, 2007, 247259. merrington, g., winder, l., green. i.: the bioavailability of cd and zn from soils amended with sewage sludge to winter wheat and subsequently to the grain aphid sitobion avenae. sci. tot. environ., 205, 1997, 245-254. nemati, k., bakar, n.k.a., sobhanzadeh, e., abas, m.r.: a modification of bcr sequential extraction procedure to investigate the potential mobility of copper and zinc in shrimp aquaculture sludge. j. microchem., 92, 2009, 165-169. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:19 utc nova biotechnologica et chimica 11-2 (2012) 175 niskavaara, h., reimann, c., chekushin, v., kashulina, g.: seasonal variability of total and easily leachable element contents in topsoils (0-5 cm) from eight catchments in the european arctic (finland, norway and russia). environ. pollut., 96, 1997, 261-274. rudd, t., cmapbell, j.a., lester, n.j.: the use of model compounds to elucidate metal forms in sewage sludge. environ. pollut., 50, 1988, 225 stover, r.c., sommers, l.e., silviera, d.j.: evaluation of metals in wastewater sludge. j. water pollut., 48, 1976, 2165. tessier, a., campbell, p.g.c., bisson, m.: j. anal. chem., 51, 1979, 844. tica, d., udovic, m., lestan, d.: immobilisation of potentially toxic metals using different soil amendments. chemosphere, 85, 2011, 577-583. ure, a.m., davidson, c.m., thomas, r.p.: single and sequential extraction schemes for trace metal speciation in soil and sediment. tech. instrum. anal. chem., 17, 1995, 505-523. ure, a.m., quevauviller, p., muntau, h., griepink, b.: speciation of heavy metals in soils and sediments. an account of the improvement and harmonization of extraction techniques undertaken under the auspices of the bcr of the commission of the european communities. int. j. environ. anal. chem., 51, 1993, 1-4. van hullebusch, e.d., utomo, s., zandvoort, m.h., lens, p.n.l.: comparison of three sequential extraction procedures to describe metal fractionation in anaerobic granular sludges. talanta, 65, 2005, 549–558. vojteková, v., krakovská, e.: frakcionačná analýza sedimentovlimitácie selektivity sekvenčného lúhovania. chem. lett., 100, 2006, 1096-1104 (in slovak). wilber, w.g., hunter, j.v.: the impact of urbanization on the distribution of heavy metals on bottom sediments of the sadlle river. j. water resour., 15, 1979, 790-799. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 13:19 utc nova biotechnologica et chimica 13-1 (2014) 57 doi 10.2478/nbec-2014-0007 © university of ss. cyril and methodius in trnava radiocesium adsorption by zeolitic materials synthesized from coal fly ash lucia remenárová, martin pipíška, eva florková, jozef augustín, marián rozložník, stanislav hostin, miroslav horník department of ecochemistry and radioecology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (l.remenarova@ucm.sk) abstract: brown coal fly ash derived from the combustion of brown-coal in power plant eno nováky (slovak republic) was used as raw material for synthesis of zeolitic materials zm1 and zm3 by hydrothermal alternation with 1m naoh and 3m naoh, respectively. fly ash and synthesized products were characterized using xrf and sem-edx analysis. subsequently, zeolitic materials were tested as sorbents to remove cs+ ions from aqueous solutions using radiotracer technique. sorption of cesium by both types of zeolitic materials obeys langmuir adsorption isotherm model. the maximum sorption capacities qmax at ph 6.0 calculated from langmuir isotherm were 1203 ± 65 μmol cs+/ g for zm1 and 1341 ± 66 μmol cs+/ g for zm3. the results showed that alkali treated fly ash can be used as effective sorbent for radiocesium removal from contaminated solutions. key words: 137cs, adsorption, fly ash, hydrothermal zeolitization, zeolitic materials 1. introduction the development of nuclear science and technology, nuclear weapons testing, generation of electricity in nuclear power plants and the use of radionuclides in a research and medicine have led to the increase production of both liquid and solid wastes containing radionuclides and release of artificial radionuclides into the environment. radiocesium 134cs and 137cs are one of the most abundant radionuclide in nuclear fission products which has a relatively long half-life and is considered as hazardous element (khan, 2003) due to its high solubility what enables easy migration into the environment. effective separation technologies, e.g. fractional crystallization, solvent extraction, ion exchange using inorganic and organic ion exchangers have been developed for removal of radionuclides from liquid radioactive wastes. the most widely used methods for water treatment to remove radionuclides (cs, sr, u, pu) are those based on co-precipitation and sorption (milyutin et al., 2012). many researchers have reported about utilization of zeolite and bentonite type minerals for removal of cs+ ions (yildiz et al., 2011; abd el-rahman et al., 2006; atun and bodur, 2002). ion exchange properties of zeolites have been received great attention, especially for application in radioactive liquid waste treatment (abd el-rahman et al., 2006; el-kamash et al., 2005). the cationic radioisotopes, present in the liquid effluents of low and intermediate level liquid waste, can be removed by the ion exchange with the na+ ions of the zeolites. these inorganic materials possess high exchange capacity, selectivity and specificity, good resistant to radiation, and have proven advantages with respect to immobilization and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc 58 remenárová, l. et al. final disposal when compared with organic ion exchangers (goni et al., 2006; li et al., 2005). while the particular zeolitic development may take thousands of years in order to form natural zeolites (koukouzas et al., 2010), formation of synthetic zeolitic materials can be speeded up in the laboratory. in that case the activation solution is an alkaline one, usually naoh or koh. the classical alkaline conversion of fly ash (fa) is based on the combination of different [activation solution]/ [fly ash] ratios. the methodologies developed on this field aim at the dissolution of al-si bearing phases of the fa and the subsequent precipitation of the zeolitic material (blissett and rowson, 2012; ahmaruzzaman, 2010; hui and chao, 2006; moutsatsou and protonotarios, 2006; querol, 2002; iyer and scott, 2001; hollman, 1999; amrhein et al., 1996; shigemoto et al., 1993). in addition, the application of fly ash can contribute to the solution of another environmental problem, namely accumulation of fly ash produced by burning of coal in power stations. fly ash is a massing waste material containing hazardous substances which may be released to the environment from dumping places which are still enlarged. in spite of the fact that there exist many ways of utilization of this toxic pollutant (e.g. civil engineering, metallurgy, mining, agriculture, etc.) it is necessary to increase the amount of fly ash being re-utilized. single fly ash is known for low sorption capacities. however, after its alternation, products with better ion-exchange properties and several fold-enlarged surfaces comparable with natural zeolites can be obtained. of the many applications of fly ash, zeolite synthesis has been reported to be of great interest due to the fact that zeolites have wide industrial application (musyoka et al., 2013). in our work the brown coal fly ash was utilized for synthesis of two types of zeolitic materials. the main motivation for this work was inspired by the need for effective sorbents for radiocesium that are essential for purifying the radioactive contaminated aqueous solutions. the key objectives of our research are as follows: (a) to characterize fly ash and products of its hydrothermal alternation using scanning electron microscopy connected with edx and x-ray fluorescence analysis; (b) to quantify sorption capacity of fly ash and zeolitic materials, and (c) to investigate the major mechanisms of cesium ions sorption. equilibrium isotherm models according to langmuir and freundlich were used for mathematical description of sorption equilibrium in single systems. to obtain reliable experimental sorption data within a broad concentration range, radiotracer technique with 137cscl was used. 2. material and methods 2.1 sorbent preparation brown-coal fly ash (density ρ = 2 850 kg/m3) was obtained from power station nováky (eno) (zemianske kostoľany, slovak republic) and was stored in sealed containers before use. fluid fly ash was washed in deionised water (0.054 μs/cm simplicity 185, millipore, usa) and oven-dried for 24 h at 60°c. dried ash was used bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc nova biotechnologica et chimica 13-1 (2014) 59 as the raw material for zeolite synthesis according to the method of querol et al. (2001). fly ash (100 g) was resuspended in 300 ml of 1.0 m or 3.0 m naoh for zm1 and zm3 preparation, and the mixture was boiled under reflux for 18 h. the products of hydrothermal zeolitization were thoroughly washed 3 times in surplus of deionised water, oven-dried for 24 h at 60°c and were stored in sealed containers before use in sorption experiments. 2.2 sorbent characterization the surface structure analysis of sorbents (fly ash, zm1 and zm3) before and after cs+ sorption and edx microanalysis were performed by scanning electron microscope vega 2 sem (tescan inc., czech republic) coupled with quantax qx2 detector (rontec, germany) for electron dispersive x-ray analysis. prior to the sem and edx analysis, the samples were dried for 24 h at 80°c and stuck to a sample holder using conductive adhesive (ag). samples were then coated with au using bp 343.7 evaporator (tesla elmi inc., czech republic). the analyses were performed at voltage 30 kv and vacuum pressure 9.0×10-3 pa. elemental analysis of fly ash and zeolitic materials was performed by x-ray fluorescence analysis using high performance x-ray fluorescence spectrometer x-lab 2000, spectro, germany. for determination of specific surface area (ssa) zeolitic material was subjected to n2 adsorption using bet method (brunauer et al., 1938). 2.3 adsorption experiments batch adsorption experiments were carried out by suspending the sorbent (2.5 g/dm3, d.w. of fly ash, zeolitic material zm1 and zm3) in solutions with concentrations of 4000 μmol/dm3 of cscl (analytical grade, sigma aldrich, germany) spiked with 137cs at ph adjusted to 6.0. the content was agitated on reciprocal shaker (120 rpm) at 22 °c. in time periods (5, 30, 60, 120, 240, 360 and 1440 min.) aliquot samples of solution were taken from individual flasks for radiometric determination of 137cs. the cs+ uptake was calculated as: m v ccq eqeq )( 0 −= (1) where q is the uptake (μmol/g), c0 and ceq are the initial and the final cs + concentrations in solution (μmol/dm3), v is volume (dm3) and m is the amount of dried sorbent (given in grams). for determination of the adsorption capacity of sorbents batch equilibrium experiments were carried out in solutions of cscl in concentration range 100-6000 μmol/dm3, spiked with 137cs. ph was adjusted to 6.0. the content was agitated on a reciprocal shaker (120 rpm) for 6 h at 22°c. to evaluate the effect of the ph on the uptake of cs by studied sorbents, experiments were carried out in cscl solutions (4000 μmol/dm3) spiked with 137cs at ph ranging from 2.0 to 8.0. the content was agitated on reciprocal shaker (120 rpm) for 6 h at 22 °c. at the end of the experiments bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc 60 remenárová, l. et al. the sorbent was filtered out, washed in deionized water and the radioactivity of both the sorbent and the liquid phase was measured. 2.4 radiometric analysis the gamma spectrometric assembly using the well type scintillation detector 54bp54/2-x, nai(tl) (scionix, the netherlands) and the data processing software scintivision 32 (ortec, usa) were used for obtaining quantitative data of cs+ sorption by sorbents at the energy of γ – photons: 137cs – 661.64 kev. standardized 137cscl solution (5.4 mbq/cm 3, cscl 20 mg/dm3 in 3 g/dm3 hcl) was obtained from the czech institute of metrology, prague (czech republic). 2.5 data analysis the langmuir (eq. 2) and freundlich (eq. 3) adsorption models were used for describing equilibrium data. the integral equations are as follows: eq eq eq bc cbq q + = 1 max (2) )/1( neqeq kcq = (3) where qmax represents the maximum sorption capacity upon complete saturation of the sorbent, b is a constant related to the energy of adsorption. k and 1/n values are the freundlich constants referring to adsorption capacity and intensity of adsorption, respectively. the qmax values and the corresponding parameters of adsorption isotherms were calculated using the non-linear regression analysis performed by origin 7.0 professional (originlab corporation, northampton, usa). the theoretical cs speciation in solution was calculated using mineql+ chemical equilibrium modeling system (version 4.6 for windows). 3. results and discussion 3.1 synthesis of zeolitic materials and their characterization the chemical composition of zeolitic material zm1 and zm3 prepared from brown coal fly ash by hydrothermal activation is given in table 1. the main components of fly ash are sio2 (54.7%) and al2o3 (19.2%) which present elementary building blocks for preparation of synthetized zeolites. concentration of these components was further decreased in both zeolitic materials (sio2 from 54.7 to 50.1% (zm1) and 42.2% (zm3)) as a result of dissolution within zeolitization in naoh. moreover, during the hydrothermal activation, both alumina and silicate ions are condensed and formed aluminum–silicate gel. evident increase in na2o amount from 1.0 (coal fly ash) to 6.2% (zm1) and 9.8% (zm3) is due to entrapment of sodium ions to neutralize the negative charge on aluminosilicate gel (table 1). naoh causes differentiation in the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc nova biotechnologica et chimica 13-1 (2014) 61 distribution of the electric charge between the al-o and si-o bonds and the enhancement of the chemically active centres in the lattice. as was mentioned by other authors (koukouzas et al., 2010) generation of terminal groups such as ≡si-oh, ≡si-ona, ≡si-o-, (≡si-o)3al-oin the presence of naoh lead to the formation of zeolitic materials. fig. 1. scanning electron micrographs of zeolitic material zm3 – magnification: (a) 100×; (b) 500×; (c) 1000× and (d) 2000×. the sem microscopy was used for surface characterization of raw fly ash and synthesized zm1 and zm3. although the synthesized zeolitic materials keep the original spherical shape of raw fly ash, pseudomorphs (zeolitic aggregates) on the surface of fly ash particles were created (fig. 1a-d). similar formation of pseudomorphs after hydrothermal reaction was observed by ríos et al. (2009). murayama et al. (2002) have synthetized zeolites in solutions of na2co3 and/or koh. they observed that the crystal of zeolite p synthesized in na2co3 solution a b c d bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc 62 remenárová, l. et al. covers like a thin film on the fly ash surface. on the other hand, in the case of koh solution, chabazite crystal grows as an ellipsoid on the fly ash surface. table 1. the chemical composition of brown coal fly ash and synthesized zeolitic materials zm1 and zm3 (wt. %). component (%) sio2 al2o3 fe2o3 cao mgo mno tio2 k2o na2o p2o5 so3 raw fly ash 54.7 19.2 9.88 5.39 2.07 0.09 0.73 2.30 1.00 0.19 0.58 zm1 50.1 18.8 8.38 4.16 2.10 0.09 0.61 2.06 6.20 0.13 0.09 zm3 42.2 18.9 8.94 5.85 2.25 0.10 0.67 1.63 9.80 0.06 0.08 3.2 influence of ph on cesium adsorption the ph value presents one of the crucial parameters in the sorption of radionuclides heavy metals by zeolites and zeolitic materials. aqueous liquid radioactive waste is generated during nuclear reactor operations and during industrial and institutional application of radioisotopes. the chemical compositions and radioactivity levels of the generated wastes depend on the conducted operation (abdel-rahman et al., 2011). also the ph of aqueous radioactive waste depends on its origin. therefore, to investigate the influence of ph on sorption of cs+ ions, experiments were conducted in the ph range from 2.0 to 8.0. 2 3 4 5 6 7 8 0 20 40 60 80 100 q eq ( % ) ph sorption of cs+ onto zm1 sorption of cs+ onto zm3 fig. 2. effect of initial ph on sorption of cs+ (c0 = 4000 µmol/dm3 cscl, 60 kbq/dm3 137cs) by zeolitic material zm1 and zm3 (cs = 2.5 g/dm3, d.w.) from single metal solution. error bars represent standard deviation (sd) of the mean (n = 2). the effect of initial ph on cs sorption is depicted in fig. 2. it was observed that cesium ions´ binding is ph dependent and that the amount of sorbed metal ions increased with increasing ph of the solution. increasing ph value causes that the netbereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc nova biotechnologica et chimica 13-1 (2014) 63 electronegativity of zeolitic materials increases as a result of deprotonation of active sites present on surface. the highest uptake was observed at ph ranging from 5.0 to 6.0 and reached 57% (zm1) and 61% (zm3) respectively. observed negligible sorption at ph 2.0 is closely related to competition between h+ and cs+ ions for occupancy of the active sites. the slight decrease of cs sorption at ph 8.0 is attributed to the abundance of ohthat results in an increased hindrance to the diffusion of cesium ions. rao et al. (2006) pointed out that mineral acids affect the structure of zeolites in the zeolite framework, the si-o-al is weaker than si-o-si and can easily be attached by h+ ions affecting the zeolite structure. this defect is more pronounced in the case of zeolites with low si/al ratios such as zeolite a and x (rao et al., 2006), as well as studied zeolitic materials zm1 and zm3. the extent of the damage to their structure depends on the ph of the solution. the structure of zeolites with low si/al ratios may collapse in the presence of acids with ph lower than 5.0, but the severity would be more below ph value of 3.0 (el-kamash, 2008). aluminosilicate structure is vulnerable also to strong basic environments since strong bases can lead to desilication (mishra and tiwari, 2006). these morphological changes could influence the zm sorption capacity. the ph of the solution also influences the ionization and speciation of metals in aqueous solutions. metals can form various ionic forms depending on the ph of solution. it is generally known that zeolites have a very little affinity to anionic complexes due to their negative framework structure (ziyath et al., 2011). according to speciation calculation (visual minteq ver. 3.0) cesium, in ph from 2.0 to 8.0, exists exclusively in two forms cs+ (>99.9%) and cscl (0.1%) indicating that speciation has no significant influence on cesium uptake by zeolitic materials. due mentioned facts, all further experiments in present study were carried out at initial ph value of 6.0. 3.3 cs+ sorption kinetics by zeolitic materials and fly ash zeolitic materials (zm1 and zm3) prepared from coal fly ash represent well sedimenting material with a large specific surface area, suitable for sorption of dissolved solutes primarily metal ions and radionuclides. the kinetic studies on the adsorption of cesium ions from single system by zeolitic materials and brown coal fly ash were performed to determine the minimum time needed to reach the sorption equilibrium. results showed that the cs sorption by zeolitic materials is a rapid process (fig. 2). maximum uptake, 933 µmol/g (59%) in case of zm1 and 1015 µmol/g (63%) in case of zm3 at sorbent dosage 2.5 g/dm3, initial cesium concentration c0 = 4000 µmol/dm 3 and 22 °c, was recorded after 30 min and equilibrium was established within 60 min. after this time there was no significant increase during next 23 hours, although a slight decrease of cs uptake in case of zm1 was observed. at the initial phase, rapid uptake of cesium ions onto surface of zeolitic materials occurs. subsequently, the cs+ ions diffused into the crystal structure through the micropores followed by chemical and physical adsorption at the active sites of the pores. contrary, the equilibrium sorption of cs+ ions by raw fly ash was obtained after 60 min and the cesium uptake reached only 4 µmol/g, indicating negligible affinity of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc 64 remenárová, l. et al. fly ash towards cesium ions. el-naggar et al. (2008) also observed very fast sorption of cesium ions by naa-x zeolite blend from fly ash. the largest amount of cesium ions was adsorbed within the first 90 min. maximum sorption of cs+ ions by turkish clays (bentonite, zeolite, sepiolite and kaolinite) was reached within 4h (bayülken et al., 2010). similarly, the kinetic studies of faghihian et al. (2013) showed that the process of cs+ and sr2+ sorption onto magnetic zeolite nanocomposite was quite rapid and 90% of equilibrium capacity was achieved within 30 min. on the contrary, yildiz et al. (2011), who studied the sorption behavior of cs+ ions on clay minerals and zeolite from radioactive waste, found that in all cases, equilibrium was reached in about 2 days. 0 200 400 600 800 1000 1200 1400 1600 0 200 400 600 800 1000 1200 q eq [μ m ol g -1 ] t [min] fly ash zeolitic material zm1 zeolitic material zm3 fig. 2. sorption kinetics of cs+ ions by coal fly ash (c0 = 1000 µmol/dm3 cscl, 60 kbq/dm3 137cs, cs = 2.5 g/dm3, d.w.) and zeolitic materials zm1, zm3 (c0 = 4000 µmol/dm3 cscl, 60 kbq/dm3 137cs, cs = 2.5 g/dm3, d.w.) at 22°c and ph 6.0. 3.4 sorption isotherm study the cesium sorption equilibrium is shown in fig. 3. on the basis of obtained results we found that sorption capacity for cesium was 287 times higher (zm1) and 316 times higher (zm3) in comparison with raw coal fly ash. due to the fact that we observed only negligible sorption capacity for cesium in case of fly ash, this sorbent was not used in further experiments. the langmuir (eq. 2) and freundlich (eq. 3) isotherms were fitted to the equilibrium data for cs+ sorption on zm1 and zm3. parameters of the models determined from the experimental data using non-linear regression analysis are reported in table 2. in both cases the langmuir isotherm fits experimental data better than the freundlich isotherm, as is demonstrated by the more homogeneous standard deviation of each observed parameter obtained and by the akaike´s information criterion values (table 2).the maximum sorption capacity qmax of zeolitic materials for cesium obtained from langmuir isotherm was 1203 ± 65 µmol/g for zm1 and 1341 ± q eq [μ m ol /g ] bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc nova biotechnologica et chimica 13-1 (2014) 65 66 µmol/g for zm3, indicating slightly higher sorption capacity of zm3 for cesium cations. affinity constant b indicate similar affinity of both materials for cs+ ions at low concentrations (zm1 b = 0.0042; zm3 b = 0.0036). the difference in qmax values is related to reaction conditions of zeolite preparation. for preparation of zeolitic material zm3, by hydrothermal synthesis of fly ash, higher naoh concentration (3 m) was used. this fact relates with higher molar content of exchangeable na+ ions in the final product in comparison with zm1 and raw coal fly ash (table 1). the difference in maximum sorption capacities of studied materials was more visible in case of cd2+ ions sorption. the qmax were 696 ± 22 μmol/g for zm1 and 1160 ± 44 μmol/g for zm3 indicating higher capacity of zm3 for cadmium (remenárová et al., 2014). 0 1000 2000 3000 4000 5000 6000 0 200 400 600 800 1000 1200 1400 q eq [μ m ol /g ] ceq [μmol/l] fly ash zeolitic material zm1 zeolitic material zm3 fig. 3. the equilibrium sorption of cs+ ions onto: (-■-) fly ash, (-●-) zeolitic material zm1 and (-▲-) zeolitic material zm3 (2.5 g/dm3, d.w.) after 6 h at 22°c, initial ph 6.0. table 2. langmuir and freundlich isotherm model parameters (± sd) for sorption of cs+ ions onto zeolitic material zm1 and zm3 at ph 6.0 (6 h, 22 °c) calculated using non-linear regression analysis. isotherm model qmax [μmol/g] b [dm3/μmol] k [dm3/g] 1/n r 2 akaike´s weight zeolitic material zm1 langmuir 1203 ± 65 0.0042 ± 0.0008 0.989 97.58 freundlich 76.4 ± 31.4 0.34 ± 0.06 0.951 2.42 zeolitic material zm3 langmuir 1341 ± 66 0.0036 ± 0.0006 0.992 97.93 freundlich 62.9 ± 26.1 0.37 ± 0.06 0.960 2.07 it can be seen from table 2 that the freundlich intensity constant n are greater than unity for both studied zeolitic materials. this has physicochemical significance with bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc 66 remenárová, l. et al. reference to the qualitative characteristics of the isotherms, as well as to the interactions between metal ions species and zeolite (el-kamash, 2008). value n > 1 indicates that the zeolitic material has an increasing tendency for sorption with increasing sorbent concentration. this should be attributed to the fact that with progressive surface coverage of adsorbent, the attractive forces between the metal ion species such as van der waals forces, increases more rapidly than the repulsive forces, exemplified by short-range electronic of long-range coulombic dipole repulsion, and consequently, the metal ions manifest a stronger tendency to bind to the zeolitic materials (abd el-rahman et al., 2006; mohan and singh, 2002). table 3 compares the maximum sorption capacities qmax of various inorganic sorbents for removing of cesium cations. comparison of qmax values obtained in our work with those of other authors indicates that studied synthetic zeolitic materials prepared from coal fly ash belong to the group of sorbents with significantly higher sorption capacity for cesium. table 3. comparison of qmax values of different inorganic sorbents for cs+ ions. sorbent qmax [µmol/g] references magnetic zeolite 1725 faghihian et al. (2013) zeolite a 1561 el-kamash (2008) bentonite j15 850 galamboš et al. (2010) bentonite k15 950 galamboš et al. (2010) bentonite l15 550 galamboš et al. (2010) montmorillonite k10 280 galamboš et al. (2010) zeolite (fine) 1610 bayülken et al. (2010) zeolite (coarse) 671 bayülken et al. (2010) coal fly ash 4* present study zeolite zm1 1203 present study zeolite zm3 1341 present study * maximum sorption capacity qmax determined experimentally 3.5 sorption mechanism due to the fact that obtained results indicate higher capacity of zm3 for cesium ions, sorption mechanism will be discussed only for zm3. the specific surface area was determined from the n2 adsorption isotherm by applying the brunauer-emmettteller equation (brunauer et al., 1938). the specific surface area (ssa) of zm3 evaluated from bet isotherm equals to 9.24 m2/g. in the literature is often found an inconsistence in determination of specific surface area of zeolites and zeolitic materials. significantly higher values of ssa were obtained in case of cancrinite-type zeolite synthetized from fly ash (278.9 m2/g) produced by qiu and zheng (2009), naa-x zeolite blend from fly ash (593.6 m2/g) (el-naggar et al., 2008) and zfa (278.9 m2/g) (qiu and zheng, 2009). this inconsistency could be associated with differences in the properties of zeolites, their origin and the method of study (chojnacki et al., 2004). in case of synthetized zeolites, the differences in ssa are also related with the original fired coal, type of used combustion technology and also bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc nova biotechnologica et chimica 13-1 (2014) 67 the type of used additives (e.g., for stabilization of flame). wang et al. (2009) reported that the removal of heavy metals increased with the increase in the specific surface area of zeolite. however, some studies have reported that the increase in specific surface area does not necessarily increase the sorption capacity (oren & kaya, 2006; leyva-ramos et al., 2004). ssa value of zm3 was rather low indicating that the material either has low sorption capacity or the dominating mechanism of sorption is different than physical adsorption. maximum sorption capacity acquired rather high values indicating that the physical adsorption is not the main binding mechanism. table 4. predicted values of pka constants and site concentration of functional groups on zeolitic material zm3. potentiometric titration of zm3 (3 g/dm3) was performed by 0.1 m naoh in 0.1 m nacl at 25 °c. calculated by protofit ver. 2.1 software. functional group pka can [mmol/g] phzpc 1 0.00 – 1.46 0.020×10-3 2 5.76 – 5.95 0.013 3 9.55 – 10.60 1.050 4 11.10 – 11.20 1.910 10.9 ∑ 2.97302 on the basis of the chemical nature of zeolites, most studies (remenárová et al., 2014; merrikhpour and jalali, 2013; qiu and zheng, 2009; sprynskyy et al., 2006; erdem et al., 2004) expect that the dominating mechanism of metal cations sorption is ion-exchange in which cations in the zeolite framework are replaced by metal cations present in water. fig. 4. potentiometric titration of zeolitic material zm3 prepared from coal fly ash (3.0 g/dm3) expressed in the form of first derivation according to gran. titration performed with 0.1 m naoh at background electrolyte 0.1 m nacl and 25 °c. 2 3 4 5 6 7 8 9 10 11 12 0 5 10 15 20 25 δ ph δ pr íd av ok n ao h ph δ ph /δ ad di tio n of n ao h ph bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc 68 remenárová, l. et al. fig. 5. scanning electron micrographs of zeolite zm3: (a,b) before and (c-f) after cesium sorption (6000 µmol/dm3 cscl) at ph 6.0; magnification 150. edx elemental mapping of na and k on zeolite zm3: (a, b) before and (c, d) after cesium sorption; and (e, f) elemental mapping of na, k and cs after cesium sorption. a a b c d e f bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc nova biotechnologica et chimica 13-1 (2014) 69 to identify the types and the amount of functional groups present on the zm3 surface, potentiometric titration of zm3 was performed. for calculation of pka values of functional groups and corresponding site concentrations can from titration curve behavior the modeling software protofit using the non-electrostatic model (nem) was used (table 4). it was found that a four-site model provided a good fit of titration data and the results revealed the presence of four functional groups. their detailed chemical identification was not possible. fig. 4 illustrates potentiometric titration curve of zm3. total ion exchange capacity of zeolitic material zm3 was 2.97 mmol/g, giving the theoretical maximum sorption capacity of zm3 for cs ions 2970 µmol/g. in previous experiments (remenárová et al., 2014) we confirmed by sem edx analysis and elemental mapping that the main mechanism of cd2+ ions binding onto zeolitic materials was ion-exchange. similar results were obtained also in case of cs sorption. evident decrease of sodium and also potassium and calcium peaks after cs sorption was observed on the edx spectra indicating the key role of ion-exchange mechanism on cs+ ions sorption by zeolitic materials (data not shown). the expected replacement of sodium and potassium ions by cesium is evident from fig. 5a-f. it has to be pointed out that beside ion-exchange also other mechanisms such as physisorption and chemisorption partly participate on cs binding by zeolitic materials. this statement was also confirmed by slight decrease of sorption capacity at ph 8.0 which cannot be interpreted with a pure ion-exchange mechanism because phdependent surface sites would be expected to retain more cesium as the ph was elevated (bayülken et al., 2010). 4. conclusions zeolitic materials based on brown coal fly ash produced by power plant eno nováky (slovak republic) were successfully synthesized by hydrothermal alternation. the results showed that zeolitic materials present excellent adsorbents for the removal of cesium ions from liquid radioactive wastes. sorption capacities of studied zeolitic materials for cs+ ions removal are more than 287 times higher in case of zm1 and more than 316 times higher in case of zm3 compared with the sorption capacity of the original coal fly ash. zeolitic materials demonstrate high sorption capacities and rapid uptake of cesium ions from aqueous solutions. the values of qmax calculated from langmuir isotherm (qmax) were 1203 ± 65 μmol/g for zm1 and 1341 ± 66 μmol/g for zm3 indicating slightly higher capacity of zm3 for cesium. sem-edx analysis confirmed surface retention of cesium and the participation of ion-exchange mechanism. reported results indicate 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of two methods for the synthesis of fly ash-based sodium and potassium type zeolites. fuel, 88, 2009,1403-1416. shigemoto, n., hayashi, h., miyaura, k.: selective formation of na–x zeolite from coal fly ash by fusion with sodium hydroxide prior to hydrothermal reaction. j. mater. sci., 28, 1993, 4781–4786. sprynskyy, m., buszewski, b., terzyk, a.p., namieśnik, j.: study of the selection mechanism of heavy metal (pb2+, cu2+, ni2+, and cd2+) adsorption on clinoptilolite. j. colloid interface sci., 304, 2006, 21-28. wang, c., li, j., sun, x., wang, l.: evaluation of zeolites synthetized from fly ash as potential adsorbents for wastewater containing heavy metals. j. environ. sci., 21, 2009, 127-136. yildiz, b., erten, h.n., kiş, m.: the sorption behaviour of cs+ ion on clay minerals and zeolite in radioactive waste management: sorption kinetics and thermodynamics. j. radioanal. nucl. chem., 288, 2011, 475-483. ziyath, a.m., mahbub, p., goonetilleke, a., adebajo, m.o., kokot, s., oloyede, a.: influence of physical and chemical parameters on the treatment of heavy metals in polluted stormwater using zeolite – a review. j. water resour. protect., 3, 2011, 758-767. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:11 utc nova biotechnol chim (2017) 16(1): 32-41 doi: 10.1515/nbec-2017-0005  corresponding author: hornik@ucm.sk nova biotechnologica et chimica imaging of photoassimilates transport in plant tissues by positron emission tomography denisa partelováa, klára kuglerováa, yevheniia konotopb, miroslav horníka,, juraj lesnýa, marcela gubišovác, jozef gubišc, peter kováča,d and ildikó matušíkováa a department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic b department of plant biology, educational and scientific center “institute of biology and medicine”, taras shevchenko national university of kyiv, volodymyrska 64, kyiv, 016 01, ukraine c plant production research institute, national agricultural and food centre, bratislavská cesta 122, piešťany, sk-921 68, slovak republic d biont inc., karloveská 63, bratislava, sk-842 29, slovak republic article info article history: received: 15th december 2016 accepted: 15th june 2017 keywords: 2-[18f]fdg pet photoassimilates transport plant tissues giant reed abstract the current findings show that positron emission tomography (pet), primarily developed for medical diagnostic imaging, can be applied in plant studies to analyze the transport and allocation of wide range of compounds labelled with positronemitting radioisotopes. this work is focused on pet analysis of the uptake and transport of 2-deoxy-2-fluoro[18f]-d-glucose (2-[18f]fdg), as a model of photoassimilates, in tissues of giant reed (arundo donax l. var. versicolor) as a potential energy crop. the absorption of 2-[18f]fdg and its subsequent transport in plant tissues were evaluated in both acropetal and basipetal direction as well. visualization and quantification of the uptake and transport of 2-[18f]fdg in plants immersed with the root system into a 2-[18f]fdg solution revealed a significant accumulation of 18f radioactivity in the roots. the transport rate in plants was increased in the order of plant exposure through: stem > mechanically damaged root system > intact root system. pet analysis in basipetal direction, when the plant was immersed into the 2-[18f]fdg solution with the cut area of the leaf of whole plant, showed minimal translocation of 2-[18f]fdg into the other plant parts. the pet results were verified by measuring the accumulated radioactivity of 18f by direct gamma-spectrometry.  university of ss. cyril and methodius in trnava introduction among the important issues that currently affect the whole world are mainly food shortage also in direct connection with climate changes and environment pollution. plant growth and yields are dependent on photosynthetic fixation of carbon, and optimal allocation of carbon to growing sink tissues (karve et al. 2015). to achieve increase in yields on available arable land, it must be developed a mechanistic understanding of photosynthetic c-fixation, c-transport and c-allocation to different tissues, and the regulatory system that controls photosynthesis and c-allocation (evans 2013). also, a long-standing aim for plant biologists is capturing and interpreting water and sugar flow in plants because of their vital importance for plant bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:26 utc mailto:hornik@ucm.sk nova biotechnol chim (2017) 16(1): 32-41 33 growth and life (hubeau and steppe 2015; fiorani et al. 2012). in this way, non-invasive imaging methods that analyze the photoassimilates transport and allocation in plants on appropriate spatial and temporal scales can play an important role. it is generally known and accepted that radiotracer imaging is one of the few methods that enable to study and analyze the dynamics of substances in a living plant body, without sampling or fixation of plant tissues (kawachi et al. 2016). one of these radiotracer imaging systems and approaches is positron emission tomography (pet). pet represents a non-invasive in vivo and real-time imaging technique that can achieve spatial resolution on the order of millimetres and provides quantitative information about dynamic physiological processes over a relatively large field of view (karve et al. 2015). such systems have been developed to give the medical world a tool to measure the activity of organs, cell aggregates, or brain zones, and primarily for the diagnosis of human cancer diseases. in addition to these biomedical or medical diagnosis applications, pet can be used to study the regulation of plant growth and development via metabolites transport (karve et al. 2015). the most frequently used pet positron emitters are carbon-11 (11c), nitrogen-13 (13n), oxygen-15 (15o), and fluorine-18 (18f) due to their short half-lives between 3 and 110 min (mcrae et al. 2009). the radiotracers, i.e. labelled molecules with positron emitters, are absorbed via normal metabolism and distributed throughout the plant body. in vivo pet analysis involves the γ-rays detection after annihilation process of an emitted positron with a nearby electron producing two 511 kev photons and enables to track the transport and distribution of the radiotracers in the plant as a function of time (kiser et al. 2008). for plant biologists, the advantages of pet systems mainly include the capability of providing real-time dynamic images of positron-emitting isotopes or the movement of labelled molecules under in vivo conditions without damage to the plants, the possibility to obtain 3d images, and measurements in absolute units of radioactivity with high precision. the previous papers (partelová et al. 2014; 2016) deal with the evaluation of application potential of a commercial micropet system in visualization and quantification of uptake and transport of 2-deoxy-2-fluoro[18f]-d-glucose (2-[18f]fdg) within the tissues of tobacco plants (nicotiana tabacum l.) showing very thin anatomical structures. discussed are also the effects of various factors (including the design of experiments, initial conditions of experiments, and the morphological and physiological parameters of plants or plant organs) on visual or quantitative side of the obtained 3d pet images in connection with the distribution of 2-[18f]fdg within the plant tissues. a radioactive 2-[18f]fdg is a chemical analogue of d-glucose (substitution of oh for f at the c-2 position), but only in terms of the transport pathways of d-glucose. the 2-[18f]fdg is transported across the cell membrane by d-glucose transport systems, but the metabolic fate of 2-[18f]fdg will be different from d-glucose (plathow and weber 2008). it is commonly used in medical diagnostics and animal studies to trace the uptake and metabolism of d-glucose in metabolically active tissues, such as brain tissue or cancer cells, however is rarely used in plant imaging studies to trace sugar dynamics (fatangare et al. 2014). giant reed (arundo donax l.), belonging to poaceae, is a low-maintenance, multipurpose crop providing biomass for energy, fibre, pulp and other raw materials. a typical and notable characteristic of giant reed (a. donax l.) plants is the storage of large amounts of carbohydrates in their root system. currently, considering the fact that giant reed plants are tolerant to diverse soil types and even to adverse environmental conditions, such as supranormal concentration of salts, drought or low availability of nutrients, giant reed represents a fast-growing and high-yielding crop with high application potential in bioenergy (mack 2008). it could become an important energy crop due to its high yield (the highest multiyear dry biomass production 400 t per ha) and capacity to grow in marginal land, therefore not competing with the arable land used for food production (czakó and márton 2010). cultivation of giant reed has a positive energy balance and compares favourably to other biomass crops, including c4 species, because of its exceptionally high water bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:26 utc nova biotechnol chim (2017) 16(1): 32-41 34 utilization efficiency and high photosynthetic activity at hot climate despite its c3 anatomy (rossa et al. 1998). however, until challenges concerning giant reed breeding and cultivation practices as well as pollution of the environment by emission from direct combustion of giant reed will be solved, bioethanol production from lignocellulose appears to be the best way to utilise the giant reed biomass (pilu et al. 2012). moreover, properties of this crop including adaptability to high water, salinity, nutrient inputs, and tolerance to pollutants, allow phytoremediation application combined with biomass production (czakó and márton 2010). the aim of this work was to visualize and quantify the uptake and distribution of 2-[18f]fdg, as a model of photoassimilates, in tissues of giant reed (a. donax l. var. versicolor) plants, as a potential energy crop, by positron emission tomography. the processes of absorption of 2-[18f]fdg and its subsequent transport in tissues of giant reed plants were evaluated in acropetal and basipetal direction as well. experimental seedlings of giant reed seedlings of giant reed (arundo donax l. var. versicolor) were obtained by vegetative propagation under in vitro conditions in the following steps: (i) lateral buds were cultivated in an ms medium (murashige and skoog 1962) with an addition of 2–4 mg/dm3 of bap (6-benzylaminopurine) as a cytokinin-type growth regulator for induction of shoots development; (ii) the rooting of regenerated shoots was achieved on ms medium without an addition of growth regulators, and (iii) the pre-cultivation of giant reed seedlings before the experiments was carried out in a diluted nutrient solution (25 % strength) according to hoagland (1920). the plants were cultivated at a photoperiod of 16 h light / 8 h dark (maximum light intensity was 11 450 lx), temperature of 28 °c (light) / 18 °c (dark) and a relative humidity of 60–80 %, which were provided by the plant growth chamber (kbwf 720; binder, germany). the composition of the full strength hoagland medium (100 % hm) was (in mg/dm3): mgso4.7h2o – 370; kno3 – 404; cacl2 – 444; nah2po4.2h2o – 292; na2hpo4.12h2o – 46.5; feso4.7h2o – 17.9; nano3 – 340; nh4cl – 214; nh4no3 – 160; h3bo3 – 8.5; na2moo4.2h2o – 0.06; mnso4.5h2o – 5.0; znso4.7h2o – 0.66; cuso4.5h2o – 0.8. production of 18f and synthesis of 2-[18f]fdg positron emitter 18f (t1/2 = 109.5 min) in the form of fanions was produced via the reaction 18o (p, n) 18f using h2 18o and the cyclotron cyclone 18/9 (iba, belgium). the important intermediate 1,3,4,6-tetra-o-acetyl-2-[18f]fluoro-dglucopyranose for the production of 2-[18f]fdg was synthesised from the precursor 1,3,4,6-tetra-oacetyl-2-o-trifluoromethanesulfonyl-β-d-mannopyranose, the phase-transfer catalyst aminopolyether-potassium complex (kryptofix® 222) and produced [18f]f-. the final product 2-[18f]fdg was obtained by the acid hydrolysis of mentioned intermediate. the production of 18f and synthesis of 2-[18f]fdg were carried out by the company biont inc. (bratislava, slovak republic). the physico-chemical parameters of the final solution of 2-[18f]fdg supplemented with nacl (9.0 g/dm3) and produced by the mentioned procedure as radiopharmaceutical product under the trade name biontfdg are summarized in the table 1. the solution of 2-[18f]fdg applied in experiments was diluted with deionised water at a volume ratio of 1 : 9. experimental setup and pet analysis for the experiments, uniform seedlings of giant reed (a. donax l. var. versicolor) were chosen in terms of their growth phase, height (approx. 15 cm), weight (approx. 0.4 g; w.w.) and number of leaves. the visual and quantitative analysis of 2-[18f]fdg uptake and distribution in tissues of giant reed plants were carried out in two experimental configurations. in the first experimental setup, the uptake of 2-[18f]fdg by giant reed was evaluated in the acropetal direction by immersion of intact root system, damaged root system (cut root capillaries) or stem bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:26 utc nova biotechnol chim (2017) 16(1): 32-41 35 table 1. physico-chemical parameters of produced 2-[18f]fdg solution. analyzed parameter determined/evaluated value/character volume gravimetrically 0.2–0.5 cm3 opalescence organoleptically clear, colourless solution ph colorimetrically 6.8–7.2 specific volume radioactivity ionization chamber 1.6–2.8 gbq/cm3 half-life decay ionization chamber 109.5 min concentration of 2-[18f]fdg liquid chromatography < 0.0016 mg/cm3 kryptofix® 222 concentration thin-layer chromatography < 0.22 mg/cm3 radiochemical purity of 18f in the form of 2-[18f]fdg and 2-[18f]fdm* thin-layer chromatography 97 %–99 % glucose concentration liquid chromatography 0.0762 mg/cm3 acetonitrile concentration gas chromatography 0.03 mg/cm3 ethanol concentration gas chromatography 0.037 mg/cm3 * 2-[18f]fdm = 2-deoxy-2-[18f]fluoro-d-mannose. (removed root system by cutting with a scalpel blade) in small plastic vials containing of 2-[18f]fdg solution with known specific 18f radioactivity and composition in the given volume (see table 1). to avoid spillage and evaporation of the solution, the plastic vials were covered with parafilm over the neck of the vial. in the second type of the experiments, the uptake of 2-[18f]fdg was analyzed in the opposite (basipetal) direction as in the first type of experiments. the absorption of 2-[18f]fdg by giant reed plants was realized through the leaf blade (excised using a scalpel in the middle part of the leaf) and by immersion of cut area of the leaf of whole plant into a 2-[18f]fdg solution (see table 1). simultaneously, the root system of giant reed plant was immersed in a 25 % hoagland medium without addition of 2-[18f]fdg. the plant exposure and pet analyses were carried out during 120 min under laboratory conditions (temperature between 23–25 °c and a relative humidity of 40–45 %). at the end of the exposure period, the root system or immersed plant part were washed with deionised water to remove any unabsorbed 2-[18f]fdg. the plants were then carefully placed on the scanning bed of the micropet system explore vista pre-clinical pet scanner (ge healthcare, spain) and gently fixed with strips of parafilm. acquisition of primary data was carried out in a static mode during 2 min for every length of the scanning bed position (2 cm). the total number of scanning bed positions was chosen depending on the length of the analyzed giant reed plant. the existence of stray signals originating from escaping positrons annihilated in the plastic material of the commercial scanning bed, was eliminated by the replacement of commercial scanning bed with a cardboard plate which showed a low ability to annihilate escaping positrons. the used micropet system consists of 18 detection modules, whereby modules were arranged in a circle and the diameter of the ring was 11.8 cm. each detection module was represented by 13 × 13 arrays of 1.45 mm × 1.45 mm × 7.0 mm lutetium oxyorthosilicate (lso) crystals and 1.45 mm × 1.45 mm × 8.0 mm gadolinium orthosilicate single (gso) crystals and was placed in time coincidence with the seven opposite modules to give an effective transverse field-of-view of 6.7 cm and an axial field-of-view of 2.0 cm. the performance capabilities of the micropet system used showed a reconstructed central spatial resolution of 1.5 mm, and the absolute central point source sensitivity was 1.9 % for the 250–700 kev energy window (required in this study). for the control of micropet system, the acquisition of primary data, as well as the reconstruction of pet images (voxel size 0.3875 mm × bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:26 utc nova biotechnol chim (2017) 16(1): 32-41 36 0.3875 mm × 0.775 mm) the program explore vista ver. 3.1 (ge healthcare, spain) was used. the process of reconstruction involved the conversion of acquired 3d data sets to 2d sinograms with the fourier rebinning (fore) algorithm and the reconstruction of 2d sinograms with a 2d ordered subset expectation maximization (2d osem) reconstruction algorithm as well. within the reconstruction process, the 2d sinograms were also corrected for accidental coincidences, radioactive decay and dead-time. the quality of visualization of regions with relatively lower accumulated radioactivity in reconstructed pet images was enhanced by adjusting the threshold using the program amide.exe ver. 1.0.4. gamma-spectrometric analysis for the precise determination of 18f radioactivity accumulated in the individual parts of giant reed plants, a well-type scintillation detector assembled with a nai(tl) crystal (54bp54/2; scionix, the netherlands) and scintivision-32 software (ortec, usa) were used. a library of radionuclides was built from characteristic γ-ray peaks of 109cd (eγ = 88.04 kev), 18f (eγ = 511.00 kev) and 137cs (eγ = 661.66 kev) for the energy and efficiency calibration. the radioactivity of individual samples was corrected on the basis of the 18f half-life (t1/2 = 109.5 min) to the time of pet analysis completion. higher values of 18f radioactivity (> 1.105 bq) of applied 2-[18f]fdg solutions or samples of individual parts of giant reed plants were also analyzed by ionisation chamber (curiementor; 3ptw, germany). results and discussion giant reed plants have not yet been studied using non-invasive imaging techniques, such as positron emission tomography (pet). previously, hattori et al. (2008) and karve et al. (2015) applied the pet in the analysis of the uptake and distribution of radioindicator in the tissues of sorghum (sorghum bicolour l.) plants also belonging to poaceae like giant reed plants. in this work, we evaluated the possibility to visualize and quantify the uptake and distribution of 2-[18f]fdg, as a model of photoassimilates, in tissues of giant reed (a. donax l. var. versicolor) plants using positron emission tomograph. the processes of absorption of 2-[18f]fdg and its subsequent transport in the tissues of giant reed plants were analyzed in acropetal and basipetal direction as well. moreover, in acropetal mode of 2-[18f]fdg transport, the role of root system, as a natural selective barrier determining the solute transport, was also studied in the case of mechanically damaged root system (cut root capillaries) or removed root system. to obtain the visual as well as quantitative data, the positron emission tomograph primarily developed for animal objects and direct gamma-spectrometry were used. in the pet analysis, the process of visualization and quantification of accumulation of 18f radioactivity in the form of 2-[18f]fdg in the plant tissues included the measurements of the number of true coincidences (numc) corresponding to two gamma quantum of 511 kev emitted under 180° with respect to each other by annihilation of positron and negatron. in the first series of experiments, we focused on studying and comparison of the distribution and accumulation of 18f radioactivity in the form of 2-[18f]fdg in tissues of giant reed plants after 120 min of their exposure by immersion with the intact root system, damaged root system or stem into a 2-[18f]fdg solution with known 18f radioactivity and d-glucose concentration. individual parts of plants were distributed within the 9 (fig. 1a), 12 (fig. 1b) or 11 (fig. 1c) scanning bed positions (1 bed position = 2 cm of axial length), where the first positions represent the root system or immersed part of the stem and the last positions represent the topmost leaves. initial conditions of experiments and obtained data from pet analysis of giant reed plants are summarized in table 2. the obtained pet images depicted in the fig. 1a show that the applied 2-[18f]fdg was minimally translocated from the intact root system to the nonimmersed parts of plant. measurements of numc confirmed that up to 91 % of detected 18f bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:26 utc nova biotechnol chim (2017) 16(1): 32-41 37 fig. 1. visualisation of 2-[18f]fdg distribution in tissues of giant reed plants (a. donax l. var. versicolor) analysed by micropet system after 120 min of exposure of the plant immersed with the intact root system (a), damaged root system (b; cut root capillaries) or stem (c; removed root system by cutting with a scalpel blade) into a 2-[18f]fdg solution (initial radioactivity 80 mbq) containing 0.00762 mg/cm3 of d-glucose. the acquisition time (2 min/bed position) was: a. 18 min for 9 scanning bed positions; b. 24 min for 12 scanning bed positions; c. 22 min for 11 scanning bed positions. index 1 – pet image; index 2 – the number of analysed coincidences (numc) within the pet analysis of plant for individual scanning bed positions. table 2. initial conditions of experiments and obtained data from pet analysis of giant reed plants (a. donax l. var. versicolor) after the exposure of individual parts of plant in a 2-[18f]fdg solution. for details see fig. 1 and 2. parameter uptake of 2-[18f]fdg by root uptake of 2-[18f]fdg by damaged root uptake of 2-[18f]fdg by stem uptake of 2-[18f]fdg by cut leaf mp (g; w.w.) 0.321 0.453 0.379 3.63 a0 (mbq) 80 80 80 274 numc 10,762 7,869 1,301 3,526 ev (cm3) 3.53 2.19 1.02 60.1 pv 30,316 18,811 8,753 516,000 tgv 38,818*103 24,630*103 3,977*103 10,759*103 maxpv 33,012 59,934 17,376 1,050 tfc 0.084 0.251 2.93 – mp – fresh weight of the plant (g); a0 – initial applied radioactivity of 2-[ 18f]fdg solution (mbq); numc – number of analysed coincidences within the pet analysis; ev – estimated volume within the pet analysis (cm 3); pv – pixel volume; tgv – total gray value; maxpv – maximum pixel value; tfc – coincidence transfer factor defined as the ratio of the numc in the non-immersed parts of the plant to the numc in the parts of the plant immersed into a 2-[18f]fdg solution. coincidences correspond to the accumulated radioactivity in the intact root system. a similar result was described by partelová et al. (2014) for the 2-[18f]fdg uptake in whole tobacco plants. these authors found that a significant part of the accumulated 18f radioactivity was allocated in the root system of tobacco, while a minimal transport of 2-[18f]fdg into the aboveground parts was observed. this result can be explained by the fact that the natural role of the root system is to form a selective barrier for the transport of substances from the roots to the aerial parts of plant. according to fatangare and svatoš (2016), this barrier is likely to constitute by casparian strips in the radial and transverse cell walls of the endodermis. partelová et al. (2014) also found that on the quantitative side of the 2-[18f]fdg transport from the root system to the aerial parts of plant, the concentration of d-glucose as a carrier will play a decisive role. to eliminate the function of root system as a selective barrier of solute transport, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:26 utc nova biotechnol chim (2017) 16(1): 32-41 38 in the second type of the experiment about half of the root system was removed by cutting the root capillaries (damaged root system) prior to the immersion into a 2-[18f]fdg solution. by this root treatment, we expected the reduction of the surface of the root system as an area with enhanced 2-[18f]fdg accumulation and the suppression of the activity of casparian strips as well. we found that the 2-[18f]fdg was minimally translocated from the immersed root to the non-immersed parts of plant (to stems or leaves) (fig. 1b), but in a higher extent than in the first type of the experiment (fig. 1a). in the damaged root system of giant reed plants, approx. 75 % of analyzed 18f coincidences from the total accumulated radioactivity were measured. fatangare et al. (2014) also studied the uptake and distribution of 2-[18f]fdg in tissues of arabidopsis thaliana plants by combined positron emission tomography with computed tomography (pet/ct). their results showed a comparable proportion of 18f radioactivity accumulated in the aboveground plant parts to the accumulated radioactivity in the root system, whereby the highest specific radioactivities were mainly analyzed in the nodal stems and root system. according to values of coincidence transfer factor tfc (table 2), it was found that approx. 4-fold higher amount of 2-[18f]fdg was accumulated in damaged root system than in the aboveground parts of plant (tfc = 0.25) and approx. 12-fold higher amount of 2-[18f]fdg was accumulated in the intact root system (tfc = 0.08) under the same experimental conditions. thus, damaging the root system caused a 3-fold increase in the translocation of 2-[18f]fdg into the aerial parts of giant reed plant in comparison with the plant immersed into a 2-[18f]fdg solution by intact root system. in general, a very low level of 2-[18f]fdg translocation into leaves can be related to low transpiration rate of giant reed plants (found by independent experiments). in the last type of the experiment, the uptake and transport of 2-[18f]fdg were evaluated for giant reed plant without root system with the aim to confirm the role of the root and the function of casparian strips as selective barriers controlling the 2-[18f]fdg transport into aboveground parts of plant. fig. 1c shows that root system removal resulted in enhanced distribution of 2-[18f]fdg along the stem, probably due to capillary action of water in the xylem pathway. as in the case of previous experiments, the visually evaluable pet image in terms of visualization of 2-[18f]fdg distribution within the whole plant body was not obtained. this fact can be related with the formation of “hot spots” representing high levels of specific radioactivity accumulated in individual plant parts (e.g. root or stem segments) that represent problems in the detection of relatively low levels of radioactivity in other parts of the plant. this phenomenon could be explained by the technical nature and performance characteristics of pet systems, which are designed to accurately visualize regions with high accumulation of radioactivity and reject regions with low accumulation of radioactivity. however, it was found that approx. 3-times higher 18f radioactivity was analyzed in the non-immersed parts of plant than radioactivity accumulated in stem (tfc = 2.93). the significant 2-[ 18f]fdg accumulation in the leaves was also verified and confirmed by direct gamma-spectrometry. the highest 18f radioactivity (bq) value was detected in the case of young developed leaves (data not shown). in relation to this finding, it is interesting that despite of high 18f radioactivity accumulated in young developed leaves these regions were not visualized within the pet record. contrary to this finding, the stem showing a slightly lower accumulation of 18f radioactivity was visualized in obtained pet image. this fact can be explained by different specific 18f radioactivities (in bq/cm2 for 2d pet image or bq/cm3 for 3d pet image) accumulated in individual plant parts, when the highest specific 18f radioactivities are allocated in stem as area and volume smaller objects than leaves. therefore, the regions with the highest specific 18f radioactivities are visualized in pet images, but the regions with lower specific 18f radioactivities are eliminated within the reconstruction of pet images. in the second series of experiments, the uptake and transport of 2-[18f]fdg were bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:26 utc nova biotechnol chim (2017) 16(1): 32-41 39 analyzed in basipetal direction, when the plant was exposed to the 2-[18f]fdg solution through the cut area of the leaf (in the middle part of the leaf). fig. 2. a. visualisation of 2-[18f]fdg distribution in tissues of giant reed plant (a. donax l. var. versicolor) analysed by micropet system after 120 min of exposure of the plant immersed with the cut area of the leaf (in the middle part of the leaf) of whole plant into a 2-[18f]fdg solution (initial radioactivity 274 mbq) containing 0.00762 mg/cm3 of d-glucose. simultaneously, the root system of giant reed plant was immersed in a 25 % hoagland medium without addition of 2-[18f]fdg. the acquisition time was 24 min for 12 scanning bed positions (2 min/bed position). b. the number of analysed coincidences (numc) within pet analysis of plant for individual scanning bed positions. in the literature focused on the analysis of uptake and translocation of compounds in plant tissues using pet, the application of radiotracer on the cut leaf tip of plants represents a relatively common method. for example, hattori et al. (2008) using this approach found that 2-[18f]fdg was uptaken from the leaf tip and it wastranslocated to the basal part of the shoots from where it moved further to the roots, the tillers and the sheaths of sorghum plant. as it can be seen in the fig. 2, the immersion of cut leaf in our study did not resulted in significant translocation of 2-[18f]fdg into the other plant parts. initial conditions of experiment and obtained data from pet analysis are presented in the table 2. we suppose a more significant translocation of 2-[18f]fdg into other plant parts would require a longer-term exposure, but the application of 18f as a positron emitter with short half-life decay (t1/2 = 109.5 min) is a limiting factor in this way. previously, partelová et al. (2016) have explained a fast initial uptake of 2-[18f]fdg in tobacco leaves through the cut petiole as a result of physico-chemical process driven by capillary action of water in the xylem pathway. fatangare et al. (2015) have noted that 2-[18f]fdg as a radioactive analogue of d-glucose can also be transported by phloem, while the behaviour and metabolism of 2-[18f]fdg in plant tissues remain still unclear. the distribution of 18f radioactivity in giant reed plant after the 2-[18f]fdg administration through the cut leaf was also analyzed by direct gammaspectrometry (fig. 3). obtained results are in agreement with the pet analysis. it was confirmed that more than 98 % of 18f radioactivity accumulated by plant was allocated in the immersed cut leaf. also, the translocation of 2-[18f]fdg in the direction of the root system represents a more likely process than the translocation of 2-[18f]fdg in the direction of topmost leaves (fig. 3b). fig. 3. a. 18f radioactivity analysed in individual parts of giant reed plant (a. donax l. var. versicolor) by direct gamma-spectrometry. for details of experiments see fig. 2. the numbering of leaves (l): 1. the oldest developed leaf, 5. the youngest developed leaf, 3. exposed leaf immersed into a 2-[18f]fdg solution; and stem (s): 1. lower stem, 3. top stem; k – root system. b. detailed distribution of 18f radioactivity analysed in individual parts of giant reed plant without exposed leaf l3. conclusions the obtained results show that commercial micropet systems primarily developed for animal objects and 2-deoxy-2-fluoro[18f]-d-glucose (2-[18f]fdg) can be used for analysis of wholeplant photoassimilates allocation in energy crops. visualization and quantification of the uptake and transport of 2-[18f]fdg in giant reed plant immersed with the root system into a 2-[18f]fdg solution revealed a significant accumulation of 18f radioactivity in the roots, confirming the natural function of this organ as a selective barrier determining the solute transport. the 2-[18f]fdg transport expressed as coincidence transfer factor numc [10 3] a b b a a [bq] a [bq] bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:26 utc nova biotechnol chim (2017) 16(1): 32-41 40 tfc = [ 18f]non-immersed plant part : [ 18f]immersed plant part was increased in the order of plant exposure through: stem (removed root system by cutting with a scalpel blade) > mechanically damaged root system (cut root capillaries) > intact root system. thus, the complete removal of the root system with casparian strips as selective barriers of solute transport into the aboveground parts of plant caused a significant increase in 2-[18f]fdg allocation into the aerial parts of giant reed plants, especially into the young developed leaves. pet analysis of the uptake and transport of 2-[18f]fdg in basipetal direction, when the plant was immersed with the cut area of the leaf of whole plant into a 2-[18f]fdg solution, showed minimal translocation of 2-[18f]fdg into the other plant parts. however, it was found that the translocation of 2-[18f]fdg from the cut leaf into the root system represents a more likely process than the translocation of 2-[18f]fdg in the direction of topmost leaves. the possibility of visualization and quantification of carbon allocation or photoassimilate transport dynamics under real-time and in vivo conditions can be important for functional genomic studies to reveal the mechanisms controlling their allocation and transport in energy crops, which will provide crucial insights for improving yields. however, as was demonstrated in this study, the physical properties of positron-emitting isotopes, performance characteristics or capabilities of pet systems as well as the design of experiments can restrict the pet application in plant biology. acknowledgement this work was supported by the slovak research and development agency under the contract no. apvv-150098. financial support for the stay of yevheniia konotop at ucm in trnava is provided by slovak academic information agency (saia). references czakó m, márton l (2010) subtropical and tropical reeds for biomass. in: halford ng, karp a (eds.), energy crops, rsc publishing, cambridge, uk: 322-340. evans jr (2013) improving photosynthesis. plant physiol. 162: 1780-1793. fatangare a, gebhardt p, saluz h, svatoš a (2014) comparing 2-[18f]fluoro-2-deoxy-d-glucose and [68ga]gallium-citrate translocation in arabidopsis thaliana. nucl. med. biol. 41: 737-743. fatangare a, paetz c, saluz h, svatoš a (2015) 2-deoxy-2fluoro-d-glucose metabolism in arabidopsis thaliana. front. plant sci. 6: 935. fatangare a, svatoš a (2016) applications of 2-deoxy-2fluoro-d-glucose (fdg) in plant imaging: past, present, and future. front. plant sci. 7: 483. fiorani f, rascher u, jahnke s, schurr u (2012) imaging plants dynamics in heterogenic environments. curr. opin. biotechnol. 23: 227-235. hattori e, uchida h, harada n, ohta m, tsukada h, hara y, suzuki t (2008) incorporation and translocation of 2-deoxy-2-[18f]fluoro-d-glucose in sorghum bicolour (l.) moench monitored using a planar positron imaging system. planta 227: 1181-1186. hoagland dr (1920) optimum nutrient solution for plants. science 52: 562-564. hubeau m, steppe k (2015) plant-pet scans: in vivo mapping of xylem and phloem functioning. trends plant sci. 20: 676-685. kawachi n, yin y-g, suzui n, ishii s, yoshihara t, watabe h, yamamoto s, fujimaki s (2016) imaging of radiocesium uptake dynamics in a plant body by using a newly developed high-resolution gamma camera. j. environ. radioact. 151: 461-467. karve aa, alexoff d, kim d, schueller mj, ferrieri ra, babst ba (2015) in vivo quantitative imaging of photoassimilate transport dynamics and allocation in large plants using a commercial positron emission tomography (pet) scanner. plant biol. 15: 273. kiser mr, reid cd, crowell as, phillips rp, howell cr (2008) exploring the transport of plant metabolites using positron emitting radiotracers. hfsp j. 2: 189-204. mack rn (2008) evaluating the credits and debits of a proposed biofuel species: giant reed (arundo donax). weed sci. 56: 883-888. mcrae r, bagchi p, sumalekshmy s, fahrni cj (2009) in situ imaging of metals in cells and tissues. chem. rev. 109: 4780-4827. murashige t, skoog f (1962) a revised medium for rapid growth and bio assays with tobacco tissue cultures. physiol. plant. 15: 473-497. partelová d, uhrovčík j, lesný j, horník m, rajec p, kováč p, hostin s (2014) application of positron emission tomography and 2-[18f]fluoro-2-deoxy-d-glucose for visualization and quantification of solute transport in plant tissues. chem. pap. 68: 1463-1473. partelová d, horník m, lesný j, rajec p, kováč p, hostin s (2016) imaging and analysis of thin structures using positron emission tomography: thin phantoms and in vivo tobacco leaves study. appl. radiat. isot. 115: 87-96. pilu r, bucci a, badone fc, landoni m (2012) giant reed (arundo donax l.): a weed plant or a promising energy crop? afr. j. biotechnol. 11: 9163-9174. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:26 utc nova biotechnol chim (2017) 16(1): 32-41 41 plathow ch, weber wa (2008) tumor cell metabolism imaging. j. nucl. med. 49: 43s-63s. rossa b, tuffers av, naidoo g, von willert dj (1998) arundo donax l. (poaceae) – a c3 species with unusually high photosynthetic capacity. bot. acta. 111: 216-221. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:26 utc microsoft word jarabkova et al., revised.doc nova biotechnologica et chimica 14-2 (2015) 117 doi 10.1515/nbec-2015-0021 © university of ss. cyril and methodius in trnava phage endolysin: a way to understand a binding function of c-terminal domains a mini review veronika jarábková, lenka tišáková, andrej godány department of biology, faculty of natural sciences, university of ss. cyril and methodius, trnava, sk-917 01, slovak republic (lenka.tisakova@ucm.sk) abstract: endolysins are bacteriophage-encoded peptidoglycan hydrolases, which are synthesized in the end of phage reproduction cycle, in an infected host cell. usually, for endolysins from phages that infect gram-positive bacteria, a modular structure is typical. therefore, these are composed of at least two separate functional domains: an n-terminal catalytic domain (ead) and a c-terminal cell wall binding domain (cbd). specific ligand recognition of cbds and following peptidoglycan (pg) binding mostly allows a rapid lytic activity of an ead. here we briefly characterize phage endolysin cbds in conjuction with their domain architecture, (non)necessity for the following lytic activity and a high/low specificity of their ligands as well. such an overall assessment of cbds may help to find new ways to widen opportunities in their protein design to create ‛designer recombinant endolysins’ with diverse applications. key words: bacteriophage, endolysin, cell wall binding domain (cbd), ligand/receptor, peptidoglycan (pg) 1. introduction bacteriophage (phage) is a virus that precisely infects bacterial hosts. after the completion of a replication inside the infected bacterial cell, newly formed phage particles need to be released outside the cell with the help of lytic enzymes (young, 1992; 2014). these lytic enzymes endolysins, along with holins, are encoded by a phage and located in so called ‛lytic module’ in the phage genome. endolysins belong to a group of enzymes peptidoglycan hydrolases (pghs) that break down the peptidoglycan (pg) structure. another efficacy of endolysin is ‛lysis from without’, by which pg scaffold could be cleaved from the outside of a cell (fischetti, 2008; loessner, 2005). thorough studies of endolysin mechanism should bring a promising option for the various applications in practice (tišáková and godány, 2014). theoretically, just a small amount of a recombinant endolysin added externally to gram-positive cells could immediate a rapid and irreversible lysis of these cells. conversely, for gram-negative cells, this action is limited due to the presence of an outer membrane (om) (schmelcher et al., 2012). depending on the origin, phage endolysins can be divided into several groups according to their molecular structure. our focus is on the architecture of grampositive endolysins, known as modular structure, and is composed of at least two distinct functional domains: the enzymatically active domain (ead) at the n-terminus and cell wall binding domain (cbd) at the c-terminus of an endolysin (nelson et al., 2012). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 08:37 utc 118 jarábková, v. et al. here we evaluate key facts about phage endolysins, and especially about their binding domains aimed at their diversity, variety of their ligands and possible manipulation (genetic and protein) with these domains. as it is getting to be favourable for such manipulations, there are some endolysin features outstanding, which place them in the category of irreversible inhibitors with diverse applications from without the bacterial cell, such as more or less specific binding of cbds to the receptors, along with predominantly rapid lytic activity of eads. 2. endolysins as representatives of peptidoglycan hydrolases in both gram-positive and gram-negative bacteria, peptidoglycan (pg) is an essential basic component of the cell wall that gives to bacteria: shape and physical integrity (schleifer and kandler, 1972; vollmer et al. 2008; humann and lenz, 2009). pg is a unique and covalent macromolecular structure (polymer) of linear glycan strands cross-linked by short peptides (rogers et al., 1980). the glycan strands are formed by alternating n-acetylmuramic (murnac) and nacetylglucosamine (glcnac), where (murnac) residues are linked by β(1→4) bonds. in general, peptidic chains differ in conformation through species (schleifer and kandler, 1972; chapot-chartier and kulakauskas, 2014). the stem peptides are cross-linked by an (l-ala)2 or l-ala-l-ser interpeptide bridge between the amino group of the l-lys of one stem peptide and carboxylate of the d-ala of another stem peptide (karakawa and krause, 1966; reinscheid et al., 2002; pritchard et al., 2004). basically, the pg structure is moderately conserved, so there are limited types of covalent bonds (glycoside, amide and peptide bonds) which are accessible to be cleaved by endolysins (loessner et al., 1997). 2.1 peptidoglycan hydrolases peptidoglycan hydrolases (pghs) are lytic enzymes that target and degrade bonds found in the pg structure. the result of their catalytic activity is a disruption of the cell envelope and a subsequent cell lysis (shockman and höltje, 1994). advantage of phgs is a huge diversity of sources including animals, insects, plants, prokaryotic organisms (bacteria) and non-cellular organisms (viruses) (parisien et al., 2008). firstly, bacteria possess a high number of pghs and it appears that they have redundant roles (höltje and tuomanen, 1991; smith et al., 2000). secondly, pghs may have more than one function – for instance escherichia coli have five nacetylmuramyl-l-alanine amidases, six membrane-bound lytic transglycolases and three peptidoglycan endopeptidases. all of them contribute at variable extent to cleave the bonds (heidrich et al., 2001, 2002; vollmer et al. 2008). one of the essential functions is the ability of pghs to inhibit the growth of other bacterial species. in this case, they are called bacteriocins, in this means pghs would affect bacterial pathogenicity (they cause bacterial lysis). generally, pghs describe a wide range of lytic enzymes that can be classified into sundry groups, while their categorization is based on an individual author, for instance to: (i) lysozymes; (ii) autolysins; (iii) exolysins (bacteriocins); and (iv) endolysins (nelson et al., 2012). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 08:37 utc nova biotechnologica et chimica 14-2 (2015) 119 2.2 phage endolysins and their tasks a lytic enzyme endolysin disrupts the host cell wall itself and new virions can be released into the external environment (thiel, 2001). in bacteriophage genome, endolysins along with holins are part of the lytic module. significantly, endolysins are phage-encoded pghs produced in phage-infected bacterial cells in the end of their lytic cycle. they are accumulated in the cytoplasm of the infected cell while new phage particles are matured (young, 1992). endolysins miss the signal peptide sequence, so they are not transferred through the cytoplasmic membrane. but this process is under control of holins (wang et al., 2000). holins are expressed at a genetically predetermined time during the late phase of a phage infection and disrupt the inner membrane (bacterial cytoplasm). subsequently, the pg structure is exposed and cleaved by an endolysin (loessner et al., 1997). their ability to lyse bacteria is often limited to specific bacterial species therefore endolysins mostly have a narrow host spectrum of the lytic activity (fischetti, 2008). 2.3 endolysin enzymatic and recognition tasks the primary action of this enzyme is a lysis from within (from inside the infected cell), so it is referred as endolysin. fig. 1. schematic representation of phage endolysin mechanism – how to access and breach the pg and release virions layer through the holin-endolysin lytic system. in gram-positive bacteria, the pg layer is thicker (up to 40 layers) thereby a surface for catalytic activity of an endolysin is increased (schleifer and kandler, 1972; bernhardt et al., 2001). in contrast, gram-negative pg lies in bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 08:37 utc 120 jarábková, v. et al. periplasmic space protected by the outer membrane; it is relatively thin and not included surface proteins and carbohydrates. this outer membrane effectively prevents from lytic enzymes to directly access the pg structure. however, if the outer membrane is impaired (e.g. by edta, detergents), the cells become more sensitive to potential exogenous application of endolysin (loessner et al., 2005). 3. variable modular structure of endolysins as mentioned above, endolysins possess two basic functions: (i) binding with its specificity and (ii) enzymatic hydrolysis (khosla and harbury, 2001). these two functions are allowed by domains (or modules) that form the structure of these lytic enzymes. usually, endolysins of gram-negative-infecting phages are globular proteins, most frequently composed of a single catalytic domain (15-20 kda) (nelson et al., 2012). nevertheless, kz144 and el188d, pseudomonas aureginosa phage endolysins, were shown as two endolysins, containing also an ead, in which first 83 amino acids were shown to be responsible for the proper binding to the cell wall (briers et al., 2009). the occurrence of modular structure was also confirmed in endolysins encoded by phages: 201, phie202, phi52237, phirsa1, phie12-2, bcp3, bcep781, bcep43 and bcep1 (briers et al., 2008; oliveira et al., 2013). despite of, the sequence analysis of available endolysins of gram-negative-infecting phage has shown that their modular structure is really unique. on the contrary, gram-positive endolysins mostly use well-defined modular organization (díaz et al., 1990; oliveira et al., 2012) with one or more enzymatically active domains (ead) at the n-terminus and a cell wall binding domain (cbd) at the c-terminus, separated by a flexible interdomain linker sequence (korndörfer et al., 2006). for instance, endolysins of staphylococcal bacteriophages phi11, twort, 187, p68, phiwmy have the typical modular structure, which consists of ead (with enzymatic activity as d-alanyl-glycyl endopeptidase, l-muramyl-l-alanine amidase, n-acetyl-glucosaminidase) and cbd (loessner et al., 1998; navarre et al., 1999) (fig. 2). in addition, eads are also occasionally present in phage tail-associated pghs; and both cbds and eads can be localized in some bacterial autolysins included in cell division and cell death (nelson et al., 2012). fig. 2. scheme of a typical two-domain modular organization of phage endolysin. n = amino terminus of the protein, c = carboxy-terminus of the protein, l = linker, linking the inter-domain region connecting the functional domains. ead, predominantly located at the n-terminus, is responsible for endolysin catalytic activity that is based upon the degradation of the pg by the (hydro)lysis of the layers in pg. more specifically, it is targeted directly to individual receptors in pg bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 08:37 utc nova biotechnologica et chimica 14-2 (2015) 121 (vollmer et al., 2008). in the most cases of modular structure, ead does not achieve sufficient catalytic activity of the cell wall without being connected to cbd, because of the weak affinity to the substrate. proposed higher specificity and adequate affinity of the enzyme is typically mediated by its cbd (loessner et al., 2002). frequently, cbds can be responsible for the rising strainor speciesspecific binding (schmelcher et al., 2010). in general, the size of endolysins is between 25-45 kda for dna phages infecting gram-positive bacteria (fischetti, 2008). the exception is plyc (114 kda), a streptococcal endolysin that is unique because of its composition of two separate gene products (plyca, plycb) (nelson et al., 2006). actually, the modular structure has been experimentally demonstrated by several means, e.g. deletion analyses or design of chimeric proteins (garcía et al., 1990; croux et al., 1993; morita et al., 2001). because of the modular structure, it seems to be plausible that different domains could be swapped and the result is an endolysin with altered enzymatic efficacy and specificity. this was accomplished for the first time by garcia et al. (1990), where an ead of streptococcus pneumoniae phage endolysin was swapped but its cbd was preserved so that the enzyme was able to cleave a different bond in pg structure. the domain cpl-7 of pneumococcal amidase is one of the earliest identified cbds specifically in endolysins (garcía et al., 1990). a lot of conserved domains have already been described (hermoso et al., 2007; tišáková et al., 2014), including the domain lysm (garvey et al., 1986; visweswaran et al., 2011) that belongs to the most frequently occurring domains of pghs (ohnuma et al., 2008). many authors have dealt with the characterization of endolysin properties through mutation analysis (cheng and fischetti, 2007; mayer et al., 2011; schmelcher et al., 2012). for instance, mayer et al. (2011) described a method how to improve some enzymatic attributes – if the point mutation didn’t affect the catalytic activity, changes would occur in the species specificity, in comparison with the original protein. 4. the fashion of endolysin cbds the cell wall of gram-positive bacteria is characterized by the absence of the om, which typically consists of a lipid complex, such as in the case of gram-negative bacteria. in fact, cbd of any endolysin allows targeting and directly binding to a pg layer of the host bacterium (fischetti, 2008). 4.1 cell wall surface receptors endolysins have been evolving over millions of years and so their character has focused on recognizing many different ligands within the cell wall. a ligand (receptor) of a cbd could represent a variation of molecules on the cell wall of host organism and has a function as a specific ligand (loessner, 2005). moreover, binding ability of endolysin cbds requires disparate type of ligands. unfortunately, ligands recognized by cbds of different endolysins have not been sufficiently identified yet. however, expected ligands predominantly belong to carbohydrates in the cell wall, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 08:37 utc 122 jarábková, v. et al. and their function in binding was confirmed by several studies (ohnuma et al., 2008; schmelcher et al., 2010, 2012). ligands can represent different subunits of the pg, like carbohydrates, proteins, choline, as well as teichoic and lipoteichoic acids (oliveira et al., 2013). the knowledge of variability among cbds brings new information that all the components of the bacterial cell wall (carbohydrates, protein, lta, etc.) may serve as ligands (eugster et al., 2011). for example, the choline-binding sites anchoring of protein to choline group of teichoic acids were found for endolysin cpl-1 of the pneumococcal phage cp-1 (garcía et al., 1990; hermoso et al., 2003, 2007). further, endolysin lyb5 of phage φ pyb5 infecting lactobacillus fermentum includes a lysm domain that is supposed to be largely responsible for binding to the pg (hu et al., 2010). several endolysins of phage infecting the genus listeria have been characterized (loessner et al., 2002; korndörfer et al., 2006; schmelcher et al., 2010), but the exact identity of ligands recognized by their cbds is under cover. such analyses will undoubtedly require additional complex and more detailed study. 4.2 (non)specificity of the cbds endolysins have gained their substrate specificity in the process of evolution. this feature is based upon the selective interaction of the enzyme with a small group of substances or just with a particular substrate. the cbd is responsible for specificity and high affinity of the endolysin. cbd recognizes and binds to specific ligands on the cell wall of bacteria thereby affinity of the enzyme to the pg is increased. the obvious effect of substrate specificity was proved in two basic examples: for ply118 and ply500 listeria monocytogenes phage endolysins, wherein an irreversible system of binding to a ligand in the surface of the host bacteria was developed (loessner et al., 2002). again, the cbd, located mostly at the c-terminus, targets and binds to specific ligands of a certain bacterial type of cell wall (it might be a part of pg or the other molecules of the cell wall) and these bonds have really important impact on range of the enzymatic activity (increasing affinity of enzyme to the pg substrate (loessner et al., 2002; schmelcher et al., 2010). although cbds are usually located at the c-terminus as well as they can be located at n-terminal end. in some cases their location might be marked as central domains, for instance lysm, cpl-7 and pg-1 (e.g. streptococcal bacteriophage smp with following structure nlpc/cpl7/cpl-7/gluco) (oliveira et al., 2013). some molecular studies suggested that the molecular basis of interactions between cbds and their ligands is in charge of their cell wall. therefore it leads to the high affinity of the individual bacterial substrate (loessner et al., 2002; briers et al., 2009). for cbds of endolysin expressed by phage infecting the genus listeria, the equilibrium of constants of binding affinity (in picoand nanorange) was established. those values are comparable or higher than affinity of antibody-antigen complex (lopez and garcía, 2004; schmelcher et al., 2010). in some cases, an increase of the lytic activity was observed in the exact moment when cbd was absent (mayer et al., 2011). the same or similar features were observed while cbd was removed; thereby the higher lytic efficacy of endolysin was achieved (loessner bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 08:37 utc nova biotechnologica et chimica 14-2 (2015) 123 et al., 1998; gaeng et al., 2000; low et al., 2005; cheng and fischetti, 2007). in other words, cbd likely helps endolysin with binding to the cell wall of the host bacterium (sanz et al., 1992). conversely, cbd with its peptide structure may sometimes hinder the action of ead, as soon as endolysin connects with the cell wall. efficiency of endolysins also depends on the binding kinetics of cbd. but, on the other hand, there is an evidence of endolysins in which their lytic activity was destroyed after removing cbd from their structural sequence (sanz et al., 1992; loessner et al., 2002; kikkawa et al., 2007; porter et al., 2007; sass and bierbaum, 2007). these exact cases confirm that cbd is required for ead´s activity and, in most cases, these two domains together initiate the disruption of the bacterial cell wall (mayer et al., 2001). 4.3 diversity of the cbds endolysin cbds can often show recognition affinity to more types of strains of the single bacterial species. consequently, the spectrum of the binding specificity of bacterial cell wall receptors is higher than the host range for phage particle itself, encoding a definite endolysin (schmelcher et al., 2012). in comparison, endolysin plyv12, encoded by enterococcal phage phi1 (infecting enterococcus faecalis, strain v12), has a wider range of the catalytic activity as phi1 itself. endolysin plyv12 exhibits the lytic activity against 14 e. faecalis strains and e. faecium and against pathogenic streptococci, including streptococcus pyogenes (group a, b, c) as well, and its catalytic activity was also demonstrated against staphylococcus aureus (yoong et al., 2004). endolysin lyb5, encoded by phage phipyb5 infecting lactobacillus fermentum, is also known to exhibit lytic activity against gram-positive bacteria (s. aureus, bacillus subtilis and few species of the lactic acid bacteria) and against gram-negative bacteria (escherichia coli and salmonella typhimurium) (wang et al., 2008). the distribution of cbds of phages infecting gram-positive bacteria was also analyzed by in silico approaches (fig. 3). fig. 3. frequency of domain distribution of modular phage endolysins. the focus is on the diversity of different types of cbds from endolysins targeting various gram-positive and gram-negative species (adapted from oliveira et al., 2013). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 08:37 utc 124 jarábková, v. et al. it is necessary to note that there is increasing evidence about the multiple-domain architecture, important for binding specificity of endolysins. interestingly, modular endolysins of phages infecting lactobacillus, lactococcus and bacillus strains represent the broadest diversity of their cbds (pg-1, sh3-3, sh3-5, sh3b, chw, lysm a spor). an example of endolysin with three cbds is the endolysin of phage a2 infecting lactobacillus (domain arrangement is: ami2/chw/chw/chw) or the endolysin of phage phiat3 infecting the same bacterial genus, including three different cbds (domain arrangement is: gh25/sh3-5/lysm) (khosla and harbury, 2001). 4.4 categorization of the endolysin cbds in the large and diverse group of cbds, some common domain motifs are comprised: lysm, pg-1, sh3, cpl-7 (tab. 1). all of them have also been described and characterized by in vitro experiments (buist et al., 2008; loessner et al. 2002; hu et al., 2010; walmagh et al., 2012). (i) lysm (lysin motif) (garvey et al., 1986) is one of the most common domains that are present among pghs (buist et al., 2008; visweswaran et al., 2011). lysm polypeptide modules are considered to bind to the widest range of receptors; more than 4,000 proteins in prokaryotic and eukaryotic organisms have been identified. it is known that this domain may bind to glcnac residues in the sugar backbone of the pg (ohnuma et al., 2008). lysm often occurs in double-domain or three-domain architecture, an example of this type of organization in molecule is endolysin of phage lb338-1 infecting lactobacillus (gh25/lysm/lysm/lysm) (oliveira et al., 2013). (ii) pg binding (peptidoglycan binding domain) targets specifically to d-asn in pg cross-bridges. it is also a part of two different endolysins lclys and lylys2 that have been identified in prophages present in the genome of lactobacillus casei bl23, targeting at d-asn interpeptide bridge of the pg (regulski et al., 2013). pg_binding_1 (peptidoglycan binding domain type one) is mainly found in the structure of endolysin of gram-positive-infecting phage (walmagh et al., 2012; oliveira and melo, 2013). exceptions are endolysins of gram-negative-infecting phages: phage pvp-se1 infecting genus salmonella and phages phikz, el, 201phi21 and finally obp infecting genus pseudomonas. one or more cbds may be uniquely placed at the n-terminus. pg_binding_1 consists of three alpha-helices and its binding activity has been examined f. e. in briers et al. (2007), as well as in tišáková et al., (2013). pg_binding_3 type cbd is generally present in endolysins of gram-negativeinfecting phage. interestingly, endolysin shows the modular structure, as it composed of two distinct polypeptide modules (separated functional domains gh108/pg-3) (grundling et al., 2006; oliveira and melo, 2013). (iii) fog (friend of gata – zinc finger protein), slap (src-like a dapter protein), lgfp, spor (sporulation related domain) belong to the group of uncommon cbds motifs that may represent alternative types of domains for stronger binding and their catalytic activity can directly target against muropeptides or changes in species specificity (oliveira et al., 2013). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 08:37 utc nova biotechnologica et chimica 14-2 (2015) 125 (iv) cpl-1 (the choline-binding modules) (garcía et al., 1988) recognizes the choline-containing teichoic acids in the cell wall of streptococcus pneumoniae (hermoso et al., 2007). cpl-1 is a type of cbds that binds to the pneumococcal cell wall and its binding activity is independent on the presence of choline (diaz et al., 1991; bustamante et al., 2010). (v) chw (clostridial hydrophobic with conserved tryptophan w) domains belong to the protein family that targets to the cell wall of clostridium acetobutylicum (sullivan et al., 2007). oliveira et al. (2013) demonstrated that nine domains of chw are also present in three endolysins of phage infecting genus lactococcus and six endolysins of bacteriophage infecting genus lactobacillus. (vi) sh3 (src homology 3 – domain binds to proline-rich ligands) (mayer et al., 1988; stahl et al., 1988) is common in autolysins and phage endolysins, often as sh3b, sh3-3 or sh3-5 (grundling et al., 2006; lu et al., 2006). in the study by buist et al. (2008) it was shown that sh3 mostly occurs in the modular structure of endolysins encoded by phages infecting genus staphylococcus. table 1. representation of identified binding domains of gram-positive and gram-negative endolysins (adapted from oliveira et al., 2013). binding domains conserved domains phage example pg_binding_3 pf09374/ipr018537 vibrio phage vp882 lysm pf01476/ipr018392 enterococcus phage phief11 sh3_3 pf08239/ipr013247 lactobacillus phage lv-1 sh3_5 pf08460/ipr013667 staphylococcus phage phi2958pvl pg_binding_1 pf01471/ipr002477 mycobacterium phage hertubise salmonella phages phikz, el, 201phi21 chw pf07538/ipr006637 lactococcus phage 949 cpl-7 pf08230/ipr013168 streptococcus phage smp lgfp pf08310/ipr013207 nocardia phage nbr1 sh3-r (related) supfam0051050 listeria phage a500 fog cog5263 listeria phage b054 sh3b smart00287 lactococcus phage p087 spor pf05036/ipr007730 bacillus phage ap50 slap pf0321/ipr004903 bacillus phage 0305phi8-36 4.5 endolysin cbds as enzymatic activity regulators binding activity of cbds is their primary feature, but sometimes they can play determining roles in the enzymatic activity associated with the manner to allow substrate to achieve its catalytic site. carbohydrate-cleaving hydrolases, such as xylanases and cellulases have a similar modular architecture as endolysins (khosla and harbury, 2001) and their activity increase if there is enzyme-substrate proximity (bolam et al., 1998). in the case of truncated version of cbd from clostridium phage phi3626 endolysin, an entirely abolished activity has been achieved, accentuating the important role of cbd in the enzyme activity (zimmer bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 08:37 utc 126 jarábková, v. et al. et al., 2002). structural analysis of plyl suggests that cbd not only targets the endolysin to the specific cell wall but also it may be significant for the lytic activity. it has been demonstrated that with the absence of cbd in the modular structure of endolysin, the activity has been inhibited (low et al., 2005). 5. sh3 domain universal though specific regularly, sh3 domain occurs in the modular structure of staphylococcal endolysins, but it is possible to be found in other endolysins, such as streptococcal. this unique cbd may be exploited for the enhancement of anti-staphylococcal efficacy (for instance in protein fusion constructs) (becker et al., 2009). at first, sh3 domain (src homology 3) was described in eukaryotic proteins involved in cellcell communication and intracellular signalization from the surface to the nucleus. moreover, this domain prefers to bind to the proline rich sequence (mayer and eck, 1995). extensive description of its presence in phage endolysin was firstly presented by ponting et al., (1999), and whisstock and lesk (1999). sh3 domain is a small module of 50 to 60 amino acids (weng et al., 1995). in the pfam domain database (http://www.sanger.ac.uk/software/pfam/), three groups of sh3b are possible to be found, respectively: sh3_3, sh3_4 and sh3_5 (lu et al., 2006). the sh3 domain of lysostaphin was one of the first identified binding domains at all. a critical 92 amino acid region at the c-terminus of lysostaphin was proved as essential for a correct specificity of binding to the staphylococcal surface through fusion of this region to ligands (baba and schneewind, 1996). successfully, this fusion strategy has also been used to verify sh3 for other pghs: lyta (phi 11 endolysin) (baba and schneewind, 1996), ba02 endolysin (low et al., 2005), ply500 and ply118 endolysin (loessner et al., 2002). becker et al. (2009) presented the first comprehensive comparison of all staphylococcal sequences of sh3_5 domains obtained from the publicly available databases. the maintenance of sh3 binding domain in modular structure of endolysin has been optimized through evolution and gives the added value for these enzymes, although this fact has not been confirmed in every study (becker et al., 2009). for some endolysins, sh3b domain is indispensable, especially for the binding of an endopeptidase domain to the pg of staphylococcus aureus (baba and schneewind, 1996) and ale-1 (lu et al., 2006). moreover, many studies have been directly focused on the phage endolysin infecting various staphylococci and streptococci. in this association, it has been shown that after the removal of the sh3 domain, the catalytic activity still carried on (donovan et al., 2006). 6. how to exploit the cbds for the bacterial detection currently, detection methods of bacterial pathogens, causing a variety of human and animal infectious diseases, are often slow and limited. the same stands for a rapid identification of contaminating bacteria in food products as well. endolysins may be used for specific detection of bacteria (kretzer et al., 2007; schmelcher et al., 2010). using the enzyme features (as the specificity of the binding domain) may help in the selection and proper separation of the bacterial cell (kretzer et al., 2007). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 08:37 utc nova biotechnologica et chimica 14-2 (2015) 127 for example, schuch et al. (2002), focused on detection of bacillus anthracis by endolysin plyg with direct results in 15 minutes, based on the detection of substances that are produced during the bacterial lysis. in another study, magnetic balls were used for detection of selected bacterial hosts, where these special balls were coated with a layer of binding domain of ply188 and ply500 endolysins of phage infecting genus listeria. by this strategy the effective separation of the cells listeria in contaminated food was approached (kretzer et al., 2007). it was also proved by in vitro experiments that cbds of bacteriophage endolysin infecting bacillus cereus (loessner et al., 1997) and clostridium perfringens (zimmer et al., 2002) may also bind and mediate the immobilization of these bacteria (kretzer et al., 2007). an efficient non-destructive monitoring of the bacterial host can be also mediated by the fusion of a target protein with gfp (green fluorescent protein) from jellyfish aequorea victoria (chalfie et al., 1994; andersen et al., 1998). gfp can be expressed in a variety of organisms (bacteria, yeasts, insects, mammals) and its characteristic property green fluorescence is used in the studied system at the level of individual cells. according to some authors (loessner et al., 2002; schmelcher et al., 2010), fluorescent proteins are useful, for instance in the detection of listeria monocytogenes (loessner et al., 2002; schmelcher et al., 2010; tolba et al., 2012). in recent years, bacteria causing serious infections have become resistant to a broad spectrum of antibiotics. examples are strains of mrsa (methicillin-resistant staphylococcus aureus) that are resistant to β-lactam antibiotics such as penicillin. endolysin therapy is one way how to ameliorate bacterial resistance of s. aureus borysowski et al., 2011; nelson et al., 2012). linden et al. (2015) identified endolysin plygrcs, which shows antimicrobial activity against mrsa s. aureus that are planktonic and active in biofilm formation. the occurrence of bacterial resistance to endolysins is highly unlike because the phage lytic system has been evolving for millions of years. the result of phage evolution is highly evolved enzyme endolysin that focuses on specific molecules (ligands) in the pg layer of the bacterial cell wall, while these specific molecules are important for the viability of particular bacteria (young, 1992; fischetti, 2008). for instance, choline like ligand is important for binding activity of endolysins of pneumococcal phages and for endolysins of phages infecting streptococci it is typical to bind to a ligand polyrhamnose (yamashita et al., 1999; fischetti, 2003). to sum up, the use of the feature of cbds to distinguish specific ligands could without a doubt bring new detection methods (schmelcher and loessner, 2014) and opportunities to create novel ‛designer recombinant endolysins’. 7. conclusion according to reviewed information, phage-encoded endolysins represent a promising tool to control bacterial cells and especially those strains that cause infections, as well as reduce food quality by contamination, which has been showing a considerable health, social and economic importance. endolysins from phages infecting gram-positive bacteria show a well-defined modular organization, mostly comprising functional separate domains: n-terminal catalytic (eads) and c-terminal bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 08:37 utc 128 jarábková, v. et al. cell wall binding ones. whereas cbds target their endolysins to bind to the bacterial cell wall ligands, eads contribute to peptidoglycan (hydro)lysis and so to cell wall degradation and bacterial death. to emphasize, recombinant endolysin-based strategies do not have to be convenient just in elimination of bacteria by the external application of eads to the cells. as we have shown, a huge diversity of endolysin cbds (e.g.) pg-1, sh3-3, sh3-5, sh3b, chw, lysm, spor) that determine endolysin specificity in terms of cbd ligand recognition might be effective agents themselves. so to understand the whole potential of endolysins, it is also important to study cbd binding specificity and ligands. it is prudent to stress out that cbds can be favourable in various methods of bacterial detection. to sum up, using molecular engineering to design recombinant protein chimeras can improve and enhance both binding and lytic activities of bacteriophage endolysins for their potential applications. references andersen, j.b., sternberg, c., lars, k.p., bjorn, s.p., givskov, m., molin, s.: new unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. appl. environ. microbiol., 64, 1998, 22402246. baba, t., schneewind, o.: target cell specificity of a bacteriocin molecule: a cterminal signal directs 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zimmer, m., vukov, n., scherer, s., loessner, m. j.: the murein hydrolase of the bacteriophage phi3626 dual lysis system is active against all tested clostridium perfringens strains. appl. environ. microbiol., 68, 2002, 1531115317. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 08:37 utc 148 nova biotechnologica et chimica 13-2 (2014) doi 10.1515/nbec-2015-0005 © university of ss. cyril and methodius in trnava some environmentally relevant reactions of cerium oxide pavel janoš, jakub ederer, marek došek faculty of the environment, university of jan evangelista purkyně, králova výšina 7, 40096 ústí nad labem, czech republic (pavel.janos@ujep.cz) abstract: reactive forms of cerium oxide were prepared by a thermal decomposition of various precursors, namely carbonates, oxalates and citrates, commercially available nanocrystalline cerium oxide (nanoceria) was involved in the study for comparison. scanning electron microscopy (sem) and x-ray diffraction analysis (xrd) were used to examine the morphology and crystallinity of the samples, respectively, whereas the brunauer-emmett-teller (bet) method of nitrogen adsorption was used to determine surface areas. interactions of cerium oxide with some phosphorus-containing compounds were investigated. some of the examined samples, especially those prepared by annealing from carbonate precursors, exhibited an outstanding ability to destroy highly toxic organophosphates, such as pesticides (parathion methyl), or nerve agents (soman, vx). there were identified some relations between the degradation efficiency of cerium oxides and their crystallinity. it was also shown that cerium oxide is able to destroy one of widely used flame retardants – triphenyl phosphate. a phosphatase-mimetic activity of various cerium oxides was examined with the aid of a standardized phosphatase test. key words: cerium oxide; reactive sorbent; toxic organophosphates; chemical warfare agents 1. introduction cerium oxide belongs to the most important rare earths with numerous applications in such diverse areas as electrochemistry (hrizi et al., 2014), glass polishing (janoš and petrák 1991) or ceramics (markmann et al., 2002). specifically designed forms of cerium oxide have found widespread applications in heterogeneous catalysis (benadda et al., 2013), mainly as a part of so-called three-way catalysts for the control of gaseous exhaust emissions. zhai et al. (2007) demonstrated a photocatalytic activity of cerium oxide and its ability to destroy synthetic dyes. in recent time, a pharmacological potential of cerium oxide has been discovered (celardo et al., 2011) and its ability to interact with phosphate ester bonds in biological systems were studied (kuchma et al., 2010) with the goal to find a new kind of artificial enzymes – nanozymes (wei and wang, 2013; xu and qu, 2014). an ability of cerium oxide to destroy highly dangerous organophosphate compounds (pesticides, nerve agents) was demonstrated in our recent papers (janoš et al. 2014a, janoš et al., 2014b). the unique properties (enhanced catalytic activity, enzyme-mimetic ability) of cerium oxide are attributed usually to its nanosized forms – nano-ceria. various sophisticated procedures have been used to prepare the catalytically active forms of nano-ceria, including sol-gel methods (ansari et al., 2008), thermolysis and homogeneous hydrolysis (liu and zhong, 2010). it was shown that conventional precipitation/calcination methods may be also adopted to produce a highly reactive material capable to destroy toxic organophosphates; a simple and easily scalable bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc nova biotechnologica et chimica 13-2 (2014) 149 procedure involves the precipitation of sparingly soluble cerium carbonate (either with an ammonium bicarbonate solution or with a gaseous mixture of co2 and nh3) with subsequent calcination in the presence of oxygen (air) (janoš et al., 1989; janos et al., 2014b). in this paper, three synthetic routes were used to prepare cerium oxide from: i) carbonate precursor, ii) oxalate precursor and iii) citrate precursor by annealing at 500°c. morphology and crystalline structure of the annealed samples were studied using scanning electron microscopy (sem) and x-ray diffraction analysis (xrd), respectively, the surface areas were determined based on nitrogen adsorption/desorption isotherms, using the brunauer-emmett-teller (bet) method for evaluations. the reactivities of various cerium oxides were compared using the hydrolytic degradation of parathion methyl (organophosphate pesticide) in organic solvent as a model system. the enzyme-mimetic abilities of the cerium oxides were compared using a standardized phosphatase test. an ability to destroy other organophosphate compounds, such as highly toxic nerve agents soman and vx, as well as triphenyl phosphate (flame retardant) was also demonstrated. 2. material and methods 2.1 chemicals, preparation of cerium oxide samples cerium(iii) nitrate, ce(no3)3·6h2o, was obtained from sigma-aldrich (steinheim, germany) as a reagent-grade preparative with purity 99.9% (trace metal basis), ammonium bicarbonate, 99.5%, oxalic acid dihydrate, >99%, and citric acid, 99%, were obtained from the same supplier. parathion methyl and triphenyl phosphate, as well as their degradation products 4-nitrophenol, diphenyl phosphate, phenol, were obtained from sigma-aldrich as chromatographic standards. if not stated otherwise, hplc-grade organic solvents and deionized water were used for preparing the solutions including mobile phases for liquid chromatography. synthetic routes used for preparation of the ceo2 samples are summarized in table 1, more detailed description follows (if not stated otherwise, the preparation started with the solution containing 0.05 mol ce): the carbonate precursor was prepared by precipitation of aqueous solution of cerium(iii) nitrate (0.2 mol/l) with an excess of ammonium bicarbonate (0.5 mol/l) under stirring, completeness of the precipitation was checked by reaction with oxalic acid. after an addition of the last portion of ammonium bicarbonate, the agitation continued for one more hour and then the precipitate was left to stay until the next day. then the precipitate was separated by filtration, washed with water and dried overnight at 110°c. alternatively, cerium carbonate was precipitated from the cerium nitrate solution by gaseous mixture of co2 and nh3 as follows: under continuous stirring, gaseous nh3 was introduced through a glass frit until ph >4, and then gaseous co2 was introduced along with nh3. absorption of co2 was not complete under given conditions (on contrary to the nh3 absorption), and thus co2 was used in an over-stoichiometric amount – the molar ratio of co2 : nh3 was 5 : 1. during precipitation, ph of the mixture was kept at ca. 7.2. when the precipitation of cerium carbonate was completed, the ph value raised suddenly up to 9.5. the nh3 introduction was stopped and the mixture was saturated bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc 150 janoš, p. et al. with co2 for one more hour, and then it was left to stay until the next day. the precipitate was separated by filtration, washed with water and dried at 110 °c. the oxalate precursor was prepared by precipitation of aqueous solution of cerium nitrate (0.2 mol/l) with an excess of oxalic acid solution (saturated) under stirring. after an addition of the last portion of oxalic acid, the agitation continued for one more hour and then the precipitate was left to stay until the next day. then the precipitate was separated by filtration, washed with water and dried overnight at 110° c. the citrate precursor was prepared by the sol-gel method (alifanti et al., 2003) as follows: aqueous solution of cerium nitrate (0.5mol/l) was mixed with an equal volume of aqueous solution of citric acid (0.5mol/l) and agitated at 60°cfor 4 hours, then additional citric acid solution was added (30 ml) and agitation continued until the next day. the transparent viscous gel was placed on a petri dish and dried at 80°c overnight. the ceo2 samples were prepared from the above precursors by annealing at 500°c for 2 hours in an open crucible in a muffle furnace. for comparison, a commercially available nanocrystalline cerium oxide mkn-025 (mknano, missisauga, usa) was involved in the study. table 1. ceo2 samples used in this study – overview of synthetic routes. sample preparation route carb-s precipitation of cerous carbonate with nh4hco3 solution, annealing at 500°c carb-g precipitation of cerous carbonate with gaseous co2 and nh3, annealing at 500°c oxal precipitation of cerous oxalate with solution of oxalic acid, annealing at 500°c citr citrate sol-gel procedure, annealing at 500°c mkn-025 commercial sample, without further treatment 2.2 methods of characterisation and chemical analyses a jeol jsm 6510 l scanning electron microscope (sem) was used to examine the morphology of the ceo2 samples. x-ray diffraction (xrd) measurements were performed using a panalyticalxpert pro diffractometer. the surface areas of the samples were determined based on nitrogen adsorption/desorption isotherms using a coulter sa3100 instrument (beckman), and the brunauer-emmett-teller (bet) method was used for the surface area calculations. the pore size distribution was determined using the barrettjoyner-halenda (bjh) method. the liquid chromatographic determinations of parathion methyl and its degradation products were performed using a lachrom hplc system (merck/hitachi) equipped with a uv/vis detector operating at 230 nm and a luna column (phenomenex, torrance, ca, usa) 150 × 4.6 mm packed with 5µm pfp stationary phase. the mobile phase consisted of an 80/20 (v/v) mixture of methanol (hplc-grade, labscan, dublin, ireland) and water. determinations of triphenyl phosphate were performed using a kinetex column (phenomenex) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc nova biotechnologica et chimica 13-2 (2014) 151 100 × 2.1 mm packed with 2.6-µm biphenyl 100a stationary phase, mobile phase consisted of an 65/35 (v/v) mixture of methanol and formic acid (1%). a varian gc 3800 gas chromatograph coupled with an ion-trap mass spectrometer (varian 4000) and a fused-silica capillary column (vf-5; 20 m × 0.25 mm id × 0.25 μm film thickness) was used to confirm the identities of the target analytes. an agilent 6890 gas chromatograph with an hp-5 column (5% phenyl methyl siloxane, 30 m × 0.32 mm id × 0.25 μm film thickness) was used to follow the degradation of the nerve agents vx and soman. 2.3 testing the reactivity of cerium oxides an ability to destroy toxic organophosphates was examined using the testing procedure derived from previously used procedures for assessing the degradation of chemical warfare agents, as described in our previous work (janos et al., 2014a): equal amounts (50 mg) of cerium oxide were placed into a series of glass vials (supelco, 4 ml), and exact volumes (150 μl) of the pesticide solution (6660 mg/l) were added to each vial (corresponding to a dosage of 1 mg of pesticide per 50 mg of ceo2). the vials were sealed with caps and covered with aluminium foil to shield the reaction mixture from ambient light. at pre-determined time intervals, the reaction was terminated by the addition of 2-propanol, and solid phase was immediately separated by centrifugation. the supernatant was decanted into a 50 ml volumetric flask, and solid phase was re-dispersed in 4 ml of methanol and centrifuged again. the extraction with methanol was repeated three times. all the supernatants were combined in the same volumetric flask, brought to volume with methanol, and analysed immediately by liquid chromatography and gas chromatography. all of the degradation experiments were performed at a laboratory temperature of 22±1°c in an air-conditioned box. the degradation of soman and vx agents was assessed using essentially the same procedure: gas chromatography was used to follow the course of degradation. if not stated otherwise, heptane served as the solvent and as a reaction medium in the degradation of parathion methyl, and nonane was used in the degradation tests for soman and vx. degradation of triphenyl phosphate was examined in a batch arrangement using acetonitrile as reaction medium: 200 mg of cerium oxide was suspended in 1 ml of acetonitrile and mixed with 3 ml of solution containing 5000 mg/l of triphenyl phosphate; the reaction mixture was agitated using a horizontal shaker. at pre-determined time intervals, small amounts of mixture were withdrawn and diluted immediately with mobile phase acidified with formic acid. solid phase was separated by centrifugation and the supernatant was decanted into a 25 ml volumetric flask. then the solid phase was extracted with methanol repeatedly in a similar way as described above, the combined extracts/supernatants were analysed by liquid chromatography. the phosphatase-mimetic activity was assessed using the commercially available alp-meg l-500 (erbalachema, brno, czech republic) phosphatase test, in which the reaction with 4-nitrophenyl phosphate in an aqueous buffer solution was used to compare the activities of the samples. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc 152 janoš, p. et al. 3. results and discussion 3.1 characterization of the samples it was shown in our previous work that the reactivity of cerium oxide is governed mainly by the calcination temperature – it was also found that the samples annealed at temperatures below ca. 600°c exhibit the highest degradation efficiency towards organophosphate compounds (janos et al., 2014a). therefore, all the ceo2 samples were prepared by calcination at the same temperature 500°c. as follows from thermogravimetric analyses (fig. 1), this temperature is sufficient for a conversion of the respective precursors to cerium oxides. 0 200 400 600 800 1000 80 90 100 t g ( % ) temperature (°c) tg dtg dta exo m as s flo w co2 release h 2 o release 0 200 400 600 800 1000 40 60 80 100 t g ( % ) temperature (°c) m as s flo w 0 200 400 600 800 1000 25 50 75 100 t g ( % ) temperature (°c) m as s flo w fig. 1. thermogravimetric analyses of precursors used for preparation of cerium oxides. upper row: ms detection of released gases. a) cerium carbonate prepared by precipitation with aqueous nh4hco3 (cerium carbonate precipitated by co2(g) and nh3(g) exhibited almost identical behaviour); b) cerium oxalate; c) cerium citrate. xrd analyses confirmed the presence a single crystalline phase in all the examined samples, corresponding to cerium oxide with its characteristic fluorite-like structure (space group fm-3m). the cell unit parameters were nearly identical in the examined samples (table 2). however, the shapes of the xrd patterns differed markedly. table 2. physico-chemical characteristics of the ceo2 samples. sample cell parameter a (nm) crystallite size (nm) surface area (m2/g) micropore area (m2/g) total pore volume (ml/g) micropore volume (ml/g) carb-s 0.5413 9.8 57.4 6.35 0.074 0.00234 carb-g 0.5414 9.7 116 21.0 0.114 0.00829 oxal 0.5423 5.6 138 14.8 0.174 0.00516 citr 0.5413 19.4. 19.4 0 0.094 0 mkn-025 0.5412 62.6 9.8 0 0.079 0 c) b) a) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc nova biotechnologica et chimica 13-2 (2014) 153 as can be seen from fig. 2, the most developed crystalline structure was detected in the commercial cerium oxide mkn-025.the narrow and sharp diffraction peaks indicate the presence of a more organised crystal structure in this sample, which may be related to the crystallite sizes as calculated from the peak broadening using the scherrer formula (johnson, 1987) see table 2. 20 40 60 80 0.0 5.0x103 1.0x104 1.5x104 2.0x104 2.5x104 3.0x104 in te ns ity ( a u ) 2 theta carb-s carb-g oxal citr mkn-025 fig. 2. xrd patterns of cerium oxides. fig. 3. pore size distribution of the examined cerium oxides. the cerium oxides prepared from the carbonate and oxalate precursors exhibited the highest surface area (table 2) with certain amounts of micropores (fig. 3). as can be seen from sem images (fig. 4), the ceo2 samples prepared by annealing from carbonate and oxalate precursors consist of relatively large clusters with layered bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc 154 janoš, p. et al. structure, which is clearly visible in a more detailed view in fig. 4b. aggregates of the ceo2 crystals obtained from the citrate precursor (fig. 4e) exhibit an even finer structure resembling a crumpled silk scarf, whereas the commercial nanocrystalline cerium oxide consists of small sub-micron particles forming aggregates with a complex structure (fig. 4f). fig. 4. sem images of ceo2. a, b) carb-s; c) carb-g; d) oxal; e) citr; f) mkn-025. 3.2 degradation of parathion methyl degradation of parathion methyl was examined in organic solvent heptane using all the examined samples of ceo2; the respective kinetic dependencies are shown in c ba d e f bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc nova biotechnologica et chimica 13-2 (2014) 155 fig. 5. experimental data for the parathion methyl degradation were fitted to the pseudo-first order kinetic equation in the form: ∞+−= qtkqqt )exp( 11 (1) generally, a variable q indicates a dimensionless fraction of the reactant with respect to its initial amount. specifically, qt represents the residual quantity of parathion methyl at time t, q1 is the fraction of parathion methyl degraded during the experiment, q∞ is the residual fraction of parathion methyl at the end of the reaction in the event that the destructive capacity of the reactive sorbent is insufficient for complete degradation, and k1 is the degradation rate constant. the model parameters obtained by non-linear regression are listed in table 3. fig. 5. kinetic dependencies for degradation of parathion methyl on various ceo2.a) carb-s; b) carb-g; c) oxal; d) citr; e) mkn-025. c) b) e) d) a) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc 156 janoš, p. et al. table 3. model parameters describing kinetics of the parathion methyl degradation on various ceo2 samples sample y∞ a) k1 (min -1) a) t1/2 r 2 carb-s 0.026 (0.065) 0.073 (0.026) 9.50 0.896 carb-g 0.458 (0.029) 0.045 (0.010) 15.4 0.958 oxal 0.645 (0.023) 0.021 (0.003) 33.0 0.985 citr 0.699 (0.020) 0.025 (0.005) 27.7 0.969 mkn-025 0.831 (0.015) 0.031 (0.008) 22.4 0.920 a) standard errors given in parentheses as can be seen from the degradation curves, the ceo2 sample prepared from carbonate precursor carb-s exhibited the superior degradation efficiency with the degree of conversion approaching 100% whereas the efficiency of the commercial nanocrystalline cerium oxide mkn-025 was rather poor, with the degree of conversion of about 20%. different efficiencies of these samples may be related to the differences in their surface areas, the presence (absence) of micropores and mainly to the differences in their crystallinity. the differences in the degradation efficiency between the samples carb-s and carb-g may be attributed to the differences in their surface area and porosity. it should be noted that the high surface area by itself does not guarantee high degradation efficiency – note a relatively low efficiency of the sample citr. during the degradation experiments, an increase in the concentration of 4-nitrophenol was measured simultaneously with the decrease in the parathion methyl concentration (fig. 5). it follows from a mass balance that an amount of 4-nitrophenol liberated is almost identical to an amount of parathion methyl disappeared. no other reaction products were identified by gc-ms. it was hypothesized that nucleophilic substitution (sn2) is the main mechanism responsible for cleavage of the p-o-aryl bond in the pesticide molecule (janos et al., 2014a). the –oh groups on the surface of the sorbent play a crucial role in the degradation process, acting as a very strong nucleophile toward p atom. analogously to hydrolytic reactions in aqueous solutions (khare et al,. 2012), the possible reaction pathway involve the nucleophilic attack to the central phosphorus atom with a subsequent liberation of 4-nitrophenol (leaving group). it is assumed that metal ions are involved in the degradation process as well. possible intermediate and transition states are depicted in fig. 6. 3.3 degradation of nerve agents based on the measurements presented above, the carb-s sample prepared from carbonate precursor was selected to examine its efficiency in degrading the extremely dangerous nerve agents vx (o-ethyl s-[2-(diisopropylamino)ethyl] methylphosphonothioate) and soman (gd, o-pinacolylmethylphosphonofluoridate). the experimental procedure was essentially the same as that described in the previous section, except that the reactions were performed in nonane. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc nova biotechnologica et chimica 13-2 (2014) 157 metal ion as lewis acid nucleophilic attack of –oh group combined effects   p x r1o l r2o h o ce ce ce     h o ce ce ce p x r1o l r2o       p x r1o l r2o h o ce ce ce   fig. 6. modes of interaction of cerium oxide with organophosphates. based on an analogy with hydrolysis of organophosphates in the presence of metal complexes and metalloenzymes: (khare et al. 2012, zhao et al. 2012, naik and shinde 2013). x=s (alternatively o), l – leaving group (4-nitrophenolate in the case of parathion methyl). as can be seen from fig. 7, cerium oxide was very effective in the degradation of these organophosphorus nerve agents: it was capable of destroying vx agent nearly completely in less than one hour, and a substantial degree of conversion was achieved within the first ten minutes. a total degree of conversion about 80% was achieved also for soman. 0 20 40 60 80 100 120 0.0 0.2 0.4 0.6 0.8 1.0 c t /c 0 time (min) vx soman fig. 7. degradation of soman and vx using cerium oxide carb-s. 3.4 degradation of triphenyl phosphate phosphorus flame retardants were introduced as an alternative to brominated flame retardants with (supposedly) less negative impacts to the environment and human bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc 158 janoš, p. et al. health (van der veen and de boer, 2012). some of them, however, exhibit toxic effects to living organisms (waaijers et al., 2013) and allergic effects to humans (camarasa and serra-baldrich, 1992). the degradation of triphenyl phosphate, as a representative of halogen-free phosphorus flame retardants, was investigated in a batch arrangement using the carb-s sample as a reactive sorbent. it was found that triphenyl phosphate was destroyed relatively quickly in organic aprotic solvent (acetonitrile) phenolic groups are gradually released by the mechanism of nucleophilic substitution to form diphenyl phosphate and subsequently phenol – fig. 8. 0 50 100 150 0.0 0.5 1.0 1.5 c t /c 0 time (min) ph tpp dpp fig. 8. degradation of triphenyl phosphate using cerium oxide carb-s. tpp – triphenyl phosphate; dpp – diphenyl phosphate; ph .phenol. 3.5phosphatase-mimetic activity of cerium oxide an ability of cerium oxide to interact with biologically relevant molecules may show promise in new applications in medicine, but it may also pose a threat of damage to important chemical bonds in biological systems. the enzyme-like ability of various ceo2 samples to hydrolyse (accelerate hydrolysis) the organophosphate compounds was examined in aqueous solutions using a standardised phosphatase test (alp-meg l 500); 4-nitrophenyl phosphate was used as the substrate. as shown in fig. 9, the commercial nanoceria mkn-025 exhibited the lowest activity again, but the differences between examined samples were not so pronounced. it is supposed that this activity belongs to unique properties of certain forms of cerium oxide, as other metal oxides, such as magnetite, maghemite or photocatalytically active tio2 gave a significantly lower response (janoš et al., 2015). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc nova biotechnologica et chimica 13-2 (2014) 159 carb-s carb-g oxal citr mkn-025 0 5 10 p ho sp ha ta se -li ke a ct iv ity ( a u ) fig. 9. phosphatase-mimetic activity of various ceo2 samples. 4. conclusions it was shown that cerium oxide exhibits a remarkable ability to interact with important phosphorus-containing compounds. the reactivity of cerium oxide depends strongly on its physicochemical characteristics, such crystallinity, surface area, surface chemistry (presence of functional groups). these characteristics may be adjusted by a proper selection of the preparation route. it was proven that relatively simple procedures, such as a precipitation/calcination route, or sol-gel process, can be used to obtain highly reactive kinds of cerium oxide. however, much more must be done to establish simple relations between a synthetic route, physicochemical characteristics and reactivity in the given system. interactions of cerium oxide (and engineered nanoparticles in general) are important not only for technical applications of these materials, but also for their (potential) ability to interact with many compounds having vital functions in living organisms. acknowledgement: financial support from the czech science foundation (grant no. p106/12/1116) is gratefully acknowledged. additional financial support was obtained from the student grant agency of the university of jan evangelista purkyně in ústí nad labem. the thermogravimetric analyses were performed in the central laboratory of the institute of chemical technology in prague. ph.d. students jiří henych, petr vomáčka, michaela slušná and martin šťastný are thanked for their assistance with the sem observations and method development. references alifanti, m., baps, b., blangenois, n., naud, j., grange, p., delmon, b.: characterization of ceo2 −zro2 mixed oxides. comparison of the citrate and sol−gel preparation methods. chem. mater., 15, 2003, 395–403. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc 160 janoš, p. et al. ansari, a. a., kaushik, a., solanki, p. r., malhotra, b. d.: sol–gel derived nanoporous cerium oxide film for application to cholesterol biosensor. electrochem. commun., 10, 2008, 1246–1249. benadda, a., djadoun, a., guessis, h., barama, a.: effect of the preparation method on the structural and catalytic properties of mnox-ceo2 manganese cerium mixed oxides, proceedings of the international conference nanomaterials: applications and properties. sumy state university, 2013, 3–5. camarasa, j. g., serra-baldrich, e.: allergic contact dermatitis from triphenyl phosphate. contact dermat., 26, 1992, 264–265. celardo, i., pedersen, j. z., traversa, e., ghibelli, l.: pharmacological potential of cerium oxide nanoparticles. nanoscale, 3, 2011, 1411–20. hrizi, f., dhaouadi, h., touati, f.: cerium carbonate hydroxide and ceria micro/nanostructures: synthesis, characterization and electrochemical properties of ceco3oh. ceram. int., 40, 2014, 25–30. janos, p., kuran, p., kormunda, m., stengl, v., grygar, t. m., dosek, m., stastny, m., ederer, j., pilarova, v., vrtoch, l.: cerium dioxide as a new reactive sorbent for fast degradation of parathion methyl and some other organophosphates. j. rare earths, 32, 2014a, 360–370. janoš, p., hladík, t., kormunda, m., ederer, j., šťastný, m.: thermal treatment of cerium oxide and its properties: adsorption ability versus degradation efficiency. adv. mater. sci. eng., 2014b, article id 706041. janoš, p., kuráň, p., pilařová, v., trögl, j., šťastný, m., pelant, o., henych, j., bakardjieva, s., životský, o., kormunda, m., mazanec, k., skoumal, m.: magnetically separable reactive sorbent based on the ceo2/γ-fe2o3 composite and its utilization for rapid degradation of the organophosphate pesticide parathion methyl and certain nerve agents. chem. eng. j., 262, 2015, 747-755. janoš, p., novák, j., broul, m.: preparation of ceria-based polishing agents by thermal decomposition of rare earth carbonates (in czech). chem. prum., 39/64, 1989, 419–425. janoš, p., petrák, m.: preparation of ceria-based polishing powders from carbonates. j. mater. sci. 26, 1991, 4062–4066. johnson, m.: cerium dioxide crystallite sizes by temperature-programmed reduction. j. catal., 103, 1987, 502–505. khare, s. d., kipnis, y., greisen, p., takeuchi, r., ashani, y., goldsmith, m., song, y., gallaher, j. l., silman, i., leader, h., sussman, j. l., stoddard, b. l., tawfik, d. s., baker, d.: computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis. nat. chem. biol., 8, 2012, 294–300. kuchma, m. h., komanski, c. b., colon, j., teblum, a., masunov, a. e., alvarado, b., babu, s., seal, s., summy, j., baker, c. h.: phosphate ester hydrolysis of biologically relevant molecules by cerium oxide nanoparticles. nanomedicine, 6, 2010, 738–44. liu, k., zhong, m.: synthesis of monodispersed nanosized ceo2 by hydrolysis of the cerium complex precursor. j. rare earths, 28, 2010, 680–683. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc nova biotechnologica et chimica 13-2 (2014) 161 markmann, j., tschöpe, a., birringer, r.: low temperature processing of dense nanocrystalline yttrium-doped cerium oxide ceramics. acta mater., 50, 2002, 1433–1440. naik, v., shinde, c. p.: degradation of organophosphorus compounds by hydrolysis in presence of meta cations. sci. technol. j. aisect univ., 2013. van der veen, i., de boer, j.: phosphorus flame retardants: properties, production, environmental occurrence, toxicity and analysis. chemosphere, 88, 2012, 1119–1153. waaijers, s. l., hartmann, j., soeter, a. m., helmus, r., kools, s. a. e., de voogt, p., admiraal, w., parsons, j. r., kraak, m. h. s.: toxicity of new generation flame retardants to daphnia magna. sci. total environ. 463-464, 2013, 1042–1048. wei, h., wang, e.: nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. chem. soc. rev., 42, 2013, 6060–6093. xu, c., qu, x.: recent progress of rare earth cerium oxide nanoparticles applied in biology. sci. sin. chim., 44, 2014, 506. zhai, y., zhang, s., pang, h.: preparation, characterization and photocatalytic activity of ceo2 nanocrystalline using ammonium bicarbonate as precipitant. mater. lett., 61, 2007, 1863–1866. zhao, m., zhao, c., jiang, x.-q., ji, l.-n., mao, z.-w.: rapid hydrolysis of phosphate ester promoted by ce(iv) conjugating with a β-cyclodextrin monomer and dimer. dalton trans., 41, 2012, 4469–76. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:32 utc microsoft word augustin nbc 2015.doc 96 nova biotechnologica et chimica 14-1 (2015) doi 10.1515/nbec-2015-0019 © university of ss. cyril and methodius in trnava magnetostructural relationships for fe(iii) spin crossover complexes peter augustín1, roman boča2 1institute of inorganic chemistry, faculty of chemical and food technology, slovak university of technology, sk-812 37 bratislava, slovakia 2department of chemistry, university of ss. cyril and methodius in trnava, sk-917 01 trnava, slovakia (roman.boca@stuba.sk) abstract: structural data for fifteen complexes of fe(iii) of a general formula [fel5x], with pentadentate schiff-base ligands l5 and unidentate coligands x−, were subjected to a statistical analysis. the multivariate methods such as pearson correlation, cluster analysis and principal component analysis split the data into two clusters depending upon the low-spin and/or high-spin state of the complex at the temperature of the x-ray experiment. some of these complexes exhibit a thermally induced spin crossover. the numerical analysis of the magnetic susceptibility and magnetization data for an enlarged set of fe(iii) spin crossover systems yields the enthalpy δh and entropy δs of the transition along with the transition temperature t1/2 and the solid state cooperativeness. the thermodynamic data show a mutual relationship manifesting itself by linear δs vs δh and t1/2 vs δh correlations. key words: spin crossover, fe(iii) complexes, structural and magnetic data, magnetostructural relationships 1. introduction a number of studies confirm a presence of thermally induced spin crossover between the low-spin (ls, s = 1/2) and high-spin (hs, s = 5/2) states in fe(iii) complexes (e.g. halcrow et al., 2013). this process can be viewed as a unimolecular, entropy driven reaction for which δh > 0 and δs > 0 is characteristic. the minimum value of δs is represented by a pure electronic spin contribution ln(6 / 2)s rδ = = 9.1 j k-1 mol-1 that is enriched by the vibrational contribution. the transition temperature, when the mole fraction obeys xls = xhs = 0.5, is given by the ratio t1/2 = δh/δs and in order to get a sufficiently high value, say t1/2 > 100 k, the lowest estimate for the enthalpy of the spin transition is δh > 0.9 kj mol-1. these thermodynamic data can be measured by calorimetry (sorai, 2002) but they are available also from an analysis of magnetic data such as temperature dependence of the magnetic susceptibility (boča et al., 2003; pavlik et al., 2013). it was reported recently that the spin transition can be tuned by varying coligands in the basic skeleton of the complexes of [feiiil5x] type; here l5 stands for a pentadentate schiff base whereas x− is a monodentate pseudohalido coligand (krüger et al., 2013; krüger et al., 2015; masárová et al., 2015; nemec et al., 2011; šalitroš et al., 2009). although chemists try to rationalize δh by changing the ligand field strength, the aspects influencing the δs cofactor are unclear so far. to our hypothesis some magnetostructural relationships could bring an answer of how to get the spin crossover with a desired transition temperature. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:18 utc nova biotechnologica et chimica 14-1 (2015) 97 2. material and methods the pentadentate ligands h2l 5 were prepared by a schiff condensation between the substituted salicylaldehyde (y-sal) and the asymmetric triamine pet = 1,6-diamino-4azahexane as described elsewhere (krüger et al., 2013; nemec et al., 2011). their fe(iii) complexes [fe(y-salpet)x] were prepared by published recipes (see fig. 1) where the fe(iii) salt was combined with h2l 5 and pseudohalide x− ligands. the elemental analysis confirmed the composition of the complexes. n oh n oh nh y y o oh y n h nh2 nh22 + + fe(iii) + x− fig. 1. synthetic route for preparing fe(iii) complexes of the [fel5x] type; y = cl and br, x− = cl−, n3−, nco−, ncs− and ncse−. single crystals were subjected to the x-ray structure analysis and the results can be retrieved from the cambridge structural database. the measured interatomic distances within the {fen2n’o2x} chromophore are listed in table 1. table 1. structural parameters for feiii complexes under study.a compound class fe-nam fe-nim fe-o fe-x σ α 1 [fe(lbr)(cl)]·0.5h2o h 2.188 2.088 1.959 2.352 62.3 63.1 2 [fe(lbr)(ncs)] h 2.207 2.099 1.927 2.111 56.3 67.7 3 [fe(lbr)(n3)]·meoh h, sc 2.183 2.113 1.942 2.048 72.3 35.0 4 [fe(lbr)(ncse)] l, sc 2.013 1.952 1.884 1.944 27.8 85.9 5 [fe(lcl)(cl)]·0.25h2o h 2.199 2.095 1.958 2.361 66.9 62.2 6 [fe(lcl)(nco)] h 2.205 2.093 1.954 1.996 55.4 69.0 7 [fe(lcl)(ncs)] l, sc 2.000 1.932 1.879 1.944 27.1 84.5 8 [fe(lcl)(ncse)] l, sc 2.001 1.932 1.881 1.952 26.9 85.5 9 [fe(lcl)(cn)]·h2o l 2.012 1.934 1.903 1.959 19.3 65.9 10 [fe(l)(cn)]·ch3oh l 2.000 1.922 1.880 1.962 24.6 76.0 11 [fe(3-but,5-me-l)(ncs)] h 2.191 2.096 1.916 2.100 56.0 75.6 12 [fe(lm)(nco)]·meoh h 2.218 2.078 1.946 2.081 54.0 73.3 13 [fe(lm)(n3)] h 2.211 2.085 1.936 2.085 57.4 58.1 14 [fe(lm)(cn)]·meoh l 2.028 1.935 1.904 1.971 26.0 69.1 15 [fe(le)(cn)]·h2o l 2.010 1.944 1.904 1.975 20.8 66.4 a distances in å, angles σ and α in deg. class – classification according to the temperature of the x-ray experiment in comparison with spin state: h – high-spin, l – low spin, sc – spin crossover. octahedral distortion angle σ is calculated from twelve cis angles within the chromophore. angle α – between planes of aromatic rings. abbreviations: l = salpet, lcl = 5-cl-salpet, lbr = 5-br-salpet, lm = 3-meo-salpet, le = 3-eto-salpet. references: krüger et al., 2013; krüger et al., 2015; masárová et al., 2015; nemec et al., 2011; šalitroš et al., 2009. the squid magnetometer (quantum design, mpms-xl7) was used for measurements of the magnetic data (temperature dependence of the magnetic bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:18 utc 98 augustín, p. and boča, r. susceptibility, and field dependence of the magnetization). the magnetic data for feiii complexes referring either to the low-spin (ls) or the high-spin (hs) state was analyzed in terms of the curie-weiss law; to this end the g-factor, weiss constant θ, and the van vleck term α were obtained. some systems exhibit a thermally induced spin crossover and those data was analyzed by using ising-like model with vibrations (equivalent to the thermodynamic regular solution model) giving rise the enthalpy δh and entropy δs of the spin transition along with the transition temperature t1/2 and the solid-state cooperativeness γ (boča et al., 2003; pavlik et al., 2013). these data is listed in table 2 along with some literature sources. table 2. thermodynamic parameters for spin crossover complexes under study.a compound type t1/2/k δh/kj mol-1 δs/j k-1 mol-1 (γ/kb)/k 1 [fe(lcl)(ncs)] sc 280 5.77 20.6 180 2 [fe(lcl)(ncse)] sc 293 6.54 22.3 197 3 [fe(lbr)(n3)]·ch3oh sc 142 1.64 11.5 76 4 [fe(lbr)(ncse)] sc 326 6.03 18.5 215 5 [fe(le)(ncs)] sc 84, 82 2.07 (5.0) 90 6 [fe(5cl-saldptn)py]bph4 sc* 78 0.44 5.61 63 7 [fe(3m-saldptn)py]bph4 sc 273 4.54 16.6 90 8 [fe(saldptn)py]bph4 sc 310 5.42 17.5 150 9 [fe(napet)(n3)]⋅meoh sc 122, 117 1.53 11.2 99 10 [fe(napet)(ncs)]⋅mecn sc 151 1.92 12.5 87 11 [fe(napet)(ncs)] sc 180 0.90 (5.0) 135 12 [fe(napet)(nco)] sc 155 2.54 16.3 102 13 [fe(napet)(ncse)]⋅mecn sc 170 2.29 13.3 99 a sc – spin crossover between spins 1/2 5/2, sc – with hysteresis. sc* spin crossover between spins 3/2 5/2. abbr. for ligands: saldptn = n,n'-bis(2-hydroxybenzyliden)-1,7-diamino-4-azaheptane, napet = n,n'-bis(2-hydroxynapthylidene)-1,6-diamino-4-azahexane. references: boča et al., 2000; krüger et al., 2013; krüger et al., 2015; masárová et al., 2015; nemec et al., 2011; šalitroš et al., 2009. 3. results and discussion the structural data was analyzed by using the statistical tools of the multivariate methods (statgraphics centurion xv, 2006): the cluster analysis (ca), the principal component analysis (pca), and the pearson correlation (pc). the cluster analysis (using ward’s method, distance according to the square euclidean norm) shows that the data is split into two principal clusters according to their similarity (fig. 2). centroids of the two clusters are listed in table 3. it can be seen that all distances in the cluster 1 are higher than in cluster 2 which allows concluding that the cluster 1 involves high-spin complexes (complexes which are high-spin at the temperature of the x-ray experiment). then the cluster 2 contains lowspin complexes. table 3. centroids of the parameters according to the cluster analysis. cluster fe-nam fe-nim fe-o fe-x σ α 1 2.200 2.093 1.942 2.142 60.07 63.0 2 2.009 1.936 1.891 1.958 24.64 76.2 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:18 utc nova biotechnologica et chimica 14-1 (2015) 99 0 20 40 60 80 100 d is ta nc e 1 2 3 45 6 7 8 91011 12 13 1415 0 10 20 30 40 50 d is ta nc e fe n am fe x fe n im fe o s um al ph a fig. 2. dendograms of the cluster analysis. left – for complexes; right – for distances and angles. data according to table 1. the pca method performs a linear transformation of the original parameters into principal components that bear the maximum information about the variability of data. here the first component involves 79 % and the second one 11 % of the variability, in total 90 %. the diagram (fig. 3) shows which variables correlate, anticorrelate, or do not correlate. it follows that the interatomic distances fe-nam, fe-nim, fe-o mutually correlate and also they correlate with the parameter σ. on the contrary, α-parameter does not correlate with the others. fex feo alpha -3 -2 -1 0 1 2 3 component 1 -3 -2 -1 0 1 2 3 c om po ne nt 2 fenam fenim sum component 1 c om po ne nt 2 cluster h l 4 10 7 8 9 15 14 11 12 2 6 13 1 5 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 3 fig. 3. results of pca. left – a biplot with ray diagram, right – individual complexes classified into two clusters. pca also brings information about the distribution of the original objects and the corresponding graph is also display in fig. 3. it unambiguously identifies that the cluster 1 (squares) contains high-spin complexes whereas the cluster 2 (circles) the low-spin ones (at the temperature of the x-ray experiment). finally, the pc analysis offers the pair correlation coefficients as listed in table 4. now it is seen that the greater fe-nam, the greater fe-nim and fe-o. also the octahedral distortion parameter σ increases with distance of the amino nitrogen fe-nam and also imino nitrogen fe-nim. the angle of the aromatic rings α does not display any significant correlation with the other structural parameters. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:18 utc 100 augustín, p. and boča, r. table 4. pair correlation (pearson) coefficients for structural parameters. fe-nam fe-x fe-nim fe-o σ α fe-nam 0.677 0.984 0.900 0.942 -0.515 fe-x 0.677 0.683 0.757 0.732 -0.372 fe-nim 0.984 0.683 0.885 0.972 -0.569 fe-o 0.900 0.757 0.885 0.869 -0.639 σ 0.942 0.732 0.972 0.869 -0.613 α -0.515 -0.372 -0.569 -0.639 -0.613 the recorded temperature dependence of the effective magnetic moment for a number of complexes is displayed in fig. 4 for a fixed pentadentate ligand l5 and varied coligand x−. it can be seen that with increasing crystal field strength of the coligand x− (according to the well-known spectrochemical series) the spin states of complexes move from the high-spin behavior through the spin crossover behavior to the low-spin state. t/k 0 50 100 150 200 250 300 350 400 μ e ff/ μ b 0 1 2 3 4 5 6 [fe(lcl)(cl)] [fe(lcl)(nco)] [fe(lcl)(ncs)] [fe(lcl)(ncse)] [fe(lcl)(cn)] t/k 0 50 100 150 200 250 300 350 400 μ e ff/ μ b 0 1 2 3 4 5 6 [fe(lbr)(ncs)] [fe(lbr)(n3)] [fe(lbr)(ncse)] fig. 4. comparison of the spin crossover in [feiiil5x] type complexes for a fixed schiff-base ligand and varied coligand. for references see table 2. t/k 0 50 100 150 200 250 300 350 400 μ e ff/ μ b 0 1 2 3 4 5 6 [fe(5br-salpet)(ncs)] [fe(3meo-salpet)(ncs)] [fe(5cl-salpet)(ncs)] t/k 0 50 100 150 200 250 300 350 400 μ e ff/ μ b 0 1 2 3 4 5 6 γ-[fe(1anapet)(ncs)] α-[fe(1anapet)(ncs)] [fe(2anapet)(ncs)] fig. 5. comparison of the spin crossover in [feiiil5x] type complexes for a varied schiff-base ligand and fixed coligand. for references see table 2. the spin state of the complex can be influenced also by the schiff-base ligand with a fixed coligand as shown in fig. 5 for x− = ncs−. in order to find relationships among the thermodynamic parameters governing the spin crossover the multivariate statistical methods were applied: ca, pca and pc. for bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:18 utc nova biotechnologica et chimica 14-1 (2015) 101 this purpose the data listed in table 2 were employed. the results of the cluster analysis are presented in fig. 6. it can be seen that they span two clusters with centroids characterized in table 5. the cluster 1 involves complexes (no 1, 2, 4, 7, and 8) for which nearand above-room temperature spin crossover has been observed. dendrogram ward's method,squared euclidean 0 3 6 9 12 15 d is ta nc e 1 2 34 5 67 8 910 1112 13 dendrogram ward's method,squared euclidean 0 0.5 1 1.5 2 2.5 3 d is ta nc e t h s j fig. 6. dendograms of the cluster analysis. left – for complexes, right – for thermodynamic parameters. data according to table 2. table 5. centroids of the parameters according to the cluster analysis. cluster t = t1/2 h = δh s = δs j = γ 1 296.4 5.66 19.1 166.4 2 134.9 1.67 10.1 93.9 the pca analysis yields the diagrams presented in fig. 7. it can be seen that the transition temperature t1/2 (abbr. t) correlates with the enthalpy of the transition δh (abbr. h) whereas the correlation with the remaining parameters δs (abbr. s) and γ (abbr. j) is much weaker. the diagram on the right shows the objects classified into individual clusters. h j -3 -2 -1 0 1 2 3 component 1 -2 -1 0 1 2 c om po ne nt 2 t s component 1 c om po ne nt 2 cluster 1 2 7 8 1 4 2 6 5 11 9 3 10 13 -3 -2 -1 0 1 2 3 -2 -1 0 1 2 12 fig. 7. results of pca. left – a biplot with ray diagram, right – individual objects classified to the clusters (numbering according to table 2). the pearson correlation yields the pair correlation coefficients listed in table 6. it is confirmed numerically that the highest correlation coefficient refers to the tc – δh pair. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:18 utc 102 augustín, p. and boča, r. table 6. pair correlation (pearson) coefficients for thermodynamic parameters. t = t1/2 h = δh s = δs j = γ t 0.922 0.826 0.824 h 0.922 0.890 0.826 s 0.826 0.890 0.675 j 0.824 0.826 0.675 the values of δs were plotted versus the δh and the corresponding graph is presented in fig. 8 along with the linear regression curve and confidence intervals (95 %). analogously, the values of t1/2 correlate with δh. these correlations were replotted on the right side of fig. 8 when points with a problematic value of δs < 9.1 j k-1 mol-1 (complexes 5 and 11) were excluded form the correlation. the correlation is much improved. δh /kj mol-1 0 1 2 3 4 5 6 7 δ s /j k -1 m ol -1 0 5 10 15 20 25 δh /kj mol-1 0 1 2 3 4 5 6 7 t 1/ 2/ k 0 50 100 150 200 250 300 350 400 δh /kj mol-1 0 1 2 3 4 5 6 7 δ s /j k -1 m ol -1 0 5 10 15 20 25 δh /kj mol-1 0 1 2 3 4 5 6 7 t 1/ 2/ k 0 50 100 150 200 250 300 350 400 fig. 8. linear correlations t1/2 vs δh and δs vs δh. right – reduced data omitting two problematic points. 4. conclusions it has been demonstrated that the structural parameters within the {fen2n’o2x} chromophore of the [feiiil5x] type complexes mutually correlate at a significant level. the complexes are classified into two clusters depending upon the low-spin and/or high-spin state of the complex at the temperature of the x-ray experiment. the thermodynamic data that characterize the spin crossover in the given class of fe(iii) complexes again show a mutual correlation confirmed by the multivariate methods. again two principal clusters are visible, one for the systems exhibiting the spin crossover nearand above-room temperature, and the second for those showing the spin crossover below the room temperature. the enthalpy of the spin transition can be tuned by the coligand x− and consequently also the transition temperature is a function of δh. to this end, the coligands spanning the right side of the spectroscopic series bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:18 utc nova biotechnologica et chimica 14-1 (2015) 103 cause an increase in the enthalpy of the spin transition, and consequently a rising of the transition temperature towards and above the room temperature. acknowledgements: slovak grant agencies (vega 1/0233/12, vega 1/0522/14 and apvv14-0078) are acknowledged for the financial support. references boča, r., linert, w.: is there a need for new models of the spin crossover? monats. chemie, 134, 2003, 199-216. boča, r., fukuda, y., gembický, m., herchel, r., jaroščiak, r., linert, w., renz, f., yuzurihara j.: spin crossover in mononuclear and binuclear iron(iii) complexes with pentadentate schiff−base ligands. chem. phys. lett., 325, 2000, 411-419. cambridge structural database, cambridge crystallographic data centre, cambridge, cb2 1ez, united kingdom. halcrow, m. a. (ed.): spin-crossover materials: properties and applications. wiley, new york, 2013. krüger, c., augustín, p., nemec, i., trávníček, z., oshio, h., boča, r., renz, f.: spin crossover in iron(iii) complexes with pentadentate schiff-base ligands and pseudhalido coligands. eur. j. inorg. chem., 2013, 902-915. krüger, c., augustín, p., dlháň, ľ., pavlik, j., moncoľ, j., nemec, i., boča, r., renz, f.: iron(iii) complexes with pentadentate schiff-base ligands: influence of crystal packing change and pseudohalido coligand variations on spin crossover. polyhedron, 87, 2015, 194-201. masárová, p., zoufalý, j., moncoľ, j., nemec, i., pavlik, j., gembický, m., trávníček, z., boča, r., šalitroš, i.: spin crossover and high spin electroneutral mononuclear iron(iii) schiff base complexes involving terminal pseudohalido ligands. new. j. chem., 39, 2015, 508-519. nemec, i., herchel, r., boča, r., trávníček, z., svoboda, i., fuess, h., linert, w.: tuning of spin crossover behaviour in iron(iii) complexes involving pentadentate schiff bases and pseudohalides. dalton trans., 40, 2011, 10090-10099. pavlik, j., boča, r.: established static models of the spin crossover. eur. j. inorg. chem., 2013, 697-709. sorai, m.: calorimetric investigations of phase transitions in which electrons are directly involved. j. chem. thermodyn., 34, 2002, 1207-1253. statgraphics centurion xv. statpoint inc., 2006. šalitroš, i., boča, r., dlháň, ľ., gembický, m., kožíšek, j., linares, j., moncoľ, j., nemec, i., perašínová, l., renz, f., svoboda, i., fuess, h.: unconventional spin crossover in dinuclear and trinuclear iron(iii) complexes with cyanido and metallacyanido bridges. eur. j. inorg. chem., 2009, 3141-3154. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 17:18 utc nova biotechnol chim (2017) 16(2): 147-151 doi: 10.1515/nbec-2017-0020  corresponding author: renata.gasparova@ucm.sk nova biotechnologica et chimica nmr spectroscopic properties of furo[2',3':4,5]pyrrolo[1,2-d][1,2,4]triazine derivatives ivana zemanová and renata gašparová department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic article info article history: received: 12 th june 2017 accepted: 4 th july 2017 keywords: chemical shift furo[3,2-b][pyrrole nmr spectroscopy abstract the 1 h and 13 c nmr spectroscopic properties of a series of furo[2',3':4,5]pyrrolo[1,2-d][1,2,4]triazin-8(7h)-ones and -thiones were investigated. the influence of various electron donating as well as electron withdrawing substituents at c-5 or n-7 on 1 h nmr chemical shifts as well as 13 c chemical shifts at c8 were observed. the 5-chloromethyl group had a little influence on the chemical shift of h-7 proton and the 8-thione group causes deshielding of h-7 as well as h-5 protons in comparison with the c-8 carbonyl group.  university of ss. cyril and methodius in trnava introduction furo[3,2-b]pyrroles are isosteres of the indole ring system in which the benzene ring is replaced by the furan ring. efficient synthetic routes to these heterocycles are of great interest (krutošíková et al. 1992; beccalli et al. 2008; zhao et al. 2014) as the furo[3,2-b]pyrrole core has been found in compounds with diverse biological activities (sparey et al. 2008; fehér et al. 2010) or they are used as the fluorescent dyes (umezawa et al. 2008). heterocyclic compounds containing five and six-membered nitrogen heterocyclic rings have also attracted the attention due to the fact that they exhibit many biological interactions (astakhina et al. 2016). in addition, 1,2,4-triazin-6-one is structural system found in numerous natural and synthetic biologically active compounds with a wide range of biological activity including antiinflammatory (george et al. 2015) or anticonvulsant (el-gendy et al. 2008). we have recently reported the synthesis of furo[2',3':4,5]pyrrolo[1,2-d][1,2,4]triazin-8(7h)ones with interesting antibacterial activity (zemanová et al. 2017), but more complex spectroscopic studies were realised only on benzothieno[3,2-d]-1,2,3-triazines (lauria et al. 2014) or 7-aryl-7h-pyrazolo[3,4-d]-[1,2,3]triazin4-ols (khutova et al. 2012). therefore we report here on the nmr spectroscopic properties of the full series. results and discussion the title compounds 1a-1c (fig. 1) are well accessible from methyl 4h-furo[3,2-b]pyrrole-5carboxylate via its hydrazinolysis and subsequent cyclization of hydrazides with orthoesters (triethyl orthoformate, triethyl orthochloroacetate or triethyl orthoacetate) in dimethylformamide. resulting furo[2',3':4,5]pyrrolo[1,2-d][1,2,4]triazin-8(7h)ones 1 were obtained in 69-75%. the carbonyl group at c-8 of triazine ring of 1a can be easily thionated by reaction with phosphorus pentasulfide in pyridine to give thione 2a in 85% yield alkylation or acylation of triazines 1a and 2a with alkylhalides (ch3i, n-c6h9br, clch2co2me) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:20 utc nova biotechnol chim (2017) 16(2): 147-151 148 fig. 1. structure of furo[2',3':4,5]pyrrolo[1,2-d][1,2,4]triazin-8(7h)-ones 1 and thiones 2. or acetic anhydride in dimethylformamide in the presence of sodium hydride provided 7-substituted triazinones 1d-1h or thiones 2b, 2c (fig.1) in 45-72 % yields (zemanová et al. 2017). 1 h nmr spectroscopy the influence of various substituents (c5)-r and (n7)-r 1 on 1 h nmr chemical shifts of the protons in triazinones 1 and triazinethiones 2 were investigated (table 1).the influence of c-5 substituent (h, ch2cl, ch3) on chemical shifts of the protons of derivatives 1a-1c showed the slight increasing of the chemical shift value of h-7 proton in case of electron withdrawing group (ch2cl) at c-5 (fig. 2). table 1. 1 h nmr chemical shifts of furo[2',3':4,5]pyrrolo[1,2-d][1,2,4]triazin-8(7h)-ones 1a-1h and thiones 2a-2c in dmso-d6. x r r1 1a o h h 1b o ch2cl h 1c o ch3 h 1d o h ch3 1e o h n-c4h9 1f o h ch2ph 1g o h ch2co2ch3 1h o h coch3 2a s h h 2b s h ch3 2c s h ch2co2ch3 chemical shift (ppm) h-2 h-3 h-5 h-7 h-9 other 1a 8.00 7.11 8.75 11.81 7.14 1b 7.97 6.67 12.09 6.88 5:09 (ch2) 1c 7.99 7.15 11.65 7.10 2.60 (ch3) 1d 7.99 7.09 8.81 7.14 3.56 (ch3) 1e 7.99 7.09 8.82 7.13 3.99 (t,ch2), 1.73 (sextet, ch2), 1.37 (sextet, ch2), 0.93 (t, ch3) 1f 8.01 7.09 8.83 7.19 5.17 (ch2) 1g 8.01 7.09 8.83 7.20 4.77 (ch2), 3.67 (ch3) 1h 8.09 7.11 8.83 7.39 2.59 (ch2) 2a 8.09 7.15 9.16 13.29 7.29 2b 8.31 7.34 10.98 7:76 4.17 (ch3) 2c 8.11 7.15 9.24 7.36 5.32 (ch2), 3.67 (ch3) fig. 2. the influence of substituent on c-5 of 1a-1c on the chemical shifts of protons in 1 h nmr. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:20 utc nova biotechnol chim (2017) 16(2): 147-151 149 as it is shown in table 1, the presence either electron withdraving either electron donating substituents at n-7 has no significant influence on the chemical shifts of protons in compounds 1d-1h in comparison with n-7 unsubstituted derivative 1a. the values of h-2 shifts are in 7.99-8.09 ppm region, h-3 resonate in 7.09-7.11 ppm. in case of h-5 the range is only 8.81-8.83 ppm. the chemical shifts of h-9 are in 7.14 7.39 ppm range (fig. 3).when the carbonyl group at c-8 was replaced by thione, the chemical shifts of h-2 and h-3 were not significantly influenced, but the shifts of h-5 significantly deshielded to 9.16 10.98 ppm. deshielding of h-7 protons is not so remarkable, except 2b with value 7.76 ppm (fig. 4). 13 c nmr spectroscopy there was very little variation in the c-8 (c=o) signal of triazinones 1a-1h (table 1). the range was only 153.7-154.6 ppm. c-8a carbons resonate at 123.6-125.7 ppm and c-5 carbons resonate at 127.7-35.7 ppm, respectively. when the carbonyl group is replaced by thione, the value of c-8 signal of 2a-2c has deshielded and appear at 173.1-162.3 ppm region, signals of c-8a appear at 130.5-130.9 ppm and c-5 at 131.6-144.8 ppm region, respectively (table 2). fig. 3.the influence of substituent on n-7 of 1a, 1d-1h on the chemical shifts of protons in 1 h nmr. fig. 4. the influence of c-8 thione group of 2a-2c on the chemical shifts of protons in 1 h nmr in comparison with c-8 carbonyl compounds 1a, 1d and 1g. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:20 utc nova biotechnol chim (2017) 16(2): 147-151 150 table 2. 13 c nmr chemical shifts of furo[2',3':4,5]pyrrolo[1,2-d][1,2,4]triazin-8(7h)-ones 1a-1h and thiones 2a-2c in dmso-d6. chemical shift (ppm) c-2 c-3 c-3a c-5 c-8 c-8a c-9 c-9a 1a 149.5 100.0 123.5 127.7 155.2 124.8 92.4 148.4 1b 1c 149.8 101.7 123.9 135.7 155.3 125.6 92.9 148.9 1d 149.9 100.6 123.8 127.8 154.6 124.9 93.0 149.2 1e 150.0 100.6 123.8 128.0 154.3 124.9 93.3 149.3 1f 149.7 100.1 123.5 128.3 153.9 124.2 93.2 148.7 1g 149.7 99.8 123.5 127.5 153.7 123.6 93.2 148.3 1h 151.6 100.7 124.3 128.6 154.4 125.7 97.6 149.2 2a 150.5 100.2 123.9 131.7 173.1 130.5 95.3 149.4 2b 157.7 106.1 125.9 144.8 162.3 130.9 99.7 156.5 2c 150.9 100.4 127.9 131.6 167.4 130.6 96.8 150.9 the 13 c nmr spectrum was unmeasurable because of the low solubility of 1b. conclusions the chemical shifts in 1 h and 13 c nmr spectra of series of furo[2',3':4,5]pyrrolo[1,2d][1,2,4]triazin-8(7h)-ones were investigated. among the c-5 substituted compounds the little influence of electron withdraving chloromethyl group on the chemical shift of h-7 proton was observed, while the substitution on n-7 has no significant influence on the chemical shifts of protons. thione group at c-8 causes deshielding of h-7 as well as h-5 protons in comparison with the c-8 carbonyl group. 13 c nmr spectra of 1a-1h showed only little variation of signals, the significant influence of thione group of 2a-2c on c-8, c-8a and c-5 chemical shifts deshielding in comparison with c-8 carbonyl was observed. acknowledgement this work was supported by the slovak research agency under the contract no. vega 1/0534/16. references astakhina v, voievudskyi m, kharchenko o, novikov v, komarovska-porohnyavets e, petukhova o (2016) synthesis and biological activity of novel ethyl esthers of 4-r-6,8-dimethyl-1-oxo-1,2-dihydropyrrolo[1,2d]triazine-7-carboxylic acids. j. heterocycl. chem. 53: 421-428. beccalli em, broggini g, martinelli m, sottocornola s (2008) microwave-assisted intramolecular cyclistion of electron-rich heterocycle derivatives by a palladiumcatalyzed coupling reaction. synthesis 2008: 136-140. el-gendy aa, said mm, ghareb n, mostafa ym, el-ashry esh (2008) synthesis and biological activity of functionalized indole -2-carboxylates, triazinoand pyridazino-indoles. arch. pharm. 341: 294-300. george dm, breinlinger ec, friedman m, zhang y, wang j, argiriadi m, bansal-pakala p, barth m, duignan db, honore p, lang q, mittelstadt s, potin d, rundell l, edmunds jj (2015) discovery of selective and orally bioavailable protein kinase cθ (pkcθ) inhibitors from a fragment hit. j. med. chem. 58: 222-236. lauria a, alfio a, bonsignore r, gentile c, martorana a, gennaro g, barone g, terenzi a, almerico am (2014) new benzothieno[3,2-d]-1,2,3-triazines with antiproliferative activity: synthesis, spectroscopic studies, and biological activity. bioorg. med. chem. lett. 24: 3291–3297. khutova bm, klyuchko sv, gurenko ao, vasilenko an, balya ag, rusanov eb, brovarets vs (2012) conversions of 7-aryl-7h-pyrazolo[3,4-d][1,2,3]triazin-4-ols by the action of phosphorus pentoxide, pentasulfide, and oxychloride. chem. heterocycl. comp. 48: 1251-1262. krutošíková a, dandárová m, chylová j, vegh d (1992) condensed o-, n-heterocycles by the transformation of azidoacrylates. monatsh. chem. 123: 807-815. fehér d, barlow r, mcatee j, hemscheidt tk (2010) highly brominated antimicrobial metabolites from a marine pseudoalteromonas sp. j. nat. prod. 73: 1963–1966. sparey t, abeywickrema p, almond s, brandon n, byrne n, campbell a, hutson ph, jacobson m, jones b, munshi s, pascarella d, pike a, prasad g s, sachs n, sakatis m, sardana v, venkatraman s, young mb (2008) the discovery of fused pyrrole carboxylic acid as novel, potent d-aminoacid oxidase (dao) inhibitors. bioorg. med. chem. lett. 18: 3386-3391. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:20 utc nova biotechnol chim (2017) 16(2): 147-151 151 umezawa k, nakamura y, makino h, citterio d, suzuki k (2008) fluorescent dyes in the visible-near-infrar region. j. am. chem. soc. 130: 1550-1551. zemanová i, gašparová r, kraic f, kružlicová d, maliar t, boháč a, addová g (2017) synthesis of furo[2',3':4,5]pyrrolo[1,2-d][1,2,4]triazine derivatives and their antibacterial activity. arkivoc part iv: 184-193. zhao h, koenig sg, dankwardt jw, singh sp (2014) practical nonazide synthesis of d-amino acid oxidase inhibitor via sequential erlenmeyer-plochl reaction and ligand-free copper (i) amination protocol. org. process res. dev. 18: 198-204. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:20 utc nova biotechnol chim (2017) 16(2): 99-104 doi: 10.1515/nbec-2017-0014  corresponding author: bardacova.monika@gmail.com  nova biotechnologica et chimica variable dynamics of cadmium uptake and allocation in four soybean cultivars monika bardáčová1,, yevheniia konotop2, zuzana gregorová3, miroslav horník1, jana moravčíková3, ján kraic4 and ildikó matušíková1 1 department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic 2 department of plant biology, educational and scientific center “institute of biology and medicine”, taras shevchenko national university of kyiv, volodymyrska 64, kyiv, 016 01, ukraine 3 institute of plant genetics and biotechnology, centre of biology and biodiversity slovak academy of sciences, akademická 2, p.o. box 39a, nitra, sk-950 07, slovak republic 4 department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic article info article history: received: 12th october 2017 accepted: 16th december 2017 keywords: cadmium distribution in tissue glycine max metal intake radiotracers abstract cadmium is a serious environmental pollutant and its uptake by plant represents a serious health risk. uptake, accumulation as well as sensitivity of soybean plants to metals have been shown to vary with genotype, while the dynamics of this uptake has rarely been studied. here we studied the uptake and accumulation of cd2+ ions in different parts of soybean plants of four cultivars moravians, gallec, kent and cardiff. the plants at early developmental stage were immersed in hoagland nutrient solution in the presence or absence of 50 mg.l-1 and the isotope of 109cd2+ to monitor its accumulation continuously at 24 h intervals for 10 days. our results showed that the uptake rate varied among the cultivars, being the highest in roots of the cv. moravians and the lowest in the cv. gallec. we also observed a non-even distribution of radioactivity within the entire plants of individual cultivars. the most of cd2+ isotope was translocated into primary leaves and leaves in the cvs. kent and moravians; on the contrary, relatively less in the cvs. cardiff and gallec. the results were fitted with genetic potential, growth as well as defense parameters such as proline accumulation. combining uptake dynamics and biochemical data are indicative for different tolerance strategies of soybeans.  university of ss. cyril and methodius in trnav introduction contamination of soils represents a serious concern of sustainable environment but also food production. heavy metals, released to the environment from natural sources (e.g. volcanic activity, weathering of rocks) but in great manner also due to various anthropogenic activities (mining, industry etc.), endanger plants but also animals including humans. therefore, metal contamination of farmland has widely been discussed due to its potential risk for food safety (zhao et al. 2017). cadmium toxicity represents one of the most severely discussed safety issue in many european countries. for example in slovakia, an average bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:00 utc nova biotechnol chim (2017) 16(2): 99-104 100 concentration of 1.24 mg cd per kg of soil was reported (granec and šurina 1999; six and smolders 2014) comparing to 0.4 mg cd.kg-1 worldwide. the exposed organisms are primarily plants, which have to face metal-induced membrane disruption and electrolyte leaking, disruption of water balance, oxidative stress, inhibition of enzymes and others, leading to impaired plant processes and finally reduced yields. the workable strategies to address cd toxicity in plants include selection of genotypes that either demonstrate low uptake or are hyperaccumulators (jha and bohra 2016). for several crop species including rice, wheat and soybean have already been identified genetic markers that allow for selection of genotypes with low genetic potential to accumulate cadmium (jegadeesan et al. 2010). in addition to many transporters and channels on the plasma membrane (van kerkhove et al. 2010), the metal uptake largely depends on the composition and properties of cell walls since they affect metal binding (immobilizing) and filtering capacity, restricting metal entrance to the cells (parrotta et al. 2015). the metal uptake and its distribution in plants has widely been studied, however, reports on dynamics of these processes are rather rare. this work was devoted to studying the dynamics of cd uptake in four soybean cultivars, and to reveal its distribution within the plants. our findings show that the uptake of cd by soybean roots is variable among the cultivars and might significantly contribute to the ability of soybeans to cope metal toxicity. experimental plant material and experimental conditions soybean (glycine max l.) seeds of four cultivars moravians, cardiff, gallec and kent were obtained from matex, s.r.o (veľké kapušany, slovakia). seeds were sterilized with 0.5 % (w/v) sodium hypochlorite for 5 minutes and germinated on wet filter paper in petri dishes. after 2 days, uniforomly germinated seeds were placed into 5 ml vial tubes with ¼ hoagland solution with presence or absence of 50 mg.ml-1 cd2+ in the form of cdcl2. the seedlings were incubated in dark at 23 °c for 48 hours. at daily time intervals the roots were subjected to analyses. at the end of experiment (10 days) the tolerance indexes (ti) were determined for each cultivar as ratio of dry weight of control to metal-exposed tissue x 100. all determinations were performed in triplicate. after verification of normal distribution and variance homogeneity the data were analysed by t-test. determining the genetic potential for cd accumulation the simple sequence repeat (ssr) genetic markers amplifying the cda1 locus for low cd accumulation capacity (jegadeesan et al. 2010) were used. dna was isolated according to békésiová et al. (1999). the pcr conditions were described in socha et al. (2015). reproducible presence/absence of amplicons in at least two of the 3 marker primers satk147, satk 149 and satk 150 (jegadeesan et al. 2010) was taken for evaluation of accumulation potential. content of proline content of proline in plant tissues upon homogenization with liquid nitrogen was determined spectrophotometrically as described by bates et al. (1973). radiometric analysis determinations of radioisotope 109cd in liquid samples of culture medium (expressed as bq/ml) or in solid samples of washed plant biomass (expressed as bq/g tissue) was implemented by scintillation gammaspectrometry with well type nai(tl) crystal, using a scintillation gammaspectrometer, type 76bp76/3 (scionix, the netherland). calibration of the instrument and calculation of radioactivity were realized using a library of analyzed radionuclides and the program scintivision-32 (ortec, usa). results the genetic potential for accumulation of cadmium was determined for the tested varieties. the data showed that the cda1 locus was present in only the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:00 utc nova biotechnol chim (2017) 16(2): 99-104 101 variety gallec, which therefore we assigned as of low cd accumulation potential (fig. 1). the tolerance indexes for growth in presence of cd showed highest values for the cultivars kent and gallec, while the variety moravians was the most sensitive one (table 1). to estimate the stressed nature of tissues we measured the levels of proline as typical multifunctional metabolite accumulating fig. 1. pcr-detection of the locus cda1 for low cd accumulation capacity. three primers including satk150 (a) were used, and reproducible presence of corresponding amplicons for at least two of them conditioned the assignment of a genotype as a low metal-accumulating type (b). table 1. relative values of individual parameters in soybeans exposed to 5 mg/l of cadmium. variety genetic potential to accumulate cd tolerance index (%) proline sd moravians high 62 1.1780* 0.5434 cardiff high 75 1.2243 1.5104 gallec low 78 1.7488** 4.5375 kent high 80 1.3125*** 5.2231 statistically significant differences are marked * at p ≤ 0.05 and *** p ≤ at 0.001. in plants under metal stress. significant elevation was observed in each cultivar, except for the variety cardiff (table 1). with radiological analyses, we studied the uptake, accumulation and allocation of cadmium in different parts of soybeans. the plants were immersed in hoagland nutrient solution in the presence or in the absence of the defined amount of the isotope 109cd2+. first, its depletion from solution by roots was measured continuously at 24 h intervals for 10 days. our results showed that the uptake rate greatly differs among cultivars. fastest and most intense cadmium uptake (removal) from the solution was typical for the cv. cardiff already after 1 day and moravians after 2 days (fig. 2a), in contrast to the cvs. gallec and kent. in the latter two varieties the uptake was clearly delayed and ~10 times lower after 2 days than in the cv. moravians. the difference in the uptake rates from environment (solution) among the varieties moravians/cardiff and gallec/kent decreased with time, nevertheless still remained more than ~ 2 fold at the end of the experiment (fig. 2a). the two former cultivars removed up to 85% of cadmium from the solution, while the latter ones only 50 – 60% (fig. 2). part of cadmium taken up by plants from the solution accumulated in the plant tissue. due to different physiology we again detected differences among tested cultivars. highest radioactivity representing the amount of cadmium accumulated in the tissue was detected in the cv. cardiff (fig. 2b). the cd contents in the other three cultivars (including the cv. moravians) were comparable to each other, but obviously lower comparing to the cv. cardiff (fig. 2b). the differences in metal deposition in tissue among the cultivars were not as pronounced as in case of the uptake rate from solution; while up to only 5 fold difference was observable after 2 days, at the end of experiment the amount of cadmium deposited in tissues of all bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:00 utc nova biotechnol chim (2017) 16(2): 99-104 102 cultivars was in a range of 120 – 170 µg/g of tissue (fig. 2b). the total amount of cd accumulated in the plant tissue during 10 days was determined. highest total amount of cadmium accumulated in the cv. cardiff, in contrast the lowest amount was detected in the cv. moravians (fig. 3a). fig. 2. the dynamics of uptake (depletion) of 109cd2+ from the growth solution by the root system of four soybean cultivars (cardiff, gallec, moravians, kent) after 9 days of culturing in the liquid hoagland medium. intake of cd2+ by the tissue is expressed in % of the initial amount of cd2+ solution (a) and in mg/g (dry weight) (b). data represent the average values obtained from 3 independent experiments. the metal was distributed within the soybean plants, while the allocation pattern depended on cultivar. the cv. moravians retained most of metal in roots, in contrast the cv. kent translocated a considerable amount of cd2+ into aerial parts – mainly to the stem and to primary leaves (fig. 3b). discussion sensitivity/tolerance of plants (including soybean) to cadmium results partly from genetic potential. therefore, uptake of the metal from environment and the ability of plants to alleviate toxicity vary among genotypes/cultivars. chromosome loci have been identified (benitez et al. 2010; jegadeesan et al. 2010) that can explain to some extent the variability in tolerance to cadmium. in our study, the locus for low cadmium accumulation potential cda1 was present in the cultivar gallec (fig. 1), and confirmed (radio)analytically (fig. 3a). in this cultivar, the metal uptake is probably governed mainly by the transporters (and other genes) on the chromosome 9. however, comparable (or even lower) amounts of metal did accumulate in the other tested cultivars, in which the cda1 locus was absent. this is in agreement with the fact that the given locus explains ~60% of observed variability for soybean tolerance to cadmium (jegadeesan et al. 2009; socha et al. 2015), and suggests for these cultivars another mechanisms regulating the metal uptake. the dynamics of cadmium entrance to the root cells (and their compartments) can vary, depending on several factors including ph of soils and metal speciation (rodriguez-serrano et al. 2009), or metal concentration in the growth media (zhao et al. 2017). at given dose we observed a gradually increasing uptake of cd with time during the 10 days of experiment. the uptake of metal from solution was very rapid in case of the cvs. cardiff and moravians (fig. 2a), but the metal amount measured in the tissue was correspondingly high only for cv. cardiff (fig. 2b). interestingly, this cultivar appears as relatively tolerant to cd, while under similar conditions we observed lack of induction of defense components such pr protein synthesis (bardáčová et al. 2016) or proline accumulation (this study). in addition, some part of cadmium was transported to the areal parts of plants (fig. 3b). we propose that the cv. cardiff might have an efficient system for detoxification and/or intracellular sequestration of cadmium ions, e.g. high level of phytochelatins, glutathion or organic acids (schützendübel and polle 2002). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:00 utc nova biotechnol chim (2017) 16(2): 99-104 103 fig. 3. cadmium allocation in four soybean cultivars (cardiff, gallec, moravians, kent). total amounts of cd2+ deposited in soybean plants were measured (a) and the relative abundances within different plant parts was calculated (b). the data represent average values of three independent experiments from plants after 10 days of culturing in liquid medium with 50 mg/l cd2+ and spiked with 109cdcl2 (141.7 kbq/l). among the tested soybeans, the cv. moravians took up cd2+ from solution as rapidly as cardiff, however, accumulated cd in the tissue like the cvs. gallec and kent (fig. 2b).moreover, after 9 days we quantified in this cultivar the lowest amounts of cd of all tested genotypes. noteworthy that gallec is genetically (socha et al. 2015; this study) and also practically (this study) a low-cd accumulator. therefore, we conclude that part of cd ions likely binds to the cell walls on the surface of roots (cataldo et al. 1983; piršelová et al. 2012) but never enters the root. this fits with relatively high tolerance index of this cultivar, too. the allocation pattern of cd in soybean plantlets points to even higher complexity of metal transport in plats than expected. furthermore, metal distribution and allocation within plants reflect to different strategies applied by soybeans to withstand negative impacts, as suggested previously (arao et al. 2003). the cv. gallec exerted low genetic predisposition for cadmium accumulation, and the both uptake dynamics and real accumulation rate supported the molecular prediction. in contrast, the cv. moravians with relatively slow but intensive uptake and translocating the metal to shoot revealed the poorest strategy resulting in high sensitivity to cadmium. dynamics of uptake as well as deposition rate and allocation of metals within a plant might be indicative to defense efficiency and strategy of a plant genotype to cope metal toxicity. conclusions sensitivity of soybean plants to cadmium can partly be explained by genetic determinants. however, uptake of the metal from environment varies among cultivars. furthermore, metal distribution and allocation within plants reflect different strategies applied by (soybean) plants to withstand negative impacts. the cv. gallec exerted low genetic predisposition for cadmium accumulation and the both uptake dynamics and accumulation rate supported the molecular prediction. in contrast, the cv. moravians with relatively slow but intensive uptake and translocating the metal to shoot revealed the poorest strategy resulting in high sensitivity to cadmium. dynamics of uptake as well as deposition rate and allocation of metals within a plant might be indicative to defense efficiency of a plant genotype to cope metal toxicity. acknowledgement this work was supported by the bilateral project sk-bg2013-0007 and by the project apvv-15-0051. we gratefully acknowledge the financial support for the stay of yg at ucm bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:00 utc nova biotechnol chim (2017) 16(2): 99-104 104 in trnava provided by slovak academic information agency saia. references arao t, ae n, sugiyama m, takahashi m. (2003). genotypic differences in cadmium uptake and distribution in soybeans. plant soil 251: 247-253. bardáčová m, maglovski m, gregorová z, konotop y, horník m, moravčíková j, kraic j, mihálik d, matušíková i (2016) the activity of cell-wall modifying β-1,3-glucanases in soybean grown in presence of heavy metals. nova biotechnol. chim.15: 114-121. bates ls, waldren rp, teare id (1973) rapid determination of free proline for water-stress studies. plant soil 39: 205-207. békésiová i, nap jp, mlynárová l (1999) isolation of high quality dna and rna from leaves of the carnivorous plant drosera rotundifolia. plant mol. biol. rep. 17: 269-277. benitez er, hajika m, yamada t, takahashi k, oki n, yamada n, nakamura t, kanamaru k (2010) a major qtl controlling seed cadmium accumulation in soybean. crop sci. 50: 1728-1734. cataldo da, garland tr, wildung re (1983) cadmium uptake kinetics in intact soybean plants. plant physiol. 73: 844-848. granec m, šurina b (1999) atlas pôd sr [atlas of soils of slovak republic]. bratislava: výskumný ústav pôdoznalectva a ochrany pôdy, 1999. 60 p. [in slovak] jegadeesan s, yu k, poysa v, gawalko e, morrison mj, shi c, cober e (2010) mapping and validation of simple sequence repeat markers linked to a major gene controlling seed cadmium accumulation in soybean glycine max (l.) merr. theor. appl. genet. 121: 283294. jha uc, bohra a (2016) genomics enabled breeding approaches for improving cadmium stress tolerance in plants. euphytica 208: 1-31. parrotta l, guerriero g, sergeant k, cai g, hausman jf (2015) target or barrier? the cell wall of earlyand laterdiverging plants vs. cadmium toxicity: differences in the response mechanisms. front. plant sci. 6: 133. piršelová b, mistríková v, libantová j, moravčíková j, matušíková i (2012) study on metal-triggered callose deposition in roots of maize and soybean. biologia 67: 698-705. rodriguez-serrano m, romero-puertas mc, pazmino dm, testillano ps, risueno mc, del rio la, sandalio lm (2009) cellular response of pea plants to cadmium toxicity: cross talk between reactive oxygen species, nitric oxide, and calcium. plant physiol. 150: 229-243. schützendübel a, polle a (2002) plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. j. exp. bot. 53: 1351-1365. six l, smolders e (2014) future trends in soil cadmium concentration under current cadmium fluxes to european agricultural soils. sci. total environ. 485-486: 319-328. socha p, bernstein n, rybanský l, mészáros p, gálusová t, spieß n, libantová j, moravčíková j, matušíková i (2015) cd accumulation potential as a marker for heavy metal tolerance in soybean. israel j. plant sci. 62: 160166. van kerkhove e, pennemans v, swennen q (2010) cadmium and transport of ions and substances across cell membranes and epithelia. biometals 23: 823-855. zhao y, zhang sj, wen n, zhang c, wang j, liu z (2017) modeling uptake of cadmium from solution outside of root to cell wall of shoot in rice seedling. plant growth regul. 82: 11-20. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:00 utc nova biotechnologica et chimica 15-1 (2016) 77 doi 10.1515/nbec-2016-0008 © university of ss. cyril and methodius in trnava preliminary results on growing second generation biofuel crop miscanthus x giganteus at the polluted military site in ukraine valentina pidlisnyuk1, josef trögl1, tetyana stefanovska2, pavlo shapoval3, larry erickson4 1department of technical sciences, faculty of environment, jan evangelista purkyně university in ústí nad labem, králova výšina 3132/7, 400 96, ústí nad labem, czech republic; (josef.trogl@ujep.cz) 2department of plant protection, national university of life and the environmental sciences, herojiv oboronu 13, 03040, kyiv, ukraine 3department of analytical chemistry, national university “lvivska polytechnika”, sv.yura square 9, 79013 lviv, ukraine 4department of chemical engineering, kansas state university, 1005 durland hall, manhattan ks 66506, usa abstract: the semi-field research on using second-generation biofuel crop miscanthus x giganteus for restoration of former military site in kamenetz-podilsky, ukraine was carried out during two vegetation seasons. despite high metal pollution of soil, in particular, by fe, mn, ti, and zr, no growth inhibition was observed. the concentrations followed pattern soil > roots > stems > leaves. accumulation of particular metals in roots was different: fe, mn and ti were accumulated rather palpably after the first vegetation season and less tangible after the second one. cu, pb and zn were less accumulative in both vegetation seasons, and for as and pb the accumulative concentrations were very small. accumulations in the aboveground parts of the plant in comparison to roots were significantly lower in case of fe, ti, mn, cu, zn, sr and even statistically comparable to zero in case of as, pb and zr. calculated translocation ratio of metals in the plant’s parts preferably indicated lack of metals’ hyper accumulation. generally, no correlations were observed between concentrations of metals in the soil and in the upper plant’s parts. the research confirmed the ability of miscanthus x giganteus to grow on the military soils predominantly contaminated by metals. key words: miscanthus x giganteus, phytotechnology, restoration of military contaminated soil, translocation ratio 1. introduction one of the significant environmental problems in the eastern european countries including ukraine are abandoned military contaminated sites widely dispersed and intensively polluted by metals, oil, and products of their decomposition. they constantly pose health risks and negatively affect soil and water resources as well as biodiversity. some former military sites may be used for agriculture, increasing risks of toxicity, while others are without plants and lack proper land management, allowing soil and water erosion. revitalization of those sites is an important task for the region’s sustainable development. the use of bioenergy plants for the restoration of polluted soils is an innovative strategy to derive additional benefits (i.e. phytoproducts) from those remediation bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:14 utc 78 pidlisnyuk, v. et al. activities (gomes, 2012; mosa et al., 2016; tripathi et al., 2016). plants applicable for bioenergy and phytoremediation of contaminated soils have to be fast growing and showing high biomass, possessing an extensive root system, easy to harvest and tolerate (wenzel, 2009; guldanova et al., 2010; kulakow and pidlisnyuk, 2010; prasad, 2016). synergistic bonding by using potential energy crops in phytoremediation programs would be useful to generate new bioenergy resources along with remediation of contaminated soil (pandey et al., 2016). among the bioenergy crops second generation biofuel crop miscanthus x giganteus is considered to be attractive and useful for the goals of phytoremediation (nsanganwimana et al., 2014; masters et al., 2016) or for combination of phytoremediation process with biomass production (pidlisnyuk et al., 2014a; kołodziej et al., 2016 ). m. x giganteus was introduced in europe and exhibited good production properties while used at the brownfield sites, former mining sites and contaminated agricultural lands (kahle et al., 2001; kocon and matyka, 2012; nsanganwimana et al., 2015; mosa et al., 2016). this plant is a c-4 perennial grass, has a high biomass productivity accompanied by good water use efficiency and low nutrient demands (pidlisnyuk et al., 2014a). m. x giganteus has the potential to improve soil quality by adding organic matter to soil, while as a perennial grass, it has advantages to reduce water and wind erosion (usda, 2011). the plant can be harvested and used in place for combustion heating including in a pellet form, or processed to liquid biofuels (nsanganwimana et al., 2014). we have initiated investigation on possibility to use m. x giganteus for restoration of former military sites (pidlisnyuk, 2012; pidlisnyuk and stefanovska, 2013; davis et al., 2014). the plant showed the good ability to grow at the model soil, artificially contaminated by heavy metals (pidlisnyuk et al., 2014b). the next step was to test ability of miscanthus x giganteus to grow at the abandoned military soil collected directly from the contaminated site and to explore translocation coefficients. experiment was done using moderately contaminated soil taken from abandoned military site located in kamenetz-podilsky, ukraine during two vegetation seasons, 2014 and 2015, as a greenhouse pot experiment. 2. materials and methods the contaminated research site was located in city kamenetz-podilsky, ukraine and had the following coordinates: latitude-48.680910n; longitude-26.58025e. the contaminated site was used as a military storage of former soviet union army and it is placed close to the central city park. the soil was collected on may 20th, 2014. soil sampling was done in accordance with the standard approach (gost, 1984). briefly, one testing square was selected at the site with the size of 5m × 5m; from the square five samples were taken from the depth of 0-0.3 m using ”envelope methods”, mixed and used in the pot experiment. general agronomic characteristics of the soil were determined using standard procedures (table 1). rhizomes of m. x giganteus were obtained from the agricultural station of the institute of bioenergy and sugar beetroot, ukraine and were one year old at the time bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:14 utc nova biotechnologica et chimica 15-1 (2016) 79 of planting. in each pot two rhizomes of m. x giganteus were planted. the vegetation season in 2014 started on june 6th and finished on november 19th when stems and leaves withered and were cut down; the pieces of rhizomes were sampled from each pot for the analysis. for the winter season pots with rhizomes were stored in the dark, dry conditions. the second year of the experiment started on april 25th, 2015, when the first sprouts appeared. that day pots were taken back to the light in the greenhouse. the growing year ended on october 15th, 2015 when stems and leaves withered. table 1. agronomic data of the soil from the research site parameter value method ph 6.90 ± 0.15 dstu iso 10390-2001 n-no3 [mg/kg] 11.6 ± 2.3 dstu 4729-2007 n-nh4 + [mg/kg] 35.2 ± 1.8 dstu 4729-2007 humus [%] 2.84 ± 0.16 dstu 4289-2004 preparation of soil samples for analysis was done in accordance with the standard iso 11464-2001. the soil sample was dried at 105ос to a constant mass. the dry sample was put on the clean sheet of paper, and small stones, plant particles and other inclusions were removed. bigger soil clods were ground in a porcelain mortar and mixed with the main part of the soil sample. then average soil sample was prepared for the analysis using the following approach. thoroughly mixed soil was put on the clean paper in the form of a square and divided in four equal parts by spatula. two opposite parts were removed, and two others were combined, mixed again and taken further in the analysis. this average sample was additionally sieved (0.25 mm pore size). the bigger particles were milled if necessary. preparation of roots, stems and leaves samples for analysis was done in accordance with the standard dstu iso 11464-2001 and dstu iso 11465 -2001. the samples of roots were carefully cleaned by distilled water and first dried in the open air and after dried at 105 °c to a constant mass, cooled in desiccators for 1 hour and weighed. for further roentgen-fluorescence analysis that examples were combusted at 450 °c, 4 hours, cooled during 1 hour in desiccators and weighed. analysis of heavy metals in the soil, roots, stems and leaves were carried out by roentgen-fluorescence analysis using analyser expert-3l (inam, ukraine, http://inam.kiev.us/contact-information). statistical evaluation of data was carried out using microsoft excel and statistica software pack at the significance level α = 0.05. extreme values were excluded using the inner-fence test (altman, 1990). 3. results and discussion the research soil, likely due to former intensive army activities, was contaminated with metals (table 2), especially iron, manganese, strontium, titanium and zirconium. concentrations of as, cu, pb and zn were also elevated compared to the inherent soil in the area (medvedev, 2001). the variability of metal concentrations in soils was bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:14 utc 80 pidlisnyuk, v. et al. not high, maximal relative deviation was ± 33% around average. also with contribution of this low variability the correlations between metal concentrations in soil and in aboveground plant parts were either insignificant (as, fe, mn, sr, ti, zr) or occasional (cu, pb, zn) (supplementary material tables s1a-i). this enabled to consider all variants labelled 1 to 5 as equal and to compare them together in order to increase significance of statistical comparisons. table 2. concentrations of selected metals in soil samplings (1-5) taken from the research site (in mg/kg dwtdry weight). c [mg/kg dwt] 1 2 3 4 5 as 75±5 165±85 115±35 70±0 75±5 cu 180±10 120±20 125±25 155±5 255±45 fe 140 955 ±5 715 135 140 ±14 580 139 010 ±13 870 131 530 ±8 570 136 115 ±1 515 mn 5 020±1 580 5 210±40 5 835±115 4 305±375 7 205±1 245 pb 395±85 185±85 150±50 230±10 450±50 sr 795±25 935±65 700±10 655±115 1 055±135 ti 19 815±1 475 17 640±1 370 19 160±1 960 20 265±1 115 19 755±775 zn 560±30 540±0 515±15 505±15 585±15 zr 1 910±140 1 515±235 1 165±65 1 070±230 1 115±145 despite high concentrations of metals pollution in the soil the growth of m. x giganteus seemed not affected (supplementary material, fig. s1a and s1b) and plant height was comparable to regular plantation growing at the clean agricultural land with similar climates (hanzhenko et al., 2015). two years observation confirmed high adaptability of m. x giganteus for growth on marginal and metal-contaminated soils. these preliminary results therefore extend list of possible non-agricultural sites needing reclamation which are suitable for plantation of m. x giganteus. accumulation of metals in the plants took place predominantly in the roots; translocation to upper parts was order of magnitude lower (tables 3 and 4). it has to be stressed that accumulation in roots was different: fe, mn and ti were accumulated rather palpably after the first vegetation season and less tangible after the second one. cu, pb, zn were less accumulative in both vegetation seasons, and for as and pb the accumulative concentrations were very small. accumulations in the above-ground parts in comparison to roots were significantly lower in case of fe, ti, mn, cu, zn, sr and statistically comparable to zero in case of as, pb and zr. these phenomena may be attributed to annual accumulation of nutrients in rhizomes, since m. x giganteus above grounds parts fade at the end of each season. shoot/root coefficients were however significantly lower than 1 (with exception of zn in year 1) indicating no hyperaccumulation of metals in the plant. the absolute totals of metals accumulated in roots, stems and leaves were higher in year 1 than 2; however overall the values were statistically comparable (sign test, p>0.05). individually significant decrease of concentrations between year 1 and 2 was detected for fe and ti (in stems and leaves), and mn and sr (in stems only). contrary, concentrations of cu (stems and leaves) and zn (stems) significantly increased bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:14 utc nova biotechnologica et chimica 15-1 (2016) 81 between year 1 and 2. for as and pb, the concentrations in above-ground parts were statistically comparable to zero (t-test, α = 0.05); the same phenomena was observed for concentrations of zr in stems. table 3. accumulation of metals in the different parts of m. x giganteus at the end of first and second vegetation seasons (average ± std. deviation, n = 10). letters indicate overlapping of confidence intervals based on mutual comparisons (α = 0.05), i.e. values with the same letters can be considered comparable; bolded values indicate values significantly higher than zero (t-test, α = 0.05). c (mg/kg dwt) year 1 year 2 soil roots stems leaves roots stems leaves as 83±24c 7±7b 0±0a 0±0a 8±4b 0±0a 0±0a cu 152±35e 55±32d 4±1a 10±11ab 57±13d 8±3b 14±4c fe 136 550±10 641f 27 162±18 187e 316±146b 5 227±3 529d 20 238±3 034e 130±62a 1 107±251c mn 5 189±963e 953±552cd 128±32b 445±260cd 638±265d 46±23a 176±65bc pb 282±134c 60±60b 1±1a 1±2a 21±13b 0±0a 0±0a sr 788±128e 158±93d 7±3a 39±17c 95±4d 16±7b 29±12bc ti 19 327±1 668f 4 067±2 629e 67±24b 913±641d 2 800±360e 28±17a 158±34c zn 541±34e 138±75cd 18±8a 114±54cd 163±47d 49±15b 75±9c zr 1 355±364e 269±194d 1±1ab 19±13c 112±53d 0±0a 2±1b table 4. translocation ratios1 of metals in m. x giganteus parts measured after first and second vegetation seasons (average ± std. deviation, n = 10). letters indicate overlapping of intervals (α = 0.05, see table 3); bolded values indicate significantly non-zero values (t-test, α = 0.05). year 1 year 2 stems/roots leaves/roots leaves/stems stems/roots leaves/roots leaves/stems as 0±0a 0.08±0.18a 0±0a 0.01±0.03a 0.07±0.14a 2.53±2.53a cu 0.11±0.09b 0.05±0.07a 2.70±3.12bcd 0.12±0.03b 0.24±0.06c 1.76±0.34d fe 0.02±0.02 0.29±0.31ab 15.06±11.03c 0.01±0a 0.05±0.02b 7.94±2.73c mn 0.26±0.22ab 0.88±0.95ab 3.10±1.78bc 0.07±0.04a 0.29±0.16b 4.26±1.57c pb 0.03±0.04a 0.01±0.01a 6.75±9.18a 0±0a 0±0a 0±0a sr 0.10±0.10a 0.39±0.38a 5.97±3.25d 0.18±0.06ab 0.35±0.16b 1.93±0.75c ti 0.03±0.02bc 0.37±0.46abc 16.12±14.63d 0.01±0.01b 0.05±0.01c 5.66±1.73d zn 0.23±0.19a 1.04±0.86ab 5.55±2.47c 0.31±0.07a 0.42±0.07a 1.64±0.39b zr 0±0a 0.13±0.16ab 22.15±24.16ab 0±0a 0.02±0.02a 2.67±1.38ab 1generally the leaves/roots ratio divided by stems/roots ratio should be equal to leaves/stems ratio, which was predominantly observed. nevertheless, due to high data variability and elimination of extremes concentration values by inner-fence test, sometimes the values differ. the leaves/stems ratio was calculated directly from the concentration values and not from two other ratios. the metal accumulation data confirm the desired pattern required for simultaneous phytoremediation and biomass production. on the one hand, m. x giganteus extracted some metals from soil and thus it carried out slow soil decontamination. on the other hand, these metals are deposited predominantly in roots, which preserve upper plant parts relatively clean and thus enable their use as energetic biomass. nevertheless, m. x giganteus is a perennial plant and supposed to be grown for far longer time than two years (pidlisnyuk et al., 2014a,b). described promising results from two vegetation periods (2014 and 2015) need to be therefore verified during experiment, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:14 utc 82 pidlisnyuk, v. et al. which is expected to be continued for 2016-2017. the results can illustrate the overall picture for the long-term growing of m. x giganteus at the former military contaminated site. the further research has also to be concentrated on interconnection between m. x giganteus biomass quality and quantity, nature and concentrations of contaminants at the military sites including those newly appeared at the east of ukraine. the results indicate that production of energy biomass from m. x giganteus is attractive and might be developed into a profit making operation. 4. conclusions the greenhouse pot experiment with miscanthus x giganteus during two vegetation seasons using soil taken from the abandoned military site located in kamenetzpodilsky, ukraine and moderately contaminated by metals confirmed the ability of the research plant to grow on the marginal contaminated land. accumulation of metals in m. x giganteus was observed predominantly in roots and order of magnitude less in stems and leaves preserving upper parts usable as energy biomass. while the experiment continues these preliminary results indicate applicability of this crop for simultaneous phytoremediation and energy biomass production. acknowledgement: the research is supported by nato myp sps project g4687 and kansas state university, usa. supplementary materials: additional illustrative materials are presented at supplementary material tables s1a-i nad figures s1a and s1b. references altman, d.g.: practical statistics for medical research. chapman & hall, london, 1990, 624 pp. davis, l., erickson, l., hattiarachchi, g., mengarelli, j., pidlisnyuk, v., roozeboom, k., stefanovska, t., tatarina, n. phytoremediation with miscanthus produced for bioenergy. proceed. of 11th international phytotechnologies conference, heraklion, greece, 2014, 313, isbn 978-960-6865-81-7. dstu iso 10390-2001 (state standard of ukraine): soil quality. determination of ph (iso 10390:1994,idt). 2003, 8 pp.(in ukrainian). dstu iso 11464-2001(state standard of ukraine): soil quality. preliminary preparation of samples for physical-chemical analysis, 2001,13 pp. (in ukrainian) dstu iso 11465-2001 (state standard of ukraine): soil quality. determination of dry matter and moisture on the mass base. gravimetric method, 2001,7 pp.(in ukrainian). dstu 4289-2004 (state standard of ukraine): soil quality. methods for determination of organic substances, 2005,18 pp (in ukrainian). dstu 4729-2007 (state standard of ukraine): soil quality. determination of nitrates’ and ammonium nitrogen using modification of institute of soil science and agrochemistry named after o.n.sokolovskyi, 2006, 18 pp.(in ukrainian). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:14 utc nova biotechnologica et chimica 15-1 (2016) 83 gomes, h. i.: phytoremediation for bioenergy: challenges and opportunities, environ. technol. rev., 1, 2012, 59-66. gost 17.4.4.02-84 (state standard of the ussr): nature protection. soils. methods for sampling and preparation of soils for chemical, bacteriological, helminthological analysis, standard-inform, moscow, russia.1984, 8 pp.(in russian). guldanová, j., horník, m., marešová,j., pipíška, m., augustín,j.: bioaccumulation and distribution of 137cs in tobacco cultivated under hydroponic conditions. nova biotechnol., 10-2, 2010, 95-106. hanzhenko, m., humentik, ya., kvak, v.: solid biofuels production from miscanthus. bioenergy, 2, 2015,16-19 (in ukrainian). kahle, p., beuch, s., boelcke, b., leinweber, p., schulten, h.-r.: cropping of miscanthus in central europe: biomass production and influence on nutrients and soil organic matter. eur. j. agron., 15, 2001, 171-184. kocon, a., matyka, m.: phytoextractive potential of miscanthus x giganteus and sida hermaphrodita growing under moderate pollution of soil with zn and pb. j. food agric. environ., 10, 2012, 1253-1256. kołodziej, b., antonkiewicz, j., sugier, d.: miscanthus×giganteus as a biomass feedstock grown on municipal sewage sludge. ind. crop prod., 81, 2016, 72-82. kulakow, p.a., pidlisnyuk, v.v.: application of phytotechnologies for clean up of industrial, agricultural and waste water contamination, springer, dordrecht, 2010. 198 pp. masters, m.d., black, c.k., kantola, i.b., woli, k.p., voigt, t., david, m.b., delucia, e.h.: soil nutrient removal by four potential bioenergy crops: zea mays, panicum virgatum, miscanthus×giganteus, and prairie. agr. ecosyst. environ., 216, 2016, 51-60. medvedev, v,v.: monitoring of soil in ukraine, kharkiv, antikva, 2002. 428 pp. (in russian). mosa, k.a., saadoun, i., kumar,k., helmy, m., dhankher, p.o.: potential biotechnological strategies for the cleanup of heavy metals and metalloids. front. plant sci., 7, 2016, art.no.303. nsanganwimana, f., pourrut, b., mench, m., douay, f. suitability of miscanthus species for managing inorganic and organic contaminated land and restoring ecosystem services. a review. j. environ. manag. 143, 2014, 123-134.. nsanganwimana, f., pourrut, b., waterlot, c., louvel, b., bidar, g., labidi, s., fontaine, j., muchembled, j., lounes-hajd sahraoui, a., fourrier, h., douay, f.: metal accumulation and shoot yield of miscanthusxgiganteus growing in contaminated agricultural soils: insights into agronomic practices. agr. ecosyst. environ., 213:12, 2015, 61-71. pandey, v.c., bajpai, o., singh, n.: energy crops in sustainable phytoremediation, renew. sust. energ. rev., 54, 2016, 58-73. pidlisnyuk, v.: expanding the potential of second generation biofuels crops by using for phytoremediation of sites contaminated by heavy metals: laboratory stage. scientific bulletin of kremenchuk national university, 74, 2012, 104-108. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:14 utc 84 pidlisnyuk, v. et al. pidlisnyuk, v., stefanovska, t.: method for growing plants in heavy metals contaminated soils. claim for the invention of ukraine, #12471, 2013 (in ukrainian). pidlisnyuk, v.v., stefanovska, t.r., lewis, e.e., erickson, l.e., davis, l.c.: miscanthus as a productive biofuel crop for phytoremediation. critical rev. plant sci., 33:1, 2014a, 1-19. pidlisnyuk, v., erickson, l., kharchenko, s., stefanovska, t.: sustainable land management: growing miscanthus in soils contaminated with heavy metals, j. environ. protect., special issue in environmental remediation, 5, 2014b, 723-730. prasad, m.n.v. (edt). bioremediation and bioeconomy, elsevier science, 2016. 698 pp. isbn 978-0-12-802830-8. tripathi, v., edrisi, s.a., abhilash, p.c.: toward the coupling of phytoremediation with bioenergy production. renew. sust. energ. rev., 57, 2016, 1386-1389. usda (us department of agriculture), natural resources conservation service, plant materials program: planting and managing giant miscanthus as biomass energy crop, technical note no.4, 2011, 33pp. wenzel, w.w.: rhizosphere processes and management in plant-assisted bioremediation (phytoremediation) of soils. plant soil, 321, 2009, 385-408. received 24 may 2016 accepted 27 june 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:14 utc nova biotechnologica et chimica 13-1 (2014) 1 doi 10.2478/nbec-2014-0001 © university of ss. cyril and methodius in trnava anthocyanins in wheat seed – a mini review michaela havrlentová1, ivana pšenáková2, alžbeta žofajová1, ľubomír rückschloss1, ján kraic1,2 1national agricultural and food center, plant production research institute piešťany, bratislavská cesta 122, sk-92168 piešťany, slovak republic (havrlentova@vurv.sk) 2department of biotechnology, university of ss. cyril and methodius, nám. j. herdu 2, sk-91701 trnava, slovak republic abstract: improving the micronutrients in food has become an important field of the second green revolution. in recent years, minor bioactive compounds such as polyphenols, pigments and carotenoids, have attracted more and more interest from both researchers and food manufactures as health-promoting and disease-preventing effects in both in vitro and in vivo studies. one of plant pigments, wheat anthocyanins as plant phenolics are increasingly attractive as natural compounds positively affecting consumer´s health and condition moreover wheat is staple food source consumed usually daily. for a purple, blue, or red colour of wheat seed are responsible glycosylated cyanidins, delphinidins, malvinidins, pelargonidins, petunidins, and peonidins located in aleurone layer or pericarp, respectively. other than white seed colour is not natural for common hexaploid wheat but this trait can be introduced from donors by aimed breeding programs. the way of wheat anthocyanins to provide positive effects for consumer´s physiology is limited due to their specific occurrence in seed parts usually removed during grain milling practice and lower stability during processing to foods. key words: phenolics, flavonoid, anthocyanins, wheat grain 1. introduction anthocyanins are chemical compounds of plant origin well known as pigments responsible for blue, purple, red, or orange coloration of plant tissues and organs. from the structural point of view anthocyanins are glycosides composed of hydroxyled or methoxyled 2-phenylbenzopyrilium skeleton with hydroxyl and methoxy groups in the b-ring. the structural variation extend bounded sugars (the most frequent are pentoses xylose, arabinose, rhamnose, fructose and hexoses galactose, glucose), aliphatic, and aromatic acids. the consequence of molecular variability is more than six hundreds known natural anthocyanins. six the most frequented are cyanidins, delphinidins, malvinidins, pelargonidins, petunidins, and peonidins (iwashina, 2000). anthocyanins are phenolic phytochemicals classified within flavonoids together with flavonols, flavones, flavanols, flavanones, and isoflavonoids (liu, 2004). their biosynthesis initializes conjugation of malonyl-coa and p-coumaroyl-coa derived from phenylpropanoid biosynthetic pathway under operation of seven enzymes chalconsynthase, chalconisomerase, flavanon-3-hydroxylase, flavonoid-3hydroxylase, dihydroflavonol-4-reductase, anthocyanin synthase, and flavonoid-3-oglucosyltransferase (holton and cornish, 1995; schijlen et al., 2004). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:51 utc 2 havrlentová, m. et al. anthocyanins are generally specified as bioactive, non basicly-nutritional compounds, responsible for antioxidant and uv/photoprotective functions (ryan et al., 2001), playing also role in plant reproduction (kong et al., 2003). anthocyanins participate in the formation of non-specific disease resistance in plants (treutter, 2006) such as pre-harvest sprouting where the red pigment of red-grained wheat is synthetized through the flavonoid biosynthetic pathway, in which the dihydroflavonol4-reductase gene (dfr) is one of the genes involved in anthocyanin synthesis (bi et al., 2014). specific properties make anthocyanins attractive in prophylaxis and therapy (anonymus, 2014) due to their antitumor, cardioand hepatoprotective, antimutagenic, antiulceral, uv-protective, and other health beneficial effects (kong et al., 2003; kay, 2006; prior and wu, 2006; hodgson and croft, 2006). it appears that these phytochemicals are responsible for the reduced risk of various diseases associated with oxidative stress, such as cancer and cardiovascular and neurodegenerative diseases (jacobs and steffen, 2003). compared to vitamins c and e which are absorbed in the upper segments of the intestine, anthocyanins are dietary antioxidants presented in the colon in valuable concentrations (manach et al., 2004). seeds of cereals are not typical sources of anthocyanins nevertheless blue, red, and purple seeds attract consumers, food producers, and plant breeders (guo et al., 2012), therefore relevant knowledge has been cumulated also in maize, wheat, barley, oat, and rice as the most important world food sources. coloured-grain wheat varieties with good genetic stability, excellent stress resistance and high yield are still required (guo et al., 2012). 2. anthocyanins in wheat seeds isolation, identification, and analyses of wheat anthocyanins have begun long time ago. dedio et al. (1972) detected cyanidine-3-glucoside and peonidine-3-glucoside (fig. 1) as the main anthocyanins of pericarp in purple wheat seeds. cyanidine-3glucoside as the dominant anthocyanin in purple wheat seeds was detected also by hosseinian et al. (2008) and abdel-aal and hucl (2003) together with cyanidine-3-galactoside (fig. 1), peonidine-3-glucoside, and others unidentified ones. as the most frequent anthocyanins in blue wheat seeds were declared delphinidine-3glucoside and delphinidine-3-rutinoside (abdel-aal et al., 2006; fig. 1), cyanidine-3-glucoside and peonidine-3-glucoside (abdel-aal and hucl, 2003). the blue wheat seeds contain more anthocyanins in flour then in bran, in comparison with purple seeds (abdel-aal and hucl, 1999). on the other hand, in hungarian hard red winter wheat varieties and blue-grained varieties, the anthocyanin content of the grind was 21-157 mg kg-1, while that of the flour was only 5.3-17.4 mg kg-1 (varga et al., 2013) so most of the anthocyanin content was in the bran. hu et al. (2007) detected in the wheat cultivar hedong wumai also minor anthocyanins cyanidine-3-galactoside, pelargonidine-3-glucoside (fig. 1), peonidine 3-glucoside, and ferulic acid (fig. 1). red-coloured wheat seeds contain incomparably lower content of anthocyanins in comparison with blue and purple ones. relationships between content of catechins, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:51 utc nova biotechnologica et chimica 13-1 (2014) 3 tannins, and red colour of wheat seeds published miyamoto and everson (1958). winkel-shirley (2001) inclined to the opinion that pigmentation causes proanthocyanins (condensed tannins). cyanidine-3-glucoside peonidine-3-glucoside delphinidine-3-glucoside pelargonidine-3-glucoside cyanidine-3-galactoside delphinidine-3-rutinoside ferulic acid fig. 1. main anthocyanins of coloured wheat grains. it is known that environment affects production of polyphenols (eticha et al., 2011; khlestkina, 2013) as well as anthocyanins (varga et al., 2013) and abiotic stresses like high light intensity, low temperature, high salinity and/or drought stress, and others increase their amounts (chalker-scott, 1999). cold stressed coloured wheat genotypes show either reduced, similar or increased anthocyanin content compared to unstressed plants caused possibly by the allelic state of the rc genes responsible for the seed colour (gordeeva et al., 2013). according to authors, the changes in anthocyanin content correlate negatively with the changes of growth parameters in response to cold stress, suggesting the presence of some stressdependent regulation of anthocyanin biosynthesis in wheat coleoptiles. changes in anthocyanin content are associated with physiological maturity of the plant and grain, respectively. location of grains in spike affects anthocyanin content by decreasing at distal positions reflecting positive response to higher source-sink ratio bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:51 utc 4 havrlentová, m. et al. (bustos et al., 2012). in the experiments of žofajová et al. (2012) genotypes with purple pericarp reached the highest total anthocyanins concentration on the 22nd day post anthesis with increasing and decreasing before and after sampling time, respectively. pre-anthesis halving increased anthocyanins content by 54 and 37%, while post-anthesis halving increased this trait by 31 and 29%, respectively (bustos et al., 2012). promising to increase anthocyanins in purple wheat is the combination of early harvesting and magnesium fertilization (bustos et al., 2012). content of total phenolic compounds in wheat seeds can be also affected by growing condition (vaher et al., 2010) or by application of selenium into the soil (chu et al., 2010). 3. origin and genetic control of the seed colour the blue and purple colours are not natural for seeds of common wheat. anthocyanins responsible for blue pigmentation are located in aleurone layer and for purple colour in pericarp, respectively. according to zeven (1991) the blue colour was introduced into wheat from blue coloured diploid wild einkorn wheat triticum boeoticum, agropyron tricholphorum, agropyron glaucum or tall wheatgrass agropyron elongatum, and purple colour from tetraploid emmer triticum dicoccum. zeller et al. (1991) specified that hexaploid wheat received the blue aleurone colour through chromosomal substitutions of 4a and 4b chromosomes with 4a chromosomes of diploid einkorn wheat triticum monococcum or triticum boeticum, respectively. “black” (deep purple) hues of wheat kernel colour can be due to the combination of anthocyanin genes for blue pericarp and blue aleurone (li et al., 2005). results of guo et al. (2012) showed that the colour inheritance of purplegrained wheat follows a maternal inheritance pattern and that the blue-grained wheat expresses xenia in most cases. nevertheless, the genetics of wheat anthocyanins has been unlocking more than fifty years. knott (1958) published that partially dominant gene/genes with different effects were responsible for the blue seed colour. hurd (1959) supported responsibility of two partially dominant genes. later, keppenne and baenziger (1990) presented that blue colour of wheat aleurone is controlled by single dominant gene. nowadays, the ba (blue aleurone) gene originated from the 4e chromosome of thinopyrum ponticum is considering as the incompletely dominant gene responsible for the blue wheat aleurone (dubcovsky et al., 1996). the concept of single dominant gene has been published recently, too (knievel et al., 2009). results on mapping of anthocyanins pigmentation genes in wheat along with the observation of previous studies related were presented by khlestkina et al. (2008). the purple colour of pericarp originated from purple ethiopian tetraploid and hexaploid wheat and purple abyssinian wheat (triticum aethiopicum jakubz.) was firstly described long ago (wittmack, 1906). mcintosh and baker (1967) published hypothesis that the purple colour of wheat pericarp is controlled by two duplicated genes. two independent responsible genes were located later at the 3a and 7b chromosomes (piech and evans, 1979), griffin (1987) defined these genes as dominantly duplicated. arbuzova et al. (1998) and arbuzova and maystrenko (2000) located genes for purple seeds in the triticum aestivum cultivar purple feed at bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:51 utc nova biotechnologica et chimica 13-1 (2014) 5 chromosomes 7b (gene pp1) and 6a (pp2), and in the purple varieties at chromosomes 7b (pp1) and 2a (pp3). dobrovolskaya et al. (2006) located three genes pp1, pp2, and pp3 and relabelled at once them as pp1 (at the 7bl chromosome), and pp3b, pp3a (chromosome 2a). the work of tereshchenko et al. (2012) demonstrated that the d genome of bread wheat purple carries one of two complementary genes determining purple grain colour and this gene was mapped on the short arm chromosome 7d 2.5 cm distal to the locus rc-d1 determining red coleoptile colour. this position is highly comparable with that of the pp1 gene mapped earlier in tetraploid triticum durum. recently, knievel et al. (2009) concluded that genetic control of purple coloured wheat pericarp is more complicated and authors also presented design of three dominant alleles. their results also indicate that wheat breeding programmes aimed to blue seed colour should be feasible, moreover supported by available genetic maps of wheat chromosomes 2a and 7b, mapped genes pp3 and pp1, and linked molecular markers (khlestkina et al., 2010; tereshchenko et al., 2012). red pigmentation of wheat seeds is also based on the content of polyphenols. according to himi et al. (2005), expression of four genes (chalkonsynthase, chalkonisomerase, flavanon-3hydroxylase, and dihydroflavanol-4-reductase) in white coloured wheat seeds was reduced in comparison with red coloured ones. this suggests that three r genes (r1, r2, r3) encoding the seed colour and located at the chromosomes 3a, 3b, and 3d (mcintosh et al., 1998; khlestkina et al., 2011) are transcription activators (myb transcription factors) of genes in flavonoid synthesis (himi and noda, 2005). 4. coloured wheat seeds for nutrition coloured wheat seeds are natural source of pigments as phytochemicals and may impart desirables colour and stability for commercial food products (giusti and wrolstad, 2003), especially for some slightly alkaline ones (cabrita et al., 2000). results of mazzaracchio et al. (2012) indicate a presence of strong interaction between structure of anthocyanins and gluten molecule by playing an important role in pigments adsorption by gluten or its fraction, whereby delphinidine is more adsorbed than cyanidine. occurrence of anthocyanins in foods and beverages, their transformation during processing, and their bioavailability are described by clifford (2000). they are beneficial for human health and fitness, possessing several unique traits and functions in organism including anti-obesity effects and influencing of brain activities (prior and wu 2006). anthocyanins are competent for effective elimination of oxidative stress in human body by balancing between oxidants and antioxidants (temple, 2000). coloured seeds in comparison to colourless equivalent contain higher level of anthocyanins and phenolic compounds and their extracts have stronger antioxidant competence (liu et al., 2010). this confirmed choi et al. (2007) in rice and millet, and liu et al. (2005) in wheat where purple and blue coloured seeds had higher capability to scavenge free radicals than white ones. total amount of anthocyanins in pigmented wheat seeds is relatively high, abdel-aal and hucl (1999) quantified their amount on average 15.7 mg kg-1 and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:51 utc 6 havrlentová, m. et al. 45.8 mg kg-1 in flour and bran of blue coloured wheat, respectively. on the opposite, iqbal et al. (2007) detected in extracts from red wheat´s bran much lower content of anthocyanins (3.0-3.8 mg kg-1), nevertheless their occurrence relates to oxygen radical absorbance capacity, radical scavenging activity, and total phenolic content. there are several specific traits of wheat anthocyanins including the location in seed. due to occurrence in seed pericarp and aleurone about 80% of phenolic compounds and flavonoids are in bran or wholemeal (liu, 2007) and the free radical scavenging capacity of endosperm is much lower (liyana-pathirana and shahidi, 2007; siebenhandl et al., 2007). wheat particle size effect the extraction of phytochemicals. the coarse treatment (unmilled whole bran) exhibit significantly higher antioxidant properties compared to fine treatment, but on the other hand, the fine treatment was higher in anthocyanin content (brewer et al., 2014). common disadvantage of anthocyanins in food processing is the sensitivity to different factors such as temperature (ibanoğlu, 2002), light intensity, storage conditions, ph, metal ions, enzymes, oxygen, sulphur dioxide, ascorbic acid, sugars, and co-pigments (mazza and brouillard, 1987; cabrita et al., 2000; bąkowska-barczak, 2005), nevertheless their colour stability can be improved by acylation (bąkowska-barczak, 2005). the antioxidant capacity can be affected during the thermal processing by both, the extraction solvent and matrix composition (li et al., 2007). analysis of nutrient composition of purple wheat shows that the amount of 40 kinds of nutrients is higher than those of the control (guo et al., 2012; guo et al., 2013). for example, the amounts of sodium and manganese in purple wheat are higher than the standards by 312 2018% and 548 733%, respectively. the contents of beta plus gama-vitamin e were higher by 300% and zinc, iron, magnesium, and potassium were higher than the control by 100%. in biofortification breeding and functional food development there are also useful significant positive correlations between anthocyanins and molybdenum and glutamic acid concentration observed. on the other hand, significant negative correlations between anthocyanin´s concentration and free tryptophan and calcium have been found, too (guo et al., 2013). on the opposite, in comparison with other typical anthocyanin sources (e.g. fruits and vegetables) cereals seeds can be stored easily and treated to stable products (abdel-aal et al., 2006). improvement of anthocyanin content in wheat relates to available genetic resources and breeding programmes. desirable sources of seed phenolic compounds could be wheat landraces where were also new unique phenolic substances identified (dinelli et al., 2009). coloured wheat has been traditionally used like purple hexaploid wheat for special bread baking in the new zealand. the wheat cultivar indigo with purple seeds designed for special food products has been released in austria in the year 2006 (eticha et al., 2011). indigo possess about 200 mg kg-1 of anthocyanins in grains what is the amount comparable with the red wine. the wheat cultivar ps karkulka with purple grains has been registered in the year 2014 as results of the breeding program "rainbow wheats" in slovakia. more than twenty years running effort in breeding of coloured wheat led to the development of black-grained wheat (bgw) cultivar in china. the bgw possess also high content of proteins, selenium, and amino acids, but has lower bread-making quality (li et al., 2006). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 15:51 utc nova biotechnologica et chimica 13-1 (2014) 7 the number of registered coloured wheat cultivars is very limited and those available ones have low baking quality and agronomic value (vaculová et al., 2010). 5. conclusions information presented in this mini-review is based on our own experiments as well as published results in topic of wheat anthocyanins. there are both known, the chemical structure of major anthocyanins of the wheat seed as well as their location in specific part of the seed. from the genetic point of view, the final opinion about the number and location of genes is still developing. different donors of purple, blue, and red colour of the seed have been found and used for incorporation these traits into wheat cultivars. following challenge for wheat breeders is to create modern wheat cultivars with coloured seeds but also adapted to local growing conditions: materials with accepted agronomic characteristics, technological quality traits, and therefore profitable for producers. currently, based on accumulated knowledge also genetic maps and molecular markers linked to relevant loci can effectively support molecular breeding programmes. the interest in creation of new wheat cultivars with seeds coloured by presence of anthocyanins results mainly from their positive effects on health and wellness of the consumer. on the other side, there are also problems to include wheat anthocyanins into food chain due to their unstability during the food processing. presented information about wheat anthocyanins can initiate further related challenges e.g. improvement of anthocyanin production in wheat seed, modification of wheat agricultural practice and analyses of environmental influences, redirecting of synthesis or expression of anthocyanin to other seed part that aleurone and pericarp, development of new processing methods more protecting biological properties of anthocyanins. acknowledgements: this work was supported by the operational programme research and development for the project “systems biology for protection, reproduction and use of plant resources of slovakia (itms 26210120039)” co-financed from the resources of the european union fund for regional development. references abdel-aal, e.-s.m., young, j.c., rabalski, i.: anthocyanin composition in black, blue, pink, purple, and red cereal grains. j. agric. food chem., 54, 2006, 4696-4704. abdel-aal, 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tian et al., 2009). polyphenols easily protect cells against the damaging effects of reactive oxygen species, such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxynitrite. an imbalance between antioxidants and reactive oxygen species results in oxidative stress induced cytotoxicity (duthie et al., 1997; skaper et al., 1997). in addition, these compounds show a wide spectrum of action involving antitumor, antiviral, antibacterial, cardioprotective, prooxidant and antimutagenic activity (caballero, 2010; fan et al., 2011; markovits et al., 1989). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:01 utc 38 kruzlicova, d. et al. according to the chemical structure, flavonoids can be categorized into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones, etc. the basic structure of flavonoids allows a variety of substitution patterns in the benzene rings. the capacity of flavonoids to act as antioxidants depends upon their molecular structure. the position of hydroxyl groups and other features in the chemical structure of flavonoids are important for their antioxidant and free radical scavenging activities. quercetin, the most common dietary flavonol, is a potent antioxidant, because it has all the right structural features for free radical scavenging activity. our work concerns about new synthetised derivatives of quercetin, that may be potentially useful in the prevention of human disease attributed to free radical damage. the observation of antioxidant activity referring to various substituents may lead to the discovery or synthesis of novel flavonoids as preventive or therapeutic agents against human diseases associated with free radicals. a computing technique of artificial neural networks (ann) was applied for prediction of antioxidant activity of quercetin derivatives. 2. materials and methods the structures of all 10 investigated derivatives of quercetin are listed in table 1. the main characteristic of an antioxidant is its ability to trap free radicals. dpph (dawidowitz et al., 2012) and abts (floegel et al., 2011) test were applied for determination of the inhibitory potencies of flavonoids. a rapid, simple and inexpensive method to measure antioxidant activity of compounds involves the use of the free radical, 2,2-diphenyl-1-picrylhydrazyl, dpph (sigma-aldrich gmbh, de). (s)-(-)-6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid, trolox (sigma-aldrich gmbh, germany, de), was used as a reference standard for this purpose. 12 mg of dpph was diluted in 100 ml of 100% methanol (vitrum spol s r.o., czech republic, cz). trolox and 10 new synthetised derivatives as well as quercetin were diluted in 100% dimethylsulfoxide, dmso (sigma-aldrich gmbh, germany, de), to appropriate concentration (312.5 μm 5 mm). 50 μl of sample, 100 μl of 100% methanol and 100 μl of dpph solution were pipetted into microplate well (greiner bio-one gmbh, germany, de). the reaction of samples with dpph solution lasted for 10 min at room temperature in dark. after that, the absorbance changes were measured spectrophotometrically (multiscan fc microplate photometer, thermo fisher scientific inc., united states, us) at 520 nm. inhibitory concentrations (ic50) were expressed as the quantity of sample necessary to react with one half of the dpph (table 2). abts test was also used to determine the total antioxidant activity of synthetic compounds. the assay measures abts radical cation formation induced by metmyoglobin and hydrogen peroxide. trolox serves as a positive control inhibiting the formation of the radical cation in a dose dependent manner. quercetin, its derivatives and trolox were diluted in dimethylsulfoxide (dmso) to appropriate concentrations (from 8 mm to 15.6 µm). cationradical abts˙+ was prepared by reaction between 7 mm 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (abts) in phosphate buffered saline (0,1 m, ph 7.4) and 2.45 mm potassium bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:01 utc nova biotechnologica et chimica 11-1 (2012) 39 table 1. structures of quercetin and investigated quercetin derivatives. r1 r2 r3 r4 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:01 utc 40 kruzlicova, d. et al. persulfate in phosphate buffered saline (0.1 m, ph 7.4) in the rate of 1:1. this mixture stayed at room temperature in the dark for 12 hours. solution of cationradical abts˙+was diluted with dmso (1.5 ml of abts˙+ was pipetted into 60 ml of dmso) to get an absorbance of 0.700 at 734 nm. then 1.95 ml of diluted abts˙+ was added to 0.05 ml of sample. reaction mixture was incubated 7 min at room temperature in the dark. thereafter the absorbance was measured at 734 nm. the inhibition percentage was calculated for each concentration relative to a blank absorbance (dmso). value of ic50 was stated (the concentration, which eliminates the half of radical present) for each sample (table 2). table 2. antioxidant activity determined by the radical scavenging activity of antioxidants against the 2,2’azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (abts) and 1,1-diphenyl-2-picrylhydrazyl (dpph) radical. ic 50 ± sd (µm) no. name abts dpph 1. quercetin 2.5 ± 0.1 16.2 ± 1.1 2. tri(diprenylcaffeoyl) quercetin 8.4 ± 0.6 > 200.0 3. di(diprenylcaffeoyl) quercetin 5.5 ± 0.1 160.9 ± 13.2 4. di(diacethylcaffeoyl)mono(monoacethylcaffeoyl) quercetin 10.4 ± 0.7 > 200.0 5. monoacethyl-di(diacethylcaffeoyl) quercetin 4.1 ± 0.4 29.2 ± 0.2 6. tri(trimethylgalloyl) quercetin 16.0 ± 2.1 > 200.0 7. tri(triacethylgalloyl) quercetin 32.4 ± 2.8 52.8 ± 4.5 8. tri(acethylcaffeoyl) quercetin 5.8 ± 0.6 35.4 ± 2.7 9. tri(acethylferuloyl) quercetin 6.1 ± 0.4 > 200.0 10. di(prenylferuloyl) quercetin 5.6 ± 0.6 82.8 ± 12.8 11. monoacethylferuloyl quercetin 24.9 ± 1.5 > 200.0 12. trolox 8.7 ± 0.5 34.7 ± 1.0 for qsar purposes (kartasasmita et al., 2009; nemeček et al., 2006; todeschini, 2009), the 3d structures of quercetin and its derivatives were first constructed using software hyperchem v. 8.0.6 (hypercube inc., united states, us), then were optimized using austin model 1 (am1). for optimization of each structure 2000 maximum iterations were applied, followed by conjugate gradient minimization to a root mean square (rms) energy gradient of 0.1 kcal/(å mol). after optimization, seven qsar properties were obtained for each structure (table 3). the inhibitory concentrations (ic50) were predicted using the artificial neural network techniques (statistica v. 8; statsoft inc., united states, us) and these were compared with measured values (table 4). ann is modeled after the (hypothesized) processes of learning in the cognitive system and the neurological functions of the brain, and capable of predicting new observations (on specific variables) from other observations (on the same or other variables) after executing a process of so-called learning from existing data (bocaz-beneventi et al., 2002; gasteiger, 2006; kvasnička et al., 1997). the artificial neural network itself consists of neurons arranged in array-like structures known as the input, hidden and output layers. each layer consists of at least one bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:01 utc nova biotechnologica et chimica 11-1 (2012) 41 neuron. each neuron is represented by a mathematical function which transforms the incoming signals from the layer below into an outgoing signal. this process is repeated till the artificial neural network output (prediction) is produced (kruzlicova et al., 2009). 3. results and discussion ten derivatives of quercetin were characterized by seven descriptors, which have been used in the ann training process. a large amount of networks with different architectures were examined during the training step. a variety of algorithms for different neural network types were automatically tested and the best alternatives were selected. the number of input neurons was set by the number of descriptors (7); output neuron (1) represented calculated ic50 value (expressed in log(1/ic50) form (thakur et al., 2004)). the optimal choice of hidden neurons depends on the problem domain and can be related to the number of the inputs. the final number was found by examining several types of the three layer perceptron (3-mlp) with regard to the corresponding final root mean squared (rms) error. table 3. values of qsar properties for quercetin (no. 1) and ten derivatives (no. 2-11) calculated after structure optimization by software hyperchem. no. hydration energy log p refractivity polarizability molecular mass lumo homo (kcal/mol) (a3) (a3) (amu) (ev) (ev) 1. -32.82 0.28 73.43 28.54 302.24 -1.069324 -8.770832 2. -18.77 12.55 348.48 131.44 1197.39 -1.004567 -8.677681 3. -23.21 8.46 257.46 97.14 899.00 -0.997896 -8.591627 4. -30.79 3.80 252.35 96.32 998.86 -1.310604 -8.890491 5. -23.64 2.27 209.11 79.99 836.72 -1.299082 -8.889960 6. -30.57 3.09 222.43 85.53 884.80 -0.969415 -9.153621 7. -29.54 0.86 264.45 102.82 1136.89 -1.523262 -9.381587 8. -29.20 3.59 261.79 100.08 1040.90 -1.572512 -9.412628 9. -28.45 4.33 247.78 94.31 956.87 -1.367721 -9.197675 10. -25.59 5.97 219.23 82.84 790.82 -0.967023 -8.808840 11. -30.67 1.63 132.88 50.47 520.45 -1.185066 -8.984444 input neurons received the input data variables for each qsar property, the output neuron provided predicted value of the studied objects (inhibitory concentration). statistica’s automatic network search function (ans) was used for primordial ann building. data set was split into 60:40 (60% of samples were utilizing the model and 40% of samples were used for further validation of the model). multi-layer perceptron (mlp) network type was chosen with the specification from 1 to 3 hidden neurons. the learning process was initialised by the bfgs (or quasi-newton) training algorithm. the number of cycles (epochs) was 200 (the network error was calculated bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:01 utc 42 kruzlicova, d. et al. in each training cycle and was used to adjust the weights so that the error was further reduced). weight decay value was set to 10-6 for hidden layer and 10-5 for output layer. a number of 1000 neural networks were trained for (a) prediction of ic50 obtained from abts test as well as for (b) prediction of ic50 determined by dpph test. the network with best performance was found for each case. the optimal network architecture mlp 7-3-1 was found according to the root mean squared error in case of log(1/ic50)abts calculation. the hyperbolic tangent function (tanh) was used for hidden neurons activation as well as for output neuron. training performance reached 0.9996 with the error 0.0000 and the test performance was 1.0000 with the validation error 0.0124. in case of neural network for log(1/ic50)dpph prediction, mlp 7-3-1 with exponential function for hidden neurons activation and with logistic function for output neuron activation was the network with the best prediction performance. training performance was 0.9969 with training error 0.0005; validation performance was 0.8591 with the validation error 0.0268. computed values for recalculated ic50 are listed in table 4. table 4. comparison of measured and predicted values of antioxidant activity expressed as inhibitory concentration (ic50). abts and dpph tests were used for experimental determination of ic50 values. and calculations of predicted values were made by using artificial neural networks. ic 50 (µm) abts test dpph test name measured predicted rsd (%) measured predicted rsd (%) tri(diprenylcaffeoyl) quercetin 8.4 8.3 b) -1.0 200.0 200.0 b) 0.0 di(diprenylcaffeoyl) quercetin 5.5 5.3 a) -3.4 160.9 153.4 a) -4.7 di(diacethylcaffeoyl)mono(monoacethylcaffeoyl) quercetin 10.4 6.7 b) -35.4 200.0 200.0 b) 0.0 monoacethyldi(diacethylcaffeoyl) quercetin 4.1 4.1 a) 0.0 29.2 29.7 a) 1.7 tri(trimethylgalloyl) quercetin 16.0 16.1 a) 0.7 200.0 200.0 a) 0.0 tri(triacethylgalloyl) quercetin 32.4 32.3 a) -0.2 52.8 52.5 a) -0.6 tri(acethylcaffeoyl) quercetin 5.8 6.7 b) 15.8 35.4 72.5 b) 104.8 tri(acethylferuloyl) quercetin 6.1 6.2 b) 2.0 200.0 117.8 b) -41.1 di(prenylferuloyl) quercetin 5.6 5.8 a) 3.6 82.8 95.1 a) 14.8 monoacethylferuloyl quercetin 24.9 24.7 a) -0.9 200.0 198.7 a) -0.7 a) samples were used for building of the prediction model b) samples were used for validation of the model the fact, that models are suitable for prediction of antioxidant activity of untested derivatives, is confirmed by predictive squared correlation coefficient q2 (consonni et al., 2010). the value of q2 was 0.9745 for log(1/ic50)abts predictions and 0.9907 for log(1/ic50)dpph predictions. it is important to notice, that the use of ten derivatives is not a lot for data set build up, thus better results can be obtained with the study of data matrix containing more derivatives prepared in the future. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:01 utc nova biotechnologica et chimica 11-1 (2012) 43 4. conclusions it has been demonstrated that technique of artificial neural network can be a very useful tool for study of relationship between structure and antioxidant activity. they provide a new alternative to the most commonly used methods for developing predictive models, which are often limited by strict assumptions of normality, linearity, variable independence etc. logarithm of inhibitory concentration is one of the most common forms of the antioxidant activity used as dependent variable for the neural network model. main advantages of these modern approaches are (a) robust performance in dealing with noisy or incomplete input patterns, (b) ability to detect complex nonlinear relationships between dependent and independent variables, (c) ability to detect all possible interactions between predictor variables, (d) and the availability of multiple training algorithms. these advantages make anns superior and applicable in various predictions for the most of biological and clinical data. acknowledgement: research is supported by the agency of the ministry of education, science, research and sport of the slovak republic for the structural funds of eu, project no. itms 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223231. fan, d., zhou, x., zhao, ch., chen, h., zhao, y., gong, x.: antiinflammatory, antiviral and quantitative study of quercetin-3-o-β-d-glucuronide in polygonum perfoliatum l. fitoterapia, 82, 2011, 805-810. floegel, a., kim, d.-o., chung, s.-j., koo, s.i., chun, o.k.: comparison of abts/dpph assays to measure antioxidant capacity in popular antioxidant-rich us foods. j. food comp. anal., 24, 2011, 1043-1048. gasteiger, j.: the central role of chemoinformatics. chem. int. lab. sys., 82, 2006, 200-209. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:01 utc 44 kruzlicova, d. et al. kartasasmita, r.e., herowati, r., gusdinar, t.: docking study of quercetin derivatives on inducible nitric oxide synthase and prediction of their absorption and distribution properties. j. app. sci., 23, 2009, 3098-3104. kruzlicova, d., mocak, j., balla, b., petka, j., farkova, m., havel, j.: classification of slovak white wines using artificial neural networks and discriminant techniques. food chem., 112, 2009, 1046-1052. kvasnička, v., beňušková, ľ., pospíchal, j., farkaš, i., tiňo, p., & kráľ, a.: introduction to theory of neural networks. iris publisher, bratislava, 1999. markovits, j., linassier, c., fosse, p., couprie, j., pierre, j., jacquemin-sablon, a., saucier, j.m., le pecq, j.b., larsen, a.k.: inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian dna topoisomerase ii, cancer res., 49,1989, 5111-5117. nemeček, p., ďurčeková, t., mocák, j., lehotay, j., waisser, k.: štúdium vzťahov medzi biologickou aktivitou a fyzikálnochemickými vlastnosťami potenciálnych antituberkulotík. nova biotechnol., vi-i, 2006, 37-47. pick, a., müller, h., mayer, r., haenisch, b., pajeva, k.i., weigt, m., bönisch, h., müller, e.ch., wiese, m.: structure–activity relationships of flavonoids as inhibitors of breast cancer resistance protein (bcrp). bioorg. med. chem. 19, 2011, 2090-2102. skaper, s.d., fabris, m., ferrari, v., dalle carbonare, m., leon, a.: quercetin protects cutaneous tissue-associated cell types including sensory neurons from oxidative stress induced by glutathione depletion: cooperative effects of ascorbic acid. free radic. biol. med., 22, 1997, 669-678. tian, x.-j., yang, x.-w., yang, x., wang, k.: studies of intestinal permeability of 36 flavonoids using caco-2 cell monolayer model. int. j. pharm., 367, 2009, 58-64. thakur, a., vishwakarma, s., thakur, m.: qsar study of flavonoid derivatives as p56lck tyrosinkinase inhibitors. bioorg. med. chem., 12, 2004, 1209-1214. todeschini, r., consonni, v., gramatica, p.: 4.05 chemometrics in qsar. comprehensive chemometrics. chemical and biochemical data analysis, vol. 4, 2009, 129-172. presented at the 3rd international scientific conference “applied natural sciences 2011”, october 5–7, 2011, častá papiernička, slovak republic. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 12:01 utc << /ascii85encodepages false 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can be opened with acrobat and adobe reader 5.0 and later.) >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [2834.650 2834.650] >> setpagedevice microsoft word machalova et al. nbc 2015-2.doc 158 nova biotechnologica et chimica 14-2 (2015) doi 10.1515/nbec-2015-0024 © university of ss. cyril and methodius in trnava comparison of cd2+ biosorption and bioaccumulation by bacteria – a radiometric study linda machalová, martin pipíška, zuzana trajteľová, miroslav horník department of ecochemistry and radioecology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (martin.pipiska@ucm.sk) abstract: in this work, bioaccumulation and biosorption characteristics of cd2+ ions by both dead and living non-growing biomass of gram-positive bacteria kocuria palustris and micrococcus luteus isolated from spent nuclear fuel pools were compared. the radioindicator method with radionuclide 109cd was used to obtain precise and reliable data characterizing cd compartmentalization in bacterial cells. the following cellular distribution of cd in living non-growing biomass after 4 h incubation in solutions containing different concentration of cd2+ ions (100, 250, 500, 750 and 1000 µmol/l) spiked with 109cdcl2 under aeration at 30 °c were obtained: in m. luteus almost 85 % of cd was localized on the cell surface and 15 % in cytoplasm. similarly, in k. palustris 83 % of cd was localized on the cell surface and 17 % in cytoplasm. the data were obtained by gamma spectrometry of extracts and solids after sequential extraction of biomass with 5 mm ca(no3)2 and 20 mm edta. biosorption of cd by non-living bacterial biomass is a rapid process strongly affected by solution ph and as was confirmed by ftir analysis beside carboxylate ions also other functional groups such as amino and phosphate contribute to cd binding by bacterial cell surfaces. maximum sorption capacities qmax (μmol/g) calculated from the langmuir isotherm were 444 ± 15 μmol/g for m. luteus and 381 ± 1 μmol/g for k. palustris. key words: 109cd, bioaccumulation, biosorption, micrococcus, kocuria, 1. introduction rapid industrialization and urbanization have resulted in the generation of large quantities of aqueous effluents, many of which contain high level of toxic pollutants such as heavy metals, organic compounds and radionuclides (remenárová et al., 2012). from current research activities is evident that various technologies based on interactions between pollutants and biological systems in a contaminated environment are investigated (chojnacka, 2010; singh and tripathi, 2007). biosorption and bioaccumulation have been investigated as promising processes of toxic metal removal. such processes appear as ideal candidates for replacing conventional methods for metal removal from wastewaters such as chemical precipitation, electrowinning, membrane separation, evaporation and ion-exchange, especially in cases when low concentrations of metals are present in wastewaters (remenárová et al., 2012). biosorption is a surface phenomenon where one or more physico-chemical mechanisms are involved in metal uptake by both dead and living biomass. however, bioaccumulation is an intracellular metabolically dependent metal accumulation and involves metal binding on intracellular compounds, intracellular precipitation, methylation and other mechanisms. bioaccumulation can also be regarded as a second bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc nova biotechnologica et chimica 14-2 (2015) 159 part of the metal sequestering process by living biomass (kaduková and virčíková, 2005). cambel et al. (2002) stated that the formation of complexes between metals and anionic functional groups on cell surfaces is viewed as a prerequisite for uptake of metals by the organism. once surface sorption occurs, the metal may be transported into the cytoplasm. both processes can be considered as two major biological techniques for the removal of toxic metals. from this point of view the increased interest of microorganisms application in metal sequestration was recently observed (chojnacka, 2007). however, only a few experimental studies dealing with comparison of metal biosorption and bioaccumulation by microrganisms can be found. kaduková and virčíková (2005) revealed that the cu binding capacity of living cells of algae chlorella kessleri is significantly lower than the capacity of dead cells. on the contrary vargas-garcía et al. (2012) observed that fungi isolated from compost showed a higher efficiency against cd, pb, ni, cr, zn and the predominant removal mechanism was intracellular accumulation, which made growing cells more efficient than dead biomass as detoxifying agents. also uptake of the thallium by fungus neosartorya fischeri is highly enhanced when the active biomass is used (urík et al., 2010). alam and ahmad (2013) found that biosorption of cd2+, ni2+, cu2+ and zn2+ ions was higher with the non-growing biomass of bacteria exiguobacterium sp. zm-2 compared to the dried biomass. it is evident that discussion whether to employ the living or dead cells for bioremediation still take place in literature. the objectives of the present study were to determine effects of cd2+ ions on growth of gram-positive bacteria kocuria palustris and micrococcus luteus isolated from spent nuclear fuel pools and to investigate the differences between cd2+ ions biosorption and bioaccumulation by dead and live non-growing bacteria. the radioindicator method with radionuclide 109cd was used to obtain precise and reliable data characterizing cd compartmentalization in bacterial cells. 2. material and methods 2.1 bacteria isolation and cultivation gram-positive bacteria were isolated from pool water in the interim spent nuclear fuel storage facility in javys a.s. in jaslovské bohunice, slovak republic. isolates were further identified using 16s rdna methods as kocuria palustris and micrococcus luteus (tišáková et al., 2013). bacteria were grown on dev nutrient agar at 22 ± 2°c (merck, germany) and maintained at 4°c. bacteria were cultivated in nutrient broth (peptone 10 g/l, beef extract 10 g/l, nacl 5 g/l, ph 7.4 ± 0.2) on a rotary shaker (biosan es 20) for 24 32 h at 30 °c. subsequently, cells were harvested at stationary phase by centrifugation (4500 rpm, 5 min) and rinsed two times with deionized water. such bacterial pellet was used in bioaccumulation experiments. bacterial pellet was also dried for 24 h at a maximum of 60°c to avoid the degradation of binding sites. dried biomass was crushed into a fine powder, sieved and used in biosorption experiments. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc 160 machalová, l. et al. 2.2 inhibition activity a micro dilution method was performed in 96-well sterile microplates to determine inhibition activity and id50 values of cd 2+ ions for k. palustris and m. luteus. briefly, 100 µl of steriled deionized water was added to each well. to wells in column 1 was added 100 µl of cdcl2 solution (4 mmol/l) and mixed well with a micropipette. subsequently, 100 µl of solution from column 1 was transferred into column 2 yielding two-fold serial dilution. procedure was repeated down to column 12. actively growing bacteria (100 μl) from subculture was added to each of the wells except the row 1 which serves as control. the microplate was placed on rotary shaker (30 °c, 140 rpm) and cultivated for 48 h. in time intervals, the optical density (od) was measured at λ= 600 nm using microplate reader elx800 (biotek, usa). suspensions of m. luteus or k. palustris were prepared from 72 h subculture in fresh nutrient medium (10 g peptone, 10 g beef extract, 5 g nacl per liter). subculture (5 ml) was put into 30 ml of fresh nutrient broth and placed on rotary shaker (30°c, 140 rpm) for 30 min. 2.3 bioaccumulation and cell compartmentalization of cd2+ ions living bacterial cells harvested from stationary phase (see section 2.1) were added to 10 ml of cd solution (100 µmol/l cdcl2, ph 6.0) spiked with 109cd (37.1 kbq/l) in erlenmayer flasks. flasks were shaken at 150 rpm (rotary shaker biosan es 20) at 25°c for 4 h. in time intervals the content of the flasks was centrifuged (4 500 rpm, 4 min), bacterial pellet was washed in deionized water and radioactivity of cells was measured using scintillation gamma-spectrometry. biomass dry weight was estimated after drying for 24 h at 60 °c. cadmium uptake was calculated according to (1): m v ccq f )( 0 −= (1) where q is the cd uptake (μmol/g), c0 and cf are the initial and the final cd concentrations in solution (μmol/l), v is volume (l) and m is the amount of bacterial biomass (d.w.; given in grams). similarly, living bacterial cells harvested from stationary phase were added to 10 ml of cd solution with initial concentration c0 = 100, 250, 500, 750, 1000 µmol/l of cdcl2 labelled with 109cd (37.1 kbq/l) in erlenmayer flasks and ph was adjusted to 6.0. flasks were shaken at 150 rpm at 25°c. after 1 h the content of the flasks was centrifuged (4 500 rpm, 4 min), bacterial pellet was washed in deionized water and radioactivity of cells was measured. biomass dry weight was estimated after drying for 24 h at 60 °c. metal uptake was calculated according to equation (1). cell compartmentalization of cd was determined using modified sequential extraction procedure according to pabst et al. (2010) and huang et al. (2014). metals associated with exchange sites on the cell surface were extracted by resuspending the cell pellet in 10 ml of 5 mm ca(no3)2 for 15 min with gentle shaking at 40 rpm. the suspension was centrifuged (4 500 rpm) and the radioactivity of cells was measured. subsequently, the cell pellet was treated with 10 ml of 20 mm bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc nova biotechnologica et chimica 14-2 (2015) 161 edta (tetrasodium salt dehydrate) for 1 min to solubilise. the edta remove cd tightly bound to cell surface. after centrifugation (4 500 rpm, 4 min) supernatant radioactivity was measured. the radioactivity of remaining pellet was also measured and represents intracellularly localized cd. all tubes with biomass were weight between extraction steps and at the end of experiments dried to constant weight. cadmium uptake was calculated according to (1). all experiments were performed in duplicate series. the langmuir model (see below) was used to analyse the distribution of cd associated with cell compartments of k. palustris and m. luteus. 2.4 biosorption kinetics dried biomass of k. palustris and m. luteus was added to 8 ml of cd solution (1000 µmol/l cdcl2, 109cd 58.1 kbq/l). solution ph was adjusted to 6.0 and flasks were incubated on rotary shaker (150 rpm) at 25°c. at the time intervals 60, 120, 240, 360, 1440 min the content of the flask was centrifuged (4 500 rpm, 4 min) and the biomass radioactivity was measured. all experiments were performed in duplicate series. the cadmium uptake was calculated according to (1). 2.5 biosorption equilibrium dried bacterial biomass was added to 8 ml of cd solution with initial concentrations c0 = 100, 500, 1000, 2000 and 4000 µmol/l cdcl2 labelled with 109cd (37.1 kbq/l) and ph was adjusted to 6.0. flasks were incubated on rotary shaker (150 rpm) at 25°c. after 4 h the content of the flasks was centrifuged (4 500 rpm, 4 min) and the biomass radioactivity was measured. all experiments were performed in duplicate series. the cadmium uptake was calculated according to (1). equilibrium data were analysed using adsorption langmuir (2) and freundlich (3) isotherm models. eq eq eq bc cbq q + = 1 max (2) )/1( n eqeq kcq = (3) where qmax represents the maximum cd sorption capacity of bacterial biomass, b is a constant related to the energy of sorption. k and 1/n values are the freundlich constants referring to sorption capacity and intensity of sorption, respectively. to calculate the qmax values and the corresponding parameters of isotherms non-linear regression analysis was performed by origin 7.0 professional (originlab corporation, northampton, usa). 2.6 effects of ph to analyze the influence of ph, dried bacterial biomass was shaken in cd2+ solutions (c0 = 1000 μm) of desired ph spiked with 109cd (48.3 kbq/l) for 4 h on a bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc 162 machalová, l. et al. rotary shaker at 150 rpm and 25°c. in order to eliminate interference of buffer components on cd biosorption, the non-buffered solutions in deionised water were adjusted to the desired ph values by adding 0.5 m hcl or 0.1 m naoh. 2.7 speciation modeling prediction of the speciation of cadmium in the aqueous systems as a function of total salt concentration and solution ph was performed using the visual minteq (version 3.0) program. visual minteq is a chemical equilibrium program that has an extensive thermodynamic database for the calculation of metal speciation, solubility and equilibria (gustaffson, 2004). all data sets were calculated considering the carbonate system naturally in equilibrium with atmospheric co2 (pco2 = 38.5 pa). 2.8 radiometric analysis the gamma spectrometric assembly using the well type scintillation detector 54bp54/2-x, nai(tl) (scionix, the netherlands) and the data processing software scintivision 32 (ortec, usa) were used for 109cd determination at the energy of γ photons 109cd – 88.04 kev. standardized 109cdcl2 solution (3.857 mbq/ml, cdcl2 50 mg/l in 3 g/l hcl) was obtained from the czech institute of metrology, prague (czech republic). 2.9 ftir analysis ftir analysis was carried out to identify chemical functional groups on dried bacterial biomass and to explain the cd biosorption mechanism. ftir analysis was performed by affinity 1 spectrometer (shimadzu, japan). samples of dried k. palustris and m. luteus biomass before and after cd biosorption (c0 = 4 mm cdcl2; ph 6.0; 4 h) were mixed with kbr at a ratio 1:100 for making pellets and the ftir spectra were obtained within the range 400-4000 cm-1. 3. results and discussion 3.1 effect of cd2+ ions on bacterial growth the toxic effects of an increasing concentration of cd2+ ions on the growth of g+ bacteria k. palustris and m. luteus, isolated from deionized water in interim spent nuclear fuel facility in javys a.s., jaslovské bohunice, slovak republic (tišáková et al., 2013), were studied using the microplate dilution method. 3d diagrams of bacterial growth curves in the presence of various cd2+ ions concentrations in cultivation medium are shown on fig. 1a, b. such representation helps finding detailed description of the growth difference between k. palustris and m. luteus. without the presence of cd2+ ions both species showed logarithmic growth during the majority of the incubation time (not shown). both bacteria were able to grow at 0.063 mm cdcl2, while concentrations from 0.125 to 2 mm were highly toxic bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc nova biotechnologica et chimica 14-2 (2015) 163 and significantly inhibited the bacterial growth in comparison with growth of control culture. 05 001 000 150 020 002 500 t (min) 2 0.2 0.02 c0 cd mm 0 0 0.08 0.08 0.16 0.16 0.24 0.24 0.32 0.32 0.4 0.4 a t a 0 60 0 nm a t a 0 60 0 nm 05 001 000 150 020 002 500 t (min) 2 0.2 0.02 c0 cd mm 0 0 0.05 0.05 0.1 0.1 0.15 0.15 0.2 0.2 0.25 0.25 0.3 0.3 0.35 0.35 0.4 0.4 a t a 0 60 0 nm a t a 0 60 0 nm fig. 1. 3d diagrams of growth curves of bacteria kocuria palustris (a) and micrococcus luteus (b) in nutrient broth with cd2+ ions. the x-axis is the cd2+ concentration (0.063 – 2 mm cdcl2) in medium, the yaxis is the incubation time and the z-axis is the optical density measured at λ = 600 nm. cultivation in microplate on rotary shaker (140 rpm) at 30°c. a0 = 0.122 ± 0.008 (k. palustris) and a0 = 0.100 ± 0.003 (m. luteus). individual points represent mean (n = 3). points represent experimental data of absorbance (at – a0). in case of k. palustris, the long lag phase was observed at 0.125 mm cdcl2, while cadmium concentrations from 0.25 to 2 mm fully inhibited the bacterial growth (fig. 1a). from fig. 2b it is evident that cd concentrations from 0.125 to 0.5 mm reduced the lag phase and m. luteus grew slowly during a studied cultivation period. almost complete growth inhibition of m. luteus was observed at concentrations 1 and 2 mm cdcl2, respectively. similarly, the major elongation of lag phase of bacillus cereus rc-1 under higher cadmium concentrations was observed by huang et al. (2014). the inhibition concentration ic50 values (mm) of cd obtained after 48 h cultivation are shown in table 1. cd ic50 for m. luteus was 5 times higher than the ic50 for k. palustris indicating slightly higher resistance of m. lutues toward cd ions. ic50 values of cd for both isolates are comparable with ic50 for e. coli (adam et al., 2014) and significantly lower in comparison with cd ic50 values for stenotrophomonas sp. and ochrobactrum sp. isolated from metal acclimatized activated sludge (bestawy et al., 2013). table 1. inhibition of bacterial growth by cdcl2 as ic50 (mm) concentrations after 48 h cultivation at 30 °c. experimental data see in fig. 1. bacteria ic50 (mm) references kocuria palustris 0.02 this work micrococcus luteus 0.10 this work escherichia coli nctc 13216 0.04 adam et al. (2014) stenotrophomonas sp. 2.8 bestawy et al. (2013) ochrobactrum sp. 1.3 bestawy et al. (2013) 3.2 cd2+ uptake by living bacteria when living bacteria are used for metal removal the uptake process consists of metabolism independent extracellular binding (biosorption) and metabolism dependent a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc 164 machalová, l. et al. intracellular uptake (bioaccumulation). to obtain precise quantitative data characterizing the location of cd in bacterial cell compartments a radioindicator method with radionuclide 109cd was used in our experiments. at the ph 6.0, cd was present in solutions predominantly as the free ions cd2+ (87.5 %) and as a complex cdcl+ (12.6 %) (determined by visual minteq speciation program). 0 1 2 3 4 0 20 40 60 80 100 120 140 160 180 q t ( μ m ol /g d .w .) t (h) total removal of cd surface cd biosorption intracellular cd bioaccumulation m. luteus fig. 2. kinetics of cd removal (c0 = 100 µmol/l cdcl2, 37.1 kbq/l 109cdcl2) by living non-growing cells of m. luteus at ph 6.0 and 25 °c. error bars represent standard deviation of the mean (± sd, n = 2). cd2+ ions uptake by living non-growing cells of g+ bacteria k. palustris and m. luteus was time (fig. 2 and 3) and concentration (fig. 4 and 5) dependent process. initial rapid phase of cd uptake followed by the slower phase was observed in both bacteria. sequential extraction of bacterial biomass (5 mm ca(no3)2 and 40 mm na2edta) used for cd cell compartmentalization indicated that the prevailing part of uptaken cd2+ ions is associated with cell surface in both bacteria (fig. 2, 3). therefore surface complexation and electrostatic attractions played a key role in cd removal from solution by living non-growing bacterial cells. however, both bacteria exhibit also intracellular accumulation of cd2+ ions. at initial cd concentration of 100 μmol/l, after 4 h incubation up to 35 µmol/g (m. luteus) and 25 µmol/g (k. palustris) of cd2+ ions were localized in cytoplasm. in case of k. palustris (fig. 3) a slight decrease of cd bound on cell surface from 139 ± 9 µmol/g to 113 ± 4 µmol/g and simultaneous increase of cd in cytoplasm from 18 ± 1 µmol/g to 25 ± 4 µmol/g was observed. although the total removal of cd (extracellular + intracellular sequestration) by living non-growing bacterial cells at initial cd concentration of 100 µmol/l after 4 h incubation was higher in k. palustris (138 µmol/g) in comparison with m. luteus (100 µmol/g), m. luteus exhibited higher intracellular cd accumulation. this indicates higher resistance of m. luteus towards cd what is in conformity with the obtained cd ic50 values (table 1). observed differences in uptake capacities are not surprising and can be attributed primarily to differences in cell morphology of k. palustris and m. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc nova biotechnologica et chimica 14-2 (2015) 165 luteus and also to different strategies of resistance to cd2+ ions. hrynkiewicz et al. (2015) pointed out that the ability of soil bacteria to accumulate cd2+ ions is strain specific and the variation within a species can exceed the variation between different species or genera. 0 1 2 3 4 0 20 40 60 80 100 120 140 160 180 200 220 q t ( μ m ol /g d .w .) t (h) total removal of cd surface cd biosorption intracellular cd bioaccumulation k. palustris fig. 3. kinetics of cd removal (c0 = 100 µmol/l cdcl2, 37.1 kbq/l 109cdcl2) by living non-growing cells of k. palustris at ph 6.0 and 25 °c. error bars represent standard deviation of the mean (± sd, n = 2). cd biosorption and bioaccumulation relationship were also investigated as a function of the cd initial concentration in solution. to describe the association of cd2+ ions with extracellular binding sites (biosorption) and cytoplasm (bioaccumulation) the langmuir isotherm model (eq. 2) was used. results demonstrated that with increasing concentration of cd2+ ions in solution extracellular binding (biosorption) of cd increased in both bacteria (fig. 4 and 5). obtained curves are typical for metal biosorption by dead biomass of bacteria, fungi, algae and others (hetzer et al., 2006; hrynkiewicz et al., 2015). m. luteus showed significantly higher uptake values at higher cd initial concentrations (500 – 1000 µmol/l) than k. palustris (fig. 4 and 5). maximum cd surface binding capacities qex max calculated from langmuir model were 775 ± 111 µmol/g d.w. for m. lutues and 430 ± 37 µmol/g d.w. for k. palustris (table 2). qex values indicate that up to 85 % (m. luteus) and 83 % (k. palustris) of cd was associated with exchangeable binding sites and/or non-covalently bound to cell surface polymers including proteins and membrane associated lipocarbohydrates of g+ bacteria. the role of functional groups in cd binding will be discussed separately. according to literature review also cd microprecipitation on cell surface in the form of cadmium hydroxides and phosphates can not be neglected (kotrba et al., 2010; guibaud et al., 2006). intracellular accumulation of cd2+ ions (qin) increased only slightly with increasing initial cd concentration (fig. 4 and 5). the amount of cd in m. luteus cytoplasm was 2 times higher than that of k. palustris in the studied cd concentration bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc 166 machalová, l. et al. range (100 to 1000 µmol/l). qin max values calculated from langmuir model were 157 ± 25 µmol/g d.w. for m. lutues and 74.9 ± 7.1 µmol/g d.w. for k. palustris (table 2). 0 100 200 300 400 500 600 0 100 200 300 400 500 600 700 m. luteus ceq (μmol/l) q ( μ m ol /g ) qex extracellular biosorption qin intracellular bioaccumulation fig. 4. langmuir isotherms of cd associated with cell surface and cd localized in intracellular space of living non-growing cells of m. luteus. individual points represent experimental data; curves represent the calculated values from langmuir model. error bars represent standard deviation of the mean (± sd, n = 2). 0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 700 800 q ( μ m ol /g ) ceq (μmol/l) k. palustris qex extracellular cd biosorption qin intracellular cd bioaccumulation fig. 5. langmuir isotherms of cd associated with cell surface and cd localized in intracellular space of living non-growing cells of k. palustris. individual points represent experimental data; curves represent the calculated values from langmuir model. error bars represent standard deviation of the mean (± sd, n = 2). as has been previously reported, bacteria accumulated cd2+ ions via uptake systems for essential divalent metals. e.g. in escherichia coli, cd2+ ions enter cells via a zn2+ transport system (laddaga and silver, 1985) and in g+ bacteria (lactobacillus plantarum, bacillus subtilise) through transport systems for mn2+ ions bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc nova biotechnologica et chimica 14-2 (2015) 167 (hao et al., 1999). pabst et al. (2010) observed that 90 % of the initial cd was associated with the surface of the cells of gbacteria pseudomonas putida corvallis and pseudomonas putida kt2440 and only minor part was observed in cytoplasm. in another study with g+ bacteria, huang et al. (2014) revealed that when cd solution concentrations were less than 165 µmol/l intracellular accumulation of cd by growing bacillus cereus rc-1 was higher than surface adsorption. above this concentration the cd surface sorption increased significantly. authors hypothesized that at higher cd concentrations the efflux mechanism might be functional in order to maintain intracellular cd below a toxic threshold. kiesling (1997) proposed that some bacteria (pseudomonas, klebsiella, arthrobacter) used polyphosphates localized in granules to detoxify cadmium transported into cytoplasm. however, we suppose that at lower cd concentration the cell surface binding (biosorption) of cd2+ ions could be the main detoxification mechanism in both bacteria studied. these findings are in an agreement with results obtained by hrynkiewicz et al. (2015). table 2. langmuir sorption isotherms and parameters for cd2+ ions in specific cell compartments of k. palustris and m. luteus calculated using non-linear regression analysis. bacteria compartmentalization of cd qmax [μmol/g] b [l/μmol] r 2 % cd associated in cell compartments extracellular 430 ± 37 0.046 ± 0.020 0.860 85.2 kocuria palustris intracellular 74.9 ± 7.1 0.056 ± 0.027 0.818 14.8 extracellular 775 ± 111 0.014 ± 0.006 0.927 83,2 micrococcus luteus intracellular 157 ± 25 0.059 ± 0.038 0.670 16.8 3.3 cd2+ uptake by non-living bacteria 3.3.1 cd2+ uptake kinetics the kinetic studies were realized also using dried bacterial biomass (fig. 6) and as expected we found that the biosorption of cd2+ ions by k. palustris and m. luteus is a rapid process. at initial phase driving force is high and available high affinity binding sites on biomass are occupied. in case of m. luteus residual sites with lower affinity are occupied slowly during the next 2 h. a slight decrease of cd uptake was observed in case of k. palustris. in both bacteria the final equilibrium was reached within 200 min and after this time there was no considerable increase until the end of experiments. m. luteus exhibited slightly higher cd sorption (266 ± 3 µmol/g d.w.) in comparison with k. palustris (235 ± 4 µmol/g d.w.). based on obtained results, subsequent cd sorption experiments were realized with an equilibration time 4 h. these findings are in agreement with other studies of cd biosorption by bacterial surfaces. alam and ahmad (2013) revealed that cd2+ ions biosorption by dried biomass of gram-positive exiguobacterium sp. zm-2 was rapid during the first 15 min bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc 168 machalová, l. et al. and equilibrium was reached after 120 min. full sorption of cd by thermophilic bacterium anoxybacillus flavithermus occurred within 45 min (burnett et al., 2006). the fast rate of cd uptake suggests that the biosorption is dependent solely on passive cadmium-sequestering processes such as electrostatic and chemical attraction between cd2+ ions and the negatively charged functional groups present on the bacterial cell wall, microprecipitation and physical entrapment. 0 200 400 600 800 1000 1200 1400 1600 0 50 100 150 200 250 300 q eq (μ m ol /g d w ) t (min) micrococcus luteus kocuria palustris fig 6. kinetics of cd (1000 µmol/l cdcl2, 58.1 kbq/l 109cd) biosorption by dried biomass of k. palustris biomass (2.5 g/l d.w.) and m. luteus (2.5 g/l d.w.) at 20 °c and ph 6.0. error bars represent standard deviation of the mean (± sd, n = 2). 3.3.2 effect of ph the experimental data show that biosorption increased with increasing ph and maximum uptake of cd by both bacteria occurred at ph 7.0 (k. palustris 256 ± 2 µmol/g d.w.; m. luteus 273 ± 5 µmol/g d.w.). slightly lower biosorption was observed from ph 4.0 to 6.0 and negligible at ph 2.0. with increasing ph, deprotonation of binding sites increased and cd2+ cations are electrostatically attracted by negatively charged functional groups on bacterial cell wall. at lower ph values a strong competition between h+ and cd2+ during occupation of binding sites occurs. moreover, extreme ph values (low and high) can damage the structure of dried bacterial biomass and therefore decrease cd uptake. boyanov et al. (2003) using x-ray adsorption spectroscopy revealed that cd2+ ions biosorption onto g+ bacterium bacillus subtilis at ph 3.4 is predominated by phosphoryl ligands, whereas carboxylic ligands are the dominant binding sites in the ph range of 5.0 to 7.8. similarly, the maximum uptake of cd by gbacterium acidiphilium symbioticum h8 was observed at ph 6.0 (chakravarty and banerjee, 2012). however, ph affects not only binding site dissociation on bacterial surfaces, but also the solution chemistry of the cadmium. calculation by visual minteq speciation program showed that cadmium predominantly exists as cations cd2+ (~88%) and cdcl+ (~10%) within ph ranging from 2.0 to 7.5 (fig. 7). also other cadmium ionic forms such as cdoh+, cd2oh 3+, cd(oh)3 and cd(oh)4 2are present bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc nova biotechnologica et chimica 14-2 (2015) 169 in solution between ph 8.0 and 12.0. the concentration of cd2+ starts to decrease at ph > 8.0 and the precipitation of cd started at ph > 9.0. maximum cd biosorption was observed at ph 7.0 when cd2+ cations represent 87.5 % of total ionic forms of cadmium. however, cdcl+ is a complex that forms under experimental conditions studied (~10%) and it is expected to adsorb onto negatively-charged functional groups too. mclean et al. (2013) confirmed the uptake of cdcl+ complexes onto cell surface of pseudomonas putida. 2 4 6 8 10 12 0 20 40 60 80 100 m. luteusk. palustris ph [m e] fo rm /[m e] to ta l*1 00 ( % ) cd2+ cdcl+ precipitation forms of cd other ion forms of cd biosorption cd 0 50 100 150 200 250 300 q eq (μm ol/g dw ) fig. 7. effect of ph on the biosorption of cd (1000 µmol/l, 43.8 kbq/l 109cdcl2) by dried biomass of k. plaustris (2.5 g/l, dw) and m. luteus (2.5 g/l, dw) and cd speciation in solution. error bars represent standard deviation (sd) of the mean (n = 2). theoretical cd speciation was calculated using visual minteq version 3.0 with initial conditions: 1000 μmol/l cdcl2, t = 20°c, pco2 = 38.5 pa. 3.3.3 equilibrium cd2+ biosorption generally known langmuir (eq 2) and freundlich (eq 3) isotherms were fitted to the equilibrium data for cd2+ ions biosorption by non-living biomass of k. palustris and m. luteus. isotherm curves and parameters of the models determined from the experimental data using non-linear regression analysis are reported in fig. 8a, b and table 3. the langmuir isotherm fits the data of cd2+ ions biosorption by both bacteria better than the freundlich isotherm, as is demonstrated by higher values of the coefficient of determination (r2), by the lower of the sum of squares (rss) values obtained and by the more homogeneous standard deviation of each observed parameter (table 3). also other authors found that the sorption of cd2+ ions by pseudoalteromonas sp. scse709-6 (zhou et al., 2014), bacillus cereus rc-1 (huang et al., 2013), acidiphilium symbioticum h8 (chakravarty and banerjee, 2012) and activated sludge (remenárová et al., 2012) was well described using the langmuir isotherm. the maximum sorption capacity qmax of m. luteus obtained from the langmuir isotherm for cd2+ was 444 ± 15 μmol/g. a lower value of qmax was observed in the case of k. palustris biomass, i.e. 381 ± 1 μmol/g. as was mentioned in the case of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc 170 machalová, l. et al. living bacteria, observed differences in uptake capacities can be attributed to differences in cell morphology of k. palustris and m. luteus. the affinity constant b of the isotherms corresponds to the initial gradient, which indicates the bacterial biomass affinity at low concentrations of cd ions. a higher initial gradient corresponds to a higher affinity constant b (sheng et al., 2007). from fig. 8a and 8b it is evident that both isotherms have similar behaviour at lower equilibrium concentrations. the difference in the b values 0.0042 ± 0.0005 l/μmol (m. luteus) and 0.0083 ± 0.0001 l/μmol (k. palustris), indicates higher affinity of k. palustris for cd ions, although m. luteus exhibited higher maximal capacity (table 3). 0 500 1000 1500 2000 2500 3000 0 50 100 150 200 250 300 350 400 450 langmuir freundlich q eq ( μ m ol /g s . h m .) ceq (μmol/l) a 0 500 1000 1500 2000 2500 3000 3500 0 50 100 150 200 250 300 350 400 450 langmuir freundlich q eq ( m m ol /g s .h m ) ceq (μmol/l) b fig. 8. adsorption isotherm of cd2+ by dried biomass of m. luteus and k. palustris (2.5 g/l) at 20 °c and ph 6.0 according to langmuir and freundlich with experimental points. error bars represent the standard deviation of the mean (± sd, n = 2). table 3. langmuir and freundlich parameters for the biosorption of cd2+ ions by dried biomass of m. luteus and k. palustris obtained by non-linear regression analysis. langmuir freundlich bacteria qmax [μmol/g] b [l/μmol] r 2 rss k [l/g] 1/n r 2 rss m. luteus 444 ± 15 0.0042 ± 0.0005 0.995 523 33.0 ± 20.25 0.32 ± 0.08 0.888 10747 k. palustris 381 ± 1 0.0083 ± 0.0001 0.999 6.8 52.0 ± 26.0 0.25 ± 0.07 0.880 9153 despite the fact that the langmuir isotherm offers no insights into the biosorption mechanism (liu and liu, 2008) it is still a convenient tool for comparing data on a quantitative basis (qmax, b). obtained results show that the maximum specific uptake capacities qmax of cd were higher for living non-growing biomass (table 2) in comparison with non-living (dried) biomass (table 3) of both bacteria. similar to our findings, alam and ahmad (2013) demonstrated higher efficiency of non-growing biomass of exiguobacterium sp. zm-2 to adsorb cd2+ ions than dried biomass. authors pointed out that some part of cd2+ ions was transported into cytoplasm of non-growing cells what was also confirmed in our study (fig. 4 and 5). contrary, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc nova biotechnologica et chimica 14-2 (2015) 171 huang et al. (2013) based on qmax and b values inspection revealed that dead biomass of bacillus cereus rc-1 was more efficient in adsorbing cd2+ ions than live biomass. 3.4 the role of functional groups of cell components in cd2+ binding ftir analysis was performed to identify the surface nature of dried k. palustris and m. luteus, as well as to identify major functional groups and to confirm their participation in cd uptake. obtained spectra reflect a complex character of both bacterial surfaces and illustrate significant changes in transmittance of characteristic peaks after cd biosorption (fig. 9 and 10). bands in the ftir spectrum were assigned to various groups according to wave numbers (table 4) as reported in literature (huang et al., 2014; zhou et al., 2014; garip et al., 2009). table 4. main functional groups of k. palustris and m. luteus with corresponding wave numbers obtained using ftir analysis. kp kp-cd ml ml-cd wavenumber (cm-1) wavenumber (cm-1) vibration type functional type 3 325 3 331 3 360 3 377 stretching vibration of oh -oh of polysaccharides and proteins 2 929 2 929 2 924 2 922 asymetric vibration of –ch2 lipids 1 653 1 651 1 679 1 651 strong vibration of c=o and c-n (primary amide) proteins (peptidic bond) 1 541 1 537 1 539 1 537 stretching vibration of c-n and deformation vibration of n-h (secondary amide) proteins (peptidic bond) 1 379 1 379 1 392 1 384 c=o symetric stretching of coocarboxylates and carboxylic acids 1 236 1 232 1 238 1 236 asymetric vibration of po2phospholipids 1 060 1 064 1 066 1 060 symetric vibration of po2phospholipids kp – unloaded k. palustris; ml – unloaded m. luteus; kp-cd – cd loaded k. palustris, ml-cd – cd loaded m. luteus. ftir spectra of unloaded and cd loaded k. palustris biomass is shown on fig. 9. it is obvious that asymetric vibration of po2 group (phospholipids) at 1236 cm-1 and symetric vibration of po2 group at 1060 cm-1 were shifted after cd biosorption. although, significant shift of absorption band corresponding to coo(1 379 cm-1) was not detect, changes in peak intensity was observed after cd treatment. the reason is probably the presence of ca and mg bound with carboxyl and phosphoryl groups of k. palustris, which have been replaced by cd2+ ions in the process of ion exchange. we suppose that carboxylate ions may coordinate to cd2+ ions as chelating complexes. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc 172 machalová, l. et al. 55 1, 64 13 8 10 60 ,8 48 8 12 36 ,3 71 06 13 79 ,1 03 44 15 41 ,1 23 98 16 52 ,9 95 31 29 29 ,8 71 5 33 25 ,2 78 78 39 03 ,9 23 58 55 7, 42 78 2 10 64 ,7 06 43 12 32 ,5 13 42 13 79 ,1 03 44 15 37 ,2 66 35 16 51 ,0 66 5 29 29 ,8 71 5 33 31 ,0 65 233 89 4, 27 95 4000 3600 3200 2800 2400 2000 1600 1200 800 400 -80 -60 -40 -20 0 20 40 60 80 100 % t cm-1 4000 3600 3200 2800 2400 2000 1600 1200 800 400 -80 -60 -40 -20 0 20 40 60 80 100 cd loaded k. palustris % t unloaded k. palustris fig. 9. ftir spectrum of k. palustris biomass before and after cd sorption. 54 9, 71 25 6 59 9, 86 17 8 10 66 ,6 35 25 12 38 ,2 99 87 13 92 ,6 05 15 15 39 ,1 95 17 16 79 ,9 98 74 29 24 ,0 85 06 33 59 ,9 97 47 38 94 ,2 79 5 50 5, 34 97 9 53 8, 13 96 6 10 60 ,8 48 8 12 36 ,3 71 06 13 84 ,8 89 89 15 37 ,2 66 35 16 51 ,0 66 5 21 13 ,9 82 34 29 22 ,1 56 24 33 77 ,3 56 82 4000 3600 3200 2800 2400 2000 1600 1200 800 400 -80 -60 -40 -20 0 20 40 60 80 100 % t cm-1 cd loaded m. luteus 4000 3600 3200 2800 2400 2000 1600 1200 800 400 -40 -20 0 20 40 60 80 100 % t unloaded m. luteus fig. 10. ftir spectrum of m. luteus biomass before and after cd sorption. the main differences between spectra of unloaded and cd loaded m. luteus biomass are associated with the primary amide (c=o) vibrations and c=o symetric e f bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc nova biotechnologica et chimica 14-2 (2015) 173 stretching of coo-. evident shifts of absorption band from 1651 cm-1 to 1679 cm-1 and 1384 cm-1 to 1392 cm-1 after cd uptake were observed (table 4, fig. 10). as seen from fig. 10, the intensity of bands at 1 655, 1 537 and 1 384 cm-1 decreased considerably after cd biosorption. beside carboxylate ions also other functional groups such as amino and hydroxyl may contribute to cd binding by cell surfaces. girault et al. (1997) observed strong cd binding to membrane phospholipids. using 113cd and 31pnmr confirmed that cd interactions with membrane phospholipids are electrostatic in nature and the phosphate moiety is proposed as a binding site. although there are differences in cd sorption capacities of k. palustris and m. luteus we expected that the functional groups of teichoic and teichuronic acids that are characteristic for g+ bacteria mediated cd uptake onto cell surface by both live and dead bacterial biomass. 4. conclusions using radioindicator method with 109cd we confirmed that living non-growing cells of g+ bacteria k. palustris and m. luteus effectively taken up cd ions. up to 85 % (m. luteus) and 83 % (k. palustris) of cd was associated with exchangeable binding sites and/or non-covalently bound to cell surface polymers. intracellular accumulation of cd2+ ions increased only slightly with increasing initial cd concentration. the maximum specific cd uptake capacities qmax obtained from the langmuir isotherm were higher for living non-growing biomass in comparison with non-living (dried) biomass of both bacteria. the biosorption of cd2+ ions by non-living biomass of k. palustris and m. luteus is strongly affected by ph and initial cd concentration. the maximum sorption capacities qmax were 444 ± 15 μmol/g for m. luteus and 381 ± 1 μmol/g for k. palustris. ftir analysis indicates that beside carboxylate ions also other functional groups such as amino and phosphate contribute to cd binding by bacterial cell surfaces. the present results suggest that beside dead bacterial biomass also living non-growing bacterial cells may be applied for biosorption purposes especially in low concentrations of cd in water. references adam, v., chudobova, d., trnejova, k., cihalova, k., krizkova, s., guran, r., kominkova, m., zurek, m., kremplova, m., jimenez, a.m.j., konecna, m., hynek, d., pekarik, v., kizek, r.: an effect of cadmium and lead ions for methallothionein (mt-3) revealed by electrochemistry. electrochim. acta, 140, 2014, 11-19. alam, m.z., ahmad, s.: multi-metal biosorption and bioaccumulation by exiguobacterium sp. zm-2. ann. microbiol., 63, 2013, 1137-1146. bestawy, e.e., helmy, s., hussein, h., fahny, m., amer, r.: bioremediation of heavy metal-contaminated effluent using optimized activated sludge bacteria. appl. water. sci., 3, 2013, 181-192. burnet, p.g.g., daughney, ch.j., peak, d.: cd adsorption onto anoxybacillus flavithermus: surface complexation modelling and spectroscopic investigations. geochim. cosmochim. acta, 70, 2006, 5253-5269. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc 174 machalová, l. et al. boyanov, m.i., kelly, s. d., kemner, k. m., bunker, b. a., fein, j. b., fowle, d. a.: adsorption of cadmium to bacillus subtilis bacterial cell walls: a ph-dependent x-ray absorption fine structure spectroscopy study. geochim. cosmochim. acta, 67, 2003, 3299–3311. garip, s., gozen, a.c., severcan, f.: use of fourier transform infrared spectroscopy for rapid comparative analysis of bacillus and micrococcus isolates. food chem., 113, 2009, 1301-1307. girault, l., boudou, a., dufourc, e.j.: 113cd-, 31p-nmr and fluorescence polarization studies of cadmium(ii) interactions with phospholipids in model membranes. biochim. biophys. acta, 1414, 1998, 140-154. guibaud, g., hullebusch, e., bordas, f.: lead and cadmium biosorption by extracellular polymeric substances (eps) extracted from activated sludges: phsorption edge tests and mathematical equilibrium modelling. chemosphere, 64, 2006, 1955-1962. gustaffson, j.p. visual-minteq, version 3.0. 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2007. tisakova, l., pipiska, m., godany, a., hornik, m., vidova, b., augustin, j.: bioaccumulation of 137cs and 60co by bacteria isolated from spent nuclear pools. j. radioanal. nucl. chem., 295, 2013, 737-748. urik, m., kramarova, z., sevc, j., cernansky, s., kalis, m., medved, j., littera, p., kolencik, m., gardosova, k.: biosorption and bioaccumulation of thallium(i) and its effect on growth of neosartorya fischeri strain. pol. j. environ. stud., 19, 2010, 457-460. vargas-garcia, m.c., lopez, m.j., suarez-estrella, f., moreno, j.: compost as a source of microbial isolates for the bioremediation of heavy metals: in vitro selection. sci. total environ., 431, 2012, 62-67. zhou, w., dongsheng, l., zhang, h., kong, w., zhang, y.: bioremoval and recovery of cd(ii) by pseudoalteromonas sp. scse709-6: comparative study on growing and grown cells. bioresour. technol., 165, 2014, 145-151. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:02 utc nova biotechnologica et chimica 13-2 (2014) 109 doi 10.1515/nbec-2015-0002 © university of ss. cyril and methodius in trnava natural organic matter in ecosystems a review przemysław kosobucki*, bogusław buszewski chair of environmental chemistry and bioanalytics, faculty of chemistry, nicolaus copernicus university, 7 gagarin street, 87 – 100 toruń, poland (pkosob@chem.umk.pl) abstract: one of the most essential parameters limiting the potential use of the ecosystem (soil, water) is the content of the organic matter. the natural organic matter (nom) is a ubiquitous component of the lithosphere and hydrosphere that constitutes one of the largest reservoirs of the carbon in the environment. natural organic substances play several important functions in ecosystems and they are necessary for their normal functioning. despite many years of the research and using many advanced analytical techniques, their structure has not been fully explained. the main aim of this review is to present the actual state of the knowledge about the natural organic matter and provide a comprehensive overview of the research that has explored up to date in this matter. the additional attention was focused on the relations within and between humic and fulvic acids in terrestrial and aquatic environments. special attention is focused on the analytical methods used to analysis natural organic matter. key words: natural organic matter, humic acids, fulvic acids, humins, ecosystem, ecoanalytics 1. introduction the carbon is the most important element on the earth, its resources are stable and limited. therefore, there is the continual cycle of processes in which this element is involved. from the environmental point of view, the human life, the carbon cycle has an essential importance in the biochemical way in which living organisms are actively involved. it is estimated that the geochemical cycle is up to 1000 times slower than the biochemical cycle. the human has been actively participating in the cycle of the matter (including the carbon cycle) from the moment appeared on the earth. however, as a result of the anthropogenic human activity this impact is becoming bigger and bigger and it leads to the disruption of natural biogeochemical cycles (for example increase of co2 in the atmosphere). the natural organic matter is a ubiquitous element of the lithosphere and hydrosphere constituting one of the biggest reservoirs of the carbon in the environment (the estimated amount of the carbon in the organic matter reaches 5 × 1015 kg). basic carbon cycle is presented on fig. 1. the natural organic substances fulfill a range of important functions in ecosystems and they are essential for their proper functioning. however, their presence is sometimes undesirable for example in the water absorbed by the water treatment plants for the nutritional needs of the population. a certain amount of the organic matter of the new quality appears in the form of the compost, sludge and waste due to the growth of the human ecological awareness by conscious actions. this matter does not appear in a fully natural way, so determination of the natural organic matter name makes the sense in the context of the processes that it undergoes for example the soil organic matter (mineralization and humification). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc 110 kosobucki, p. and buszewski, b.   fig. 1. basic carbon cycle in environment (epstein, 1997). 2. natural organic matter the natural organic matter (nom) is a highly heterogeneous mixture. it consist degradation products of plant and animal tissues as well as substances resulting from biochemical changes (biodegradation, resynthesis) of plant and animal origin substrates and their transitional products (nissen et al., 2001; gu et al., 2002). it appears in the soil as the soil organic matter (som), surface waters, both fresh and salt water, ground water (in the soil solution) as the dissolved organic matter (dom), in such parts of the environment as sediments, sewage sludge, organic fertilizers, composts made on the basis of the biomass, organic waste and sewage sludge, etc. the composition of the natural organic matter, its chemical and physicochemical properties depend on the quality of the substrate and environmental conditions in which it appears. three main fractions were identified that are part of the natural organic matter: high molecular chemical substances with recognized structure that could be classified in one of the structural groups in the organic chemistry such as polysaccharides and proteins, chemical compounds with well-explored chemical structure such as amino acids, carbohydrates and other low molecular organic compounds, humic substances, the final product of the transformation of plant and animal residues. this definition is very general although it is very illustrative. however, six main fractions of the organic matter: fats, waxes, oils, resins, hemicellulose, cellulose, protein and lignin-humus complexes can be identified (waksman, 1936). a lot of misunderstanding and confusion has accumulated in relation to the definition of the soil organic matter (som). this term should be used in the context of the whole soil organic substrate including living organisms. the soil organic matter is the amount of dead organic compounds (mostly plant) occurring in the soil beginning bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc nova biotechnologica et chimica 13-2 (2014) 111   from fresh, plant and animal residues that are not decomposed to amorphous products of the decomposition and re-synthesis excluding living organisms. in the scientific literature it is common to use the term of the som in terms of the soil organic substance or humic substances (also known as humus). the individual fractions of the soil organic matter according to gonet (2003) can be presented on fig. 2. fig. 2. partition of the soil organic matter (gonet, 2003). both the qualitative and quantitative composition of the natural organic matter depends mostly on the chemical composition of its source – a substrate. some differences in the chemical structure of the natural organic matter substrates (lynch, 1986; epstein, 1997; khil'ko et al., 2011) were identified (within the same substrate e.g grass or straw). the composition of the natural organic matter also depends on environmental factors and it may be subject to considerable changes during the time. therefore the criterion for defining the main fractions of the natural organic matter may be the susceptibility to the microbiological decomposition of organic substances (epstein, 1997). three groups of substances and organic compounds were identified according to their susceptibility to the decomposition (biodegradation): substances that are easily decomposed in the microbiological way (polysaccharides (starch, glycogen, pectin)) fatty acids and glycerol, lipids and fats, amino acids and proteins, nucleic acids, difficult degradable substances (hemicellulose, cellulose, chitin), substances resistant to the decomposition (lignin cellulose). in the case of organic composts, seven main groups of chemical substances included in the compost matter should be listed: carbohydrates and sugars; proteins; fats; hemicellulose; cellulose; lignin and mineral matter. the substances belonging to the first three groups are decomposed relatively fast , as a source of easily digestible nutritious elements for the flora and fauna of i.e. the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc 112 kosobucki, p. and buszewski, b.   soil environment. the hemicellulose, cellulose and lignin decompose slower. the last group is the mineral matter of the compost that is not biodegradable (epstein, 1997). the organic matter of composts and fertilizers make a highly amorphous mixture of the raw organic waste, the products of their decomposition and re-synthesis. during the composting process, the organic matter was divided into three fractions: soluble in water (dissolved organic matter dom), undergoing the acid hydrolysis, non-hydrolysis. the first group consists of water-soluble carbohydrates, proteins, fulvic acids. the second group consists of hemicellulose, cellulose and fats. the non-hydrolysis in the acid environment fraction includes complexes of lignin, humic acids and humin. 3. humic substances in the terrestrial environment humic substances are one of the main fractions of the natural organic matter that have a significant ecological role in the natural environment (stevenson, 1994). humic substances are the most common organic substances in the world. because of its changeability they are highly resistant to the biodegradation, they are found in almost all components of the natural terrestrial and marine environment. they constitute 85-90% of the general amount of the organic matter in the soil, regulating a range of its fundamental physicochemical properties mainly connected with the fertility (epstein, 1997; sun et al., 2006). humic substances, otherwise known as peculiar humus substances (in relation to the soil organic matter) are a complex of brown and yellow organic substances that can be extracted from alkaline solutions, neutral salts or organic solvents (kononowa, 1968). these substances are formed in the biochemical processes of the decomposition and re-synthesis of the organic material of different origins with the help of microorganisms. moreover, they have a strong heterogeneous nature, they are a mixture of polymolecular and polydispersive copolymers with the unknown chemical structure (epstein, 1997; jones et al., 1998). the definition of humic substances includes their division into three main fractions based on their behavior in the acid-alkaline environment (kononowa, 1968; stevenson, 1994): humic acids the fraction of humic substances, insoluble in water in the acid conditions below ph 2, but they are soluble at the higher ph values, fulvic acids the fraction of soluble humic substances in the whole ph range, humins the fraction of humic substances insoluble in water in the whole ph range (fig. 3). creon and apocreon acids were distinguished as subgroups of the fulvic acids fraction, hymatomelanic acid regarded as the initial product in the biosynthesis of humic acids, ulmic acids and ulmins substances belonging to the same faction as humic acids and humins (kononowa, 1968). in the literature we can find the division according to which the fraction of humic substances consists of two subgroups: brown humic acids and gray humic acids. due to the fact that the differentiation of humic substances is based on the fractionation, it should be treated as a test which means that the status of humic substances may be quite different in the home environment (schiavon et al., 2010; qi et al., 2012). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc nova biotechnologica et chimica 13-2 (2014) 113   fig. 3. divide of the humic substances (kononowa, 1968). 4. natural organic matter in the aqueous environment the term of the dissolved organic matter (dissolved organic matter dom) or the dissolved organic carbon (dissolved organic carbon doc) concerns the fraction of the organic matter (organic carbon) separated from a soil sample (or other solid material) by the centrifugation (gonet, 2003). these terms are also widely used in the studies of natural organic substances found in surface waters (rivers, lakes and sea), groundwater and the fraction of the organic matter of solid samples (sediment, soil) extracted with water (cheftez et al., 1998). the method of the concentration and fractionation on adsorption gels and ion-exchange resin is used in the analysis of the natural organic substances dissolved in water (kosobucki, 2011). the methods using processes of freezing, the precipitation, the solvent extraction, the reverse osmosis and the ultrafiltration of the water sample for the isolation of organic substances can be found in the literature (zbytniewski et al., 2005). the ddissolved organic matter can be separated into two main groups: hydrophobic and hydrophilic, each of them can be further divided into three next fractions (leenher, 1981): hydrophobic basic (hob), hydrophobic acid (hoa), hydrophobic neutral (hon), hydrophilic base (hib), hydrophilic acid (hia) and hydrophilic neutral (hin). the composition of individual fractions of the dissolved organic matter was confirmed after connecting the extraction and/or the fractionation techniques of the dissolved organic matter and the 13c nmr technique (leenher, 1981; cheftez et al. 1998). hob fraction is the carbon in the aliphatic combinations, hydrocarbons and amines, hoa consists of tannins, polyphenols and organic complexes of humic substances, hon contains highly nonpolar connections like humins. the hydrophilic fraction named hib consists of protein substances, peptides, amino sugars, hia has highly oxidized organic compounds like humic substances of the low atomic mass, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc 114 kosobucki, p. and buszewski, b.   hin is polysaccharides and oligosaccharides of the plant and microbiological origin (cheftez et al., 1998). a group of substances of the dissolved organic matter of the crucial, ecological importance for the aquatic ecosystem is the fraction of humic substances like in the case of the terrestrial environment (siyanitsa et al., 2007; veprikova et al., 2007). although the natural organic matter in the marine environment may be both the autochthonous and alochthonous origin, it is assumed that most of the humic substances dissolved in fresh surface water come from the basin (kosobucki, 2011). however, significant differences in the properties of humic substances from the sea water and from the soil were indicated 95% humic acids are synthesized in the sea (klucakova et al., 2012). 5. carbon changes in natural environment the natural organic matter in the environment changes constantly in the quantity and quality way. in the course of biochemical transformations, the organic matter reduces in the environment. easily absorbed by the microfauna substances such as polysaccharides, fatty acids, lipids and fats, amino acids and proteins, nucleic acids are a source of the carbon and energy for organisms. humic substances are formed from the remaining part as a result of the product re-synthesis of the transformation of plant and animal residues (hsu et al., 1999). the first direction of the transformations is the mineralization. it is connected with simple inorganic compounds such as co2, h2o, nh3 and ions so4 2-, no3 -, hpo4 2-. the second direction, which is a synthesis of humic substances, is the humification (epstein, 1997). the changes that associate with the mineralization, transformation and decomposition of the organic matter in three main stages: -the initial phase includes various processes occurring in the dead parts of plants, mainly the oxidation and hydrolysis. the biggest changes are in aromatic compounds and proteins, the phase of the mechanical decomposition – grinding of the organic material occurs here under the influence of the soil microfauna and macrofauna activity and the inclusion of particulate residues to the soil mass, the phase of the microbiological decomposition – the organic material completely without the tissue structure undergoes the enzymatic decomposition becoming the energy resource and building resource of soil microorganisms. the microflora and microfauna preferentially decompose plant residues due to the availability of the organic matter contained in them. water-soluble carbohydrates are decomposed into the basic components: the starch and protein which resorbed by the organism are partly used to build their bodies. next, fats and waxes are decomposed, the cellulose and hemicellulose, and lignin that can be biodegradable under the influence of the proper groups of microorganisms, mainly fungi and actinomycetes (epstein, 1997). these phenomena occur with different intensity within the time and space, they are dependent on internal factors of the environment in which the organic matter is present as one of its components (for example the soil type) and external factors such as the atmospheric phenomena, climate, human activity. the greatest bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc nova biotechnologica et chimica 13-2 (2014) 115   dynamics of these changes can be observed in systems where there is a fresh substrate, for example during maturing of the compost. a lot of studies show a multidirectional course of processes of the mineralization, but basically it can be noticed that the ratio of the organic substances susceptible to the microbiological decomposition (polysaccharides, fatty acids, lipids and fats, amino acids, nucleic acids) to substances resistant to the decomposition (cellulose, chitin, lignincellulose) increases (zbytniewski et al., 2005; kosobucki et al., 2006) and is very much dependent on the tested substrate. the fraction of the organic matter soluble in water has the particular importance for the environment, living organisms and for the transport of pollutants. generally, it can be noticed that the amount of the carbon of this fraction decreases when the process of the transformation of the organic matter progresses (leita et al., 1991; inbar et al., 1993). some specific maximum of the carbon content was observed in the extract at the beginning of the transformation of various organic materials (harvey et al., 1983; zbytniewski et al., 2002). the amount of the carbon in the fraction undergoing the acid hydrolysis in the relation to the carbon of the non-hydrolysis fraction decreases during the process of composting (pare et al., 1998). in the case of the dynamics of changes of the fraction of the organic matter susceptible to the oxidation under the influence of dilute solutions of oxidants observed that the systematic manuring the soil increases the part of this fraction compared to the organic matter of the soils that are not fertilized or fertilized with mineral fertilizers. cultivated soils are richer in the fraction of the organic matter that is susceptible to the oxidation than the forest soils (szombathova, 1999). agrotechnical activities in the long term reduce the amount of the matter of the oxidizable fraction in soils (coneth et al., 1999). 6. humification – formation and transformation of the humic substances humic substances are formed as a result of biochemical transformations of the natural organic matter, plant residues and animal residues (polikreti and christofides, 2009). the humification process proceeds parallel to the processes associated with the decomposition of the chemical connections and the mineralization (fourti et al., 2010). it is preceded by three mentioned earlier phases of the decomposition of the organic material. the humification proceeds essentially in two stages: vthe decomposition of the organic substrate to some simpler elements, more or less complicated synthesis and re-synthesis of these simpler substances that cause the appearance of humic substances (savel'eva et al., 2010). despite the various methods, the researchers claim that the course of these processes depends on the organic substrate, type and environmental conditions (kosobucki et al., 2011). the main source of the organic matter in soils is the plant substrate in which lignin, polypeptides and polysaccharides dominate (inbar et al., 1989). thus, it is commonly believed that the formation of humic substances from the residues of plant organisms (and animal residues) can occur according to four different mechanisms (fig. 4) (stevenson, 1994). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc 116 kosobucki, p. and buszewski, b.   fig. 4. different mechanisms of soil organic matter formation (stevenson, 1994). the first of them – the lignin theory of waksman (fig. 4 way 1) says that humic substances are formed from the lignin modified by the activity of microorganisms. modifications involve the de-methylation increasing the number of phenolic groups and shortening the side chains of the carbon to carboxyl groups. next, the modified lignin are enriched in the nitrogen as a result of the abiotic condensation with polypeptides according to form schiff’s bases. humic acids formed in this way undergo fragmentation to fulvic acids. according to the second and third mechanism (figure 4, way 2, 3) polyphenols that appear due to the demethylation and the enzymatic oxidation of the lignin or cellulose are oxidized to quinones. enzymes phenol oxidases play an active role here. quinones undergo the condensation with polypeptides that are produced by soil microorganisms, thereby they enrich the appearing products (humic acids) in the nitrogen. many systems that catalyze the processes of the oxidation of polyphenols to quinones other than the activity of microorganisms were found in the soil environment. these are clay minerals, hydroxides and amphoteric metal ions (fe, mn, al) (stepanov, 2008). the fourth way of the formation of humic substances in the terrestrial environment is the condensation of simple sugars and amino acids thanks to maillard’s reaction (steinberg, 2008). glucosamines appear in the huge amount of the substrates that next undergo a series of reactions including the dehydration leading to the formation of dark-coloured polymers with features similar to humic acids (stevenson, 1994; sivakova et al., 2011). humic acids may be also formed by the auto-oxidation of unsaturated fatty acids a lipid model (inbar et al., 1989). these processes are typical for the surface layer of sea areas, where fatty acids of the algae origin accumulate. this mechanism is catalyzed by light and ions of heavy metals. humic acids can be formed only in the microbiological way. it has been proven that sulfur bacteria and archaea are capable to produce the brown biopolymers containing elements of an aromatic carbon with properties similar to the humic acids in its structure from the aliphatic substrates (maryganova et al., 2013). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc nova biotechnologica et chimica 13-2 (2014) 117   observing the process of the humification of the organic matter in composts, many researchers show that the content of the fraction of humic substances increase relatively, but generally there are more humic acids and less fulvic acids (cheftez et al., 1996; hsu et al., 1999). however, at the same time other studies show that the total fraction of humic substances does not change in the whole period of composting – the humification. other authors show that the direction of changes of the carbon content of the fraction of the humic acids and fulvic acids is ambiguous (cheftez et al., 1996) or even that the fraction of humic acids is decrease during maturing of the compost (veeken et al., 2000). these significant discrepancies of the obtained results show the diversity of organic materials undergoing composting. 7. structure of the nom studying the organic matter of the soil, of the composts, of the sludge or water it is very important to describe the structure that clearly reflects the physicochemical properties of the tested substance. the problem of the chemical structure of naturally occurring chemical substances has been bothering researchers for nearly two centuries and it has still remained in many aspects clearly not solved. particular difficulties appear during attempting to determine the structure of humic substances. during the past 50 years, a lot of models of the structure have appeared. among them couple of them should be distinguished: a model of the structure of dragunov’s humic acids from 1948, kasatoczkin’s from 1951, the model of the structure of fulvic acids presented by schnitzer and khan in 1972 and by buffle, the model of humic acids according to stevenson from 1982 (stevenson, 1994) and a model of schulten, plage and schnitzer presented in 1991 (schulten et al., 1991; yarkova and gyul'maliev, 2012). these structures are shown in fig. 5. they differ greatly because of the elementary composition, the molecule shape, the size of structural units and the nature of the connections. it is assumed that humic substances are a mixture of a large number of chemical individuals of a polymeric feature. the latest studies also indicate the alkyl-aromatic structure of humic acid molecules (gonet, 1993; chaikovskaya et al., 2008). but the aromatic rings of the hydroxyphenol type and the aliphatic structures of the type of carbohydrate residues, proteins and amino acids are connected with oxygen bridges (-o-), nitrogen (-n-), sulfur (-s-) and -ch2-,-nhgroups and other (gonet, 1993; benzosikov and lodygin, 2009; efanov and chernenko, 2010). the functional groups: carboxyl, phenolic and alcohol, quinones and ketones, methoxy, amino and amide are a very important element of the structure that determines a range of properties of humic substances. they are chemically connected mainly with the aromatic part through, as it could be seen thanks to the example of schulten and co-workers (fig. 5), they are also partly connected with the alkyl group. the shape of the humic acid molecule in the solution strongly depends on the composition of the solvent (concentration, ph and ionic strength). it is assumed that they are spherocolloids with the porous (spongy) structure, the molecular weight from 2000 to about 300 000 g/mol. the low molecular fractions constitute fulvic acids and the high molecular fractions constitute humic acids (kowalkowski, 2009; kulikova and perminova, 2010a; kulikova and perminova, 2010b). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc 118 kosobucki, p. and buszewski, b.     fig. 5. models of the humic substances structure (schulten et al., 1991; gonet, 1993; stevenson, 1994). to conclude, the natural organic matter has the elementary composition similar to other natural polymers (table 1). table 1. elemental analysis of soil organic matter (dziadowiec, 1993; pshinko, 2009; klucackova, 2010). element content [%] c h o n humic acids 52.0-62.0 3.0-5.5 30.0-33.0 3.5-5.0 fulvic acids 44.0-49.0 3.5-5.0 44.0-49.0 2.0-4.0 peptides 50.0-55.0 6.5-7.3 19.0-24.0 15.0-19.0 cellulose 44.4 6.2 49.4 sugars 45.4 6.1 48.5 lignin 62.0-69.0 5.0-6.5 26.0-33.0 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc nova biotechnologica et chimica 13-2 (2014) 119   the elementary composition of the natural organic substances is a feature that can be used to their identification and the conclusion of the structure, origin and energy or sorption (dziadowiec, 1993; pshinko, 2009; klucackova, 2010). 8. significance of the nom in environment from all ecosystems in the terrestrial environment the role and the importance of the organic matter for the soil are the most known. the soil organic matter consisted of about 90% with humic substances affects the main physical, chemical and biological features taking part in all processes occurring in the soil. the key functions of the soil organic matter commonly include (dziadowiec, 1993; epstein, 1997): the participation in the formation of soils and shaping their properties, the participation in the processes of ion exchange, the participation in the biological cycle of elements, providing the energy and carbon for soil microorganisms, providing biogenic elements for higher plants, influencing on the plant growth and development. the organic matter affects the sorption and buffer abilities of soils. it regulates the concentration of soil solutions and their reaction. specific organic substances (humic substances) are an important part of the sorption complex. their absorptive ability denoted as the cation exchange capacity (cec) is higher than the sorption capacity of its mineral part (klavins and purmalis, 2010). sorption properties of the organic matter are determined by the presence of functional groups such as carboxyl and hydroxyl groups, constant and the degree of their dissociation, mainly in the structure of fractions of humic substances. the chemical composition and the energy value of humic substances of the soil organic matter indicate that they may be an interesting source of the energy, biogens (carbon and nitrogen) for plants and microorganisms. however, due to their great diversity and uniqueness of the chemical structure, it is a source that is difficult to reach and it is only for a specialized group of microorganisms. the significance of the soil organic matter fraction for higher plants is conditioned on the effects both indirect and direct (kowalkowski, 2001). the organic matter affecting the physicochemical properties of the soil environment forms favorable conditions for the plant growth. it is assumed that the plant growth and plant development are conditioned by the availability of nutrients, water, air to the roots as well as light and temperature. but most of these conditions are regulated by the presence of the humus soil and its quality. many studies have clearly shown that humic substances also affect directly the germination, the seedling growth, the growth of roots and stem (gonet, 2003). the soil organic matter regulates the bioavailability of nutrients to microorganisms and higher plants through their impact on weathering processes, the properties of ion exchange, chelating and buffer properties of the soils. on the other hand, organic matter fractions, in particular low molecular humic acids can penetrate the plant through the root system (or leaves as a result of agrotechnical practices involving spraying humic preparations) and participate in a series of metabolic processes of the plant (dziadowiec, 1973; kowalkowski 2001). humic substances after penetrating the plant form redox bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc 120 kosobucki, p. and buszewski, b.   systems affecting the metabolism of plant cells. in addition, they also influence on the membrane potential of cells by facilitating taking food elements including the nitrogen in the ammonium form and iron (dziadowiec, 1993). the increased respiration, the increase of the activity of the enzymes and the intensity of the photosynthesis and the increase of the resistance to unfavorable conditions of life were observed among the tested plants. the mechanisms according to which humic substances impact directly on the plants have not been explained yet. the activity of humic substances as for example the growth hormones may be connected with the inhibition of the oxidase of 3-indoleacetic acid and counteracting its decomposition. maintaining a high activity of this compound has a positive effect on the plant growth (gonet, 2003). the physicochemical properties (quality) of the organic matter have a significance for many effects connected with the soil fertility and plant growth. it was noticed for example that the soil fertility depends on the quality of the organic matter. 9. soil organic matter analytical methods measuring of som directly is difficult. it is substituted by measurement of soc. a classical procedure to calculate som is by multiplying the percentage of organic carbon by a factor. conversion factors change between 1.4 and 3.3 (lal, 1979; rasmussen et al., 1991) and this large range is connected with the origin of the soils. a conversion factor of 1.72 is most often used. therefore, to ensure consistency and allow reliable comparison of data, it is advantageous to report results as soc rather than as som. som studies have included: 1) detailed study of humus chemistry to prediction of the chemical structure of som via fractionation procedures, 2) empirical methods to assessment effects of som by evaluating field experiments, 3) modeling by soil models (korschens et al. 1998). the most common methods used to characterize the humic substances in the half of xx centuries were destructive testing methods (wet or dry red-ox processes pyrolysis). later, a number of non-destructive methods have been developed. these include elementary analysis, the assay of carbon, oxygen, nitrogen containing components. the properties of humic substances can be described with the classical instrumental analytical methods (potentiometric and conductometric titration, and within the category of spectral methods, uv and vis, fluorescent, infrared, nmr and esr spectroscopy). but, different methods may also be used, including for instance x-ray diffraction, surface tension measuring, determination of molecular weight, steam pressure and membrane osmometry, size excluded chromatography (sec), field flow fractionation (fff), electrophoresis, ultracentrifuge, viscometry and mass spectrometry. other methods worth mentioning include light dispersion, x-ray dispersion, electron microscopy and ultrafiltration (hsu et al., 1999; peuravuori et al., 1998; keller et al., 2000; karlen et al., 2003; zbytniewski et al., 2005; kowalkowski, 2010; zav'yalova, 2012). sometimes, the simplest methods i.e.. elemental analysis (chn), give fundamental information about structure of analyzed compounds (table 1). between humic and fulvic acids, peptides, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc nova biotechnologica et chimica 13-2 (2014) 121   cellulose, sugars and lignins huge differences in content of carbon, hydrogen, oxygen and nitrogen are observed (kalembasa et al., 1973). size exclusion chromatography (sec) and gel permeation chromatography (gpc) with uv-vis detection is separation technique often used in analysis high molecular analytes (polymers, humic substances). few selected wavelengths (280 nm, 472 nm, 664 nm) are used to detection. humic substances possess no maximum absorption in uv-vis spectrum. from gpc analysis molecular mass of analytes can be assign (lodygin et al., 2012). nuclear magnetic resonance (nmr) spectroscopy, especially 13c nmr and 1h nmr has been used to predict structure of humic substances. from nmr analysis, distribution of functional groups (aliphatic/aromatic) in molecules is easy to perform. in solid state nmr main disadvantage is amount and purity of sample (chung et al.,2012). spectroscopic techniques, especially infrared spectroscopy (ir) combined with fourier transform (ft) having many advantages in analysis of organic molecules (hss also). the main advantage are: high sensitivity and s/n ratio, many spectra library, small amount sample necessary and simplicity of measure. in the result of ftir analysis, identification of functional groups in humic substances molecules is easy to carry out (patrakov et al., 2010). electrophoretic techniques, capillary zone electrophoresis (cze), isotachophoresis (itp) and others, has been used to provide chemical “fingerprints” of humic substances. after electrophoretic analysis, determination of humic substances origin is possible. additionally different electrophoregram profiles and migration times of humic and fulvic acids have been used to distinguish between young, old, autochthonous and allochthonous natural organic matter. sometimes, especially isotachophoresis is used to isolation of humic substances fractions (takahashi et al., 2009, nagyova et al., 2001). many information about molecular structure of humic substances can be obtaining from mass spectrometric (ms) techniques (especially electrospray ionization (esims) mode). both modes (negative and positive) have provided information on changes in molecule structure during photodegradation. uv spectroscopy do not detect any changes. coupling of systems (ms/ms, triple or quadrupole) and chemometric methods (library’s) permit identified compounds in samples: benzene, phenol, sugars, organic acids (piccolo et al., 2010). molecular weight distribution of humic substances can be performed by field flow fractionation (fff). fff offers many advantages in comparison to sec. main are separating molecules by their size (hydrodynamic) in solvents, without sieve effect in chromatographic column (easy to perform in pratice) (kowalkowski, 2010). soil organic carbon can by determined by different, classical methods (moody et al., 1997; moskvin et al., 2005). acidic wet oxidation (with dichromate) is well known method as the walkley-black method (no heating) or heanes method (externally heated and addition of concentrated sulphuric acid). results obtained by the walkley-black method are not repeatable (recoveries can vary from 56% to 100%). it is result of origin of soil sample. this problem can be solve by dry combustion methods, where all organic carbon is converted to co2 in the oxygen or bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc 122 kosobucki, p. and buszewski, b.   air atmosphere (zomeren et al., 2008). kalembasa and jenkinson compare both (dry and wet) methods, and they state that dry method were more accurate (kalembasa et al., 1973). later, proidakov (proidakov, 2009) recommended application of infrared detector (nir) to measurement of content of co2 in combustion gases. table 2 shows most often used analytical techniques used in humic substances analysis. table 2. analytical techniques most often used in humic substances analysis. analytical methods literature high performance liquid chromatography (hplc) hutta et al., 2003 gas chromatography (gc) fabrri et al., 2002 gel electrophoresis (page) baxter et al., 1992 gel permeation chromatography (gpc) chin et al., 1991 lodygin et al., 2012 field flow fractionation (fff) kowalkowski, 2010 yohannes et al., 2005 capillary zone electrophoresis (cze) baxter et al., 1992 takahashi et al., 2009 abramov and bezzubov, 2007 lucio and schmitt-koplin, 2006 isoelectric focusing (ief) curvetto et al., 1974 capillary electrochromatography (cec) andre and guillaume, 2008 micellar electrokinetic chromatography (mekc) de moraes et al., 2005 isotachophoresis (itp) kopacek et al., 1991; nagyova et al., 2001 electron paramagnetic resonance (epr) peurovuari et al., 1998 nuclear magnetic resonance (nmr) keller et al., 2000 chung et al., 2012 elemental analysis (chn) kalembasa et al., 1973 lishtvan et al., 2013 mass spectrometry (ms) piccolo et al., 2010 x-ray diffraction (xrd) thieme et al., 1998 uv-vis spectroscopy zbytniewski et al., 2005 hsu et al., 1999 kucerik et al., 2009 leopold et al., 2012 ir spectroscopy hsu et al., 1999 patrakov et al., 2010 gostishcheva et al., 2009 moros et al., 2008 fluorescence spectroscopy divya et al., 2009 plaza et al., 2007 titration khil'ko et al., 2011 quentel and fillela, 2011 thermal analysis rotaru et al., 2008 kucerik et al., 2006 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc nova biotechnologica et chimica 13-2 (2014) 123   10. conclusions the organic matter is present in all elements of the ecosystem (soils, rivers, oceans). it is very reactive, it is involved in many geochemical cycles and it is essential to the life on the earth. the soil organic matter is a source of food for the soil fauna and it contributes to the biological diversity of the soil, because it is a source of nutrients such as the nitrogen, phosphorus and sulfur; the soil fertility depends on it at a great rate. the organic carbon reinforces the soil structure, it improves the physical environment, so it allows roots to penetrate the soil. the organic matter absorbs water (it is able to hold water in an amount equivalent to six times of its weight), so that it is a very important source of life for the flora present in naturally dry and sandy soils. soils containing the organic matter have a better structure that improves the infiltration of the water and reduces the susceptibility to the compaction and erosion. their structure has not been explained in a full way, despite many years of research and using many advanced analytical techniques. to explain the formation and structure of humic substances many models and hypothetic structures have been elaborated. still occur degradation of organic matter is problem, and next investigation will be conducted with this processes. understanding of interactions of natural organic matter (humic substances) with environment (terrestrial, aquatic) is fundamental to comprehend the nature of studied material. development of technology (new analytical methods) probably solves many instrumental problems. but problem with standards of humic, fulvic substances and humins will be still actual (no repetably). acknowledgements: this paper was supported from the budget of kuyavia and pomerania and the european regional development fund under the rop for the years 2007 – 2013: project no. rpkp.05.04.00-04-003/13 references abramov, e.g., bezzubov, a.a.: electrosorptive separation of humic substances. j. water chem. technol., 29, 2007, 125-130. andré, c., guillaume, y.c.: cec for studying the retention and separation of pesticides on a humic acid stationary phase. chromatographia, 68, 2008, 791796. baxter, r.m., malysz, j.: analysis of aquatic humic material and high molecular weight components of bleached kraft mill effluent (bkme) by gradient gel electrophoresis. chemosphere, 12, 1992, 1745-1753. beznosikov, v.a., lodygin, e.d.: characteristics of the structure of humic substances of podzolic and peaty podzolic gleyey soils. russ. agricult. sci., 35, 2009, 103-105. chaikovskaya, o.n., sokolova, i.v., sokolova, t.v., yudina, n.v., mal’tsev, e.v.: effect of humic acids on phototransformation of methylphenols in water. j. app. spectroscopy, 75, 2008, 597-602. cheftez, b., hatcher, p., hadar, y., chen, y.: chemical and biological characterization of organic matter during composting of msw. j. environ. quality, 25, 1996, 776-785. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc 124 kosobucki, p. and buszewski, b.   cheftez, b., hatcher, p., hadar, y., chen, y.: characterization of dissolved organic matter extracted from composted municipal waste. soil sci. soc. american j., 62, 1998, 326-332. chin, y.p., gschwend, p.m.: the abundance, distribution, and configuration of porewater organic colloids in recent sediments. geochim. cosmochim. acta, 55, 1991, 1309-1317. chung, t.l., chen, j.s., chiu, c.y., tian, g.: 13c-nmr spectroscopy studies of humic substances in subtropical perhumid montane forest soil. j. forest res., 17, 2012, 458-467. coneth, a., blair, g.j., lefroy, r., whitbread, a.: labile organic carbon determined by permanganate oxidation and its relationships to other measurements of soil organic carbon. humic sub. environ., 1, 1999, 3-15. curvetto, n.r., balmaceda, n.a., orioli, g.a.: isotachophoresis and isoelectric focusing of soil humic substances in polyacrylamide gel. j. chromatography, 93, 1974, 248-253. de moraes, s.l., rezende, m.o.o.: behavior of humic acid as a micellar phase in micellar electrokinetic chromatography (mekc). microchim. acta, 151, 2005, 115-122. divya, o., venkataraman, v., mishra, a.k.: analysis of metal ion concentration in humic acid by excitation-emission matrix fluorescence and chemometric methods. j. app. spectr., 76, 2009, 864-875. dziadowiec, h.: ekologiczna rola próchnicy glebowej. zesz. prob. post. nauk rol., 411, 1993, 269-282. efanov, m.v., chernenko, p.p.: preparation of nitrogen-containing humic preparations from peat. solid fuel chem., 44, 2010, 61-64. epstein, e.: the science of composting. technomic publishing company, inc. lancaster, pensylvania, 1997, 45-58. fabrri, d., vassura, i., snape, c.e.: simple off-line flash pyrolysis procedure with in situ silylation for the analysis of hydroxybenzenes in humic acids and coals. j. chromatogr. a, 967, 2002, 235-242. fourti, o., jedidi, n., hassen, a.: humic substances change during the cocomposting process of municipal solid wastes and sewage sludge. world j. microbiol. biotechnol., 26, 2010, 2117-2122. , gonet, s.s.: struktura substancji humusowych. zesz. prob. post. nauk rol., 411, 1993, 189-194. gonet, s.s.: próchnica, substancje humusowe, węgiel organiczny – definicje, komentarze i metody oznaczania. in: substancje humusowe w glebach i nawozach. problemy badań, dębska b., gonet s.s. 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spectroscopic properties. bioresour. technol., 4, 2005, 471-478. zbytniewski, r., buszewski, b.: characterization of natural organic matter (nom) derived from sewage sludge compost. part 2, multivariate techniques in the study of compost maturation. bioresour. technol., 4, 2005, 479-484. zbytniewski, r., kosobucki, p., kowalkowski, t., buszewski, b.: the comparison study of compost and natural organic matter samples. environ. sci. poll. res., 1, 2002, 68-74. zomeren, a., weij-zuiver, e., comans, r.n.j.: development of an automated system for isolation and purification of humic substances. anal. bioanal. chem., 381, 2008, 2365-2370. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:24 utc 30 nova biotechnologica et chimica 12-1 (2013) doi 10.2478/nbec-2013-0003 © university of ss. cyril and methodius in trnava optimization of the antioxidant extraction from eleutherococcus senticosus roots by response surface methodology miroslav ondrejovič1,2, daniela chmelová3, tibor maliar1 1department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (miroslav.ondrejovic@ucm.sk) 2food research institute, department biocentrum, kostolna 5, modra, sk-900 01, slovak republic 3department of biochemistry and biotechnology, faculty of biotechnology and food sciences, slovak university of agriculture, tr. a. hlinku 2, nitra, sk-949 76, slovak republic abstract: eleutherococcus senticosus is known as adaptogen with benefits in general health promotion. the aim of this study was to investigate the effect of major extraction parameters on extraction yield of antioxidants measured by 2,2-diphenyl-1-picrylhydrazyl (dpph) radical-scavenging activity. secondly, content of total polyphenols was evaluated. optimal conditions of the extraction were processed by response surface methodology. the independent variables of extraction were proposed as temperature, solid–liquid ratio and solvent composition. for the optimal antioxidant extraction, e. senticosus is suitable to extract by 23 % (v/v) aqueous ethanol at 70 °c in ratio 53 ml of extraction solvent per g of plant material. the optimal conditions calculated for the extraction of total polyphenols were very similar (70 °c, 22 % (v/v) aqueous ethanol) expect solid-liquid ratio which indicates need of increasing of solid-liquid ratio to 91 ml of extraction solvent per g of plant material. key words: extraction, eleutherococcus senticosus, antioxidant activity, polyphenols, response surface methodology 1. introduction eleutherococcus senticosus, known as siberian ginseng or acanthopanax senticosus, belong to the araliaceae family, is distributed in china, korea and russia. e. senticosus has been known in traditional chinese medicine due to putative benefits in general health promotion, vitality, stamina, restoration of homeostasis, chemoprevention, wound healing, longevity and other indications (kitts and hu, 2000). by detailed research, it was found that the active compounds of e. senticosus include ginsenosides, polysaccharides, peptides, polyacetylenes, phenols and enzymes (xiang et al., 2008). the major active compounds are acanthoside d, eleutherosides i, k, l, m (triterpenic saponins), eleutherosides b, b1, d, e (phenylpropane derivates) (kitts and hu, 2000), senticoside, β-sitosterol, sesamin (davydov and krikorian, 2000; lee et al., 2004; li et al., 2006). today, the extracts from different parts of e. senticosus are studied for their biological and pharmacological bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:25 utc nova biotechnologica et chimica 12-1 (2013) 31 effects include antioxidant, antidiabetes, anticancer, anti-inflammatory, imunoregulatory and immunomodulating, antimicrobial and antiviral activities (fujikawa et al., 1996; hibasami et al., 2000; yi et al., 2002; jung et al., 2003). several works are focused on the medical efficacy of e. senticosus mediated via its antioxidant actions, e.g. inhibiting lipid peroxidation and oxidative injury to dna (lee et al., 1998; naval et al., 2007; lee et al., 2008; chen et al., 2010; lee et al., 2011). except for protection against oxidative reaction, the antioxidant activity of e. senticosus is associated with their traditional using as adaptogen by the prevention of human body against faster organ aging. antioxidants from e. senticosus belonging to polar and less polar components can be extracted by various extraction solvent at different extraction conditions. determination of optimal values of extraction parameter is significant point in design of extraction process for obtaining of biological active compounds from plant material. in this regard, the response surface methodology (rsm) is a powerful tool that can provide a complete optimal condition to improve a various technology processes (box and wilson, 1951). it is a collection of statistical and mathematical techniques and it has important applications in the design, development and formulation of new products as well as in the improvement of existing products (myers and montgomery, 1995). the aim of this study was to evaluate the effects of the extraction temperature, solvent composition and solid-liquid ratio on the extraction yield of antioxidants. secondary, total polyphenol content was determined to find possible relations between this parameter and antioxidant activity. this work provides preliminary data for making products from e. senticosus with high antioxidant activity which would be used as food additives or nutraceutical supplement with specific effects to human health. 2. material and methods 2.1 chemicals ethanol 96 %, methanol, folin-ciocalteu reagent and sodium carbonate were obtained from mikrochem (sk). trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid), 2,2-diphenyl-1-picrylhydrazyl (dpph) and gallic acid were obtained from sigma-aldrich (d). 2.2 plant material the roots of e. senticosus (f-dental hodonin, cz) were an aliquot amount of the tested material cut at particle size < 0.1 cm. 2.3 extraction procedure for determination of optimization ranges, 1 g of dried roots of e. senticosus was extracted by extraction solvent (aqueous ethanol with concentration 0, 10, 30, 50, 70, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:25 utc 32 ondrejovič, m. et al. 90 and 96 % (v/v) of ethanol) in solid-liquid ratio (10, 25, 50, 75 and 100 ml per gram of plant material) at various temperatures (20, 30, 50 and 70 °c) during 60 minutes. after 60 minutes, the solution was centrifuged for 2 minutes at 4,000 rpm and the supernatant was used for analysis. 2.4 experimental design three factors, five level experiment was carried out with tested, independent variablestemperature (22, 34, 52, 70 and 82 °c), solvent composition (3.3, 10, 20, 30 and 36.7 % (v/v) ethanol) and solid-liquid ratio (8.2, 25, 50, 75 and 91.8 ml/g). real variables values were transformed into non-dimensional coded form (table 1). table 1. independent variables in original and coded form and experimental results for response variables teac [mg/g plant material] and total polyphenols [mg/g plant material]. stand. order temperature [°c] solvent composition [% etoh] solid-liquid ratio [ml/g] teac [mg/g plant material] total polyphenols [mg/g plant material] 1 34 (-1) 10 (-1) 25 (-1) 15.0 8.8 2 52 (0) 20 (0) 50 (0) 30.9 18.5 3 34 (-1) 30 (1) 75 (1) 20.5 15.9 4 70 (1) 30 (1) 25 (-1) 31.5 17.5 5 70 (1) 10 (-1) 75 (1) 26.1 21.8 6 70 (1) 10 (-1) 25 (-1) 18.9 13.0 7 34 (-1) 30 (1) 25 (-1) 14.7 9.5 8 70 (1) 30 (1) 75 (1) 27.4 21.5 9 52 (0) 20 (0) 50 (0) 30.9 18.0 10 34 (-1) 10 (-1) 75 (1) 24.4 10.7 11 52 (0) 3.3 (-1.682) 50 (0) 18.2 9.8 12 52 (0) 20 (0) 91.8 (1.682) 23.3 20.2 13 82 (1.682) 20 (0) 50 (0) 26.4 12.8 14 52 (0) 20 (0) 8.2 (-1.682) 10.1 6.2 15 22 (-1.682) 20 (0) 50 (0) 17.9 9.1 16 52 (0) 36.7 (1.682) 50 (0) 14.3 12.3 17 52 (0) 20 (0) 50 (0) 28.9 17.5 measured dependent variables were trolox equivalent antioxidant capacity (teac) in mg/g of plant material and total polyphenols in mg/g of plant material. experimental data were fit by the polynomial regression of the second order (eq. 1), and regression coefficients (bi) were calculated. (1) where xi are independent variables responsible for response y and bi are regression coefficients, describing relations of the measured properties to coded levels of the ∑∑∑∑ − < = === +++= 1 1 2 2 11 0 k ji i j jiiji k i ii k i ii xxbxbxbby bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:25 utc nova biotechnologica et chimica 12-1 (2013) 33 selected parameters (table 1). for computer and statistical processing, the statgraphics plus 5.1 (statpoint technologies, usa) was applied. 2.5 analysis of the response variables 2.5.1 trolox equivalent antioxidant capacity teac was determined using the method proposed by yen and chen (1995) by with modification to microplate form. dpph (0.012 %; w/v) was dissolved in pure methanol. the radical stock solution was prepared fresh daily. the dpph solution (100 μl) was added to 25 μl extract. the mixture was shaken and allowed to stand at room temperature for 10 minutes. the decrease in absorbance was monitored at 540 nm. the results were corrected for dilution and expressed in mg trolox per gram of plant material. 2.5.2 total polyphenols total polyphenol content was measured using folin–ciocalteu colorimetric method (singleton et al., 1999). plant extracts (20 μl) were mixed with 20 μl folin– ciocalteu reagent and incubated at room temperature for 5 minutes. following the addition of 200 μl 20 % na2co3 (w/v) to the mixture, total polyphenols were measured at 690 nm. the results were expressed as gallic acid equivalents, mg per gram of plant material. 2.6 statistical analyses all experiments were realized in triplicate and statistical analysis was calculated by statgraphics plus 5.1. 3. results and discussion 3.1 selection of optimization ranges the extraction efficiency of natural compounds from the plant material is dependent on some parameters such as temperature, time, solid-liquid ratio, solvent composition and others. all these parameters were put in optimization aimed to maximal antioxidant activity. the polyphenol content in plant extracts is often employed to content of antioxidant constituents (wu et al., 2004). this dependence was evaluated also in our work. the influence of some above-mentioned parameters was partly reported in literature (chen et al., 2010; lee et al., 2011; park et al., 2006; wang, 2012), but optimization for the extraction of antioxidants and polyphenols from e. senticosus using rsm have not been reported. in the literature, for antioxidant extraction, various extraction solvents such as water, methanol, acetone or their water solutions was used (park et al., 2006; wang, 2012). for this purpose, methanol appeared to be an effective extraction solvent. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:25 utc 34 ondrejovič, m. et al. the temperature plays an important role in the extraction of active substances from plant materials. in this work, we studied antioxidant extraction from e. senticosus at four different temperatures: 20, 30, 50 and 70 °c by measuring kinetics of extraction within 1 hour because it was necessary to determine the extraction time as a fixed parameter in the optimization. after one hour of extraction, the antioxidant activity of prepared extracts no longer increases (fig. 1). therefore, in all experiments, we used extraction parameter – time as fixed parameter with value one hour. according to fig. 1, the most suitable temperature for extraction of antioxidants from e. senticosus was 70 °c. in the literature, the extraction of antioxidants was carried out at 70 °c by distillated water during 3 hours (park et al., 2006). on the other hand, kim et al. (2002) and lee et al. (2011) have done extraction antioxidant activity at laboratory temperature. on the basis of this information, we have been selected the temperature range of 34 – 70 °c. transport of active compounds from plant material to extraction solvent is diffusion process affected by various factors, but primary attribute of extraction is the solubilization of extracted compounds. solubilization of target compounds is affected also by solubilization of ballast compounds. therefore, selection of extraction solvent volume needed for extraction target compounds is very important. the highest antioxidant activity was measured at up to 50 ml/g of plant material. in the literature, authors used various solid-liquid ratios as 13.3 (lee et al., 2011), 35 (wang, 2012) and 100 ml/g of plant material (park et al., 2006). therefore, it can be considered that optimal value of parameter solid-liquid ratio will be located between values 25 – 75 ml/g of plant material. fig. 1. the kinetic of antioxidant extraction by various temperature (20, 30, 50 and 70 °c) during 1 hour by 50 ml to g of plant material expressed as mg of teac per g of plant material. in the context with solubilization of target compounds, solvent polarity is also important. for extraction of active compounds from e. senticosus, authors of other studies recommend using of polar extraction solvent such as ethanol or methanol and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:25 utc nova biotechnologica et chimica 12-1 (2013) 35 their water mixtures. because methanol is toxic and unauthorized for food industry, aqueous solution of ethanol with concentrations of ethanol varied between 0 and 96 % (v/v) was used for antioxidant extraction from e. senticosus. the highest antioxidant activity was determined in extracts prepared by 10 and 20 % (v/v) aqueous ethanol. opposed to the work of lee et al. (2011) was the highest antioxidant activity in extracts prepared from e. senticosus by 40 – 80 % (v/v) aqueous ethanol. based on the measured results, the optimization ranges of solvent composition were selected 10 – 30 % (v/v) aqueous solution of ethanol. 3.2 extraction optimization by response surface methodology (rsm) optimal conditions of the extraction were calculated by the software statgraphics plus 5.1., processed by rsm approach. in table 1, teac and polyphenols are presented. based upon the regression analysis results, we can state, that compared dependent variable expressed self-independent relation. 3.2.1 multiple linear regression for the purpose of the fitting the presented results in table 1, polynomial regression of the second order (eq. 1) had regression coefficient r2 = 0.95 for teac as parameter y1, r2 = 0.91 for total polyphenols as parameter y2. 3.2.2 regression coefficient analysis regression coefficients of the model for teac and total polyphenols obtained by multiple polynomial regression are presented in table 2. dependent variable in coded form (table 1) allow direct interpretation of the effect (linear, quadratic and interaction) of the independent variables to dependent variables and visualization by 3d surface plots (fig. 2) assisted visualization of the statistically important factors (marked as bold in the table 2) obtained from statistical analysis. table 2. regression coefficients of the model polynomial regression of the second order for dependent variables teac [mg/g plant material] and total polyphenols [mg/g plant material]. model parameters teac total polyphenols constant effect -34.11 -20.8457 temperature [°c] (a) 0.673912 0.653699 solvent composition [% etoh] (b) 1.30007 0.83962 linear effect solid-liquid ratio [ml/g] (c) 1.03881 0.213897 a × a -0.00558012 -0.0052778 b × b -0.0390238 -0.0166475 quadratic effect c × c -0.00598639 -0.00143291 a × b 0.0125748 -0.00116015 a × c -0.00335388 0.00123142 interaction effect b × c -0.00736462 -0.000160227 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:25 utc 36 ondrejovič, m. et al. 3.3 determination and experimental validation of the optimal conditions optimal values of the parameters for extraction teac and total polyphenols from e. senticosus are presented in table 3. predicted value for extraction teac (70 °c, 53 ml per g of plant material, 23 % (v/v) aqueous ethanol) and total polyphenols (70 °c, 91 ml per g of plant material, 22 % (v/v) aqueous ethanol) from e. senticosus as dependent parameters were comparable with experimentally measured value at the level of the statistical significance at p < 0.05. achieved results confirm the possibility to predict the course of the extraction of active substances from e. senticosus by the model under particular experimental conditions. fig. 2. the relation of the dependent variables from the temperature and solvent composition at constant solid-liquid ratio 50 ml per g of plant material; a – teac [mg of teac per g of plant material]; b – total polyphenols [mg of gallic acid per g of plant material]. table 3. optimal extraction parameters for maximizing yield of teac and total polyphenols and comparison of the predicted values of the dependent variables and experimentally measured values at these optimal conditions. optimal extraction parameters temperature [°c] 70 70 solvent composition [% etoh] 23 22 solid-liquid ratio [ml/g] 53 91 teac [mg/g plant material] total polyphenols [mg/g plant material] predicted values 32.1 22.8 experimental values 31.4 21.3 a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:25 utc nova biotechnologica et chimica 12-1 (2013) 37 in the work lee et al. (2011) not found the optimal conditions for antioxidant extraction from e. senticosus. they describe that both concentration ethanol in extraction solvent and extraction time weren´t affected antioxidant extraction. in comparison with our experiment, they used low value of solid-liquid ratio, which cannot allow achieving sufficient antioxidant extraction. conclusions the response surface methodology was successfully employed to optimize the antioxidant extraction from e. senticosus. the second-order polynomial model gave a satisfactory description of the experimental data. the optimal conditions for antioxidant extraction were 70 °c, 53 ml per g of plant material, 23 % (v/v) aqueous ethanol and the optimal conditions for the extraction of total polyphenols were 70 °c, 91 ml per g of plant material and 22 % (v/v) aqueous ethanol. predicted values of dependent variables were comparable with the experimentally measured values. acknowledgement: this work was supported by grant no.: apvv-vmsp-ii-0021-09. references box, g.e.p., wilson, k.b.: on the experimental attainment of optimum conditions. j. r. stat. soc. series b, 13, 1951, 1-45. chen, c.y.o., ribaya-mercado, j.d., mckay, d.l., croom, e., blumberg, j.b.: differential antioxidant and quinone reductase inducing activity of american, asian and siberian ginseng. food chem., 119, 2010, 445451. davydov, m., krikorian, a. d.: eleutherococcus senticosus (rupr. & maxim.) maxim. (araliaceae) as an adaptogen: a closer look. j. ethnopharmacol., 72, 2000, 345-393. fujikawa, t., yamaguchi, a., morita, i., takeda, h., nishibe, s.: protective effects of acanthopanax senticosus harms from hokkaido and its components on gastric ulcer in restrained cold water stressed rats. biol. pharm. bull., 19, 1996, 1227-1230. hibasami, h., fujikawa, t., takeda, h., nishibe, s., satoh, t., fujisawa, t., nakashima, k.: induction of apoptosis by acanthopanax senticosus harms and its component, seamin in human stomach cancer kato iii cells. oncol. rep., 7, 2000, 1213-1216. jung, h.j., park, h.j., kim, r.g., shin, k.m., ha, j., choi, j.w., kim, h.j., lee, y.s., lee, k.t.: in vivo anti-inflammatory and antiociceptive effects of liriodendrin isolated from the stem bark of acanthopanax senticosus. planta med., 69, 2003, 610-616. kim, l.h., han, s.s., choi, y.s.: antioxidant effects of the extracts of acanthopanax senticosus. korean j. pharmacogn., 32, 2002, 359-363. kitts, d., hu, c.: efficacy and safety of ginseng. public health nutr., 3, 2000, 473485. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:25 utc 38 ondrejovič, m. et al. lee, b.m., lee, s.k., kim, h.s.: inhibition of oxidative dna damage, 8-ohdg, and carbonyl contents in smokers treated with antioxidants (vitamin e, vitamin c, beta-carotene and red ginseng). cancer lett., 132, 1998, 219-227. lee, s., son, d., ryu, j., lee, y.s., jung, s.h., kang, j., lee, s.y., kim, h.s., shin, k.h.: anti-oxidant activities of acanthopanax senticosus stems and their lignan components. arch. pharm. res., 27, 2004, 106-110. lee, s.r., shin, h.h., jeong, j.h., hwang, k.t., kim, t.y.: effect of ethanol concentrations and extraction time on acanthoside-d and total polyphenol contents and antioxidant activities in ethanol extracts of eleuthero. j. med. plant res., 24, 2011, 5700-5705. lee, y.j., chung, h.y., kwak, h.k., yoon, s.: the effects of a. senticosus supplementation on serum lipid profiles, biomarkers of oxidative stress and lymphocyte dna damage in postmenopausal women. biochem. biophys. res. commun., 375, 2008, 44-48. li, q., jia, y., xu, l., wang, x., shen, z., liu, y., bi, k.: simultaneous determination of protocatechuic acid, syringin, chlorogenic acid, caffeic acid, liriodendrin and isofraxidin in acanthopanax senticosus harms by hplc-dad. biol. pharm. bull., 29, 2006, 532-534. myers, r.h., montgomery, d.c.: response surface methodology: process and product optimization using designed experiments. wiley, new york, 1995, 704 pp. naval, m.v., goméz-serranillos, m.e., carretero, m.e., villar, a.m.: neuroprotective effect of a ginseng (panax ginseng) root extract on astrocytes primary culture. j. ethnopharmacol., 112, 2007, 262-270. park, h.r., park, e., rim, a.r., jeon, k.i., hwang, j.h., lee, s.c.: antioxidant activity of extracts from acanthopanax senticosus. afr. j. biotechnol., 5, 2006, 2388-2396. singleton, v.l., orthofer, r., lamuela-raventos, r.m.: analysis of total phenols and other oxidation substrates and antioxidants by means of folinciocalteu reagent. methods enzymol., 299, 1999, 152-178. wang, b.: antioxidant activity of acanthopanax senticosus extract and derivatives. adv. mat. res., 531, 2012, 524-527. wu, x., beecher, g.r., holden, j.m., haytowitz, d.b., gebhardt, r.l., prior, r.l.: lipophilic and hydrophilic antioxidant capacities of common foods in the united states. j. agric. food chem., 52, 2004, 4026-4037. xiang, y.z., shang, h.c., gao, x.m., zhang, b.l.: a comparison of the ancient use of ginseng in traditional chinese medicine with modern pharmacological experiments and clinical trials. phytother. res., 22, 2008, 851858. yen, g.c., chen, h.y.: antioxidant activity of various tea extracts in relation to their antimutagenicity. j. agric. food chem., 43, 1995, 27-32. yi, j.m., hong, s.h., kim, j.h., kim, h.k., song, h.j., kim, h.m.: effect of acanthopanax senticosus stem on mast cell-dependent anaphylaxis. j. ethnopharmacol., 79, 2002, 347-352. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 14:25 utc nova biotechnol chim (2017) 16(2): 76-85 doi: 10.1515/nbec-2017-0011  corresponding author: fargasova@fns.uniba.sk  nova biotechnologica et chimica plant stress activated by chlorine from disinfectants prepared on the base of sodium hypochlorite agáta fargašová department of environmental ecology, faculty of natural sciences, comenius university in bratislava, ilkovičova 6, bratislava, sk-842 15, slovak republic article info article history: received: 14th july 2017 accepted: 12th november 2017 keywords: growth inhibition phytotoxicity pigment production sodium hypochloride water content abstract in this study, the phytotoxicity of disinfectants prepared on the base of sodium hypochlorite was determined. for our tests two commercial products, savo and dom amor, as well as 10% naclo solution were used. while savo contained only naclo, dom amor contained naclo and earthworm enzymes. products on the base of naclo are used in households for cleaning and disinfection of floors, furniture, sanitary and kitchen equipment. savo may be used for the disinfection of drinking waters as well. products with naclo are also used for bacteria, algae and pathogens reduction in irrigation waters. as a subject, young seedlings of mustard sinapis alba l. were used for the study of chronic toxicity. the observed parameters of the inhibition of roots and shoots growth, dry (dm) and fresh (fm) mass as well as photosynthetic pigments production (chlorophyll a, b, carotenoids) and water content in the plants were determined. the results point out that dom amor was the most toxic for s. alba seedlings growth and the rank order of the fac contents for both plant parts was arranged as: dom amor > savo > naclo. all disinfectants reduced the dm and fm of roots; however, they stimulated biomass production in the shoots. on the basis of the obtained results it could be concluded, that disinfectants stimulated photosynthetic pigments production and reduced water content mainly in the roots. dom amor did not significantly reduced the water content in the shoots and for this parameter the following rank orders of inhibition for roots and shoots could be arranged as naclo > dom amor > savo and naclo > savo > dom amor, respectively. all commercial products increased chlorophyll a (chla) and the carotenoids (car) content in the shoots. as significant increase was confirmed first for chla whose content in the presence of naclo at concentration 24 ml/l overextended that in the control by 3.5 times. the rank orders of stimulation for chla and car were naclo > savo > dom amor and dom amor > naclo > savo, respectively.  university of ss. cyril and methodius in trnava       introduction chlorine is classified as a plant micronutrient essential for proper plant growth and all crops require it in small quantities. it is important for photosynthesis acts as co-factor (hajrasuliha 1980), it is involved in the opening and closing of stomata and plays some important role in osmotic adjustment and plant disease suppression (slabu et al. 2009). it is also important for enzyme activity regulation in the cytoplasm, acts as a counter anion to stabilize membrane potential, and is involved in turgor and ph regulation (xu et al. 2000; white and broadley 2001). chlorine bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:51 utc nova biotechnol chim (2017) 16(2): 76-85 77 deficiency evokes chlorosis and necrosis. necrosis appears along leaves margins and tips, leaves are smaller than usual and plant growth is also reduced (montero et al. 1998; marschner and marschner 2012). symptoms are usually seen first on older leaves. high chloride concentration can reduce the yield (albacete et al. 2008). crops differ both in their chloride requirements as well as in their tolerance to chloride toxicity. since chloride is an anion, it does not adsorb to soil particles and moves readily with the water in the soil. therefore, water quality and irrigation management are the major factors that affect chloride concentration in soil. other chloride sources in the soil are some fertilizers. chloride toxicity is often accompanied or complicated by salinity or infiltration problems which appear when salinity is low. the most common toxicity is from chloride in irrigation water. as chloride is not adsorbed to soil, it moves readily with the soil water and is taken up by the crop, moves in the transpiration steam, and accumulates in the leaves. however, toxic symptoms appear when the chloride content in the leaves achieves 0.3 to 1.0% on dry weight, and sensitivity depends on crop. as presented by xu et al. (2000) and white and broadley (2001) the critical concentration for chloride toxicity is estimated to be 4-7 mg/l for cl sensitive and 15 – 50 mg/l for cltolerant species. chloride transport and exclusion from shoots is correlated with salt tolerance in many species. crop tolerance to chloride is not nearly so well documented as crop tolerance to salinity, however, its concentration is important for salt tolerance (tavakkoli et al. 2010; teakle and tyerman 2010). chlorine inputs to soils occur mainly as a result of cldeposition from rainwater, fertilizer applications (kcl), irrigation water, sea spray, dust and air pollution. cldeposition from human activity belongs mainly to irrigation and fertilization. however, some agricultural establishments try using water for irrigation from reservoirs or ponds, such water recycling may disperse plant pathogens into crops (hong and moorman 2005). bush et al. (2003) presented that recycled irrigation water from harbors contained substantial densities of pathogens and hong and moorman (2005) reported the presence of 17 phytophthora sp., 26 pythium sp., 27 genera of fungi, 8 species of bacteria, 10 viruses and 13 species of plant parasitic nematodes in irrigation water from ponds, rivers, canals, streams, lakes, runoff water, etc. these facts explain why chlorine is used for disinfection of irrigation water (cayanan et al. 2009). at present, it is widely used in agriculture, chemical, paintand lime, food, glass, paper, pharmaceutical, synthetic and water disposal industries. in the textile industry, it is used as a bleacher and chlorine compounds could also be used for electrochemical treatment of water contaminated with dyes (valica and hostin 2016). sometimes naclo is added to industrial waste waters to reduce odor. hypochlorite can be used to prevent algae and shellfish growth in cooling towers and in water treatment, it is used for water disinfection. in households, it is frequently used for the purification and disinfection of the house. however, water with commercial sodium hypochlorite products released into a water environment has relatively low concentrations enough, strong toxic effects on freshwater invertebrates and bacteria may appear (fargašová 2017). by adding sodium hypochlorite to water, hypochlorous acid (hclo) and hypochlorite ions (clo-) are formed. the equilibrium of these two forms depends only on water ph (pka for hclo/clois ~ 7.4). naclo in water is gradually depleted for oxidation of inorganic and organic compounds (mohammadi 2008). however, naclo is very effective and low cost biocide used for water disinfection (amin et al. 2013), hypochlorite is a highly destructive, selective oxidant that reacts easily with all biomolecules. moreover, dissolved chlorine dissociated into hypochlorite and hypochlorous ions, which penetrate cell membrane, may result in the formation of genotoxic, mutagenic, and/or carcinogenic disinfection byproducts (dpbs) (sapone et al. 2016) and are also associated with adverse reproductive outcomes (nieuwenhuijsen et al. 2000). as described by white and broadley (2001), chlorine occurs in the soil predominantly as a clanion, which does not form complexes readily, and is repelled from predominantly negatively charged mineral surfaces of many soil particles. this fact changes clmovement in the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:51 utc nova biotechnol chim (2017) 16(2): 76-85 78 soil and the consequence of its slight adsorption to soil components is not chemically altered by soil organisms. its movement within the soil is largely determined by water flows. that’s why is often used as a tracer for soil water movement. during our study, attention was focused on free available chlorine – the fac (mg/l) effect of naclo and two commercial disinfectant products, prepared on naclo base, on some physiological processes in crop sinapis alba. chlorine enters the plants through the roots by a symplastic pathway and is mobile within the plant. its fluxes across membranes and between tissues limited growth, water translocation in the plant and interferes with photosynthesis through inhibition of photosynthetic pigments production. in an effort to avoid chlorine movement through soils and the assurance of direct fac effect on plant growth and physiological activity, only water solutions of sodium hypochlorite products were used. experimental disinfectans the following commercial products on the base of sodium hypochlorite (naclo) were investigated with regard to their phytotoxic effects – savo (produced by bochemie a.s., bohumín, czech republic) and dom amor (produced by boos biologické substancie, košice, slovak republic). savo contained only naclo; while dom amor contained both naclo and earthworm enzymes. the content of effective ingredients (cei) of products did not exceed 5%. the toxicity of sodium hypochlorite (naclo) (10% solution) was also determined. the active substance of naclo in water is hypochlorous acid (hclo) and hypochlorite ions (clo-). fresh samples were used for each test due to high chlorine volatility. the concentration of free available chlorine (fac) of products was determined by the reaction of the tested products with phosphate buffer and potassium iodide, followed by titration of thiosulphate on starch (añasco et al. 2008). the results of the analyses for three tested products are presented in table 1. however, only free active chlorine concentrations, being the most active and dominant form, are presented here. doses of the disinfectants used in the tests were decided after preliminary experiments performed in the laboratory according to the corresponding guide (oecd 208 2006). table 1. content of effective ingredients – cei (%) and the concentration of free available chlorine – fac (mg/l) in tested substances. cei (%) fac (mg/l) savo 5 39.90 dom amor 5 32.21 naclo 10 108.86 the dissipation tests were conducted before the experiments due to chlorine volatility. to provide a constant chlorine level, the solutions were replaced every 24 hours. under these conditions, fac concentration at the end of the experiments did not decrease below 90%. the typical levels of free chlorine in drinking water ranged from 0.2 – 2.0 mg/l, though regulatory limits allow levels as high as 4.0 mg/l. sinapis alba growth inhibition test mustard sinapis alba l. seeds were germinated in petri dishes with 17-cm diameter, laboratory filter paper no. 1 disks and plastic nets on the bottom. nine different concentrations from each sample (table 2), designed on the base of orientation tests, were prepared in dechlorinated tap water (80 mg/l ca, 27 mg/l mg, ph = 7.3±0.05). each petri dish contained 50 ml of tested solution and 20 healthy looking and similar size mustard seeds spread on the plastic net situated on the filter paper laid down on the bottom of the dish. as a control, only tap water was used in the same way. dishes were put in a dark thermostat at 22±1°c and plastic nets with germinated seeds were replaced every 24 hours to fresh tested solutions. after 72 hours the roots and shoots length was measured and ic50 concentrations were calculated by using probit analysis (fargašová and lištiaková 2009). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:51 utc nova biotechnol chim (2017) 16(2): 76-85 79 photosynthetic pigments and water content determination after 72 hours plastic nets with seedling were transferred to hydroponic dark containers (250 ml) with control or disinfectants solutions, situated in laboratory box with a day-light cycle (16/8), and the temperature was maintained at 23±2 °c. afterwards, the dishes were shielded from the direct sunlight and the cultivation continued for the next 7 days. during this period, the tested solutions were changed every 24 hours in the same way as was described before to provide a constant chlorine concentration during the entire experiments. during this cultivation shoots were not in direct contact with the disinfectant solutions. after 10 days of growth, the plants were harvested and the roots and shoots were separated. fresh mass (fm) was weighed immediately after organ separation. the pigment contents (chlorophyll a, b, total table 2. concentrations of savo, dom amore and naclo used during the tests with s. alba seedlings expressed as the fac concentrations in mg/l and the volume of solution ml/l (used concentrations were selected on the base of preliminary tests). compound concentration savo ml/l naclo 2.0; 4.0; 6.0; 8.0; 12.0; 16.0; 24.0; 32.0; mg/l fac 0.08; 0.16; 0.24; 0.32; 0.48; 0.64; 0.96; 1.28; dom amore ml/l naclo 2.0; 4.0; 6.0; 8.0; 12.0; 16.0; 24.0; 32.0; mg/l fac 0.064; 0.13; 0.19; 0.26; 0.39; 0.52; 0.77; 1.03; naocl ml/l naclo 2.0; 4.0; 6.0; 8.0; 12.0; 16.0; 24.0; 32.0; mg/l fac 0.22; 0.44; 0.65; 0.87; 1.31; 1.74; 2.6; 3.48; carotenoids) were determined in the fresh shoots after extraction in 95% ethanol (w/v) (1 g of fresh shoots per 6 ml of ethanol). pigment extraction continued until all of the homogenized plant mass was white. after a brief centrifugation (2 min at 2 900 x g), the pigment content was measured spectrophotometrically at 665, 649 and 470 nm in supernantant (lichtenthaler and wellburn 1983). photosynthetic pigments amount was calculated by the following equations: chla = 13.95 (a665) – 6.88 (a649) chlb = 24.96 (a649) – 7.32 (a665) car = [1 000 (a470) – 2.05 (chl a) – 114.8 (chl b)]/245 (chla – chlorophyll a concentration, chlb – chlorophyll b concentration, car – carotenoids concentration; in µg/ml of plant extract). afterwards plant material – roots and shoots, were separately dried at a temperature of 40 °c to a constant mass and then weight. from the obtained values of fresh (fm) and dry mass (dm), the water content was calculated (drazic and mihailovic 2005): wc = (fm – dm)/dm (wc – water content, fm – fresh mass, dm – dry mass) in g/g dm of s. alba seedling organs and the effect of tested solutions on wc in the roots and shoots was determined as a standardized values of the reference control. statistical analysis all phytotoxicity tests were carried out in triplicate, and they included a control in tap water. quality control data were considered acceptable according to control charts and other established criteria. the adstat 2.0 statistical software was used for statistical evaluation. a t-test was used to assess the significant difference between the controls and other treatments. results and discussion all results obtained by the use of the above mentioned methods were evaluated according to an ecological risk assessment framework which implies the examination of risks from natural, human and industrial activities (fargašová 2016). experiments with the crop s. alba confirmed that chlorine ions are generally toxic to plants’ growth at relatively low concentrations and may cause irreversible damage of their development. free bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:51 utc nova biotechnol chim (2017) 16(2): 76-85 80 chlorine is often added to irrigation water for algae, fungi, and bacteria destruction. chlorination is used mainly for surface irrigation water from rivers, canals, reservoirs and ponds, as well as for the prevention of clogged piping by organic material (cayanan et al. 2009; parke and fisher 2012). as we presented before, the usage of disinfectant prepared on the base of naclo is very effective for such prevention, because free chlorine levels which destroy algae are at least 10-times lower than those which reduced plant growth by 50% (fargašová 2017). the inhibitory concentrations (ic) which reduced roots and shoots growth of mustard by 50% together with their confidence intervals (ci) are introduced in table 3. from these results, it is evident that the roots of s. alba were more sensitive to disinfectants than shoots, and the commercial product dom amor enriched with earthworm table 3. ic50 concentrations and their 95% confidence intervals (ci) for roots and shoots growth inhibition (values are introduced as volume of compound in ml/l as well as the concentration of free available chlorine in mg/l fac). compound roots growth shoots growth ic50  (ci) (ml/l) ic50  (ci) (mg/l fac) ic50  (ci) (ml/l) ic50  ci) (mg/lfac) savo 12.68 (12.20 – 13.25) 0.51 (0.49 – 0.53) 19.98 (18.45 – 25.59) 0.80 (0.74 – 1.02) dom amor 6.85 (6.25 – 7.33) 0.22 (0.20 – 0.24) 18.58 (17.32 – 20.38) 0.60 (0.55 – 0.65) naclo 5.16 (4.96 – 5.33) 0.56 (0.54 – 0.58) 8.84 (8.65 – 9.05) 0.97 (0.94 – 0.99) enzymes had the strongest inhibitory effect on both plant parts. for inhibition, the following rank orders for fac content should be arranged as so: roots growth: dom amor  savo ≥ naclo; shoots growth: dom amor  savo  naclo. all tested compounds reduced the growth more for roots than shoots. this might be explained by greenway and thomas (1965) conclusion, which assume that the shoots initially contained no cland therefore, the toxic effects of chlorine don’t appear mainly in young seedlings. according to the growth responses of plants of high clconcentration in the environment plants could be divided into four categories. the differences between them are often related to the ability to restrict cltransport to shoots (white and broadley 2001). large differences between the chlorine content in various plant parts and its relation to the manifestation toxic effects, was also confirmed (greenway and munns 1980) long before. chlorine, as an essential element, supporting plant growth is taken into the xylem and thereby delivered to the shoots (white and broadley 2001). there are two pathways for clanion intake into the xylem – the symplastic (cytoplasmic) and the apoplastic (extracellular). the intake process influences fluxes and accumulation of the chlorine into the plant and its distribution within the plant. toxic effects of chlorine on plants development were confirmed also by carrilo et al. (1996) to radish and lettuce seedlings after the application of a commercial dioxide product (hallox) with 2.6 mg/l of active chlorine. hallox was in this case used for direct irrigation on a soil surface at a level of 1 : 1000. as presented by cayanan et al. (2009) chlorine had no phytotoxic or growth effects on all plants and growth inhibition depends on free active chlorine content. in addition to growth, dry and fresh mass production could also be affected and this was also confirmed by our experiments. plant growth is very closely connected with crop production expressed as dry (dm) and fresh (fm) mass production. both these parameters are very strongly influenced by chlorine fluxes and accumulation within the roots and its translocation into the shoots (white and broadley 2001). chlorine intake is closely connected with water uptake and translocation through plants and the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:51 utc nova biotechnol chim (2017) 16(2): 76-85 81 fig.1. water content in the roots and shoots of s. alba after 10 days’ application of savo, dom amor and naclo disinfectants introduced as standardized values of the reference control (water content in control = 1). statistical significance: * p < 0.05; ** p < 0.01. results from these observations are shown on fig. 1. the presented results indicate that all products in all used concentrations reduced more water reception by the roots than its translocation into the shoots. plants try to supply chlorine toxicity by its movement through water into the upper part of the plant and in this way, attempt to reduce chlorine toxicity in the roots which are responsible for nutrients’ reception. as presented by white and broadley (2001), plants support their growth by loading chlorine into the xylem and thereby deliver it to the shoots. this is in accordance with the results we obtained during the determination of water content and dry and fresh mass (fig. 1, 2). these authors showed that there are two pathways by which anions might reach the xylem: (1) anions enter root cells through a plasma membrane, are then transferred from cell to cell through plasmodesmatal connections, and are exported across the plasma membrane of cells into the stele (2) anions are taken extracellularly through the cell wall and water spaces to reach the stele – this is a relatively non-selective process governed by the transport of water through solvent drag. tested compounds significantly reduced water content only in the roots while the water content in the shoots was stimulated or reduced only slightly. however, dom amore increased the water content in the shoots in all of the tested concentrations; naclo indicated a reduction which did not exceed 64%. the following rank orders of water content reduction could be arranged as so: for roots naclo  dom amor  savo, for shoots naclo  savo  dom amor. water reception from the solvents with naclo, including the two commercial disinfectant products savo and dom amor, is very closely connected with dry (dm) and fresh (fm) production by the roots and its translocation to upper plant parts (fig. 2) from the obtained results, it can be concluded that while dm and fm production of the roots was reduced in the presence of chlorine from all tested compounds, dm and fm of the shoots production was stimulated and this is in accordance with the calculations and equations by white and broadley (2001), who confirmed the close relation between the root uptake of chlorine and a plant’s relative growth and biomass production. as was previously presented, chlorine is an essential micronutrient for higher plants and its minimal requirement for crop growth is 1 g/kg dm. however, high chlorine concentration in tissues can bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:51 utc nova biotechnol chim (2017) 16(2): 76-85 82 fig. 2. roots and shoots dry (dm) and fresh (fm) mass production in the presence of tested disinfectants after 10 days’ cultivation introduced as standardized values of the reference control (water content in control = 1). statistical significance: * p < 0.05; ** p < 0.01. be toxic to a crop and restrict the agriculture mainly in saline regions. many important cereals, vegetables and fruit crops are susceptible to cl toxicity during cultivation (xu et al. 2000), and this statement was confirmed also for the mustard crop during our study. the last observed parameter during our experiments was the determination of photosynthetic pigments content. these results are presented on fig. 3, and suggest a significant increase of chla content in the presence of all disinfectants. its production increased with the disinfectant’s concentration, and maximal stimulation was confirmed during naclo application when the chla content in the naclo concentration of 24 ml/l overextended its content in the control by 3.5 times. for this pigment stimulation, the following rank order may be arranged: naclo  savo  dom amor. however, chla is the most abundant pigment in plant and absorbs red wavelengths of light; chlb is not as abundant and probably evolved later. in presence of active chlorine, its concentration did not significantly change. cars are a class of accessory pigments and in plants; they contribute to the photosynthetic machinery and protect them against photo-damage. in the presence of all the tested disinfectants, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:51 utc nova biotechnol chim (2017) 16(2): 76-85 83 carotenoids concentration in the shoots were in higher applied concentrations significantly increased, however, did not overextend their content in the control by to 2.5 times for dom amor product. the stimulatory rank order for this case was: dom amor  naclo  savo. chlorophyll content and fluorescence have been used to investigate the adverse effects of various environmental factors in experiments under controlled conditions. as presented by khaleghi et al. (2012), drought is one of the factors affecting photosynthesis and chlorophyll content. the measurement of chlorophyll content and fluorescence has become established as a sensitive method for assessing the efficiency of psii photosynthetically, as well as its role in environmental perturbations of photosynthesis. some researchers have reported that chlorophyll content might estimate the influence of environmental stress on growth because these parameters were closely correlated with the rate of carbon exchange (figueroa et al. 1997; guo and li 2000). as presented by khaleghi et al. (2012), kiani et al. (2008) and guerfel et al. (2009), chlorophyll content (chla, chlb and total chlorophyll = chla + chlb) decreased under stress elicited by water toxicity. these statements can explain why the content of chlorophylls and carotenoids increased in the presence of free available chlorine in the concentrations used during our experiments with three disinfectant products. chlorine is classified as a plant micronutrient and is important for photosynthesis (slabu et al. 2009 marschner and marschner 2012). in a high concentration, chlorine reduces plant growth, reduce yield (albacete et al. 2008) and interfere with photosynthesis (harjasuliha 1980). the critical concentration for chloride toxicity is estimated as 4 – 7 mg/l fac for sensitive plants. in our tests, the fac concentration which was used was 10-times lower, and adverse effects developed mainly in the roots. shoots were more than twotimes less sensitive than roots and their production of fm and dm increased. the water content in the shoots increased or was reduced only slightly, and fig. 3. disinfectants effect on the production of chlorophyll a (chla), chlorophyll b (chlb) and carotenoids (car) in the shoots of s. alba after 10 days’ cultivation (µg/ml). statistical significance was in all cases at least p < 0.05. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:51 utc nova biotechnol chim (2017) 16(2): 76-85 84 this could explain why the photosynthetic pigment content also increased. conclusions in low concentrations chlorine is classified as a plant micronutrient and the phytotoxic effects of its anion begin to appear only in higher concentrations. the obtained results confirmed that in s. alba crop roots are more sensitive to free active chlorine. all tested products – naclo and the two commercial products savo and dom amor, reduced roots growth more than dry (dm) and fresh mass (fm) production. dm and fm production correlate also with a strong reduction of water content (wc) in the roots. slight shoots growth decreases as well, since dm and fm stimulation support photosynthetic pigments production, mainly that of chlorophyll a (chla). acknowledgement this study was performed with the support of the scientific grant agency vega 1/0098/14 and kega 029uk-4/2016 references albacete a, ghanem, me, martinez-andujar c, acosta m, sanchez-bravo j, martinez v, lutts s, dodd ic, perezalfocea f (2008) hormonal changes in relation to biomass partitioning and shoot impairment in salinized tomato (solanum lycopersicum l.) plants. j. exp. bot. 59: 4119-4131. amin mm, hashemi h, bovini am, hung yt (2013) a review on wastewater disinfection. int. j. environ. health. eng. 2: 22-30. añasco nc, koyama j, imai s, nakamura k (2008) toxicity of residual chlorines from hypochlorite-treated seawater to marine amphipod hyale barbicornis and estuarine fish oryzias javanicus. water air soil pollut. 195: 129136. bush ea, hong c, stromberg ei (2003) fluctuation of phytophthora and phythium spp. in 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(2012) evaluation of chlorophyll content and chlorophyll fluorescence parameters and relationships between chlorophyll a, b and chlorophyll content index under water stress in olea europea cv. dezful. agric. biosystems engr. 6: 636-639. kiani sp, maury p, sarrafi a, grieu p. (2008) qtl analysis of chlorophyll fluorescence parameters in sunflower (helianthus annuus l.) under well-watered and waterstressed conditions. plant sci. 175: 565-573. lichtenthaler hk, wellburn ar (1983) determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. biochem. soc. trans. 603: 591-592. marschner h, marschner p (2012) marschner's mineral nutrition of higher plants, 3rd ed. elsevier/academic press, london; waltham, ma, 672 p. mohammadi z. 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(2006) terrestrial plant test: seedling emergence and seedling growth test. organization for economic co-operation and development, paris, 19 p. parke j, fisher p (2012) treating irrigation water. digger 56: 41-45. sapone a, canistro d, vivarelli f, paolini m (2016) perturbation of xenobiotic metabolism in dreissena polymorpha model exposed in situ to surface water (lake trasimene) purified with various disinfectants. chemosphere 144: 548-554. slabu c, zörb c, steffens d, schubert s. (2009) is salt stress of faba bean (vicia faba) caused by na+ or cltoxicity? j. plant nutr. soil sci. 172: 644-650. tavakkoli e, rehgasamy p, mcdonald gk (2010) high concentration of na+ and clions in soil solution have simultaneous detrimental effects on growth of faba been under salinity stress. j. exp. bot. 61: 4449-4459. teakle nl, tyerman sd (2010) mechanisms of cltransport contributing to salt tolerance. plant cell environ. 33: 566-589. valica m, hostin s. (2016) electrochemical treatment of water contaminated with methylorange. nova biotechnol. chim. 15: 55-64. white pj, broadley mr (2001) chloride in soils and its uptake and movement within the plant: a review. ann. bot. 88: 967-988. xu g, magen h, tarchitzky j, kafkafi u (2000) advances in chloride nutrition of plants. adv. agron. 68: 97-150. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:51 utc nova biotechnol chim (2017) 16(2): 124-131 doi: 10.1515/nbec-2017-0017  corresponding author: wipharatc@nu.ac.th  nova biotechnologica et chimica microextraction based on solidified floating organic drop coupled with etaas for the determination of lead in herbs arnon thongsaw, ratana sananmuang, gareth m. ross and wipharat chuachuad chaiyasith department of chemistry, research center for academic excellence in petroleum, petrochemical and advanced materials, faculty of science, naresuan university, phitsanulok, thailand 65000 article info article history: received: 19th july 2017 accepted: 27th november 2017 keywords: etaas herb samples lead solidified of floating organic droplet microextraction abstract a rapid, inexpensive and practical solidified of floating organic droplet microextraction (sfodme) prior to electrothermal atomic absorption spectrometry (etaas) was proposed for lead (pb) determination in herb samples. for sfodme procedure, 1-(2-pysidylazo)-2-naphthol was used as a complexing agent. analytical parameters influencing the extraction efficiency, i.e. types and volume of extracting solvent, concentration of 1-(2-pyridylazo)-2-naphthol, ph, extraction temperature and time were optimized. under the optimized conditions, lod and loq were 0.064 and 0.214 µg l-1, respectively, and an enrichment factor was achieved at 18.71 with the relative standard deviation ranging from 1.3 to 2.5% (n=6). the proposed method was effectively applied to the determination of lead in spinach leaves (srm-1570a) and thai herb samples with acceptable results.  university of ss. cyril and methodius in trnava           introduction in recent years, consumption of herbs for health benefits are not only common in asia but are also widely used around the world. they can also have used for phytotherapy, which is using plants for relieve and treat of diseases. medicinal plants have been increasingly used due to their mild and low side effects (rates 2001; oviedo et al. 2013). manufacturers are only required to carry out the analysis of contaminants such as hazardous metals in the raw plant materials. however, the extracts normally do not define the quality and amount of contaminate present. in order to help the quality estimation of these products, the analysis of contaminates can reduce health risk, especially by toxic metals. therefore, it is necessary to monitor the amount of toxic heavy metals in these samples (khan et al. 2008). lead (pb) results in harmful effects to the renal, endocrine, digestive, cardiovascular, reproductive systems as well as affecting the development of neurologic system (solidum 2014). the world health organization (who) has suggested 10 mg kg-1 as the maximum acceptable concentration of pb in therapeutic plants. literature review found that pb was mostly found in a variety of herbs and showed pb contents between 0.2 to 2.7 mg kg-1 (divrikli et al. 2014). consequently, traceable and reliable measurement of pb is essential for the safety and risk assessment for the wide use of traditional and medicinal herbs. currently, there are some variety methods related to modern instrumentation that have been widely used for heavy metal determination; these include icp (ms and aes) (zhao et al. 2012; sorbo et al. 2014; deng et al. 2015; tai et al. 2016) and aas (demirtaş et al. 2015; batista et al. 2016; bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:11 utc nova biotechnol chim (2017) 16(2): 124-131 125 zhong et al. 2016). however, trace determination of pb in herb samples is relatively inconvenient because of the matrix effect and low concentration of pb. to answer this problem, separation or pre-concentration techniques are required. numerous techniques have been examined for pre-concentration/determination of trace pb, namely liquid phase extraction (ikeda et al. 1998; dapaah et al. 1999; zendelovska et al. 2001) and solid phase extraction (dadfarnia et al. 2007; ghaedi et al. 2007; tharakeswar et al. 2012). although, conventional extraction methods can be obtaining reliable results, they usually have some disadvantages such as required long time, labor intensive and/or required large volumes of extracting solvent. in order to find a solution to pre-concentration, microextraction techniques, namely single drop microextraction (sdme) (maltes et al. 2008; manzoori et al. 2009), solid phase microextraction (spme) (zhang et al. 1994; górecki and pawliszyn 1996; djozan and assadi 2004) dispersive liquid liquid microextraction (dllme) (lópez-garcía et al. 2014; jalbani and soylak 2015; rosa et al. 2015) and hollow fiber liquid phase microextraction (hflpme) (xia et al. 2007; jiang et al. 2009; saleh et al. 2009) have been reported. solidified of floating organic droplet microextraction (sfodme) was developed in 2007 (lam et al. 2010). this microextraction methodis different from other liquid phase microextraction that uses a microliter volume of the extractant )melting point around 10 – 30 °c( delivered to the sample solution, stirred for an appropriate time, and then relocated into an ice bath. after the extracting solvent becomes solid,it is transferred into another vial and melted immediately. finally, the solution is directly determined the analyte concentration. from the literature, there are only few reports related to separation/pre-concentration of pb in herb samples (solidum 2014; tai et al. 2016, lam et al. 2010; yavuz et al. 2016). therefore, sfodme technique combination with etaas for the pb determination in herb samples was conducted in this work. we selected 1-(2-pyridylazo)-2-naphthol as the complexing agent and the several parameters influencing the extraction efficiency were optimized. experimental standard solution of pb 1,000 mg l-1 was obtained from avs titrinorm (vwr international, belgium) and working standard solutions were freshly prepared with deionized water. chelating reagent was prepared by dissolving an appropriate amount of 1-(2-pyridylazo)-2-naphthol (acros organics, us) in ethanol. 1-undecanol was purchased from merck (ohg, germany). buffer solutions of ammonium acetate (ph 4, 0.2 mol l-1), phosphate (ph 7, 0.2 mol l-1), and ammonium chloride (ph 9, 0.2 mol l-1) were used to adjust the ph in the range of 3.0 – 12.0. the laboratory glassware were soaked in nitric acid (10%) for 24 h and rinsed with deionized water before use. the certified reference material was used for method validation: spinach leaves (srm-1570a) was purchased from national institution of standard and technology. the experiments were performed with an electrothermal atomic absorption spectrometer (varian model spectraa 220z) and the instrument parameters were demonstrated in table 1. the ph of solution were measured by ph meter (metrohm, 827 ph lab) combined glass electrode. for sfodme extraction procedure, blank solution, 15.00 μg l-1 of standard (pb) or sample solution (13.0 ml) was adjusted to ph 6 with phosphate buffer and 0.5 ml of 1-(2-pyridylazo)-2-naphthol (3.0 mmol l-1) were mixed in a screw-cap glass vial (15 ml). then, 90 μl of 1-undecanol was added to the aqueous solution and the solution was stirred for 40 min at 750 rpm at room temperature. after this procedure, the glass vial was transferred into an ice bath for 5 min and the organic phase eventually turned to solid. this solvent was simply relocated into the etaas vial by a small spatula and the droplet was melted immediately. finally, the organic drop was diluted to 500 μl with ethanol and determined by etaas. thai herb samples were collected from phitsanulok (thailand) and dehydrated with oven (70°c) for one hour. after that, samples were sieved through a 100 µm sieve (oviedo et al. 2013). 4 ml of concentrated hno3 and h2o2 at a ratio 3:1 was added and thoroughly mixed with 0.01 g of the sieved sample. the solution was heated on the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:11 utc nova biotechnol chim (2017) 16(2): 124-131 126 hotplate at 120 °c until the red brown smoke disappeared. the residue was filtrated and diluted to 25 ml with volumetric flask. this solution was then used for extraction by sfodme technique. the proposed method was validated with recovery study from spiked known amount of pb standard solution at the concentration of 5.00 and 10.00 µg l-1 to herb samples before digestion. table 1. instrumental parameters and furnace heating program for determination of pb. instrumental parameter value lamp current (ma) 10 wavelength (nm) 283.3 spectral resolution (nm) 0.5 background correction zeeman background correction injected sample volume (µl) 10 chemical modifier pd(no3)2 1000 mg l-1 5 µl furnace program stage temperature (°c) time (s) drying 95 15.0 ashing 400 19.0 atomization* 2 100 2.9 cleaning 2 100 2.0 *reading step results and discussion 1-(2-pyridylazo)-2-naphthol is a ligand that can interact with many metal ions to form complexes, such as, cd(ii) or pb(ii) (tharakeswar et al. 2012), as shown in fig. 1. 1-(2-pyridylazo)-2-naphthol is relatively soluble in aqueous condition but its complexes have low polarity. also, in the preliminary experiment, the pb-1-(2-pyridylazo)-2naphthol complexes are well soluble in 1-undecanol and this ligand can be used for the separation and extraction of pb by sfodme method followed by etaas detection. in order to get high extraction efficiency, the effect of different parameters was considered and optimized. selection of extracting solvent in the sfodme technique, the suitable organic solvent has a remarkable effect on the formation of metal-ion complexes. furthermore, it must have a melting point around 10 to 30°c (near room temperature), low toxicity, and high enrichment factor (yavuz et al. 2016). in this experiment, two extracting solvents, 1-dodecanol and 1-undecanol, were compared and 1-undecanol was chosen as it gave the highest relative absorbance. since the volume of organic solvent could affect the pre-concentration factor (li et al. 2006), therefore, different volumes (20 – 150 μl) of 1-undecanol were investigated in the extraction procedure. when increasing the volume of 1-undecanol, the relative absorbance increased until 90 μl and then relative absorbance decreased. this could be owing to the transportation of analyte, which is associated with increasing the surface contact between the layers of two liquids. from the results, 90 μl of 1-undecanol was considered as optimized extraction volume. ph value generally, the extraction efficiency depends on ph value of the aqueous solution and have a significant effect to the complex formation of pb-1-(2-pyridylazo)-2-naphthol and subsequently the sfodme extraction. in this experiment, ph of solution was controlled in the range of 3.0 – 12.0 fig. 1. chemical structure of metal-pan complex. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:11 utc nova biotechnol chim (2017) 16(2): 124-131 127           fig. 2. effect of the ph. extraction conditions: sample volume – 13 ml; extraction solvent – 1-undecanol; pan concentration – 3.0 mmol l-1 – 0.5 ml; extraction time – 30 min; stirring rate – 625 rpm at room temperature. using different buffer solutions. the result is illustrated in fig. 2 and it is clearly seen that the best relative absorbance was obtained at ph 9. at this ph, pb-1-(2-pyridylazo)-2-naphthol complexes were effectively extracted into the organic solvent. therefore, ph 9 was chosen for further experiments. 1-(2-pyridylazo)-2-naphthol concentration due to the amount of 1-(2-pyridylazo)-2-naphthol can affect to the extraction efficiency, the concentration of 1-(2-pyridylazo)-2-naphthol was consequently investigated. the result showed that the relative absorbance was increased when increasing the concentration of 1-(2-pyridylazo)-2naphthol up to 3.0 mmol l-1 and this continued constant even as the amount of 1-(2-pyridylazo)-2naphthol is increased further. therefore, 3.0 mmol l-1 of 1-(2-pyridylazo)-2-naphthol was selected for further studies. extraction time in order to obtain high sensitivity, good precision and speed up the extraction process, it is required to investigate the effect of the extraction time to ensure the completion of extraction between organic phase and sample solution. the extraction time was studied between of 10 – 120 minutes. the result revealed that the relative absorbance was increased and steadied after 40 minutes (fig. 3). therefore, 40 minutes of extraction time was selected for subsequent experimentations. fig. 3. effect of the extraction time. conditions are the same as in fig. 2. except extraction time, at ph 9 and concentration of pan, 3.0 mmol l-1. stirring rate to reduce the time required to reach the equilibrium between the extracting solvent and aqueous solution (li et al. 2006), the effect of stirring rate was studied between of 250 – 1250 rpm. for the optimized experimental conditions, 750 rpm was selected. extraction temperature at higher temperatures, the extraction kinetics influence the mass transfer of the analyte between the aqueous solution and the extracting solvent (ghanbarian et al. 2013). extraction temperature was studied over a temperature range of 20 – 80°c. from the results, the relative absorbance increased until 50°c, after that relative absorbance decreased when increasing extraction temperature. this phenomenon may be due to the solubility of organic solvent increased and the breakdown of organic solvent at high temperature (bidabadi et al. 2009). therefore, the extraction temperature was controlled at 35°c. table 2. effect of interfering ions on the quantitative recovery of 10 μg l-1 pb. ions mole ratio (ion/analyte) na+, k+, cr3+, no3-, so42-, po43 10 000 cd2+, mg2+, fe3+, as3+ 100 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:11 utc nova biotechnol chim (2017) 16(2): 124-131 128 table 3. application of presented method in certified reference material and thai herb. samples certified (µg g-1) added (µg l-1) concentration in digested solution (µg l-1 ± sd) concentration in sample (µg g-1 ± sd) recovery (% ± sd) srm-1570a (spinach leaves) 0.2 – – 0.20 ± 0.01 100.0 ± 1.1 zingiber officinale – 3.91 ± 0.55 0.48 ± 0.07 – 5.00 9.43 ± 0.67 – 110.5 ± 1.7 10.00 14.19 ± 0.41 – 102.9 ± 3.3 coscinium fenestratum – 0.76 ± 0.02 0.09 ± 0.01 – 5.00 5.74 ± 0.19 – 99.5 ± 4.2 10.00 10.80 ± 0.39 – 100.3 ± 3.9 curcuma longa – 1.72 ± 0.34 0.20 ± 0.04 – 5.00 6.62 ± 0.28 – 98.1 ± 1.3 10.00 11.96 ± 0.47 – 102.4 ± 3.7 piper retrofractum – 2.64 ± 0.68 0.31 ± 0.09 – 5.00 7.79 ± 0.97 – 103.1 ± 4.6 10.00 13.01 ± 0.89 – 103.7 ± 9.1 tinospora crispa – 1.54 ± 0.26 0.18 ± 0.03 – 5.00 6.77 ± 0.18 – 104.7 ± 1.0 10.00 12.00 ± 0.34 – 104.6 ± 3.1 cryptolepis buchanani – 2.32 ± 0.26 0.27 ± 0.03 – 5.00 7.43 ± 0.25 – 102.3 ± 0.8 10.00 12.78 ± 0.30 – 104.6 ± 1.4 butea superba – 1.42 ± 0.20 0.16 ± 0.02 – 5.00 6.44 ± 0.23 – 100.4 ± 0.7 10.00 11.48 ± 0.19 – 100.6 ± 1.0 moringa oleifera – 2.14 ± 0.38 0.25 ± 0.05 – 5.00 7.26 ± 0.34 – 102.4 ± 1.9 10.00 12.33 ± 0.78 – 101.9 ± 5.0 kaempferia parviflora – 1.65 ± 0.02 0.19 ± 0.01 – 5.00 6.80 ± 0.23 – 103.1 ± 1.9 10.00 11.79 ± 0.21 – 101.4 ± 4.3 addition of salt the addition of salt in aqueous solution can change the physical properties of the nernst diffusion film and reduce the rate of diffusion of the interest analytes into the drop (sobhi et al. 2009). also, the effect of salt addition on sfodme technique was studied by adding different quantities of nacl and kcl at 0–5% (w/v). the results exhibited that when increasing amount of salt, the relative absorbance decreased. consequently, the extraction procedures were accomplished without salt addition. interfering ions in the sfodme procedure (wang et al. 2011; chen et al. 2013; dadfarnia et al. 2013), many other heavy metal ions can form complexes with the chelating reagent and can co-extraction with interested metal. to investigate the selectivity of the proposed procedure, the effect of interfering ions on pb determination in herb samples by sfodme was investigated considering from the percentage recovery. these coexisting ions were considered to interfere in pb determination if the percentage recovery was over ±5%. the results were demonstrated in table 2. analytical performance under the optimized condition for sfodme, the analytical performance for pb determination was evaluated. the linear range of pb concentration was 0.5 – 30.0 µg l-1 (y = 0.0131x + 0.0100, where y is the concentration of analyte) with r2 = 0.9996. the lod and loq were 0.064 and 0.214 µg l-1, respectively and %rsds varied from 1.27% to 2.48% (n=6). the enrichment bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:11 utc nova biotechnol chim (2017) 16(2): 124-131 129 table 4. comparison of analytical performance of the proposed method with other methods for determination of pb. method sample lod (μg l-1) linear range (μg l-1) rsd (%) reference cpe-gfaas water 0.08 up to 30.0 2.8 chen et al., 2005 sdme-etaas water 0.2 1.0 – 15.0 4 maltes et al., 2008 dllme-etaas water 0.02 0.05 – 1.00 2.5 naseri et al., 2008 spe-etaas seawater 0.12 0.1 – 10.0 3.2 alonso et al., 2006 sfodme-etaas water and infant formula 0.058 0.2 – 10.0 8.8 chamsaz et al., 2013 sfodme-etaas herbs 0.064 0.5 – 30.0 2.48 this work factor, ef (dadfarnia et al. 2007; durukan et al. 2011) defined by the division of the slope of calibration equation after pre-concentration and that of previous pre-concentration by etaas was accomplished at 18.71 for 13 ml of sample solution. to guarantee the accuracy of the method, the determination of certified reference material srm-1570a (spinach leaves) was used for method validation through recovery experiments. the results are presented in table 3 and it was indicated that a found concentration agreed well with certified value. satisfactory recoveries revealed that sfodme-etaas method was effective for the pre-concentration/determination of lead. a comparison of analytical performance of the proposed method with others is shown in table 4 and indicates its higher effectiveness to pb determination at lower detection limit and better precision than previously reported methods. determination of pb in thai herb samples the procedure was applied to the determination of pb in thai herb samples. the samples were prepared as described and 13 ml of it was treated according to the same procedure. the proposed method was validated with recovery study from spiked known amounts of pb standard solutions. recoveries from spiked standard solution to the samples are shown in table 3. it was found that recoveries were in the range of 98 – 110.5% which confirms the validity and accuracy with acceptable results. conclusions sfodme-etaas method was assessed for the pre-concentration and determination at trace μg l-1 level of pb in thai herb samples. this method provided several advantages such as simple to operate, minimum consumption of extraction solvent and the solidified organic phase is simply collected and separated from the aqueous sample. additionally, on-going research related to the application of sfodme method for pre-concentration/determination of other heavy metals in different types of sample matrices using new chelating reagents are subjects on ongoing research. acknowledgement financial support from the science achievement scholarship of thailand and research center for academic excellence in 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anal. 24: 46-55. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:11 utc microsoft word 4_urgeova nova biotechnologica et chimica 15-2 (2016) 133 doi 10.1515/nbec-2016-0014 © university of ss. cyril and methodius in trnava comparison of enzymatic hydrolysis of polysaccharides from eggshells membranes eva ürgeová, katarína vulganová department of biology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk917 01, slovak republic (eva.urgeova@ucm.sk) abstract: ahyaluronic acid (ha) is part of the extracellular matrix of connective, epithelial and neural tissues, as well as the synovial fluid, skin, and cartilage. it is composed of repeating disaccharide units of d-glucuronic acid and n-acetyl glucosamine. hyaluronic acid is used in abdominal surgery, ophthalmology, dermatology, rhinology; it is usable for the osteoarthritis treatment. the membranes of eggshell are a natural source of hyaluronic acid, collagen, glycosaminoglycan and collagenous proteins. in paper, we tested the possibility of extraction hyaluronic acid from the eggshell membranes by enzymatic hydrolysis. we identified optimal conditions of hydrolysis with trypsin at reaction temperature of 37 °c and ph 8; with pepsin at 40 °c and ph 3, as well as with papain at 60 °c and ph 7.5. the content of hyaluronic acid in samples was determined spectrophotometrically using the carbazole method. the experimental results showed a yield of ~ 4 -4.5 % hyaluronic acid per 1 g of dry eggshell membranes. key words: enzymatic hydrolysis, papain, pepsin, trypsin, hyaluronic acid 1. introduction hyaluronic acid is a biodegradable linear polysaccharide, which is a component of connective tissue and capillary walls present in the body of higher organisms (illiás et al., 2011). it is non-sulphated glycosaminoglycan. hyaluronic acid is composed of alternating residues of β-(1, 3)-d-glucuronic acid and β-(1, 4)-n-acetyld-glucosamine via 1-3 and 1-4 bonds. the characteristic high molecular weight of its depended on natural material and the way of isolation (roig et al., 2013; ünlüer et al., 2013; šlezingrová et al., 2012). it is a very hydrophilic compound that binds water and creates a viscoelastic solution (slíva and minárik, 2009). hyaluronic acid, thanks to its properties, has found application in medicine, cosmetics and clinical praxis (ünlüer et al., 2013; collins and birkinshaw, 2013, kanchana et al., 2013). the main biological function includes hydration connective tissue and supporting their viscoelasticity, it acts as a lubricant in the knees, hips (poláková, 2012; podškubka et al., 2006). hyaluronic acid has a certain role in the processes of tumours growth (lukáčová and lukáč, 2008). as a signal molecule it participates in immunological processes. long chains of hyaluronic acid effectively regulate immune processes, stimulate angiogenesis and initiate the synthesis of proinflammatory cytokines (illiás et al., 2011). its insoluble form can be used in medicine as a matrix in drugs (benešová et al., 2006). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:51 utc 134 ürgeová, e. and vulganová, k. it promotes wound healing, has bacteriostatic and antiseptic properties, protects tissues wound healing, has bacteriostatic and antiseptic properties, protects tissues against ingress of substances of high molecular weight, and regulates migration of fagocytes into inflammatory areas (poláková, 2012). hyaluronic acid is extracted from rooster combs, fish tissues, human umbilical cord, or produced by fermenting streptococcus equi and s. zoopidemicus (collins and birkinshaw, 2013; sadhasivam et al., 2013; murado et al., 2012; saranraj et al., 2011; price et al., 2007; girish and kemparaju, 2007). this polysaccharide was also isolated from the synovial fluid, skin or vitreous fluid of the eye (ünlüer et al., 2013). eggshell membranes have a fibrous structure. they are rich sources of biologically active substances such as hyaluronic acid, collagen, glucosamine, chondroitin sulphate, and glycoprotein’s sulphate (khanmohammadi m. et al., 2014; ruff et al., 2012; d´souza et al., 2013; ruff et al., 2009; zhao and chi, 2009; zhao and chi, 2008; nakano et al., 2003). extraction hyaluronic acid from the biological material is carried out enzymatically; hyaluronic acid is isolated from the tissues with enzymes, e.g. alcalase (panagos et al., 2014), tryptase (nagyery et al., 2011; zhao and chi, 2008), papain (sadhasivam et al., 2013; pi et al., 2011; volpi and maccari, 2003), trypsin (zhao et al., 2008), pepsin (slíva and minárik, 2009; zhao and chi, 2009) or chemically; hyaluronate can be isolated changing the ph (tommeraas and melander, 2008) by ultrasound (slíva and minárik, 2009), by sodium acetate (vázqeuz et al., 2013), and ethanol with the addition of hydrochloric acid (nimptsch et al., 2010; yu and gao, 2007). in our paper, we present the isolation of the hyaluronic acid from the eggshell membranes by enzymatic hydrolysis using pepsin, papain, and trypsin. the basis for our work was results published by zhao and chi (2008) and pi et al. (2011). 2. materials and methods dry eggshell membranes, provided by biomin a.s., cífer, were used as a source of hyaluronate. homogenized eggshell membranes were hydrolysed by pepsin (ec 3.4.23.1, 0.8 fip-u/mg, applichem), trypsin (ec 3.4.21.4, 334 u/mg, amresco) and papain (ec 3.4.22.2, 31523 usp u/mg, applichem) in universal britton and robinson buffer. we investigated the influence of ph, temperature and time of hydrolysis for extraction of hyaluronic acid, and ratio of material to fluid (of solid to liquid). a centrifuged liquid after enzymatic hydrolysis was subsequently determined for the concentration of hyaluronic acid by carbazole method of rosa et al. (2007). the method is based on the colour development due to the action of organic components, carbazole reagent, after the hydrolysis of hyaluronic acid with h2so4. the absorbance was measured at λ = 525 nm and the content of hyaluronic acid were expressed in mg of hyaluronic acid per gram of dry matters of eggshell membranes. sodium hyaluronate was used as the standard. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:51 utc nova biotechnologica et chimica 15-2 (2016) 135 3. results and discussion the aim of work was extraction of hyaluronic acid from homogenized dry membranes of eggshell by enzymatic hydrolysis, using pepsin, trypsin and papain. we investigated the effect of ph, temperature, amount of enzymes, ratio of eggshell membranes and liquid and time of hydrolysis. in the beginning, we tested the influence of ph level on the hydrolysis. enzymatic hydrolyses were at 37 °c using enzymes for 5 hours in ratio of solid to liquid 1 : 40, amount of enzymes 100 mg/g dm of pepsin, and 1 mg/g dm of trypsin and papain. we tested conditions of enzymatic hydrolyse the eggshell membranes at ph 2; 2.5; 3; 4; 5, using pepsin (80 fip-u/g dm); at ph 7; 7,5; 8; 8,5 using trypsin (33 u/g dm); and at ph 3.5; 4; 4.5; 5; 5.5; 6; 6.5; 7 and 7.5, using papain (3152 usp u/g dm). the best results of pepsin hydrolysis were showed at ph 3; this finding consistent with work of zhao and chi (2008), in which was the indicated ph 3 as the optimal for hydrolysis using pepsin. the content of hyaluronic acid was 34.91 mg/g dm. for hydrolysis by trypsin zhao and chi (2008) indicated as the optimal ph 8.5, our results were different. the most hyaluronic acid we isolated by trypsin at ph 8. amount of hyaluronic acid in trypsin hydrolysate at ph 8 were 40.13 mg/g dm, it was more, than in experiments of zhao and chi (2008) at ph 8.5. at ph 7.5, we obtain the optimal condition for isolation of hyaluronic acid by papain; these results correspond with the best extracting condition at ph 7.3 in accordance pi et al. (2011). in following experiments we proceed with investigation of the effect of reaction temperature on hydrolysis and yield of the hyaluronic acid in the hydrolysate. hydrolysis was carried out 5 hours for all enzymes; amount of enzymes and ratio of solid to liquid were the same as in the previous experiment. other conditions of hydrolysis using pepsin were ph 3, temperature at 30 °c, 37 °c, 40 °c and 50 °c, using trypsin were ph 8, temperature at 30 °c, 37 °c, and 40 °c, using papain were ph 7.5 and temperature at 40 °c, 50 °c, 60 °c and 70 °c (fig. 1). fig. 1. content of hyaluronic acid at different temperatures of hydrolysis by enzymes pepsin, trypsin and papain. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:51 utc 136 ürgeová, e. and vulganová, k. the content of hyaluronic acid in the hydrolysates at different reaction temperature shows fig. 1. we obtained the optimal temperature 40 °c for hydrolysis by pepsin, at temperature 30 °c was the content of hyaluronic acid by 21 % less, and at 37 °c by 10.5 % less than at 40 °c. this results show different optimal temperature as in work by zhao and chi (2008), who published optimal temperature 37 °c, using pepsin. the optimal temperature of trypsin was obtained at 37 °c, in opposite of zhao and chi (2008) work, who obtained the optimal temperature for trypsin hydrolysis 50 °c. in our experiments content of hyaluronic acid at 40 °c decreased about 23 % in comparison with content at 37 °c. the yielding rate of hyaluronic acid was 39.02 mg/g dm in hydrolysate at 60 °c using papain, this finding corresponded with vopii and maccari (2003) work. the amount of hyaluronic acid at 50 °c, optimal temperature used by pi et al. (2011), was 34.5 % less. at the temperature 70 °c the yield of hyaluronic acid decreased about 9.5 %. in further experiments, we observed the influence of the enzyme amount on the hydrolysis and content of hyaluronic acid in hydrolysate. we tested the effect of added enzymes in conditions as follows: pepsin the 80 fip-u/g, 40 fip-u/g at 40 °c, ph 3; trypsin the 17 u/g, 33 u/g, 50 u/g at 37 °c, ph 8; and papain the 1576 usp u/g, 3152 usp u/g, 9457 usp u/g, 15762 usp u/g at 60 °c, ph 7.5. table 1. effect of amounts of enzyme on content of hyaluronic acid. enzyme and its activity hyaluronic acid [mg/g dm] pepsin 80 fip-u/g 38.79 40 fip-u/g 29.70 trypsin 17 u/g 38.45 33 u/g 40.13 50 u/g 44.83 papain 1576 usp u/g 34.76 3152 usp u/g 39.02 9457 usp u/g 38.45 15762 usp u/g 34.42 table 1 shown yield of hyaluronic acid at optimal ph and temperature of hydrolysis by using various amounts of pepsin, trypsin and papain. the best result was proved after the addition of pepsin in amount 80 fip-u/g, 38.79 mg/g. at the same conditions, with amount of pepsin 40 fip-u/g was determined 29.07 mg hyaluronic acid per 1 g eggshell membranes. the yield rate, comparing with the double quantity of pepsin, was decreased by 25.1 %. the best results of hydrolysis by trypsin were obtained at 50 u/g dm in other conditions: ph 8 at 37 °c. the content of hyaluronic acid was 44.82 mg/g dm. by the use of papain 3152 usp u/g dm, we received 39.02 mg/g dm. the content of hyaluronic acid by using 1576 usp u/g dm of papain was decreased of 11 %, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:51 utc nova biotechnologica et chimica 15-2 (2016) 137 and using the amount of enzyme15762 usp u/g dm was decreased by 12 % at the same temperature, ph and ratio of solid to liquid. in following phase of the experiments, we followed the influence of amount of buffer added on hydrolysis, ratio of material-fluid (solid-liquid). we used amounts of universal britton and robinson buffer as follows: 20 ml, 30 ml and 40 ml per gram of the eggshell membranes at the best conditions from previous experiments. the highest content of hyaluronic acid was obtained by using ratio of materialfluid 1 : 40, i.e. addition 40 ml buffer per gram of dry eggshell membranes. by the ratio of solid to liquid 1 : 20 we obtained by 20 % less hyaluronic acid in average, and by using ratio of solid to liquid 1 : 30 was yield of hyaluronic acid by 45 % less in comparison with the use of ratio of solid to liquid 1 : 40. in the next, we tested the impact of hydrolysis time on yield of hyaluronic acid. the conditions of experiments were under optimal ph, temperature, ratio of solid to liquid, and amount of enzymes. the hydrolysis time was varied: three, five and eight hours. after the time of hydrolysis we compared the content of hyaluronic acid in samples by standard procedures (fig. 2). fig. 2. effect of hydrolysis time on content of hyaluronic acid. fig. 2 shows results in various time of hydrolysis. the highest yield of hyaluronic acid we received at time of hydrolysis 5 hours, in the times of hydrolysis 3 and 8 hours was the content by 11 – 23 % less. the best extracting conditions of hyaluronic acid from eggshell membranes were followed: time of hydrolysis 5 hours, ratio of solid to liquid 1 : 40 generally. other optimal conditions for hydrolysis were: amount of pepsin 80 fip-u/g dm, ph 3 at temperature 40 °c; ph 8, temperature 37 °c, and amount of enzyme 50 u/g dm using trypsin; and ph 7.5, temperature 60 °c, and amount of enzyme 3152 usp u/g dm using papain. in the final experiment, we compared effectiveness of hydrolysis by different enzymes at the standard, optimized parameters. fig. 3 shows the highest yield of hyaluronic acid using trypsin, 42.82 mg/g dm. the content of hyaluronic acid in hydrolysate by other enzymes was about the same, 13 % for papain or 13.5 % bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:51 utc 138 ürgeová, e. and vulganová, k. for pepsin less. in generally the yielding rate of hyaluronic acid was higher in all hydrolysis, minimal 38.79 mg/g dm, than reported zhao and chi (2008), by trypsin 13.29 mg/g dm, by pepsin 24.49 mg/g dm or and pi et al. (2011) 12.2 mg/g dm by papain. fig. 3. comparison of content of hyaluronic acid using pepsin, trypsin and papain. 4. conclusions the paper presents the results of extraction of the hyaluronic acid from the membranes of eggshell using various enzymes. for isolation of hyaluronic acid, enzymes pepsin, trypsin, and papain were used. extraction in all cases was most effective in ratio of solid (eggshell membranes) to liquid (buffer) 1 : 40; time of hydrolysis 5 hours. the best hydrolysing conditions of hyaluronic acid were as followed: amount of pepsin 80 fip-u/g dm, reaction temperature 40 °c, ph 3. under the condition, the hyaluronic acid yielding rate reached 38.79 mg/g dm eggshell membranes. hydrolyse of eggshell membranes by papain shows the highest amount of hyaluronic acid under conditions: amount of papain 3152 usp u/g dm, reaction temperature 60 °c and ph 7.5. content of hyaluronic acid under these conditions was 39.02 mg/g dm. the optimal parameters of trypsin were obtained and shown as follows: amount of trypsin 50 u/g dm, reaction temperature 37 °c, ph 8. therefore, the yield of hyaluronic acid was found to be 44.82 mg/g dry eggshell membranes. this indicated that effect of the yielding rate with trypsin was better than pepsin or papain. in our work we obtained approximately 4 – 4.5 % of hyaluronic acid per gram of eggshell membranes by enzymes hydrolysis. this result are slightly different from the results obtained zhao and chi (2008). they proved content of hyaluronic acid in one gram membranes of eggshell about 2.5 %. alike pi et al. (2011) in their work describe yield of hyaluronic acid less, 1.2 %. it was shown that enzymatic hydrolysis of the eggshells membranes offers an opportunity to obtain biological active substances such as hyaluronic acid. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:51 utc nova biotechnologica et chimica 15-2 (2016) 139 references collins, m.n., birkinshaw, c.: hyaluronic acid solution-a processing method for efficient chemical modification. j. app. polym. sci., 10, 2013, 145-152. d´souza, s.f., kumar, j., jha k.s., kubal, b.s.: immobilization of the urease on eggshell membrane and its application in biosenzor. mat. sci. eng., 33, 2013, 850-854. girish, k.s., kemparaju, k.: the magic glue hyaluronan and its eraser hyaluronidase a biological overview. life sci., 80(21), 2007, 1921-1943. illiás, a., liliom, k., kleinlercher, b.g., reitinger, s., lepperdinger, g.: unbinding of hyaluronan accelererates the enzymatic activity of bee hyaluronidase. j. 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a.e., szabo, j.r., schauss, a.g.: safety evaluation of a natural eggshell membrane – derived product. food chem. toxicol., 50, 2012, 604-611. ruff, k.j., winkler, a., jackson, r.w.: eggshell membrane in the treatment of pain and stiffness from osteoathritis of the knee: a randomized, multicenter, double-blind, placebo-controlled clinical study. clin. rheumatol., 28, 2009, 907914. sadhasivam, g., muthuvel, a., pachaiyappan, a., balasubramanian, t.: isolation and characterization of hyaluronic acid from the liver of marine stingray aetobatus narinari. int. j. biol. macromolec., 54, 2013, 84-89. saranraj, p., sivakumar, s., sivasubramanian, j., geetha, m.: production, optimization and spectroscopic studies of hyaluronic acid extracted from streptococcus pyogenes. int. j. pharm. biol. arch., 2(3), 2011, 954-959. slíva, j., minárik, j.: hyaluronát – nejen pasivní pozorovatel, nýbrž aktivní modulátor imunitných reakcií. new eu magaz., 2(1), 2009, 75-79. šlezingrová, k., šmejkalová, d., 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clinic. res., 4, 2007, 1240-1248. zhao, y., chi, y.: extraction and partial characterization of hyaluronic acid from eggshell membrane. sci. technol. food ind., 2008. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:51 utc nova biotechnologica et chimica 15-2 (2016) 141 zhao, y., chi, y.: characterization of collagen from eggshell membrane. biotechnology, 8(2), 2009, 254-258. zhao, y., han, l., chi, y.: extracting hyaluronic acid from eggshell membrane with enzyme. food res. dev., 2008. received 22 november 2016 accepted 8 december 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:51 utc separation of enantiomers of selected sulfur-containing amino acids by using serially coupled achiral-chiral columns nova biotechnologica et chimica 14-1 (2015) 1 doi 10.1515/nbec-2015-0009  university of ss. cyril and methodius in trnava separation of methionine enantiomers by using teicoplanin and cyclofructan columns katarína hroboňová1, zuzana deáková1,2, jakub moravčík1, jozef lehotay1,3, daniel w. armstrong4, anna lomenová1 1institute of analytical chemistry, faculty of chemical and food technology, slovak university of technology, radlinského 9, 812 37 bratislava, slovakia (katarina.hrobonova@stuba.sk) 2institute of medical chemistry, biochemistry and clinical biochemistry, faculty of medicine, comenius university, sasinkova 2, 811 08 bratislava, slovakia 3department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, nam. j. herdu 2, trnava, sk-917 01, slovakia 4department of chemistry, university of texas at arlington, 700 planetarium place, tx, 76019 arlington, usa abstract: methionine is a naturally occurring amino acid. its enantiomeric separation by using high performance liquid chromatography on various types of chiral stationary phases was studied. the effect of mobile phase composition on enantioselectivity and retention was considered. the separation of the enantiomers was attained in different separation modes – reversed phase mode for the macrocyclic antibiotic chiral stationary phases (teicoplanin, teicoplanin aglycone), normal phase and polar organic phase modes for the isopropyl carbamate cyclofructan 6 chiral stationary phase. it was shown that the hydrogen bonding, dipole interactions, steric effects between methionine molecules and stationary phases play an important role in the separation of enantiomers. key words: methionine, enantiomers, cyclofructan 6-based column, teicoplanin-based column, mobile phase composition, hplc 1. introduction methionine (fig. 1) is a sulfur-containing essential amino acid that participates in protein synthesis. it plays an important role as a precursor for the synthesis of other sulphur containing amino acids (cysteine and taurine) (hardy, 1985). some studies have indicated that d-methionine is poorly utilized and excreted in the urine (steging et al., 1986). cooh nh 2 s fig. 1. the chemical structure of methionine. methionine is continuously converted to homocysteine and adenosine. significant amounts of methionine are subsequently regenerated via the remethylation of bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:44 utc 2 hroboňová, k. et al. homocysteine. this enzymatic reaction is catalyzed by methionine synthase (visser et al., 2011; kuo et al., 2005; miner et al., 1997). many studies are focused on enantiomeric separations of amino acids by hplc. direct analysis of amino acids or their derivatives is by chiral ligand-exchange chromatography (ilisz et al., 2006) or by a chiral stationary phase (csp) based on cyclodextrins (wang et al., 2010; remsburg et al., 2008), crown-ether (chen et al., 2006; lee et al., 2010), macrocyclic glycopeptides (poplewska et al., 2007; guillen-casla et al., 2010), polysaccharide derivatives (lee et al., 2008), and proteins (winkler et al., 2009). macrocyclic glycopeptides are chiral selectors mainly used in separation techniques such as hplc (xiao et al., 2004; ilisz et al., 2006) and capillary electrophoresis (armstrong et al., 1997). the most frequently utilized glycopeptide chiral selector for amino acids enantioseparation are teicoplanin and teicoplanin aglycone (poplewska et al., 2008; ilisz et al., 2009). the variety of stereoselective interactions of the teicoplanin (also other macrocyclic glycopeptides) chiral selector is related to the presence of many stereogenic centers and the heterogeneity of its functional groups. the structure of these chiral selectors (fig. 2) indicates that interactions including hydrogen bonding, - complexation, dipole stacking, steric interactions, hydrophobic interactions, electrostatic interactions, and inclusion are present. macrocyclic antibiotic csps can be operated in all common separation modes, reversed phase (aqueous polar organic mobile phase; rp), normal phase (little polar alkane alcohol mobile phase; np), and polar organic phase (nonaqueous organic mobile phase; po) (armstrong et al., 1995). cyclofructans (cfs) are macrocyclic oligosaccharides consisting of six or more β(21) linked d-fructofuranose units (from six to eight; cf6, cf7, cf8). each fructofuranose unit contains four stereogenic centers and three hydroxyl groups that can be derivatized in order to improve the enantioseparation performance of cf csps. cyclofructan based csps are the newest approach used for chiral hplc (sun et al., 2009). the development of derivatized cyclofructan csps is based on aliphatic or aromatic functionalization of a native chiral selector. it was reported, that aliphaticderivatized cf6 are suitable for separation of racemic primary amines. these csps can be operated in all separation modes (np, rp, po) (sun et al., 2010; janečková et al., 2011). this work is focused on comparison of hplc separation and interaction abilities of two different types of csps and three separation modes for the resolution of methionine enantiomers. these csps included derivatized cyclofructan 6 and macrocyclic antibiotic chiral selectors. the influence of the mobile phase composition on various chromatographic parameters (retention factor, resolution) was investigated. 2. material and methods 2.1 chemicals l-methionine and dl-methionine were purchased from sigma-aldrich. organic solvents of hplc gradient grade, acetonitrile, methanol, ethanol, and n-hexane were a bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:44 utc nova biotechnologica et chimica 14-1 (2015) 3 product of merck. glacial acetic acid (100 % purity), trifluoroacetic acid, and triethylamine of analytical grade purity were purchased from merck. a stock solutions of racemic mixture and l-enantiomeric form were prepared in water (doubly deionized water, < 18 m cm-1) at concentration 1.0 mg ml-1. working solutions were obtained by appropriate dilutions and were prepared weekly. the solutions were filtered through a 0.45 m nylon membrane filter before the hplc analysis. 2.2 instrumentation and chromatographic conditions i) the hplc system consisted of an isocratic pump (deltachrom sds 030, watrex), an injection valve (rheodyne), and electrochemical detector (coulochem ii, esa). detector was composed of guard cell model 5020 and analytical cell model 5010a (esa). analytical cells had potential set to + 0.65 v (e1), + 0.9 v (e2) and + 1.4 v (e0) according to garaiova et al. (2013). enantiomeric separations were performed on teicoplanin (chirobiotic t) or teicoplanin aglycone (chirobiotic tag) columns (4×250 mm i.d, 5 m) (astec). the mobile phases for consisted of methanol or acetonitrile and water in different volume ratios. the flow rate was 0.6 ml min-1. a nylon filter (0.45 m) was used for the mobile phases filtering. the columns were thermostated with a jet stream ii plus hplc column thermostat (wo industrial electronics) at 25 °c. system peak obtained by injection of methanol or acetonitrile to the studied separation system served for determination of the dead time. the elution order of enantiomers was l-form, d-form in all cases. ii) the agilent technologies hplc (series 1200) consisted from a binary pump, an injection valve (rheodyne), a column thermostat, a diode array and polarimetric detector (chiralizer, ibz messtechnik) were used for the assay. the chiral column ipcf6 (4×250 mm i.d, 5 m) (astec) was used for separation (the chiral selector was isopropyl carbamate cyclofructan 6). the temperature was maintained at 23 or 0 c. the mobile phases for polar organic mode consisted of methanol, acetonitrile, acetic acid, and triethylamine. in all experiments the acid and basic additive percentages in the mobile phase were fixed at 0.3/0.2 (v/v). the mobile phases for normal phase mode were composed of n-hexane and ethanol in different volume ratios, small addition of trifluoroacetic acid (0.1 %, v) was used. a nylon filter (0.45 m) was used for the mobile phases filtering. the flow rate was 0.8 ml min-1 and the injection volume was 20 l. uv detection of the analytes was performed at 210 nm. system peak obtained by injection of methanol (po mode) or ethanol (np mode) to the studied separation system served for determination of the dead time. the elution order of enantiomers was l-form, d-form in all cases. the all measurements were done in three replicates. the retention factor (k) values of first and second eluted enantiomer were calculated as the ratio of differences between retention time of enantiomer (tr) and dead time (tm) to dead time. the resolution values (rs) of land denantiomers were calculated by ratio of difference between retention times of enantiomers (trd, trl2) to sum of peak widths at half peak height (w05d, w05l) as follow: rs = 1.18 * (trd-trl) / (w05d + w05l). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:44 utc 4 hroboňová, k. et al. 3. results and discussion the purpose of this study was to select suitable chromatographic conditions to directly separate methionine enantiomers (fig. 1). two types of chiral stationary phases were tested with the aim to obtain complementary hplc conditions from the point of view of the separation mode used. the interaction abilities of tested csps were studied. 3.1 hplc enantioseparation in reversed phase mode teicoplanin based csps (fig. 2) were used for enantioseparations in the reversed phase separation mode. enantiomeric recognition of the methionine was studied in a mobile phase composed of water and organic solvent (methanol or acetonitrile). different organic modifier/water ratios were tested as shown in table 1 and fig. 3. a b or o ro o or 6 r = o nh ch3 3h c c fig. 2. structure of tested chiral selectors. teicoplanin (a), isopropyl carbamate cyclofructan 6, ip-cf6 (b), teicoplanin aglycone (c) (armstrong et al., 1995; berthod et al., 2000; sun et al., 2009). the retention factor vs. methanol/water or acetonitrile/water ratio showed a ushaped retention curve, which indicates that different interactions take place at different solvent compositions. increased retention and selectivities were obtained when the mobile phase contain more than 40 % methanol or more than 50 % acetonitrile. higher retention (higher values of retention factors) is probably the result of higher solubility of the selected amino acid in water in comparison to organic bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:44 utc http://www.trc-canada.com/structures/t015500.png nova biotechnologica et chimica 14-1 (2015) 5 solvent. table 1 data also indicates that the retention is higher in methanol containing mobile phase then in acetonitrile containing mobile phase. the same trend was observed for resolution values of enantiomeric forms (fig. 3). higher rs values were obtained in the mobile phases containing methanol as an organic modifier. it can be supposed that the formation of hydrogen bonds supported in methanol may increase the enantioselectivity. the suitable mobile phase composition for separation of methionine enantiomers on teicoplanin chiral stationary phase was methanol/water (70/30 v/v). the separation of methionine enantiomers was also studied on a column containing the teicoplanin aglycone (fig. 2) as a chiral selector. this csp is similar to teicoplanin based csp, but without saccharide moieties (berthod et al., 2000). the separations were accomplished with methanol/water or acetonitrile/water mobile phases. among these two organic modifiers better enantioselectivity and resolution was observed using methanol as the modifier. figure 3 and table 1 show that the concentration of methanol has a significant effect on enantiomeric separation of methionine enantiomers. table 1. effect of methanol and acetonitrile percentage on the values of retention factors (k) of land dmethionine in the reversed phase modea by the teicoplanin and teicoplanin aglycone csps. teicoplanin csp methanol (%) 5 10 20 40 50 70 85 k1 2.4 2.3 2.1 2.1 2.2 2.6 3.5 k2 2.9 2.9 2.8 3.2 3.5 4.5 7.1 acetonitrile (%) 5 10 20 40 50 70 80 k1 2.1 1.7 1.6 1.4 1.7 3.1 6.5 k2 2.4 1.9 1.8 1.6 2.0 3.9 8.6 teicoplanin aglycone csp methanol (%) 5 10 20 40 50 70 80 k1 1.7 1.8 1.8 1.9 2.0 2.3 2.5 k2 2.5 2.7 3.1 3.9 4.5 6.2 7.3 a mobile phase: methanol/water or acetonitrile/water (v/v); column temperature: 25 °c; flow rate: 0.6 ml min-1; rsd  5 % the comparison of chromatographic results obtained on the teicoplanin and teicoplanin aglycone csps may show the effect of saccharide moieties in enantiorecognition. the  values of methionine are 1.2-1.6 times higher on the aglycone csp than on the native teicoplanin csp. the higher resolutions (rs  3.9) were observed for aglycone csp in comparison with teicoplanin csp (rs  0.7) obtained in tested methanol percentage interval. these differences suggest that the saccharide units of the native teicoplanin molecule have a negative influence on chiral bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:44 utc 6 hroboňová, k. et al. recognition process of methionine enantiomers. this also indicates that the aglycone part of teicoplanin molecule is dominant in enantiorecognition. it is probably the effect of steric hindrance, where the sugar parts occupy the inside of “basket” and limits the accessibility of other molecules to binding sites, i.e., blocking the possible interaction on the aglycone surface (berthod et al., 2000) the retention factor vs. methanol/water ratio dependencies (table 1 and fig. 3) showed that the overall polarities of the stationary phases used in this study are similar. teicoplanin csp teicoplanin aglycone csp 0 20 40 60 80 0 2 4 6 8 k ,  , r s methanol (%) k 1  r s 0 20 40 60 80 0 2 4 6 8k ,  , r s methanol (%) k1  r s fig. 3. effect of methanol concentration on enantioseparation of methionine in reversed phase mode a by the teicoplanin and teicoplanin aglycone csps. a mobile phase: methanol/water (v/v); column temperature: 25 °c; flow rate: 0.6 ml min-1. the tested teicoplanin and teicoplanin aglycone csps are extremely selective for the enantiomers of methionine. figures 4a and 4b show chromatograms of methionine on the two csps with the methanol/water 70/40 (v/v) mobile phase. the l-enantiomeric forms elute first and d-form elute second in all tested mobile phases. 3.2 hplc enantioseparation in polar organic mode in 2009, sun et al. demonstrated that the polar organic separation mode was preferred for separation of enantiomers of primary amines on novel class of cyclofructan based csps. the derivatization on the chiral selector proved to be essential for enantioselectivity. several different binding sites have been demonstrated on isopropyl carbamate cyclofructan 6 (ip-cf6) (fig. 2) including hydroxyl and carbamate groups which may have interactions with analyte via hydrogen bonding or dipolar interactions. the possible existing interactions also include steric effects in the chiral groove. (sun et al., 2009) the combination of interactions may have a different effect on the stabilization of the complex csp-enantiomer. ip-cf6 csp and mixture of methanol/acetonitrile/acetic acid/triethylamine as mobile phase were used for hplc separation of methionine enantiomers. the influence of mobile phase composition on the retention and enantioresolution on ip bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:44 utc nova biotechnologica et chimica 14-1 (2015) 7 cf6 csp was first studied to obtain an effective enantioseparation (rs  1.0). the concentration of methanol in mobile phases increased from 10 % to 85 % and acidic and basic modifier concentrations were kept the same (0.3 % of acetic acid, 0.2 % of triethylamine). the results shown in figure 5 documented that increasing the concentration of methanol decreases the resolution values from 2.6 to 0.3. the retention factor values plots of the first and the second eluted enantiomers exhibited an (a) teicoplanin csp (b) teicoplanin aglycone csp (c) ip-cf6 csp (d) ip-cf6 csp fig. 4. chromatograms of l(1) and d(2) methionine in rp separation mode by the teicoplanin (a) and teicopanin aglycone (b) csps, po separation mode (c) and np separation mode (d) by the ip-cf6 csp. conditions: (a) mobile phase: methanol/water (70/30 v/v), temperature: 25 °c, flow rate: 0.6 ml min-1, electrochemical detection; (b) mobile phase: methanol/water (70/30 v/v), temperature: 25 °c, flow rate: 0.6 ml min-1, electrochemical detection; (c) mobile phase: methanol/acetonitrile/acetic acid/triethylamine (75/25/0.3/0.2 v/v/v), temperature: 23 °c, flow rate: 0.8 ml min-1, polarimetric detection; (d) mobile phase: ethanol/n-hexane/trifluoroacetic acid (50/50/01 v/v/v), temperature: 0 °c, flow rate: 0.8 ml min-1, spectrofotometric detection at 210 nm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:44 utc 8 hroboňová, k. et al. observable dependence with varying methanol concentration. the differences in the retention factors with change methanol concentration could be explained by competition of methanol and methionine for hydrogen bonding sites on ip-cf6 csp. the suitable mobile phase for separation of methionine enantiomers was mixture methanol/acetonitrile/acetic acid/triethylamine (75/25/0.3/0.2 v/v/v/v). the highest value of resolution was observed with 20 % methanol, resulting from longer retention. figure 4c shows the chromatogram of methionine separation on ip-cf6 column in polar organic separation mode. the l-enantiomeric form elutes first and d-form elutes second. the previous results demonstrated that decreasing the column temperature can increase selectivity and resolution (moravčík et al., 2013). 20 40 60 80 0 2 4 6 8 10 k 1 ,  , r s methanol (%) k 1  r s fig. 5. effect of methanol concentration on enantioseparation of methionine in polar organic mode a by the ip-cf6 csp. amobile phase: methanol/acetonitrile/0.3 % acetic acid/0.2 % triethylamine, column temperature: 23 °c, flow rate: 0.8 ml min-1. 3.3 hplc enantioseparation in the normal phase mode since the short retention of methionine enantiomers on ip-cf6 csp in the polar organic separation mode was observed (retention times from 6 to 7 min), the normal phase separation mode was tested. the mobile phases consist of n-hexane, ethanol, and trifluoroacetic acid. the amount of acidic modifier was constant, 0.1 % (v) in all measurements. the different n-hexane/ethanol ratios (50-95 % of ethanol) were tested as it is shown in figure 6. the increased retention and selectivity were obtained when the mobile phase contained less than 65 % of ethanol. also the increasing of ethanol concentration caused decreasing of resolution values from 1.1 to 0.5. the retention factor and the resolution values vs. n-hexane/ethanol ratio plot indicate that in the interval 95-65 % of ethanol is favorable influence the hydrogen donation groups from mobile phase and therefore probably the hydrogen bonding interactions are contribute to the enantiomeric separation of methionine complexes with ip-cf6 csp. in mobile phases containing lower than 65 % of ethanol decreases the portion of available hydrogen donation groups in the mobile phase, probably are preferred interactions in the stationary phase, which results increase in retention and also in enantioresolution. there is no significant influence of different mobile phase composition on the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:44 utc nova biotechnologica et chimica 14-1 (2015) 9 selectivity factors ( = 1.42 – 1.48). figure 4d shows the chromatogram of methionine on the ip-cf6 csp with the n-hexane/ethanol/trifluoroacetic acid 50/50/0.1 (v/v/v) mobile phase. the l-enantiomeric form elutes first and d-form elutes second in all tested mobile phases. 50 60 70 80 90 0,0 0,4 0,8 1,2 1,6 2,0 k 1 ,  , r s ethanol (%) k 1  r s fig. 6. effect of ethanol concentration on enantioseparation of methionine in normal phase modea by the ipcf6 csp. a mobile phase: n-hexane/ethanol/trifluoroacetic acid v/v/0.1, column temperature: 0 °c, flow rate: 0.8 ml min-1. the tested ip-cf6 csp in np separation mode is so more selective resolving enantiomers of methionine in comparison to po mode. 4. conclusions the hplc separation conditions (stationary phase, mobile phase composition) were proposed for separation of methionine enantiomers. the separation of enantiomers was achieved on macrocyclic antibiotic (teicoplanin, teicoplanin aglycone) csps in reversed phase separation mode and on newly developed cyclofructan-based (isopropyl carbamate cyclofructan 6) csp in normal phase and polar organic separation modes. it appears that hydrogen bonding, steric and dipole interactions are of great importance for these separations. the proposed separation systems are complementary and may be used depending on the type of sample matrix. acknowledgements: this study was financially supported by the scientific grant agency of the ministry of education, science, research and sport of the slovak republic and of the slovak academy o f sciences (grant vega no. 1/0499/14). references armstrong, d.w., nair, u.b.: capillary electrophoretic enantioseparations using macrocyclic antibiotics as chiral selectors. electrophoresis, 18, 1997, 23312342. armstrong, d.w., youbang, l., ekborgott, k.: a covalently bonded teicoplanin chiral stationary phase for hplc enantioseparations. chirality, 7, 1995, 474-497. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:44 utc 10 hroboňová, k. et al. berthod, a., chen, z., kullman, j.p., armstrong, d.w., gasparrini, f., d`acquarica, i., villani, c., carotti, a.: role of the carbohydrate moieties in chiral recognition on teicoplanin-based lc stationary phases. anal. chem., 72, 2000, 1767-1780. chen, j.j., zhang, d.t., shen, b.c., zhang, x.j., xu, b.j., xu, x.z.: enantioseparation of d,l-α-amine acid on crown ether chiral stationary phases. chin. j. anal. chem., 34, 2006, 1535-1540. garaiova, i., muchová, j., nagyová, z., mišľanová, c., oravec, s., dukát, a., wang, d., plummer, s.f., ďuračková, z.: effect of a plant sterol, fish oil and b vitamin combination on cardiovascular risk factors in hypercholesterolemic children and adolescents: a pilot study. nutr. j., 12, 2013, 1-8. guillen-casla, v., leon-gonzalez, m.e., perez-arribas, l.v., polodiez, l.m.: direct chiral determination of free amino acid enantiomers by twodimensional liquid chromatography: application to control transformations in ebeam irradiated foodstuffs. anal. bioanal. chem., 397, 2010, 63-75. hardy, p.m.: in chemistry and biochemistry of amino acids; g.c. basrrett, ed., chapmen and hall: new york, 1985, p. 6. ilisz, i., berkecz, r., peter, a.: hplc separation of amino acid enantiomers and small peptides on macrocyclic antibiotic-based chiral stationary phases: a review. j. sep. sci., 29, 2006, 1305-1321. ilisz, i., berkecz, r., peter, a.: retention mechanism of high-performance liquid chromatographic enantioseparation on macrocyclic glycopeptide-based chiral stationary phases. j. chromatogr. a, 1216, 2009, 1845-1860. ilisz, i., tourwe, d., armstrong, d.w., péter, a.: high-performance liquid chromatographic enantioseparation of unusual secondary amino acids on a dpenicillamine-based chiral ligand-exchange column. chirality, 18, 2006, 539-543. janečková, l., kalíková, k., vozka, j., armstrong, d.w., bosáková, z., tesařová, e.: characterization of cyclofructan-based chiral stationary phases by linear free energy relationship. j. sep. sci., 34, 2011, 26392644. kuo, h.k., sorond, f.a., jen-hau chen, j.h., hashmi, a., milberg, w. p., lipsitz, l.a.: the role of homocysteine in multisystem age-related problems: a systematic review. j. gerontology, 60, 2005, 1190-1201. lee, t., lee, w., hyun, m.h., park, j.h.: enantioseparation of β-amino acids on an 18-crown-6-tetracarboxylic acid-bonded silica by capillary electrochromatography. j. chromatogr. a, 1217, 2010, 1425-1428. lee, k.a., yeo, s., kim, k.h., lee, w., kang, j.s.: enantioseparation of nfluorenylmethoxycarbonyl α-amino acids on polysaccharide-derived chiral stationary phases by reverse mode liquid chromatography. j. pharm. biomed. anal., 46, 2008, 914-919. miner, s.e.s., evrovski, j., cole, d.e.c.: clinical chemistry and molecular biology of homocysteine metabolism: an update. clin. biochem., 30, 1997, 189201. moravčík, j., hroboňová, k.: high-performance liquid chromatographic method for enantioseparation of underivatized -amino acids using cyclofructanbased chiral stationary phases. nova biotechnol. chim., 12, 2013, 108-119. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:44 utc nova biotechnologica et chimica 14-1 (2015) 11 poplewska, i., kramarz, r., piatkowski, w., seidel-morgenstern, a., antos, d.: behavior of adsorbed and fluid phases versus retention properties of amino acids on the teicoplanin chiral selector. j. chromatogr. a, 1192, 2008, 130-138. poplewska, k.r., pitkowski, w., seidel-morgenstern, a., antos, d.: influence of preferential adsorption of mobile phase on retention behavior of amino acids on teicoplanin chiral selector. j. chromatogr. a, 1173, 2007, 58-70. remsburg, j.w., armstrong, d.w., péter, a., tóth, g.: lc enantiomeric separation of unusual amino acids using cyclodextrin-based stationary phases. j. liq. chromatogr. rel. technol., 31, 2008, 219-230. stegink, l.d., bell, e.f., filer, l.j., ziegler, e.e., andersen, d.w., seligsonäž, f.h.: effects of equimolar doses of l-methionine, d-methionine and l-methionine-dl-sulfoxide on plasma and urinary amino acid levels in normal adult humans. j. nutr., 116, 1986, 1185-1192. sun, p., armstrong, d.w.: effective enantiomeric separations of racemic primary amines by the isopropyl carbamate-cyclofructan6 chiral stationary phase. j. chromatogr. a, 1217, 2010, 4904-4918. sun, p., wang, c., breitbach, z.s., zhang, y., armstrong, d.w.: development of new hplc chiral stationary phases based on native and derivatized cyclofructans. anal. chem. 81, 2009, 10215-10226. visser, w.f., verhoeven-duif, n.m., ophoff, r., bakker, s., klomp, l. w., berger, r. de koning, t.j.: a sensitive and simple ultra-highperformance-liquid chromatography–tandem mass spectrometry based method for the quantification of d-amino acids in body fluids. j. chromatogr. a, 1218, 2011, 7130-7136. wang, y., young, d.j., tan, t.t.y., ng, s.c.: click preparation of hindered cyclodextrin chiral stationary phases and their efficient resolution in high performance liquid chromatography. j. chromatogr. a, 1217, 2010, 7878-7883. winkler, m., klempier, n.: enantioseparation of nonproteinogenic amino acids. anal. biomed. chem., 393, 2009, 1789-1796. xiao, t.l., armstrong, d.w.: enantiomeric separations by hplc using macrocyclic glycopeptide-based chiral stationary phases. chiral separations methods in molecular biology, 243, 2004, 113-171. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 16:44 utc http://link.springer.com/book/10.1385/1592596487 http://link.springer.com/bookseries/7651 nova biotechnologica et chimica 15-1 (2016) 55 doi 10.1515/nbec-2016-0006 © university of ss. cyril and methodius in trnava electrochemical treatment of water contaminated with methylorange martin valica, stanislav hostin department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic (martin.valica.mv@gmail.com) abstract: this study examines electrochemical degradation of water artificially contaminated by azo dye methyl orange (mo). degradation is based on chemical electro-oxidation of mo molecules. graphite was used as an electrode material for electrochemical oxidation of mo. in this work, the different operative parameters (electric current, nacl content) and their effect on effectiveness as well as the treatment time/duration of mo degradation were tested. the highest dye removal (91.0 %) was obtained during the electrolysis at current density 3.032 ma/cm2, electrolyte with the content of nacl 4 g/dm3 (nacl) and the treatment time 35 min. key words: dyes, methyl orange, electrochemical oxidation, active chlorine, water contamination, degradation. 1. introduction environmental pollution is a global issue. textile and printing industries are regarded as the most polluting sectors due to their high discharge volume of dyescontained wastewater, especially azo-dye with approx. 50–70% of the world dye production. these dye effluents are typical by high organic content, intense color and stable chemical structure due to the presence of azo groups in molecules (kar et al., 2009; ma and zhou, 2009; shakir et al., 2010). wastewater contained dyes can be toxic and mutagenic. dyes are highly soluble and persistent in polluted water, because they have long degradation time in the environment (azami et al., 2011). pretreatment processes have been found important for significant decrease of organic content and color of dye polluted water. the commonly used pretreatment processes include adsorption, chemical oxidation, and ion exchange (kong et al., 2011). in recent years, electrochemical treatment processes, especially electrochemical oxidation and electrocoagulation, have been studied as alternatives for degradation of various types of organic pollutants in wastewater (panizza et al., 2013; araujo et al., 2014; sirés et al., 2014; brillas and martínez-huitle, 2015; santos et al., 2015). electrochemical oxidation is especially important because no chemical reagents are used in the process. electrochemical oxidation is the process of oxidizing pollutants on the electrode surface, directly through the formation of disinfectants such as highlyoxidative oxygen species from water itself (eq. 1-4) or in combination with substances producing electrogenerated active chlorine compounds (eq. 5-7) if chloride ions are present in the solution (sirés et al., 2006; carter and farrell, 2008; chaplin et al., 2009): 2h2o+ 2e −→ 2oh−+h2 (1) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:10 utc 56 valica, m. and hostin, s. 2h2o + o2+4e −→ 4oh− (2) 3h2o → o3 + 6e –+ 6h+ (3) o2 + 2h2o + 2e −→ h2o2 + 2oh − (4) 2cl– → cl2 + 2e – (5) cl2 + h2o → h + + cl–+ hocl (6) hocl→ h++ ocl– (7) electrochemical degradation as a pretreatment process significantly improves further water purification. moreover, the simple setup, easy control, ambient operation and complete degradation during electrochemical enhanced process further broadens its attractiveness for practical implementation (bonin et al., 2004; valero et al., 2010; sala and gutierrez-bouzan, 2012). the aim of this work is to study the different electrochemical degradation behaviours of the model azo-dye on carbon electrodes through investigation of main operative parameters (time of electrolysis, amounts of nacl in electrolyte and current density). methyl orange (mo) was selected as the model azo-dye (containing –n=n– bonds), because it is widely used in the textile industry and has harmful effect on human health. to avoid the accumulation of mo in the environment it is necessary to develop effective methods for degradation or mineralization of mo (azami et al., 2011; zhou et al., 2011; ramírez et al., 2013). these requirements may be provided by electrochemical water treatment, because organics can be completely mineralized to carbon dioxide by the hydroxyl radical and active chlorine is generated during the electrolysis of nacl in the water solution. 2. materials and methods 2.1 chemicals methyl orange (mo) dye (lachema, czech republic) and nacl (purity > 98 %; chemapol, czech republic) of analytical grade were used in experiments. the chemical structure of mo is shown in the fig. 1.all solutions were prepared in distilled water with the conductivity 10 µs/cm. fig. 1. chemical structure of methyl orange. 2.2 experimental device and procedures electrochemical experiments were carried out at laboratory temperature in an undivided electrochemical cell under conditions of batch system. equipment for bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:10 utc nova biotechnologica et chimica 15-1 (2016) 57 electrochemical water treatment is shown in the fig. 2. the cell contained two graphite electrodes (elektrokarbon, slovak republic) positioned vertically and parallel to each other with an inter-electrode distance of 1.5 cm. the geometric area of the anode and the cathode was 15.7 cm2. the model wastewater was prepared with the known content and volume of mo solution and the supporting electrolyte of nacl. the solution was stirred by circulation performed with a pumping device to maintain a uniform concentration of the electrolyte solution within the system. also, this device pumped wastewater into the spectrophotometer (spekol 11, carl zeiss jena, germany) to analyse the remaining concentration of mo by recording results into a computer in time intervals of two seconds. because output values of spectrophotometer were in electric current (measured by digital voltcraft vc820 with software digiscop for windows ver. 2.06), it was necessary to convert these values to absorbance using the calibration curve between these two variables. fig. 2. electrochemical wastewater treatment equipment: 1. photovoltaic cell/; 2. laboratory electric source; 3. amperemeter; 4. voltmeter; 5. electrolytic cell; 6. graphite electrodes; 7. pump; 8. spectrophotometer; 9. flow cuvette; 10. voltmeter; 11., 12. pc for recording results. all degradation processes were carried out under the galvanostatic conditions for the wastewater volume of 4 dm3. the electric power was supplied with a regulated dc power supply (elektrolyser, veb mlw labortechnik ilmenau, germany). after ph adjustment, the degradation was initialized and the current was set at the desired value. during electrolysis, the values of electric current and voltage transferred through the electrolyte were recorded on a computer in time intervals of two seconds. for this measurement, two separate digital multimeters (metex m-3850d with software scopeview ver. 1.06; and axio met ax-18b with software pc link soft) were used. electrochemical water treatment was finished, when the lowest and constant values of the absorbance of wastewater were obtained. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:10 utc 58 valica, m. and hostin, s. 2.3 analytical methods mo concentrations were measured using the spectrophotometer spekol 11 at the maximum absorption peak of 465 nm. this wavelength was determined from the absorption spectrum of mo in water solution measured with the uv-vis spectrophotometer (spectroquant® pharo 300 merck, germany) in the wavelength range between 200 to 800 nm. the decolorization ratio (η) was calculated using the following equation (eq. 8): %100 0 0 × − = c cc tη (8) where c0 and ct are concentrations of model wastewater measured at wavelength 465 nm and at the initial and the given time t. the remaining concentration of mo in wastewater was determined from the equation of the linear dependence between the concentration of mo and absorbance, for solutions with the known concentration of mo (0.4 – 4.4 μg/cm3). 3. results and discussion 3.1 effect of current density the effect of current density on wastewater decolorization was analysed at different current densities within the range of 1.011 4.042 ma/cm2 under the constant concentration and volume of nacl electrolyte (4 g/dm3) and the initial content of mo in the wastewater (approx. 4 μg/cm3). this range of current densities was chosen from point of view of the energy consumption i.e. economic efficiency of water treatment. the dependence of the residual content of mo in wastewater from treatment time is shown in the fig. 3. 0 5 10 15 20 25 30 35 40 45 50 55 60 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 co nc en tr at io n of m o [ μg /c m 3 ] time [min] 1.011 ma/cm2 2.021 ma/cm2 3.032 ma/cm2 4.042 ma/cm2 fig. 3. electrochemical decolorization of mo under different current densities (conditions of the experiment: initial concentration of nacl 4 g/dm3; ph 6). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:10 utc nova biotechnologica et chimica 15-1 (2016) 59 it is evident that the dye degradation on graphite electrodes was more efficient at higher values of current density. also, the treatment time needed for electrochemical degradation of mo was shorter at higher current density (table 1). as demonstrated in the table 1 and fig. 4, the decolorization ratio η linearly increased with treatment time and current density. the key role of current density is shown for instance, at the treatment time of 400 s when under current density of 4.042 ma/cm2 the decolorization ratio was about 80%. at the same time, decolorization ratio under 1.011 ma/cm2current density was only 18%. table 1. efficiency of mo degradation obtained under various operating parameters. parameters concentration of mo current density [ma/cm2] concentration of nacl [g/dm3] initial [μg/cm3] final [μg/cm3] decolorization ratioη [%] treatment time [min] 1.011 4 3.86 0.50 87.1 55 2.021 2 3.97 0.75 81.3 78 2.021 4 4.00 0.44 89.1 39 2.021 6 3.89 0.43 89.0 32 3.032 4 3.87 0.36 91.0 35 4.042 4 3.97 0.47 88.6 24 0,1 0,2 0,3 0,4 0 10 20 30 40 50 60 70 80 90 de co lo riz at io n ra tio η [% ] current density [ma/cm2] 100 s 200 s 300 s 400 s fig. 4. dependence of the decolorization ratio η from current densities on electrodes, conditions of the experiment: initial concentration of mo aprox. 4 µg/cm3 and nacl 4 g/dm3, ph 6. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:10 utc 60 valica, m. and hostin, s. similar results were found in various research papers (ma et al., 2007; carvalho et al., 2011; yusuf et al., 2016), where a slower response of mo to electrochemical decolorization at lower current densities was observed, but an increase of applied current densities caused a sharp increase in the percentage of color removal. at different condition of current density was reached same level of the total dye removal efficiency (87.1 91.0 % dye removal). this could be ascribed to the fact that the amounts of the produced oxidants, such as chlorine, hypochlorite, oxygen species are very similar, however they are produced in different electrochemical treatment times. this assumption is supported by the faraday's law making known that the amount of electrochemical products is proportional to the current and is constant at a constant applied current (yusuf et al., 2016). 3.2 effect of supporting electrolyte in experiments, nacl was chosen as the supporting electrolyte to enhance the degradation efficiency and shorten the treatment time according to findings of other authors (panizza et al., 2000; kong et al., 2009). the different supporting electrolytes such as nacl, kcl, kno3 and na2so4 were studied by other authors (lorimer et al., 2001; kong et al., 2011, yusuf et al., 2016).their results showed that the presence of chloride ions played an important role. almost total decolorization was obtained with kcl, when a very low color removal was obtained with na2so4 and no decolorization was achieved with kno3. electrochemical degradation of mo was provided by the direct anodic oxidation, but the most important role played the indirect oxidation with active chlorine compounds produced in electrolysis of nacl/kcl. in the absence of chloride ions, neither any oxidative species are produced nor does any direct anodic oxidation of the dye occur (yusuf et al., 2016). in article li et al. (2013) was observed from changes of ftir spectra evidences of mo degradation and intermediate formation. most of the characteristic bands of mo, e.g., the characteristic band of benzene rings (1600 cm-1) and the band of n=n (1520 cm-1), significantly decreased or disappeared after 4 h treatment, while two new bands at 1263 cm-1 and 800 cm-1 appeared. possible mechanism of electrochemical degradation of mo (fig. 5) was studied by lc–ms (ramírez et al., 2013) and gcms analysis (du et al., 2011). results from lc-ms indicated the formation of seven various oxidation products from the cleavage of the –n=n– group of the dye, followed by deamination, formation of a nitro group and/or desulfonation of the resulting aromatics (ramírez et al., 2013). in the presence of chloride, the electrolysis was able to oxidise the dye with partial mineralisation of carbon, nitrogen and sulfur into co2, no3 and so4 2-(du et al., 2011). dye removal was found to be dependent of nacl concentration in electrolyte (initial concentration of nacl in wastewater was in the range of 2 6 g/dm3) during the experiment with the constant current density (2.021ma/cm2) and initial mo concentration (approx. 4 μg/cm3). these conditions were chosen with the aim to find the correlation between the concentration of nacl and the treatment time needed for mo degradation. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:10 utc nova biotechnologica et chimica 15-1 (2016) 61 n naso3nn h3c h3c no3 so4 -n2 ohclnhn cl o h2n oh o oh o o o h2o cl/ oh/ ch3 co2 o h3c h3c ch3 ohcnh2n+2 ohinorganic ions oh fig. 5. proposed electrochemical degradation pathways for mo (adopted from du et al., 2011). 0 10 20 30 40 50 60 70 80 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 co nc en tr at io n of m o [μ g/ cm 3 ] time [min] 2g/dm3 nacl 4g/dm3 nacl 6g/dm3 nacl fig. 6. electrochemical decolorization of mo at various concentrations of nacl, conditions of the experiment: initial concentration of mo approx. 4 µg/cm3; ph 6; current density 2.021 ma/cm2. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:10 utc 62 valica, m. and hostin, s. as can be seen from fig. 6,the total dye removal efficiency was almost the same for decolorization running in the solutions with 4 6 g/dm3 nacl (decolorization or η = 89 %). very similar decolorization rate and the total treatment time at higher concentrations of nacl (<4 g/dm3) was observed by yusuf et al. (2016), where kcl solution was used as supporting electrolyte (<0.1 m). efficiency of decolorization was almost the same and small differences were probably caused by different electrode material (polymer electrode).lower efficiency was obtained in 2 g/dm3 nacl solution, where decolorization ratio was only 81.3%. this difference can be explained by the deficient production of active chlorine due to the low concentration of chlorine ions in the model wastewater. mo degradation treatment time increased with the decreasing concentration of nacl. these results correspond to the observations other authors (rajkumar et al., 2007; martínezhuitle and brillas, 2009). 4. conclusions the methylorange (mo) degradation by electrochemical oxidation on two carbon electrodes was analysed in this study. electrochemical degradation of mo was tested at various operative parameters. our results demonstrated that concentration of nacl, time of electrolysis and the applied current density on electrodes played an important role in the efficiency of dye removal from the model wastewater. at different current densities, probably due to the amount of the produced active chlorine, decolorization ratio was the same, and the rate of the production of the active chlorine played the key role in degradation of mo. the presence of nacl increased the degradation of mo due to a synergic effect of anodic and basal oxidation, which greatly improved the decolorization rate. the obtained results also showed that the efficiency of mo degradation increased along with the concentration of nacl electrolyte. the results of this work offer an effective alternative method for enhancing degradation of dyes in liquid solutions and wastewaters. references araujo, e.g., santos, a.j., silva, d.r., salazar, r., martínez-huitle c.a.: cysteic acid-modified glassy carbon electrode for monitoring oxalic acid (oa) concentration during its electrochemical oxidation at ti/pt anode. electroanalysis, 26, 2014, 748-755. azami, m., bahram, m., nouri, s., naseri, a.: a central composite design for the optimization of the removal of the azo dye, methyl orange, from waste water using the fenton reaction. j. serb. chem. soc., 76, 2011, 1-21. bonin, p.m.l., bejan, d., schutt, l., hawari, j., bunce. n.j.: electrochemical reduction of hexahydro-1,3,5-trinitro-1,3,5-triazine in aqueous solutions. environ. sci. technol., 38, 2004, 1595-1601. brillas, e., martínez-huitle c.a.: a central composite design for the optimization of the removal of the azo dye, methyl orange, from waste water using the fenton reaction. appl. catal. b-environ., 166-167, 2015, 603-643. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:10 utc nova biotechnologica et chimica 15-1 (2016) 63 carter, k.e., farrell, j.: oxidative destruction of perfluorooctane sulfonate using boron-doped diamond film electrodes. environ. sci. technol., 42, 2008, 6111-6118. carvalho, d.a., bezerra rocha, j.h, fernandes, n.s, da silva, d.r., martínez-huitle. c.a.: application of electrochemical oxidation as alternative for removing methyl green dye from aqueous solutions. lat. am. appl. res., 41, 2011, 127-133. chaplin, b.p., schrader, g., farrell, j.: electrochemical oxidation of nitrosodimethylamine with boron-doped diamond film electrodes. environ. sci. technol., 43, 2009, 8302-8310. du, l., wu, j., qin, s., hu, c.:degradation mechanism of methyl orange by electrochemical process on ruo(x)-pdo/ti electrode. water sci. technol., 43, 2011, 1539-1545. kar, a., smith, y.r., subramanian, v.: improved photocatalytic degradation of textile dye using titanium dioxide nanotubes formed over titanium wires. environ. sci. technol., 43, 2009, 3260-3268. kong, y., yuan, j., wang, z.l.: application of expanded graphite/attapulgite composite materials as electrode for treatment of textile wastewater. appl. clay sci., 46, 2009, 358-362. kong, y., wang, z., wang, y., yuan, j., chen, z.: degradation of methyl orange in artificial wastewater through electrochemical oxidation using exfoliated graphite electrode. new carbon mater., 26, 2011, 459-464. li, s., zhao, y., chua, j., li, w., yu, h., liu, g.: electrochemical degradation of methyl orange on pt–bi/c nanostructured electrode by a square-wave potential method. electrochim. acta, 92, 2013, 93-101. lorimer, p., mason, t.j., plattes, m., phull, s.s., walton, d.j.: degradation of dye effluent. pure appl. chem., 73, 2001, 1957-1968. ma, h., wang, b., luo, x.: studies on degradation of methyl orange wastewater bycombined electrochemical process. j. hazard. mater, 149, 2007, 492-498. ma, x.j., zhou, m.h.: a comparative study on azo dye decolorization by electrofenton in two common electrolytes. j. chem. technol. biot., 84, 2009, 1544-1549. martínez-huitle, c.a., brillas e.: decontamination of wastewaters containing synthetic organic dyes by electrochemical methods: a general review. appl. catal. environ.,87, 2009, 105-145. panizza, m., barbucci, a., delucchi, m., carpanese, m.p., giuliano, a., cataldo-hernandez, m., cerisola g.: electro-fenton degradation of anionic surfactants. sep. purif. technol., 118, 2013, 394-398. panizza, m., bocca, c., cerisola, g.: electrochemical treatment of wastewater containing polyaromatic organic pollutants. water res., 34, 2000, 2601-2605. rajkumar, d., song, b.j., kim, j.g.: electrochemical degradation of reactive blue 19 in chloride medium for the treatment of textile dyeing wastewater with identification of intermediate compounds. dyes pigments, 72, 2007, 1-7. ramírez, c., saldaña, a., hernández, b., acero, r., guerra, r., garcia-segura, s., brillas, e., peralta-hernández, j.m.: bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:10 utc 64 valica, m. and hostin, s. electrochemical oxidation of methyl orange azo dye at pilot flow plant using bdd technology. j. ind. eng. chem., 19, 2013, 571-579. sala, m., gutierrez-bouzan, c.m.: electrochemical techniques in textile processes and wastewater treatment. int. j. photoenergy, 9, 2012, 1-8. santos, e.v., sáez, c., martínez-huitle, c.a., cañizares, p., rodrigo, m.a.: the role of particle size on the conductive diamond electrochemical oxidation of soil-washing effluent polluted with atrazine. electrochem. commun., 55, 2015, 26-29. shakir, k., elkafrawy, a.f., ghoneimy, h.f., beheir, s.g.e., refaat, m.: removal of rhodamine b (a basic dye) and thoron (an acidic dye) from dilute aqueous solutions and wastewater simulants by ion flotation. water res., 44, 2010, 1449-1453. sirés, i., cabot, p.l., centellas, f., garrido, j.a., rodríguez, r.m., arias, c., brillas, e.: electrochemical degradation of clofibric acid in water by anodic oxidation: comparative study with platinum and boron-doped diamond electrodes. electrochim. acta, 52, 2006, 75-82. sirés, i., brillas, e., oturan. m.a., rodrigo, m.a., panizza, m.: electrochemical advanced oxidation processes: today and tomorrow. a review. environ. sci. pollut. r., 21, 2014, 8336-8367. valero, d., ortiz, j.m., exposito, e., montiel, v., aldaz, a.: electrochemical wastewater treatment directly powered by photovoltaic panels: electrooxidation of a dye-containing wastewater. environ. sci. technol., 44, 2010, 5182-5188. yusuf, a.h., redha, m.z., baldock, s.j., fielden, p.r., goddard, n.j.: an analytical study of the electrochemical degradation of methyl orange using a novel polymer disk electrode. microelectron. eng., 149, 2016, 31-36. zhou, m., särkkä, h., sillanpää, m.: a comparative experimental study on methyl orange degradation by electrochemical oxidation on bdd and mmo electrodes. sep. purif. technol., 78, 2011, 290-297. received 10 november 2015 accepted 16 june 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:10 utc microsoft word 10_lomjansky et al nova biotechnologica et chimica 15-2 (2016) 200 doi 10.1515/nbec-2016-0020 © university of ss. cyril and methodius in trnava redetermination of zero-field splitting in [co(qu)2br2] and [ni(pph3)2cl2] complexes dominik lomjanský1, filip varga1, cyril rajnák1, ján moncoľ2, roman boča1, ján titiš1 1 department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic (lomjanskyd@gmail.com) 2 institute of inorganic chemistry, fchpt, slovak university of technology, bratislava, sk-812 37, slovak republic abstract: a mononuclear coii complex, [co(qu)2br2], and niii complex, [ni(pph3)2cl2], (qu = quinoline, pph3 = triphenylphosphine) have been reinvestigated. their crystal and molecular structures are reported along with ir and uv-vis spectra. magnetism of both complexes has been studied by using the dc squid magnetometry. these complexes exhibit a moderate magnetic anisotropy expressed by zero-field splitting parameter d. the d-value is positive for both complexes with d/hc = +5.94 cm-1 and d/hc = +12.76 cm-1, that is also confirmed by ab initio calculations. key words: tetracoordinate complexes, electronic spectra, magnetic susceptibility, magnetization, zero-field splitting 1. introduction detection of static and dynamic magnetic properties of mononuclear 3d metal complexes is an important task in the emerging field of molecular magnetism. single-molecule magnets (smms), as the main objects of interest, show slow magnetic relaxation as a consequence of the energy barrier (eb ≈ |d|s 2, where d is axial zerofield splitting (zfs) parameter) for the magnetization reversal between the two lowest ms = ±s states (gatteschi et al., 2006; frost et al., 2016). smms are a major scientific target because of their potential applications in high-density magnetic memories and quantum-computing devices. the zero-field splitting represents a fine structure of the electronic energy levels that appears as a result of the splitting of the crystal-field terms into crystal-field multiplets. it is characterized by the axial zfs parameter d that is related to the energy gap between the ground and excited states (boča, 2004; boča, 2006). despite the relatively wide range of knowledge, a rational design of the d-parameter is still far from the routine, though there is a need of this when tuning the properties of the smms. the sign of the d-parameter is either negative or positive and it adopts values between 10-2 – 102 cm-1. some insights to this target brought the magnetostructural d-correlations that bring a linear relationship between the axial distortion of the octahedron and the d-parameter in a series of niii hexacoordinate complexes (titiš and boča, 2010). for hexacoordinate coii complexes the magnetostructural d-correlation has also been outlined (titiš and boča, 2011). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:07 utc 201 lomjanský, d. et al. the case of tetracoordinate complexes is no less interesting. tetrahedral ligand environments produce weaker crystal-fields in comparison with its octahedral counterparts. this results to the enhanced second-order perturbation (as a consequence of the spin-orbit coupling) of the ground crystal-field term, and thus to the significantly larger d-values (idešicová et al., 2013). the present communication compares magnetic behavior of two different systems, the tetracoordinate coii (kramers ion, s = 3/2) and tetracoordinate niii (non-kramers ion, s = 1). reinvestigation of complexes [co(qu)2br2] and [ni(pph3)2cl2] (qu = quinoline, pph3 = triphenylphosphine), hereafter 1 and 2, using the dc magnetometry and contemporary methods of quantum chemistry represents the aim of this study. in tetracoordinate coii complexes the ground term 4a2(td) is split into the ground and excited multiplets (each referring to a kramers doublet) separated by 2d. in reference td symmetry the ground term of the ni ii ion is the orbitally degenerate 3t2. this term in lower symmetry (d2d) is split into the ground 3a2 and excited 3e terms. spin-orbit coupling further split the 3a2 ground term into ms = 0 and ms = ±1 multiplets. difference between them is equal to d. 2. materials and methods 2.1. synthesis the compound 1, [co(qu)2br2], has been prepared as follows. a solution of 0.218 g (1 mmol) cobr2 in 10 cm 3 of acetonitrile was added to the solution of 0.258 g (2 mmol) of qu in 10 cm3 of acetonitrile under an intense stirring (the molar ratio qu:cobr2 = 2:1). a mixture was stirred for 3 h at room temperature. the filtrate was left for 3 days for a spontaneous evaporation. the dark blue crystals were separated on büchner funel. the compound 2, [ni(pph3)2cl2], has been prepared as follows. a solution of 0.238 g (1 mmol) of nicl2⋅6h2o in 20 cm 3 of distilled water and 5 cm3 of acetic acid was added. a solution of 0.525 g (2 mmol) of pph3 in 2.5 cm 3 of acetic acid was slowly added in solution of nicl2⋅6h2o using a pasteur pipette. deep blue crystals were obtained after 20 hours standing. 2.2. physical measurements elemental analysis was carried out on flashea 1112 (thermofinnigan). ir spectra were measured using the atr method (magna ftir 750. nicolet) in the 400 – 4 000 cm−1 region. electronic spectra were measured in nujol mull (specord 200, analytical jena) in the range 9 000 – 50 000 cm−1. magnetic data were taken with the squid magnetometer (mpms-xl7. quantum design) using the rso mode of detection. the susceptibility data were scanned in the temperature range 2 – 300 k at the applied field of b = 0.1 t. the magnetization has been measured at t = 2.0 and 4.6 k. raw data were corrected for the underlying diamagnetism using estimate of χdia/(10 −12 m3 mol−1) = −5m[g mol−1]. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:07 utc nova biotechnologica et chimica 15-2 (2016) 202 2.3. quantum-chemical calculations ab initio calculations were performed with orca 3.0.3 computational package at the experimental geometry determined by the x-ray diffraction for the mononuclear entity (neese 2012). relativistic effects were included in the calculations by zero order regular approximation (zora) together with the scalar relativistic contracted version of tzvp basis functions. the calculations of zfs parameters were based on state average complete active space self-consistent field (sa-casscf) wave functions complemented by nevpt2 theory (angeli et al., 2001; angeli et al., 2002; angeli et al., 2004; atanasov et al., 2011). the calculations utilized the ri approximation with appropriate decontracted auxiliary basis set and the rijcosx approximation to exact exchange. increased integration grids (grid4) and tight scf convergence criteria were used. the zfs parameters were calculated through quasi-degenerate perturbation theory (qdpt) in which an approximation to the breit-pauli form of the spin-orbit coupling operator (somf) and the effective hamiltonian theory was utilized (neese, 2005; ganyushin and neese, 2006; neese, 2007). table 1. selected crystal data. compound 1, [co(qu)2br2] 2, [ni(pph3)2cl2] chemical formula c18h14br2con2 c36h30cl2nip2 m(g mol−1) 477.06 654.15 crystal system space group triclinic p -1 monoclinic p21/c t (k) 293(2) 298(2) a (å) 8.685(4) 11.620(4) b (å) 9.637(5) 8.202(2) c (å) 11.232(5) 17.387(7) α (°) 80.671(4) 90 β (°) 74.592(4) 107.009(4) γ (°) 73.296(4) 90 v (å3) 864.45(8) 1583.80(10) z 2 2 radiation type mo κα (λ=0.71073) mo κα (λ=0.71073) µ (mm−1) 5.614 0.907 f(000) 466 676 index ranges -11≤h≤11,-12≤k≤12, -14≤l≤14 -8≤h≤14,-10≤k≤10, -21≤l≤19 ρ calc.(g cm−3) 1.833 1.372 final r indexes [f2 > 2σ(f2)], wr2(f2) r1=0.0321 wr2=0.0738 r1=0.0322 wr2=0.0742 r indices (all data) r1=0.0453 wr2=0.0804 r1=0.0453 wr2=0.0812 data / restrains / parameters 3814/0/208 3232/0/186 goodness of fit 1.029 1.033 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:07 utc 203 lomjanský, d. et al. 2.4. x-ray crystallography data collection and cell refinement were carried out using a κ-axis diffractometer gemini r ccd (oxford diffraction, 2010) with graphite monochromated mo kα radiation. the diffraction intensities were corrected for lorentz and polarization factors. the structures were solved by direct methods using sir-97 (altomare, 1999) or shelxs-97 (sheldrick, 2008) and refined by the full-matrix leastsquares procedure with shelxl-97 (clark, 1995). the analytical or multi-scan absorption corrections were made by using crysalis-red (oxford diffraction 2010). geometrical analyses were performed with shelxl-97. crystal data and conditions of data collection and refinement are reported in table 1. 3. results and discussion the molecular structures with thermal ellipsoids of 1 and 2 are presented in fig. 1. the coordination environment of 1 consists of two donor nitrogen atoms of two quinoline ligands completed by two bromide anions. the coordination sphere of 2 consists of two donor phosphorus atoms of triphenylphospine ligands completed by two chloride anions. in both cases the chromophore, {con2br2} and {cop2cl2}, refers to a distorted tetrahedron. important structural parameters, metal-ligand bond distances and bond angles, for the studied complexes are collected in table 2. fig. 1. molecular structure of complexes 1 and 2 (thermal ellipsoids shown at 50 % of probability level, hydrogens omitted for clarity). we note that compounds under study have already been structurally characterized and are deposited in structural database (ccdc, 2016) under refcodes tusqab (korchagin et al., 2015) and cltpni (garton et al., 1963). by comparing the structural characteristics of these structures with complexes studied in this work we note marked similarities especially for the coii complex. for the niii complex the differences are more pronounced. one can found a shorter ni-p distances (2.277 å) and longer ni-cl distances (2.274 å) in the cltpni structure. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:07 utc nova biotechnologica et chimica 15-2 (2016) 204 furthermore, the bond angles in cltpni (p-ni-p, 116.6° and cl-ni-cl, 123.2°) also shown significant deviations from those in 2. table 2. selected bond lengths (å) and angles (°) in 1 and 2. bond lengths angles 1, [co(qu)2br2] co(1)-br(1) 2.377(5) co(1)-br(2) 2.382(6) co(1)-n(1) 2.032(2) co(1)-n(2) 2.059(2) br(1)-co(1)-br(2) 113.27(2) n(1)-co(1)-br(1) 109.39(7) n(1)-co(1)-br(2) 107.75(7) n(1)-co(1)-n(2) 111.90(10) n(2)-co(1)-br(1) 107.98(7) n(2)-co(1)-br(2) 106.57(7) 2, [ni(pph3)2cl2] ni(1)-p(1) 2.366(5) p(1)-ni(1) 2.366(5) ni(1)-cl(1) 2.202(6) cl(1)-ni(1) 2.202(6) p(1)-ni(1)-p(1) 112.00(3) cl(1)-ni(1)-p(1) 104.95(2) cl(1)-ni(1)-p(1) 103.82(2) cl(1)-ni(1)-p(1) 103.82(2) cl(1)-ni(1)-p(1) 104.95(2) cl(1)-ni(1)-cl(1) 127.25(4) important intermolecular contacts have been identified in the crystal structure of 1 (fig. 2). these contacts (c7-h7…br1, 3.006 å and c1…br2, 3.545 å) interconnect the adjacent molecular units that create linear chains along the a crystallographic axis. in case of the crystal structure of 2, no intermolecular contacts shorter than the sum of the van der waals radii have been found. fig. 2. intermolecular contacts in 1. the solid state electronic spectra of 1 and 2 are shown in fig. 3. for complex 1 a number of d-d transitions can be identified in the range of 9 000 – 24 000 cm−1 which are followed by an intense charge transfer band. the first visible transition is a broad band at ca 9 000 cm−1, the second is split into three peaks between 15 000 – 17 000 cm−1. furthermore, a weak band at ca 20 000 cm−1 is also visible. in the spectrum of 2, three relatively intense bands are present at ca 11 000, 18 000 and 24 000 cm−1. the second band is broad and asymmetric suggesting the presence of a number of d-d transitions. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:07 utc 205 lomjanský, d. et al. since the structure of the complexes is derived from a tetrahedron, the interpretation of the spectra can be done in the td group of symmetry in the first approximation. the estimate for the crystal-field strength for tetrahedral systems is dq(td) = (4/9)dq(oh) = (4/9)(1/6)f4(r) (boča, 2006). the first transition in 1 is 4a2 → 4t2 that lies in the nir region. it has not been detected by the used hardware. the first observed band refers to the 4a2 → 4t1(f) transition and its energy is about 18dq = 9 000 cm−1. an estimate for the next allowed d-d transition 4a2 → 4t1(p) is 12dq + 15b. using dq = 500 cm −1 the racah parameter of coii is estimated as b = 733 cm−1. this value is lowered relative to the free-ion value (b0 = 980 cm −1) owing to the nephelauxetic effect (lever, 1984). the assignment of the individual transitions within the band in the 15 000 – 17 000 cm−1 region can be done in the more realistic d2d group of symmetry (or c2v) where the 4t1g(p) mother term is split into { 4a2, 4e} daughter terms. in such a case also a spin-forbidden transitions can be considered since the close-lying 2e(g), 2a1(g), 2t1(g) and 2t2(g) terms might borrow the intensity from the spin-allowed transitions. the spin-orbit coupling is also in the play and it further modifies the term scheme. wavenumber/cm-1 9000 12000 15000 18000 21000 24000 27000 in te ns ity /a .u . 0.0 0.3 0.6 0.9 1.2 1.5 ni(ii) complex co(ii) complex gaussian deconvolution fig. 3. solid state electronic spectra (nujol mull) of complexes under study. deconvolution of the second band of complex 2 using three gaussian primitives (dot-dashed lines). in complex 2, we expect three principal d-d transitions considering the td group of symmetry. the first transition, 3t1 → 3t2 (3 500 cm −1), lies in the ir region (using the electronic structure parameters 10dq = 3 850 cm−1, b = 725 cm−1). the second one appears at 6 550 cm−1, 3t1 → 3a2. the third transition is located in 14 250 – 15 240 cm−1 region, 3t1 → 3t1(p). this assignment is, however, hypothetical since the real symmetry of 2 is lower than td, most likely c2v. thus the 3t1(f,p) and 3t2 terms are split into { 3a2, 3b1, 3b2} and { 3a1, 3b1, 3b2} terms, respectively. furthermore, the configuration interactions between ground and excited bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:07 utc nova biotechnologica et chimica 15-2 (2016) 206 terms are effective causing the shift of the transitions. to elucidate the nature of detected spectral bands we have decompose the second one into gaussian primitives. it was found that in this region three transitions are hidden (see fig. 3). these are the transitions to 3a2, 3b1 and 3b2 (c2v) terms. according to these results, we assume that the first visible band refers to the 3t1 → 3a2 (td) transition shifted to a higher energy. the third most intense band at ~24 000 cm−1 hides the charge-transfer transitions. the molar magnetic susceptibility for 1 and 2, corrected for the underlying diamagnetism, has been converted to the effective magnetic moment μeff that is displayed in fig. 4. t/k 0 50 100 150 200 250 300 μ e ff/ μ b 0 1 2 3 4 5 6 b/t 0 1 2 3 4 5 6 7 m m ol /( n a μ b ) 0 1 2 3 0 10 20 30 40 χ m ol /( 10 -6 m 3 m ol -1 ) 0 2 4 6 8 10 t = 2.0 k b = 0.1 t t = 4.6 k t/k 0 50 100 150 200 250 300 μ e ff/ μ b 0 1 2 3 4 b/t 0 1 2 3 4 5 6 7 m m ol /( n a μ b ) 0 1 2 0 10 20 30 40 χ m ol /( 10 -6 m 3 m ol -1 ) 0 1 2 3 4 5 6 t = 2.0 k b = 0.1 t t = 4.6 k fig. 4. magnetic functions for complexes 1 (upper panel) and 2 (bottom panel). left – temperature dependence of the effective magnetic moment (molar susceptibility as inset), right – field dependence of the magnetization. circles – experimental data. lines – fitted. dashed lines for 2 refer to simulation with the use of negative d value (d = −12 cm−1). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:07 utc 207 lomjanský, d. et al. for complex 1, at the room temperature the value of μeff = 4.85 μb matches the s = 3/2 spin system with some orbital contribution to the g-factor (g ~ 2.5). on cooling the effective magnetic moment slightly decreases along a straight line which is a fingerprint of some temperature-independent paramagnetism. below 10 k the decrease is more rapid until 3.17 μb at 1.9 k; this reflects some zero-field splitting (the splitting of the ground 4b1(d2d) term into two kramers doublets). the magnetization data taken at t = 2.0 k indicate a saturation at b > 7.0 t. the magnetization per formula unit at b = 7 t is only m1 = mmol/(naμb) = 2.6 which again reflects the zero-field splitting. magnetic data for complex 2 are different. at the room temperature the value of μeff = 3.32 μb. this value corresponds to the s = 1 spin system with some orbital contribution. estimated value of the g-factor is g ~ 2.3. on cooling the effective magnetic moment decreases until ~10 k, than the decrease is more rapid until 1.41 μb at 1.9 k. here again we expect the zero-field splitting. susceptibility data show a clear plateau at low temperatures, thus we expect positive d value in this case. with increasing field the magnetization increases rapidly. at both temperatures the behaviour is almost identical. at b = 7.0 t the magnetization per formula unit is m1 = mmol/(naμb) = 1.39 and does not show a saturation feature. the experimental magnetic data have been fitted by assuming the spin hamiltonian in the following form: 2 2 2 2 2 2 , 1 b ˆ ˆ ˆˆ ( / 3) ( ) ˆ ˆ ˆ( sin cos sin sin cos ) k l z x y m x k l x y k l y z k z h d s s e s s b g s g s g sμ ϑ ϕ ϑ ϕ ϑ − − − = − + − + + + r h h h (1) where d is the axial and e is the rhombic zero-field splitting parameter, respectively. the second contribution is the spin zeeman term. the minor improvements refer to the molecular-field correction zj and the uncompensated temperature-independent magnetism χtim: molcorr tim2 a 0 b mol1 ( / )zj n χ χ χ μ μ χ = + − (2) table 3. fitted magnetic parameters for complexes under study. compound 1, [co(qu)2br2] 2, [ni(pph3)2cl2] gx 2.643 2.284 gy 2.544 – gz 2.427 2.203 d (cm−1) +5.94 +12.76 e (cm−1) 1.24 – χtim (10 −9 m3 mol−1) 5.23 9.98 zj (cm−1) −0.01 – r (χ) 0.022 0.021 r (m) 0.034 0.067 an advanced fitting procedure accounts simultaneously for the two data-sets: χ = f(t, b0 = 0.1 t) and m = f(b, t0) with t0 = 2.0 and 4.6 k, respectively. the powder bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:07 utc nova biotechnologica et chimica 15-2 (2016) 208 average has been done by averaging 210 points of polar coordinates ( , )i iϑ ϕ distributed over the top hemisphere. it converged to a set of magnetic parameters listed in table 3. the quality of the fits is good as expressed by discrepancy factors for the susceptibility r(χ) and magnetization r(m). for both complexes the d value is positive. the sign of the d-parameter arises from the assignment of the lowest crystal-field multiplet. for 1, the kramers doublet 1 / 2sm = ± is ground state and 3 / 2sm = ± refers to the excited state, then 2d > 0 holds true. in case of 2, the ground state is the crystal-field multiplet with ms = 0 and the excited one with ms = ±1. thus, the energy difference between these multiplets refers to d > 0. positive d value has a specific effect on the progress of magnetic functions of the niii complexes. the susceptibility of the complex 2 appears a plateau at low temperatures which is often attributed to the presence of ferromagnetic interaction between the complex molecules. in case of 2, however, simulation of the magnetic functions (see fig. 4) clearly confirmed that this behaviour is the result of the positive d-parameter. also the almost identical progress of the magnetization at different temperatures is a consequence of the ms = 0 ground state. table 4. calculated magnetic parameters. compound 1, [co(qu)2br2] 2, [ni(pph3)2cl2] g1 2.222 2.243 g2 2.275 2.339 g3 2.284 2.379 d (cm−1) +5.45 +20.06 e/d 0.03 0.17 the zero-field splitting has also been studied by ab initio calculations. we have applied the cas(n,5)/nevpt2 (n = 7 for 1, n = 8 for 2) methodology that results to magnetic parameters collected in table 4. comparing the experimental and calculated values we conclude that the correlation between them is acceptable. for complex 1 the agreement between the fitted and calculated d is excellent. however, the g-factor components are quite low, possibly due to the formation of magnetic chains in the solid state. for complex 2 the calculated d is somewhat larger than the experimental value. considering the earlier reported high-frequency and high-field electron paramagnetic resonance (hfepr) spectroscopy results (d = +13.20 cm−1) (krzystek et al., 2002) it is evident that just the experimental d-value must be taken as the more realistic one. overestimation of the calculated d may be associated with the omission of some important solid state effects that in such calculations cannot be included. contributions to the d-values that come from individual electronic transitions are presented in fig. 5. one can see that the most important contributions are those at low transition energies (< 10 000 cm−1). in complex 1, the largest contribution (d = −14.5 cm−1) comes from the transition at 4 818 cm−1. this sizeable negative value is compensated by significant positive contributions mainly at 3 870 and 4 507 cm−1. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:07 utc 209 lomjanský, d. et al. in 2 the situation is analogous, however, with more pronounced d contributions. the d for the transition at 8 365 cm-1 is negative and very large (−48 cm-1). however, the contributions from transitions at lower energies dominate at summation giving the moderate positive total d-value. by using this method, the origin of the positive d-parameter of the studied complexes can be explained. transition energy/cm−1 5000 10000 15000 20000 c on tr ib ut io n to d /c m − 1 -60 -40 -20 0 20 40 complex 1 complex 2 fig. 5. calculated contributions to the d-parameter from individual electronic transitions. 4. conclusions we have prepared two tetracoordinate complexes, namely [co(qu)2br2] and [ni(pph3)2cl2] where qu = quinoline and pph3 = triphenylphosphine are n-donor and p-donor ligands, respectively. from structurally point of view these complexes are not new, since such structures are in disposal in structural database. we have redetermined the structure of these complexes and studied their spectral and magnetic properties. static magnetism has been studied by using the dc squid magnetometry. we confirmed that the studied complexes exhibit a moderate magnetic anisotropy expressed by zero-field splitting parameter d. the d-value is positive for both complexes with d/hc = +5.94 cm-1 for the coii and d/hc = +12.76 cm-1 for the niii complex. these results have been supported by ab initio calculations. based on these results, we do not expect the slow magnetic relaxation for these complexes. acknowledgments: slovak grant agencies (apvv-14-0078, apvv-14-0073 and vega 1/0534/16) are acknowledged for the financial support. references altomare, a., burla, m.c., camalli, m., cascarano, g.l., giacovazzo, c., buagliardi, a., moliterni, a.g.g., polidori, g., bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:07 utc nova biotechnologica et chimica 15-2 (2016) 210 spagna, r.: sir97: a new tool for crystal structure determination and refinement. j. appl. chryst., 32, 1999, 115-119. angeli, c., cimiraglia, r., evangelisti, s., leininger, t., malrieu, j.p.: introduction of n-electron valence states for multireference perturbation theory. j. chem. phys., 114, 2001, 10252. angeli, c., cimiraglia, r., malrieu, j.p.: n-electron valence state perturbation theory: a spinless formulation and an efficient implementation of the strongly contracted and of the partially contracted variants. j. chem. phys., 117, 2002, 9138. angeli, c., borini, s., cestari, m., cimiraglia, r.: a quasidegenerate formulation of the second n-electron valence state perturbation theory approach. j. chem. phys., 121, 2004, 4043. atanasov, m., ganyushin, d., pantazis, d.a., sivalingam k., neese, f.: detailed ab initio first-principles study of the magnetic anisotropy in a family of trigonal pyramidal iron(ii) pyrrolide complexes. inorg. chem., 50, 2011, 74607477. boča, r.: zero-field splitting in metal complexes. coord. chem. rev., 248, 2004, 757-815. boča, r.: magnetic function beyond the spin-hamiltonian. in: mingos, d.m.p. (ed.), structure and bonding, springer berlin heidelberg, 2006 1-264. clark, r.c., reid, j.s.: the analytical calculation of absorption in multifaceted crystals. acta chryst. a, 51, 1995, 887-897. ccdc (cambridge crystallographic data centre): http://www.ccdc.cam.ac.uk/. 2016. frost, j.m., harriman, k.l.m., murugesu, m.: the rise of 3-d single-ion magnets in molecular magnetism: towards materials from molecules. chem. sci., 7, 2016, 2470-2491. garton, g., henn, d.e., powell, h.m., venanzi, l.m.: tetrahedral nickel(ii) complexes and the factors determining their formation. part v. the tetrahedral co-ordination of nickel in dichlorobistriphenylphosphinenickel. j. chem. soc., 1963, 3625-3629. ganyushin, d., neese, f.j.: first-principles calculations of zero-field splitting parameters. j. chem. phys., 125, 2006, 24103. gatteschi, d., sessoli, r., villain, j.: molecular nanomagnets, oxford university press, 2006. 395 p. idešicová, m., titiš, j., kryzstek, j., boča, r.: zero-field splitting in pseudotetrahedral co(ii) complexes: a magnetic, high-frequency and -field epr, and computational study. inorg. chem., 52, 2013, 9409-9417. korchagin, d.v., shilov, g.v., aldoshin, s.m., morgunov, r.b., talantsev, a.d., yureva, e.a.: halogen atom effect on the magnetic anisotropy of pseudotetrahedral co(ii) complexes with a quinoline ligand. polyhedron, 102, 2015, 147-151. krzystek, j., park, j.h., mark, w., meisel, w.m., michael, a., hitchman, a.m., stratemeier, h., brunel, c.l., telser, j.: epr spectra from “epr-silent” species: high-frequency and high-field epr bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:07 utc 211 lomjanský, d. et al. spectroscopy of pseudotetrahedral complexes of nickel(ii). inorg. chem., 41, 2002, 4478-4487. lever, a.b.p.: innorganic electronic spectroscopy, elsevier, new york, 1984, 863 p. neese, f.: orca – an ab initio, density functional and semi-empirical program package, version 3.0.3. neese, f.j.: efficient and accurate approximations to the molecular spin-orbit coupling operator and their use in molecular g-tensor calculations. j. chemical physics., 122, 2005, 34107. neese, f.j.: calculation of the zero-field splitting tensor on the basis of hybrid density functional and hartree-fock theory. j. chemical physics., 127, 2007, 164112. neese, f.: the orca program system. rev. comput. mol. sci., 2, 2012, 73-78. oxford diffraction: crysalis–ccd and crysalis-red, oxford diffraction ltd, abingdon, england, 2010. sheldrick, m.: a short history of shelx. acta chrystallogr. a, 64, 2008, 112122. titiš, j., boča, r.: magnetostructural d correlation in nickel(ii) complexes: reinvestigation of the zero-field splitting. inorg. chem., 49, 2010, 3971-3973. titiš, j., boča, r.: magnetostructural d correlations in hexacoordinated cobalt(ii) complexes. inorg. chem., 50, 2011, 11838-11845. received 16 november 2016 accepted 5 december 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:07 utc nova biotechnol chim (2017) 16(1): 20-25 doi: 10.1515/nbec-2017-0003  corresponding author: martincova21@uniba.sk nova biotechnologica et chimica transient expression of green fluorescent protein in parasitic dodder as a tool for studying of cytoskeleton peter kaštiera, michaela martinčováa,, roderik fialab and alžbeta blehováa a department of plant physiology, faculty of natural sciences, comenius university in bratislava, ilkovičova 3278/6, bratislava, sk-841 04, slovak republic b institute of botany, plant science and biodiversity centre sas, dúbravská cesta 9, bratislava, sk-845 23, slovak republic article info article history: received: 22nd march 2017 accepted: 16th may 2017 keywords: actin agrobacterium tumefaciens cuscuta europaea gfp parasitic plants transient transformation abstract dodder (cuscuta) species cause severe agricultural damage in many countries throughout the world. to establish strategies for control of its growth and spreading it is important to study its life cycle and survival strategies. for these efforts genetic modification would represent a powerful tool. here we report on agrobacteriummediated transformation of dodder using green fluorescent protein (gfp) fused to actin-binding protein as a vital marker. since the shoot of germinating c. europaea contains a functional apical meristem and grows quickly comparing to the root-like structure, the shoot apex was used here as explant. the transgene expression was only transient, nevertheless it enabled to detect allocation of actin filaments and studying the cytoskeleton organization in dodder shoot apex. transient expression of gfp appears to be a suitable method for studying cuscuta development through cytoskeleton organisation that is presently largely unexplored.  university of ss. cyril and methodius in trnava introduction parasitic cuscuta (dodder) genus (convolvulaceae) contains 194 species divided into four subgenera, namely monogynella, cuscuta, grammica and pachystigma (costea et al. 2015). they are spread nearly worldwide (garcía et al. 2014). dodders are twining herbs with reduced vegetative organs (leaves are minute scales). dodder species cause severe agricultural damage in many countries throughout the world resulting in yield decrease of important crops such as tomatoes and potatoes (heide-jørgensen 2011). in spite of their agricultural importance, many questions remain unanswered and an efficient transformation system would greatly accelerate the progress in understanding host-parasite relationships. research on mechanisms of haustorium formation as of key organ necessary for dodder survival is a prerequisite for developing efficient strategies to control its development and spreading. understanding the interaction between host plants and parasitic dodders at the ultrastructural and molecular levels may generate knowledge applicable to control plant-parasitic interactions and/or engineer dodder-resistant plants. though efficient transformation protocols have been described for dodder calli (borsics et al. 2002; švubová and blehová 2013), these have never resulted in regeneration of whole plants (ichihashi et al. 2015). here we report on transformation of excised dodder shoot explants via agrobacterium that resulted in transient expression of the vital marker gfp gene. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:36 utc mailto:martincova21@uniba.sk nova biotechnol chim (2017) 16(1): 20-25 21 to study the transgenic nature and development of transformed tissue we used a gfp protein fused in frame to a protein that binds to actin. actin as a component of the cytoskeleton coordinates numerous cellular processes required for plant development, including cell division and cell expansion (wasteneys and ambrose 2009), targeted-vesicle transport to establish hormone gradients (muday 2000), polarity determination to direct localized wall biosynthesis (baluška et al. 2003), and organelle positioning in response to environmental cues (šamaj et al. 2006). in cuscuta species, the presence of α-tubulin, though in quantities lower than in roots, has been associated with the degradation processes of the root-like structures (sherman et al. 2008). further, indirect immunolocalization of actin as well as tubulin has indicated important role in prehaustorial cells in c. europaea (kaštier et al. 2015). the techniques used in those studies, however, do not allow to study the dynamic changes in cytoskeleton organization in real-time in living cells, therefore would largely benefit from a use of a vital marker like gfp (švubová and blehová 2013; blehová et al. 2015). experimental plant material parasitic dodder seeds (c. europaea) were collected in ivanka pri dunaji (2014, slovakia, latitude 48°19´, longitude 17°22´) and their host plant was urtica dioica. the seeds were scarified at 4 °c and then treated with concentrated h2so4 for 60 min at room temperature in order to disrupt the seed coat integration. acid was washed-off by sterile distilled water. the seeds were then sterilized with 5 % savo (0.24 % naclo) solution for 15 min and thoroughly washed with sterile distilled water. dodders were cultivated in vitro on ms medium (murashige and skoog 1962) containing agar (7 g/l), ph 5.8, in the growth chamber (16/8 h long-day photoperiod) at 23±2 oc, 100 μmol/m2s par. bacterial strains and cultivation conditions agrobacterium tumefaciens lba4404 carrying the plasmid pcb302 (voigt et al. 2005) was used for dodder transformation. the t-dna of the pcb302 contained a single gfp fused with n-terminus of arabidopsis thaliana fimbrin actin-binding domain that is driven by constitutive camv 35s promoter. as a plant selection marker, the neomycin phosphotransferase ii gene (nptii) was used under control of the nos promoter. a. tumefaciens/pcb302 was cultivated on lb (luria-bertani) medium, ph 6.9. bacterial suspension (300 µl) and 100 mg/l of kanamycin were added into a liquid lb medium and cultivated for 12 hours on a shaker (shaker dos-20l; 120 rpm) in the dark at 23 °c. bacterial transformation segments of c. europaea stems (7–8 mm shoot apices) were infected by a. tumefaciens/pcb302 in the liquid lb medium for 10 min and subsequently co-cultivated on ms medium without antibiotics overnight in the dark at 23±2 °c. infected segments were washed by a liquid ms medium containing cefotaxime (200 mg/l). after drying on a sterile paper, explants were grown on a selective ms medium containing agar and antibiotics – kanamycin (100 mg/l) and cefotaxime (200 mg/l). they were inoculated on fresh selective media in one week sub-cultivation intervals. to monitor plant growth, shoot lengths were measured every week for six weeks of in vitro cultivation. microscopy of dodders light microscopy of c. europaea seedlings was carried out using a stereomicroscope (leica m165 fc). gfp positive dodder stem segments were examined by a confocal microscope(olympus fv1000) at an excitation wavelength 488 nm. gfp fluorescence and plastid autofluorescence were detected using 505 – 550 nm and 655 – 755 nm barrier emission filters, respectively. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:36 utc nova biotechnol chim (2017) 16(1): 20-25 22 results and discussion technical advances in transformation techniques have facilitated research in parasitic plants, too. transgenic dodder calli or roots derived from a. rhizogenes-mediated transformations still were able to form lateral haustoria that penetrated the host (fernández-aparicio et al. 2011; ishida et al. 2011). however, stable and heritable transformation of cuscuta plants, enabling molecular genetics studies of these species, have not been established yet, in part due to challenges in developing transformation protocols, but also because regeneration of a parasitic organism in vitro is difficult (borsics et al. 2002; švubová and blehová 2013). since the c. europaea seedlings germinate as cotyledonless and leafless shoots with ephemeral root-like structure, transformation of shoot apex can be used as an alternative for agrobacterium-mediated transformation of monocotyledonous crop species (gould et al. 1991; this study). fig. 1. growth and development of c. europaea seedlings in vitro. a. germinating dodder seeds on ms medium on 1st day after germination. b. germinating seed with tuberous radicle and root tip (circle). c. detail of the root tip (circle) with hairs. d. dodder leafless stem with shoot apex (rectangle) on 7th day after germination. e. early degradation (7th day after germination; asterisks) of root-like structure with visible xylem (arrow). f. detail of the degrading rootlike structure (asterisks) on 7th day after germination. g. progressing degradation (asterisks) of the root-like structure on 14th day after germination, xylem (arrow). h. detailed view on root-like structure in the late stage of degradation (asterisks) on 14th day after germination. scale bars: a = 2 cm; b, d, e, g = 2 mm; c, f, h = 0.5 mm. dodder seeds were transferred to in vitro conditions. according to some authors, stratification by cold and scarification are essential for their successful germination (švubová and blehová 2013; furuhashi et al. 2016). variability of seed coat structure among different dodder species determines their germination characteristics impacting adaptation to natural environment (behdarvandi et al. 2015). the seeds started to germinate 2–3 days after sowing (fig. 1a). a visibly swollen radicle of c. europaea emerged from the seeds as the first structure (fig. 1a and b) and this was followed by the emergence of a stem (fig. 1d). since the first day after germination, differentiation of root hairs at the radicle tip was observed (fig. 1c). in contrast, in the case of c. campestris, the bulges resembling root hairs are absent and the surface of the radicle-like structure is smooth (lyshede 1985). according to sherman et al. (2008), these root hairs do not elongate and have a different ultrastructure compared to the root hairs of typical dicots. in addition, the root-like structure of dodder is considered to be a remnant of the true shoot that was significantly modified during evolution (sherman et al. 2008). for example, in c. europaea it lacks both root cap and root apical meristem (fig. 1b and c) as observed by other authors (lyshede 1986; lee et al. 2000; behdarvandi et al. 2015). several days after germination this root-like organ degrades through a senescence-like process from apex towards the stem base (fig. 1e – h), possibly due to low abundance of microtubules in its cells and lack of mitotic division (sherman et al. 2008). nevertheless, this structure is still capable of interacting with microorganisms, therefore behdarvandi et al. (2015) consider this organ to be a modification of true root. moreover, xylem strands with non-lignified cell walls were clearly visible even under a light microscope (fig. 1e and g) indicating to functional translocation of some nutrients into the rapidly growing shoot (sherman et al. 2008). the development of c. europaea plants shows a remarkable degree of plasticity to a large extent attributed to shoot apical meristem. in contrast to the pale root-like structure c. europaea stem tips are green (fig. 1b and d). several photosynthesisrelated proteins have been described in dodder stems (e.g. rbcl, thf1, d1) indicating that bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:36 utc nova biotechnol chim (2017) 16(1): 20-25 23 fig.2. lengths of dodder shoot explants during 35 days of in vitro cultivation. gfp positive (full line) shoot explants transformed by a. tumefaciens/pcb302. dot dotted line represents the length of control shoot explants. dodders in this non-parasitic developmental stage are photosynthetically active (švubová et al. 2013). in addition, they contain much more actin and microtubule elements compared to roots (sherman et al. 2008) enabling rapid growth of dodder shoot explants on hormone free ms media (fig. 2). dynamic reorganization of the cytoskeleton in combination with photosynthetic activity of young dodder seedlings is likely responsible for ability to survive without host plants for up to 3 weeks (heide-jørgensen 2008). transformation of excised shoot explants was performed using the construct pcb302 which has successfully been used for actin visualization in transgenic arabidopsis plants (voigt et al. 2005; wang et al. 2007). after transformation, dodder tissue was cultivated and its growth was monitored. we observed strong gfp fluorescence, especially in the shoot tips (fig. 3a). since the marker protein (gfp) was fused to actin-binding protein, higher magnification images of cells in the transformed shoot’s apical zone, revealed a network of fine f-actin surrounding the nuclei (fig. 3b) as described previously for other plant species (chiu et al. 1996; wang et al. 2007). however, fig. 3. gfp fluorescence of c. europaea shoot explants analysed by confocal microscopy. a) gfp positive dodder shoots on 7th day after transformation, arrow indicates stronger gfp positive signal in the meristematic region of the shoot apex. b) gfp fluorescence of the stem tip epidermal cells. c) the positive signal in the cells of elongation zone on 7th day after transformation. d) the elongating cells with lowered gfp fluorescence on 14th day after transformation. e) autofluorescence of gfp negative dodder stem on 35th day after transformation. f) autofluorescence of control stem of non-transformed c. europaea. scale bars: a, f = 200 µm; b, c, d = 50 µm; e = 25 µm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:36 utc nova biotechnol chim (2017) 16(1): 20-25 24 in the distal elongation zone the gfp fluorescence turned considerably weaker (fig. 3c–e). this observation is consistent with significantly reduced growth of the transformed dodder stems in comparison with the control explants (fig. 2). nevertheless, the exact reason for this phenomenon is unclear and might also include problems with the transformation procedure (e.g. gradual degradation of un-incorporated t-dna, gradual silencing of the transgene expression, recalcitrant dodder genotype etc.) or failing of the binding of the fusion protein in the tissue, or both. nevertheless, fluorescence of the transgene product in dodder tissue (cultivated in presence of selection agent for 7–14 days), reflects to transient transgene expression that allows for non-invasive observation of cytoskeleton organisation in developing shoot tissue of the parasite. the presence and activity of the transgene in transformed dodder tissue needs to be verified in more detail at molecular level. further, since dodder shoots appeared as gfp positive only for a relatively short period of time, further optimization of the transformation procedure using e.g. sonication followed by vacuum treatment (fernández-aparicio et al. 2011). enhancement of assays for transient gene expression will probably involve optimization of factors promoting t-dna transfer e.g. over-expression of bacterial virulent vire and virg genes can enhance transient expression (wroblewski et al. 2005). also vird that probably plays a major role in targeting tstrands to the nucleus through α importins localized in nuclear envelope (gelvin 2012). in addition, it is also beneficial to apply agrobacterium strains that carry specific p19 helper plasmids as silencing suppressors (voinnet et al. 2003; canto et al. 2006), what will be a challenge for future. conclusions the shoot of germinating c. europaea contains a functional apical meristem and grows quickly comparing to the root-like structure, therefore shoot apex was used here for genetic transformation via a. tumefaciens. the fluorescence pattern of the marker gene product suggests only a transient transformation. fusion of gfp with actin-binding element appeared as feasible for not only confirming the presence of transgene product in host tissue but also for studying the organization and of cytoskeleton in the vital dodder tissue. this is the first report on (transient) transgene expression in vegetative tissue of parasitic dodder. acknowledgements this work was supported by the slovak research and development agency under the contract no. apvv-160051. references baluška f, šamaj j, wojtaszek p, volkmann d, menzel d (2003) cytoskeleton plasmamembrane-cell wall continuum in plants. emerging links revisited. plant physiol 133: 482-491. behdarvandi b, guinel fc, costea m (2015) differential effects of ephemeral colonization by arbuscular mycorrhizal fungi in two cuscuta species with different ecology. mycorrhiza 25: 573-585. blehová a, švubová r, lukáčová z, moravčíková j, matušíková i (2015) transformation of sundew: pitfalls and promises. plant cell tiss. org. cult. 120: 681-687. borsics t, mihálka v, oreifig as, bárány i, lados m, nagy i, jenes b, told o (2002) methods for genetic transformation of the parasitic weed dodder (cuscuta trifolii bab. et gibs) and for pcr-based detection of early transformation events. plant sci. 162: 193-199. canto t, uhrig jf, swanson m, wright km, macfarlane sa (2006) translocation of tomato bushy stunt virus p19 protein into the nucleus by aly proteins compromises its silencing suppressor activity. j. virol. 80: 9064-9072. chiu w, niwa y, zeng w, hirano t, kobayashi h, sheen j (1996) engineered gfp as a vital reporter in plants. curr. biol. 6: 325-330. costea m, garcía ma, stefanović s (2015) a phylogenetically based infrageneric classification of the parasitic plant genus cuscuta (dodders, convolvulaceae). syst. bot. 40: 269-285. fernández-aparicio m, rubiales d, bandaranayake pcg, yoder ji, westwood jh (2011) transformation and regeneration of the holoparasitic plant phelipanche aegyptica. plant methods 7: 36-46. furuhashi t, nakamura t, iwase k (2016) analysis of metabolites in stem parasitic plant interactions: interaction of cuscuta – momordica versus cassytha – ipomoea. plants 5: 43-57. garcía ma, costea m, kuzmina m, stefanović s (2014) phylogeny, character evolution, and biogeography of cuscuta (dodders; convolvulaceae) inferred from coding plastid and nuclear sequences. am. j. bot. 101: 670-690. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:36 utc nova biotechnol chim (2017) 16(1): 20-25 25 gelvin sb (2012) traversing the cell: agrobacterium t-dna´s journey to the host genome. front plant sci. 3: 52-62. gould j, devey m, hasegawa o, ulian ec, peterson g, smith rh (1991) transformation of zea mays l. using agrobacterium tumefaciens and the shoot apex. plant physiol. 95: 426-434. heide-jørgensen hs (2011) parasitic plants. in: simberloff d, rejmánek m (eds.) encyclopedia of biological invasions. university of california press, berkeley and los angeles, 504-510. heide-jørgensen h (2008) parasitic flowering plants. 1st ed. danvers, ma 01923, usa, 438 p. ichihashi y, mutuku jm, yoshida s, shirasu k (2015) transcriptomics exposes the uniqueness of parasitic plants. brief. funct. genomics 14: 275-282. ishida jk, yoshida s, ito m, namba s, shirasu k (2011) agrobacterium rhizogenes-mediated transformation of the parasitic plant phtheirospermum japonicum. plos one 6: e25802. kaštier p, švubová r, fiala r, blehová a (2015) organization of the cytoskeleton in various stages of cuscuta europaea haustorium development. in: proceedings of 3rd international conference: green for good. olomouc: palacky university of plant biotechnology, 15.6.-18.6.2015, p. 64. lee kb, park jb, lee s (2000) morphology and anatomy of mature embryos and seedlings in parasitic angiosperm cuscuta japonica. j. plant biol. 43: 22-27. lyshede ob (1985) morphological and anatomical features of cuscuta pedicellata and c. campestris. nord. j. bot. 5: 65-77. lyshede ob (1986) fine-structural features of the tuberous radicular end of the seedling of cuscuta pedicellata. ber. deutsch. bot. ges. bd. 99: 105-109. muday g (2000) interactions between the actin cytoskeleton and an auxin transport protein. in: staiger cj, baluska f, volkmann d, barlow p (eds.) actin: a dynamic framework for multiple plant cell functions. kluwer academic press, dordrecht, netherlands, 541-556. murashige t, skoog f (1962) a revised medium for rapid growth and bioassay with tobacco tissue cultures. physiol. plant. 15: 473-497. sherman td, bowling aj, barger tw, vaughn kc (2008) the vestigial root of dodder (cuscuta pentagona) seedlings. int. j. plant sci. 169: 998-1012. šamaj j, müller j, beck m, bohm n, menzel d (2006) vesicular trafficking, cytoskeleton and signalling in root hairs and pollen tubes. trends plant sci. 11: 594-600. švubová r, blehová a (2013) stable transformation and actin visualization in callus cultures of dodder (cuscuta europaea). biologia 68: 633-640. švubová r, ovečka m, pavlovič a, slováková ľ, blehová a (2013) cuscuta europaea plastid apparatus in various developmental stages: localization of thf1 protein. plant signal. behav. 8: e24037. voigt b, timmers acj, šamaj j, müller j, baluška f, menzel d (2005) gfp-fabd2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of arabidopsis seedlings. eur. j. cell biol. 84: 595608. voinnet o, rivas s, mestre p, baulcombe d (2013) an enhancement transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. plant j. 33: 949956. wang y-s, yoo c-m, blancaflor eb (2007) improved imaging of actin filaments in transgenic arabidopsis plants expressing a green fluorescent protein fusion to the cand n-termini of the fibrin actin-binding domain 2. new phytol. 177: 525-536. wasteneys go, ambrose jch (2009) spatial organization of plant cortical microtubules: close encounters of the 2d kind. trends cell biol. 19: 62-71. wroblewski t, tomczak a, michelmore r (2005) optimization of agrobacterium-mediated transient assays of gene expression in lettuce, tomato and arabidopsis. plant biotechnol. j. 3: 259-273. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:36 utc nova biotechnol chim (2018) 17(1): 29-37 doi: 10.2478/nbec-2018-0003  corresponding author: dmakris@aegean.gr nova biotechnologica et chimica the effect of 2-hydroxypropyl β-cyclodextrin on the stability of polyphenolic compounds from moringa oleifera lam leaf extracts in a natural low-transition temperature mixture ioanna karageorgou1,2, spyros grigorakis3, stavros lalas2 and dimitris p. makris1, 1school of environment, university of the aegean, mitr. ioakim street, myrina – 81400, lemnos, greece 2department of food technology, technological educational institute (t.e.i.) of thessaly, n. temponera street, karditsa – 43100, greece 3food quality & chemistry of natural products, mediterranean agronomic institute of chania (m.a.i.ch.), international centre for advanced mediterranean agronomic studies (ciheam), p.o. box 85, chania – 73100, greece article info article history: received: 3rd april 2018 accepted: 23rd may 2018 keywords: antiradical activity 2-hydroxypropyl β-cyclodextrin low-transition temperature mixtures moringa oleifera polyphenols abbreviations: hba, hydrogen bond acceptor hbd, hydrogen bond donor hp-β-cd, 2-hydroxypropyl β-cyclodextrin lttm, low-transition temperature mixture mol, m. oleifera leaves abstract polyphenol extracts from moringa oleifera leaves (mol) were obtained with a glycerol-based low-transition temperature mixture (lttm) and a combination of lttm with 2-hydroxypropyl β-cyclodextrin (hp-β-cd). the extracts were maintained at 4, 22 and 50 °c for 18 days and the antiradical activity (aar) was recorded to detect modifications in the antioxidant activity of the extracts. aar displayed a constant decline at every temperature tested, following pseudo firstorder kinetics and the decay constants suggested that the presence of hp-β-cd had a protective action, slowing down aar decline. the analysis of the polyphenolic profiles using liquid chromatography-diode array-mass spectrometry revealed that after storage for 18 days at 50 °c, the major quercetin glycosides occurring in mol were extensively degraded. based on the detection of protocatechuic acid in the stored extracts, putative pathways of flavonol glycoside degradation were proposed. it was concluded that the decomposition of these components was mainly responsible for the aar decline observed.  university of ss. cyril and methodius in trnava introduction plants have played a central role in maintaining human health for millennia. herbs have been widely used all over the world as food and medicines and nowadays research has focused on various botanicals that possess antitumor, antiinflammatory and immune-stimulating properties, which may contribute in reducing the risk of cardiovascular disease and cancer. in different herbs, a broad variety of active phytochemicals, including simple phenolics and flavonoids have been identified as the major active constituents and numerous methodologies have been developed for the effective preparation of polyphenolcontaining extracts. moringa oleifera leaves (mol) is a tissue that displays several bioactivities beneficial to human health (farooq et al. 2012) and possess a relatively high richness in bioactive polyphenols (lalas et al. 2017). for this reason, there have been some efforts pertaining to the efficient polyphenol extraction, by employing green extraction media, such as water/ethanol mixtures (wang et al. 2017). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:30 utc nova biotechnol chim (2018) 17(1): 29-37 30 however, a more recent study demonstrated a particularly high effectiveness in polyphenol extraction, using a novel, natural low-transition temperature mixture (lttm), composed of glycerol and sodium acetate (karageorgou et al. 2017). following examinations also demonstrated that combination of this lttm with hydroxypropyl β-cyclodextrin (hp-β-cd) may boost extraction, achieving even higher yields (karageorgou et al. 2018). an important issue associated with extraction of natural substances is their stability in the extraction medium over time. since many polyphenols are inherently unstable molecules, owed to their susceptibility to oxidation, the examination of their stability under specific conditions is imminent and requires detailed examination. on such a ground, this examination was undertaken to investigate the stability of mol polyphenols in extracts generated using the abovementioned extraction medium, composed of the lttm and hp-β-cd. stability in the presence and absence of hp-β-cd was assessed by monitoring the antiradical activity (aar) of the extracts over 18 days at various temperatures, and polyphenol transformations that might be linked with changes in aar were identified using liquid chromatography-diode array-mass spectroscopy. experimental plant material dried and pulverised moringa oleifera leaves (mol), with average particle diameter of 0.5 mm, were used. collection and handling of the plant material have been previously described in detail (karageorgou et al. 2017). all treatments were repeated at least twice. chemicals and reagents glycerol (99 %), 2-hydroxypropyl β-cyclodextrin (98 %) and ethanol (99.8 %) were from acros organics (geel, belgium). anhydrous sodium carbonate was from carlo erba reactifs (val de reuil, france). sodium acetate trihydrate was from penta (prague, czeck republic). folin-ciocalteu reagent was from fluka (steinheim, germany). 2,2-diphenyl-1-picrylhydrazyl radical (dpph) was from aldrich (steinheim, germany). all solvents used for liquid chromatography were hplc grade. preparation of the lttm the lttm used was prepared using the optimised conditions described previously (karageorgou et al. 2017). glycerol (hbd) was mixed with an appropriate amount of sodium acetate (hba) to give a molar ratio hbd:hba of 6:1, and the mixture was moderately heated under stirring until a transparent liquid was formed. aqueous solution 80 % (w/v) of this lttm was used for the extractions. batch extraction procedure and sample handling extraction of mol polyphenols was performed with a previously developed optimized methodology (karageorgou et al. 2018). accurately weighted 2.5 g of dried powdered material was mixed with 100 ml of 80 % (w/v) aqueous lttm containing 1 % (w/v) 2-hydroxypropyl β-cyclodextrin (hp-β-cd), to give a liquid-to-solid ratio of 40 ml g-1. extractions were carried out at 50 °c, under continuous stirring at 600 rpm, for 180 min. extractions without hp-β-cd were also performed, under the same conditions. following extraction, samples were centrifuged in a table centrifuge (hermle, wehingen, germany) at 10,000 × g for 10 min, and the clear extract was used for all subsequent procedures. stability test the centrifuged extracts were divided into 30-ml portions in screw-cap glass vials and stored in a freezer (4 °c), in a dark temperature-controlled chamber (22 °c) and in a thermostated water bath (50 °c). sampling was carried out at regular intervals, within 18 days. determinations total polyphenols were determined with the folinciocalteu methodology and expressed as gallic acid bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:30 utc nova biotechnol chim (2018) 17(1): 29-37 31 equivalents. antiradical activity (aar) was estimated with the dpph and expressed as μmol dpph per g dry weight (paleologou et al. 2016). qualitative liquid chromatography-diode arraymass spectrometry (lc-dad-ms) for the analyses, a finnigan mat spectra system p4000 pump, a uv6000lp diode array detector and a finnigan aqa mass spectrometer were employed. chromatography was performed on a fortis rp-18 column, 150 × 2.1 mm, 3 μm, at 40 °c. details of the analytical procedure were given elsewhere (paleologou et al. 2016). statistics at least two experiments were carried out for the extractions and stability tests. analyses were performed in triplicate and values given are means. linear regressions were established at least at a 95 % significance level. for all statistics, microsoft excel™ 2010 and sigmaplot™ 10 were used. results and discussion kinetics of aar evolution and the effect of hp-β-cd to appraise stability of the extracts at various temperatures and to illustrate the role of hp-β-cd, aar evolution was traced over 18-days period. aar was chosen as a safe criterion to detect modifications of extracts status, as it is linked with polyphenol structure (huang et al. 2005) and therefore it was hypothesised that any polyphenol modification would be readily reflected on the aar. it was revealed that aar showed a declining tendency, described by first-order kinetics (eq. 1): aar(t) = aar(0) e-kt (1) where aar(t) is the aar at any time t; aar(0) the initial aar value and k the first-order decay constant. the linearised form of eq. 1 is given as follows (eq. 2): ln(aar(t)) = ln(aar(0)) – kt (2) rearrangement of eq. 2 would give (eq. 3): = – kt (3) thus k (days-1) could be determined graphically from the slope of the straight line obtained after plotting against t. the half-life of aar decrease could then be estimated as shown below (eq. 4): t½ = – (4) fig. 1. first-order kinetics of aar decay in mol extracts stored at 4, 22 and 50 °c. lttm with (rectangles) and without 2-hydroxypropyl β-cyclodextrin (cd) (circles). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:30 utc nova biotechnol chim (2018) 17(1): 29-37 32 table 1. kinetic parameters estimated for aar decay in mol extracts. as can be seen in fig. 1 and table 1, kinetics at 4 °c was identical for extracts stored either in the presence or in the absence of hp-β-cd. however, differentiations appeared upon storage at 22 and 50 °c, where aar exhibited apparently higher stability in the presence of hp-β-cd. from the data given in table 1, it was illustrated that the pseudo-first order decay constant for the aar increased by almost 22 % from 4 to 50 °c, in the extracts containing hp-β-cd. to the contrary, the increase in the extracts lacking hp-β-cd was approximately 34 %. this outcome highlighted the role of hp-β-cd in aar stability. bibliographic data on polyphenol stability in des are scarce and therefore no concrete conclusions could be drawn. regarding green tea polyphenols, it was recently demonstrated that they had better stability in a des than in conventional solvents, including aqueous mixtures of methanol and ethanol (jeong et al. 2017). no clear explanation for such a phenomenon has been provided, but it could be presumed that hydrogen bonds developed between polyphenols and the hba could act in a stabilizing manner, since polyphenols may act as hbds (bi et al. 2013). however, polyphenol instability in a medium such as the des used in this study may stem from the intrinsic basicity of the solvent, owed to the presence of the acetate anion. at this point, the presence of hp-β-cd might be crucial. quercetin glycosides and other flavonol aglycones may form complexes with cyclodextrins, such as β-cyclodextrin (β-cd) and hp-β-cd (alvarez-parilla et al. 2005; jullian et al. 2007; lucas-abellán et al. 2008) and it has been demonstrated that flavonoid/hp-β-cd complexes provided improved stability of the encapsulated molecule, as opposed to the free (non-encapsulated) one (nguyen et al. 2013). such a finding was ascribed to the protective effect of hp-β-cd cavity (liu et al. 2006). studies on polyphenol-containing extracts (mourtzinos et al. 2008; kalogeropoulos et al. 2010) were in the same line. investigations on rosmaric acid inclusion complexes by various cyclodextrins (çelik et al. 2011) gave an explanation concerning the correlation of polyphenol stability with enhanced antioxidant activity. it has been proposed that once a polyphenol radical is formed and engulfed in hp-β-cd hydrophobic cavity, it could be better stabilised through resonance, by intramolecular hydrogen bonding. as a consequence, the redox potential between the aroxyl radical and the reduced polyphenol may be lowered, endowing the polyphenol higher antioxidant activity. indeed, several examinations showed that polyphenols such as chlorogenic acid (shao et al. 2014), quercetin and rutin (alvarez-parilla et al. 2005), rosmarinic acid (çelik et al. 2011; medronho et al. 2014), and quercetin and glycosides therof (çelik et al. 2015), encapsulated in cyclodextrins may behave as more potent antioxidants, compared with the non-encapsulated molecules. thus, the slower aar decay recorded for mol extracts in the presence of hp-β-cd could be owed to effective inclusion and higher polyphenol stability. modifications in the polyphenolic profile to ascertain if alterations in the polyphenolic profile were responsible for the changes in aar observed, lc-dad-ms was performed for the samples stored at 50 °c, which had the highest decay rate. in the chromatogram traced at 340 nm for the initial extract obtained with the lttm/hp-β-cd (fig. 2, upper chromatogram), eight polyphenolic substances could be tentatively identified. the extract obtained only with the solvent temperature [°c] 4 22 50 k t½ k t½ k t½ lttm + cd 0.0297 23.3 0.0344 20.1 0.0379 18.3 lttm 0.0297 23.3 0.0351 19.7 0.0448 15.5 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:30 utc nova biotechnol chim (2018) 17(1): 29-37 33 lttm (no hp-β-cd) had identical profile (data not shown). this was confirmation of previous results, which demonstrated that hp-β-cd acted merely by boosting extraction yield. based on published data, peak #1 was identified as chlorogenic acid, while peaks #2 and 3 as chlorogenate derivatives (table 2). peak #4 was assigned to multiflorin b, peaks #5 – 7 to quercetin glycosides and peak #8 to a kaempferol glycoside (karageorgou et al. 2017). when the extracts were analysed after 18 days of storage at 50 °c, it was revealed that the polyphenolic profile was fundamentally altered (fig. 2, lower chromatogram). peaks #1 and 2 completely disappeared, while peaks #5 – 8 suffered extensive degradation. however, this modification did not result in glycoside hydrolysis, as no quercetin or kaempferol aglycones were detected. this finding led to the conclusion that decomposition of these substances would have involved cleavage of the flavonoid skeleton. to ascertain such a hypothesis, chromatograms were also recorded at 280 nm, to detect the formation of compounds that could have emerged through quercetin or kaempferol degradation. indeed, two products associated with quercetin oxidative degradation were detected, protocatechuic acid (pa) and the depside 2-(3,4-dihydroxybenzyloxy)-4,6-dihydroxybenzoic acid, termed as quercetin oxidation product (qop) (fig. 3). these compounds did not occur in the initial (untreated) extract and consisted sound evidence of quercetin decomposition. in fact, pa may derive from quercetin via thermal degradation (makris and rossiter 2000), free-radical oxidation (makris and rossiter 2002) and enzyme-mediated oxidative cleavage (osman et al. 2008). however, the generation of qop would require the existence fig. 2. chromatogram of mol extracts before (day 0) and after storage at 50 °c (day 18). the chromatograms were monitored at 340 nm. peak assignment is as in the table 2. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:30 utc nova biotechnol chim (2018) 17(1): 29-37 34 table 2. uv-vis and mass spectrometry data for the main polyphenols and some degradation products thereof, detected in the extracts of m. oleifera leaves. peak rt [min] uv-vis [m + h]+ [m/z] other ions (m/z) tentative identity 1 14.90 244, 318 355 377[m + na]+, 163 chlorogenic acid 2 18.88 246, 320 513 427, 377, 355, 163 chlorogenate derivative 3 19.50 242, 318 703 733[m + h + meoh]+, 695, 545, 409 chlorogenate derivative 4 22.12 270, 340 595 617[m + na]+, 457, 379 apigenin rutinoside (multiflorin b) 5 26.90 256, 356 465 487[m + na]+, 303 quercetin glucoside or galactoside 6 27.82 256, 360 551 573[m + na]+, 303 quercetin glycoside 7 28.46 266, 346 877 791, 551, 303 quercetin rhamnoside derivative 8 29.58 264, 350 535 577[m + na]+, 287 kaempferol malonylglucoside pa 12.38 258, 292 153* protocatechuic acid qop 15.73 260, 296 305* 153 quercetin oxidation product * negative ionisation of a quercetin dioxygenated derivative and therefore it was suspected that the oxidative degradation followed the pathway proposed for room temperature quercetin autoxidation (zenkevich et al. 2007). initially, quercetin hydrolysis may occur giving rise to quercetin aglycone. following this step, quercetin radicals may be formed, most probably due to the presence of transition metal ions (e.g. fe or cu), which then may react with the di-radical o2 (fig. 4). fig. 3. chromatogram of mol extracts before (day 0) and after storage at 50 °c (day 18). the chromatogram was monitored at 280 nm. peak assignment is as in the table 2. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:30 utc nova biotechnol chim (2018) 17(1): 29-37 35 fig. 4. putative mechanism of quercetin glucoside degradation, resulting in the formation of pa and qop. after a cascade of reactions leading to the depside 2-(3,4-dihydroxybenzyloxy)-4,6-dihydroxybenzoic acid, pa may be formed through depside hydrolysis. the fact that extracts stored at 50 °c for 18 day displayed identical profile irrespective of the hp-β-cd presence (data not shown) indicated that hp-β-cd did not interfere with the oxidation mechanism, but it merely acted as protective agent retarding polyphenol degradation, as shown by the kinetic investigation. on the other hand, the clear evidence regarding flavonol degradation involving cleavage of the flavonoid skeleton was a strong background to justify aar decline of mol extracts. this is because quercetin degradation products including pa and phloroglucinol carboxylic acid have been proven to be far less effective antioxidants than the parent molecule (makris and rossiter 2001). therefore, oxidative decomposition of the quercetin glycosides in mol extracts inevitably led to loss of aar. conclusions lttms are novel solvents of unpredictable extraction behaviour and thus their characterisation in this regard is a matter of case experimentation. in the extraction of antioxidant polyphenols using lttms their stability is of high significance because polyphenols may undergo reactions, such as hydrolysis and oxidation. the polyphenolic profile of mol extracts, obtained with either bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:30 utc nova biotechnol chim (2018) 17(1): 29-37 36 lttm/hp-β-cd or only lttm, was demonstrated to be subject to modifications when stored for 18 days at 50 °c, characterised by flavonol glycoside degradation, which included cleavage of the flavonoid skeleton. given that flavonoids were shown to be far superior antioxidants than their degradation products, the decline in aar seen in the stored mol extracts could be attributed to the disappearance of these substances. however, the kinetic study showed that the incorporation of hp-β-cd in the extraction solvent may provoke significant decrease in aar decay. this outcome emphasized the importance of hp-β-cd for polyphenol stability in lttms, but additional examinations are needed to illustrate the mechanisms implicated. this is anticipated to lead in more effective hp-β-cd use in foods, pharmaceuticals and cosmetics. nomenclature aar, antiradical activity [μmol dpph g -1; dw] aar(0), initial antiradical activity [μmol dpph g -1; dw] aar(t), antiradical activity at time t [μmol dpph g -1; dw] k, pseudo first-order decay constant [days-1] t, time [days] t½, half-time [days] references alvarez-parrilla e, laura a, torres-rivas f, rodrigo-garcia j, gonzález-aguilar ga (2005) complexation of apple antioxidants: chlorogenic acid, quercetin and rutin by β-cyclodextrin (β-cd). j. inclus. phenom. macrocycl. chem. 53: 121-129. bi w, tian m, row kh (2013) evaluation of alcohol-based deep eutectic solvent in extraction and determination of flavonoids with response surface methodology optimization. j. chrom. a 1285: 22-30. çelik se, özyürek m, güçlü k, apak r (2015) antioxidant capacity of quercetin and its glycosides in the presence of β-cyclodextrins: influence of glycosylation on inclusion complexation. j. inclus. phenom. macrocycl. chem. 83: 309-319. çelik se, özyürek m, tufan an, güçlü k, apak r (2011) spectroscopic study and antioxidant properties of the inclusion complexes of rosmarinic acid with natural and derivative cyclodextrins. spectrochim. acta part a: mol. biomol. spectr. 78: 1615-1624. farooq f, rai m, tiwari a, khan aa, farooq s (2012) medicinal properties of moringa oleifera: an overview of promising healer. j. med. plants res. 6: 4368-4374. huang d, ou b, prior rl (2005) the chemistry behind antioxidant capacity assays. j. agric. food chem. 53: 1841-1856. jeong km, ko j, zhao j, jin y, yoo de, han sy, lee j (2017) multi-functioning deep eutectic solvents as extraction and storage media for bioactive natural products that are readily applicable to cosmetic products. j. clean. prod. 151: 87-95, jullian c, miranda s, zapata-torres g, mendizábal f, oleaazar c (2007) studies of inclusion complexes of natural and modified cyclodextrin with (+) catechin by nmr and molecular modeling. bioorg. med. chem. 15: 32173224. kalogeropoulos n, yannakopoulou k, gioxari a, chiou a, makris dp (2010) polyphenol characterization and encapsulation in β-cyclodextrin of a flavonoid-rich hypericum perforatum (st john's wort) extract. lwtfood sci. technol. 43: 882-889. karageorgou i, grigorakis s, lalas s, makris dp (2017) enhanced extraction of antioxidant polyphenols from moringa oleifera lam. leaves using a biomoleculebased low-transition temperature mixture. eur. food res. technol. 243: 1839-1848. karageorgou i, grigorakis s, lalas s, mourtzinos i, makris dp (2018). incorporation of 2-hydroxypropyl β-cyclodextrin in a biomolecule-based low-transition temperature mixture (lttm) boosts efficiency of polyphenol extraction from moringa oleifera lam leaves. j. applied res. med. arom. plants, 9: 62-69. lalas s, athanasiadis v, karageorgou i, batra g, nanos gd, makris dp (2017) nutritional characterization of leaves and herbal tea of moringa oleifera cultivated in greece. j. herbs spices med. plants: 24: 320-333. liu j, qiu l, gao j, jin y (2006) preparation, characterization and in vivo evaluation of formulation of baicalein with hydroxypropyl-β-cyclodextrin. inter. j. pharm. 312: 137-143. lucas-abellán c, fortea m, gabaldón j, núñez-delicado e (2008) complexation of resveratrol by native and modified cyclodextrins: determination of complexation constant by enzymatic, solubility and fluorimetric assays. food chem. 111: 262-267. makris dp, rossiter jt (2000) heat-induced, metal-catalyzed oxidative degradation of quercetin and rutin (quercetin 3-o-rhamnosylglucoside) in aqueous model systems. j. agric. food chem. 48: 3830-3838. makris dp, rossiter jt (2001) comparison of quercetin and a non-orthohydroxy flavonol as antioxidants by competing in vitro oxidation reactions. j. agric. food chem. 49: 3370-3377. makris dp, rossiter jt (2002) hydroxyl free radicalmediated oxidative degradation of quercetin and morin: a preliminary investigation. j. food compos. anal. 15: 103-113. medronho b, valente aj, costa p, romano a (2014) inclusion complexes of rosmarinic acid and cyclodextrins: stoichiometry, association constants, and antioxidant potential. colloid polymer sci. 292: 885-894. mourtzinos i, makris dp, yannakopoulou k, kalogeropoulos n, michali i, karathanos vt (2008) thermal stability of anthocyanin extract of hibiscus sabdariffa l. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:30 utc nova biotechnol chim (2018) 17(1): 29-37 37 in the presence of β-cyclodextrin. j. agric. food chem. 56: 10303-10310. nguyen ta, liu b, zhao j, thomas ds, hook jm (2013) an investigation into the supramolecular structure, solubility, stability and antioxidant activity of rutin/cyclodextrin inclusion complex. food chem. 136: 186-192. osman a, makris dp, kefalas p (2008) investigation on biocatalytic properties of a peroxidase-active homogenate from onion solid wastes: an insight into quercetin oxidation mechanism. process biochem. 43: 861-867. paleologou i, vasiliou a, grigorakis s, makris dp (2016) optimisation of a green ultrasound-assisted extraction process for potato peel (solanum tuberosum) polyphenols using bio-solvents and response surface methodology. biomass conver. bioref. 6: 289-299. shao p, zhang j, fang z, sun p (2014) complexing of chlorogenic acid with β-cyclodextrins: inclusion effects, antioxidative properties and potential application in grape juice. food hydrocol. 41: 132-139. wang y, gao y, ding h, liu s, han x, gui j, liu d (2017) subcritical ethanol extraction of flavonoids from moringa oleifera leaf and evaluation of antioxidant activity. food chem. 218: 152-158. zenkevich ig, eshchenko ay, makarova sv, vitenberg ag, dobryakov yg, utsal va (2007) identification of the products of oxidation of quercetin by air oxygenat ambient temperature. molecules 12: 654-672. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:30 utc microsoft word malinicova proofs.doc nova biotechnologica et chimica 11-1 (2012) 1 doi 10.2478/v10296-012-0001-4 ©university of ss. cyril and methodius in trnava bioinformatic analysis of prophage endolysins and endolysin-like genes from the order lactobacillales lenka maliničová, peter pristaš, peter javorský institute of animal physiology, slovak academy of sciences, šoltésovej 4-6, košice, slovak republic (malinicova@saske.sk) abstract: endolysins belonging to the group of peptigoglycan hydrolases, which are able to cleave peptidoglycan in bacterial cell walls, become an extensively studied group of enzymes. thanks to their narrow target specificity and low probability of resistance they are considered to be an appropriate alternative to conventional antibiotics. the present paper concerns the occurrence of endolysin and endolysin-like genes in genomes of bacteria belonging to the order lactobacillales. using bioinformatic programmes we compared and analysed protein sequences of catalytic and cell wall binding (cwb) domains of these enzymes, their preferred combinations, their phylogenetic relationship and potential occurence of natural „domain shuffling“. the existence of this phenomenon in selected group of enzymes was confirmed only in limited range, so we assume that the natural trend is the distribution of „well-tried“ combinations of catalytic and cwb domains of endolysin genes as a whole. key words: endolysin, lactobacillales, bioinformatic analysis, domain shuffling 1. introduction bacterial peptidoglycan hydrolases (pghs) form a vast and highly diverse group of enzymes of different origin capable of cleaving bonds in polymeric peptidoglycan, which is a major component of the bacterial cell envelope in both gram-positive and gram-negative bacteria (vollmer et al., 2008; humann and lenz, 2009). activity of these enzymes results in a cell wall disruption and consequent bacterial cell lysis. one subgroup of pghs are endolysins, which are encoded in genomes of bacteriophages and expressed in the host cells to digest the bacterial cell wall for bacteriophage progeny release at the terminal stage of the phage reproduction cycle. endolysins as well as the most of other pghs are composed of at least two clearly separated functional domains: with few exceptions, the n-terminal domain contains the catalytic activity of the enzyme, whereas the c-terminal cell wall binding domain (cwb) directs the enzyme to a specific substrate (fischetti, 2005; loessner, 2005). according to the specific bond that is split down in the peptidoglycan, catalytic domains can be divided into several classes: (1) glycosidases (glucosaminidases and muramidases) hydrolyze β-1→4 bonds between disaccharide units of glycan chains, (2) peptidases (endopeptidases and carboxypeptidases) cleave short peptide bridges cross-linking glycan chains, and (3) amidases cleave the amide bond between glycan chains and peptide bridges (layec et al., 2008). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:40 utc 2 maliničová, l. et al. there is also variability among cwb domains given the fact that any compounds present on bacterial cell wall (peptidoglycan subunits, saccharides, proteins, lipoteichoic acid, etc.) could serve as their receptor. correct binding of whole enzyme molecule to the cell wall of sensitive bacteria requires these specific receptors. cwb domains thus offer to the enzymes a high degree of specificity, since these receptors are only found in enzyme-sensitive bacteria (loessner, 2005). one of the favourable features of endolysins is their narrow target specificity – these enzymes are uniquely specific to their host (or a small number of closely related bacteria), therefore they might represent an effective way to control specific pathogens without disturbing the normal microflora (lópez et al., 2004). although endolysin and endolysin-like genes are usually present in genomes of bacteriophages, they can also be found in bacterial genomes, whether as a result of the presence of prophages or as their residua. this work is focused on study of endolysins from the order lactobacillales, group of gram-positive lactic acid bacteria that are ubiquitous in nature. these bacteria have a role as commensals on mucosal surfaces and skin and inhabit the digestive tract of many animal species and humans (strompfová and lauková, 2004). they have also been the focus of substantial research because of their economic importance in food fermentation. some strains are used as the starter cultures in the cheese industry, other ones are used in the production of yogurt. an important problem in industrial milk fermentation is bacteriophage attack. bacteriophage infection leads to the lysis of the starter cells and thereby interrupts the fermentation of milk sugar (lactose) into lactic acid by the starter bacteria (brussow, 2001). fortunately, we can also find several favourable features of bacteriophages and enzymes encoded by their genomes (as mentioned above), which made them an interesting tool for medicine. decades of antibiotic misuse have resulted in increasing bacterial resistance to many modern antibiotics and generating potentially dangerous multiresistant strains of lactic acid bacteria. this antibiotic resistance can cause significant danger for many people with common bacterial infections (mathur and singh, 2005). infections caused by antibiotic resistant bacterial strains could be treatable by pghs, including bacteriophage endolysins. using bioinformatic analysis of protein sequences of pghs obtained from genbank database we studied natural trends in combinations of their individual domains and potential occurence of natural „domain shuffling“. 2. material and methods in the first step we searched genbank database (benson et al., 2011) for protein sequences of endolysins using pair of key words “endolysin” and “lactobacillales” (or “lysin” and “lactobacillales”). every obtained sequence we used as the query in the protein blast search (altschul et al., 1990) and this way we obtained several more sequences of endolysin-like proteins from the order lactobacillales. from all sequences obtained in the first genbank database search and subsequent protein blast search (sequences with similarity score 500 or more) we chose those with known type of both catalytic and cwb domain and this way we created a set of sequences for consequent analyses. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:40 utc nova biotechnologica et chimica 11-1 (2012) 3 cdd outputs from protein blast, graphic representation of conserved domains (marchler-bauer et al., 2011) we used for determination of protein domain boundaries. using mega 4 software and its component tree explorer (tamura et al., 2007) we performed the phylogenetic analysis, we constructed two phylogenetic trees (maximum parsimony method) of studied proteins, individually according to the sequences of catalytic and cwb domains. finally we compared these trees to verify the degree of difference that is proportional to the intensity of natural "domain shuffling". all sequences were analysed as one set regardless of their origin (whether they were obtained from phage or bacterial gemomes). 3. results and discussion domain structure of pghs represents the potential for constructing new recombinant enzymes with artificially created combinations of catalytic and cwb domains ("domain shuffling"). such approach allows one to prepare novel enzymes with desired target specificity (provided by cwb domain) and concrete catalytic activity targeting selected type of chemical bonds in the peptidoglycan (croux et al., 1993; sanz et al. 1996). in our work we analysed the selected protein sequences of endolysins and endolysin-like genes from order lactobacillales and using bioinformatic software we compared their domain structure (table 1). we identified 116 sequences from 33 bacterial species; the most of sequences was obtained from genomes of streptococcus pyogenes (23 sequences), lactobacillus reuteri (16 sequences) and enterococcus faecalis (10 sequences). on the other hand, genomes of several species (e.g. enterococcus faecium, lactobacillus helveticus or streptococcus gordonii) were especially poor of endolysin-like sequences. the results of sequence analysis revealed 9 types of catalytic domains (glycosidases: glucosaminidase, gh25_muramidase and lysozyme_like; amidases: amidase_2, amidase_3, amidase_5 and chap; peptidases: endopeptidase and scp_bacterial) and 5 types of cwb domains (the most frequent were lysm and sh3_5 domain). several analysed protein sequences contain one catalytic and two different cwb domains and vice versa. the most preferred domain combinations were glycosidase catalytic domain with lysm (∼35%), glycosidase catalytic domain with sh3_5 (25%) and amidase catalytic domain with sh3_5 (∼20%). while glycosidase and amidase domains were combined with both lysm and sh3_5 cwb domains, peptidase domains were present only in combination with lysm domain (∼17% of all sequences). we observed some differences between preferred domain combinations among individual bacterial species. enterococci for example prefer combinations of glycosidase domain with lysm or amidase domain with sh3_5; in lactococcal sequences we found only lysm domain in combination with glycosidase or peptidase domains; in contrast streptococci prefer sh3_5 domain with glycosidase or amidase domains. the greatest variability in domain combinations was observed among lactobacilli. given the small number of sequences analysed it is not clear, how significant these differences are. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:40 utc 4 maliničová, l. et al. table 1. domain structure of endolysin and endolysin-like proteins from the order lactobacillales. domains id source catalytic cwb lysm zp_02184645 carnobacterium sp. at7 amidase_3 pg_binding_1 lysm zp_02185877 carnobacterium sp. at7 amidase_2 pg_binding_1 aaa67325 enterococcus faecalis glucosaminidase lysm lysozyme_likebag12399 enterococcus faecalis amidase_5 sh3_5 np_814147 enterococcus faecalis v583 gh25_muramidase lysm np_814543 enterococcus faecalis v583 glucosaminidase lysm np_815016 enterococcus faecalis v583 amidase_2 sh3_5 np_815207 enterococcus faecalis v583 amidase_2 sh3_5 np_815299 enterococcus faecalis v583 glucosaminidase lysm np_815667 enterococcus faecalis v583 gh25_muramidase lysm np_816267 enterococcus faecalis v583 glucosaminidase lysm np_816427 enterococcus faecalis v583 gh25_muramidase lysm zp_00602624 enterococcus faecium do glucosaminidase lysm p39046 enterococcus hirae glucosaminidase lysm ay581208 enterococcus sp. phage 1 amidase_5 sh3_5 yp_194209 lactobacillus acidophilus ncfm gh25_muramidase slap yp_795546 lactobacillus brevis atcc 367 endopeptidase lysm yp_805691 lactobacillus casei atcc 334 gh25_muramidase lysm sh3_5 yp_805885 lactobacillus casei atcc 334 gh25_muramidase lysm yp_807109 lactobacillus casei atcc 334 gh25_muramidase lysm yp_001986445 lactobacillus casei bl23 gh25_muramidase lysm sh3_5 yp_001986643 lactobacillus casei bl23 gh25_muramidase lysm sh3_5 yp_001987288 lactobacillus casei bl23 gh25_muramidase lysm yp_812161 lactobacillus delbrueckii atcc baagh25_muramidase slap yp_812257 lactobacillus delbrueckii atcc baaendopeptidase lysm yp_812312 lactobacillus delbrueckii atcc baaglucosaminidase slap yp_618265 lactobacillus delbrueckii atcc 11842 gh25_muramidase slap yp_618351 lactobacillus delbrueckii atcc 11842 endopeptidase lysm yp_001843302 lactobacillus fermentum ifo 3956 gh25_muramidase lysm yp_001843358 lactobacillus fermentum ifo 3956 endopeptidase lysm yp_001844636 lactobacillus fermentum ifo 3956 glucosaminidase lysm yp_814010 lactobacillus gasseri atcc 33323 gh25_muramidase sh3_5 yp_814525 lactobacillus gasseri atcc 33323 gh25_muramidase sh3_5 yp_815282 lactobacillus gasseri atcc 33323 gh25_muramidase cpl-7 yp_001577714 lactobacillus helveticus dpc 4571 gh25_muramidase slap np_964172 lactobacillus johnsonii ncc 533 gh25_muramidase sh3_5 sh3_5 bae02832 lactobacillus plantarum gh25_muramidase lysm sh3_5 np_784445 lactobacillus plantarum wcfs1 gh25_muramidase lysm sh3_5 np_785858 lactobacillus plantarum wcfs1 gh25_muramidase lysm np_786050 lactobacillus plantarum wcfs1 glucosaminidase sh3_5 np_786398 lactobacillus plantarum wcfs1 gh25_muramidase sh3_5 np_786644 lactobacillus plantarum wcfs1 endopeptidase lysm np_964349 lactobacillus prophage lj928 gh25_muramidase sh3_5 np_965218 lactobacillus prophage lj928 gh25_muramidase sh3_5 aay86796 lactobacillus reuteri endopeptidase lysm aay86900 lactobacillus reuteri endopeptidase lysm bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:40 utc nova biotechnologica et chimica 11-1 (2012) 5 continuation of table 1. domains id source catalytic cwb aay86914 lactobacillus reuteri glucosaminidase lysm yp_001842732 lactobacillus reuteri f275 glucosaminidase lysm yp_001841599 lactobacillus reuteri jcm 1112 endopeptidase lysm yp_001842145 lactobacillus reuteri jcm 1112 endopeptidase lysm yp_001842697 lactobacillus reuteri jcm 1112 endopeptidase lysm zp_03072312 lactobacillus reuteri 100-23 gh25_muramidase sh3_5 zp_03072387 lactobacillus reuteri 100-23 gh25_muramidase lysm zp_03072513 lactobacillus reuteri 100-23 gh25_muramidase sh3_5 zp_03072609 lactobacillus reuteri 100-23 endopeptidase lysm zp_03072731 lactobacillus reuteri 100-23 gh25_muramidase sh3_5 zp_03073251 lactobacillus reuteri 100-23 endopeptidase lysm zp_03073284 lactobacillus reuteri 100-23 glucosaminidase lysm zp_03073901 lactobacillus reuteri 100-23 gh25_muramidase lysm zp_03074398 lactobacillus reuteri 100-23 endopeptidase lysm zp_03212068 lactobacillus rhamnosus hn001 gh25_muramidase lysm zp_03212389 lactobacillus rhamnosus hn001 gh25_muramidase lysm yp_396046 lactobacillus sakei subsp. sakei 23k glucosaminidase lysm yp_534994 lactobacillus salivarius ucc118 endopeptidase lysm yp_535201 lactobacillus salivarius ucc118 gh25_muramidase lysm yp_535698 lactobacillus salivarius ucc118 gh25_muramidase lysm yp_001031636 lactococcus lactis mg1363 glucosaminidase lysm yp_001031859 lactococcus lactis mg1363 glucosaminidase lysm yp_001032179 lactococcus lactis mg1363 gh25_muramidase lysm yp_808554 lactococcus lactis subsp. cremoris sk11 glucosaminidase lysm yp_809109 lactococcus lactis subsp. cremoris sk11 gh25_muramidase lysm yp_811362 lactococcus lactis subsp. cremoris sk11 gh25_muramidase lysm np_266428 lactococcus lactis subsp. lactis il1403 glucosaminidase lysm np_266697 lactococcus lactis subsp. lactis il1403 glucosaminidase lysm yp_001727510 leuconostoc citreum km20 scp_bacterial lysm yp_817821 leuconostoc mesenteroides atcc 8293 scp_bacterial lysm yp_817823 leuconostoc mesenteroides atcc 8293 scp_bacterial lysm yp_818129 leuconostoc mesenteroides atcc 8293 endopeptidase lysm yp_810395 oenococcus oeni psu-1 endopeptidase lysm yp_810757 oenococcus oeni psu-1 glucosaminidase lysm yp_810956 oenococcus oeni psu-1 chap (amidase) lysm sh3_5 yp_804483 pediococcus pentosaceus atcc 25745 gh25_muramidase lysm yp_804659 pediococcus pentosaceus atcc 25745 endopeptidase lysm glucosaminidasezp_00787586 streptococcus agalactiae cjb111 amidase_3 lysm amidase_5np_688827 streptococcus agalactiae 2603v/r glucosaminidase cpl-7 amidase_3abv55414 streptococcus dysgalactiae subsp. equisimilis chap (amidase) lysm yp_001449532 streptococcus gordonii ch1 chap (amidase) lysm zp_02920165 streptococcus infantarius atcc baachap (amidase) lysm np_720819 streptococcus mutans ua159 chap (amidase) lysm glucosaminidaseyp_598205 streptococcus pyogenes mgas10270 chap (amidase) sh3_5 glucosaminidaseyp_059383 streptococcus pyogenes mgas10394 chap (amidase) sh3_5 yp_060515 streptococcus pyogenes mgas10394 chap (amidase) sh3_5 yp_060862 streptococcus pyogenes mgas10394 chap (amidase) sh3_5 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:40 utc 6 maliničová, l. et al. continuation of table 1. domains id source catalytic cwb amidase 5 yp_602773 streptococcus pyogenes mgas10750 glucosaminidase cpl-7 glucosaminidasenp_664535 streptococcus pyogenes mgas315 chap (amidase) sh3_5 glucosaminidasenp_664726 streptococcus pyogenes mgas315 chap (amidase) sh3_5 amidase_5 np_664900 streptococcus pyogenes mgas315 glucosaminidase cpl-7 glucosaminidasenp_665012 streptococcus pyogenes mgas315 chap (amidase) sh3_5 np_665215 streptococcus pyogenes mgas315 chap (amidase) sh3_5 yp_282364 streptococcus pyogenes mgas5005 chap (amidase) sh3_5 amidase_5 np_606641 streptococcus pyogenes mgas8232 glucosaminidase cpl-7 glucosaminidasenp_607527 streptococcus pyogenes mgas8232 chap (amidase) sh3_5 glucosaminidaseyp_596324 streptococcus pyogenes mgas9429 chap (amidase) sh3_5 glucosaminidasenp_268942 streptococcus pyogenes m1 gas chap (amidase) sh3_5 glucosaminidasenp_269522 streptococcus pyogenes m1 gas chap (amidase) sh3_5 yp_002285797 streptococcus pyogenes nz131 chap (amidase) sh3_5 glucosaminidaseyp_002286426 streptococcus pyogenes nz131 chap (amidase) sh3_5 yp_598455 streptococcus pyogenes phage 315.5 chap (amidase) sh3_5 np_801715 streptococcus pyogenes ssi-1 chap (amidase) sh3_5 np_802383 streptococcus pyogenes ssi-1 chap (amidase) sh3_5 glucosaminidaseyp_001128106 streptococcus pyogenes str. manfredo chap (amidase) sh3_5 amidase_5yp_001128256 streptococcus pyogenes str. manfredo glucosaminidase cpl-7 yp_001034311 streptococcus sanguinis sk36 chap (amidase) lysm zp_00874987 streptococcus suis 89/1591 chap (amidase) lysm yp_819956 streptococcus thermophilus lmd-9 chap (amidase) lysm yp_138970 streptococcus thermophilus lmg 18311 chap (amidase) lysm consecutively we performed a detailed phylogenetic analysis of catalytic and cwb domain sequences and the resulting phylogenetic trees we compared with each other by connecting the catalytic and cwb domains together representing one protein (fig. 1). the results indicate that the degree of relatedness of the catalytic domains of proteins in most cases corresponds to the degree of relatedness of their cwb domains (approximately parallel flowlines between clusters of phylogenetic trees). figure 2 illustrates more detailed view to phylogenetic trees from fig. 1 and demonstrates one example of domain relatedness, as mentioned above. seven proteins from four different bacterial strains, and thus regardless of their origin (see fig. 2 caption) show very similar relatedness of their catalytic domains (gh25_muramidase) and cwb domains (lysm), which belong to the same phylogenetic clusters. on the other hand, there are few exceptions. as you can see in fig. 1 several flowlines between clusters of phylogenetic trees show remarkably different direction from other groups of flowlines. these examples represent few proteins with proven natural “domain shuffling”. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:40 utc nova biotechnologica et chimica 11-1 (2012) 7 fig. 1. comparation of phylogenetic trees of catalytic and cwb domains of selected endolysins and endolysin-like proteins from order lactobacillales. groups of approximately parallel flowlines between clusters of phylogenetic trees demonstrate that degree of relatedness of the catalytic domains of proteins in most cases corresponds to the degree of relatedness of their cwb domains. individually running flowlines with different direction between clusters demonstrate few proteins representing exceptions with proven natural domain shuffling. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:40 utc 8 maliničová, l. et al. from these examples we can mention two pairs of proteins: (1) yp_001844636 from lactobacillus fermentum ifo 3956 and yp_795546 from lactobacillus brevis atcc 367, which both have the same type of cwb domains (lysm) belonging to the same phylogenetic clustre and exhibiting 55% sequence similarity, but their catalytic domains (glucosaminidase and endopeptidase, respectively) show no significant similarity; (2) proteins yp_001844636 from lactobacillus fermentum ifo 3956 and np_786050 from lactobacillus plantarum wcfs1 have the same type of catalytic domains (glucosaminidase) from the same phylogenetic clustre and exhibiting 68% sequence similarity, but they differ in the type of cwb domains (lysm and sh3_5, respectively) with no significant similarity. fig. 2. more detailed view to phylogenetic trees from fig. 1. seven proteins from four different bacterial strains (yp_809109 and yp_811362 from lactococcus lactis subsp. cremoris sk11, yp_001032179 from lactococcus lactis mg1363, np_814147, np_815667 and np_816427 from enterococcus faecalis v583, and yp_535698 from lactobacillus salivarius ucc118) show very similar relatedness of their catalytic domains (gh25_muramidase) and cwb domains (lysm), which belong to the same phylogenetic clusters. 4. conclusions in present work we analysed domain structure of endolysins and endolysin-like proteins from the order lactobacillales. understanding the system of the natural domain distribution is the base for consequent experiments with artificial domain bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:40 utc nova biotechnologica et chimica 11-1 (2012) 9 shuffling and production of novel recombinant pghs. based on our findings we conclude, that the natural trend is distribution of endolysin genes as a whole and natural “domain shuffling” occurs in this case only in limited degree. nevertheless, an artificial "domain shuffling" may be regarded as an indispensable method in enzyme engineering. acknowledgement: this publication is the result of the project no.26220120043 implementation supported by the research & development operational programme funded by the erdf. references altschul, s.f., gish, w., miller, w., myers, e.w., lipman, d.j.: basic local alignment search tool. j. mol. biol. 215, 1990, 403-410. benson, d.a., karsch-mizrachi, i., lipman, d.j., ostell, j., sayers, e.w.: genbank. nucleic acids res. 2011, 39(database issue): d32-7. brüssow, h.: phages of dairy bacteria. annu. rev. microbiol. 55, 2001, 283-303. croux, c., ronda, c., lópez, r., garcía, j.l.: interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcalclostridial cell wall lytic enzyme. mol. microbiol. 9, 1993, 1019-1025. fischetti, a.v.: bacteriophage lytic enzymes: novel anti-infectives. trends. microbiol. 13, 2005, 491-496. humann, j., lenz, l.l.: bacterial peptidoglycan degrading enzymes and their impact on host muropeptide detection. j. innate immun., 1, 2009, 88-97. layec, s., decaris, b., leblond-bourget, n.: diversity of firmicutes peptidoglycan hydrolases and specificities of those involved in daughter cell separation. res. microbiol. 159, 2008, 507-515. loessner, m.j.: bacteriophage endolysins current state of research and applications. curr. opin. microbiol. 8, 2005, 480-487. lópez, r., garcía, e., garcía, p.: enzymes for anti-infective therapy: phage lysins. drug discov. today ther. strateg. 1, 2004, 469-474. marchler-bauer, a., lu, s., anderson, j.b., chitsaz, f., derbyshire, m.k., deweese-scott, c., fong, j.h., geer, l.y., geer, r.c., gonzales, n.r., gwadz, m., hurwitz, d.i., jackson, j.d., ke, z., lanczycki, c.j., lu, f., marchler, g.h., mullokandov, m., omelchenko, m.v., robertson, c.l., song, j.s., thanki, n., yamashita, r.a., zhang, d., zhang, n., zheng, c., bryant, s.h.: cdd: a conserved domain database for the functional annotation of proteins. nucleic acids res. 2011, 39(database issue): d225-9. mathur, s., singh, r.: antibiotic resistance in.food lactic acid bacteria a review. int. j. food microbiol. 105, 2005, 281-295. sanz, j.m., garcía, p., garcía, j.l.: construction of a multifunctional pneumococcal murein hydrolase by module assembly. eur. j. biochem. 235, 1996, 601-605. strompfová, v., lauková, a.: antibiotic resistance of lactic acid bacteria from canine faeces. bull. vet. inst. pulawy 48, 2004, 215-218. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:40 utc 10 maliničová, l. et al. tamura, k., dudley, j., nei, m., kumar, s.: mega4: molecular evolutionary genetics analysis (mega) software version 4.0. mol. biol. evol. 24, 2007, 1596-1599. vollmer, w., joris, b., charlier, p., foster, s.: bacterial peptidoglycan (murein) hydrolases. fems microbiol. rev., 32, 2008, 259-286. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 16.01.20 11:40 utc << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (iso coated) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.3 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent 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can be opened with acrobat and adobe reader 5.0 and later.) >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [2834.650 2834.650] >> setpagedevice microsoft word 1_havrlentova et al nova biotechnologica et chimica 15-2 (2016) 107 doi 10.1515/nbec-2016-0011 © university of ss. cyril and methodius in trnava can β-d-glucan protect oat seeds against a heat stress? michaela havrlentová1,2, ľubomíra deáková1, ján kraic1,2, alžbeta žofajová1 1 national agricultural and food centre – research institute of plant production, bratislavská cesta 122, piešťany, sk-921 68, slovak republic (havrlentova@vurv.sk) 2 department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic abstract: plants have evolved to live in environments where they are often exposed to different stress factors. being sessile, they have developed specific mechanisms that allow them to detect precisely environmental changes and respond to complex conditions, minimizing damage while conserving valuable resources for growth and reproduction. the cell wall polysaccharide β-d-glucan observed in some species of poales can determine responses to various environmental factors in specific plant developmental stages. it is located in the outer epidermal layer, at the place of stress attack and therefore its metabolism could relate to response of plant to environmental factors within moderate, physiological range. putative protective role of β-d-glucan during heat stress was indicated through naked oats with higher content of β-d-glucan. it appeared that oats with higher β-d-glucan content are better adapted to stress conditions. the presented article discusses the β-d-glucan as a possible protective mechanism in oat during (heat) stress conditions. key words: barriers, β-d-glucan, cell wall, heat stress, oat, protective effect 1. introduction the (1→3)(1→4)-β-d-glucan is a linear homopolysaccharide of d-glucopyranose residues bonded with mixture of β-(1-3) and β-(1-4)-glycosidic linkages (mantovani et al., 2008) in cereals and some grass species of the order poales. the most important sources of β-d-glucan among the main agricultural crops are seeds of barley and oat (havrlentová and kraic, 2006). naked oats contain usually higher amount of β-d-glucan in comparison with hulled ones (havrlentová and kraic, 2006). content of β-d-glucan content in oat is generally in the range of 0.27 – 0.58 (holthaus et al., 1996), and is affected by both genetic and environmental factors (ames et al., 2006). interactions of genotype with environment are also significant sources of variability in β-d-glucan content (yalcin et al., 2007), nevertheless genotype is superior to the environment (peterson et al., 1995). at the utmost extent β-d-glucan is located in the inner aleurone and subaleurone layer of the cell walls of endosperm and surrounding tissues (brown et al., 1997), however it was observed in leaves and roots, too (inouhe et al., 1997). carpita et al. (2001) found the polysaccharide to be distributed uniformly across the thin walls of the mesophyll cells and inner facing walls of the epidermis but concentrated primarily on the innermost portion of the outer facing walls of the epidermis. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:40 utc 108 havrlentová, m. et al. 2. function of β-glucans in plants beta-d-glucan plays an important role in cell wall architecture (virkki et al., 2005) and plant development (buckeridge et al., 2004). its primary role in plant cell wall is formation of thin surface layer on the cellulose microfibrils (virkki et al., 2005) which consequently interact with other cellulose-interlaced glycans during growth (carpita et al., 2001). the cell wall represents a key determinant of overall plants form, growth and development, and response of plant to abiotic and biotic stresses (farrokhi et al., 2006). plant exposed to physical, chemical, or biological stress factors must be able to respond by hardening of cell surfaces and related tolerance or resistance (hoson, 2002). polysaccharides of the cell wall, including the β-d-glucan, determine responses to various environmental factors in specific plant developmental stages (hrmova and fincher, 2001). the β-d-glucan located in the outer epidermal layer within the family of the gramineae is located at the place of stress attack (buckeridge et al., 2004). therefore metabolism of β-d-glucan could relate to response of plant to environmental factors within moderate, physiological range (hoson, 1998) what accents its special role at least in cereals. quick reinforcement of the cell wall may reduce the success of pathogen penetration. such reinforcements occur through the accumulation of callose (beta-1,3-glucan) (piršelová and matušíková, 2013), β-d-glucan (buckeridge et al., 2004) and proteins in the area between the cell wall and the cell membrane. 3. β-glucans under heat stress abiotic, or biotic forms of stress can induce changes in the plant metabolism, as in the context of production of primary metabolites, so in the secondary metabolites production, too. as one of the first are generated reactive oxygen species (hydrogen peroxide, singlet oxygen, superoxide radical, etc.), or nitrogen oxide, which triggers a cascade of defense reactions such as lipid peroxidation and synthesis of specific proteins (hématy et al., 2009). in the category of defensive molecules (defensive proteins) – active especially during fungal pathogenesis the important are so called pr (pathogenesis-related) proteins. their antimicrobial properties have previously been detected in vitro as well as in planta (hammond-kosack and jones, 1996). the subgroup of these proteins, beta-1,3-glucanases (pr-3), are enzymes that hydrolyze polysaccharides of fungal cell walls while their role in plant defense has been proven in transgenic plants (sundaresha et al., 2010). typical symptoms of pathogens toxicity are also reduction of photosynthetic pigments, changes in chlorophyll fluorescence and also changes in the activity of antioxidant active enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) (redondo-gómez, 2013). in one of our experiments, the objective was to expose oat seeds with high and low content of β-d-glucan to high temperature. temperature was used as a stress factor and relations between stress expression in the seed and the content of β-d-glucan as a potentially protective tool was monitored. mature seeds of eight oats bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:40 utc nova biotechnologica et chimica 15-2(2016) 109 of slovak provenience (with the content 2.55 – 5.63 % of β-d-glucan) were exposed to temperature 20 ºc, 30 ºc, 40 ºc, 50 ºc, and 60 ºc for 7 and 14 days, respectively. the amount of β-d-glucan was determined using the method of mccleary and codd (1991) (aoac 995.16) via an assay kit (megazyme inc., bray, ireland). viability of oat seeds, as an indicator of plant damage, was determined according to relevant national technical norm (stn 46101114, 2005). the results showed that heat treatment did not influence significantly the content of β-d-glucan in oat seeds, neither after 7 nor 14 days of heat treatment. evaluation of heat stress impact to seed viability revealed statistically significant negative correlation between seed viability and heat treatment for both the naked (r = -0.98**, p < 0.05) and hulled oats (r = -0.79**, p < 0.05). analysis showed also that time of exposition to elevated temperature, as well as oat genotype, significantly (p ≤ 0.01) influenced viability of hulled oat seeds. statistically significant source of variability in viability of naked oat seeds was only genotype represented in our study by the content of β-d-glucan. despite the fact that correlation between content of β-d-glucan and seed viability after heat treatment was not statistically confirmed (moderate correlation in naked oats, r = 0.55 and low correlation in hulled oats, r = 0.10) it could be speculated on putative protective role of the cell wall polysaccharide β-d-glucan against heat stress, at least in naked oats. environmental signals, including elevated temperature, are able to elicit responses of plant such as changes in the cell walls where the β-d-glucan is an essential substance, especially in oat. the β-d-glucan is a highly hygroscopic cell wall component. thus, seeds with higher β-d-glucan content should retain more water, water loses have mild expression due to higher mobility of water, seeds show higher rate of drying, and due to strong desiccation the cracking of seeds is absenting (fast seefeldt et al., 2007). high temperature during growing season and drought before grain harvest (savin and molina-cano, 2001) could increase the content of β-d-glucan in oat grains. the same effect has been observed in barley ehrenbergerová et al. (2003). the authors concluded that the contents of β-d-glucan and arabinoxylans were significantly affected by genotype, environmental conditions during the vegetation period and by their interactions. moreover, higher temperature increased the β-d-glucan content in barley seeds. on the other side, short exposition of barley plants to very high temperature may partially reduce the content of β-d-glucan (savin et al., 1997). relationship between higher content of β-d-glucan and elevated temperature could relate in grasses to pre-transport of some substances within the plant body in direction to grains (dupont and altenbach, 2003) where the β-d-glucan accumulates in cells not only as structural element of walls but also as endosperm storage material (buckeridge et al., 2004). very important is timing of stress factors such as heat, drought, or humidity (macnicol et al., 1993). other conditions such as nitrogen content in soil and precipitation also influence content of β-d-glucan in cereal grains (güler, 2003). generally, the plant cell wall containing polysaccharides, including the β-d-glucan as its major component, is a dynamic structure responding to external stimuli such as bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:40 utc 110 havrlentová, m. et al. abiotic and biotic stresses (farrokhi et al., 2006). subsequently, either the breakdown or the accumulation of these polysaccharides determine the composition and properties of cell walls (hoson, 1998) and hereby response to external stimuli. previously published studies were focused to synthesis, mobility, and accumulation of the β-d-glucan during plant growth and seed development (reference). our study was concerned to relations between content of β-d-glucan and viability of mature oat seeds stressed by high temperature. heat stress parameters, i.e. temperatures between 40 °c to 60 °c should simulate a condition in field during the summer when the final stage of oat seed maturation occurs. the mean content of β-d-glucan analyzed in mature seeds before heat treatment was 4.54 % in naked and 3.50 % in hulled oats. content of the β-d-glucan in seeds exposed to high temperatures for 7 and 14 days, respectively, was more balanced in naked in comparison to hulled oats and has been changed (increased or decreased) only slightly in both types (hulled and naked) oats in comparison to non-treated control seeds. oliveira et al. (2012) have reported that content of the β-d-glucan in mature oat seeds was affected by air drying at temperature above 50 °c, and the extracted β-d-glucan physically changed after drying at above 75 °c. simultaneously, water holding capacity of the β-d-glucan was decreased at temperature above 50 °c and water retention capacity decreased at more than 75 °c. high temperature may have result to degradation of β-glucosidic linkages in β-d-glucan and its disintegration to low molecular weight fragments or depolymerization of the linear structure, thus changing the content and properties of the β-d-glucan (butt et al., 2008). on the other hand, high temperatures (50 °c – 60 °c) can counteract against decreasing of β-d-glucan content as detected by fastnaught (2001) during process of β-d-glucan extraction where these temperatures may not be sufficient to inactivate endogenous β-glucanases. content of β-d-glucan could be related to thickness and consequently to insulation capacity of cell walls. correspondence between higher content of β-d-glucan and greater cell walls thickness has been reported in barley by bhatty et al. (1991). similar attribute of β-d-glucan in stress response to low temperature has been reported by takeda et al. (2011) in barley in mutants lacking (1,3;1,4)-β-d-glucan. they were more sensitive to chilling, probably due to thinner cell walls providing weaker temperature protection. specific association between the role of β-d-glucan and cell protection during heat stress (58 °c) has been demonstrated in bacteria (stack et al., 2010). genetically modified strain of probiotic lactobacillus paracasei nfbc 338 endogenously produced β-d-glucan. heat stress assays revealed that production of this polysaccharide was associated with significantly increased protection during heat stress (60-fold). another factor affecting stability and content of β-d-glucan in heat stressed seeds is inhibition of hydrolytic enzymes such as xyloglucan transglycosylases/hydrolases abundant in the apoplastic space (rose et al., 2002). further examples include hydrolysing xyloglucan backbones, β-glucanases, and other endo-type enzymes with β-glucanase activity (hrmova and fincher, 2001) breaking β-glucosidic linkages in β-glucans. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:40 utc nova biotechnologica et chimica 15-2(2016) 111 4. conclusions the cell wall polysaccharide β-d-glucan plays an important role in the cell wall architecture and plant development, too. according to some studies its metabolism could relate to response of plant to environmental factors within moderate, physiological range. this fact accents a special role of the β-d-glucan at least in cereals. exposure to temperatures of 20 ºc, 30 ºc, 40 ºc, 50 ºc, and 6 0ºc for 7 and 14 days, respectively, did not influence the content of β-d-glucan in seeds. however, significant negative correlation was observed between seed viability and heat treatment for both naked (r = -0.98**, p < 0.05) and hulled oats (r = -0.79**, p < 0.05). this indicates heat treatment as a stress factor. time of exposition and oat genotype significantly (p ≤ 0.01) influenced life cycle in hulled oats. in naked seeds, only genotype was the source of variability. the putative protective role of the cell wall polysaccharide β-d-glucan against heat stress, at least in naked oats, however, requires further investigations. acknowledgements: this work was supported by the operational program research and development: development of new types of genetically modified plants with farm traits (itms 26220220189) provided by the european regional development fund. references ames, n., rhymer, c., rossnagel, b.: genotype and environment effects on oat β-glucan, total dietary fiber and antioxidant activity. agriculture agri-food canada, 15, 2006, 1-9. aoac method 995.16: β-d-glucan in barley and oats, streamlined enzymatic method. aoac international. maryland, 2000. bhatty, r.s., macgregor, a.w., rossnagel, b.g.: total and acid-soluble β-glucan content in hulless and its relationship to acid-extract viscosity. cereal chem., 68, 1991, 221-227. buckeridge, m.s., rayon, c., urbanowicz, b., tiné, m.a.s., carpita, n.c.: mixed linkage (1→3),(1→4)-β-d-glucans of grasses. cereal chem., 81, 2004, 115-127. butt, m.s., tahir-nadeem, m., khan, m.k.i., shabir, r., butt, m.s.: oat: unique among the cereals. eur. j. nutr., 47, 2008, 68-79. carpita, n.c., tierney, m., campbell, m.: molecular biology of the plant cell wall: searching for the genes that define structure, architecture, and dynamics. plant mol. biol., 47, 2001, 1-5. dupont, f.m., altenbach, s.b.: molecular and biochemical impacts of environmental factors on wheat grain development and protein synthesis. j. cereal sci., 38, 2003, 133-146. ehrenbergerová, j., vaculová, k., psota, v., havlová, p., šerhantová, v.: effects of cropping system and genotype on variability in important phytonutrients content of the barley grain. plant soil environ., 49, 2003, 443-450. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:40 utc 112 havrlentová, m. et al. farrokhi, n., burton, r.a., brownfield, l., hrmova, m., wilson, s.m., bacic, a., fincher, g.b.: plant cell wall biosynthesis: genetic, biochemical and functional genomics approaches to the identification of key genes. plant biotechnol. j., 4, 2006, 145-167. fastnaught, c.: barley fibre. in: cho, s.s., drecher, m.l. 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(in slovak). sundaresha, s., manoj kumar, a., rohini, s., math, s.a., keshamma, e., chandrashekar, s.c., udayakumar, m.: enhanced protection against two major fungal pathogens of groundnut, cercospora arachidicola and aspergillus flavus in transgenic groundnut over-expressing a tobacco β 1-3 glucanase. eur. j. plant pathol., 126, 2010, 497-508. virkki, l., johansson, l., ylinen, m., maunu, s., ekholm, p.: structural characterization of water-insoluble nonstarchy polysaccharides of oats and barley. carbohydr. polym., 59, 2005, 357-366. yalcin, e., çelik, s., akar, t., sayim, i., köksel, h.: effect of genotype and environmental on β-glucan and dietary fiber contents of hull-less barleys grown in turkey. food chem., 101, 2007, 171-176. received 12 october 2016 accepted 7 december 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:40 utc nova biotechnol chim (2018) 17(1): 58-65 doi: 10.2478/nbec-2018-0006  corresponding author: jose.isagani.janairo@dlsu.edu.ph nova biotechnologica et chimica a machine learning approach in predicting mosquito repellency of plant – derived compounds jose isagani b. janairo, gerardo c. janairo and frumencio f. co de la salle university, 2401 taft avenue, manila 0922, philippines article info article history: received: 10th may 2018 accepted: 7th july 2018 keywords: ensemble learning quantitative structure-activity relationship quantum descriptors abstract the increasing prevalence of mosquito – borne diseases has prompted intensified efforts in the prevention of being bitten by the vector. among the various strategies of vector control, the application of repellents provides instant and effective protection from mosquitoes. however, emerging concerns regarding the safety of the widely used repellent, deet, has led to initiatives to explore natural alternatives. in order to fully realize the potential of natural repellents, focusing on the discovery of natural compounds eliciting repellency is of paramount importance. in this paper, machine learning was utilized to establish association between the mosquito repellent activity of 33 natural compounds using 20 chemical descriptors. individually, the descriptors had insignificant monotonic relationship with the response variable. but when optimized, the formulated model through boosted trees regression exhibited reliable predictive ability (r2 train = 0.93, r 2 test = 0.66, r 2 overall = 0.87). the findings presented have also introduced new descriptors that exhibited association with repellency through ensemble learning such as heat capacity, log p, entropy, enthalpy, gibb’s free energy, energy, and zero-point energy.  university of ss. cyril and methodius in trnava introduction vector – borne diseases are serious health burdens, which account for one – sixth of the illnesses suffered by the global population (who 2014). in particular, mosquitoes are carriers of dreaded diseases such as malaria, dengue, zika, and chikungunya. aside from being a health concern, mosquito – borne diseases are also associated in aggravating poverty (suaya et al. 2009). considering the negative multi-faceted impact of these diseases, the prevention of being bitten by the vector is of paramount importance. among the various strategies available to prevent being bitten by mosquitoes, the application of repellents is considered safe, and provides instant and effective protection. repellents also serve as the first line of defense in cases where the mosquito-borne disease, such as zika, has no established therapies and personal protection is the best approach to alleviate the disease burden (gulland 2016; wong et al. 2016). repellents exert their effect by preventing the binding of attractant odors to odorant – binding proteins (obps), that leads to the disruption of signal transduction pathways related to odor recognition (pellegrino et al. 2011). obps play a critical role in odor perception given that they act as carriers to the bound odor across the mucus barrier to initiate the physiological olfactory response (pelosi 1994). in addition, repellents have also been shown to block the electrophysiological responses of sensory neurons of mosquitoes toward attractive odors (ditzen et al. 2008). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:23 utc nova biotechnol chim (2018) 17(1): 58-65 59 the synthetic repellent, n-n-diethyl-meta toluamide (deet) is a widely used insect repellent, and is considered to be the most broad spectrum (katz et al. 2008). however, issues concerning the toxicity of deet to humans (diaz 2016) as well as being a pollutant to the environment (dsikowitzky et al. 2014) have led to initiatives to explore natural repellents as alternatives. however, most natural repellents come in the form of crude botanical extracts, such as lemongrass (oyedele et al. 2002), and thyme (park et al. 2005). it is therefore evident that identification of the compounds eliciting repellency can significantly enhance the formulation and efficacy of repellents. a good example is citronellal, which is a major component of several botanical repellents (trongtokit et al. 2005). when tested individually, citronellal exhibited promising repellency dose (rd50) against the malaria vector anopheles gambiae, which is comparable with deet (omolo et al. 2004). thus, natural compound screening is crucial in the discovery of more nature-derived mosquito repellents. economic and practical considerations are major impediments in compound screening, since testing each compound within a botanical extract for repellency is tedious, and entails serious financial support. a viable strategy in increasing the efficiency of drug discovery activities involves computation – driven approaches. doing so can help identify promising leads, group together compounds with similar mechanisms of action, or provide other relevant information (miszta et al. 2013). however, data – driven studies about mosquito repellents have mostly been confined to deet and deet-like compounds (katritzky et al. 2006; natarajan et al. 2008; katritzky et al. 2008). thus, employing data science and statistical strategies to examine natural repellents will accelerate and promote discovery and development for these natural compounds. in this study, a predictive model for mosquito repellency against anopheles gambiae of natural compounds was established though ensemble learning. the results presented may help accelerate the identification of repellent leads from natural sources, and provide novel insights regarding their biological activities. experimental the repellent activities (rd50) against a. gambiae of the natural compounds examined in this study were taken from omolo et al. (2004). rd50 refers to the minimum concentration of the compounds table 1. summary of transformations carried out on selected variables in order to minimize variations due to scale. variable transformation range of values rd50 (response variable) log (1/rd50) 2.23 to 5.00 energy [kj/mol] log (-energy) 6.01 to 6.24 homo [ev] none -6.90 to -5.23 lumo [ev] none -1.86 to 1.60 chemical potential [ev] none -4.22 to -2.19 hardness [ev] none 2.49 to 4.30 electrophilicity [au] none 0.59 to 3.40 dipole [debye] none 0.05 to 4.34 solvation [kj/mol] none -17.13 to 11.12 weight [g/mol] log (weight) 2.13 to 2.34 area [a2] log (area) 2.26 to 2.44 volume [a3] log (volume) 1.25 to 2.42 polar surface area, psa [a2] log (psa+1) 0 to 1.40 ovality none 1.24 to 1.47 log p none 1.71 to 4.28 polarizability [kj/mol] log (polarizability) 1.73 to 1.79 zero point energy, zpe log (zpe) 2.71 to 2.98 enthalpy, h log (-h) 2.59 to 2.82 heat capacity, cv log (cv) 2.21 to 2.44 entropy, s log (s) 2.57 to 2.69 gibb’s free energy, g log (-g) 2.59 to 2.82 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:23 utc nova biotechnol chim (2018) 17(1): 58-65 60 needed to elicit repellency to half of the mosquito population. thus lower rd50 values indicate better repellency. the most stable conformers of the 33 compounds of the library were determined using the merck molecular force field (mmff) in spartan ’10 (wavefunction, inc.). after the most stable conformers were identified, the optimum geometries, and molecular descriptors were obtained through density functional theory b3lyp / 6-311++g**. the 33 compounds are: camphene, beta-pinene, p-cymene, alpha-terpinene, gamma-terpinene, alpha-terpinolene, alphe-pinene, perillyl alcohol, cis-verbenol, cis-carveol, geraniol, alpha-terpineol, eugenol, terpinen-4-ol, linalool, citronellal, perillaldehyde, camphor, verbenone, fenchone, carvone, caryophyllene oxide, limonene oxide, 1,8-cineole, alpha-fenchyl alcohol, borneol, myrtenol, geranyl acetate, thujone, myrtenal, aromadendrene, 4-isopropylbenzaldehyde. the molecular descriptors energy, homo energy, lumo energy, dipole moment, solvation energy, molecular weight, area, volume, polar surface area, ovality, log p, polarizability, zero-point energy (zpe), constant volume heat capacity at 298 k (cv), enthalpy, entropy, and gibb’s free energy were directly obtained from the dft calculations. the quantum chemical descriptors of chemical potential, electronegativity, chemical hardness, chemical softness, and electrophilicity were calculated using various reactivity equations founded on dft, as reviewed by kaya and kaya (2015). the calculated descriptors were transformed in order to minimize their variations in terms of scale and magnitude. the transformations carried out are summarized in table 1 (raw and transformed datasets are available in the supporting information). the transformed descriptors were then subjected to correlation analysis using r version 3.5.0 (r core team 2018). all transformed descriptors, including the response variable were tested for normality of distribution using the shapirowilk test. following this test, isotonic relationships that may exist between the transformed descriptors and the response variable, were evaluated using the appropriate correlation function. after correlation analysis, the transformed descriptors were used to formulate a predictive model for mosquito repellent activity using machine learning. for support vector machine (svm) regression, the radial basis function (rbf) kernel was utilized. as a result, the best gamma and c parameters were automatically selected. 75 % of the data set was table 2. correlation analysis summary between the transformed response variable and the transformed descriptors. descriptor correlated with rd50 shapiro-wilk (p-value) spearman’s rho p-value kendall’s tau p-value energy <0.01 0.189 0.293 0.154 0.209 homo 0.014 -0.368 0.035 -0.240 0.052 lumo 0.033 -0.202 0.259 -0.130 0.292 chemical potential <0.01 -0.327 0.063 -0.226 0.065 hardness 0.071 – – – – electrophilicity <0.01 0.236 0.187 0.163 0.183 dipole 0.253 – – – – solvation 0.079 – – – – weight <0.01 0.217 0.236 0.187 0.153 area <0.01 0.246 0.167 0.171 0.163 volume <0.01 0.228 0.203 0.1444877 0.239 psa <0.01 – – – – ovality <0.01 – – – – log p 0.069 – – – – polarizability <0.01 0.231 0.196 0.156 0.204 zpe <0.01 0.038 0.833 0.030 0.804 h <0.01 0.190 0.290 0.156 0.204 cv <0.01 0.254 0.154 0.190 0.121 s <0.01 0.276 0.120 0.205 0.094 g <0.01 0.189 0.292 0.152 0.215 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:23 utc nova biotechnol chim (2018) 17(1): 58-65 61 used for training, while the remaining 25 % served as the test set. the results were validated by applying a 10-fold cross validation. for random forest regression, the random test data proportion was set to 0.30, and 0.5 for the subsample proportion. the stopping parameters were set as follows: minimum n cases = 5, maximum n cases = 10, minimum n child in node = 5, maximum n of nodes = 100. for the boosted trees regression, the learning rate was set to 0.1, with the following conditions: number of additive terms = 200, random test data proportion = 0.30, subsample proportion = 0.50. the stopping parameters were set as follows: minimum n of cases = 5, maximum n of levels = 10, minimum n in child node = 1, maximum n of nodes = 3. for both the random forest, and boosted trees regression, 60 % of the data was dedicated to train the algorithm while the remaining 40 % was used for model testing. the predictive ability of the models was assessed based on the goodness of fit between the observed and predicted values of the rd50 for the compounds. results and discussion predictive modelling is an important component of drug design and discovery. predictive models establish association with the response variable, usually the biological activity, with a set of molecular properties or descriptors using statistical tools. common statistical methods used in establishing association is multiple linear regression, but the utilization of machine learning has recently gained traction due to its versatility and effectivity in establishing predictive models (gertrudes et al. 2012). correlation analysis was used to aid in the selection of transformed descriptor to be included in the formulation of the predictive models. a prerequisite to correlation analysis is the test for normality distribution, which was done through the shapirowilk test. in this test, a p-value greater than significance level of 0.05 indicates a normal distribution of the data. the transformed response variable had a p-value of 1.16e-5, indicating a non-normal distribution. this means that the usual pearson correlation is not applicable for analysing isotonic relationships with the response variable. the shapiro-wilk test was further conducted for the transformed variables, and the results are shown in table 2. the descriptors hardness, dipole, solvation had p-values greater than 0.05, indicating a normal distribution, while the other descriptors had non-normal distribution. thus, hardness, dipole, and solvation were excluded in the succeeding correlation analysis using spearman, and kendall. these aforementioned correlation tests are used for data with a non-normal distribution, wherein the data are converted into ranks prior to correlation. as a consequence, psa, ovality, and log p were also excluded from the correlation analysis since ties in the rankings of the compounds were observed. hence, the remaining descriptors were subjected to both spearman, and kendall correlation tests. the results of the tests showed that the transformed descriptors had insignificant isotonic relationship with the response variable, as demonstrated by p-values greater than 0.05. as a consequence of these results, other approaches in selecting relevant descriptors to predict the mosquito repellency should be explored, such as backward elimination (dudek et al. 2006). thus, all 20 transformed descriptors were initially used to formulate the predictive models. the next part of the study involves identifying the suitable algorithm for the given data set, since the type of machine learning algorithm affects the performance of the predictive model. a model to predict the repellent activities of the 33 natural compounds using the 20 descriptors was preliminarily established through boosted trees, support vector machine, and random forest. the three aforementioned machine learning techniques are different supervised predictive algorithms, each possessing different characteristics. boosted regression trees is a sequential model-building classifier wherein weak predictors in the previous model are given more weight or boosted, to improve prediction performance in the next model. random forest is a bagging algorithm wherein bootstrapped samples with replacement are taken from the data set and predictions are obtained from each of these samples. final predictions are then obtained from the average predictions of these samples. this way, the model variance may be improved but not bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:23 utc nova biotechnol chim (2018) 17(1): 58-65 62 the predictive power. boosting such as boosted regression trees reduces bias and therefore improves prediction while bagging such as random forest reduces variance. random forests, unlike boosted trees, are less sensitive to outliers. boosted trees has another advantage in terms of speed and table 3. comparison of the performance of various machine learning algorithms in predicting the repellent activity of the 33 natural compounds using all calculated molecular descriptors. fig. 1. performance of the predictive models based on relative mean squared error (rmse, top graph) and correlation coefficient (bottom graph). the broken line represents values for the training set, the dotted line for the test set, and the solid line for the overall set. simplicity over random forests since the former performs better with smaller trees than the latter. boosted trees is also insensitive under monotone transformations of the predictors such as logarithmic transformation. in svm, optimal hyperplanes are constructed in a high-dimensional space for classification or regression. for a good separation, a hyperplane with the largest distance from the nearest training data point is chosen in order to achieve a lower error rate. svm, like boosted trees, is also susceptible to overfitting that could be avoided by properly tuning its hyperparameters. the formulated predictive models utilized transformed descriptors and response variables (table 1) in order to have a narrower range of values among the variables. various goodness of fit measures between the observed and predicted repellent activity values were used for model diagnostics, as shown in table 3. it is thus evident that boosted trees regression (using gradient boosting algorithm) is the ideal algorithm to be used for this particular dataset since it had the lowest deviations, and strongest correlations between the observed and predicted values. the adeptness of boosted trees for this particular dataset can be possibly rooted in its robustness to outliers, irrelevant variables, correlated variables, and log-transformed predictors (hastie et al. 2008). model optimization was thus confined to boosted tree regression, wherein a stepwise back elimination of the predictors was conducted based on the calculated predictor importance. this is necessary since the results of the correlation analysis (table 2) failed to show strong association between the descriptors and the response variable. for example, the least important descriptor for the full-descriptor model 1 was hardness. hence boosted trees support vector machine random forest train test overall train test overall train test overall mean square error 0.134 0.133 0.133 0.487 0.271 0.428 0.455 0.244 0.372 mean absolute error 0.274 0.262 0.269 0.647 0.472 0.599 0.562 0.452 0.519 mean relative squared error 0.014 0.014 0.014 0.044 0.024 0.038 0.045 0.024 0.037 mean relative absolute error 0.090 0.088 0.089 0.193 0.141 0.179 0.177 0.142 0.164 correlation coefficient 0.862 0.616 0.807 0.431 0.393 0.413 0.508 0.316 0.351 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:23 utc nova biotechnol chim (2018) 17(1): 58-65 63 in model 2, only 19 descriptors were used for the modelling since hardness was removed. this process was repeated until the remaining descriptors had importance scores of greater than or equal to 0.90 (the predictors and their corresponding importance for each model is available in the supporting information). the resulting performance of the formulated predictive models following this optimization method is shown in fig. 1. fig. 2. summary of boosted trees showing the optimum number of trees prior to data overfitting. the best model was judged to be model 14, since the remaining descriptors all had importance scores of greater than 0.90 (table 4). model 14 also exhibited the lowest rmse values, and highest correlation coefficients in all sets (training, test, and overall sets). model 14 was found optimal in terms of average squared error using 32 trees for the test set (fig 2). the formulated regression models were validated using 40 % of the data. while no general rule exists regarding the optimum division of the data between training and testing (roy et al. 2008), a 60–40 split seems appropriate in this case to balance training and validation of the relatively small population of the data set. the most important chemical descriptor is log p, which relates the hydrophobicity of the molecule. this result resonates with the property of obps that possess a hydrophobic cavity to which odors are bound (murphy et al. 2013). majority of the descriptors are thermodynamic properties of the molecules, which are known to heavily influence ligand binding to proteins (bostrom et al. 1988). table 4. descriptors of model 14 used to predict the repellent activity of 33 natural compounds. all descriptors had importance scores of greater than 0.90. transformed descriptor predictor importance score log p 1.00000 log (cv) 0.991466 log (psa +1) 0.973521 log (s) 0.956767 log (-energy) 0.938961 log (-h) 0.938961 log (-g) 0.938961 log (zpe) 0.927269 aside from establishing a satisfactory predictive model for mosquito repellency, the present findings have introduced new descriptors that showed association with repellency within the context of ensemble learning. previous quantitative structure – activity relationship models reported the descriptors of boiling point, molecular surface area, total charge of the other substituents on the cyclic backbone, and dipole moment were correlated with the repellent activity of a set of terpenoid compounds (wang et al. 2008). another model showed that the repellent activity of a class of terpenoid compounds showed correlation with the lumo energy, minimum valence of the o atom, principal moment of inertia, and the shadow area of the repellent (song et al. 2013). molecular topological indices have also been demonstrated to exhibit association with mosquito repellency (garcia-domenech et al. 2010). it is interesting to note that the present study has provided new descriptors in which repellency can be predicted, which are heat capacity, log p, entropy, enthalpy, gibb’s free energy, energy, and zpe. past studies that attempted to predict mosquito repellency mostly applied multiple linear regression on a compound library with limited diversity. the presented results thus demonstrate the utility of machine learning in bioactivity predictive modelling. individually, the descriptors had insignificant correlation with repellency. but when these descriptors were processed through boosted trees, a satisfactory predictive model was constructed. it should be noted that further validation of the model is still needed. although the algorithm already exhibits reliable predictive power, increasing the general applicability and robustness of the model requires incorporating bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:23 utc nova biotechnol chim (2018) 17(1): 58-65 64 more compounds to the data set. however, this can be challenging at this point considering that most of the reports on a. gambiae repellency focus on crude botanical extracts (odalo et al. 2005; deletre et al. 2013), or use a different metrics to measure repellency, such as oviposition activity index (kweka et al. 2010), and % repellency (logan et al. 2010). conclusions a model to predict mosquito repellency of natural compounds for the anopheles gambiae using boosted trees regression was established. dft calculated descriptors were used to predict repellency, wherein log p, heat capacity, polar surface area, entropy, energy enthalpy, gibb’s free energy, and zero-point energy are new descriptors found to be associated with repellency. overall the findings presented are expected to promote and accelerate the discovery of natural mosquito repellents, as well as to forge a deeper understanding on the molecular properties responsible for eliciting mosquito repellency. acknowledgements this study was funded by the national academy of science and technology, philippines, with support from the department of science and technology – innovation council (dost-pcieerd) through the data science track. references bostrom j, norrby p-o, lilijefors t (1998) conformational energy penalties of protein-bound ligands. j. comput. aided mol. des. 12: 383-396. deletre e, martin t, campagne p, bourget d, cadin a, menut c, bonafos r, chandre f (2013) repellent, irritant and toxic effects of 20 plant extracts on adults of malaria vector anopheles gambiae mosquito. plos one. 8: e82103. 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wang z, song j, chen j, song z, shang s, jiang z, han z (2008) qsar study of mosquito repellents from terpenoid with a six-member-ring. bioorg. med. chem. lett. 18: 2854-2859. wong ss-y, poon rw-s, wong sc-y (2016) zika virus infection – the next wave after dengue? j. formos. med. assoc. 115: 226-242. world health organization (2014) a global brief on vector – borne diseases. geneva: who press. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:23 utc https://www.r-project.org/ https://www.r-project.org/ nova biotechnologica et chimica 15-1 (2016) 23 doi 10.1515/nbec-2016-0003 © university of ss. cyril and methodius in trnava metagenomic analysis of slovak bryndza cheese using next-generation 16s rdna amplicon sequencing matej planý1,2, tomáš kuchta2, katarína šoltýs3, tomáš szemes3, domenico pangallo4, peter siekel1,2 1department of biology, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (matejpln@gmail.com) 2department of microbiology and molecular biology, food research institute nafc, priemyselná 4, 824 75 bratislava 26, slovak republic 3department of molecular biology, comenius university, 842 15 bratislava 4, slovak republic 4institute of molecular biology, slovak academy of sciences, dúbravská cesta 21, 845 51 bratislava, slovak republic abstract: knowledge about diversity and taxonomic structure of the microbial population present in traditional fermented foods plays a key role in starter culture selection, safety improvement and quality enhancement of the end product. aim of this study was to investigate microbial consortia composition in slovak bryndza cheese. for this purpose, we used culture-independent approach based on 16s rdna amplicon sequencing using next generation sequencing platform. results obtained by the analysis of three commercial (produced on industrial scale in winter season) and one traditional (artisanal, most valued, produced in may) slovak bryndza cheese sample were compared. a diverse prokaryotic microflora composed mostly of the genera lactococcus, streptococcus, lactobacillus, and enterococcus was identified. lactococcus lactis subsp. lactis and lactococcus lactis subsp. cremoris were the dominant taxons in all tested samples. second most abundant species, detected in all bryndza cheeses, were lactococcus fujiensis and lactococcus taiwanensis, independently by two different approaches, using different reference 16s rrna genes databases (greengenes and ncbi respectively). they have been detected in bryndza cheese samples in substantial amount for the first time. the narrowest microbial diversity was observed in a sample made with a starter culture from pasteurised milk. metagenomic analysis by high-throughput sequencing using 16s rrna genes seems to be a powerful tool for studying the structure of the microbial population in cheeses. key words: lactic acid bacteria, bryndza cheese, next-generation sequencing, metagenomics, 16s rrna genes 1. introduction bryndza is a cheese traditionally produced in the mountainous regions of slovakia, by ripening, salting and grinding of ewes’ cheese, or a mixture of ewes’ and cows’ cheese. based on the current legislation, the minimum content of the ewes’ component must be more than 50%. since 2008, slovak bryndza is on the list of european cheeses with protected geographical indication status. the quality of bryndza cheese depends on many factors, e.g. the production period, quality of ewes’ milk and if the starter culture was used. may bryndza cheese is the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:02 utc 24 planý, m. et al. highest valued variant of bryndza, due to its distinctive flavour (sádecká et al., 2014). ewes’ milk, in comparison to cows’ milk, contains higher amounts of proteins, carbohydrates, traces of copper, panthotenic acid, biotin and vitamin b12 (burdová, 1997). composition and activity of microflora are believed to be important for flavour and aroma of bryndza cheese. study of lactic acid bacteria present in traditional fermented foods and their diversity is necessary to understand their quality and, eventually, for starter culture selection if pasteurisation of the substrate is necessary for safety reasons. the use of starter or adjunct cultures is also useful to control the fermentation process and to standardize the end product (leroy and de vuyst, 2004). however, this approach often leads to a loss of the uniqueness of the original product and the loss of the characteristics that have made the product popular (caplice and fitzgerald, 1999). metagenomics deals with the investigation of the whole genetic material isolated from environmental samples to characterise their microbial consortia composition. metagenome is nowadays mostly sequenced using the technique of next-generation sequencing (ngs), which allows sequencing of huge numbers of different dna strands at the same time. the use of 16s rrna genes in targeted metagenomics is suitable for bacteria identification, and their taxonomy and phylogeny analysis because of their presence in almost all prokaryotes and their relatively short length (approximately 1500 bp). moreover, 16s rdna sequence contains nine hypervariable regions, which are often conserved within species but different between species. the microbial composition investigation in dairy products by ngs represents an innovative approach, which provides extensive data while requiring less labour and time. this technology has been used to study the bacterial diversity of various cheeses, such as irish artisanal cheeses (quigley et al., 2012), latin-style cheeses (lusk et al., 2012), traditional polish cheeses (alegria et al., 2012), water buffalo mozzarella cheese (ercolini et al., 2012) and danish semi-hard cheese (masoud et al., 2012). the composition of bryndza cheese microflora was previously studied by various culture-based and culture–independent methods. enterococci in bryndza cheese were previously identified on the genus and species level by phenotypic methods and confirmed by pcr using species-specific primers for ddl genes (jurkovič et al., 2006). laurenčík et al. (2008) investigated the occurrence and diversity of yeast and filamentous fungi in bryndza cheese collected during four months of the summer production period by conventional taxonomy and sequence comparision of d1/d2 region from 26s rrna genes. mostly lactobacilli were characterized using microbiological and biochemical testing, genotyping by ramp, eventually by 16s rdna sequencing by berta et al. (2009). the knowledge about the bryndza cheese microflora was extended by chebeňová-turcovská et al. (2011) using the universal prokaryotic and fungal primers to amplify rdna internal transcribed spacer regions. diversity and dynamics of prokaryotes and eukaryotes in selected stages of the may bryndza cheese production were studied by pangallo et al. (2014) using culture-based and non-culture approach based on amplification of 16s rdna and internal transcribed spacer region, coupled to denaturing gradient gel electrophoresis and sequencing. also occurrence of pathogenic bacteria e. coli (holko et al., 2006) and s. aureus (mašlanková et al., 2009) in bryndza cheese were previously bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:02 utc nova biotechnologica et chimica 15-1 (2016) 25 studied. in this article, the most progressive culture-independent technique based on ngs 16s rdna amplicon sequencing was used for the first time to analyse microbial consortia in bryndza cheese. 2. materials and methods 2.1 sample preparation four samples of bryndza cheese from different manufacturers and regions of slovakia were analysed. their origin, region of production, ewes´ milk content, use of starter culture, production season and the type of manufacturing process are summarized in table 1. table 1. samples of bryndza cheese in this experiment used, and their properties. sample/origin region of production ewes´ milk content starter culture use production season manufacturing process 1./store senica 50% yes winter industrial 2./store zvolenská slatina 50% yes winter industrial 3./store liptovský mikuláš 50% yes winter industrial 4./ farm (salaš) pružina 100% no spring (may) artisanal dna was isolated from each sample by chaotropic solid-phase extraction using dneasy food kit mericon, (qiagen, hilden, germany) according to the standard protocol of the manufacturer attached to the kit (sample size, 200 mg). 16s rdna were amplified in six replicates (to increase the amount of pcr product) using primers 27f (5´-aga gtt tga tcm tgg ctc ag-3´) and 1062r (5´-aca gcc atg cag cac ct-3´) to amplify v1-v6 hypervariable regions (ghyselinck et al., 2013). into the mastermix with total volume of 22 μl (0.3 μl of each primer, 2.5 μl cheetah taq dilution buffer (biotium, hayward, california), 2.5 μl 25 mm magnesium chloride (biotium), 4 μl 10 mm dntp (2.5 mm each); 0.3 μl 500 u cheetah hotstart taq dna polymerase (biotium) and 12.1 μl pcr grade water, 3 μl of template dna solution were added. the pcr program was composed of initial denaturation step (95°c, 2 min), followed by 35 cycles (94 °c for 1 min.; 54 °c for 1 min.; 72 °c for 2 min) and final polymerization step (72 °c for 10 min). success of pcr was checked by 1.5 % agarose gel electrophoresis using 100 bp dna ladder (biolabs, ipswich, ma, usa). products of pcr were pooled and purified by qiaquick pcr purification kit (qiagen). 2.2 massively parallel sequencing and sequence data processing purified pcr products were diluted to equimolar concentration suitable for library preparation and were used as template for library preparation using transposon based bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:02 utc 26 planý, m. et al. nextera library preparation kit (illumina, san diego, ca, usa) according to standard protocol. four samples were analysed using paired end sequencing on illumina miseq sequencing system (illumina) in two separate runs targeting at least 250 000 reads and at least 2 x 200 bp read lengths. sequence data (sequence lists composed of reads) in fasta format were imported into clc genomics workbench version 7.5 (qiagen, hilden, germany). the obtained 2 x 250 bp long paired reads from samples 1-3, and 2 x 200 bp paired reads from sample 4 were merged and pre-processed. before merging, sequence lists of given samples 1-4 contained 2 970 530, 2 202 668, 1 885 378 and 285 398 reads, respectively. overlapping pairs in paired reads of each sequence list representing a given sample were merged, using default parameters (alignment scores: mismatch cost 2; gap cost 3; maximum unaligned end mismatches 0; minimum score 8. after merging, two datasets are produced: merged and not merged). reads in sequence lists were then trimmed based on their length and quality. limit of trimming using quality score was set to 0.001 and reads shorter than 150 nucleotides were discarded. reads with ambiguous nucleotides were discarded with the maximum number of ambiguities set to 2. 2.3 data analysis and results interpretation sequence data were analysed using miseq reporter software (msr; illumina) on the genus level using greengenes 16s rrna gene database (desantis et al., 2006) integrated in metagenomics analysis module. reads were identified based on their homology to reference 16s rrna genes in ncbi database using basic local alignment search tool (blast) (altschul et al., 1990) with following parameters: number of threads 1; low complexity filter; expectation value 10 e-50, word size 50; match/mismatch: match 2, mismatch -3; gap costs: existence 5, extension 2; maximum number of hit sequences 3. blast results were exported to metagenome analyzer (megan v5) (huson et al., 2011), and interpreted using ncbi taxonomy. 3. results and discussion 3.1 sequence data analysis and pre-processing sequence data quality analysis showed that all raw sequence data, obtained from each sample without any pre-processing step, were of relatively high quality. the percentage of reads with average phred score (ewing and green, 1998) above 30 in samples senica, zvolenská slatina, liptovský mikuláš and pružina were more than 89%, 88%, 87 % and 97%, respectively. phred score or quality score q, is defined by the equation: q= -10 log (p), where p is an error probability. higher quality values (q) correspond to lower error probabilities, and conversely. if, for example, quality value is 30, the error probability (p) is 1:1000. after raw data merging, the number of reads in each sequence list was reduced and the maximum length of reads (in the case of, for example, 250 bp) increased bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:02 utc nova biotechnologica et chimica 15-1 (2016) 27 approximately twice (492 bp). the average phred score percentage of each sample increased as well. it is apparent that, for accurate assignment to reference 16s rrna genes, longer reads including more hypervariable regions of the 16s rrna gene are of greater information value. 3.2 metagenomic interpretation using msr msr results in the form of tables with a percentage of reads assigned to each taxonomic rank (from kingdom to species) were transformed to pie charts representing microbial composition of each bryndza cheese sample on the genus level (fig. 1). fig. 1. percentage of reads assigned to bacteria on genus level (first 8 most abundant) evaluated using msr and greengenes database. the results from msr showed that the largest percentage of reads, assigned on the genus level, belonged in all bryndza samples to lactococci, followed by the unclassified reads (sample 1-3), which were not assigned to any reference 16s rrna gene present in greengenes database. the exception was sample 4, where the second most abundant genus was streptococcus. streptococci and lactobacilli, as the third and fourth dominant genera in order, were observed in samples 1 and 2 in similar bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:02 utc 28 planý, m. et al. amounts (16%; 6.1% and 15.1; 3.2, respectively). lactobacilli constituted only 0.3% of assigned reads in sample from liptovský mikuláš. lactobacilli were not detected by msr on the genus level in the sample from pružina, which confirmed that amount of lactobacilli depends on the ripening stage of the cheeses and maturation (quigley et al., 2012). the another significant group of lactic acid bacteria observed, were enterococci in amounts of 2.7%; 1.7%; 1.5%; and 1.15% in the samples 1-4, respectively. the detailed analysis (not illustrated) on the species level showed that the most abundant lactococci were lactococcus lactis in all samples (22.9%; 20.1%; 37.7%; and 8.6 in samples 1-4, respectively) followed by lactococcus fujiensis in samples 1, 2 and 3 (in the amounts of 5.4%; 5.3%; and 8.6%, respectively) and, in the sample 3 (pružina), s. thermophillus (3.22%) and l. fujiensis (2.54%) on the 2nd and 3rd places. lactococcus fujiensis was detected in bryndza cheese for the first time. 3.3 metagenomic interpretation using megan 5 monitoring of microbial diversity on genus level can be used to trace major changes in the structure of the microbial community in foods during fermentation but, in such cases, ngs sequencing at the genus level is not very informative (ercolini, 2013). the phylogeny relations between taxonomic units of microbial consortia in bryndza cheese samples are illustratively described by taxonomic trees (fig. 2, 3, 4, 5), which are composed of nodes and roots. each node represents one taxonomic unit. the size of the node depends on the number of reads assigned. firmicutes were the predominant phylum in all four samples tested. various species of lactic acid bacteria was observed. the species lactococcus lactis predominated in all 4 samples, followed by lactococcus taiwanensis, which was detected in bryndza cheese for the first time. based on results obtained from msr, no lactococcus taiwanensis was observed, which could have been caused by the different databases used. after the node uncollapsing to the lowest taxonomic level (showed only in fig. 4), lactococcus lactis subsp. lactis dominated, followed by lactococcus lactis subsp. cremoris and lactococcus taiwanensis in all bryndza cheese samples tested. lactococcus taiwanensis seems to be an alternatively assigned operational taxonomic unit instead to lactococcus fujiensis in the case of msr. the third most abundant species observed was streptococcus thermophillus. lactococcus lactis and streptococcus thermophilus were observed in most bryndza chees samples as major species, regardless of whether the cheese was made from pasteurised or nonpasteurised milk (chebeňová-turcovská et al., 2011). in sample 1, the species of lactococcus, streptococcs, lactobacillus, enterococcus and leuconostoc genera predominated (fig. 2). this finding was equivocally displayed in results from both approaches (msr and blast + megan 5). although msr uses greengenes database and a proprietary algorithm that provides species-level classification for paired-end reads, the percentages of lab on the genus level in sample 1 correlated, except for the amount of unknown reads. only in this sample of winter bryndza cheese, a significant group of enterococci was observed. these results are different from those of jurkovič et al. (2006), which showed that the prevalence of enterococcus species was highest in samples from the winter season. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:02 utc nova biotechnologica et chimica 15-1 (2016) 29 fig. 2. taxonomic tree of microflora in bryndza cheese sample from senica (megan 5). in sample from zvolenská slatina (fig. 3), lots of gammaproteobacteria were detected including enterobacter, kleibsella, shigella, salmonella and yersinia. these human pathogens could be present in milk before pasteurisation. it must be noted that ngs allows us to detect also bacteria that cannot be cultivated in laboratory conditions, including unviable and dead microorganisms, which cannot affect human health. the group of lactic acid bacteria present in this sample was composed mostly of lactococcus, streptococcus enterobacter and lactobacillus species. the narrowest microbial diversity was observed in the sample from liptovský mikuláš (fig. 4). only 4 different species including 3 species of lactococci with dominance of l. lactis followed by l. taiwanensis and l. raffinolactis, with contrast to msr results (figure 1, liptovský mikuláš sample), where genera of lactococcus, enterococcus, streptococcus, candidatus biochmannia, lactobacillus, leuconostoc and clostridium (79,6%; 1,5%; 1,2%; 0,7%; 0,3%; 0,2% 0,1%, respectively) were detected. loss of some reads could have taken place due to the use of strict assignment identification parameters and reads pre-processing condition. to confirm this, we bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:02 utc 30 planý, m. et al. additionally analysed raw sequence data, and the taxonomic tree (fig. 4 b) showed the presence of some small amounts of enterococci and streptococci. short reads, or reads with low quality, could be assigned incorrectly, and therefore could produce false positive results (chen et al., 2014). all pre-processed reads were assigned in the sample 3 (fig. 4) while in msr results, 15.7% of the total reads were unclassified. fig. 3. taxonomic tree of microflora in bryndza cheese sample from zvolenská slatina (megan 5). the bryndza cheese sample from pružina differed from the others in the production season and manufacturing process. apart from the sample number 3, the taxonomic tree of sample from pružina (figure 5) seems relatively similar to samples 1 and 2. therefore we couldn’t definitely confirm that the microbial diversity increased in artisanal products in comparison to industrially manufactured cheeses (coppola et al., 2001), also because our samples of bryndza cheese were produced in different seasons. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:02 utc nova biotechnologica et chimica 15-1 (2016) 31 fig. 4. taxonomic tree of microflora in bryndza cheese sample from liptovský mikuláš (megan 5). a) pre-processed reads collapsed on strain level; b) raw reads collapsed on strain level. fig. 5. taxonomic tree of microflora in bryndza cheese sample from pružina (megan 5). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:02 utc 32 planý, m. et al. a relatively huge part of microflora was composed of gammaproteobacteria, but much less in comparison to sample 2. some bacteria belonging to gammaproteobacteria were observed in the previous study, mainly during the early stages of may bryndza cheese maturation, namely the strains belonging to the genera acinetobacter, pseudomonas, enterobacter, kluyvera and raoultella (pangallo et al., 2014). 4. conclusions three samples of bryndza cheese purchased in a store (containing 50% ewes´ lump cheese made from pasteurised milk, produced in winter season), and one sample of bryndza cheese purchased in a farm (containing 100% ewes´ lump cheese made from unpasteurised milk, produced in may) were analysed and compared by metagenomics analysis using 16s rdna amplicons for ngs sequencing. the results showed some differences in the microflora composition in cheeses made from pasteurised and unpasteurised milk but, on the obtained amount of data, it was not possible to clearly determine the specific properties of these two types of bryndza cheese. the most observed genera in all samples of studied bryndza cheese were lactococci, followed by streptococci, lactobacilli and enterococci. the most abundant species observed were l. lactis, l. taiwanensis/ l. fujiensis (detected by megan 5/ msr) and s. thermophillus. l. taiwanensis and l. fujiensis were detected in bryndza cheese for the first time at all. the presence of these species in bryndza cheese could be an interesting aim of some future study. the narrowest microbial diversity was observed in pasteurised milk cheese from liptovský mikuláš. some gammaproteobacteria strains were in pasteurised and also in non-pasteurised milk bryndza cheese detected. contamination may occur after pasteurisation as well. moreover, we couldn’t recognise if the dna belonged to viable or dead pathogens. despite this, the metagenomic analysis by high-throughput sequencing using 16s rrna proved to be a very helpful and suitable tool to make overall picture of the whole microbial composition of various types of slovak bryndza cheese. acknowledgements: this work was supported by the project of the slovak research and development agency apvv-14-0025. references alegria, a., szczesny, p., mayo, b., bardowski, j., kowalczyk, m.: biodiversity in oscypek, a traditional polish cheese, determined by culturedependent and -independent approaches. appl. environ. microbiol., 78, 2012, 1890–1898. altschul, s. f., gish, w., miller, w., myers, e. w., and lipman, d. j.: basic local alignment search tool. j. mol. biol., 215, 1990, 403-410. berta, g., chebeňová, v., brežná, b., pangallo, d., valík, ľ., kuchta, t.: identification of lactic acid bacteria in slovakian bryndza cheese. j. food nutr. res., 48, 2009, 65-71. bereitgestellt von 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kolek, e., pangallo, d., valík, l., kuchta, t.: principal volatile odorants and dynamics of their formation during the production of may bryndza cheese. food chem., 150, 2014, 301-306. sulo, p., laurenčík, m., poláková, s., minárik, g., sláviková, e.: geotrichum bryndzae sp. nov., a novel asexual arthroconidial yeast species related to the genus galactomyces. int. j. syst. evol. microbiol., 59, 2009, 2370–2374. quigley, l., o’sullivan, o., beresford, t. p., ross, r. p., fitzgerald, g. f., cotter, p. d.: high-throughput sequencing for detection of subpopulations of bacteria not previously associated with artisanal cheeses. appl. environ. microbiol., 78, 2012, 5717–5723. received 27 november 2015 accepted 5 may 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:02 utc nova biotechnol chim (2017) 16(1): 61-67 doi: 10.1515/nbec-2017-0009  corresponding author: doka.otto@sze.hu nova biotechnologica et chimica rutin in buckwheat grain meal determined by uv photoacoustic spectroscopy and hplc ottó dókaa,, andrea brunorib, rezső schmidtc, dane bicanicd and györgy végvárie,f a department of physics and chemistry. faculty of mechanical engineering, electrical engineering and informatics. széchenyi istván university. egyetem sq. 1, győr, h-9026, hungary b enea, cr casaccia, sspt-bioag-probio, via anguillarese, 301, 00123 santa maria di galeria, roma, italy c institute for plant production. faculty of agricultural and food sciences. széchenyi istván university. mosonmagyaróvár vár 2, h-9200, hungary d laboratory of biophysics, wageningen university, dreijenlaan 3, wageningen, 6703 ha, the netherlands e faculty of horticultural sciences, szent istván university, budapest xl. villányi út 35-43, h-1118, hungary f kaposvár university, institute of physiology, biochemistry and animal health, kaposvár guba sándor út 40, h-7400, hungary article info article history: received: 1st january 2017 accepted: 19th may 2017 keywords: buckwheat grain rutin content uv photoacoustic spectroscopy abstract a relatively novel approach for easy and quick determination of rutin in buckwheat grain is suggested. the rutin content of the grain in seven common buckwheat (fagopyrum esculentum) and six tartary buckwheat (fagopyrum tataricum) varieties was investigated by means of uv photoacoustic spectroscopy and hplc as reference method. the lowest content was found in ’botan’ and ’bamby’ varieties, while the highest values were obtained in the variety ’emka’. rutin content in grain of all tartary buckwheat varieties was two orders of magnitude higher than in the other varieties. rutin content in f. esculentum ranges between 9 and 36 mg/100 g dry weight as compared to 921 to 2 132 mg/100 g dry weight in f. tataricum. the uv photoacoustic spectroscopy data show rather good correlations of r2=0.977 and r2=0.980 with values obtained by hplc data for all measured samples. therefore, uv photoacoustic spectroscopy can be a cheap and quick method for determining rutin content in buckwheat grain.  university of ss. cyril and methodius in trnava introduction flavonoids that constitute the largest group of phytonutrients are found in almost all fruits and vegetables. like carotenoids, they are responsible for vivid and various colors in fruits and vegetables. one of them, rutin, occurs in high concentrations in buckwheat (fagopyrum esculentum). most rutin is accumulated in the inflorescence, stalks and upper leaves (kreft et al. 2006). it is also found in leaves and petioles of rheum species and asparagus, in the fruits and flowers of pagoda tree, fruits and fruit rinds mainly of citrus fruits (like orange, grapes, lemon, lime) as well as in ash tree fruits and in berries such as mulberry and cranberries (sánchez-salcedo et al. 2015). buckwheat is a major source of natural rutin (gupta et al. 2011). rutin was detected in the whole buckwheat plant including leaves, flowers, stalks and seeds. nevertheless, plants contain the highest concentration of rutin bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:41 utc mailto:doka.otto@sze.hu nova biotechnol chim (2017) 16(1): 61-67 62 at the beginning of flowering stage (choi et al. 1996). however the amount of rutin at different parts of the plant is greatly dependent on plant species and its geographical origin (kitabayashi et al. 1995). regarding the varieties, the rutin content was much higher for fagopyrum tataricum than for fagopyrum esculentum (fabjan et al. 2003; park et al. 2004). buckwheat is mainly consumed as a seed. in addition to rutin, buckwheat is a rich source of starch and also contains many other valuable compounds such as proteins, antioxidative substances, trace elements and dietary fibre (park et al. 2000; bonafaccia et al. 2003). whole buckwheat groats contain 55 % starch, 12 % protein, 4 % lipid, 2 % soluble carbohydrates, 7 % total dietary fibre, 2 % ash and 18 % other components (organic acids, phenolic compounds, tannins, etc.) (eggum et al. 1980; thacker et al. 1983; vojtíšková et al. 2012). rutin is an important food component beneficial for human health since it affects positively the capillary fragility and permeability (kamalakkannan and stanely mainzen 2006). it has a protective effect against development of diabetes and mitigates the effects on the consequences of diabetes (je et al. 2002; srinivasan et al. 2005). moreover, it has antioxidative and anti-platelet formation properties as well (afanas’eva et al. 2001; sheu et al. 2004). in addition, it has a mitigating effect on cardiovascular diseases (he et al. 1995), it has also anticancer activity (giménez-bastida and zieliński 2015) and antimutagenic activity (brindzova et al. 2009). many sensitive analytical methods for determination of rutin have been developed including capillary electrophoresis (lu et al. 2008; zhang et al. 2013), chemiluminescence (cheng et al. 2013), fourier transform infrared spectroscopy (ftir) and uv-vis spectrophotometry (rajan and muthukrishnana 2013; xu et al. 2010) and high-performance liquid chromatography (hplc) (deineka et al. 2004; šatínský et al. 2013). however, some of the above mentioned methods, for example the hplc, are time-consuming, expensive, need complicated pretreatments (e.g. multisolvent extraction) and require trained technicians to conduct the study. here we suggest detection of rutin by photoacoustic spectroscopy (pas). this technique offers several advantages above other techniques: it is non-destructive, it requires a minimum of preparation and can be readily applied to samples that are difficult to analyze like optically opaque powders (yasa et al. 1982; dóka et al. 2004). the main objective of the research undertaken in this work was to explore the feasibility of pas for quantification of rutin content of thirteen whole grain buckwheat meals. the data obtained was compared to those acquired from the same samples by hplc that served as golden standard. experimental fagopyrum esculentum varieties were grown in the summer of 2005 at camigliatello silano on the high plain of sila in the region of calabria (southern italy). grain samples were obtained from both, an agronomic trial for variety assessment and seed multiplication plots. in addition, six f. tataricum varieties grown on the nearby massif of pollino in the region of basilicata, were included. grains of the following varieties were analysed: i.) f. esculentum varieties ’bamby’, ’emka’, ’jana’, ’kora’, ’botan’, ’hruszkowska’, ’lena’ from an agronomic trial in camigliatello silano; ii.) f. tataricum varieties ’donan’, ’golden’, ’ishisoba’, ’chumoa’, ’hei qiao-4’ and ’hei feng’ from an agronomic trial on the massif of pollino. hplc analysis the quantification of rutin was carried out by waters hplc (waters corp., milford, massachusetts, usa) equipped with a plus autosampler, a binary hplc pump and a dual absorbance detector operating at 350 nm wavelength. whole meal was prepared from the grain using a foss tecator cyclotec 1093 sample mill. samples of 1 g each were extracted with 10 ml methanol for 24 h at room temperature and in darkness. the volume of methanol for f. tataricum varieties was doubled (20 ml) because of the expected higher rutin content. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:41 utc nova biotechnol chim (2017) 16(1): 61-67 63 fig. 1. uv-pas system for measurement of rutin. a) the scheme of the self-made system and cross-section of the pa cell. a – transparent quartz window; b – sample holder; c – metal rod; d – eccentric wheel; e – lever of the sample holder; f – o-ring; g – capillary; h – microphone; i – sample. b) detailed view to the holder and the detector. at the end of the extraction period the vials were kept for 5 minutes in an elma transsonic t420 ultrasonic bath operating at a frequency of 35 khz. the methanol fraction was recovered by centrifuging for 5 min at 15 000 rpm (hettich, tuttlingen, germany). before analysis the methanol fraction was filtered by passing through a millex hn 0.45 syringe-driven filter unit (millipore corp., billerica, massachusetts, usa). the active content was separated on a symmetry c18 5 μm 4.6×150 column. the analytical process was assisted by empower 2tm software. the analysing conditions were: 2.5 % acetic acid in water (350 ml), meoh (50 ml) and acetonitrile (100 ml) as a mobile phase. the flow-rate was 1 ml min–1and pressure on the column 1 750±10 psi. twenty μl of seed extract in methanol was used for rutin determination. two replicated analyses were run for each sample. reference was made to a solution of rutin hydrate [153-18-4] containing 2.65 mg dissolved in 25 ml of methanol (hplc quality). results were expressed in mg of rutin per 100 g of grain dry weight (dw). uv photoacoustic spectroscopy (uv-pas) in the uv-pas the sample to be investigated is irradiated by a modulated beam. the fraction of the energy absorbed by the sample is converted to heat, while the temperature of sample oscillates periodically at a frequency identical to that of the modulated radiation. generated thermal waves eventually reach sample’s surface and cause a periodic heating and cooling of the contacting layer of the surrounding gas. finally, the expansions and contractions of the gas give rise to acoustic waves; these are detected as a voltage (termed pa signal) by a microphone. the optical and thermal parameters of sample and the contacting gas all play a decisive role in the generation of pa signal. in order to eliminate the variation of the output power on the emission wavelength of the power on the emission wavelength of the illuminating source it is customary to normalize the pa signal from the sample to that obtained (under identical experimental conditions) from the carbon black powder acting as a strongly absorbing reference sample. such normalized signal ratio is expressed in arbitrary units (a. u.). four major parts of the self-made pa spectrometer used in this study are the 1000 w xe lamp (oriel technology), the monochromator (jobin-yvon, h-10, spectral resolution of 16 nm), a modulator and a self-made pa cell (fig. 1). after passing through the monochromator, the collimated beam of mechanically chopped (17 hz) radiation was collected by a quartz lens and focussed into the pa cell. radiation enters the pa cell (fig. 1) through a quartz window half inch in diameter. a) b) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:41 utc nova biotechnol chim (2017) 16(1): 61-67 64 fig. 2. characteristic hplc chromatograms for extraction from jana f.esculentum (a) and chumoa f. tataricum (b) showing that rutin content of the former group is more than 30-times higher. the chromatographic peak of rutin elutes about 2.08 min of running time for the total extract. the sampling volume and a miniature (4.2 x 4.75 mm) microphone (sennheiser ke 4-211-2) are acoustically coupled by means of a 3 mm long capillary. the pa cell is sealed (closed) when the lever of the sample holder is rotated by 180°. in such a case the metal rod presses the sample holder against the o-ring which, due to the eccentricity of the wheel, seals the pa detector. the buckwheat meal was loaded into a semispherical cavity made in the sample holder. the pa signal was processed by a preamplifier (stanford sr552) and a dual phase lock-in amplifier (stanford sr530) connected to the computer. with each sample the pa signal was recorded in the range of 250 to 600 nm. three independent measurements with each sample have been performed. results hplc measurements hplc chromatograms were obtained for samples of f. esculentum and f. tataricum varieties (fig. 2). peaks assigned to rutin appeared in the chromatograms after 2.08 min of running time for the complete extract. as expected the content of rutin in the grain of f. tataricum was over 30-times higher when compared to that of f. esculentum (table 1). an appreciable variation in the rutin content of the grains was apparent among varieties of f. esculentum, ranging from 9.0 mg for the variety ’botan’ to 36.2 mg for ’emka’ (in 100 g grain dw). similarly, in f. tataricum varieties it varied between 921 mg (’chumoa’) and 2 132 mg (’donan’). uv-pa measurements the same buckwheat grain samples were also measured using uv-pas. the spectra between 250 nm and 600 nm were recorded using the pa spectrometer described in the methods part. the obtained data (figs. 3a,b) were normalized to the power output of xe lamp. the spectra feature two specific peaks in the investigated range. one of them centered at 275 nm due to the protein content of the samples, while the other (stronger) peak at 378 nm (fig. 3b) can be attributed to rutin in samples. however, the pa signal increases across the entire wavelength range in proportion to the content of rutin in the sample. the net normalized pa signals at 378 nm plotted versus the rutin content for f. esculentum (fig. 4a) and f. tataricum (fig. 4b) varieties obtained by hplc is presented in fig. 4. the correlations of data by the two methods are linear with rather good determination coefficients for samples of both varieties (r2=0.977 and r2=0.980, respectively). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:41 utc nova biotechnol chim (2017) 16(1): 61-67 65 table 1. rutin content of buckwheat grain of seven f. esculentum varieties grown at camigliatello silano (region of calabria) and six f. tataricum varieties grown at terranova del pollino (region of basilicata) measured by hplc. varieties rutin (mg/100 g dry weight) replicated analysis averagestdv 1st 2nd f. esculentum bamby 9.2 10.1 9.70.06 botan 9.6 8.3 9.00.93 emka 38.0 34.4 36.22.61 hruszkowska 22.4 21.6 22.00.52 jana 22.4 21.0 21.71.04 kora 23.6 21.8 22.71.32 lena 29.0 29.7 29.30.45 f. tataricum donan 2 115.7 2 148.5 2 132.123.16 golden 2 107.9 2 059.4 2 083.734.29 ishishoba 1 762.1 1 660.5 1 711.371.84 hei feng 1 001.1 1 024.2 1 012.611.53 hei qiao-4 1 265.6 1 280.8 1 273.27.61 chumoa 908.8 927.5 918.29.35 discussion uv-pas was tested as a possible new anaytical method for quick and simple measurement of rutin content. the pa spectra obtained for the two varieties are very similar reflecting to relatively small differences in the content of rutin in the samples (up to only 27 mg/100 g). this latter fact explains the small slope of the calibration curve. since rutin content in f. tataricum is two orders of magnitude higher than that in f. esculentum, the measured spectra have a more pronounced absorption at the given analytical wavelength. for f. esculentum varieties the pa signal can be increased by using a more powerful laser at the analytical wavelength. our parallel hplc measurements confirmed that rutin content varies from about 10 mg to a few thousands milligrams per 100 g dry weight in the whole grain meal of buckwheat varieties. calculated lod value suggests that the method can be used successfully in the analyzed buckwheat varieties that likely can be extended to even another one. the other parts of the plant (root, stem, leaf, flower) have rutin content between 90 and 380 mg/100 g (choi et al. 1996) and so the obtained lod is enough to quantify the rutin content in different parts of the plant. fig. 3. normalized photoacoustic spectra of seven fagopyrum esculentum (a) and of six fagopyrum tataricum (b) varieties between 250 and 600 nm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:41 utc nova biotechnol chim (2017) 16(1): 61-67 66 fig. 4. the normalized pa signal (at 378 nm and 17 hz) plotted versus the rutin content of fagopyrum esculentum (a) and fagopyrum tataricum (b) as determined by hplc. data represents the averages and the standard deviations of triplicate measurements. conclusions uv-pas was explored as a candidate analytical tool for rapid and simple quantification of rutin in buckwheat. in both of the tested varieties the magnitude of measured pa signal appears to be linearly dependent on the rutin content of the samples. once a calibration curve is constructed using samples of known rutin content, buckwheat samples can routinely be analyzed directly. as no pre-treatment is required, the time for completing the analysis is reduced quite significantly. references afanas’eva ib, ostrakhovitch ea, mikhal’chik ev, ibragimova ga, korkina lg (2001) enhancement of antioxidant and anti-inflammatory activities of bioflavonoid rutin by complexation with transition metals. biochem. pharmacol. 61: 677-684. bonafaccia g, marocchini m, kreft i (2003) composition and technological properties of the flour and bran common and tartary buckwheat. food chem.: 80: 9-15. brindzova l, mikulasova m, takacsova m, mosovska s, opattova a (2009) evaluation of the mutagenicity and antimutagenicity of extracts from oat, buckwheat and wheat bran in the salmonella/microsome assay. j. food compos. anal. 22: 87-90. cheng c, huang y, wang j, zheng b, yuang h, xiao d (2013) anodic electrogenerated chemiluminescence behavior of graphite-like carbon nitride and its sensing for rutin. anal. chem. 85: 2601-2605. choi bh, kim sl, kim sk (1996) rutin and functional ingredients of buckwheat and their variations. korean j. crop. sci. 41: 69-93. dóka o, bicanic d, dicko mh, slingerland m (2004) photoacoustic approach to direct determination of the total phenolic content in red sorghum flours. j. agric. food chem. 52: 2133-2136. deineka vi, grigor’ev am, staroverov vm (2004) hplc analysis of flavonoids: determining rutin in plant extracts. pharm. chem. j. 38: 487-489. eggum bo, kreft i, javornik b (1980) chemical composition and protein quality of buckwheat (fagopyrum esculentum moench). plant food hum. nutr. 30: 175179. fabjan n, rode j, kosir ij, wang z, kreft i (2003) tartary buckwheat (fagopyrum tataricum gaertn.) as a source of dietary rutin and quercetin. j. agr. food chem. 51: 6452-6455. gupta n, sharma sk, rana jc, chauhan rs (2011) expression of flavonoid biosynthesis genes vis-a-vis rutin content variation in different growth stages of fagopyrum species. j. plant physiol. 168: 2117-2123. giménez-bastida ja, zieliński h (2015) buckwheat as a functional food and its effects on health. j. agr. food chem. 63: 7896-7913. he j, klag mj, whelton pk, mo jp, chen jy, qian mg, mo ps, he gq (1995) oats and buckwheat intake and cardiovascular disease risk factors in an ethnic minority of china. am. j. clin. nutr. 61: 366-372. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:41 utc nova biotechnol chim (2017) 16(1): 61-67 67 je hd, shin cy, park sy, yim sh, kum c, huh ih, kim jh, sohn ud (2002) combination of vitamin c and rutin on neuropathy and lung damage of diabetes mellitus rats. arch. pharm. res. 25: 184-190. kamalakkannan n, stanely mainzen pp (2006) antihyperglycaemic and antioxidant effect of rutin, a polyphenolic flavonoid, in streptozoticin-induced diabetic wistar rats. basic clin. pharmacol. 98: 97-103. kitabayashi h, ujihara a, hirose t, minami m (1995) varietal differences and heritability for rutin content in common buckwheat, fagopyrum esculentum moench. breeding sci. 45: 75-79. kreft i, fabjan n, yasumoto k (2006) rutin content in buckwheat (fagopyrum esculentum moench) food materials and products. food chem. 98: 508-512. lu qh, ba cd, chen dy (2008) investigating noncovalent interactions of rutin-serum albumin by capillary electrophoresis-frontal analysis. j. pharm. biomed. anal. 47: 888-891. park bj, park ji, kwang jc, park ch (2004) comparison in rutin content in seed and plant of tartary buckwheat (fagopyrum tataricum). in: proceeding of the 9th international symposium on buckwheat. prague, czech republic, august 18–22, 2004, 626-629. park ch, kim yb, choi ys, heo k, kim sl, lee kc, chang kj, lee hb (2000) rutin content in food products processed from groats, leaves and flowers of buckwheat. fagopyrum. 17: 63-66. rajan t, muthukrishnana s (2013) characterization of phenolic compounds in pseudarthria viscida root extract by hplc and ft-ir analysis. asian j. pharm. clin. res. 6: 274-276. sánchez-salcedo em, mena p, garcía-viguera c, martínez jj, hernández f (2015) phytochemical evaluation of white (morus alba l.) and black (morus nigra l.) mulberry fruits, a starting point for the assessment of their beneficial properties. j. func. foods. 12: 399408. šatínský d, jägerová k, havlíková l, solich p (2013) a new and fast hplc method for determination of rutin, troxerutin, diosmin and hesperidin in food supplements using fused-core column technology. food anal. methods. 6: 1353-1360. sheu jr, hsiao g, chou ph, shen my, chou ds (2004) mechanisms involved in the antiplatelet activity of rutin, a glycoside of the flavonoid quercetin, in human platelets. j. agr. food chem. 52: 4414-4418. srinivasan k, kaul cl, ramarao p (2005) partial protective effect of rutin on multiple low dose streptozotocininduced diabetes in mice. indian j. pharmacol. 37: 327328. thacker pa, anderson dm, bowland jp (1983) chemical composition and nutritive value of buckwheat cultivars for laboratory rats. can. j. anim. sci. 63: 949-956. xu h, li y, tang hw, liu cm, wu qs (2010) determination of rutin with uv-vis spectrophotometric and laser-induced fluorimetric detections using a nonscanning spectrometer. anal. lett. 43: 893-904. yasa za, jackson wb, amer nm (1982) photothermal spectroscopy of scattering media. appl. opt. 21: 21-31. vojtíšková p, kmentová k, kubáň v, kráčmar s (2012) chemical composition of buckwheat plant (fagopyrum esculentum) and selected buckwheat products. j. microbiol. biotechnol. food sci. 1: 1011-1019. zhang f, qi x, zou m, li j (2013) analysis of rutin from lespedeza virgata (thunb.) dc. by microwave-assisted extraction and capillary electrophoresis. j. chem. 2013: article id 324294. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:41 utc microsoft word luptakova et al nbc 2015.doc nova biotechnologica et chimica 14-2 (2015) 135 doi 10.1515/nbec-2015-0022 © university of ss. cyril and methodius in trnava bacterial reduction of barium sulphate by sulphate-reducing bacteria alena luptáková1, ingrida kotuličová1, magdaléna bálintová2, štefan demčák2 1slovak academy of sciences, institute of geotechnics, department of mineral biotechnologies, watsonova 45, 040 01 košice, slovak republic (kotulicova@saske.sk) 2technical university of košice, civil engineering faculty, institute of environmental engineering, vysokoškolská 4, 040 01 košice, slovak republic (magdalena.balintova@tuke.sk) abstract: acid mine drainage (amd) is a worldwide problem leading to contamination of water sources. amd are characterized by low ph and high content of heavy metals and sulphates. the barium salts application presents one of the methods for the sulphates removing from amd. barium chloride, barium hydroxide and barium sulphide are used for the sulphates precipitation in the form of barium sulphate. because of high investment costs of barium salts, barium sulphide is recycled from barium sulphate precipitates. it can be recycled by thermic or bacterial reduction of barium sulphate. the aim of our study was to verify experimentally the possibility of the bacterial transformation of baso4 to bas by sulphatereducing bacteria. applied baso4 came from experiments of sulphates removal from smolnik amd using bacl2. key words: acid mine drainage, sulphate-reducing bacteria, barite, bacterial reduction. 1. introduction acid mine drainage (amd) is a big problem in mining industry and major environmental concern. it causes surface water pollution, which may impact aquatic life as well as whole ecosystem (johnson and hallberg, 2003). acid mine waters are characterized by low ph and high content of heavy metals and sulphates. in order to minimize negative impacts of amd appropriate treatment process has to be chosen (jenčárová, 2008; mačingová, 2008). these processes are focused on neutralizing, stabilizing and removing pollutants. from this reason efficient and environmental friendly methods are needed to be developed in order to reduce heavy metals as well as sulphates (singovszká, 2013; holub, 2014). according to slovak legislation – government regulation no. 269/2010, the limit concentration of sulphates in surface and drinking water is 250 mg/l. increased content of sulphates concentrations can be remediate with various existing sulphate treatment processes (singh and chugh, 2010): precipitation of sulphates with lime and limestone, precipitation using barium salts, sulphate precipitation in the presence of aluminium and calcium ions, sulphate removal using aluminous cement, membrane processes, ion exchange process, sorption and biological sulphate removal methods using sulphate reducing bacteria (srb). biological reduction, compared to chemical treatment, has more advantages: it is effective in sulphate elimination, it does not produce bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:53 utc 136 luptáková, a. et al. amounts of waste, it is cost effective and has added advantage of removing trace metals from mine water. principle of the chemical precipitation methods using barium salts is sulphate precipitation in form of barium sulphate (insoluble compound). barium chloride, barium hydroxide and barium sulphide are used for precipitation: bacl2 + h2so4 → baso4 + 2hcl (1) ba(oh)2 + h2so4 → baso4 + 2h2o (2) bas + h2so4 → baso4 + h2s (3) because of high investment costs of barium salts, barium sulphate precipitates are transformed to the form of barium sulphide. bas can be transformed (i.e. recycled) chemically by thermic reduction of barium sulphate with carbon in the form of coal at a temperature of 1200 °c to barium sulphide (hammack and hedin, 1995): baso4 + 2c → bas + 2co2 (4) like that the recycled bas can be used in the process of the sulphates removal from waste waters again. the key factor affecting the process economy during the chemical reduction is high energy consumption. bacterial reduction of barium sulphate, to obtain barium sulphide by srb, could be used as an alternative and more economical recycling method. srb represent the bacteria that use sulphates as a terminal electron acceptor for their metabolism. these bacteria realize the conversion of sulphate to hydrogen sulphide under anaerobic conditions (odom and singleton, 1993) and utilize organic compounds which range from simple organic acids (acetate and lactate) and ethanol to long-chained fatty acids and certain aromatic compounds. the aim of our study was to verify experimentally the possibility of the bas recycling by the bacterial reduction of baso4 using srb. applied baso4 came from experiments of sulphates removal from smolnik amd using bacl2. 2. materials and methods in the experiment a culture of srb (genera desulfovibrio) was used. bacteria were isolated from natural mineral water gajdovka (source in the area of kosice north). for the isolation and cultivation of srb the selective medium dsm-63 also known as j. postgate was used. for the experiment of the sulphates reduction the modified medium dsm – 63 was used. this medium contents sodium lactate as carbon source and barium sulphate as sulphate source. applied baso4 came from experiments of sulphates removal from smolnik amd, using bacl2 (demčák, 2014). the amount of baso4 was calculated on the base of sulphates content in the selective medium dsm-63 (luptáková et al., 2010). experiments were realized in the reagent bottles at 30 °c for 40 days under static and anaerobic conditions. the inoculum of srb was 10% (v/v). the initial ph value of the modified medium was 7.2. the abiotic control was carried out without the srb application at the same conditions. composition of samples was as follows: sample srb – 180 ml modified medium dsm-63 + 20 ml srb inoculum + 0.66 g baso4; sample k (abiotic control) – 200 ml modified medium dsm-63 + 0.66 g baso4. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:53 utc nova biotechnologica et chimica 14-2 (2015) 137 sulphates content was determined by ion chromatography method. soluble sulphide was analyzed using a methylene blue method based on a colorimetric determination of dissolved h2s and hs – by n,n-dimethyl-p-phenylenediamine. the composition of precipitates was analyzed by x-ray diffraction technique. 3. results and discussion we assumed the bacterial reduction of sulphates by srb in the presence of sodium lactate as an electron donor and baso4 as an electron acceptor under anaerobic conditions according to equations (5) and (6) (baldi et al., 1996): srb na-lactate + baso4 → biomass + baco3 + bas + co2 + h2s (5) baso4 + 2h2s ⎯→ bas + so2 + 2h2o + s (6) the changes of sulphates and hydrogen sulphide concentrations are plotted in fig. 1 and fig. 2. in the case of biotic conditions the initial sulphates concentration (20.00 mg/l) was caused by the sulphates concentration from the srb inoculum. after inoculation during the first and the second days the sulphates concentration was rapidly increased. consequently the decreasing of sulphates concentration and the increasing of hydrogen sulphide concentration were recorded (eq.5). fig. 1. changes of sulphates concentration. fig. 2. changes of hydrogen sulphide concentration. the presence of sulphates was estimated in solution at abiotic conditions too. probably the culture medium was caused the dissolution of baso4 by the influence of the calcium chloride which is a basic constituent of the applied culture medium dsm63 (hugdahl et al., 2007). however, the listed culture medium influence was not observed after the tenth days of experiments. on the fifth day the hydrogen sulphide concentration maximum (18.5 mg/l) was observed. at the same time the acetate i.e. the metabolic product of srb was detected as the next carbon source for srb (fig. 3). consequently the decreasing of lactate concentration and the increasing of acetate concentration were recorded. in the experiment a culture of srb was substituted by the genera desulfovibrio. this genus cannot utilize of acetate (odom and singleton, 1993) and the process of the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:53 utc 138 luptáková, a. et al. bacterial sulphate reduction was gradually stopped. in the course of experiments the release of barium to the liquid phase was observed (fig. 4). ten times the quantity of barium was dissolved by bacteria (2.96 mg/l) than in the un-inoculated medium (0.35 mg/l). in the case of abiotic conditions, the presence of hydrogen sulphide and the acetate was not proved (fig. 2 – sample k and fig. 3 – sample k-acetate); in the course of 10 days the culture medium was caused the dissolution of baso4 probably by the influence of the calcium chloride and the final concentration of ba was 0.35 mg/l (fig. 4). fig. 3. utilization of lactate by srb and creation of acetate. fig. 4. release of barium to liquid phase. after 40 days accrued precipitates from the samples srb and k were separated from liquid phase, saved at anaerobic conditions and analysed by x-ray diffraction technique. the presence of bas was not confirmed in the precipitates composition (fig. 5). fig. 5. x-ray diffraction spectrum of precipitates (sample srb). for all the informative experiments of the sulphates elimination from amd by the recycled bas were realized. the obtained results were assigned the sulphates bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:53 utc nova biotechnologica et chimica 14-2 (2015) 139 decreasing only in the case of bas prepared at the srb influence (fig. 6). the bacterially prepared bas as the sulphates precipitating agent was effected 24.4 % decreasing of sulphates from amd. the precipitates take from abiotic conditions were induced the very minimal decreasing of sulphates from amd – 0.11%. fig. 6 sulphates elimination from amd. amd – amount of sulphates in amd before elimination (1720 mg/l); srb – amount of sulphates in amd after application of bacterially prepared bas (1400 mg/l); k amount of sulphates in amd after application of precipitates from abiotic control (1718 mg/l). baldi et al. (1996) observed that barium sulfide may form in the process of bacterial barite reduction and dissolution, it could not be positively identified in the solid phase. despite its instability in aqueous solution, probably bas is a reactive transient species stabilized on a colloidal or solid phase. polysaccharide complexes which frequently form organic coatings on suspended matter may account for barium sulfide stabilization. these organic coatings i.e. biofilms could be associated with the biomass production. the polysaccharides of bacterially cells represent the suitable specific microenvironment for the bas stabilization. 4. conclusions the main purpose of this work was to investigate the possibility of the bas recycling by the bacterial reduction of baso4 using the sulphate-reducing bacteria. the obtained results demonstrate the influence of srb (genera desulfovibrio) on the studied baso4 came from experiments of sulphate removal from smolnik amd. in spite of the presence of bas was not confirmed by the x-ray diffraction methods in the precipitates composition. for all the informative experiments of the sulphates elimination from amd by the bacterially recycled bas were assigned the sulphates decreasing. specific explanation could be associated with the research of baldi et al. (1996). probably accrued bas was stabilized on a colloidal or solid phase by the bacterially cells polysaccharides. acknowledgement: this work was supported by the slovak research and development agency under the contract no. srda-0252-10 and slovak grant agency project no. 2/0145/15. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:53 utc 140 luptáková, a. et al. references baldi, f., pepi, m., burrini, d., kniewald, g., scali, d., lanciotti, e.: dissolution of barium from barite in sewage sludges and cultures of desulfovibrio desulfuricans. appl. environ. microbial.., 62, 1996, 2398-2404. demčák, š.: odstraňovanie síranov z modelových roztokov pomocou vybraných metód. diplomová práca, technická univerzita v košiciach, stavebná fakulta, košice, 2014, 57 pp. government regulation no. 296/2010 of slovak body of laws included general requirements for surface water quality in slovak republic. hammack, r., hedin, r.: microbial sulphate reduction for the treatment of acid mine drainage: a laboratory study, us bureau of mines, pittsburgh, pennsylvania, usa, 1995, 13 pp. holub, m.: potenciál prírodných materiálov pre odstránenie ťažkých kovov z kyslých banských vôd sorpcia. juniorstav 2014. 16. odborná konference doktorského studia s mezinárodní účastí, sborník anotací : 30.1.2014, vut, brno 2014,1-7. hugdahl, m., lintott, d., ashworth, j., tindal, m.: phase 2 optimization of soluble and total barium methods. bclqaac barite task group, june 5, 2007. jenčárová, j.: the bacterially preparation of iron sulphides. acta metallur., slov., 14, 2008, 106-109. johnson, d.b., hallberg, k.b.: the microbiology of acidic mine waters. res. microbiol., 154, 2003, 466-473. luptáková, a., mačingová, e., solozhenkin, p.m.: biological transformation of alkaline earth metal sulphates. mineralia slov., 42, 2010, 329332. mačingová, e.: the possibility of the treatment of acid mine drainage. acta metallurg. slov., 14, 1, 2008, 155161. odom, j.m., singleton, j.r.: the sulphate-reducing bacteria contemporary perspectives. springer-verlag, new york, 1993. singh, g., chugh, y. p.: sulphate: occurence, problems & control measures. journal of indian school of mines, special volume, 2010, 29-48. singovszká, e.: charakterizácia kvality vody a sedimentov v potoku smolník pomocou zhlukovej analýzy. seminár doktorandov 2013, seminár doktorandov v odbore environmentálne inžinierstvo, 5. ročník, zborník príspevkov, košice, svf tu, košice, 2013, 61-63. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 09:53 utc nova biotechnol chim (2022) 21(2): e1483 doi: 10.36547/nbc.1483 1 nova biotechnologica et chimica evaluation of chromatographic performance of three c18 columns for avenanthramides separation timotej jankech1,, jozef sokol1, mária maliarová1, ivana gerhardtová1, nicholas martinka2, marcela blažková3, michaela havrlentová3 1department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, j. herdu 2, trnava 91701, slovak republic 2department of clinical biochemistry, alexander winter hospital, winterova 66, piešťany 92101, slovak republic 3department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius in trnava, j. herdu 2, trnava 91701, slovak republic  corresponding author: timotej.jankech@gmail.com article info article history: received: 21st april 2022 accepted: 1st july 2022 keywords: avenanthramides chromatographic parameters high performance liquid chromatography reversed-phase column separation efficiency abstract phenolic amides contained in oats (avenanthramides; avns) are biologically active substances with strong antioxidant activity. in this paper, we evaluated efficiency of three c18 chromatographic columns (symmetry, xbridge, cortecs) with different particle technology and particle sizes for the separation of three major avenanthramides (avn a, avn b, avn c). we compared columns in terms of retention times, retention factors of avns and in terms of other parameters such as number of theoretical plates, height equivalent to a theoretical plate, reduced plate height, resolution and in terms of peak symmetry, respectively. limits of detection and limits of quantification of avns on all columns were calculated. retention results of avns on individual columns showed a significant reduction in retention times of avns on solid core column with a particle size 2.7 μm compared to columns with particle size 3.5 μm. within columns with 3.5 μm particles, separation on symmetry c18 column appeared to be more efficient than on the xbridge c18 column. in general, results achieved on cortecs c18 column can be considered as the best in terms of both separation efficiency and retention times. introduction avenanthramides (avns) are exceptional low molecular weight polyphenol amides produced in oats as phytoalexins (secondary metabolites) (wise 2013; ishihara et al. 2014; tripathi et al. 2018). avns were first described in 1989 by collins as a group of hydroxycinnamoylanthranilate alkaloids (chen et al. 2007). avns are derivatives of hydroxycinnamic acid and anthranilic acid coupled by an amide bond. it has been described approximately 40 avns consisting of these two acids so far. among the other minor avns in oats, which are generally not included in the quantitative determinations, there are three major avns, the most important (fig. 1): avenanthramide a (2p), mailto:timotej.jankech@gmail.com nova biotechnol chim (2022) 21(2): e1483 2 avenanthramide b (2f) and avenanthramide c (2c) (dokuycu et al. 2003; ishihara et al. 2014; pridal et al. 2018; tripathi et al. 2018; li et al. 2019; hernandez-hernandez et al. 2021). they are present in higher concentrations mainly in oat grains bran and outer layers of the kernel but may also be present in other parts of the plant (boz 2015; kulichová et al. 2018). avns are relatively stable compounds in uv light and in acidic and neutral ph. in alkaline ph (7-12), avn b slightly decompose, avn c decompose to a greater extent and above ph 12 avn c is completely decomposed, but avn a is stable in this ph range and thus the stability depends on the structure (dimberg et al. 2001). in addition to natural avns, there are also their synthetic structural analogues. an example is the drug called tranilast, chemically (n-[3′,4′-dimethoxycinnamoyl]-anthranilic acid), firstly described by koda et al. in 1976. tranilast has been licensed in japan and south korea since 1982. it is mainly used in the treatment of bronchial asthma and atypical dermatitis (koda et al. 1976; darakhshan and pour 2015; perrelli et al. 2018). name (miyagawa et al. 1995) designation r1 r2 r3 n-caffeoyl-5-hydroxyanthranilic acid avn c 2c oh oh oh n-p-coumaroyl-5-hydroxyanthranilic acid avn a 2p oh h oh n-feruloyl-5-hydroxyanthranilic acid avn b 2f oh och3 oh fig. 1. structure of three main avns, where n = 1 (structure made in chemdraw professional 16.0 software). avns as natural substances, in addition to their high antioxidant activity, which has been demonstrated in vitro and in vivo, are characterized by many other potential effects such as anti-irritant and anti-inflammatory. avns as strong antioxidants are able to prevent development of diseases caused by oxidative stress (e.g., cancer) (sur et al. 2008; meydani 2009; gani et al. 2012; turrini et al. 2019). many various methods for extraction and determination of avns have been proposed (gangopadhyay et al. 2015). methanol or ethanol is most often used as the extraction agent (pridal et al. 2018). the conditions under which the three main avns are extracted to the maximum extent (extraction with 70 % methanol at 55 °c) have been described by maliarová et al. (2015). these authors used response surface methodology (rsm) to determine the optimal parameters for the extraction of avns from oat. a relatively large number of methods have been proposed for the quantification of avns in oats. reverse-phased high performance liquid chromatography (rphplc) coupled with uv detection is frequently used because of fact that avns absorb the ultraviolet light (maximum in range of 315 – 365 nm) with ε in range of 23,000 – 28,000 dm3.mol-1.cm-1. however, the disadvantage of uv detection is the limit of quantification (100 – 400 ng.ml-1). based on this, several methods have been developed using hplc-ms (using negative or positive ionization mode) or partially selective but sensitive electrochemical detection to determine avns in some types of oats (jastrebova et al. 2006; xie et al. 2017; kulichová et al. 2018; de bruijn et al. 2019). none of published papers do not refer to performance evaluation of different rp c18 stationary phases for three main avns separation (e.g., in terms of separation efficiency). the aim of this paper was to evaluate the chromatographic performance of three different endcapped c18 columns including classic silica substrate, hybrid substrate and solid core column as nova biotechnol chim (2022) 21(2): e1483 3 well for three main avns analysis in oat samples and calculate chromatographic parameters. experimental chemicals, reagents, and samples all standards of avns (a, b, c) were obtained from sigma-aldrich (darmstadt, germany). formic acid (hcooh), acetonitrile (acn) and methanol (hplc grade) were obtained from centralchem, s. r. o. (bratislava, slovakia). ultrapure water was prepared using simplicity uv device. all grinded oat samples were obtained from national agricultural and food centre, research institute of plant production (piešťany, slovakia). preparation of standards and oat samples standard stock solutions (c = 100 μg.ml-1) of avns were prepared in acidic mixture of 0.1 % hcooh in methanol and 0.1 % hcooh in water (70 : 30, v/v) and then diluted to desired concentrations. grinded oat samples (2014 avenuda, 2020 avenuda and 2014 kamil, 2020 kamil) were prepared by modifying the extraction procedure according to (maliarová et al. 2015) as follows: 0.6 g of oats was weighed into a 15 ml tube and 3 ml of acidic mixture 0.1 % hcooh in methanol:0.1 % hcooh in water (70 : 30, v/v) was added. extraction of analytes was performed at 55 °c for 15 min in ultrasonic bath. after extraction step, the samples were centrifuged at 13,000 rpm for 15 min. supernatants were stored at -15 °c until hplc analysis. supernatants were filtered through a 0.45 μm nylon filter before hplc analysis and then injected to hplc system without dilution. hplc system and hplc analysis waters hplc system consisted of 2695 separation module, 2998 photodiode array detector and empower 3 software (waters, milford, ma, usa). separations were performed on three equally long reversed-phase endcapped columns: symmetry c18 100 × 4.6; 3.5 μm (waters, milford, ma, usa), xbridge c18 100 × 4.6; 3.5 μm (waters, milford, ma, usa) and cortecs c18 100 × 4.6; 2.7μm (waters, milford, ma, usa). detailed specifications of used columns are given in table 1. all separations were performed according to previously optimized isocratic method. all chromatographic parameters were calculated from data obtained using this isocratic method: composition of the mobile phase was: (a) 0.1 % hcooh in water: (b) 0.1 % hcooh in acn in volume ratio a : b = 77 : 23; temperature of column was set at 40 °c; flow rate was 1 ml.min-1; injection volume was 10 μl and detection of analytes was carried out with pda scan in range of 210 – 400 nm. table 1. specifications of all three used columns. column dimensions [mm] particle size [μm] particle substrate particle technology surface area [m2.g-1] pore size [å] carbon load [%] symmetry c18 4.6 × 100 3.5 silica 335 100 19 xbridge c18 4.6 × 100 3.5 hybrid beh* 185 130 18 cortecs c18 4.6 × 100 2.7 silica solid core 100 90 6.6 * ethylene bridged hybrid. calculation of chromatographic parameters the retention factor (k), height equivalent to a theoretical plate (hetp), reduced plate height (h), limits of detection (lod) and limits of quantification (loq) were calculated manually according to a literature (dolan 2014; samanidou 2015; şengül 2016; barth 2018; broeckhoven and stoll 2022). other three parameters: resolution (r), tailing factor (t) and number of theoretical plates (n) were calculated using empower 3 system suitability software (waters, milford, ma, usa). nova biotechnol chim (2022) 21(2): e1483 2 results and discussion retention times and retention factors of avns the retention times of the individual avns and their retention factors were monitored at three concentration levels (1; 5; 10 μg.ml-1). table 2 gives an overview of the averages (five injections for each concentration level) of retention times and retention factors of avns on the different columns. table 2. overview of avns retention times (tr) and retention factors (k) on the different columns. retention times of avns were obtained from average of five injections at each concentration level. avenanthramide parameter column symmetry c18 xbridge c18 cortecs c18 avn c tr ± sd [min] 3.685 ± 0.002 2.623 ± 0.003 2.003 ± 0.002 k ± sd 2.802 ± 0.002 1.664 ± 0.003 1.349 ± 0.002 avn a tr ± sd [min] 5.899 ± 0.003 4.006 ± 0.004 3.031 ± 0.003 k ± sd 5.086 ± 0.003 3.068 ± 0.004 2.554 ± 0.003 avn b tr ± sd [min] 7.207 ± 0.005 4.807 ± 0.005 3.649 ± 0.004 k ± sd 6.435 ± 0.006 3.882 ± 0.005 3.279 ± 0.005 avn c eluted first because it contains only oh groups as substituents (fig. 1), which generally reduce the lipophilic character of the molecule and therefore avn c was less retained on nonpolar stationary phase. avn a, which has one hydroxy group in position r2 (fig. 1) replaced by a hydrogen atom, eluted second and avn b, which has a methoxy group attached at this position (making avn b less polar) eluted last. the elution order of avns was the same on all used columns (fig. 2). when comparing individual columns, a decreasing trend of avns retention times as well as their retention factors can be observed. the most significant reduction in avns retention times (and thus analysis times) was observed for the cortecs c18 column (fig. 2). it is important to note here that the cortecs c18 have a particle size of 2.7 μm while the other columns used have 3.5 μm. fig. 2. chromatograms of avns standards on all columns: symmetry c18 100 × 4.6; 3.5 μm (red); xbridge c18 100×4.6; 3.5 μm (blue); cortecs c18 100 × 4.6; 2.7 μm (yellow); the elution order was the same on all columns: 1. peak: avn c, 2. peak: avn a and 3. peak: avn b. 0 1 2 3 4 5 6 7 8 9 a u time [min] symmetry c18 xbridge c18 avn c cortecs c18 avn b avn a 4 nova biotechnol chim (2022) 21(2): e1483 3 however, this is not the only reason for the reduction of avns retention times, the second reason for reduction of avns retention times may be a significantly lower carbon load in the case of cortecs c18 column, which is consistent with the assertion that with decreasing carbon load are analyzes shortened. in comparison of the symmetry c18 and cortecs c18, retention times of avns were reduced by approximately 47.88 % on average, while in comparison of columns with same particle size but different surface area (symmetry c18 – xbridge c18) were retention times of avns reduced by approximately 31.40 % on average. this reduction in retention times in comparison of columns with the same particle size (symmetry c18 and xbridge c18) is due to their different surface area (table 1). in general, higher surface area provides the greater retention of analytes (lakka and kuppan 2019). chromatographic column performance and system suitability of three c18 columns as in the previous case, all chromatographic column performance parameters were calculated from five injections of three different concentrations of avns. an overview of the calculated values of individual parameters on different columns is given in table 3. table 3. overview of calculated column performance parameters. the calculation was performed from five injections of three different concentrations of avns. individual parameter values are listed with the standard deviation. column avn parameter n ± sd r ± sd t ± sd hetp ± sd [μm] h ± sd symmetry c18 c 9475.954 ± 64.671 1.275 ± 0.008 10.553 ± 0.072 3.015 ± 0.021 a 11612.732 ± 48.767 12.028 ± 0.024 1.1659 ± 0.009 8.611 ± 0.036 2.460 ± 0.010 b 11759.878 ± 61.077 5.411 ± 0.014 1.152 ± 0.014 8.504 ± 0.044 2.429 ± 0.013 xbridge c18 c 9277.356 ± 75.656 1.331 ± 0.004 10.779 ± 0.088 3.080 ± 0.025 a 10913.286 ± 94.849 10.574 ± 0.044 1.275 ± 0.008 9.164 ± 0.079 2.618 ± 0.023 b 11175.433 ± 89.350 4.794 ± 0.017 1.254 ± 0.010 8.949 ± 0.072 2.557 ± 0.020 cortecs c18 c 9591.997 ± 386.548 1.441 ± 0.010 10.442 ± 0.438 3.867 ± 0.162 a 12451.522 ± 423.197 10.779 ± 0.209 1.271 ± 0.006 8.039 ± 0.274 2.978 ± 0.102 b 13790.976 ± 426.995 5.324 ± 0.087 1.213 ± 0.005 7.258 ± 0.225 2.688 ± 0.083 in general, for a 4.6 × 100 mm, 5 μm column, n is equal to 5,000 – 8,000 (ravisankar et al. 2017). in our case, we used columns with same dimensions but with smaller particle sizes (3.5 or 2.7 μm). it follows that we should achieve higher n values on all three columns than the typical values given for a column with the same size but larger particles. all three avns had close n values. the n values of all three avns were highest on the cortecs c18 column, which means the most efficient separation was on cortecs c18 column. however, n values obtained on cortecs c18 are accompanied by the highest sd. in the case of avn c, the n values were very similar on all three columns. in case of two other avns, the differences in n values between the cortecs c18 column and two other columns were more considerable. peaks eluting later have higher number of theoretical plates, as confirmed by the data in table 3. another, more general parameter which can be used to evaluate the chromatographic column efficiency is the height equivalent to theoretical plate (hetp) (fornstedt et al. 2015), hetp values for all analytes separated on individual columns are 5 nova biotechnol chim (2022) 21(2): e1483 2 shown in table 3. based on theory (barth 2018), higher values of hetp means lower separation efficiency. it follows that n is directly proportional and hetp is indirectly proportional to the efficiency. based on this, it could be said that the column efficiency expressed by the number of theoretical plates n is in our case consistent with the column efficiency expressed by the height equivalent to a theoretical plate hetp. while for the first eluting analyte (avn c) hetp values were similar for all columns (as in previous discussed parameter). on the other hand, for the last eluting analyte (avn b) the differences between hetp values were more considerable, especially for the cortecs column which has smaller particle size (2.7 μm) and separation on this column was also most efficient in terms of this parameter. columns with different particle sizes were used in our work (two with a particle size 3.5 μm and one with a particle size 2.7 μm), we can use another parameter to compare column efficiency, namely reduced plate height (h). this parameter was designed to allow comparison of separation efficiency between columns with different particle sizes. based on the literature, values between 2 and 3 indicate the optimal separation efficiency (anderson 1995). the same principle as for hetp applies for h, the lower h values mean more efficient separation. as can be seen in table 3, only two values deviate from this theoretically optimal range. the h values were expected to be the lowest for the column with a 2.7 μm particle size, but h values were similar or overlapped with h values for the columns with a 3.5 μm particle size. this phenomenon could be caused by other factors (for example the flow rate of the mobile phase). in the case of a request to reduce the values of this parameter, it would be possible to optimize method by changing chromatographic conditions (e.g., temperature or flow rate of a mobile phase). the symmetry of the peaks is another indicator of efficiency that we have evaluated. peaks having a tailing factor t ≤ 1.5 are acceptable for a large number of applications. if the t value of any peak in the chromatogram is greater than 2 (dolan 2012), which is no longer acceptable, it is necessary to adjust the separation conditions. the tailing factors of the avns did not exceed 1.5 on any of the columns used (table 3). the best results in peak symmetry evaluation were obtained on a symmetry c18 column. slightly higher t values were observed on xbridge c18 and cortecs c18 columns, but values were still acceptable. the highest t value (1.441) was observed on a cortecs c18 column at the avn c peak. based on t values peaks of all three avns on all columns used showed slight tailing. as mentioned above, this effect can also be reduced by adjusting chromatographic conditions, either by changing the mobile phase or by adjusting a ratio of mobile phase components. the last discussed parameter in this category is the resolution (r) for which different calculation methods can be used. however, many data acquisition systems use peak widths at 50 % peak heigh to calculate resolution, as it is easier and advantageous to use with tailing peaks (dolan 2014; ravisankar et al. 2017). resolution between the individual analyte pairs was more than sufficient on all columns and exceeded 4,794 in all cases. the best r was obtained on a symmetry c18 column, where, however, avns had the highest retention times. the result on cortecs c18 column can be considered the best in terms of both separation efficiency and retention times. limits of detection (lod) and limits of quantification (loq) of avns lod and loq values were calculated from the calibration lines (avns concentration range from 250 to 2,500 ng.ml-1), with r2 ≥ 0.9996 in all cases. calculated lod and loq values for avns on all columns are shown in table 4. table 4. calculated lod and loq values of avns on all columns. column avn c avn a avn b lod [ng.ml-1] symmetry c18 91.4307 95.5642 91.4382 xbridge c18 76.3345 86.6991 68.4532 cortecs c18 96.0841 80.6298 76.7470 loq [ng.ml-1] symmetry c18 304.7689 318.5472 304.7939 xbridge c18 254.4482 288.9970 228.1774 cortecs c18 320.2802 268.7661 255.8233 6 nova biotechnol chim (2022) 21(2): e1483 3 in comparison of lod and loq of individual avns, we can observe certain differences between columns. in general, lod and loq values may appear relatively high. however, as mentioned in the theoretical part (kulichová et al. 2018) as well as other authors (jastrebova et al. 2006) avns have significantly higher lod and loq when using uv (dad) detection. lod is in the range 30 – 140 ng.ml-1 and loq was in the range 100 – 400 ng.ml-1 using this type of detection. lod and loq results for avns on all columns were in these intervals. significantly improved lod and loq should be achieved using hplcms. analysis of oat samples in the last part, we analysed real oat samples on all columns using the isocratic method described above. we randomly selected 2 different oat samples (out of more than 150 samples available in our laboratory) collected in 2014 and the same 2 oat samples collected in 2020. in both samples from 2014, we successfully separated avns from other components with minimal interference on all three columns. fig. 3 shows the chromatogram of a selected mixture of avns standards (blue solid line) and the chromatogram of oat sample – 2014 avenuda (orange dotted line) analysed on xbridge c18 column. interfering components can be seen close to avn c and avn b peaks, which could be eliminated by modifying the extraction technique or in case of determination of these components, it would be better to use gradient elution to separate not only the avns but also other components of oat samples such as phenolic acids. overall, this isocratic method was primarily designed to evaluate the efficiency of three c18 columns with different particle technologies, for possibility of use the best column with gradient elution to provide simultaneous separation and quantification of avns and phenolic compounds in oat samples. in the case of oat samples collected in 2020 (avenuda and kamil), there was slightly fewer interfering components using this isocratic method, but avns were also in much lower concentrations than in the case of oat samples from 2014. fig. 3. overlayed chromatograms of avns standard mixture (blue solid line) and 2014 avenuda sample (orange dotted line) analyzed on xbridge c18 100 × 4.6; 3.5 μm. conclusion this paper offered efficiency comparison of three c18 chromatographic columns with different particle technology, to determine biologically important substances three main avenanthramides. to simplify comparison, a pre-optimized isocratic method was used to evaluate efficiency of columns. in terms of retention times, a solid-core column with a 2.7 μm particle size (cortecs c18) appears -0,015 0 0,015 0,03 0,045 0,06 0,075 0,09 0,105 0,12 0,135 0,15 0,165 0 1 2 3 4 5 6 a u time [min] avn a avn b avn c 7 nova biotechnol chim (2022) 21(2): e1483 2 to be the best, as there was a significant reduction in tr compared to columns with a 3.5 μm particle size. overall, the retention times of all three avns on each column decreased in the following order: symmetry > xbridge > cortecs. lod and loq were similar on all columns but relatively high (which is standard for this type of detection) compared to lod and loq, which can be achieved by a more sensitive hplc-ms method. however, nowadays there are still many laboratories that do not have hplc-ms or even uhplc-ms available. therefore, it is constantly necessary to look for a suitable stationary phase and optimize the hplc-uv (hplc-dad) separation conditions. the results of columns efficiency evaluation show differences between columns. the most efficient separation of avns in terms of n and hetp parameters was achieved on a cortecs c18 column, which predetermines it as the most suitable column (of three tested columns) for avns separation. if we focus on the comparison only between columns with the same particle size of 3.5 μm (symmetry c18 vs. xbridge c18), in overall evaluation, separation was more efficient on the symmetry c18 column, on which was achieved the best results in terms of peak symmetry (follows from the column name). 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(2019) overview of the anticancer profile of avenanthramides from oat. int. j. mol. sci. 20: 4536. wise ml (2013) avenanthramides: chemistry and biosynthesis. in chu yf (eds.), oats nutrition and technology, wiley, new york, pp. 195-226. xie z, mui t, sintara m, ou b, johnson j, chu yf, o’shea m, kasturi p, chen y (2017) rapid quantitation of avenanthramides in oat-containing products by highperformance liquid chromatography coupled with triple quadrupole mass spectrometry (hplc-tqms). food chem. 224: 280-288. 9 microsoft word 7_rahman et al nova biotechnologica et chimica 15-2 (2016) 166 doi 10.1515/nbec-2016-0017 © university of ss. cyril and methodius in trnava molybdate-reducing and sds-degrading enterobacter sp. strain neni-13 m.f. rahman1, m. rusnam2,*, n. gusmanizar1,3, n.a. masdor4, c.h. lee1, m.s. shukor5, m.a.h. roslan1, m.y. shukor1 1 department of biochemistry, faculty of biotechnology and biomolecular sciences, universiti putra malaysia, serdang, upm 43400, selangor, malaysia 2 department of agricultural engineering, faculty of agricultural technology, andalas university, padang, 25163, indonesia (*rusnam_ms@yahoo.com) 3 department of animal nutrition, faculty of animal science, andalas university, padang, 25163, indonesia 4 biotechnology research centre, mardi, p. o. box 12301, kuala lumpur, 50774, malaysia 5 snoc international sdn bhd, lot 343, jalan 7/16 kawasan perindustrian nilai 7, inland port, negeri sembilan, 71800, malaysia abstract: toxicants removal through microorganism’s action is intensely being sought due to economic reasons. the aim of this paper is to isolate a bacterium that is able to reduce molybdenum blue and at the same time can grow on the detergent sodium dodecyl sulfate (sds). biochemical analysis resulted in a tentative identification of the bacterium as enterobacter sp. strain neni-13. growth on sds showed a 100 % removal at 800 mg/l sds within 12 days. the removal of sds from media was confirmed through methylene blue active substances assay. molybdenum reduction using sodium molybdate as a substrate was characterized using a microplate assay. the optimum ph and temperature for molybdenum reduction was between 6.0 and 6.5, and at 37 °c, respectively. glucose was the best electron donor for molybdate reduction. phosphate and molybdate concentrations of between 2.5 and 5.0 mm and at 15 mm, were optimal for molybdate reduction, respectively. molybdate reduction was inhibited by the heavy metals mercury, silver, copper and chromium at 2 ppm. the ability of this bacterium to detoxify molybdate and degrade the sds makes this bacterium an important tool for bioremediation of toxicants in soil. key words: molybdate-reducing, sds-degrading, molybdenum blue, enterobacter sp., heavy metals 1. introduction the improper disposal, industrial and mining activities and excessive use of agricultural chemicals have resulted in a global problem. removing these types of pollutants through bioremediation is a less costly tactic in the long run particularly at low concentrations, where other strategies for example physical or chemical methods may not be effective. industrial effluents and exhaust fumes are often the source of molybdenum pollution. for example, molybdenum in the exhaust fumes in industries in tyrol, austria are the cause of molybdenum pollution covering a 300-hectare agricultural land. cattles grazing on this region suffered debilitating diseases including deaths due to hypocuprosis (neunhäuserer et al., 2001). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc 167 rahman, m.f. et al. the molycorp mine in new mexico is a molybdenum mine. its tailing wastes found their way into rivers near the mines including the red river. portions of the river exposed to high concentration of mine wastes are declared dead (jacobs et al., 2014). in indonesia, the mine tailings from the batu hijau, sumbawa, copper, gold and molybdenum mine are dumped into the ocean with an annual amount close to several million tonnes. the sea is gradually polluted resulting in a reported reduction in fish populations (angel et al., 2013). bacteria are prime candidate for metal including molybdenum remediation. bacteria have unique enzymes that can convert heavy metals to less toxic forms (ahmad et al., 2013b). in the case of molybdenum, bacteria can convert the soluble molybdenum into insoluble molybdenum disulphide (tucker et al., 1997) or to the partially soluble and colloidal molybdenum blue (capaldi and proskauer, 1896). bioremoval columns packed with molybdenum-reducing sulphate-reducing bacteria showed good potential for the removal of molybdenum, but the toxic hydrogen sulphide gas emitted require another removal column packed with hydrogen sulphide-utilizing bacteria (tucker et al., 1997). another promising candidate is the mo-reducing bacterial system. the system utilizes bacteria that reduce molybdenum into molybdenum blue. the end product can be trapped in membrane (halmi et al., 2014), and the process does not require strict anaerobic requirements (ghani et al., 1993). numerous candidates have been isolated from the genera of klebsiella (lim et al., 2012; halmi et al., 2013b; masdor et al., 2015), bacillus (abo-shakeer et al., 2013; othman et al., 2013), pseudomonas (shukor et al., 2010; ahmad et al., 2013a), acinetobacter, serratia and enterobacter (shukor et al., 2009). aside from heavy metals, detergents are often present as co pollutants, especially in water bodies. detergents have detrimental effects to aquatic life (ambily and jisha, 2012). the anionic surfactants such as sodium dodecylbenzene sulfonate (sdbs) and sodium dodecyl sulfate (sds) show toxic effects to daphnia magna at concentrations of between 0.0025 and 300 mg/l. detergents main toxicity have been documented in invertebrates and crustaceans due to the massive amount of these detergents continuously being released into the aquatic environment (chaturvedi and kumar, 2011). several microorganisms can degrade sds and use it for growth. the enzyme alkylsulfatase is the first enzyme of the pathway to degrade and assimilate sds for microbial growth (chaturvedi and kumar, 2011). to date numerous sds-degrading bacteria have been isolated (abboud et al., 2007; hosseini et al., 2007; chaturvedi and kumar, 2011; ambily and jisha, 2012; halmi et al., 2013a). the current trend of xenobiotics detoxification research is to isolate multiple degraders or microorganisms showing multiple detoxification ability. this is especially important in polluted sites where multiple contaminants can be found (husaini et al., 2008). the versatility of such multiple-degrading microorganisms are constantly being sought. previously, two mo-reducing bacteria have been reported with ability to grow on the detergent sds (halmi et al., 2013b; masdor et al., 2015). herein, we report on the isolation of another mo-reducing bacterium that can bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc nova biotechnologica et chimica 15-2 (2016) 168 grow on sds as a sole carbon source. we also report on the first utilization of the modified gompertz model to obtain growth parameters of this bacterium on sds. 2. materials and methods 2.1. chemicals and apparatus all of the chemicals utilized in this work are of analytical grade. the uv-spectrophotometer (shimadzu 1201) was purchased from shimadzu corporation, kyoto, japan. the orbital shaker was purchased fromyihder technology co., ltd., taipei, taiwan. the microplate reader utilized in this work is a biorad 680 microplate model and was purchased from biorad, richmond, ca, usa. the centrifuge model used was the beckman gs-15r and was purchased from beckman instruments inc., fullerton, ca, usa. bacterial cells were ruptured using a sonicator (biosonik 111tm, bronwill scientific, rochester, n.y) 2.2. isolation of molybdenum-reducing bacteria water samples were taken from the danau maninjau lake in the province of padang, sumatera, indonesia in january 2009. screening and isolation of potential molybdenum-reducing bacteria were carried in a minimal salts media with phosphate content fixed at 5 mm as most of the mo-reducing bacteria isolated to date showed optimal molybdenum reduction at this concentration. the media was supplemented with sodium molybdate at 10 mm. about 0.1 ml of the water sample was spread onto a solid low phosphate agar (ph 6.5). the plates were incubated at room temperature (27 oc) for 48 hours. the composition (mm) of the reduction media is as follows: glucose anhydrous (55 mm), (nh4)2.so4 (22.7 mm), mgso4.7h2o (2.03 mm), yeast extract (0.5 %), nacl (85.56 mm), na2moo4.2h2o (10 mm) and na2hpo4 (5 mm). solid medium was prepared by adding agar (1.5 %) (masdor et al., 2015) this is a low phosphate molybdenum or lpm media. after the incubation period (48 hours), blue colonies were selected, and restreaked on the lpm agar several times to obtain pure culture. the mo-reducing bacterial isolates were then grown in 20 ml of lpm liquid media to select for the best reducer. the bacterial isolates were grown for 48 hours at room temperature (27 oc)on an orbital shaker (120 rpm, yihder, taiwan). about 1 ml of the culture media from each bacterial isolate was centrifuged (beckman gs-15r, fullerton, ca, usa) at 10,000 x g for 5 min at room temperature. the amount of molybdenum blue produced in the extracellular liquid was quantified at 865 nm using the specific extinction coefficient of 16.3 mm.-1.cm-1. a portion of the blue supernatant from culture media for the best isolate was periodically sampled and scanned from 400 to 900 nm (uv-spectrophotometer, shimadzu 1201) after undergoing the centrifugation step as before. identification of the best isolate was carried out utilizing biochemical and phenotypical methods in accordance to the bergey’s manual of determinative bacteriology (holt et al., bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc 169 rahman, m.f. et al. 1994), and followed up by using the abis online system software (costin and ionut 2015) as before (masdor et al., 2015). 2.3. bacterial resting cells preparation the mo-reducing bacterium was characterized in a microtiter plate format. the characterization include concentrations of molybdate, carbon sources, phosphate, effect of ph, heavy metals, temperature and screening of potential xenobiotics as sources of growth or as electron donors supporting molybdenum reduction (shukor and shukor, 2014; masdor et al., 2015). a volume of 1 l of high phosphate molybdate media or hpm distributed in several smaller 250 ml conical flasks was utilized for growth. the hpm has the same composition to the lpm but with the phosphate concentration increased to 100 mm. briefly, bacterial cells were grown aerobically with shaking at 120 rpm on an orbital shaker (yihder, taiwan) at room temperature (27 oc). the media utilized is a modification to the lpm in which the phosphate concentration was increased to 100 mm. this was carried out to prevent the formation of bacterial blue aggregates. cells were harvested by centrifugation at 10,000 g (beckman at 4 oc for 10 min). after rinsing with deionized water twice, the pellets were resuspended in 20 ml of lpm giving an optical density measured at 600 nm of 1.0 absorbance value. in an effort to accommodate the variations in phosphate, molybdate, carbon sources, and ph conditions throughout the characterization works, appropriate modifications to the lpm was carried out. about 180 μl of the cellular suspension was transferred into the wells of a sterile microplate, and mixed with 20 μl of sterile glucose or other carbon sources from stock solutions. the final concentration of the carbon sources was 1.0 % (w/v). the microplates were covered (corning® microplate), and then incubated at room temperature. readings at 750 nm was periodically taken using a biorad reader (model no. 680, richmond, ca). the molar extinction coefficient of 11,687 m.-1.cm-1 at 750 nm was utilized to quantify molybdenum blue production. this wavelength was chosen as it is the maximum filter available for the microplate unit (shukor and shukor, 2014). the effect of several heavy metals was studied utilizing atomic absorption spectrometry calibration standard solutions from merck. 2.4. detergents and hydrocarbons as carbon sources for bacterial growth preliminary works showed that none of the detergents and hydrocarbons tested could support molybdenum reduction. hence, these compounds were then screened for bacterial growth in the microplate format as described by replacing glucose with these xenobiotics at the final concentration of 500 mg/l for detergents. diesel and crude petroleum was initially added to the final concentration of 2 % (v/v) in 10 ml of the growth media and sonicated at 60 hz (biosonik 111tm, bronwill scientific, rochester, n.y) for 5 minutes. then 200 μl of the media was added into the microplate wells, and mixed with 50 μl of bacterial cells (see above). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc nova biotechnologica et chimica 15-2 (2016) 170 the microplate was incubated at room temperature (27 oc) for three days and the amount of molybdenum blue production was measured at 750 nm as before. the ingredients of the growth medium (1 l) were as follows: (nh4)2.so4 (22.7 mm), nano3 (23.53 mm), mgso4.7h2o (2.03 mm), yeast extract (0.01 %), nacl (85.56 mm), na2hpo4 (50 mm) and 1 ml of trace elements solution. the trace elements solution composition (mm) was as follows: cacl2 (0.363), feso4·7h2o (0.076), mnso4·4h2o (0.179), znso4·7h2o (0.069), cuso4·5h2o (0.02), cocl2·6h2o (0.021), na2moo4·2h2o (0.020). the media was adjusted to ph 7.0. then 200 μl of the media was added into the microplate wells and incubated at room temperature (27 oc) for 72 hours. bacterial growth was measured as the increase in optical density at 600 nm. 2.5. methylene blue active substances (mbas) assay sds degradation was quantified according to the mbas assay jurado et al. (2006). 100 µl of slightly acidic methylene blue reagent (ph of between 5 and 6) was added into 5 ml of the sds calibration standards or samples (appropriately diluted) in a separating funnel followed by the addition of 200 µl of sodium tetraborate ph 10.5. the mixture was then mixed vigorously and then 4 ml of chloroform was added to the mixture and vigorously stirred for 1 minute. the mixture was left to separate and settle down for several minutes. the chloroform layer was separated and the absorbance was read at 650 nm in a glass cuvette. 2.6. statistical analysis values are means ± standard deviation of three replicated experiments. statistical analyses such as anova and t-test utilized the graphpad prism software ver. 5.0 available from www.graphpad.com. 3. results 3.1. detergents as carbon sources for enterobacter sp. strain neni-13 growth utilization of detergents and hydrocarbons as potential carbon sources for enterobacter sp. growth was determined after 72 hours of incubation. the data showed that the bacterium was able to grow on the detergent sds (fig. 1). however, it was discovered that none of the detergents and hydrocarbons tested in this experiment can support molybdate reduction. sds degradation studies carried out at concentrations between 200 and 1400 mg/l under aerobic conditions showed that the fastest degradation occurred at 200 mg/l of sds, with complete degradation occurring at day-4 of incubation. a 100 % of degradation was still observed at the sds concentration of 800 mg/l. the lowest degradation after 12 days of cultivation occurred at 1400 mg/l showing 19.4 % degradation (fig. 2). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc 171 rahman, m.f. et al. 0.0 0.2 0.4 0.6 0.8 co nt ro l te rg ito l n p9 te rg ito l 1 5s 9 w itc on ol 23 01 sd bs sd s be nz et ho niu m ch lo rid e be nz al ko ni um ch lor id e di es el cr ud e p etr ol eu m a bs 6 00 n m fig. 1. growth of enterobacter sp. strain neni-13 on xenobiotics as sole source of carbon and energy. bars signify the standard deviation of triplicates. 0 20 40 60 80 100 0 2 4 6 8 10 12 incubation (day) s d s r es id u al (% ) 200 400 600 800 1000 1200 1400 fig. 2. sds degradation by enterobacter sp. strain neni-13. the bacterium was grown at various concentrations of sds in a volume of 50 ml in a 250 ml shake-flask on an orbital shaker at 120 rpm at room temperature (27 oc). bars signify the standard deviation of triplicates. 3.2. identification of molybdate-reducing bacterium strain neni-13 was a gram-negative, short rod-shaped, motile, and facultative anaerobic bacterium (table 1). identification of the bacterium through the abis online software suggested that the identity of the bacterium giving the highest similarity or homology score of 90 % and accuracy score of 100 % as enterobacter sp. strain neni-13. a more accurate molecular identification technique through phylogenetic analysis of the 16srrna gene is currently being carried out to identify this species further. however, for now the bacterium is identified as enterobacter sp. strain neni-13 in honor of the late dr. neni gusmanizar. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc nova biotechnologica et chimica 15-2 (2016) 172 table 1. overview on the presence (+) or absence (-) of individual biochemical activities for enterobacter sp. strain neni-13. motility + acid production from: pigment – alpha-methyl-d-glucoside + catalase production (24 h) + d-adonitol + oxidase (24 h) – l-arabinose + onpg (beta-galactosidase) + cellobiose + arginine dihydrolase (adh) + dulcitol + lysine decarboxylase (ldc) – glycerol + ornithine decarboxylase (odc) + d-glucose + nitrates reduction + myo-inositol + methyl red – lactose + voges-proskauer (vp) + maltose + indole production – d-mannitol + hydrogen sulfide (h2s) – d-mannose + acetate utilization + melibiose + malonate utilization + mucate + citrate utilization (simmons) + raffinose + tartrate (jordans) + l-rhamnose + esculin hydrolysis + salicin + gelatin hydrolysis – d-sorbitol + urea hydrolysis + sucrose + deoxyribonuclease – trehalose + lipase (corn oil) – d-xylose + phenylalanine deaminase – growth on kcn medium + 3.3. scanning absorption spectrum of molybdenum blue from enterobacter sp. strain neni-13 a scanning absorption spectrum of the molybdenum blue produced from this bacterium from the visible to the near infrared (ir) regions showed a peak with a maximum wavelength of 865 nm and a shoulder at approximately 710 nm (fig. 3). the molybdenum blue spectra at 24-h and 48-h of incubations were similar. 0.0 1.0 2.0 3.0 600 650 700 750 800 850 900 950 wave length (nm ) a bs 24 h 48 h fig. 3. scanning absorption spectrum of molybdenum blue from enterobacter sp. strain neni-13 at different time intervals. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc 173 rahman, m.f. et al. 3.4. carbon sources as electron donor for molybdenum reduction glucose was the best electron donor for supporting molybdenum reduction enterobacter sp. strain neni-13. this is followed in descending order of efficiency by sucrose, l-rhamnose, maltose, lactose, cellobiose, d-mannose, raffinose, mucate, d-mannitol, d-adonitol, melibiose, glycerol, d-sorbitol and l-arabinose (fig. 4). the optimal concentration of glucose was 1 % (w/v) (data not shown). 0.0 1.0 2.0 3.0 dad on ito l lar ab ino se ce llo bio se du lci to l gl yc er ol dgl uc os e my oino sit ol la cto se ma lto se dma nn ito l dma nn os e me lib ios e mu ca te ra ffin os e lrh am no se sa lic in dso rb ito l su cr os e co ntr ol a bs 7 50 n m fig. 4. the effect of various carbon sources on molybdenum blue production by enterobacter sp. strain neni-13. bars signify the standard deviation of triplicates. 3.5. molybdenum reduction at various phosphate and molybdate concentrations the optimum concentration of phosphate occurred between 2.5 and 5.0 mm with higher concentrations were strongly inhibitory to reduction (fig. 5). 0.0 0.5 1.0 1.5 2.0 0 10 20 30 40 50 phosphate (mm) a bs 7 50 n m fig. 5. the effect of phosphate concentrations on molybdenum blue production by enterobacter sp. strain neni-13. bars signify the standard deviation of triplicates. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc nova biotechnologica et chimica 15-2 (2016) 174 molybdenum blue production at various sodium molybdate concentrations was studied by varying molybdate concentrations from 0 to 70 mm. the result shows that a lag period of about 10 hours was observed before molybdenum blue production started, and a maximum molybdenum blue production of 14.8 nmol molybdenum blue after 52 hours of incubation using 15 mm sodium molybdate was observed. an inhibition to molybdenum reduction was observed at molybdate concentrations higher than 30 mm (fig. 6). 0 4 8 12 16 0 11 22 33 44 55 incubation (h) nm o le m o -b lu e 0 mm 5 mm 10 mm 15 mm 20 mm 25 mm 30 mm 35 mm 40 mm 50 mm 60 mm 70 mm fig. 6. the effect of molybdate on molybdenum blue production by enterobacter sp. strain neni-13. bars signify the standard deviation of triplicates. 0 20 40 60 80 100 120 co nt ro l si lv er ar se ni c ca dm iu m ch ro m iu m co pp er m er cu ry le ad m o ly b d at ere d u ci n g a ct iv it y (% ) fig. 7. the effect of heavy metals (2 ppm) on molybdenum blue production by enterobacter sp. strain neni13. bars signify the standard deviation of triplicates. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc 175 rahman, m.f. et al. 3.6. effects of heavy metals on molybdate reduction molybdate reduction was inhibited by the heavy metals mercury, silver, copper and chromium with both mercury and silver showing a similar strong inhibition as judged by anova analysis (fig. 7). 4. discussion although the phenomenon of molybdate reduction to molybdenum blue has been reported in e. coli more than a century ago (capaldi and proskauer, 1896), the first detailed study on this phenomenon was carried out in e. coli k12 (campbell et al., 1985). in 1993, the same phenomenon was reported in enterobacter cloacae strain 48 (ghani et al., 1993), without citing previous works, indicating the rarity of similar studies. the potential for utilization in the bioremediation of molybdenum has first beent recognized by ghani et al. (1993). to date many more mo-reducing bacteria have been isolated (table 2), including two strains that can grow on the detergent sds (halmi et al., 2013b; masdor et al., 2015). in addition, a psychrophilic mo-reducing bacterium is also isolated from antarctica. previously, two molybdenum-reducing bacterium from this genus; enterobacter cloacae strain 48 (ghani et al., 1993) and enterobacter sp. strain dr.y13 (shukor et al., 2009) have been isolated. studies on the optimal conditions for molybdenum reduction utilizes the microtiter plate format with the use of whole or resting cells allowing a high throughput characterization to be carried out (shukor and shukor, 2014; masdor et al., 2015). usage of resting cells or whole cells was first started in the bacterium enterobacter cloacae strain 48 (ghani et al., 1993). there are considerable reports on the utilization of resting cells in a microtiter plate format indicating the favorable use of this method. this include a study on polyalcohol ethoxylate biodegradation (sharvelle et al., 2008). one of the advantages of resting cells is it can give cells tolerance to toxic xenobiotics especially in the beginning of the growth process. the inhibition by heavy metals on molybdenum reduction was observed in nearly all of the molybdenum-reducing bacteria isolate to date (table 2). the presence of these heavy metals at potential bioremediation site for molybdenum could present a problem if the concentrations of the metals are above 2 ppm. the inhibitory effects of heavy metals such as mercury and copper have been reported in bacterial chromate reduction, with the target of inhibition by these metal ions suggested as the sulfhydryl group of the chromate reductase (ahmad et al., 2013b). several chemicals such as calcium carbonate, manganese oxide, and magnesium hydroxide can be added to bioremediation sites to alleviate the effect of toxic cations above as these chemicals can bind and immobilize the metal ions (hettiarachchi et al., 2000). however, the effect of the addition of these sequestering agents to molybdenum reduction remains to be studied. another option to lessen the toxicity of these cationic heavy metals to bioremediation of molybdenum, especially in aquatic bodies is to entrap the molybdenum-reducing bacterium in dialysis tubing or membrane (halmi et al., 2014). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc nova biotechnologica et chimica 15-2 (2016) 176 table 2. overview of mo-reducing bacteria isolated to date. bacteria optimal c source optimal molybdate (mm) optimal po43 (mm) heavy metal inhibitors reference klebsiella oxytoca strain aft-7 glucose 5-20 5-7.5 cu2+, ag+, hg2+ masdor et al., 2015 bacillus pumilus strain lbna glucose 40 2.5-5 as3+, pb2+, zn2+, cd2+, cr6+, hg2+, cu2+ abo-shakeer et al., 2013 bacillus sp. strain a.rzi glucose 50 4 cd2+, cr6+, cu2+,ag+, pb2+, hg2+, co2+,zn2+ othman et al., 2013 pseudomonas sp. strain dry2 glucose 15-20 5 cr6+, cu2+, pb2+, hg2+ shukor et al., 2010 pseudomonas sp. strain dry1 glucose 30-50 5 cd2+, cr6+, cu2+,ag+, pb2+, hg2+ ahmad et al., 2013a enterobacter sp. strain dr.y13 glucose 25-50 5 cr6+, cd2+, cu2+, ag+, hg2+ shukor et al., 2009 enterobacter cloacae strain 48 sucrose 20 2.9 cr6+, cu2+ ghani et al., 1993 escherichia coli k12 glucose 80 5 cr6+ campbell et al., 1985 klebsiella oxytoca strain hkeem fructose 80 4.5 cu2+, ag+, hg2+ lim et al., 2012 literature search showed a variation in the efficiency of bacterial isolates on sds degradation. sds-degrading bacteria are potential agents for aquatic and soil bioremediation of sds. to date, several sds-degrading bacteria have been isolated and characterized including cold-adapted, psychrophilic or psychrotolerant sds-degrading (margesin and schinner, 1998; halmi et al., 2013a). a bacterial consortium of pantoea agglomerans and acinetobacter calcoaceticus is able to degrade 4000 mg/l of sds in approximately 5 days (abboud et al., 2007). the sds-degrading bacteria pseudomonas betelli and acinetobacter johnsonii degrades 500 mg/l sds within 5 days of incubation (hosseini et al. 2007). klebsiella oxytoca srain dry14, degrades 80 % of 2 000 mg/l of sds within 4 days of incubation (shukor et al., 2009), while pseudomonas aeruginosa sp. degrades 100 % of 1 000 mg/l of sds within 2 days of incubations (syed et al., 2010). one of the most efficient sds-degrading bacterium isolated is a mutated strain of pseudomonas aeruginosa mtcc 10311 that degrades 1 500 mg/l of sds within two days of incubation (ambily and jisha, 2012). at high concentrations, sds disrupts cellular membrane integrity. this leads to disturbances to the ion gradients resulting in the leakage of bacterial cytosolic contents (chaturvedi and kumar, 2011). another mechanism of sds toxicity is through surface protein and enzymes denaturation (ambily and jisha, 2012). the identity of the molybdenum blue produced from bacterial reduction of molybdenum has never been identified since it was first mentioned in 1896. campbell et al. (1985) were the first to suggest that the molybdenum blue found in e. coli k12 is likely a reduced form of phosphomolybdate. they primarily based this evaluation on the molybdenum blue spectrum acquired. phoshomolybdate (molybdophosphate) was not mentioned in the works on enterobacter cloacae strain 48 by ghani et al. (1993). instead, they proposed that the mo-reducing enzyme to bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc 177 rahman, m.f. et al. molybdenum (mo5+) reduces molybdenum in the form of molybdate (mo6+) enzymatically to mo5+ before the reaction with phosphate anion formed molybdenum blue. nonetheless, there are a few problems with this mechanism. firstly, molybdenum exist as [moo4] 2or molybdate anions, and does not exist as mo6+ in solution. secondly, an acidified solution of molybdate above 1 mm will be instantaneously converted to polyions such as mo8o26 4, mo7o24 6-, and mo12o37 2-. thirdly, these polyions combine with phosphate and form the heteropolymolybdate molybdophosphate or phosphomolybdate. fourthly, enzymes such as aldehyde and xantine oxidases can catalytically reduce phosphomolybdate to molybdenum blue but not molybdate to molybdenum blue (glenn and crane, 1956). lastly, almost all of the bacterially created molybdenum blue display spectra that demonstrate near resemblance of the molybdenum blue generated from the phosphate determination method (pdm) (shukor et al., 2007; masdor et al., 2015). the heteropolymolybdate species of molybdenum blue produced in the pdm is phosphomolybdate, the latter displays a scanning spectrum with a characteristics shoulder of from 700 to 720 nm, and a peak maximum from 870 nm to 890 nm (clesceri et al., 1989). in line with all of this, we suggested a new hypothesis on the standard mechanism of molybdenum blue production in bacteria. in this new mechanism, we propose that initially, a phoshomolybdate intermediate is formed from molybdate. phosphomolybdate is then reduced by the mo-reducing enzyme to molybdenum blue in bacteria (shukor et al., 2007). when we use phosphomolybdate as the electron acceptor substrate instead of the original molybdate in the mo-reducing enzyme assay, we obtain an increase in enzyme activity of about two hundred times. utilizing this compound, the mo-reducing enzyme is purified for the first time after the phenomenon was first documented in 1896 (shukor et al., 2014). the existence of an intermediate species in molybdenum blue production is likewise noticed during heavy metal reduction of chromate. in the bacteriaum shewanella putrefaciens (now known as s. oneidensis) (myers et al., 2000), biological reduction of chromate from the 6+ to 3+ shows the occurrence of the intermediate species cr5+. this has been proven via spectroscopic and paramagnetic resonance works. this indicates that the presence of an intermediate species is not unique to molybdenum. spectroscopic analysis of the resultant molybdenum blue as a method to distinguish between existing heteropolymolybdates including silicomolybdate, sulphomolybdate and phosphomolybdate is a simple and acceptable method, and is routinely used to characterize phoshomolybdate or to differentiate between the numerous heteropolymolybdate (sims 1961). however, further investigations making use of nuclear magnetic resonance and electron spin resonance are needed for detail identification of the possible lacunary species of phosphomolybdate observed in bacterial reduction of molybdenum to molybdenum blue (sims, 1961). carbon sources such as glucose and sucrose are efficient substrate for growth, energy or electron donors for the reduction of heavy metals. as bioremediation sites are often electron donor deficient, studies on the best electron donor are very useful as an important additive in bioremediation sites. at very low concentration of cells, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc nova biotechnologica et chimica 15-2 (2016) 178 carbon sources are often utilized as carbon sources for energy, as the cell concentration increases, bacterial cells begin to produce excess reducing equivalents such as nadph and nadh that are true electron donors for metal reduction. in resting or whole cells preparation, carbon sources are converted to reducing equivalents in a much greater amount, which makes metal reduction increases in efficiency. glucose is also one of the best electron donors in many metal reductions such as chromate (llovera et al., 1993) and selenate (losi and frankenberger jr., 1997). mo-reducing bacteria isolated so far either prefer glucose or sucrose as efficient electron donor for supporting molybdenum reduction to molybdenum blue (table 2). metabolic pathways such as glycolysis, kreb’s cycle and the electron transport system are the sites where these carbon sources are converted to the reducing equivalents nadh and nadph. both of these reducing equivalents are substrates for the molybdenum reducing-enzyme (shukor et al., 2014). molybdenum reduction is impacted by phosphate concentrations, with high phosphate concentrations significantly decreasing its production in the course of bacterial reduction of molybdenum. as an example, ghani et al. revealed that phosphate concentrations greater than 2.9 mm inhibited reduction (ghani et al., 1993), whilst levels greater than 5 mm inhibited most of the mo-reducing bacteria isolated up to now (table 1). previous studies (glenn and crane, 1956) have demonstrated that high phosphate concentrations negatively affecting phosphomolybdate structure by means of sustaining the ph at neutral. the phosphomolybdate complex is extremely unstable at this ph, and it is solely stable under acidic environments (glenn and crane, 1956). mo-reducing bacteria isolated so far can tolerate and reduce molybdate at the optimal concentrations ranging from 5 to 80 mm (table 2). as molybdenum concentrations may achieve up to 900 mg/l in waters and 6,500 mg/kg in soils (stone and stetler, 2008), mo-reducing bacteria represent a meaningful tool for remediation processes. another essential prerequisite is the soil phosphate concentrations should be about 5 mm but not exceeding 20 mm for reduction to occur. fortunately, phosphate concentrations are hardly ever observed exceeding beyond these range in regular type of soils (chai et al., 2011). 5. conclusions a molybdate-reducing bacterium with the ability to grow on sds as a sole carbon source has been isolated. molybdenum blue characterization results indicate a good tolerance to high concentration of molybdenum, a requirement for low concentration of phosphate. glucose, one of the most assimilable carbon sources was the best electron donor for molybdenum reduction. the absorption spectrum of molybdenum blue indicates that molybdenum blue is likely a reduced phosphomolybdate. molybdenum reduction was inhibited by several toxic heavy metals indicating future bioremediation. acknowledgment: the company snoc international sdn bhd. financed a portion of this project. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc 179 rahman, m.f. et al. references abboud, m.m., khleifat, k.m., batarseh, m., tarawneh, k.a., almustafa, a., al-madadhah, m.: 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(eds.): uranium, mining and hydrogeology, springer berlin heidelberg, 2008, 371-380. syed, m.a., mahamood, m., shukor, m.y., shamaan, n.a.: isolation and characterization of sds-degrading pseudomonas aeruginosa sp. strain d1. aust. j. basic appl. sci., 4, 2010, 5000-5011. tucker, m.d., barton, l.l., thomson, b.m.: reduction and immobilization of molybdenum by desulfovibrio desulfuricans. j. environ. qual., 26, 1997, 11461152. received 15 december 2015 accepted 29 november 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:58 utc nova biotechnol chim (2018) 17(2): 140-149 doi: 10.2478/nbec-2018-0015  corresponding author: eman_ahmad3332@yahoo.com nova biotechnologica et chimica bioanalytical hplc method development for simultaneous determination of valsartan and co-administered clopidogrel bisulfate and fenofibrate in stroke prevention in raw materials, spiked human plasma and tablets gamal h. ragab and eman a. bahgat pharmaceutical analytical chemistry department, zagazig university, faculty of pharmacy, zagazig 44519, egypt article info article history: received: 29th june 2018 accepted: 6th september 2018 keywords: valsartan clopidogrel bisulfate fenofibrate plasma rp-hplc method tablets abstract this study reports about simple, robust and reproducible method for simultaneous bioanalytical determination of valsartan (val) and co-administered clopidogrel bisulfate (cgb) and fenofibrate (fen) in raw materials, spiked human plasma and tablets using isocratic rp-hplc method. the chromatographic separation is carried out using isocratic binary mobile phase consisting of 80 mm phosphate buffer ph 3: acetonitrile (30: 70 %; v/v) at the flow rate of 1.1 ml/min and 33 °c. a diode array detector at wavelength 214 nm was used. retention times for val, cgb and fen were 3.1, 5.1 and 6.4 min, respectively. the calibration curves obtained were linear over the concentration ranges of 2.5 – 100 μg/ml for both val and cgb and 5 –100 μg/ml for fen. the mean extraction recoveries of val, cgb and fen from spiked plasma were 75.38±1.34 %, 89.91±2.17 % and 96.92±6.02 %, respectively. the limits of detection and quantification were 0.86, 0.67, 1.11 μg/ml and 2.60, 2.03, 3.36 μg/ml for val, cgb and fen, respectively. the method was applied to the analysis of these drugs in spiked human plasma and in tablets as they are commonly used as a combination for prevention of stroke. results obtained show good accuracy, precision and acceptable recoveries from plasma samples.  university of ss. cyril and methodius in trnava introduction valsartan is an angiotensin-ii receptor antagonist, used in treatment of hypertension. it is also used for patients with heart failure who are unable to tolerate ace inhibitors (sweetman 2009). clopidogrel bisulphate is a thienopyridine antiplatelet drug, used in the treatment of thromboembolic disorders (sweetman 2009). clopidogrel bisulphate is official, have been determined in bp (the british pharmacopoeia 2017) by titration with 0.1 m sodium hydroxide then the end-point determined potentiometrically and in usp (the united states pharmacopoeia 2007) by hplc method. fenofibrate is fibric acid derivative, used for regulating the lipids of plasma and in the treatment of hyper-lipoprotein-aemias (sweetman 2009). literatures reveal different methods for determination of all the three drugs in combinations with other drugs. valsartan have been determined by using rp-hplc methods in combination with hydrochlorothiazide in tablets (tian et al. 2008) and also with amlodipine and hydrochlorothiazide in tablets and spiked human plasma (el-gizawy et al. 2012). clopidogrel-bisulfate have been determined by using rp-hplc methods in combination with aspirin in tablets (shrivastava et al. 2008), aspirin and atorvastatin calcium in capsules (londhe et al. 2011) and with bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:27 utc mailto:eman_ahmad3332@yahoo.com nova biotechnol chim (2018) 17(2): 140-149 141 pantoprazole in rat plasma (gurupadayya et al. 2014). fenofibrate have been determined by using rp–hplc methods in combination with atorvastatin-calcium in tablet dosage forms (jain et al. 2008) and in combination with atorvastatin and ezetimibe in commercial formulation (sahu et al. 2016). also a validated stability indicating rp-hplc method is used for simultaneous determination of fenofibrate with atorvastatin and folic acid in bulk and pharmaceutical dosage form have been reported (khaleel and abdul rahaman 2016). modern studies state that stroke prevention is the greatest opportunity for decreasing stroke occurrence (kernan et al. 2014). there had been a significant decrease in stroke occurrence and mortality in several nations around the world over the last decade (ovbiagele 2011; hall et al. 2012; rosengren et al. 2013; wang et al. 2013). this development in decreasing stroke incidence is mainly due to the better treatment of risk factors for stroke (gu et al. 2012; lackland et al. 2014). the systematic combination of secondary prevention drugs is one of the proposed strategies for stroke prevention. for secondary prevention of stroke, there were 3 possible recommended medication classes: antihypertensive (renin-angiotensin system modulator, β-blocker, calcium antagonist and diuretic), antithrombotic (antiplatelet or/and anticoagulation), and lipid modifier therapy (i.e., ezetimibe, statin, fenofibrate and niacin). in this study, we introduce a simple, robust and precise hplc method for simultaneous determination of a very effective combination of drugs that are used for secondary prevention of stroke which is valsartan as antihypertensive, clopidogrel bisulfate as antiplatelet and fenofibrate as lipid modifier. there is no reported method for simultaneous determination of the three drugs either in spiked human plasma or in tablets. so, the developed method has been created to determine these drug combinations in both spiked human plasma and in tablets dosage form. experimental apparatus all separations were achieved on an agilent technologies 1200 series chromatographic apparatus with autosampler injector and 100 μl volume injection loop. uv lamp (germany) and g1315d photodiode array detector (dad) connected to hp computer loaded with agilent chemstation software were used for analytes detection. chromatograms were recorded on agilent integrator. mobile phase was filtered using 0.45 µm membrane filter (millipore, ireland), degassed using agilent g1322a vacuum degasser with g1354a isocratic quaternary pump and solvent cabinets. thermo hypersil (250 mm × 4.6 mm i.d, 5 µm particle size column) was used for the analysis. for ph adjustment a digital analyzer ph meter equipped with glass electrode (hanna, made in europe, romania) was used. ultra-sound sonicator (raypa, spain) and also centrifuge (hermle labortechnik gmbh, germany) were used. reagents and materials acetonitrile, methanol and water were of hplc grade (fisher scientific, uk), orthophosphoric acid (fisher chemical® laboratory reagent grade) and potassium dihydrogen orthophosphate (fisher scientific, fair lawn, new jersey). samples valsartan was obtained from eipico (egyptian international pharmaceutical industry company), egypt while clopidogrel bisulfate and fenofibrate were obtained from sigma pharmaceutical industries, egypt. all samples should be stored in dry place, away from sunlight and moisture. the human plasma was received from peoples hospital, el-ahrar, zagazig, egypt. plasma samples were frozen immediately at –20 °c until assayed. pharmaceutical dosage forms tareg® tablets contain 40 mg valsartan per tablet (product of novartis pharm s.a.e. cairo, under license from: novartis, usa). clopex® tablets contain 75 mg clopidogrel bisulfate per tablet (product of marcyrl pharmaceutical industriesegypt) and lipanthyl supra® tablets contain 160 mg bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:27 utc nova biotechnol chim (2018) 17(2): 140-149 142 fenofibrate (product of mina pharm for pharmaceuticals & chemical industries-egypt). standard solutions stock standard solutions (1 mg/ml) of val, cgb and fen were prepared by dissolving 25 mg of each drug in 25 ml volumetric flasks in methanol, stored in the refrigerator at 4 ºc. these stock standard solutions were stable for one week. working solutions of val, cgb and fen (100 µg/ml) were prepared, daily by suitable dilution of the stock solutions with methanol, respectively. different aliquots of working solutions were transferred and diluted in 10 ml volumetric flasks with methanol to reach the concentration ranges of 2.5 – 100 μg/ml for val and cgb and 5 – 100 μg/ml for fen. chromatographic conditions for general procedures analysis process was carried out at ambient temperature using agilent thermo hypersil (250 mm × 4.6 mm i.d, 5 µm particle size column) and the mobile phase was an isocratic mixture of acetonitrile and 80mm potassium dihydrogen orthophosphate buffer (ph= 3±0.2 adjusted using orthophosphoric acid) in a ratio of (70 : 30; v/v). the analysis was done at a flow rate 1.1 ml/min and using diode array detector at wave length 214 nm with injection volume 10 μl. the mobile phase was filtered by passing through a 0.45 μm millipore membrane filter type hawp. the working standard solutions were prepared by dilution of the stock standard solution with methanol to reach the concentration ranges cited above. triplicate 10 μl injections were made for each concentration and chromatographed under the optimized conditions described above. the calibration graph was constructed by plotting peak area of each concentration against the corresponding concentration. preparation of spiked serum sample prior to the extraction process, drug free plasma samples were removed from the deep freezer and were allowed to thaw. to a constant volume of human plasma (0.2 ml), 0.5 ml of each standard solution was added into 5 ml tapered bottom centrifuge tube, and the volume was made up to 4 ml by a adding 3.3 ml of a mixture of (acetonitrile: methanol, 1:1) to reach the final concentrations of the drugs in the spiked human plasma samples. the mixture was mixed well, standing for 5 min at room temperature, finally was centrifuged at 5,000 r/min for 20 min. to another clean tube, the upper layer was transferred and then filtered through a 0.45 μm millipore syringe filter. 10 µl of the clear supernatant was directly injected into the liquid chromatographic system for analysis. preparation of pharmaceutical dosage sample ten tablets of the cited drugs were weighed, finely powdered then amount equivalent to 25 mg of each drug was accurately transferred into a 25 ml volumetric flask, dissolved in methanol and the flask was sonicated for 30 min, the volume was completed to the mark with methanol. the solution was filtered through a 0.45 μm membrane filter before injection into the column. the procedures were completed as mentioned above and the concentrations of the three drugs were obtained from the computed regression equations. method validation the proposed method was validated according to ich guidelines (international conference on harmonization 2005). it was validated for the parameters as linearity, limit of detection (lod), limit of quantitation (loq), precision, specificity and accuracy. linearity, detection and quantitation limits calibration curves were obtained by the analysis of different preparations of one sample of each of the cited drugs and then, it was constructed by plotting the values of peak areas against concentrations of the cited drugs. the limits of detection (lod) and limits of quantitation (loq) were determined in accordance with ich recommendation by the use of standard deviation of the response and the slope of the calibration bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:27 utc nova biotechnol chim (2018) 17(2): 140-149 143 curve. accuracy accuracy of the measurements of the suggested method was determined using the calibration curves of the three drugs. it was determined by analysis of seven preparations of one sample for all the studied drugs. precision intra-day precision was determined for the cited drugs through replicate analysis of two concentrations (10 and 100 µg/ml), three successive times. inter-day precision was determined through replicate analysis of two concentrations of the cited drugs on three successive days. specificity the specificity of the proposed method was determined by comparing the chromatogram of the cited drugs in their pure form with the chromatogram obtained when the cited drugs extracted from spiked human plasma samples. robustness the robustness of the proposed method was evaluated by the constancy of the peak area and the retention time upon deliberate minor variations in the method parameters; these parameters included the flow rate (1.09, 1.1 and 1.11 ml/min) and mobile phase ratio (30.5, 30 and 29.5 of phosphate buffer ph 3). analytical applications the validity of the proposed method was evaluated by their application to the determination of the cited drugs in raw material, spiked human plasma and in their separate tablets dosage forms. a statistical comparison of the results obtained by the proposed and the reported methods (gupta et al. 2010a, the british pharmacopoeia 2017 and gupta et al. 2010b) was done. results and discussion method development different chromatographic conditions (organic solvent ratio, flow rate, and ph) affecting the chromatographic separation of val, cgb and fen were carefully studied in order to recognize the most suitable chromatographic system, separate the three drugs from each other with very good resolution and to obtain sharp and symmetrical peaks and retention time in between 2 and 8 min. fig. 1. chromatogram showing the separation of the three drugs: (b) valsartan at 3.1 min, (c) clopidogrel bisulfate at 5.1 min and (d) fenofibrate at 6.3 min at concentration level 100 µg/ml. b c d bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:27 utc nova biotechnol chim (2018) 17(2): 140-149 144 table 1. spectral data for the simultaneous determination of valsartan, clopidogrel bisulfate and fenofibrate by the proposed hplc method. parameters valsartan clopidogrel bisulfate fenofibrate linearity range [μg/ml] 2.5 – 100 2.5 – 100 5 – 100 intercept (a) 51.89 -1.51 -17.08 slope (b) 35.73 17.56 22.83 correlation coefficient (r) 0.9998 0.9999 0.9999 determination coefficient (r2) 0.9997 0.9999 0.9999 lod [μg/ml] 0.86 0.67 1.11 loq [μg/ml] 2.60 2.3 3.36 table 2. evaluation of the accuracy of the proposed hplc method. val cgb fen sample taken [μg/ml] found [μg/ml] recovery [%] taken [μg/ml] found [μg/ml] recovery [%] taken [μg/ml] found [μg/ml] recovery [%] 1 100.0 99.10 99.10 100.0 100.27 100.27 100 100.34 100.34 2 70.0 71.08 101.54 70.0 69.60 99.43 50 49.36 98.73 3 50.0 50.30 100.60 50.0 49.69 99.39 30 29.75 99.18 4 30.0 30.16 100.54 30.0 30.54 101.80 10 10.36 103.58 5 10.0 9.61 96.07 10.0 9.87 98.69 5 5.17 103.46 6 5.0 4.79 95.79 5.0 5.30 100.58 7 2.5 2.47 98.65 2.5 2.48 99.11 mean 98.89 99.90 101.06 sd 2.25 1.06 2.32 rsd 2.27 1.06 2.30 table 3. precision and accuracy of intra-day and inter-day analysis. drug intra-day inter-day added [µg/ml] found ± sd [μg/ml] recovery [%] rsd [%] er [%] found ± sd [μg/ml] recovery [%] rsd [%] er [%] val 10 9.85±2.433 98.49 2.470 -1.50 10.30±2.10 103.03 2.037 3.03 100 98.80±0.292 98.81 0.296 -1.19 100.82±1.72 100.82 1.710 0.82 cgb 10 9.81±0.573 98.11 0.584 -1.89 9.84±0.57 98.11 0.584 -1.89 100 100.52±0.242 100.52 0.240 0.52 102.55±1.67 102.55 1.629 2.55 fen 10 10.42±0.600 104.19 0.576 4.19 10.42±0.61 104.19 0.576 4.19 100 100.56±0.191 100.56 0.189 0.56 100.49±0.15 100.49 0.146 0.49 optimization of mobile phase ph different mobile phase ph values were tried in order to reach the optimum separation conditions. it was found that ph value below 3 lead to co-elution of the cited drugs. on the other hand, ph value greater than 3 resulted in increasing the retention times of the three drugs without improving separation. thus, ph value of 3 was chosen as optimum ph for chromatographic separation. the change of kh2po4 concentration did not have an influence on the separation process; so concentration of 80 mm of kh2po4 was used. mobile phase composition using mobile phases containing methanol alone led to separation of the three drugs but with high noise. on the other hand, increasing the concentration of buffer to more than 50 % led to longer retention bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:27 utc nova biotechnol chim (2018) 17(2): 140-149 145 fig. 2. chromatogram of blank plasma sample. times for the three drugs, clopidogrel bisulfate appears after 10 min. so, a combination of acetonitrile: phosphate buffer at ratio 70 – 30 was found to be the ideal mobile phase for separation in this method as the retention time decreased to less than 7 min (fig. 1). column temperature the mobile phase was pumped at different column oven temperatures in the range of 25 – 35 °c. peak shapes were improved with increasing temperature without affecting resolution and peak areas. so, 33 °c was found to be the optimum temperature for the separation of the cited drugs. flow rate the effect of flow rate on the separation was studied to improve the resolution of the eluted peaks. the flow rate was changed over the range of 0.8 – 1.2 ml/min. by beginning with the flow rate of 0.8 ml/min, the total run time was exceeding 10 min due to increasing the retention times of the three drugs. so, we increase the flow rate gradually in order to decease the total run time until reach the flow rate of 1.2 ml/min where, the plasma peak slightly overlapped with the first eluting drug, val. so, we decrease the flow rate again to 1.1 ml/min to ensure optimal and complete separation between the plasma peak and the three drugs. choice of appropriate wavelength the three drugs showed main absorption peaks at 210, 214, 240, 255 and 270 nm but showed maximum absorbance at 214 nm so, setting the uv detector at 214 nm, permitting the determination of the three drugs at the same time. method validation linearity, detection and quantitation limits linear calibration curves representing the relation between the concentrations of drugs versus the peak areas were obtained in the range of 2.5 – 100 μg/ml for both val and cgb and 5 – 100 μg/ml for fen. linear regression equations were obtained and correlation coefficient, slope and intercept were calculated. results are listed in table 1. accuracy accuracy of the measurements of the suggested method was determined using the calibration standards of the cited drugs. mean recovery values of 98.89, 99.90 and 101.06 % were obtained for bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:27 utc nova biotechnol chim (2018) 17(2): 140-149 146 fig. 3. chromatogram for (a): blank plasma (b): val (5 μg/ml), cgb (15 μg/ml) and fen (2o μg/ml) in spiked human plasma. val, cgb and fen, respectively, indicating high accuracy of the method. results are listed in the table 2. precision the relative standard deviations (rsd) of intra-day assay of the cited drugs were ranged from 0.19 to 2.47 % and for the inter-day assay was from 0.15 to 2.04 %. so, the values for rsd were not exceeding 2.5 % which revealed that the precision of the suggested method is very high. the results are listed in table 3. specificity specificity is the ability of the analytical proposed method to discriminate between target analytes and other components that may be present such as excipients, additives or endogenous plasma fluid components. fig. 2 and 3 show the great specificity of the proposed method. specificity of the proposed hplc method was evaluated by its successful application to determine the cited drugs in their tablets with mean recovery of 98.29 %, 99.57 % and 101.08 % for tareg® tab, clopex® tab and lipanthyl supra® tab, respectively (table 4). table 4. determination of cited drugs in their pharmaceutical preparations using the proposed hplc method. sample tareg® tablet (val) clopex® tablet (cgb) lipanthyl supra® tablet (fen) taken [μg/ml] found [μg/ml] recovery [%] taken [μg/ml] found [μg/ml] recovery [%] taken [μg/ml] found [μg/ml] recovery [%] 1 100 99.31 99.31 100 99.89 99.89 100 100.42 100.42 2 70 68.52 97.88 70 70.28 100.39 70 71.07 101.52 3 50 48.84 97.68 50 50.17 100.34 50 51.13 102.26 4 10 9.77 97.67 20 20.30 100.13 mean 98.29 99.58 100.98 sd 0.89 1.29 0.72 rsd 0.90 1.30 0.71 b a c d bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:27 utc nova biotechnol chim (2018) 17(2): 140-149 147 table 5. determination of cited drugs in spiked human plasma. val cgb fen sample taken [μg/ml] found [μg/ml] recovery [%] taken [μg/ml] found [μg/ml] recovery [%] taken [μg/ml] found [μg/ml] recovery [%] 1 4.0 3.60 76.49 7 6.49 92.67 5 5.12 102.47 2 5.0 3.82 76.53 9 7.96 88.40 8 8.16 101.98 3 6.0 4.43 73.85 10 9.60 90.61 10 9.86 98.65 4 6.5 4.85 74.67 15 13.19 87.97 20 18.56 92.83 24 21.27 88.64 mean 75.38 89.91 96.92 sd 1.34 2.17 6.20 rsd 1.78 2.41 6.20 also, specificity of the proposed hplc method was assessed by its successful application to determine drugs in spiked plasma samples with excellent extraction recoveries (74 % – 102 %) indicating that there was no interference from endogenous plasma components. results are listed in table 5. robustness the method proved to be robust for the minor variations in the method parameters but in case of increasing these small variation as in case of flow rate (1.05, 1.1 and 1.15 ml/min), this changes had influence on both peak area and retention time. the results are listed in table 6. system suitability system suitability test parameters; column efficiency (number of theoretical plates, n), tailing factor (t), retention factor (k), selectivity (ɑ) and resolution (rs) were used to verify that the reproducibility and the resolution of the system were adequate for the analysis to be done (table 7). analytical applications the statistical comparison of the results obtained showed that student's t-test and f-test (at 95 % confidence level) values are less than the tabulated ones, which showed that there is no significant difference between the proposed and reported methods. results are listed in table 8. in addition, the suggested method was applied to analyze drugs in spiked human plasma by simple protein precipitation procedure with acetonitrile: methanol mixture (1 : 1) followed by centrifugation and direct injection of the clear supernatant containing the cited drugs and analysis directly by the chromatographic system. excellent extraction recoveries were obtained in table 5. table 6. robustness of the proposed method applied on concentration (50 µg/ml for all substances). parameter affected peak area retention time val cgb fen val cgb fen flow rate (1.10, 1.09 and 1.11 ml/min) rsd of the affected parameter 0.195 0.956 0.402 1.725 2.393 2.754 flow rate(1.10, 1.05 and 1.15 ml/min) rsd of the affected parameter 4.700 4.773 4.651 4.003 4.084 4.046 acetonitrile content (70.0, 70.5 and 69.5 %) rsd of the affected parameter 0.723 0.325 0.630 1.236 0.604 0.957 conclusions the presented hplc method is simple, precise and can be used by every drug laboratory. this new procedure is very important as these drugs are commonly used as combinations for patients after stroke recovery. also the small sample plasma volume (0.5 ml), simple steps for precipitation bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:27 utc nova biotechnol chim (2018) 17(2): 140-149 148 table 7. analytical parameters for system suitability test of hplc method. parameters val cgb fen rs (resolution) 2.40 12.20 7.47 ɑ (selectivity factor) 1.18 1.78 1.35 k (retention factor) 1.61 2.87 3.88 t (asymmetry or tailing factor) 0.63 0.80 0.82 n (column efficiency) 14103 16541 16851 height equivalent theoretical plates (hetp) [mm] 0.018 0.015 0.015 of plasma proteins, good extraction recovery from plasma, and short run-time (less than 7 min) are additional advantages of this method. finally, the method was applied to the analysis of both drugs in raw materials, spiked human plasma and tablets dosage forms and can be used for routine laboratories analysis and quality control purposes for pharmaceutical companies and are very beneficial for toxicological and drug interaction studies. table 8. determination of the studied drugs in their tablets dosage forms using the proposed method compared to reference methods. parameters tareg® tablet clopex® tablet lipanthyl supra® tablet reference methoda proposed method reference methodb proposed method reference methodc proposed method n 4 3 3 4 5 4 mean recovery 99.440 98.290 99.100 99.580 100.260 101.080 variance 0.227 0.784 0.402 1.670 0.034 0.980 ±s.d. 0.465 0.886 1.091 1.290 0.185 0.989 ±r.s.d. 0.468 0.901 1.100 1.300 0.185 0.979 student-t 2.254(2.571) * 0.583(2.571) * 1.854(2.365) * f-test 3.068(5.14)* 4.154(9.55) * 1.651(5.41) * a gupta et al. (2010a) b the british pharmacopoeia (2017) c gupta et al. (2010b) references el-gizawy sm, abdelmageed oh, omar ma, duryea sm, abdel-megied am (2012) development and validation of hplc method for simultaneous determination of amlodipine, valsartan, hydrochlorothiazide in dosage form and spiked human plasma. am. j. analyt. chem. 3: 422-430. gupta kr, wadodkar ar., wadodkar sg (2010a) uvspectrophotometric methods for estimation of valsartan in bulk and tablet dosage form. int. j. chemtech. res. 2: 985-989. gupta kr, askarkar ss, rathod pr, wadodkar sg (2010b) validated spectrophotometric determination of fenofibrate in formulation. pharm. sin. 1: 173-178. gurupadayya bm, sam s (2014) bio-analytical determination of clopidogrel bisulfate and pantoprazole by rp-hplc method in rat plasma: application to drug interaction study. asian j. pharm. clin. res. 7: 10-13. gu q, burt vl, dillon cf, yoon s. (2012) trends in antihypertensive medication use and blood pressure control among united states adults with hypertension: the national health and nutrition examination survey, 2001 to 2010. circulation 126: 2105-2114. hall mj, levant s, defrances cj (2012) hospitalization for stroke in us hospitals, 1989–2009. nchs data brief 95: 1-8. international conference on harmonization. validation of analytical procedures: text and methodology, 2005. jain n, raghuwanshi r, jain d (2008) development and validation of rp-hplc method for simultaneous estimation of atorvastatin calcium and fenofibrate in tablet dosage forms. indian j. pharm. sci. 70: 263265. khaleel n, abdul rahaman sk (2016) validated stability indicating rp-hplc method for simultaneous determination of atorvastatin, fenofibrate and folic acid in bulk and pharmaceutical dosage form. pharm. lett. 8: 13-32. kernan wn, ovbiagele b, black hr (2014) guidelines for the prevention of stroke in patients with stroke and transient ischemic 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44: 2388-2393. sahu pk, murthy pyla sr, srinivas k, swain s (2016) simultaneous rp-hplc method development and validation of atorvastatin, ezetimibe and fenofibrate. pharmaceut. reg. affairs 5: 1-6. shrivastava pk, basniwal pk, jain d, shrivastava sk (2008) concurrent estimation of clopidogrel bisulfate and aspirin in tablets by validated rp-hplc method. indian j. pharm. sci. 70: 667-669. sweetman s (2009) martindale, the complete drug reference, 36th edition, pharmaceutical press, cop., london, uk, 3694 p. tian df, tian xl, tian t, wang zy, mo fk (2008) simultaneous determination of valsartan and hydrochlorothiazide in tablets by rp-hplc. indian j. pharm. sci. 70: 372-374. the british pharmacopoeia. london, uk, her majesty’s stationary office, 2017. the united states pharmacopoeia, vol. xxiv, washington, 2007. wang y, rudd ag, wolfe cd (2013) trends and survival between ethnic groups after stroke: the south london stroke register. stroke 44: 380-387. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:27 utc nova biotechnologica et chimica 15-1 (2016) 85 doi 10.1515/nbec-2016-0009  university of ss. cyril and methodius in trnava synthesis of 4-oxo-4h-chromene derivative with fused benzodiazepine ring ivana zemanová, michaela potančoková, renata gašparová department of chemistry, university of ss. cyril and methodius, j. herdu 2, trnava, sk-917 01, slovak republic (renata.gasparova@ucm.sk) abstract: 6-acetylbenzo[b]chromeno[2,3-e][1,4]diazepin-13(6h)-one 6 was synthesized by reaction of 4oxo-4h-chromene-3-carboxaldehyde 1 with 1,2-diaminobenzene 2 followed by cyclisation of formed schiff base 3 and spontaneous oxidation of dihydrodiazepine 4 by air oxygen. finally, diazepine 5 was acetylated at n(6) by reaction with acetic anhydride. key words: 4-oxo-4h-chromene, 1,2-diaminobenzene, diazepine, fused heterocycles 1. introduction the title 4-oxo-4h-chromene-3-carboxaldehyde 1 (fig.1) plays the crucial role in various reactions as oxidation and reduction, defunctionalisation, radical and nucleophilic addition, and many types of annulations and cycloaddition reactions (ghosh and chakraborty, 2015). several strategies were developed to construct chromene derivatives fused with seven-membered carbocyclic (turner et al., 2011) or heterocyclic (kumar et al., 2000) rings. among these compounds the key diazepine derivative 5 was synthesized by several pathways, e.g. from 2-(n-methyl-n-phenylamino)-4-oxochromene-3karboxaldehyde (singh et al., 2002) or from 3-(1,3-dioxolan-2-yl)-4h-chromen-4one (ghosh et al., 1983). fig. 1. 4-oxo-4h-chromene-3-carboxaldehyde. the importance of seven-membered rings consists on their biological activity, e.g. theaflavins from black tea possess antioxidant and antiviral activity (bhuyan et al., 2013). lenthionine which occurs in the shiitake mushrooms is known for its antithrombotic activity (shimada et al., 2004). seven-membered ring is also the structural unit of many alkaloids, e.g. strychnine, colchicine or samandaridine (gašparová, 2010). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:34 utc http://www.ncbi.nlm.nih.gov/pubmed?term=shimada%20s%5bauthor%5d&cauthor=true&cauthor_uid=15630278 86 zemanová, i. et al. in the present work, benzo[b]chromeno[2,3-e][1,4]diazepin-13(6h)-one (5) was synthesized by reacting 3-{[(2-aminophenyl)imino]methyl}-4h-chromen-4-one (3) in presence of glacial acetic acid and further acetylation with acetanhydride gave 6acetylbenzo[b]chromeno[2,3-e][1,4]diazepin-13(6h)-one (6) (scheme 1). 2. experimental melting points of products were determined on a kofler hot plate apparatus and are uncorrected. 1 h nmr and 13 c nmr spectra were obtained on a 300 mhz/75 mhz spectrometer vrx-300 in cdcl3 or dmso-d6 with tetramethylsilane as the internal standard. the infrared spectra were taken on a ftir iraffinity-1 spectrophotometer using kbr technique. elemental analyses were performed on flashea 2000 chns/ooea analyser. all solvents were distilled and dried appropriately prior to use. the course of reactions was monitored by tlc in ethyl acetate–hexane. 4-oxo-4hchromene-3-carbaldehyde 1 was synthesized by method, which was described by (nohara et al., 1974). 1,2-diaminobenzene was a commercial product. scheme 1. synthesis of 6-acetylbenzo[b]chromeno[2,3-e][1,4]diazepin-13(6h)-one. 2.1 synthesis of 3-{[(2-aminophenyl)imino]methyl}-4h-chromen-4-one (3) the mixture of 4-oxo-4h-chromene-3-carboxaldehyde 1 (1 g, 5.7 mmol) and 1,2diaminobenzene 2 (0.62 g, 5.7 mmol) was refluxed in 15 ml of ethanol for 1 h. after cooling the formed solid precipitate was filtered off, washed with ethanol and purified by crystallisation from ethanol to give product 3 as red solid. yield 76 %; m.p. 214216 °c. anal. calcd. for c16h12n2o2 (264.3) c, 72.72; h, 4.58; n, 10.60. found: c, 72.61; h, 4.54; n, 10.40%. ir (kbr): 3483, 3411 (nh2); 1635 (c=o) cm -1 . 1 h nmr spectrum was unmeasurable because of the low solubility of 3 2.2 synthesis of benzo[b]chromeno[2,3-e][1,4]diazepin-13(6h)-one (5) 3-{[(2-aminophenyl)imino]methyl}-4h-chromen-4-one 4 (1.04 g, 4 mmol) was refluxed in glacial acetic acid (10 ml) for 4 h. after cooling reaction mixture was bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:34 utc nova biotechnologica et chimica 15-1 (2016) 87 poured on ice and precipitate was filtered off, washed with water and crystallized from chloroform to give yellow product 5. yield 58 %; m.p. 265-268 °c. anal. calcd. for c16h10n2o2 (262.3): c, 73.27; h, 3.84; n, 10.68. found: c, 73.08; h, 3.82; n, 10.47%. ir (kbr):  3310 (nh); 1658 (c=o) cm -1 . 1 h nmr (cdcl3): 11.79 (bs, 1h, nh); 9.40 (s, 1h, ch=n); 8.47 (dd, 1h, j = 8.9 hz, 1.7 hz, h-1); 8.02-7.81 (m, 3h, ar-h); 7.65-7.28 (m, 4h, ar-h). 2.3 synthesis of 6-acetylbenzo[b]chromeno[2,3-e][1,4]diazepin-13(6h) one (6) compound 5 (1 g, 3.84 mmol) was refluxed in acetic anhydride (3.6 ml) for 1.5 h. after cooling the solid product was filtered off, washed with acetic acid (20 ml) and crystallized from ethanol to give product 6 as yellow solid. yield 69 %; m.p. 264-265 °c. anal. calcd. for c18h12n2o3 (304.3) c, 71.05; h, 3.97; n, 9.21. found c, 70.37; h, 3.62; n, 8.94%. ir (kbr):  1662 (c=o) cm -1 . 1 h nmr (dmso-d6): 8.83 (s, 1h, ch=n); 8.17 (dd, 1h, j = 7.8, 1.5 hz, h-1); 7.95-7.89 (m, 2h, h-3, h-7); 7.81-7.74 (m, 1h, h-4); 7.70 (dd, 1h, j = 7.8, 1.5 hz, h-2); 7.62-7.56 (m, 1h, h-8); 7.47-7.41 (m, 1h, h-9); 7.28-7.24 (m, 1h, h-10); 3.31 (s, 3h, ch3). 13 c nmr (dmso-d6): 178.9, 170.2, 162,7, 151,8, 144.3, 140.7, 134.1, 129.1, 128.7, 127.6, 126.7, 125.1, 123.0, 122.7, 119.1, 118.7, 102.3, 25.7. 3. results and discussion 4-oxo-4h-chromene-3-carbaldehyde 1 was easily prepared from 2hydroxyacetophenone under vilsmeier-haack conditions in 75% yield (nohara et al., 1974). 6-acetylbenzo[b]chromeno[2,3-e][1,4]diazepin-13(6h)-one 6 was synthesized by three-step synthesis (scheme 1). in the first step 4-oxo-4h-chromene-3carboxaldehyde 1 was treated with 1,2-diaminobenzene 2 for 1h under reflux in ethanol and schiff´s base 3 was formed in 76 % yield. 1,2-diaminobenzenes as 1,4binucleophiles have been already introduced into the reactions with 4-oxo-4hchromene-3-carbaldehydes 1 (plaskon et al., 2012). although variety of products was obtained, it is obvious that imine 3 was formed initially and it was isolated if the reaction was performed under relatively mild conditions (ghosh and khan, 1980). subsequently derivative 3 undergo intramolecular cyclization at the c-2 atom of the chromone ring by reflux in glacial acetic acid for 4h to form unisolable 6,11dihydrobenzo[b]chromeno[2,3-e][1,4]diazepin-13(5ah)-one 4, which oxidized spontaneously by influence of air oxygen (el-desoky and al-shihry, 2008) to product 5 (58 %). compound 5 displays in its 1 h nmr spectrum, in addition to other signals, broad singlet of nh proton at 11.79 ppm, singlet of h-12 proton at 9.40 ppm. the important feature of the above methodology is the fact that the chromone moiety remains intact, because the synthetic utility of chromones is limited due to facile opening of the chromone ring (shutov et al., 2011) and strategies are being developed to circumvent this (borrell et al., 2001). in order to exploit the n(6)-derivatization of diazepine ring of 5, we have realized n-acetylation of 5 by method of (betakis et al., 1984) in acetic anhydride for 4h, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:34 utc 88 zemanová, i. et al. when n-acetylfuro[3,2-b]pyrrole-5-carboxylate 6 was obtained in 69% yield. compound 6 display in its 1 h nmr spectrum the singlet at 8.83 ppm spectra due to h12 proton. the methyl protons of acetyl group resonate as singlet at 3.31 ppm. finally, aromatic protons appear as multiplets at 8.17-7.26 ppm region. 13 c nmr spectrum shows signals of two carbonyl groups at 178.9 and 162.7 ppm, respectively. signals of 5a and 4a carbons appear at 170.2 and 151.8 ppm, respectively. 4. conclusion in summary, we have developed the synthesis of 6-acetylbenzo[b]chromeno[2,3e][1,4]diazepin-13(6h)-one 6 69% yield by three-step reaction of 4-oxo-4hchromene-3-carboxaldehyde 1 with 1,2-diaminobenzene 2 followed by cyclisation of formed schiff base 3 and spontaneous oxidation of dihydrodiazepine 4 by air oxygen and subsequent acetylation of diazepine 5 was at n(6) by reaction with acetic anhydride. acknowledgement: this work was supported by the slovak research agency under the contract no. vega 1/0534/16. references betakis, e., piesch, s., ried, w.: 2,3-dihydro-1h-benzodiazepine aus 2halobenzaldehyden und 1,2-ethanediamin: eine neue einfache synthese. synthesis, 1988, 820-823. bhuyan, l.p., sabhapondit, s., baruah, b.d., bordoloi, c., gogoi, r., bhattacharyya, p.: polyphenolic compounds and antioxidant activity of ctc black tea of north-east india. food chem., 15, 2013, 3744-3751. borrell, j. i., teixido, j., schuler, e., michelotti, e.: solid-supported synthetic equivalents of 3-formylchromone and chromone. tetrahedron lett. 42, 2001, 5331-5334. el-desoky, e.-s.i., al-shihry, s.s.: synthesis and reactions of some new benzopyranone derivatives with potential biological activities. j. heterocycl. chem. 45, 2008, 1855-1864. gašparová, r.: syntéza prírodných látok. fakulta prírodných vied ucm v trnave, trnava 2010, 144 pp. ghosh, c.k., bandyopadhyay, c., morin, c.: heterocyclic systems. part 14. condensation reactions of 2-(4-oxo-4h-1-benzopyran-3-yl)-1,3-dioxolane. j. chem. soc., perkin trans. 1, 1983, 1989-1994 ghosh, c.k., chakraborty, a.: chemistry and application of 4-oxo-4h-1benzopyran-3-carboxaldehyde. arkivoc, 2015 (vi), 288-361. ghosh, c.k., khan, s.: heterocyclic systems; 9. reaction of 4-oxo-4h-1benzopyran-3-carboxaldehydes (3-formylchromones) with 1,2-diamines. synthesis, 1980, 701-702. kumar, k., kapoor, r., kapur, a., ishar, m.p.s. annulation reactions of allene-derived 1,3-dipole with 3-substituted-chromones: unusual recognition of 4π-component in 3-(n-aryliminomethyl)chromones through [4 + 3] annulation. org. lett. 2, 2000, 2023 – 2025. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:34 utc http://pubs.rsc.org/en/results?searchtext=author%3achandrakanta%20bandyopadhyay http://pubs.rsc.org/en/results?searchtext=author%3achristophe%20morin nova biotechnologica et chimica 15-1 (2016) 89 nohara, a., umetani, t., sano, y.: a facile synthesis of 4-oxo-4h-1benzopyran-3-carboxaldehydes by vilsmeier reagents. tetrahedron 30, 1974, 3553-3561. plaskon, a.s., grygorenko, o.o., ryabukhin, s.v.: recyclizations of 3formylchromones with binucleophiles. tetrahedron 68, 2012, 2743-2757. shimada, s., komamura, k., kumagai. h., sakurai. h.: inhibitory activity of shiitake flavour against platelet aggregation. biofactors, 22, 2004, 177179. shutov, r.v., kuklina, e.v., ivin, b.a.: a study of the reactions of 2-aryl-4hydroxy-6h-1,3-thiazin-6-ones with chromone-3-carboxaldehydes. tetrahedron lett. 52, 2011, 266-269. singh, g., singh, l., ishar, m.p.s.: 2-(n-methylanilino)-3-formylchromone a versatile synthon for incorporation of chromone moiety in a variety of heterocyclic systems and macrocycles through reactions with bifunctional nucleophiles. tetrahedron, 58, 2002, 7883-7890. turner, p.a., griffin, e.m., whatmore, j.l., shipman, m.: tetrahydroxanthones by sequential pd-catalyzed c−o and c−c bond construction and use in the identification of the “antiausterity” pharmacophore of the kigamicins. org. lett., 13, 2011, 1056–1059. received 19 may 2016 accepted 11 july 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:34 utc http://www.sciencedirect.com/science/article/pii/s0040402001970346 http://www.sciencedirect.com/science/article/pii/s0040402001970346 http://www.ncbi.nlm.nih.gov/pubmed?term=shimada%20s%5bauthor%5d&cauthor=true&cauthor_uid=15630278 http://www.ncbi.nlm.nih.gov/pubmed?term=komamura%20k%5bauthor%5d&cauthor=true&cauthor_uid=15630278 http://www.ncbi.nlm.nih.gov/pubmed?term=kumagai%20h%5bauthor%5d&cauthor=true&cauthor_uid=15630278 http://www.ncbi.nlm.nih.gov/pubmed?term=sakurai%20h%5bauthor%5d&cauthor=true&cauthor_uid=15630278 http://www.ncbi.nlm.nih.gov/pubmed/15630278 nova biotechnol chim (2018) 17(1): 86-94 doi: 10.2478/nbec-2018-0009  corresponding author: jmohamad@alumni.ut.ac.ir nova biotechnologica et chimica oil characteristics of safflower seeds under different nutrient and moisture management mokhtar pasandi, mohsen janmohammadi, amin abasi and naser sabaghnia department of plant production and genetics, faculty of agriculture, university of maragheh, p.o. box 55181-83111, maragheh, iran article info article history: received: 21st january2018 accepted: 16th april 2018 keywords: fatty acid profile foliar feeding irrigation micronutrients soil application abstract safflower is one of the most important oilseed crops in semi-arid regions. the soil of semi-arid areas often encounters micronutrient deficiencies. however, nutrients imbalance seems to affect the quantitative and qualitative aspects of the oil as well as plant growth. current experiment was carried out to evaluate the impact of different application practices (soil application and foliar spray) of micronutrients (fe, zn, mn) on oil content, fatty acid profile and yield components of safflower under full and limited irrigations. results showed that all of investigated traits were significantly affected by fertilizer treatment and irrigation system. the highest seed protein content was recorded for plants grown by soil application of zn under limited irrigation condition. the highest oil content was achieved by soil application of zn under full irrigation condition. the water deficit significantly reduced some qualitative characteristics such as oleic acid, palmitic aid, stearic acid, linoleic acid, iodine value and saponification value. the highest head number per plant, seed number per head and seed yield recorded in plants grown by soil application of fe and zn under full irrigation condition. although the use of micronutrients improved qualitative characteristics in comparison with control, the best qualitative characteristics were achieved with the soil application of zn and fe. the elimination of micronutrient deficiencies and the balanced supply of nutrients through soil along with optimal and timely irrigation can significantly increase the efficiency of safflower production systems and improve the quality of the oil.  university of ss. cyril and methodius in trnava introduction in terms of cultivated area and nutritional importance the oil crops are the second most important crops in the world after cereals. the extracted oil and by-product have an important role in maintaining the health of the community. safflower (carthamus tinctorius l.) is an annual, highly branched, herbaceous, thistle-like plant from asteraceae family. although the safflower is considered as an oilseed, it also makes an acceptable livestock forage during the vegetative growth (danieli et al. 2011). according to fao statistics in 2016 the harvest area of safflower throughout the world was about 1,140,000 hectares and the total production was about 948,516 tones with an average yield about 832 kg ha-1. however, due to the climatic and social conditions of semi-arid regions, the main focus is on the cultivation of cereals and the utilization of cereal-fallow rotation. it seems that safflower is best suited to be grown in rotation with cereal crops while there is not enough information about safflower crop management. all of the above bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:58 utc nova biotechnol chim (2018) 17(1): 86-94 87 mentioned factors have led to slow development of safflower compared to other oil crops and safflower cultivation remained a backyard crop for personal use and as a result it remained a minor and neglected crop (emongor 2010). the use of safflower in rotation can have many benefits such as improved productivity of subsequent crops, lifting farm income, reducing the impact of disease and weeds; and producing edible and industrial quality oil and meal. their integration offers the opportunity to enhance overall environmental, production and economic sustainability (johnston et al. 2002). assessment of fatty acid composition in safflower seeds showed that two main unsaturated fatty acids are oleic (18:1) and linoleic acid (18:2). oil content varies from 20 to 40 % depended on genotypes and growth conditions. they account for about 90 % of the total fatty acids and can be also responsive to environmental factors. the remaining 10 % correspond to the saturated fatty acids, palmitic (16:0) and stearic acid (18:0). standard safflower oil contains about 6 – 8 % palmitic acid, 2 – 3 % stearic acid, 16 – 20 % oleic acid, and 71 – 75 % linoleic acid (liu et al. 2016). climate and soil are the major factors influencing plant growth. the soil condition of the semi-arid regions is not suitable for providing the all required nutrients for optimum plant growth. the high ph and alkalinity of the soils are the major constraints on production in semi-arid region (keshavarzi and sarmadian 2012). the numerous other plant nutrients such as fe, mn, zn and cu also have limited bioavailability in the alkaline ph range. although micronutrients are needed at minor amounts, their deficiencies can significantly reduce growth and yield (marschner 2012). micronutrients have critical roles in the photosynthesis, assimilation, and in optimizing the function of some key enzymes. for instance, zn plays an imperative role as a co-factor of enzymes (alcohol dehydrogenase, superoxide dismutase, carbonic anhydrase and rna polymerase) or as a functional, structural, or regulator cofactor of a large number of enzymes. fe involved in the manufacturing process of chlorophyll, electron carrier in respiration and photosynthesis, in the production and detoxification of radical oxygen and redox homeostasis (hänsch and mendel 2009). in this regard, it is worth mentioning that finding and optimization of an efficient plant nutrition system is very important necessity in semi-arid region. however, the nutrient managements have multiple and sometimes contradictory effects on quantitative and qualitative aspects and this will make managing much more complicated. application of micronutrient fertilizers may be carried out as soil or foliar utilization. it has been reported that in semiarid regions with low amount of precipitation foliar spray of micronutrients is a more suitable option for improving seed yield and oil characteristics compared with soil application (galavi et al. 2012; janmohammadi et al. 2016; yadegari 2016). however, the efficiency of micronutrient applications methods is strongly dependent on other environmental conditions, and it is not possible to recommend a consistent method for all conditions. on the other hand, water scarcity is one of the most important factors limiting the growth and yield of crops in semi-arid regions. terminal water scarcity can also affect other crop management such as fertilization. especially in the north west of iran, which has moderately cold wet winters and hot dry summers, safflower is conservatively sown in spring (march-april), which approximately corresponds with the end of the rainy season in this region (mirshekari et al. 2013). according to special agro-meteorological circumstances of semi-arid region, spring-sown safflower growth is largely restricted by terminal drought. however, some irrigation managements can partly reduce the negative effects of terminal drought stress. limited irrigation (li) is a strategy to improve water use efficiency that involves the addition of limited amounts of irrigation water to essentially rain-fed crop in the last months of the spring when rainfall has dropped sharply (reddy 2016). however, irrigation rate varies according to environmental conditions, but in the northwest region for full irrigated filed of safflower usually use for 78 × 105 liters per hectare within the dry months of growing season (may-august), while in limited irrigation only 26×105 liters per hectare are used as two irrigations during reproductive growth (june-july). it appears that restricted water supply during the mid-season or terminal dry spells can, however, play a critical role in enhancing and stabilizing the productivity bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:58 utc nova biotechnol chim (2018) 17(1): 86-94 88 of spring-sown safflower. with regards to effects of environmental conditions and agronomic managements on seed yield components, oil yield and oil composition their evaluation can provide valuable information about agronomic managements. the objective of this study was to investigate the effects of soil application and foliar spray of micronutrients on the growth parameter and qualitative aspects of safflower oil in north west of iran. experimental growth conditions the current experiment was carried out in kharajou district, maragheh, north-west of iran (46°53' east longitude and 37°31' north latitude and at altitude of 1,780 m) during growing season 2016 – 2017 on a clay loam soil. soil properties are shown in table 1. the location is suitable representative of highland semi-arid zone and its climate is classified as cold semi-arid climate (peel et al. 2007) with an average annual precipitation of 379 mm. this district is large elevated area and is located in the northern mountainous site of sahand mountain. however, most of the precipitation was recorded in winter and early spring. the metrological data of location is shown in table 2. experimental setup seeds of spring safflower (carthamus tinctorius l.) cv. ‘esfahan’ were purchased from pakan bazr company. one year before the beginning of experiment were sown in isolated filed in the maragheh region and were propagated according the sabaghnia et al. (2015). the experiment was laid out according split-plot (2 ×7), with main plots arranged as an rcbd with three replications and a net plot size as 5 × 4 m. the main plot was assigned to moisture regimes including full irrigation (fi) and limited irrigation (li). the sub-plots were allocated to fertilizer treatments which included control (no-fertilizer application), fe2+s (henceforth as fes): soil application of nano-chelated iron at rate of 1.5 kg ha-1, zn2+s (zns): soil application of nanochelated zinc at rate of 1.5 kg ha-1, mn2+s: soil application of nano-chelated manganese at rate of 1.5 kg ha-1, fe2+f: foliar spray of nano-chelated iron at rate 1,500 ppm, zn2+f: foliar spray of nanochelated zinc at rate 1,500 ppm, mn2+f: foliar spray of nano-chelated manganese at rate 1,500 ppm. micronutrient fertilizers purchased from fanavar sepehr parmans co. iran. each plot included twenty rows, 5 m long and 75 cm apart. seeds were sown 20 cm apart at 5 cm depth. the small terraces of 1.5 m in the interspaces was considered to prevent contamination by surface run-off containing fertilizer. table 1. physical and chemical characteristics of the soil in 10-20 cm depth. soil property clay [%] 40 silt [%] 28 sand [%] 22 ph 7.7 organic carbon [g kg-1] 3.8 ec [ds m-1] 1.65 total n [%] 0.046 p [g kg-1] 11.7 k [g kg-1] 182 zn [g kg-1] 0.91 fe [g kg-1] 1.26 mn [g kg-1] 8.14 there was no incidence of pest or disease on plants during the experiment. weeds were controlled over the growth period with hand hoeing. one mechanical weed control was performed after the fertilization by passing with rotary hoes between the rows. in foliar treatments micronutrient solution sprayed over the foliage to point of run-off during the stem elongation (bbch=30), flowering (bbch=30) and head growth (bbch=71). at field capacity and at the permanent wilting point, mean soil moisture content in the top 50 cm of the soil is about 35 and 13 % by weight, respectively. under full irrigation conditions, plots were irrigated when soil moisture content was reduced to 50 % of field capacity. under limited irrigation plants growth were relied on rainfall and moisture stored in the soil from previous season and only two supplemental irrigations were applied during the reproductive stage (bbch=50; head emergence and bbch= 71; head and fruit development). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:58 utc nova biotechnol chim (2018) 17(1): 86-94 89 depth of net irrigation water fraction was ~130 mm. the amount of irrigation water was calculated to restore water content in the root zone to field capacity. all other necessary cultural practices and plant protection measures were followed uniformly for all the plots during the entire period of experimentation. table 2. precipitation, mean humidity and mean temperature in crop seasons of 2016 – 2017 at evaluated site. parameters march april may june july 2016 2017 2016 2017 2016 2017 2016 2017 2016 2017 pecipitation [mm] 43 62 51 49 21 18 25.9 32 13 9 mean humidity [%] 57 63 50.2 55 40.1 51 31.7 46 30.8 34.7 total evaporation [mm] 14 19 32 36 49 52 193 171 278 288 mean temperature [°c] 8.5 10.2 13.7 12.9 19.2 17.5 23.9 24.2 26.3 28 oil extraction and analyses after plant harvest at maturity stage, seeds were separated from the head and dried, then total protein was estimated by kjeldahl method according to nosheen et al. (2016). in order to perform chemical and physical analyzes, oil extraction was carried out through cold extraction method by hexane. oil content was determined gravimetrically by extraction with technical grade hexane (sigma-aldrich) in a soxhlet apparatus (cole-parmer, germany) for 10 h at 40 °c. the extracted oil was stored at -18 °c for further investigation in polypropylene containers. iodine value of oil was determined according to standard method (cd 1-25) introduced by aocs (1993). saponification value measured according to standard method (cd 3-25) suggested by aocs (1993). determination of fatty acids was carried out by gas chromatography apparatus (agilent 6890n, usa). preparation of methyl ester of fatty acids was according to ortega et al. (2004). the determination of the fatty acids profile was carried out by gc equipped with a flame ionization detector and a tc-ffap capillary column (60 m×0.25 mm internal diameter, 0.25 µm). the temperature program was as follows: starting at 150 °c and then heating to 190 °c at 5 °c/min, after 2 min followed by heating from 190 °c to 250 °c at 5 °c/min. the final temperature (250 °c) was held for 8 min. the fatty acids used as standards for the gc analyses (palmitic, stearic, and oleic acids) were from sigma. the injector and detector temperatures were both set at 250 °c. injections of methylated sample (1 μl) were made in the splitless mode. statistics data were analyzed by two-way variance analysis using spss (15.0) and comparisons among means of factors combinations were examined based on the least significance difference (lsd) test at α = 0.05. results evaluation of plant height revealed that optimal water supply under full irrigation conditions resulted in plants higher by 31 % compared to limited irrigation conditions. the tallest plants were observed under full irrigation condition when soil was supplemented with fe2+ and zn2+ (table 3). the similar statue also was observed for some seed yield parameters such as head number per plant and seed number per head. assessment of seed yield showed that under limited irrigation it was about 24 % lower than full irrigation condition. mean comparison among fertilizer treatments showed that soil enrichment with fe2+ and zn2+ under full irrigation increased the seed yield by 12 % and 14 %, over the control. the highest yield was obtained after soil enrichment with fe2+ and zn2+ under full irrigation. the seed yield of plants grown under full irrigation was 28 % higher than of plants grown under limited irrigation. soil application of fe2+ and zn2+ increased the yield by 23 % compared with the control. in addition, regular irrigation increased the 1000-seed weight by 6 % over the limited irrigation. the heaviest seeds were recorded for plants grown in soil fertilized with fe2+ and zn2+ as well as in plants sprayed with fe2+ (table 3). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:58 utc nova biotechnol chim (2018) 17(1): 86-94 90 analysis of variance (anova) showed that seed protein content was significantly (p < 0.01) affected by fertilizer treatments and irrigation system (table 3). mean comparison between the irrigation systems revealed that protein content of plant grown under limited irrigation was 8 % higher than at full irrigation. the highest protein content was measured in plants after soil fertilization with table 3. the effect of micronutrients fertilizers as soil application and foliar spraying on oil properties and yield components of safflower under different irrigation systems. treatments pr oa iv sv ph hn sn tsw by sy full irrigation c 15.95g 6.31cde 170.66cde 182.00bc 88.00b 17.33cd 21.00cd 28.29cde 7,523e 1,265e fe2+s 17.99 de 9.12a 178.33ab 196.33a 102.33a 23.00a 25.00ab 30.33a 8,488a 1,629a fe2+f 16.86 def 7.55b 171.00cde 181.00bc 90.00b 18.00bcd 22.33bc 29.64ab 7,823c 1,511b zn2+s 19.60 b 9.22a 180.66a 193.66a 102.00a 22.00a 27.00a 29.58ab 8,560a 1,621a f 2+zn 17.63def 7.29bc 175.33bc 173.33de 88.66b 19.66b 21.00cd 28.80bc 7,691cd 1,383cd mn2+s 17.86 de 6.67bcd 174.00bc 183.33b 92.33a 19.00bc 22.33bc 28.98bc 8,244b 1,426c f 2+mn 17.31ef 6.97bcd 171.33cde 178.00cd 87.66b 17.66bcd 19.00cde 28.53cd 7,575d 1,312de limited irrigation c 18.20de 5.56e 156.00f 167.66fg 66.00d 12.00f 14.66f 27.56efg 5,603g 1,008h fe2+s 19.13 bc 6.90bcd 166.33e 170.66efg 70.50cd 17.00cd 18.00def 28.23cdef 6,292e 1,175fg fe2+f 18.30 de 6.90bcd 172.33cd 174.00de 73.80c 16.00de 15.66ef 27.79defg 5,909f 1,134fg zn2+s 22.28 a 6.46bcde 168.00de 172.00ef 73.50c 18.00bcd 17.66def 27.60efg 6,330e 1,183f f 2+zn 19.60b 6.31cde 168.00de 171.33efg 71.40cd 14.66e 18.00def 27.37g 6,045f 1,141fg s 2+mn 18.50cd 5.88de 159.33f 169.33efg 68.70cd 17.00cd 17.00ef 28.02defg 5,868f 1,108g f 2+mn 17.75def 5.46e 160.66f 166.33g 71.40cd 14.33e 16.00ef 27.47fg 5,907f 1,157fg statistical significance fertilizer (f) ** ** ** ** * ** ** ** ** ** irrigation (i) ** ** ** ** ** ** ** ** ** ** f×i * * ** * ns ns ns * ** * cv 3.24 8.93 1.83 1.72 4.79 8.06 11.12 1.66 2.14 7.53 c: control (no-fertilizer application); (s) and (f) in the subscript of the fertilizer refer to soil application and foliar feeding; pr: seed protein content [%]; oa: oleic acid [%]; iv: iodine value of oil; sv: saponification value; ph: plant height [cm]; hn: head number per plant; sn: seed number per head; tsw: thousand seed weight; by: biological yield [kg ha-1]; sy: seed yield [kg ha-1]. in each column, values with similar letter(s) are not significantly different at the 5 % level of probability. ns = not significant; * = significant at 5 % level of probability; ** = significant at 1 % level of probability. zn2+ under limited irrigation, while the lowest amounts were related to lack of any fertilizer under full irrigation conditions. evaluation of the oil content showed that the effect of irrigation and fertilizers treatment was significant. oil content is one of rare traits that can be considered both as a quantitative and qualitative characteristic. reducing irrigation frequencies under limited irrigation considerably reduced (by 9 %) the oil content in comparison with full irrigation. the highest oil content was recorded for plants grown in soil with addition of zn2+ and followed with plant grown by foliar spray of zn under full irrigation system (fig. 1). also comparison of fertilizer treatment under limited irrigation showed that the highest oil content was related to soil application of zn2+. similar, though smaller effects, were observed for fe2+. assessment of oleic acid revealed a significant effect of irrigation system and fertilizer treatment (p < 0.01). oleic acid content in limited irrigation conditions was about 19 % lower than full irrigation. the highest oleic acid content was recorded for plant grown in soil enriched by zn2+ or fe2+, both under full irrigation condition (table 3). water shortage considerably reduced iodine value of oil (by 6 %). the highest iodine value was related to plant grown in soil fertilized with zn2+ and fe2+ under full irrigation condition while the lowest value was recorded for plant grown with no-fertilizer, or foliar-applied mn2+ under limited bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:58 utc nova biotechnol chim (2018) 17(1): 86-94 91 irrigation (table 3). a similar trend also was observed for saponification values (table 3). fig. 1. effect of fertilizers with micronutrient (fe2+, zn2+, mn2+) applied into soil or by spraying (indexes s and f, respectively) on total oil content in safflower seeds under different moisture regimes. values with different superscripts are significantly different (p < 0.05). all values are mean ± sem. the irrigation system and fertilizer treatment significantly affected the content of palmitic acid (fig. 2). the content of this fatty acid under limited irrigation conditions was about 18 % lower than under full irrigation conditions. effect of fertilizers with micronutrient (fe2+, zn2+, mn2+) applied into soil or by spraying (indexes s and f, respectively) on total oil content in safflower seeds under different moisture regimes are shown on fig. 1, while values with different superscripts are significantly different (p < 0.05). anova for stearic acid (fig. 3) and linoleic acid content (fig. 4) revealed that the effects of irrigation system, fertilizer treatments as well as their interaction effects were significant. the content of linoleic acid in plants grown under limited irrigation was about 6% lower than under full irrigation. the highest values were recorded for plants fertilized through both soil and foliar spray with fe2+ under full irrigation condition (fig. 4). when regular water supply was applied, foliar spray of fe2+ and zn2+ had a better effect on linoleic acid content in comparison with other fertilization methods. cluster analysis of traits in response to the treatments (fig. 5) indicated that oil content, protein percent and palmitic acid content were impacted similarly (cluster i); the highest values of the above mentioned traits were obtained for samples of soils with applied zn2+ fertilizer. fig. 2. effect of fertilizers with micronutrient (fe2+, zn2+, mn2+) applied into soil or by spraying (indexes s and f, respectively) on palmitic acid content in safflower seeds under different moisture regimes. values with different superscripts are significantly different (p < 0.05). all values are mean ± sem. the second cluster groups of oleic acid, iodine value of oil, saponification value and agronomic traits such as seed yield, head number, plant height, biological yield, seed number per head. the highest values for these parameters were obtained for plants grown in soils with applied zn2+ and fe2+ under full irrigated condition. the third cluster consists of traits such as content of stearic acid, linoleic acid and 1000-seed weight, which were positively affected by foliar application of fe2+ and zn2+ under full irrigated condition (fig. 5). further, cluster analysis of fertilizer treatments could classify them into three groups. the first group was control (no fertilizer applied) resulting in the poorest performance of plants. the second cluster grouped the treatments of mns, fef, znf and mnf. these treatments improved the qualitative and quantitative traits in comparison with the control, however, difference among them were not significant. the third cluster includes the treatments with fes and zns that resulted in the highest improvements in the studied traits (fig. 6). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:58 utc nova biotechnol chim (2018) 17(1): 86-94 92 discussion our study confirmed that qualitative traits of seeds and oil are significantly affected by agronomic managements. our results are consistent with results of some previous works (baldini et al. 2000; movahhedy-dehnavy et al. 2009; omidi et al. 2010). although limited irrigation is done to reduce water stress and prevent loss of yield, amount and distribution of rainfall in the experimental area appear inappropriate since even limited irrigation failed to fully eliminate the impacts of water shortage on plants. fig. 3. effect of fertilizers with micronutrient (fe2+, zn2+, mn2+) applied into soil or by spraying (indexes s and f, respectively) on stearic acid content in safflower seeds under different moisture regimes. values with different superscripts are significantly different (p < 0.05). all values are mean ± sem. comparison of two moisture regimes indicated that the seed protein content of plants grown under the limited irrigation was higher than under full irrigation. water scarcity is one of the most important factors that stimulates the degradation of photosynthetic proteins, such as rubisco, then can improve seed protein biosynthesis by increasing the remobilization of amino acids from source to sink (distelfeld et al. 2014). most of micronutrients can play a role as a cofactor in enzymes structure, hence enzyme activity largely depends on supply of micronutrients. in some cases, the stimulation of the remobilization of micronutrients from the source to the sink can affect the activity of the enzymes and, further, the qualitative aspects (marschner 2012; lemoine et al. 2013). fig. 4. effect of fertilizers with micronutrient (fe2+, zn2+, mn2+) applied into soil or by spraying (indexes s and f, respectively) on linoleic acid content in safflower seeds under different moisture regimes. values with different superscripts are significantly different (p < 0.05). all values are mean ± sem. most research pointed on negative impact of water deficit on photo-assimilates metabolism and phloem loading. our results supported previous findings that water deficit strongly decreases biological yield, consequently production of fatty acids and other qualitative characteristics. in this regard, it has been reported that heat and drought stress alters the development of embryo and pericarp and result in reduction of oil content (rondanini et al. 2003). fig. 5. cluster analysis to classify quantitative and qualitative traits of safflower in terms of similarity in response to nutrients managements. for abbreviations, see table 3. the best growth and highest oil quality was recorded under growth in soil supplemented with bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:58 utc nova biotechnol chim (2018) 17(1): 86-94 93 fe2+ and zn2+. comparison of mn2+ content with other micronutrients in soil indicates that deficiencies of fe2+ and zn2+ are much more pronounced than mn2+. zinc plays critical role as a cofactor in all enzymatic groupsand, therefore can regulate the actions of a number of genes. li et al. (2017) introduced a zinc-finger protein to soybean to modulate seed oil accumulation. oil accumulation in seeds is associated with synthesis of fatty acids in plastid, and many key enzymes are involved in this process, including acetyl-coa carboxylase and 3-ketoacyl-acyl carrier protein synthase. it appears that zn is necessary for acetyl-coa carboxylase function because a zn-domain has been recognized for this enzyme. also fe as redox-active metal is involved in photosynthesis, mitochondrial respiration, nitrogen assimilation, phyto-hormone biosynthesis, protective process (hänsch and mendel 2009). therefore, its supply can directly and indirectly affect the quality of the oil. it has been reported that eliminating plant nutrient deficiencies by improving plant growth and intracellular metabolic processes can lead to increase of seed oil percentage (ravi et al. 2008; movahhedy-dehnavy et al. 2009). fig. 6. cluster analysis for classification of different fertilizer treatments in terms of similarity in influencing the studied traits in safflower. c: control (no-fertilizer application), micronutrient (fe2+, zn2+, mn2+) applied into soil or by spraying (indexes s and f, respectively). although some studies reported positive effects of foliar spray of micronutrient (movahhedydehnavy et al. 2009; galavi et al. 2012; janmohammadi et al. 2017), the results of this experiment showed that the soil application of micronutrient was more efficient than foliar spray. this could be due to high temperature at the end of growing season, thickening of the cuticle, and low absorption of micronutrients. iodine value of oil is an index for determination of the unsaturation level in fatty acids. water shortage significantly reduced iodine value and this statue can be attributed to decreasing the amount or activity of desaturase enzymes (movahhedydehnavy et al. 2009). these results are consistent with those of other studies and suggest that drought stress decreases degree of fatty acid unsaturation and reduces the proportions of linolenic(18:3) and linoleic (18:2) acids (hamrouni et al. 2001). saponification value of oil depends on the average molecular weight of the fatty acids constituent of fat. saponification value has a direct relation with the amount short chain fatty acids. the high levels of this index, as a results of soil enrichment with fe2+ and zn2+ under full irrigation condition indicates to production and of the fatty acids and loading of fatty acids on glycerol backbone. conclusions although the safflower is relatively tolerant to drought stress, the water deficiency in the limited irrigation system causes a significant reduction in growth, yield components, oil content and qualitative aspects of the oil. soil fertilization with zn2+ and fe2+ under full irrigation condition significantly increased the amount of oil content, palmitic acid, stearic acid and oil quality characteristics compared with the control. there was a significant correlation between seed weight and fatty acid with 18-carbon chain (stearic acid and linoleic acid). the treatments that improved the yield also often improved the quality of the seed oil. although the foliar spray of micronutrients could increase some qualitative and quantitative properties compared with the control, the highest performance was achieved in soil application method. from the findings of present study it can be concluded that nutrition and irrigation managements are the important factors that affect the quality of oil in safflower balanced fertilization and accurate irrigation scheduling can greatly improve the quality of seed oil in semi-arid regions. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:58 utc nova biotechnol chim (2018) 17(1): 86-94 94 acknowledgement this work was financially supported by university of maragheh. also the authors would like to thank experts of central laboratory (depended on laboratory network of strategic technologies) for their assistance. references aocs (1993) official methods and recommended practices of the american oil chemists’ society. aocs press. washington, dc. baldini m, giovanardi r, vannozzi g (2000) effect of different water availability on fatty acid composition of the oil in standard and high oleic sunflower hybrids. in proceedings of 15th international sunflower conference, toulouse, pp. a79-84. danieli pp, primi r, ronchi b, ruggeri r, rossini f, del puglia s, cereti cf (2011) the potential role of spineless safflower (carthamus tinctorius l. var. inermis) as fodder crop in central italy. ital. j. agron. 6: 4-9. distelfeld a, avni r, fischer am (2014) senescence, nutrient remobilization, and yield in wheat and barley. j. exp. bot. 65: 3783-3798. emongor v (2010) safflower (carthamus tinctorius l.) the underutilized and neglected crop: a review. asian j. plant sci. 9: 299-306. galavi m, ramroudi m, tavassoli a (2012) effect of micronutrients foliar application on yield and seed oil content of safflower (carthamus tinctorius). afr. j. agric. res. 7: 482-486. hamrouni i, salah hb, marzouk b (2001) effects of waterdeficit on lipids of safflower aerial parts. phytochemistry. 58: 277-280. hänsch r, mendel rr (2009) physiological functions of mineral micronutrients (cu, zn, mn, fe, ni, mo, b, cl). curr. opin. plant biol. 12: 259-266. janmohammadi m, amanzadeh t, sabaghnia n, ion v (2016) effect of nano-silicon foliar application on safflower growth under organic and inorganic fertilizer regimes. bot. lith. 22: 53-64. janmohammadi m, sabaghnia n, seifi a, pasandi m (2017) the impacts of nano-structured nutrients on chickpea performance under supplemental irrigation. acta univ. agric. silvic. mendel. brun. 3: 859-870. johnston am, tanaka dl, miller pr, brandt sa, nielsen dc, lafond gp, riveland nr (2002) oilseed crops for semiarid cropping systems in the northern great plains. agron. j. 94: 231-240. keshavarzi a, sarmadian f (2012) mapping of spatial distribution of soil salinity and alkalinity in a semi-arid region. ann. warsaw agricult. univ. – sggw, land reclam. 44: 3-14. lemoine r, la camera s, atanassova r, dédaldéchamp f, allario t, pourtau n, faucher, m (2013) source-to-sink transport of sugar and regulation by environmental factors. front. plant sci. 4: 272. li qt, lu x, song qx, chen hw, wei w, tao jj, bi yd (2017) selection for a zinc-finger protein contributes to seed oil increase during soybean domestication. plant physiol. 173: 2208-2224. liu l, guan ll, yang yx (2016) a review of fatty acids and genetic characterization of safflower (carthamus tinctorius l.) seed oil. am. j. chin. med. 2: 48-52. marschner h (2012) marschner's mineral nutrition of higher plants. in marschner h (ed.), 2nd edition, elsevier ltd., amsterdam. mirshekari m, majnoonhoseini n, amiri r, moslehi a, zandvakili o (2013) effects of sowing date and irrigation treatment on safflower seed quality. j. agr. sci. 15: 505-515. movahhedy-dehnavy m, modarres-sanavy sam, mokhtassibidgoli a (2009) foliar application of zinc and manganese improves seed yield and quality of safflower (carthamus tinctorius l.) grown under water deficit stress. ind. crops prod. 30: 82-92. nosheen a, bano a, yasmin h, keyani r, habib r, shah st, naz r (2016) protein quantity and quality of safflower seed improved by np fertilizer and rhizobacteria (azospirillum and azotobacter spp.). front. plant sci. 7: 104. omidi h, tahmasebi z, badi han, torabi h, miransari m (2010) fatty acid composition of canola (brassica napus l.), as affected by agronomical, genotypic and environmental parameters. c. r. biol. 333: 248-254. ortega j, lópez‐hernandez a, garcia hs, hill cg (2004) lipase‐mediated acidolysis of fully hydrogenated soybean oil with conjugated linoleic acid. j. food. sci. 69: 88-96. peel mc, finlayson bl, mcmahon ta (2007) updated world map of the köppen-geiger climate classification. hydrol. earth. syst. sci. 4: 439-473. ravi s, channal ht, hebsur ns, dharmatti pr (2010) effect of sulphur, zinc and iron nutrition on growth, yield, nutrient uptake and quality of safflower (carthamus tinctorius l.). karnataka j. agric. sci. 21: 15-24. reddy pp (2016) supplemental irrigation. in sustainable intensification of crop production, springer, singapore, 253-265. rondanini d, savin r, hall aj (2003) dynamics of fruit growth and oil quality of sunflower (helianthus annuus l.) exposed to brief intervals of high temperature during grain filling. field crops res. 83: 79-90. sabaghnia n, ahadnezhad a, janmohammdi m (2015) genetic variation in garden cress (lepidium sativum l.) germplasm as assessed by some morphological traits. genet. resour. crop evol. 5: 733-745. yadegari m (2016) effect of micronutrients foliar application and biofertilizeres on essential oils of lemon balm. j. plant nutr. soil sci. 16: 702-715. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:58 utc nova biotechnol chim (2019) 18(1): 52-58 doi: 10.2478/nbec-2019-0007  corresponding author: roman.boca@ucm.sk nova biotechnologica et chimica magnetic response of bovine spleen ľubor dlháň1, roman krylov2, martin kopáni3 and roman boča2, 1 institute of inorganic chemistry, faculty of chemical and food technology, slovak university of technology, bratislava, sk-812 37, slovak republic 2 department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, trnava, sk-917 01, slovak republic 3 institute of medicinal physics, faculty of medicine, comenius university, bratislava, sk-81372, slovak republic article info article history: received: 28th december 2018 accepted: 11th february 2019 keywords: magnetism bovine spleen squid data zfcm/fcm curves hysteresis abstract bovine spleen has been used as a sample for deep magnetochemical investigation. temperature dependence of the magnetic susceptibility and field dependence of the magnetization reveal a paramagnetic behaviour that violates the curie law. the zero-field cooled magnetization and field cooled magnetization experiments show the bifurcation point at ca tc = 20 k and the blocking temperature tb = 10 k confirming a dominating portion of ferritin along with the organic tissue. there is a remnant magnetization at temperature below 20 k and the search for the magnetic hysteresis was positive.  university of ss. cyril and methodius in trnava introduction investigation of magnetic properties of several animal and/or human organs brought important information that some deposits of the iron oxides are present in them, at least in spleen (boča et al. 2013; kopáni et al. 2015) and brain (kirschvink et al. 1992; makohusová et al. 2014; dlháň et al. 2018). these deposits consist of hematite -fe2o3 (antiferromagnetic), maghemite -fe2o3 (ferrimagnetic) and/or magnetite fe3o4 (ferrimagnetic); they coexist with wüstite feo and various iron-oxides-hydroxides such as goethite feooh and ferrihydrite 5fe2o3·9h2o. the organs with the blood circulation also contain traces of hemoglobin and some amount of ferritin. ferritin is a globular protein of the external size of ca 12 nm with an internal cavity of ca 8 nm. this space serves as iron storage container and typically it is filled by the mineral core formed of ferrihydrite, along with some other iron-oxide minerals (gálvez et al. 2008). about 4500 fe3+ ions are stored in the ferritin globule. ferritin is contained mostly in spleen, liver, and bone marrow. several groups investigated magnetic properties of ferritin (makhlouf et al. 1997; brem et al. 2006). however, the actual data can depend upon the source such as the horse spleen ferritin or human spleen. in the present study, the bovine spleen has actually been subjected to magnetochemical studies. experimental bovine spleen sample 1 sample extracted from the bovine spleen has been subjected to lyophilization. the powder-like material has been weighted into gelatine made container. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:06 utc mailto:roman.boca@ucm.sk nova biotechnol chim (2019) 18(1): 52-58 53 ferritin sample 2 ferritin extracted from the horse spleen was purchased from polysciences, inc. (usa), catalog #217. the certificated element content is n, 12.48 %; fe, 15.50 %; p, 1.15 %; protein solids (including iron) 110.33 mg.cm-3, solvent 0.15 m nacl. fe: 5.8 % in lyophilized sample (aas). magnetic measurements solid samples 1 and 2 have been weighted into gelatine made container and inserted into the measuring chamber of the squid magnetometer (quantum design, mpms-xl7). temperature dependence of the magnetic susceptibility has been taken at small external field b = 0.1 t between t = 1.9 and 300 k. magnetization measurements have been conducted at low temperature t = 2.0 and 4.6 k between b = 7 t and zero. the zfcm/fcm data was taken as follows: the sample was cooled in the zero field to t = 2.0 k, then small field of b = 0.01 t has been applied and the magnetic data was acquired until t = 300 k (zfcm curve); then the data taking continued in the cooling regime (fcm curve). finally, the hysteresis loops have been taken between b = +5, 0, –5, 0, +5 t at a set of constant temperatures. results and discussion the magnetic susceptibility data of 1 at low temperature confirms the paramagnetic response (fig. 1). though the susceptibility decreases on heating it does not follow the curie law since the susceptibility-temperature product function t is not a straight line with the zero slope. the susceptibility curve crosses zero at t ~ 150 k which has an origin in the presence of the t/k 0 50 100 150 200 250 300  m a s s /( 1 0   m 3 k g   ) 0 100 200 300 t/k 0 50 100 150 200 250 300  t /( 1 0   m 3 k g   k ) -2 0 2 1, b = 0.1 t t/k 0 50 100 150 200 250 300  m a s s /( 1 0   m 3 k g   ) 0 1000 2000 t/k 0 50 100 150 200 250 300  t /( 1 0   m 3 k g   k ) 0 10 20 30 40 2, b = 0.1 t fig. 1. magnetic susceptibility (left) and the susceptibilitytemperature product function (right) for the bovine spleen sample 1 and horse spleen ferritin 2. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:06 utc nova biotechnol chim (2019) 18(1): 52-58 54 t/k 0 5 10 15 20 25 30 m m a s s /( 1 0   j t   k g   ) 0.0 0.5 1.0 1.5 2.0 1, zfcm/fcm: b = 10 mt t/k 0 5 10 15 20 25 30 m m a s s /( 1 0   j t   k g   ) 0 10 20 30 40 2, zfcm/fcm: b = 10 mt fig. 2. zfcm/fcm data for the bovine spleen sample 1 and horse spleen ferritin 2. lines are visual guide. diamagnetic organic tissue. (near zero signals the squid magnetometer is frustrated in fitting the recorded current/voltage to the equation of the perfect magnetic dipole.) there is a small hook at t ~ 50 k that refers to the solidus-solidus phase transition of the dioxygen present in the sample/container. the above data displays a similarity with that recorded for the sample 2 (horse spleen ferritin). this sample brings only a paramagnetic response since the diamagnetism of the organic globule is suppressed by a strong paramagnetic signal of inorganic core of the ferrihydrite nature, 5fe2o3·9h2o or feooh. in a harmony with expectations, the mass susceptibility of 2 is about an order of magnitude higher relative to 1. the zfcm/fcm experiment for 1 shows that there exists a maximum at the zfcm curve confirming a presence of the blocking temperature, tb ~ 9 – 10 k, that characterizes the superparamagnetism of nanosized objects (fig. 2). this curve tends to coincide with the fcm record at critical tc ~ 20 k that is the bifurcation point. however, small divergence of zfcm/fcm curves survives until the room temperature (end of the data taking) which points to the presence of a minor portion of the ferro-/ferrimagnetic phase. the above data resembles high similarity with those recorded for the horse spleen ferritin. here the bifurcation point is exactly at tc ~ 20 k and the blocking temperature of the superparamagnetism is well identified at tb = 11 k. small discrepancies between 1 and 2 can be attributed to the super-ferromagnetism of 1: the magnetism of an ensemble of magnetically interacting super-paramagnetic nanoobjects. the magnetization data, taken at the field decreasing mode, shows that at the zero field some remnant magnetization survives (fig. 3). this is much higher for 2 relative to 1. the magnetization data at t = 7 t are far from saturation. there is an anomaly above b > 3 t and t = 2 k for 1 of unknown origin; this can be due to a multiphase composition of 1. the magnetization data was fitted by using the langevin function extended to the firstand second-order susceptibility 2 mass sat 1 2 ( , ) [coth( ) 1/ ]m b t m y y b b      (1) for the argument p b b /y m b k t (2) where mp is the magnetic moment in units of bohr magneton; msat is the magnetization at the saturation, and the quadratic term 2 accounts to the magnetic anisotropy. the optimum set of magnetic parameters is listed in table 1. this table contains also the data referring to the human spleen (3, reference sample with no diagnose, kopáni et al. 2015). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:06 utc nova biotechnol chim (2019) 18(1): 52-58 55 table 1. magnetic parameters for the magnetization. temperature parameter 1, bovine spleen 2, horse spleen ferritin 3, human spleen t = 4.6 k msat / j.t -1.kg-1 0.031 0.207 mp /b 32 731 1 / j.t -2.kg-1 0.069 0.333 2 / j.t -3.kg-1  –0.0054 –0.019 t = 2.0 k msat / j.t -1.kg-1 0.262 mp /b 259 1 / j.t -2.kg-1  0.359 2 / j.t -3.kg-1   –0.023 t = 4.6 & 2.0 k msat / j.t -1.kg-1 0.248 0.856 mp /b 340 7.26 1 / j.t -2.kg-1  0.338 0.098  2 / j.t -3.kg-1  –0.020 –0.0048 magnetization curves for the horse spleen ferritin have been analyzed by using several models (brem et al. 2006). the averaged magnetic moment of a nanoparticle mp varies with temperature and the model employed. the model of the single langevin function enriched by the linear susceptibility term, i.e. truncated (1), gave mp = 350 b for t = 50 k. this is not far from the data listed in table 1. noticeable is the fact that mp is by an order of magnitude lower for 1 and by two orders for 3 relative to 2. the presence of the remnant magnetization approves a search for the full magnetic hysteresis that was successful for both, 1 and 2 (fig. 4). the loop is more opened for 2 relative to 1. in accordance with expectations, the hysteresis loops tend to be closed on heating for both, 1 and 2. the characteristics of the hysteresis loops are listed in table 2. it is seen that on heating the remnant fig. 3. magnetization data for the bovine spleen sample 1 and horse spleen ferritin 2. solid lines – fitted with the extended langevin function for t = 4.6 k. dashed – visual guide. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:06 utc nova biotechnol chim (2019) 18(1): 52-58 56 fig. 4. hysteresis loops for the bovine spleen sample 1 and horse spleen ferritin 2 at different temperatures. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:06 utc nova biotechnol chim (2019) 18(1): 52-58 57 table 2. characteristics of the hysteresis loops. t / k 1, bovine spleen 2, horse spleen ferritin 3, human spleen remnant magnetization a coercive field bc / mt remnant magnetization a coercive field bc / mt remnant magnetization a coercive field bc / mt 2 7.10 27.0 141.00 117.0 5.70 21.0 5 4.40 33.0 100.00 79.0 b 4.20 27.0 10 1.20 9.3 31.00 16.6 1.70 13.1 20 0.16 0.9 0.29 0.2 0.28 3.0 50 0.14 0.9 0.91 1.6 0.20 1.2 100 0.12 ~0 a mr in units of 10 -3 j.t-1.kg-1 (si); b bc = 180 mt was reported by makhlouf et al. 1997. magnetization decreases progressively for both, 1 and 2 (fig. 5). however, at t = 20 k and above, small hysteresis loop survives which points to the presence of a minor magnetically ordered phase. the development of the coercive field for 1 is more complex because on passing from t = 2 to 5 k the coercive field increased. this might be due to the presence of the above-mentioned minor phase. fig. 5. temperature evolution of the remnant magnetization and the coercive field for 1 and 2. in the horse-spleen ferritin 2, the profile of the hysteresis curve adopts a wast-waisted form. this was attributed to the presence of a second ordered phase with smaller coercivity (brem et al. 2006). probably a small amount of magnetite and/or maghemite is responsible for such an effect. a comparison with the data recorded for the human spleen 3 (kopáni et al. 2015) shows a great similarity to 1: analogous values and thermal evolution of the remnant magnetization and coercive field. conclusions the sample extracted from the bovine spleen possesses the magnetic response which qualitatively resembles the properties of ferritin extracted from the horse spleen. however, there exists a remarkable difference in the coercive field that is by order of magnitude higher for 2 relative to 1. more similar properties to 1 exhibits the sample extracted from the human spleen. also, the averaged magnetic moment per nanoparticle for 1 and 3 are much lower relative to 2. acknowledgement slovak grant agencies (vega 1/0919/17, apvv-14-0078, apvv-16-0039) are acknowledged for the financial support. conflict of interest the authors declare that they have no conflict of interest. references boča r, dlháň ľ, kopáni m, miglierini m, mrázová v, čaplovičová m (2013) deposits of iron oxides in the human spleen. polyhedron 66: 65-69. brem f, stamm g, hirt am (2006) modeling the magnetic behavior of horse spleen ferritin with a two-phase core structure. j. appl. phys. 99: 123906. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:06 utc nova biotechnol chim (2019) 18(1): 52-58 58 dlháň ľ, kopáni m, boča r (2019) magnetic properties of iron oxides present in the human brain. polyhedron 157: 505-510. gálvez n, fernández b, sánchez p, cuest r, ceolín m, clemente-león m, trasobares s, lópez-haro m, calvino jj, stéphan o, domínquez-vera j (2008) comparative structural and chemical studies of ferritin cores with gradual removal of their iron contents. j. am. chem. soc. 130: 8062. kirschvink jl, kobayashi-kirschvink a, woodford bj (1992) magnetite biomineralization in the human brain. proc. natl. acad. sci. usa 89: 7683. kopáni m, miglierini m, lančok a, dekan j, čaplovicová m, jakubovský j, boča r, mrázová h (2015) iron oxides in human spleen. biometals 28: 913-928. makhlouf sa, parker ft, berkowitz ae (1997) magnetic hysteresis anomalies in ferritin. phys. rev. b 55: r14717. makohusová m, mrázová v, kopáni m, boča r (2014) magnetic deposits of iron oxides in the human brain. nova biotechnol. chim. 13: 48-56. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:06 utc nova biotechnol chim (2018) 17(2): 172-180 doi: 10.2478/nbec-2018-0018  corresponding author: daniela.chmelova@ucm.sk nova biotechnologica et chimica azonaphthalene dyes decolorization and detoxification by laccase from trametes versicolor barbora legerská, daniela chmelová and miroslav ondrejovič department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius in trnava, j. herdu 2, trnava, sk-917 01, slovak republic article info article history: received: 14th october 2018 accepted: 21st november 2018 keywords: azo dye decolorization detoxification laccase trametes versicolor abbreviations: abts, 2,2´-azino-bis-3ethylbenzothiazoline-6-sulfonic acid hbt, 1-hydroxybenzotriazole mw, molecular weight abstract the aim of the present study was to investigate the dye decolorization ability of laccase from trametes versicolor. five azonaphthalene dyes (acid violet 7, acid red 1, allura red ac, orange g and sunset yellow fcf) were used to evaluate dye decolorization. laccase from t. versicolor is capable of decolorizing dyes, namely acid violet 7 (53.7±2.3 %) and orange g (46.0±2.2 %). the less effective ability of laccase was observed at the decolorization of other selected dyes (6.9 – 18.6 %). the presence of redox mediator (1-hydroxybenzotriazole) increased decolorization percentage for all tested dyes (≥ 90.5 %). toxic effect of azo dyes and their degradation products after laccase treatment was observed on the growth of selected bacteria (micrococcus luteus, bacillus subtilis, pseudomonas syringae and escherichia coli), yeasts (candida parapsilosis and saccharomyces cerevisiae) and algae (chlorella vulgaris and microcystis aeruginosa). it was confirmed that degradation products showed lower inhibition effect compared to initial dyes. these findings suggest that laccase from t. versicolor are able to decolorize and detoxify selected azonaphthalene dyes.  university of ss. cyril and methodius in trnava introduction azo dyes represent a group of synthetic dyes used in industrial processes for their stability and colour intensity (gomez et al. 2013; rovina et al. 2016). this group belongs to a large class of synthetic dyes containing one or more azo bonds (-n=n-). these bonds link various aromatic ring structures (benzene, naphthalene). azonaphthalene dyes have naphthalene ring with delocalized conjugated bonds of carbon atoms stabilise total structure of azo dye (zhu et al. 2012). moreover, functional groups present in their structure are responsible for various physical properties such as solubility, lipophilicity or absorption (legerská et al. 2016; da costa et al. 2017). approximately 2 – 50 % of synthetic dyes used in industrial dyeing operations have been discharged into wastewater (sarkar et al. 2017). moreover, many works confirmed that azo dyes are toxic or carcinogenic for various organisms including human (yang et al. 2009; gholamiborujeni et al. 2011; axon et al. 2012; nath et al. 2016). therefore, the elimination azo dyes from water systems by appropriate methods is essential for prevention of the environment contamination. for azo dye removal, some chemical and physicochemical methods have been traditionally used. their disadvantages include sludge formation (zhu et al. 2012; lau et al. 2014; youssef et al. 2016), high operating costs and toxic degradation products (liakou et al. 1997; lopez et al. 2004; linley et al. 2012). biological elimination of synthetic azo dyes is environmentally friendly and relatively bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:37 utc nova biotechnol chim (2018) 17(2): 172-180 173 table 1. characteristics of azonaphthalene dyes used in this study. α-naphthol dyes nh coch3 ho3s oh so3h n n r1 cas number r1 λmax [nm] mw [g/mol] acid violet 7 4321-69-1 –nh–coch3 519 566.8 acid red 1 25956-17-6 – 531 537.8 β-naphthol dyes r1 ho3s n n oh r2 r3 r4 cas number r1 r2 r3 r4 λmax [nm] mw [g/mol] allura red ac 25956-17-6 – –och3 –so3h –ch3 493 496.4 orange g 1936-15-8 –so3h – – – 479 452.8 sunset yellow fcf 2783-94-0 – – –so3h – 480 452.4 inexpensive. moreover, the biological methods do not form any toxic degradation products (sathe et al. 2015; legerská et al. 2016, chmelová and ondrejovič 2016). useful enzymes for this purpose seem to be azoreductases (ec 1.7.1.6), laccases (ec 1.10.3.2) and peroxidases (1.11.1.x) (viswanath et al. 2014; singh et al. 2015). in contrast to azoreductases and peroxidases, laccases catalyse dye decolorization in the presence of oxygen. consequently, laccase dye decolorization offers more efficient degradation, low cost of enzymatic process and the production of non-toxic compounds comparing to other abovementioned enzymes (casas et al. 2007; chhabra et al. 2015). while different organisms produce laccases including bacteria, fungi, plants and insect, the most perspective producers are white-rot fungi (viswanath et al. 2014; singh et al. 2015, hazuchová et al. 2017) producing laccases with high redox potential (baiocco et al. 2003; pang et al. 2015). for example, laccases from trametes versicolor have the redox potential approximately 785 mv, which allows oxidation of hardly degradable organic compounds including various groups of dyes (kurniawati and nicell 2008; legerská et al. 2018). moreover, laccases produce non-toxic degradation products, which saprophytes can use as carbon source (gavril and hodson 2007; selvam et al. 2012). therefore, the aim of this study was to evaluate potential of laccase from t. versicolor for decolorization and detoxification of synthetic dyes from the group of α-naphthol (acid violet 7, acid red 1) and β-naphthol (allura red ac, orange g, sunset yellow fcf) azo dyes. experimental microorganisms bacterial species (micrococcus luteus ccm 1569, bacillus subtilis ccm 2218, pseudomonas syringae ccm 2114 and escherichia coli ccm 7929) and yeast species (candida parapsilosis ccm 8186, saccharomyces cerevisiae ccm 8191) were purchased from czech collection of microorganisms (brno, czech republic). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:37 utc nova biotechnol chim (2018) 17(2): 172-180 174 the algae chlorella vulgaris h 1993 was purchased from culture collection of algae (prague, czech republic) and microcystis aeruginosa pcc 7806 was obtained from culture collection of cyanobacteria and algae (brno, czech republic). dyes and chemicals laccase from trametes versicolor and selected naphthalene azo dyes (table 1) were obtained from sigma-aldrich (germany). culture media were supplied by biolife (italy). all other chemicals were purchased from mikrochem (slovak republic). decolorization of azonaphthalene dyes by laccase dye decolorization by laccase from t. versicolor was spectrophotometrically determined in the range of 300 – 700 nm (spectrophotometer v-1600pc, vwr, germany) during 5 days with or without the redox mediator 1-hydroxybenzotriazole (hbt). the reaction mixtures contained 50 mg/l of azonaphthalene dye in 0.1 mol/l phosphate buffer (ph 3.0), and laccase from t. versicolor with enzyme activity of 1.0 u/ml mixed in ratio 3 : 1 (v/v). the effect of 1.0 mmol/l hbt was also tested. control samples were run without the addition of laccase. the decolorization percentage was determined as follows: decolorization [%] = [(a0 – at)/a0]*100, where a0 is the initial absorbance and at is the absorbance of enzymatic reaction at a certain time of laccase treatment. toxicity test the effect of laccase treatment on toxicity of selected azonaphthalene dyes, namely acid violet 7 and orange g, were evaluated. the applied dye concentration range was 0.04 – 5.0 g/l. the growth inhibitions of selected microorganisms (bacteria, yeasts and algae) were assayed in the microtiter plates by dilution method during 48 and 72 h at 30 °c spectrophotometrically at 690 nm. the results were expressed as minimum inhibitory concentration (mic), which completely inhibits the growth of microorganism. production of chlorophylls and biomass was monitored for algae (c. vulgaris, m. aeruginosa) in media with selected azo dyes or their degradation products after laccase treatment. the cultivation was performed at laboratory temperature with 14 : 10 h light and dark photoperiod. after 30 days of cultivation, the concentration of chlorophylls a and b in algal biomass were expressed according sumanta et al. (2014) as follows: chlorophyll a =16.72a665nm – 9.16a652nm chlorophyll b =34.09a654nm – 15.28a665nm the results were expressed as the inhibition percentage toward the control without synthetic dye or degradation products of dye after laccase treatment. the inhibition of biomass formation was calculated as follows: biomass inhibition [%] = [(bc – bd)/bc]*100, where bc is biomass weight of control sample without azo dye and bd is biomass weight of sample with selected azo dye. the inhibition of chlorophyll production was calculated as follows: chlorophyll inhibition [%] = [(cc – cd)/cc]*100, cc is the concentration of selected chlorophyll a or b in control sample and cd is the concentration of selected chlorophyll a or b in sample with azo dye or degradation products after laccase treatment. hplc analysis the degradation mixtures of selected azonaphthalene dyes (acid violet 7 and orange g) were analysed with high performance liquid chromatography hplc (agilent technologies 1200 series, usa) on c18 column (3.5 µl, 3.0 mm x 100 mm) using mobile phase a (0.1 % (v/v) aqueous solution of formic acid) and b (0.1 % (v/v) methanolic solution of formic acid). the gradient program was set as follows: 0 – 2 min = 0 – 20.0 % b, 2 – 15 min = 20.0 – 95.0 % b, 15 – 20 min = 95.0 % b. the flow rate was 0.7 ml/min and the absorbance of eluted compounds was measured at 254 nm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:37 utc nova biotechnol chim (2018) 17(2): 172-180 175 results and discussion decolorization of selected azonaphthalene dyes laccases are often studied for their potential of dye decolorization. five azonaphthalene dyes, acid violet 7, acid red 1, allura red ac, orange g and sunset yellow fcf, were used to evaluate the dye decolorization efficiency of laccase from t. versicolor (fig. 1). laccases from t. versicolor were able to decolorize the α-naphtol dye acid violet 7 (53.7±2.3 %) (fig. 1-a). the α-naphtol dye acid red 1 was decolorized by laccase less effectively (16.7±0.1 %) (fig. 1-b). dye degradation by laccase is influenced by chemical structure of dye itself, differences in electron fig. 1. uv-vis spectrum of the reaction mixture of the naphthalene dye a – acid violet 7, b – acid red 1, c – allura red ac, d – orange g and e – sunset yellow fcf decolorized with laccase from trametes versicolor during 5 days at ph 3.0 and 22 °c (▬ 0. day, ▬ 1. day, ▬ 2. day, ▬ 3. day, ▬ 4. day, ▬ 5. day). b c d e a bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:37 utc nova biotechnol chim (2018) 17(2): 172-180 176 table 2. the effect of 1-hydroxybenzotriazole (hbt) on laccase catalysed decolorization of selected azo dyes. azo dyes decolorization [%] laccase without hbt laccase-hbt mediated system α-naphtol dyes acid violet 7 53.7±2.3 97.1±0.3 acid red 1 16.7±0.1 95.0±0.5 β-naphtol dyes allura red ac 6.9±0.2 95.8±0.1 orange g 46.0±2.2 96.8±0.7 sunset yellow fcf 18.6±1.2 95.0±1.5 distribution, number of dissociating groups and steric barriers (hsueh et al. 2009). the poorest was decolorization of acid red 1 probably because of the presence of -coch3 group bonded to the naphtol ring of acid red 1. similarly, other authors described partial decolorization of these dyes. zhang et al. (2006) tested the degradation ability of laccase from panus rudis for acid violet 7 and reported decolorization of 26.0 %. this substituent belongs to electron-withdrawing group making a ring less susceptible to enzymatic oxidation (suzuki et al. 2001). in contrast to our results, zhou et al. (2017) observed higher decolorization of acid red 1 (69.7 %) after enzymatic reaction with laccase from bacillus pumilus w3. the efficiency of the decolorization process, therefore, is obviously influenced by the choice of producer. different producers produce laccase with various redox potential resulting in variable decolorization efficiency (baiocco et al. 2003; pang et al. 2015). of the β-naphtol dyes, the highest decolorization by t. versicolor laccases was showed for orange g (46.0±2.2 %) (fig. 1-d). the other β-naphtol dyes, sunset yellow fcf and allura red ac, were decolorized less effectively (18.6±1.2 % and 6.9±0.2 %, respectively) (fig. 1-c,e). the efficient decolorization of orange g was probably caused by the presence of two sulfo groups linked to the naphtol ring (table 1), which allows simple degradation of dye chromophore. sunset yellow fcf has the similar structure as orange g, however, the presence of single –so3h group on the benzene ring significantly reduces structure stabilization. the lowest decolorization was observed in the mixture of allura red ac and laccase from t. versicolor (6.9±0.2 %). the presence of methyland methoxy groups on the benzene ring restricted decolorization. these groups stabilize the dyes and prevent their effective oxidation by laccase (chivukula and renganathan 1995; hsueh et al. 2009). the dye decolorization by laccases can be enhanced using low molecular compounds, also called redox mediators. in our work, we tested the effect of 1-hydroxybenzotriazole (hbt) as synthetic redox mediator on laccase catalysed decolorization of selected azo dyes. the results are shown in table 2. the presence of hbt increased the degree of decolorization of all tested dyes (table 2), while the decolorization percentages varied in the range of 95.0 – 97.1 %. forootanfar et al. (2016) fig. 2. hplc elution profile of azonaphthalene dyes (1) (a – acid violet 7, b – orange g) and their degradation products (2) obtained after treatment with laccase from t. versicolor. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:37 utc nova biotechnol chim (2018) 17(2): 172-180 177 found that the presence of redox mediator (hbt) with laccase from paraconiothyrium variabile increased the decolorization of azo dye and decreased the needed time for degradation. wong and yu (1999) reported that laccase from t. versicolor decolorized acid violet 7 more effectively in the presence of redox mediator (abts) in the reaction mixture. furthermore, zeng et al. (2011) reported decolorization of acid red 1 only in the laccasehbt mediated system. it seems that the selection of appropriate redox mediator can increase the efficiency of decolorization process catalysed by laccases. degradation products analysis several authors (yang et al. 2009; gholamiborujeni et al. 2011; axon et al. 2012; nath et al. 2016) describe the potential toxic effect of dye degradation products. therefore, two best decolorized dyes were assessed for the effect of laccase treatment. degradation products of synthetic dyes after enzymatic degradation can be mainly analysed by hplc or gc-ms (franciscon et al. 2012; christiane et al. 2013; yuan et al. 2016). in our work, hplc was used as a tool for analysis of dye decolorization and detection of emerging products. the results are shown in fig. 2. hplc analysis of acid violet 7 (fig. 2-a) displayed a single peak at retention time 15.457 min. after laccase treatment, the loss of the main peak indicates decolorization of initial dye. similarly, hplc analysis of orange g (fig. 2-b) displayed peaks at 25.310 min, 26.888 min, 32.070 min and 32.839 min. in reaction mixture with degradation products after laccase catalysed reaction, non-detectable peaks were observed. although, new peaks were not detected, disappearance of the main peaks in acid violet 7 (15.457 min) and orange g (25.310 min) profiles point to the effectivity of laccase from t. versicolor to decolorization azonaphthalene dyes without production of other detectable compounds. toxicity tests azo dyes belong to toxic compounds with potential toxic effect on prokaryotic and/or eukaryotic organisms (przystas et al. 2012; lade et al. 2015). the loss of dye colour after laccase treatment indicates the breakdown of parent compound. however, degradation products can also be potentially toxic. therefore, toxicity tests of selected azo dyes, namely acid violet 7 and orange g, with the highest decolorization after laccase treatment were performed (table 3). the toxicity of acid violet 7 on the growth of all bacteria (m. luteus, b. subtilis, p. syringae, e. coli) and the yeast c. parapsilosis was observed at dye concentration of 5.0 g/l, except of s. cerevisiae (>5.0 g/l). similarly, mansour et al. (2010) recorded toxicity of acid violet 7; this azo dye induced chromosomal aberrations, lipid table 3. the minimum inhibitory concentrations of azonaphthalene dyes to the growth of prokaryotic and eukaryotic organisms before and after laccase treatment. minimum inhibitory concentration [g/l] acid violet 7 orange g synthetic dye degradation products synthetic dye degradation products bacteria m. luteus 5.0 5.0 b. subtilis 5.0 5.0 e. coli 5.0 p. syringae 5.0 yeasts c. parapsilosis 5.0 s. cerevisiae – without inhibitory effect on bacterial and yeast growth in concentration range of 0.04 – 5.0 g/l. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:37 utc nova biotechnol chim (2018) 17(2): 172-180 178 table 4. the inhibitory effects of acid violet 7 and orange g and their degradation products after laccase treatment on the biomass and chlorophylls production of selected algae. inhibitory rate [%] c. vulgaris m. aeruginosa acid violet 7 synthetic dye biomass 37.8±3.2 chlorophyll a 14.5±2.8 56.7±4.6 chlorophyll b 12.9±1.2 56.1±3.7 degradation products biomass 21.9±1.8 chlorophyll a 6.9±0.4 chlorophyll b 8.8±2.9 orange g synthetic dye biomass 30.3±4.9 chlorophyll a 14.8±2.1 72.1±3.1 chlorophyll b 10.7±2.1 71.2±4.7 degradation products biomass 10.6±3.6 chlorophyll a chlorophyll b – without inhibitory effect to the growth of algae species. peroxidation and cholinesterase inhibition in mouse bone marrow. on the other hand, in our study, orange g inhibited only the growth of m. luteus and b. subtilis (table 3), its growth inhibitory effect was not observed on other tested microorganisms. this lack of toxicity of orange g is consistent with the results of mariappan et al. (2003) for bacterial species pseudomonas sp. sac03, bacillus sp. sac01 and escherichia sp. sac01, and also alcántara et al. (2017) for yeast species. previous studies have indicated that the toxicity of azo dyes strongly depends on chemical structure, functional groups and the number of azo bonds (costa et al. 2012; legerská et al. 2016; da costa et al. 2017). this was also confirmed in this study showing the relatively higher toxicity of α-naphtol dye acid violet 7 comparing to β-naphtol dye orange g. even more importantly, laccase treatment resulted in degradation products with no toxicity on tested microorganisms (table 3). the presence of synthetic dyes in environment also affects the growth of photosensitive microorganisms. therefore, we tested the impact of laccase-degraded dyes on growth of selected algae, namely c. vulgaris and m. aeruginosa. contents of chlorophyll a and b were measured. the results are shown in table 4. the presence of acid violet 7 and orange g in medium inhibited the growth of c. vulgaris as well as the production of chlorophyll a and b of both algae species (table 4). in culture media with degradation products after laccase treatment, the reduction and even disappearance of toxicity effect were observed. influence of azo dye on c. vulgaris growth was also studied in the work of hernández-zamora et al. (2014) who revealed significant decrease of biomass and chlorophyll production. el-sheekh et al. (2017) confirmed the negative influence of azo dye disperse red bs on growth of m. aeruginosa. conclusions the results from this study showed the ability of laccase from trametes versicolor to decolorize synthetic dyes from model systems. as expected, decolorization was observed in all dye reaction mixture. laccase from t. versicolor showed the highest decolorization with acid violet 7, the α-naphthol azo dye, and with orange g, the β-naphthol azo dye while the addition of hbt increased the effectivity of dye degradation. hplc analysis of decolorized solutions confirmed absence of distinguishable degradation products and complete loss of the main peak representing azo dye. the dye toxicity toward bacteria, yeasts and algae after laccase catalysed reaction was decreased. our results show that laccases from the white rot fungus t. versicolor are suitable for decolorization and detoxification of azonaphthalene dyes, while generate environmentally friendly degradation products. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:37 utc nova biotechnol chim (2018) 17(2): 172-180 179 acknowledgement this work was supported by the slovak research and development agency under contract number apvv-160088. references alcántara tap, oliveira jm, evangelista-barreto ns, marbac pas, cazetta ml (2017) aerobic decolorization o azo dye orange g by a new yeast isolate candida cylindracea sjl6. biosci. j. 33: 1340-1350. axon a, may fe, gaughan le, williams fm, blain pg, wright mc (2012) tartrazine and sunset yellow are xenoestrogens in a new screening assay to identify modulators of human oestrogen receptor transcriptional activity. toxicology. 298: 40-51. baiocco p, barreca am, fabbrini m, galli c, gentili p (2003) promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccase-mediator systems. org. biomol. chem. 1: 191197. casas n, blanquez p, gabarrell x, vincent t, caminal g, sarra m (2007) 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applications in bioremediation. enzyme res. 2014: 1-21. wong y, yu j (1999) laccase-catalyzed decolorization of synthetic dyes. water res. 33: 3512-3520. yang q, li c, li h, li y, yu n (2009) degradation of synthetic reactive azo dyes and treatment of textile wastewater by a fungi consortium reactor. biochem. eng. j. 43: 225-230. youssef na, shaban sa, ibrahim fa, mahmoud as (2016) degradation of methyl orange using fenton catalytic reaction. egypt. j. petrol. 25: 317-321. yuan x, tian g, zhao y, zhao l, wang h, ng tb (2016) degradation of dyes using crude extract and a thermostable and ph-stable laccase isolated from pleurotus nebrodensis. biosci. rep. 36: 1-10. zeng x, cai y, liao x, zeng x, li w, zhang d (2011) decolorization of synthetic dyes by crude laccase from a newly isolated trametes trogii strain cultivated on solid agro-industrial residue. j. hazard. mater. 187: 517525. zhang m, wu f, wei z, xiao z, gong w (2006) characterization and decolorization ability of a laccase from panus rudis. enzyme microb. technol. 38: 92-97. zhou w, gua zb, chen y, zhang f, cai yj, xu cw, chen xs, liao xr (2017) production of spore laccase from bacillus pumilus w3 and its application in dye decolorization after immobilization. water sci. technol. 76: 147-154. zhu n, gu l, yuan h, lou z, wang l, zhang x (2012) degradation pathway of the naphthalene azo dye intermediate 1-diazo-2-naphthol-4-sulfonic acid using fenton´s reagent. water res. 46: 3859-3867. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:37 utc nova biotechnol chim (2017) 16(1): 42-47 doi: 10.1515/nbec-2017-0006  corresponding author: nikola.siposova@upjs.sk nova biotechnologica et chimica genetic diversity of acinetobacter spp. adapted to heavy metal polluted environments nikola šipošováa,, veronika liptákováa, simona kvasnováb, petra kosorínováa and peter pristaša,c a department of microbiology, institute of biology and ecology, faculty of sciences, pavol jozef šafárik university in košice, šrobárova 2, košice, sk-041 54, slovak republic b department of biology and ecology, faculty of natural sciences, matej bel university, tajovského 40, banská bystrica, sk-974 01, slovak republic c institute of animal physiology, slovak academy of sciences, šoltésovej 4-6, košice, sk-040 01, slovak republic article info article history: received: 12th april 2017 accepted: 12th june 2017 keywords: acinetobacter heavy metals resistance bioremediation plasmids abstract multiple metallotolerant bacterial strains were isolated from soil and drainage water samples collected from three industrially heavy metals polluted areas in slovakia. obtained bacterial isolates were identified using maldi-tof mass spectrometry and bacterial isolates that belonged to the acinetobacter genus were subjected for the further study. a. calcoaceticus was found to be prevalent species among analyzed acinetobacter spp. strains, followed by a. lwoffii and a. johnsonii. a. calcoaceticus strains exhibited higher minimum inhibitory concentration to mn, zn, and cu cations compared to a. lwoffii and a. johnsonii. on the other hand minimum inhibitory concentration to co and ni were identical in all acinetobacter spp. isolates. genetic analyses demonstrated multiple plasmids presence in a. lwoffii and a. johnsonii but not in a. calcoaceticus. using eric-pcr the presence of two different genotypes of a. calcoaceticus was detected in heavy metal polluted environments in slovakia.  university of ss. cyril and methodius in trnava introduction bacteria classified as members of the genus acinetobacter are gram-negative, strictly aerobic, indole negative, oxidase negative, catalase positive and non-fermenting coccobacilli. the name of this genus is derived from the greek word “akinetos” because of its non-motility. species of this genus are subjects of interest in various fields of science, e.g. molecular microbiology, environmental microbiology, clinical microbiology, various sectors of biotechnology etc. these strains can be obtained from soil or water often contaminated by human activity – agricultural or industrial production, traffic, waste disposal equipment, mining and quarrying, building industries – and contain increased amounts of heavy metals, phenols, biphenyls or chlorinated biphenyls, crude oil, phosphate, divers dyes or another chemical compounds that have a negative impact to environment and therefore to human and animals health. these bacterial species (pathogenic or nonpathogenic) can be regularly isolated from living organisms as well. due to its ability to cause nosocomial infections they have been attracting increasing attention in clinical practice. clinical strains are often multiresistant to a broad spectrum of antibiotics. besides antibiotic multiresistance bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:31 utc mailto:nikola.siposova@upjs.sk nova biotechnol chim (2017) 16(1): 42-47 43 they are often resistant to classical disinfectants (gluconate, chlorhexidine and phenols) and to desiccation or radiation (dougrahi et al. 2011 and references therein). bacterial strains of this genus are typical by a wide range of biotechnological use. some acinetobacter species (a. calcoaceticus rag-1, a. radioresistens ka53, a. calcoaceticus bd4) produce bioemulsifiers which are used in food industry, agrochemical industry, cosmetic industry, pharmaceutical industry. another application potential is made possible by the ability of several strains to accumulate various types of biopolymers – cyanophycin, wax esters, polyhydroxyalkalonic acids etc (abdel-el-haleem 2003). species of this genus have the ability to biotransform heavy metals, phenols or other contaminants to less toxic forms. it has been found that a. calcoaceticus strain pucm 1011 reduces hexachloroplatinic acid forming platinum nanoparticles (gaidani et al. 2014); a. calcoaceticus pucm 1005 synthesizes silver nanoparticles (gaidani et al. 2013); acinetobacter sp. strain memcl4 have ability to biodegrade neurotoxic chlorpyrifos (pailan et al. 2016); acinetobacter spp. such as a. calcoaceticus ncim 2890 could biodegrade diverse textile dyes (ghodake et al. 2009); a. calcoaceticus may be involved in degradation of linear alkylbenzosulfonate and sodium dodecyl sulfate (abboud et al. 2007); some acinetobacter spp. representatives are capable of degrading diesel or heating oil (marín et al. 1995). bacteria in general and acinetobacter spp. can in some cases adapt to various extreme conditions, including high concentrations of heavy-metals. bacterial adaptation mechanisms in environment are metal sorption, accumulation and uptake, mineralization, precipitation, efflux from the cell, oxidation or reduction to less toxic or nontoxic forms (nies 1999). genes encoding proteins involved in the mechanisms of biotransformation of toxic heavy metals to less toxic forms are often present on circular plasmid dna which may be involved in the processes of horizontal gene transfer (li et al. 2015). heavy – metal tolerant bacterial strains could be used in bioremediation but before their real incorporation into the real processes we must get as much information about these strains. the present study deals with characterization of metallotolerant and alkalitolerant bacteria from anthropogenically polluted areas of slovak republic. experimental sampling and identification of bacteria bacterial samples were obtained and collected from heavy metal polluted areas of slovakia: brown sludge dump from aluminium plant near žiar nad hronom (48°35′3″n, 18°51′39″e) situated at žiarska basin, landfill waste from the production of nickel near sereď (48°17′11″n, 17°44′15″e) situated in the southwest of slovakia, and tailings impoundment near slovinky (48°52′43″n, 20°50′38″e) in the east of slovakia. samplings were realized in 2010 (žiar nad hronom), 2013 (sereď), and 2014 (slovinky) resp. numbers of cultivable bacterial strains were obtained upon cultivation on tsa agar (bd difco, usa). individual pure bacterial cultures were identified by maldi-tof ms (matrix – assisted laser desorption ionization time-of-flight mass spectroscopy) method. bacterial isolates were resuspended in 300 µl of sterilized distilled water. resuspended cells were centrifuged with 900 µl of ethanol for 2 min at maximum speed. ethanol was removed and pellets were centrifuged 2 min again. pellets were re-suspended in 50 µl of 70 % formic acid and 50 µl of acetonitrile. the tubes were centrifuged 2 min at maximum speed. 1 µl of prepared samples were pipetted on maldi plate and then 1 µl of maldi matrix (solution of 50 % acetonitrile, 47.5 % of distilled water and 2.5 % trifluoroacetic acid mixed with hcca matrix) on them (ferreira et al. 2011). all analyses were performed on microflex lt (bruker diagnostics, germany) maldi-tof ms system. the members of the genus acinetobacter were subjected to further analysis. determination of minimum inhibitory concentration (mic) species from the genus acinetobacter were tested for their heavy-metal tolerance. analyzed strains bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:31 utc nova biotechnol chim (2017) 16(1): 42-47 44 were inoculated on tsa plates with gradually increasing concentrations (1 mm, 2 mm, 4 mm, 8 mm, 15 mm, 30 mm, 60 mm) of heavy metal salts (cucl2.2h2o, zncl2, nicl2.6h2o, cocl2.6h2o, mncl2) which were filtered through microbial filter first and then added to the sterilized tsa medium (ph=7.3). inoculated bacteria were incubated at 25 °c for 24 h. bacterial ability to grow at alkaline ph bacterial strains were inoculated on tsa medium with addition of 100 mm tris-hcl to control ph (9 and 10.5) and cultivated at 25 °c for 24 h. the growth was evaluated visually. plasmid isolation and gel electrophoresis the presence of plasmids in industrial isolates was determined using alkaline lysis method. three ml of overnight grown liquid bacterial cultures were centrifuged for 3 min at 25 °c, 13 000 rpm. bacterial pellets were resuspended in 250 µl of 10 mm edta and 50 mm tris-hcl with addition of lysozyme. resuspended bacterial cells were incubated in thermostat at 37 °c, 15 min. after incubation, 250 µl of lysis solution (0.2 m naoh and 1 % sds) was added to the tubes which were mixed by inverting. subsequently, 350 µl of 3m potassium acetate (ph=5) was added to the tubes which were mixed by inverting again and incubated 15 min on ice. after 10 min centrifugation at 4 °c and 13 000 rpm supernatants with dissolved double-stranded plasmids were transferred to the new tubes. rna removal was achieved by 30 min incubation with rnase (1 µl) at 37 °c. then 1/2 volume of chloroform was added to the tubes which were centrifuged 3 min at 4 °c. the aqueous phases were transferred to the new tubes and 3/4 volume of isopropanol was added to them. the tubes were centrifuged at 4 °c, 10 min. pellets were washed with 500 µl of 70 % ethanol and centrifuged 5 min. ethanol was completely removed and the plasmid dna was dissolved in te solution (50 µl) of 10 mm tris-hcl and 1mm edta. electrophoresis on 1 % agarose gel stained with ethidium-bromide was used for plasmids separation and uv light for their visualization. the plasmids sizes were compared with fastload 1kbp dna ladder (serva). total genomic dna isolation 5 ml of luria-bertani liquid cultures of all isolates were centrifuged at 4 °c for 10 min at maximum speed. bacterial pellets were resuspended in 1 ml of set solution (25 mm edta, 20 mm tris-hcl, 75 mm nacl) with lysozyme. wrapped tubes were incubated at 37 °c for 30 min with stirring first and then with 1/5 volume of 10 % sds at 55 °c for 15 min statically. after incubation 1/3 volume of 5 mm nacl and chloroform (1:1) were added to the tubes and were incubated for 30 min at laboratory temperature with stirring. tubes were centrifuged for 10 min, 10 000 rpm. the top aqueous phases with equal amount of chloroform were centrifuged at laboratory temperature for 10 min, 10 000 rpm. then the equal amount of izopropanol was added to the aqueous phases, the tubes were incubated at laboratory temperature for 15 min and centrifuged at 4 °c for 10 min, 10 000 rpm. dna pellets were washed with 200 µl of 70 % ethanol, centrifuged at 4 °c for 5 min, 10 000 rpm, air dried, and dna was dissolved in 50 µl of te solution. enterobacterial repetitive intergenic consensus polymerase chain reaction (eric-pcr) of a. calcoaceticus isolates an eric-pcr reaction (versalovic et al. 1991) was performed in c1000 thermal cycler (bio-rad laboratories, richmond, usa) in 50 µl reaction mixture which consists of 5 µl 10x concentrated high yield buffer with 3 mm mgcl2, 200 µm of each dntp, 1 µm of primer eric 1r (5´-atgtaagctcctggggattcac-3´) and 1 µm of primer eric2 (5´-aagtaagtgactggggtgagcg-3´), 1.25 u of taq polymerase (taq core kit, jena bioscience, germany), 50 ng of dna. thermocycling conditions for eric-pcr reaction were: initial denaturation: 95 °c, 15 min; denaturation: 94 °c, 1min; annealing: 48 °c, 2 min; bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:31 utc nova biotechnol chim (2017) 16(1): 42-47 45 elongation: 65 °c, 3 min; final extension: 65 °c, 10 min (37 cycles). pcr products were separated by 1 % agarose gel electrophoresis and visualized by uv light. the pcr products were compared with fastload 1kbp dna ladder (serva). results and discussion currently, in slovakia environmental loads information database there are 1 963 areas registered: 900 as probable environmental loads, 279 as existing/affirmed environmental loads, 784 as remedied/recultivated environmental loads (http://www.minzp.sk/files/sekcia-geologieprirodnych-zdrojov/spsez_2016_2021.pdf). in our work we studied metallotolerant bacteria of the acinetobacter genus naturally present in heavy metal polluted industrial areas near žiar nad hronom, sereď and slovinky. the main goal of our research was to identify prevalent species, to analyze their genetic organization and to estimate their potential use in bioremediation as an alternative cost and eco-friendly remediation method. due to metal industry activities all analyzed sampling sites are recognized by elevated concentrations of heavy metals. despite of these extreme environmental conditions numbers of cultivable bacterial strains were isolated. seventy nine pure bacterial cultures were obtained upon classical cultivation methods on agar plates. strains were than identified by maldi-tof ms. from identified bacteria eleven belong to acinetobacter spp. and eight were analyzed (table 1). table 1. characterization of acinetobacter isolates analyzed. isolate name species sample locality source ph k1 a. lwoffii žiar nad hronom soil 10.2 k6 a. calcoaceticus k13 a. calcoaceticus nhl1 a. calcoaceticus sereď soil 8.1 nhl4 a. calcoaceticus nhl11 a. johnsonii p19 a. calcoaceticus slovinky drainage water 7.8 p20 a. calcoaceticus in similar study no strains belonging to the acinetobacter genus were detected among 24 isolates from nickel sludge disposal site near sereď (pristas et al. 2015). tests for heavy metal tolerance show that a. calcoaceticus from all analyzed areas exhibit higher mic of zinc and manganese compared to a. lwofii from žiar nad hronom and a. johnsonii from sereď. mic of copper was higher for a. calcoaceticus from žiar nad hronom (k6, k13) and for a. calcoaceticus from slovinky (p19, p20) compared to a. calcoaceticus (nhl1, nhl4) and a. johnsonii (nhl11) from sereď. the lowest mic of copper was observed in a. lwofii from žiar nad hronom (k1). tolerance to cobalt and to nickel was the same among all studied isolates (table 2). for comparison, in guheswori sewage treatment plant acinetobacter spp. strains were found resistant to cadmium (1.33 mm), copper (3.15 mm) and cobalt (3.05 mm) tolerant (rajbanshi 2008); in south india sewage water was found arsenic (mic 13 mm), cadmium (4 mm) and chromium (0.7 mm) tolerant strain a. radioresistens bc3 (raja et al. 2009); from saudi arabia industrial area (hafar al batin) a. baumanii haf-13 strain was isolated exhibiting resistance to arsenic (3.34 mm), chromium (4.81 mm), cadmium (1.78 mm), lead (0.84 mm), and mercury (0.5 mm) (el-sayed 2016). some investigations show that several strains of the genus acinetobacter can thrive on mediums with acidic or alkaline ph (yavankar et al. 2007). tests of bacterial ability to grow at alkaline ph showed that all analyzed isolates are able to grow at ph 9.0 but not at ph 10.5 (table 2). genetic variability in acinetobacter spp. is high and in different species various numbers of genetic orthologs could be observed. the presence of plasmids is one of sources of genetic diversity observed and frequently, heavy metal resistance in acinetobacters is plasmid encoded. in permafrost strains of a. lwoffii multiple plasmids encoding resistance to hg, chr, co/zn/cd, ni, and ars were detected (midlin et al. 2016). in analyzed acinetobacter spp. collection of plasmid dna were detected in a. lwoffii k1 and a. johnsonii nhl11 isolates (fig. 1) but not in a. calcoaceticus isolates (data not shown) which show higher tolerance to heavy metals. strains k1 and nhl11 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:31 utc nova biotechnol chim (2017) 16(1): 42-47 46 table 2. bacterial growth at alkaline ph and heavy metals minimum inhibitory concentrations of acinetobacter spp. isolate name species growth at ph minimum inhibitory concentration (mm) 9 10.5 mn zn cu ni co k1 a. lwoffii + 4 2 2 2 1 k6 a. calcoaceticus + 30 15 8 2 1 k13 a. calcoaceticus + 30 15 8 2 1 nhl1 a. calcoaceticus + 30 15 4 2 1 nhl4 a. calcoaceticus + 30 15 4 2 1 nhl11 a. johnsonii + 8 4 4 2 1 p19 a. calcoaceticus + 30 15 8 2 1 p20 a. calcoaceticus + 30 15 8 2 1 contain at least 4 plasmids. their sizes vary from above 2 kbp to 25 kbp. currently, in genome ncbi database (https://www.ncbi.nlm.nih.gov/genome/), there are 212 complete acinetobacter spp. plasmid sequences available. plasmid sizes vary from 1.735 kbp (acinetobacter sp. m131, pm13111; peng et al. 2014) to 398.857 kbp (acinetobacter johnsonii xbb1, pxbb1-9; zong 2014). the putative role of plasmids in our isolates will be further studied. fig. 1. plasmid dna from acinetobacter spp. isolates: a. lwoffii (k1) and a. johnsonii (nhl11). to assess genetic diversity among a. calcoaceticus isolates genome fingerprinting using eric-pcr with the primers eric 1r and erc2 was used. eric-pcr is widely used for study of genetic variability in wide spectrum of organisms, including bacteria (versalovic et al. 1991). despite different origin of a. calcoaceticus isolates two genotypes were detected among tested strains (fig. 2). while unique genotypes were detected in red and brown mud waste disposal site near žiar nad hronom and nickel sludge landfill, in mine tailing slovinky the presence of both aforementioned genotypes was detected, indicating low genetic variability of a. calcoaceticus isolates from heavy metals polluted environments in slovakia. fig. 2. eric-pcr profiles of a. calcoaceticus isolates k6, k13 (žiar nad hronom); nhl1, nhl4 (sereď); p19, p20 (slovinky). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:31 utc https://www.ncbi.nlm.nih.gov/genome/ nova biotechnol chim (2017) 16(1): 42-47 47 conclusions acinetobacter spp. represent significant part of cultivable bacteria from heavy metals polluted environments in slovakia. a. calcoaceticus was found to be prevalent species among isolated strains, followed by a. lwoffii and a. johnsonii. genetic analyses demonstrated multiple plasmids presence in a. lwoffii and a. johnsonii but not in a. calcoaceticus. despite genetic diversity among a. calcoaceticus detected using eric-pcr all a. calcoaceticus strains have similar characteristics and increased tolerance to heavy metals compared to a. lwoffii and a. johnsonii. heavy metal industry waste disposal sites could be a good source of extremophilic bacteria widely used in modern biotechnologies. acknowledgements this work was supported by a research grant from the slovak grant agency (vega 1/0229/17 and vvgs-pf2017-270). references abboud mm, khleifat km, batarseh m, tarawneh ka, almustafa a, al-madadhah m (2017) different optimization conditions required for enhancing the biodegradation of linear alkylbenzosulfonate and sodium dodecyl sulfate surfactants by novel consortium of acinetobacter calcoaceticus and pantoea agglomerans. enzyme microb. technol. 41: 432-439. abdel-el-haleem d (2003) acinetobacter: environmental and biotechnological applications. afr. j. biotechnol. 2: 71-74. dougrahi hj, ndakidemi pa, human is, benade s (2011) the ecology, biology and pathogenesis of acinetobacter spp.: an overview. microbes environ. 26: 101-112. el-sayed mh (2016) multiple heavy metal and antibiotic resistance of acinetobacter baumanii strain haf-13 isolated from industrial effluents. am. j. microbiol. res. 4: 26-36. ferreira l, sánchez-juanes f, garcía-fraile p, rivas r, mateos pf, martínez-molina e, gonzález-buitrago jm, velázquez e (2011) maldi-tof mass spectrometry is a fast and reliable platform for identification and ecological studies of species from family rhizobiaceae. plos one 6: e20223. gaidhani s, singh r, singh d, patel u, shevade k, yeshvekar r, chopade ba (2013) biofilm disruption activity of silver nanoparticles synthesized by acinetobacter calcoaceticus pucm 1005. mater. lett. 108: 324-327. gaidhani sv, yeshvekar rk, shedbalkar uu, bellare jh, chopade ba (2014) bio-reduction of hexachloroplatinic acid to platinum nanoparticles employing acinetobacter calcoaceticus. process biochem. 49: 2313-2319. ghodake g, jadhav s, dawkar v, govindar s (2009) biodegradation of diazo dye direct brown mr by acinetobacter calcoaceticus ncim 2890. int. biodeter. biodegr. 63: 433-439. li a-d, li l-g, zhang t (2015) exploring antibiotic resistance genes and metal resistance genes in plasmid metagenomes from wastewater treatment plants. front. microbiol. 6: 1025. marín m, pedregosa a, ríos s, ortiz l, laborda f (1995) biodegradation of diesel and heating oil by acinetobacter calcoaceticus mm5: its possible applications on bioremediation. int. biodeter. biodegr. 35: 269-285. mindlin s, petrenko a, kurakov a, beletsky a, mardanov a, petrova m (2016) resistance of permafrost and modern acinetobacter lwoffii strains to heavy metals and arsenic revealed by genome analysis. biomed res. int. article id 3970831. nies dh (1999) microbial heavy-metal resistance. appl. microbiol. biotechnol. 51: 730-750. pailan s, sengupta k, ganguly u, saha p (2016) evidence of biodegradation of chlorpyrifos by a newly isolated heavy metal-tolerant bacterium acinetobacter sp. strain memcl4. environ. earth sci. 75: 1019. raja ce, elvam gs, omine k (2009) isolation, identification and characterization of heavy metal resistent bacteria from sewage. in: international joint symposium on geodisaster prevention and geoenvironment in asia js-fukuoka. rajbanshi a (2008) study on heavy metal resistant bacteria in guheswori sewage treatment plant. our nature 6: 5257. peng sm, liao tl, lin ac, huang tw, lauderdale tl, chen yt (2017) sequencing and analysis of an oxacillinase-encoding plasmid from acinetobacter spp. [online; cit. 2017-02-27, www.ncbi.nlm.nih.gov/nuccore/nc_025117.1]. pristas p, stramova z, kvasnova s, judova j, perhacova z, vidova b, sramkova z, godany a (2015) non-ferrous metal industry waste disposal sites as s source of polyextremotolerat bacteria. nova biotechnol. chim. 14: 6268. versalovic j, koeuth t, lupski jr (1991) distribution of repetitive dna sequences in eubacteria and application to fingerprinting of bacterial genomes. nucleic acids res. 19: 6823-6831. yavankar sp, pardesi kr, chopade ba (2007) species distribution and physiological characterization of acinetobacter genospecies from healthy human skin of tribal population in india. indian j. med. microbiol. 25: 336-345. zong z (2017) the complete genome of acinetobacter johnsonii strain xbb1 carrying blandm-1 and blaoxa-58 from hospital sewage. [online, cit. 2017-0227, www.ncbi.nlm.nih.gov/nuccore/nz_cp010351.1]. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:31 utc nova biotechnol chim (2017) 16(2): 105-112 doi: 10.1515/nbec-2017-0015  corresponding author: zinikovskaia@mail.ru  nova biotechnologica et chimica biosorption of lead ions by cyanobacteria spirulina platensis: kinetics, equilibrium and thermodynamic study inga zinicovscaia1,2,3, , nikita yushin4,1, elena rodlovskaya5 and inna kamanina4 1 joint institute for nuclear research, joliot-curie str., 6, 1419890 dubna, russia 2 horia hulubei national institute for r&d in physics and nuclear engineering, 30 reactorului str. mg-6, bucharest magurele, romania. 3 the institute of chemistry of the academy of sciences of moldova, 3, academiei str., 2028 chisinau, r. moldova 4 dubna university, 19 universitetskaya str., 141982 г. dubna, russia 5 a. n. nesmeyanov institute of organoelement compounds of russian academy of sciences, vavilova str., 28, 119991, moscow, russia article info article history: received: 14th august 2017 accepted: 26th november 2017 keywords: biosorption lead spirulina platensis abstract the potential use of dry spirulina platensis biomass to remove lead ions from aqueous solution was investigated. effects of various parameters such as contact time, temperature, dosage of biosorbent, initial ph, and initial concentration of lead were investigated in the batch adsorption mode. the highest lead removal of 5.7 mg/g was obtained at ph 5, biomass dosage of 0.5 g, initial lead concentration of 60 mg/l. the langmuir and freundlich models fit the experimental data (r2 > 0.99), while the kinetic data was best described using the pseudo second-order kinetic model (r2 > 0.99). ftir spectra indicated that the metal removal takes place through binding to oh, c=o and p=o groups. lead was efficiently recovered from biomass by mineral acids, while using ch3cooh and naoh as eluents the biomass maintained high biosorption capacity during three cycles. this study demonstrates the potential of using spirulina platensis as biosorbent to remove lead from industrial wastewater.  university of ss. cyril and methodius in trnava       introduction among the metals, lead is widely used in industry and its release in water bodies may cause serious ecological and human health consequences. in addition to such major lead sources as gasoline vehicles, lead is used as an industrial raw material for storage battery manufacturing, printing, pigments, fuels, photographic materials and explosive manufacturing (jalali et al. 2002; bahadir et al. 2007; miranda et al. 2013). traditional technologies presently used for metal removal, precipitation, chemical absorption, oxidation-reduction, use of ion-exchange resins, have significant disadvantages, including incomplete metal removal, high cost, generation of toxic sludge or other waste products that require disposal (bahadir et al. 2007; chen et al. 2011). biosorption is one of the most promising technologies involved in the removal of toxic metal, which allows reducing metal ion concentrations to environmentally acceptable levels. the biosorption process includes physicochemical interactions between metal ions and several anionic ligands present on the biomass like carboxyl, phosphoryl, carbonyl and sulfhydryl bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:04 utc nova biotechnol chim (2017) 16(2): 105-112 106 (jalali et al. 2002). the efficiency of metal biosorption is strongly dependent on experiment parameters such as: ph, temperature, and the concentration of sorbate and sorbent (chojnacka 2010). various materials of biological origin, including bacteria, cyanobacteria, fungi and yeasts, can be used for toxic metals biosorption (bahadir et al. 2007; bhatnagar et al. 2010; miranda et al. 2013; kaduková and horváthová 2012; sadovsky et al. 2016) spirulina biomass, due to its relatively high sorption capacity and low cost, was studied in the removal of a wide range of toxic metals (chen and pan 2005; vannela and verma 2006; lodi et al. 2008; zinicovscaia et al. 2016). gong et al. (2006) investigated the effects of operational conditions (ph, contact time, biomass concentration time of interaction) on lead biosorption by intact and pretreated biomass of spirulina maxima. al-homaidan et al. (2016) studied the effect of the same parameters on the lead removal by non-living spirulina platensis biomass. the objective of the present study was to examine the effect of different operational parameters on the efficiency of lead biosorption by dry spirulina biomass. equilibrium, kinetic and thermodynamic studies, not implemented in the previously published work, were performed to describe the adsorption process. functional groups responsible for metal binding were determined by fourier transform infrared spectroscopy. the recovery of bound lead by different type of desorbents was measured. experimental reagents and materials all the chemicals used for biosorption experiments were purchased from sigma-aldrich and were of analytical grade. preparation of adsorbent spirulina platensis (s. platensis) biomass purchased from “biosolar msu” company was dried in an oven at 80°c for 24 h. then the biomass was homogenized in a homogenizer at 600 rpm for 10 min. experiment the influence of ph was studied using lead nitrate solution with an initial concentration of 10 mg pb/l and ph values varying between 2.0 and 7. the initial solution was adjusted to the desired ph with diluted or concentrated hno3 and naoh solutions before being mixed with the biosorbent. the change in the working volume due to the addition of hno3 and naoh was negligible in this experiment. the effect of biosorbent dosage on batch experiments was examined by varying its weight from 0.1 to 1 g. for the isotherm study, 3 – 400 mg pb/l was mixed with 0.5 g biomass at ph 5 and shaken at 200 rpm for 1 h. to determine the contact time required for the sorption equilibrium experiments, samples were withdrawn at predetermined time intervals (3, 5, 7, 10, 15, 30, 60 and 120 min). all the experiments were carried out under ambient conditions at 20 °c, except for the thermodynamic studies at temperature 20, 30, 40 and 50 °c, respectively. in all experiments the working volume was 50 ml. after experiment, biosorbent was separated by centrifugation and the concentration of metal ion in solution was analyzed by atomic adsorption spectrometry. the metal uptake q (mg pb/g of biosorbent) was calculated from the mass balance using the following equation: where q is the amount of metal ions adsorbed on the biosorbent, mg/g; v is the volume of solution, ml; ci is the initial concentration of metal in mg/l, cf is the final metal concentration in the solution, mg/l, and m is the mass of sorbent, g. desorption experiment in desorption experiments s. platensis biomass was firstly exposed to 30 mg pb/l solution at optimum ph conditions. next, the biomass was separated from the solution by centrifugation. the obtained pb-loaded biosorbent was then brought in contact with 50 ml of 0.1 m hno3, 0.1 m hcl, 0.1 m ch3cooh and 0.1 m naoh for 1 h on an orbital shaker at 200 rpm. the procedure was repeated for three times. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:04 utc nova biotechnol chim (2017) 16(2): 105-112 107 atomic adsorption spectrometry lead content in solution was determined in propane flame on kvant-2a spectrophotometer at a resonance line of 217.0 nm. the strength of the current was 16 ma. fourier transform infrared spectroscopy (ft-ir) ft-ir spectroscopy was used to confirm the presence of the functional groups in the samples of s. platensis and to observe the chemical modification after lead biosorption. infrared spectra were recorded in the 4 000 − 600 cm–1 region using a perkin elmer spectrum 100 ft-ir spectrometer. results and discussion effect of ph value on biosorption the ph value plays an important role in biosorption process, because it influences the speciation of metal in the aqueous solution, the ionization of surface functional groups, and the binding sites on the surface of the biomass (amini et al. 2013). in the present study, the effect of initial solution ph was examined in the range 2.0 – 7.0, and the obtained results are presented in fig. 1. the equilibrium sorption capacity had a minimum at ph 2 (52%). at ph 2.0, protons, due to their high concentration occupy most of the active sites on the biosorbent surface, thus less lead fig. 1. removal of lead ions at different initial ph (t 20 °c; c0 10 mg/l; sorbent dosage 0.5 g; adsorption time 1 h). ions could be adsorbed. the maximum lead biosorption by the biomass (0.8 mg/g) was observed at ph 3 – 5, corresponding to 88 – 89% removal efficiency. there have been similar observations for lead biosorption by spirulina sp. at ph 4 (aneja et al. 2010). at ph values higher than 5.0, a slow decrease of biosorption parameters was observed. the speciation of lead is ph dependent. at ph of 2 – 6 predominant sorbing forms of lead are pb2+ and pboh+, while above ph of 6, it is hydrolyzed to pboh+ and pb(oh)2 (qaiser et al. 2009). this was the reason for higher removal of lead in the ph range of 3 – 5. therefore, ph 5.0 was considered as the optimum ph and used in further experiments. optimal ph value range obtained in present the study is in agreement with the results of other studies: ph 3 in al-homaidan et al. (2016), ph 5 in gong et al. (2005) and kőnig-péter et al. (2015) studies. effect of sorbent dosage on biosorption the effect of spirulina biomass dosage on the biosorption of lead ions was studied using different quantities of biomass (0.1, 0.2, 0.3, 0.4, 0.5, 0.7 and 1.0 g) and maintaining constant all other experimental parameters. as it is shown in fig. 2, while the biosorbent doses increase from 0.1 to 1 g the percentages of lead adsorbed increased from 54% to 88%. at increase of biosorbents amount, more binding sites are available for metal capture and thus the removal efficiency ramps up.             fig. 2. effect of biosorbent dose on biosorption capacity and removal efficiency of lead ions by s. platensis biomass (t 20 °c; c0 10 mg/l; ph 5; adsorption time 1 h). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:04 utc nova biotechnol chim (2017) 16(2): 105-112 108 however, the amount of lead adsorbed decreased from 2.46 to 0.4 mg/g. this is due to the fact that at higher adsorbent doses the solution’s ion concentration drops to a lower value and the system reaches equilibrium at lower values of ‘q’ indicating the adsorption sites remain unsaturated (amarasinghe and williams 2007). in al-homaidan et al. (2016) study, who studied the effect of different biomass dosages (0.25, 0.5, 0.7 5, 1.0, 1.5, and 2.0 g/l) of spirulina biomass on biosoption process the optimum biomass dosage was selected as 2 g. data obtained by kőnig-péter et al. (2015) showed that at sorbent dosage of 1 g the lead removal efficiency by cyanobacteria was higher than 80 % and with increasing the biosorbent concentration to 2.0 g the adsorbed amount increased not more than 10 %. thermodynamic study industrial effluents often are discharged at relatively high temperature, thus temperature is an important parameter affecting the sorbent biosorption capacity. in the present study the solution temperature has varied in the range of 20 – 50°c. a maximum adsorption capacity was obtained at the temperature of 30°c. the thermodynamic constants namely, free energy change (∆g◦), enthalpy change (∆h◦) and entropy change (∆s◦) were calculated to evaluate the thermodynamic feasibility of the process and to confirm the nature of the adsorption process. the values of enthalpy (∆h◦) and entropy (∆s◦) were               fig. 3. dependence of lnkd vs. 1/t. evaluated from the slope and intercept of ln kd vs. 1/t plots (fig. 3). where (∆h◦), (∆s◦), t (in kelvin) and r are the enthalpy, the entropy, the temperature and the gas constant, respectively. the distribution coefficient (kd) was calculated using the following formula: where ci and ce are initial and equilibrium lead concentrations (mg/l), v is the volume of the solution (ml), and m is the mass of the biosorbent (g) (chen and gao 2009). the ∆g◦ was calculated from the equation:   the values of ∆h◦, ∆s◦ and ∆g◦ are given in the table 1. table 1. thermodynamic parameters for lead biosorption on s. platensis. temperature k ∆g◦ kj/mol ∆h◦ kj/mol ∆s◦ j/mol·k 293 -1.53 -0.83 34.8 303 -1.87 313 -2.2 323 -2.5 negative ∆g◦ values of lead biosorption by spirulina biomass indicate the spontaneity of the adsorption process. ∆g◦ was more negative with decreasing temperature, which suggested that a lower temperature makes the adsorption easier (chen et al. 2006). ∆h◦ value -0.83 kj/mol revealed   fig. 4. freundlich isotherm model. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:04 utc nova biotechnol chim (2017) 16(2): 105-112 109 that the adsorption process was exothermic in nature, which agrees with the results obtained for the other sorbents (amini et al. 2013; silvamedeiros et al. 2016). at the same time, a positive value of ∆s◦ reflects an increased randomness at the solid-solution interface during biosorption and it also indicates that ion replacement reactions occurs (liu and liu 2008). therefore, lead biosorption by spirulina biomass can be considered as a physico-chemical adsorption process rather than a pure physical or chemical adsorption. biosorption equilibrium modeling the freundlich and langmuir isotherms are commonly used in biosorption studies to predict the adsorption capacity of a biosorbent. the langmuir model considers sorption by monolayer type and supposes that all the active sites on the sorbent surface have the same affinity by the sorbate (li et al. 2010). this equation of the langmuir isotherm is expressed as follows: where qe (mg/ g) is the amount adsorbed at the equilibrium concentration ce, qm (mg/g) is the langmuir constant representing the maximum monolayer adsorption capacity and b (mol/l) is the langmuir constant related to energy of adsorption. the main advantage of this model is the possibility to evaluate maximum possible quantity of metal ions adsorbed per gram of adsorbent and constant related to the affinity of binding sites for the metal ions (miranda et al. 2013).             fig. 5. effect of contact time on the sorption of lead ions by s. platensis biomass (t 20 °c; c0 10 mg/l; ph 5; sorbent dosage 0.5 g). the freundlich isotherm is an empirical equation, which assumes a heterogeneous biosorption system with different active sites. the general freundlich equation is written as follows: where: kf and 1/n are freundlich constants, associated with adsorption capacity and adsorption intensity, respectively. the isotherm parameters have been evaluated by non-linear regression. obtained data have shown that the adsorption of lead ions by s. platensis biomass was unfavorable at the specified conditions for langmuir model. at the same time experimental data were well described by freundlich model (fig. 4, table 2). obtained r2 value for freundlich model was higher compared to r2 value obtained for langmuir model (data not shown), indicating that the adsorption of lead ions by s. platensis biomass was unfavorable at the specified conditions for langmuir model. in the freundlich model, the n values in the range of 1 – 10 indicated favorable adsorption. applicability of freundlich model for description of lead biosorption by spirulina species was shown in al-homaidan et al. (2016) and gong et al. (2005) works. biosorption kinetics as it is shown in fig. 5 the lead biosorption process by spirulina biomass took place in two stages: the first rapid stage, was achieved in 30 min, and the second equilibrium stage. lead biosorption capacity reached equilibrium value in             fig. 6. the pseudo-first and pseudo second order plots of kinetic study of lead biosorption on s. platensis. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:04 utc nova biotechnol chim (2017) 16(2): 105-112 110 about 60 min. no further increase in the level of bound lead occurred after this time. this is in accordance with the observations of other similar studies (qaiser et al. 2009). table 2. isotherm parameters for the biosorption of lead ions on s. platensis biomass. r2 0.995 kf 0.085±0.006 n 1.03 in order to analyze experimental data a pseudo-first order and pseudo-second order models were used (chen et al. 2011). the pseudofirst-order lagergren model expressed as: where q and qe are the adsorbed amounts (mg/g) at time (t) (min) and at equilibrium time, respectively, ka (min−1) is the rate constant of the first-order biosorption. the pseudo-second-order kinetics can be expressed: where q and qe are the adsorbed amounts (µg/g) at time (t) (min) and at equilibrium time, respectively, kb (g/mg*min) is the rate constant of the second-order biosorption. the correlation coefficient values obtained from the plots (fig. 6) suggest that the sorption process fit well the pseudo-first-order kinetics. r2 for the pseudo second-order kinetic model is greater than 0.99 and substantially higher than that for the pseudo first-order kinetic model. besides, the qe values calculated from the pseudo secondorder kinetic model agree well with the experimental data values. the reaction rate constant, k and equilibrium biosorption capacity, qe were determined from the slope and intercept of the straightline plot and are presented in table 3. table 3. comparison of pseudo-first and pseudo-second order models parameters. pseudo-first-order model ce, mg/l qe (exp), mg/g qe (cal), mg/g ka, min−1 r2 10 0.78 0.41 7.6*10-4 0.950 pseudo-second-order ce, mg/l qe (exp), mg/g qe (cal), mg/g kb, g/mg.min r2 10 0.78 0.8 0.92 0.999 the applicability of pseudo-second order kinetics model suggested that the lead biosorption was based on chemical reaction, involving the exchange of electrons between biosorbent and metal (qaiser et al. 2009). many researches have also concluded that the second-order kinetics model is more suitable to describe the lead biosorption kinetics (qaiser et al. 2009; miranda et al. 2013). ftir analysis the ftir analysis was used for the determination of functional groups, which may be involved in the lead biosorption process. in the control ft-ir spectrum (fig. 7) some intense characteristic bands were obtained for the functional groups presented in proteins and polysaccharides: –oh (1055, 1397.9 cm-1), –nh2, s-ch2, nhc(o)amid (1235.0), c=o (1535.0), p=o (1625.0), n-ch2 (2925.0) and nh2 (3277.0) groups. after lead biosorption the bands of 1055, 1397.9, 1537.5, and 1625.0 cm-1 were shifted to 1070.5, 1401.0, 1538.5 and 1627.5 cm-1, respectively. the change in the wave numbers of these bands suggests that oh, c=o and p=o groups can be involved in the biosorption process of lead ions onto spirulina biomass. similar results were obtained by other researchers, who studied lead biosorption by cyanobacteria. for example, gong et al. (2005) have shown that amino and hydroxyl groups were involved in the lead biosorption by spirulina maxima. hydroxyl, carboxyl and amino groups participated in lead biosorption by chroococcus multicoloratus and oscillatoria trichoides (miranda et al. 2013). desorption and reuse regeneration of biomass in a scale-up is a crucial process parameter that influences the operating bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:04 utc nova biotechnol chim (2017) 16(2): 105-112 111 cost. the desorption analysis of s. platensis biomass for the lead ions removal was performed using four different eluent solutions (0.1 m). biosorption efficiency remained almost constant when ch3cooh or naoh were used. however the desorption efficiency was not more than 20%. high removal efficiency can be explained by increase of the concentration of carboxyl and hydroxyl group under interaction of biomass with abovementioned chemicals. ftir data shows that fig. 7. ft-ir spectra of s. platensis biomass: control and pbloaded. these groups are involved in lead biosorption. the applied mineral acids showed good potential as desorption agent for lead with 65 – 67% of recovery in the first cycle (fig. 8). fig. 8. biosorption-desorption cycles. this situation is likely to occur via the exchange of protons with bound lead on cyanobacteria surface. during the next two cycles the desorbing efficiency of biomass treated with mineral acids was drastically reduced, where the percentage removal dropped to 25 – 29% while percentage recovery was 4 – 18% only. the decrease in the biomass sorption/desorption capacity can be explained by structural damage of biosorbent and blockage of binding sites by metal complex. application of 1.0 m hno3 for desorption of cr(iii), cd(ii) and cu(ii) sorbed by cyanobacteria spirulina sp. reduced metals desorption by 50% (chojnacka et al. 2005). conclusions the potential of spirulina biomass application for lead removal in aqueous solutions was demonstrated in the present work. the optimum operating conditions for lead adsorption was found to be 0.5 g biomass, at ph 5, for 30 min. biosorption equilibrium data fitted very well to both the langmuir and freundlich models. analysis of the data showed that the process involves second-order kinetics. the biosorption of lead by spirulina biomass is an exothermic and spontaneous process. the functional groups of oh, c=o and p=o were mainly involved in lead ions binding by spirulina biomass. the presence of iron ions in the solution affected lead biosorption by spirulina biomass. mineral acids showed good desorption efficiency in the first cycle, after that it was significantly reduced. thus it is necessary in further study to find more effective desorbing agent. s. platensis can be applied for lead removal from industrial effluents or wastewater posttreatment. acknowledgement the authors wish to thank olga kristavchuk for measuring ir spectra. references   al-homaidan 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progress 22: 1282-1293. zinicovscaia i, cepoi l, chiriac t, culicov o, frontasyeva m, pavlov s (2016) spirulina platensis as biosorbent of chromium and nickel from industrial effluents. desalin. water treat. 57: 11103-11110. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:04 utc nova biotechnol chim (2018) 17(2): 115-124 doi: 10.2478/nbec-2018-0012  corresponding author: vijuuu333@gmail.com nova biotechnologica et chimica antifouling activities of extracellular polymeric substances produced by marine bacteria associated with the gastropod (babylonia sp.) nadarajan viju1,, nagarajan ezhilraj1, chellamnadar vaikundavasagom sunjaiy shankar1, stanislaus mary josephine punitha1 and sathianeson satheesh2 1centre for marine science and technology, manonmaniam sundaranar university, rajakkamangalam, kanyakumari, tamilnadu, india 2department of marine biology, faculty of marine sciences, king abdulaziz university, jeddah, saudi arabia article info article history: received: 3rd april 2018 accepted: 2nd november 2018 keywords: antifouling coating biofilm biofouling extracellular polymeric substances surface-associated bacteria abstract bacteria associated with surfaces have been frequently cited as a potential source for the isolation of bioactive metabolites. in this study, bacteria associated with marine gastropod, babylonia sp. were isolated and screened for antibacterial activity against biofilm-forming bacteria. the antibiofilm and antifouling effect of the selected surfaceassociated bacterial strains were examined under in vitro and in vivo conditions. results showed that the extracellular polymeric substances (eps) of the bacterial strain cml associated with gastropod species considerably reduced the adhesion of biofilm-forming bacteria on glass coupons. besides, the antifouling coat prepared by incorporating of this eps into polyurethane varnish prevented the settlement of biofoulers on test substratum submerged in marine waters. the functional groups present in the eps were analyzed using ft-ir. the bacterium responsible for the production of the bioactive eps was identified as bacillus subtilis subsp. by 16s rrna gene sequencing. more detailed characterization of the identified bioactive eps could lead to the isolation of a novel natural antifouling product.  university of ss. cyril and methodius in trnava introduction biofouling continues to be one of the important problems in the maritime sector, which causes severe problems on the surfaces of the marine living organisms and submerged structures (almedia et al. 2017). although, highly effective biocides are used as coatings for avoiding the settlement of organisms, tbt (tributyltin) like biocides has created environmental problems to non-target organisms (thomas and brooks 2010). there is a growing need for the effective and eco-friendly antifoulants for marine applications particularly after the ban of tbt based paints; therefore research interests on natural antifoulants has been increased (satheesh et al. 2016). the marine environment is a rich source of biological and chemical diversity, and also an exceptional reservoir for unique bioactive compounds (fenical 1993). therefore, the marine organisms have been placed into focus discovering novel bioactive compounds (clare 1996). a number of bioactive metabolites have been isolated from marine sources (kijjoa and sawangwong 2004; xu et al. 2010). notably, marine bacteria have been the source for the isolation of many such compounds in medicine, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:16 utc nova biotechnol chim (2018) 17(2): 115-124 116 industry and agriculture (bowman 2007). the bacteria associated with marine algae and other organisms are reported to produce a higher percentage of bioactive compounds comparing to free floating bacteria (zheng et al. 2005). hence, a consequent attention has been paid on marine bacteria associated with the surface of invertebrates for the isolation of novel antifouling compounds (satheesh et al. 2012; viju et al. 2017). the production of compounds such as giffinisterone b, lobocompacto and dicitrinin a etc. by the symbiotic bacteria has been reported (cho 2012; cho and kim 2012). therefore, the aim of the present study was to identify novel antifouling compound from the bacteria living at the surface of marine molluscs. for the screening, fouling bacteria isolated from the acrylic panel submerged in the kudankulam coastal water (satheesh et al. 2012) were used as the target. it has been reported that the biofilm formed by such bacteria could act as a cue for the settlement of following macrofouling organisms (lau et al. 2003). therefore preventing formation of this initial biofilm (microfouling) would also prevent the subsequent macrofouling (armstrong et al. 2000). with this reasoning the selected fouling bacteria have been used for the preliminary screening of extracellular polymeric substances (eps) for antifouling property. such study might contribute to knowledge about biofouling and antifouling activities expressed by surface associated bacteria. experimental collection and isolation of surface associated bacteria the marine gastropod babylonia sp. collected from colachel coast (west coast of india) were brought to the laboratory in a sterile plastic container with seawater. the bacteria associated with the gastropod were isolated according to the method described by viju et al. (2016). in brief, the loosely attached organisms on the surface of gastropod were removed by washing with sterile seawater. following this, the bacteria associated with the surface of gastropod were scraped out using a nylon brush and suspended in 1 ml of filtersterilized seawater. this suspension was serially diluted and spread on agar plate prepared with zobell marine agar (zma). these plates were then incubated for 24 h at 37 °c for the growth of bacterial colonies. the colonies grown were isolated based on the morphology and maintained on slants at 4 oc for further studies. isolation of extracellular polymeric substance the bacterial colonies were inoculated into a beaker containing a 500 ml of zobell marine broth (zmb) and placed in a shaker at 37 °c for 72 h of incubation. after that, the culture broth was centrifuged (at 5,000 rpm for 15 min 4 oc) and the supernatant was collected. the collected supernatant was added with adequate amount of ethanol (1 : 1 ratio) and kept in a safe place (overnight) for the precipitation of eps. the collected precipitate (eps) was filtered using a membrane filter and diluted in double distilled water. this suspension was then dialyzed using a tubular dialysis membrane (8 kda) and the obtained fraction was used for further studies (rajasree et al. 2012). antimicrobial assay agar disc diffusion method described by bauer et al. (1966) was used to test the antimicrobial activity of the eps. for this, the sterile disc (6 mm, himedia, india) was loaded with 50 µl of bacterial eps and placed on mueller-hinton agar plates swabbed with target (biofilm-forming) bacteria such as gallionella sp., alteromonas sp. and pseudomonas sp. these bacteria were isolated from the artificial surfaces immersed in the seawater (satheesh et al. 2012). control discs loaded with distilled water were also maintained. all the plates were incubated at 37 °c for 48 h. following incubation, the inhibition zone appeared around the discs were measured. the presented data represent the average of two measured values. preparation of bacterial cell suspension for laboratory assays the biofilm-forming bacteria were grown at 37 °c for overnight in zobell marine broth. this broth bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:16 utc nova biotechnol chim (2018) 17(2): 115-124 117 was then centrifuged at 5,000 rpm for 15 min and the cell pellets obtained were washed with phosphate-buffered saline (10 ml pbs). following this, the washed pellet was re-suspended in the same buffer in order to obtain od540 = 0.2 and used for the further assay (jain et al. 2007). adhesion assay the bacterial adhesion assay was performed according to the procedure outlined by rajasree et al. (2014). briefly, the microscopic slides (7.5×2.5 cm) were treated with eps by placing them in beaker (500 ml) containing seawater (300 ml) and 0.5 ml of eps. after 24 hours, the slides treated with eps were taken out and placed into another beaker which contained 300 ml of sterile seawater, 3 ml of zmb and 3 ml of biofilm-forming bacterial suspension. following 5 h of incubation, the microscopic slides were retrieved and the bacteria adhered on the slides were stained with crystal violet. the stained bacterial cells were then enumerated under a microscope. the variation between the bacterial cells adhered on the control and eps treated slides were statistically analysed using student’s t-test (microsoft excel program). influence of surface-associated bacterial extract (eps) on the eps production of biofilm-forming bacteria five hundred microliter of eps was taken in a test tube containing 3 ml of biofilm forming bacterial suspension and 3 ml of zobell marine broth was added in order to provide the nutrients for bacterial growth. control tubes were prepared without the addition of bacterial eps and all the tubes were incubated at 37 °c for 24 h. after that, the culture broth was centrifuged at 5,000 rpm for 5 min at 4 °c and the supernatant was collected and filtered through whatman no.1 filter paper (0.47 µm). the filtered supernatant was added with equal volume of cold absolute ethanol and allowed for the precipitation of eps by keeping at room temperature for overnight. the precipitated eps was isolated and the carbohydrate (dubois et al. 1956) and protein (lowry et al. 1951) content of the eps were estimated (viju et al. 2013). the obtained data were subjected to student’s t-test (microsoft excel program) in order to analysis the significant influence of gastropod associated bacterial eps on the production of carbohydrate and protein by biofilm-forming bacteria. preparation of eps mixed coating for biofilm control (field assay) an insoluble matrix antifouling coating was prepared in order to study the antifouling activity of bacterial eps in the coastal waters. in brief, the commercially available binder polyurethane varnish (berger) was mixed with the eps (biocide) at 1 : 1 ratio. this mixture (coating) was coated on fiber glass plates (10 cm×6 cm) using a nylon brush. the varnish coated fiber glass plate was used as a control for the comparison of results. all these plates were fitted on to an iron frame and immersed in to the coastal water for a period of 15 days. after immersion, the plates were taken out and the settlement of biofoulers on the plates was analysed (viju et al. 2014). partial purification of the bioactive eps using thin-layer chromatography (tlc) and high performance liquid chromatography (hplc) a drop of bacterial eps was placed on a silica gelg pre-coated glass plate and placed in a tlc chamber containing the mobile phase prepared by mixing benzene, acetic acid and methanol in 1 : 1 : 3 ratio. the movement of the mobile phase on the tlc plate was watched carefully and the plate was taken out once the mobile phase reached the other end of the tlc plate. following this, the plate was air dried and placed in a glass chamber containing iodine crystal for the visualization of eps. the spot developed on the tlc plate was scraped off and mixed with adequate amount of distilled water. this mixture was then centrifuged at 5,000 rpm for 15 min and the supernatant was collected. this supernatant was then concentrated and subjected to hplc analysis. in hplc, acetonitrile and water (1 : 1) were used as the solvent system and the flow rate was fixed as 1 ml.min-1 (satheesh et al. 2012). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:16 utc nova biotechnol chim (2018) 17(2): 115-124 118 fig. 1. antibacterial activity of extracellular polymeric substances from different bacterial strains colonizing the surface of babylonia sp. activities were tested against biofilm-forming pseudomonas sp. (a) gallionella sp. (b) and alteromonas sp. (c). eps (50 µl) were loaded on sterile discs on the agar plate swabbed with target bacteria. the inhibitory activity was observed after 24 h of incubation at room temperature. analysis of the functional groups present in the eps using fourier transform infrared (ft-ir) spectroscopy the ft-ir spectrometer was used to identify the functional groups present in the eps. a small quantity of tlc purified eps was placed on the face of a highly polished kbr salt plate and another kbr plate was positioned on the top to spread the compound as a thin layer. the frequencies were measured as wave numbers over the range of 400 to 4,000 cm-1 (viju et al. 2017). identification of bacteria the bacterial identification was performed according to the procedure described by satheesh et al. (2012). briefly, the 16s rrna of the 24 h old bacterial culture was extracted using phenol chloroform method. the 16s rrna was amplified by polymerase chain reaction (pcr) using universal primers 16s (f-5`agagtttgatcctggctcag-3`) and 16s (r5`ggttaccttgttacgactt-3`). the pcr reaction steps such as denaturation, annealing and extension were maintained at 95 °c, 52 °c and 60 °c for the time duration of 30 sec, 30 sec and 4 min, respectively. the whole procedure was repeated in order to get adequate number of nucleotides (bps). following this, the obtained pcr product was purified and sequenced using the same pcr primers and other internal primers (f-5´acaaacaacgtgaaacgtcaa 3´ and r5´-aaacgaaacacggaaactt 3´) to confirm the sequence. the obtained sequence of the bacterial isolate was analyzed using basic local alignment search tool (blast) and the phylogenetic tree was constructed by comparing the sequences available in ncbi database. results antimicrobial activity of eps bacteria were isolated from the surface of babylonia sp. and single colonies were picked and cultivated individually. the eps was isolated from a total of 21 bacterial strains and tested for antibacterial activity against three biofilmforming bacterial strains alteromonas sp., gallionella sp. and pseudomonas sp., respectively. the assay revealed the growth inhibitory activity of eps isolated from 10 bacterial isolates against biofilm-forming bacteria (fig. 1). notably, the eps produced by the strains cms and cml showed maximum zone of inhibition against all the tested bacteria reproducibly in 2 independent tests (table 1). the former strain cms was selected and used for further analyses. bacterial adhesion assay the antifouling effect of eps was first tested in laboratory conditions using glass plates. the eps of cms strain considerably reduced the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:16 utc nova biotechnol chim (2018) 17(2): 115-124 119 table 1. antibacterial activity of the extracellular polymeric substances (eps) isolated from the bacterial clones associated with the surface of babylonia sp. biofilm-forming bacteria zone of growth inhibition [mm] cms cml cbo 26 27 32 51 52 53 54 alteromonas sp. 12 11 10 10 8 11 9 7 11 10 gallionella sp. 13 13 10 9 9 8 8 8 9 11 pseudomonas sp. 12 12 9 7 7 biofilm bacterial density on the microscopic slides; the number of alteromonas sp. cells adhered to glass surface was significantly reduced upon treatment with eps (student’s t-test, t stat = 2.29, p < 0.041). similar restriction of bacterial adhesion was achieved for gallionella sp. (student’s t-test, t stat = 1.66, p < 0.085) and also for the pseudomonas sp. cell density (student’s t-test, t stat = 3.59, p < 0.011) (fig. 2). influence of surface associated bacteria on eps production in biofilm-forming bacteria the antifouling agents can have direct effect on biofilm-forming bacteria by means of their growth rate (fig. 2) or metabolic performance. therefore, we analysed some metabolic products of previous experiment (fig. 2). the gastropod associated bacterium cms considerably reduced the carbohydrate and protein concentration of eps fig. 2. influence of surface – associated bacterial eps on the adhesion of biofilm-forming bacteria. eps treated glass slides were placed in a beaker containing biofilm bacteria and the slides were retrieved and stained with crystal violet. the number of cells adhered was counted under a microscope. data presented are average ± standard deviation of three replicates. * indicates significance at p < 0.05. produced by biofilm-forming bacteria. the carbohydrate concentration of eps produced by alteromonas sp. culture was significantly reduced (student’s t-test, t stat = 3.18, p < 0.016) after treated with the eps of the strain cms. similarly, there was a significant reduction in the concentration of carbohydrate produced by gallionella sp. culture treated with the eps of the strain cms (student’s t-test, t stat = 14.56, p < 6.46e-05). the carbohydrate concentration of eps produced by the pseudomonas sp. significantly reduced when treated with the eps of the strain cms (student’s t-test, t stat = 2.42, p < 0.036). in the same way, the eps of gastropod associated bacterium cms substantially reduced the protein content of eps produced by biofilmforming bacteria alteromonas sp., gallionella sp. and pseudomonas sp.; the obtained results were statistically significant (student’s t-test, t stat = 3.10, p < 0.018; t stat = 2.5, p < 0.033 and t stat = 4.87, p < 0.004, respectively) (fig. 3). fig. 3: carbohydrate and protein concentrations in the eps produced by biofilm-forming bacteria after treated with the eps from bacteria associated with the gastropod babylonia sp. data presented are average ± standard deviation of three replicates. symbol * above the bars indicate the significant differences at p < 0.05.……………………………………... bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:16 utc nova biotechnol chim (2018) 17(2): 115-124 120 fig. 4. antifouling effect of eps: control plate was coated with polyurethane varnish and experimental plate was coated with polyurethane varnish and eps. both plates were submerged in to the coastal water for a period of 15 days. the growth of biofouling in the plates was observed and it was found that the (experiment) plate coated with eps considerably reduced the settlement of biofoulers. the control plate coated with polyurethane varnish was completely covered by the biofouling growth. preparation of eps mixed coating for biofilm control (field assay) since laboratory results not necessarily reflect the possible scenarios in a natural (and often much more complex) environment, we tested the antifouling efficiency of antifouling coating prepared with eps in the coastal water against marine biofoulers using fiber plate as substratum. the fiber plate coated with the eps of strain cms considerably inhibited the settlement of marine biofoulers for a short period of 15 days (fig. 4). partial purification of the bioactive eps using tlc thin-layer chromatography was used to separate and partly purify the studied eps substance from the cms bacteria. the resulting chromatogram showed a single spot with the rf value of 0.80 cm (fig. 5). after elution of the compounds into water fig. 6. hplc spectrum of the eps fraction resolved through thin-layer chromatography analysis. hplc analysis was performed using acetonitrile and water (1 : 1) as the solvent system. and subsequent analysis by hplc, the corresponding spectrum showed two major peaks at the retention time of 3.490 and 4.390 min respectively. the heights of the peaks were 0.504 and 1.266 mv, respectively (fig. 6) fourier transform infrared (ft-ir) analysis the eps may contain the functional groups such as alcohol, carboxylic acid and esters, therefore ft-ir analysis of eluted eps was performed. the obtained spectrum showed an o-h stretch at 3600 ‒ 3200 and a c-o stretch between 300 ˗ 1000 cm-1 indicating the presence of alcohol. table 2: details of the peaks obtained from the hplc spectrum of eps isolated from the bacterial strain cms (bacillus subtilis subsp.). peaks r. time [min] area [mv] height [mv] area [%] height [%] wos [min] 1 3.490 193.330 0.504 87.0 28.5 3.76 2 4.390 28.999 1.266 13.0 77.5 0.26 total 222.330 1.770 100.0 100.0 fig. 5. thin layer chromatography analysis of the eps isolated from the strain cms. the sample was placed on silica gel coated plates and the solvent system used was benzene, acetic acid and methanol (1 : 1 : 3). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:16 utc nova biotechnol chim (2018) 17(2): 115-124 121 likewise, a c=o between 1,730 ‒ 1,700, an o-h stretch between 3,400 ‒ 2,400 and a c–o stretch at 1,320 ‒ 1,210 cm-1 indicate the presence of carboxylic acid. besides, a c=o stretch at between 1,740 ‒ 1,715 and a c–o stretch at 1,300 ‒ 100 cm-1 indicates the presence of esters. (fig. 7). it has been reported that the broad peak appeared between 3,600 ‒ 3,200 could indicate the presence of saccharides (iyer et al. 2005) while the peaks appearing between 1,000 – 1,300 could be due to the presence of polysaccharides (biswas et al. 2015). identification and phylogenetic analysis of the strain cms the gram positive bacterium (cms) associated with the surface of gastropod was identified by sequencing the 16s rrna gene. the sequence data of the bacterium cms was related to the database of bacillus subitilis subsp. available in ncbi with 99 % homology. discussion this study showed that bacteria associated with gastropods possess antagonistic activities against the biofilm-forming bacteria. ten out of 21 isolates inhibited the growth of biofilm-forming bacteria on the agar plates. it has been reported that bacteria associated with the surface of marine organisms are more pronounced reservoir of bioactive compounds than the bacteria obtained from other sources (burgess et al. 1999). zheng et al. (2005) also confirmed that at least 20 % of bacteria associated with marine invertebrates were likely to produce antibacterial compounds. there are plenty of studies which demonstrate the bioactivity of bacteria associated with marine invertebrates (shankar et al. 2015). for instance, punitha et al. (2013) reported the bioactivity of bacteria associated with the surface of marine gastropod. microorganisms naturally have the tendency to attach to surfaces which are considered as one of the initial steps leading to biofilm formation (costerton et al. 1987; simoes et al. 2010). hence, preventing initial adhesion process of bacteria would disrupt subsequent biofilm formation. our adhesion assay results showed that the adhesion of biofilm-bacteria on the glass surface was considerably reduced by the eps isolated from the bacterium cms (bacillus subtilis subsp.). generally, bacterial metabolites are reported to possess anti-adhesion or anti-attachment activity against the biofilm-forming bacterial strains. for example, the inhibitory activity of polysaccharide produced by a bacterial species was previously reported by valle et al. (2006). similarly, biofilm inhibitory activities of eps produced by surfaceassociated marine bacteria have been demonstrated by rajasree et al. (2012, 2014) and viju et al. (2016). following adhesion, bacteria produce extracellular polymeric substances which are essential for cellto-cell adhesion (azedero and oliveira 2000) cell aggregation (burdman et al. 2000) and biofilm formation (decho 2000). the eps is mainly composed of polysaccharides (fletcher and floodgate 1973; costerton et al. 1985) and proteins fig. 7. ft-ir analysis of the eps isolated from the strain cms (bacillus subtilis ssp.). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:16 utc nova biotechnol chim (2018) 17(2): 115-124 122 (danielsson et al. 1977). hence, to prevent or reduce the biofilm formation on surfaces, it is necessary to screen the natural products targeting the eps production of bacteria involving in biofilm formation. in this study, eps isolated from the bacteria associated with the gastropod affected the carbohydrate and protein concentration of the eps produced by the biofilm-forming bacteria. this observation showed that the metabolite present in the surface-associated bacterial eps has the potential to reduce the bacterial adhesion on the surface. this observation correlated with a previous study by satheesh et al. (2012) in which the authors reported that the sponge-associated bacterial extract significantly reduced the carbohydrate and protein content of the eps produced by the biofilmforming bacteria. incorporation of bacterial inhibitory compounds into a polymer matrix is a model system representing a method to discover eco-friendly antifouling coating (hellio and yebra 2009). the active compounds could be incorporated into the matrix at a concentration higher than those in the natural environment without altering the physical characteristics of the settlement surface (henrikson and pawlik 1995). previously, armstrong et al. (2000) incorporated the sponge extract into a paint matrix and it was active against barnacle larvae. similarly, in our study the eps of strain cms (bacillus subtilis subsp.) was incorporated into synthetic wood polish (varnish) and tested for antifouling activity in the coastal waters. many previous studies have also reported the antifouling performance of coatings developed with the crude bacterial extracts (satheesh et al. 2012; viju et al. 2014, 2017). however, these studies including the present study were conducted for a short period of field trials. further long-term field studies using the coatings developed with natural products may provide sufficient information on the performance of these compounds in marine waters generally, chromatography methods are used for the separation and purification of the organic compounds (iyyeparaj et al. 2014). in this study, tlc and hplc methods were used for the purification of the eps. the hplc chromatogram revealed the presence of minor and major peaks at retention time of 3.490 and 4.390, respectively. these peaks likely correspond to the polysaccharide units (sugars) such as arabinose (3.63), maltose or galactose (4.50 – 5.00). the presence of these sugars (polysaccharide) has been reported in the hplc spectrum of the exopolysaccharide produced by micromonospora sp. along with respective standards (ledezma et al. 2016). moreover, the ft-ir spectrum showed the presence of polysaccharides in the purified eps with the full stretch appeared between 1,000 – 1,300 (biswas et al. 2015). it has been reported that polysaccharides are the major components of eps (vu et al. 2009) which include homopolysaccharides (α-d-glucan, β-d-glucans, fructans and polygalactan) and heteropolysaccharides (d-glucose, d-galactose, l-rhamnose, n-acetylglucosamine and glucuronic acid) (ruasmadiedo et al. 2002). the production of polysaccharides by wide variety of bacteria have been reported (guo et al. 2010), which is based on the physiochemical factors such as ph, temperature, and medium composition (heumann et al. 1994). saccharides are considered as potential antifouling agents (almedia et al. 2017) since their hydrophilic groups have the ability to form hydrogel when they react with water (piehler et al. 1999). the antifouling activity of hydrogels has been studied extensively and reported ideal antifouling agents (rasmussen et al. 2002). for instance, in a study katsuyama et al. (2002) reported the antifouling activity of polyelectrolyte hydrogels against the spores of seaweed. it is believed that the softness of the hydrogels could be the reason for their antifouling properties, as surface roughness plays major role in biofouling (petronis et al. 2000). however, the exact antifouling mechanism of hydrogels is unclear. the bacterium responsible for the production of eps which showed promising results was identified as bacillus subtilis subsp. with 99 % homology (with the sequences available in ncbi genbank). the genus bacillus is widely distributed in the marine environment and produces a variety of useful products (arias et al. 2003). the species bacillus subtilis is known to produce compounds with biotechnological and biopharmaceutical applications (stain 2005). for instance, satheesh et al. (2012) reported the antifouling activity of bacillus sp. isolated from the surface of sponge bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:16 utc nova biotechnol chim (2018) 17(2): 115-124 123 (sigmadocia sp.). similarly, rajasree et al. (2012) studied the antifouling activity of bacillus sp. isolated from the surface of seaweed sargassum wightii. conclusions the present study demonstrated the antifouling activity of eps produced by the marine bacterium bacillus subtilis subsp. in laboratory as well field conditions. further characterization and confirming the lack of toxicity of the responsible compound in the eps would promote the isolation of novel ecofriendly antifouling agent from the marine bacterium bacillus subtilis subsp. moreover, selecting a suitable binder (matrix) for its incorporation to the antifouling coat, as well as long-term field trials of the applied coating would provide further required knowledge for the development of an effective eco-friendly antifouling coating based on marine bacterial metabolites. acknowledgements the authors would like to thank ugc for providing financial support through the major project scheme (ref. f. no: 39294/2010 sr). references almedia jr, da-silva mc, sousa e, 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extracellular polymeric substance produced by exiguobacterium sp. associated with the polychaete platynereis dumerilii. thalassas 30: 13-19. rasmussen tl, bäckström d, heinemeier j, klitgaardkristensen, d, knutz, pc, kuijpers a, lassen s, thomsen e, troelstra sr, van weering tce (2002) the faeroe – shetland gateway: late quaternary water mass exchange between the nordic seas and the northeastern atlantic. mar. geol. 188: 165-192. ruas-madiedo p, hugenholtz j, zoon p (2002) an overview of the functionality of exopolysaccharides produced by lactic acid bacteria. int. dairy 12: 163-171. satheesh s, soniambi a, shankar cvs, punitha smj (2012) antifouling activities of marine bacteria associated with sponge (sigmadocia sp.). j. ocean univ. china. 11: 354-360. satheesh s, ba-akdah ma, al-sofyani aa (2016) natural antifouling compound production by microbes associated with marine macroorganisms – a review. elect. j. biotechnol. 21: 26-35. shankar cvs, satheesh s, viju n, punitha smj (2015) antibacterial and biofilm inhibitory activities of bacteria associated with polychaetes. j. coast. life. med. 3: 495-502. simones sm, blankenship jt, weitz o, farrell dl, tamada m, fernandez-gonzales r, zallen ja (2010) rhokinase directs bazooka/par-3 planar polarity during drosophila axis elongation. develop. cell. 19: 377-388. stein t (2005) bacillus subtilis antibiotics: structures, synthesis, and specific functions. mol. microbiol. 56: 845-857. thomas kv, brooks s (2010) the environmental fate and effects of antifouling paint biocides. biofouling 26: 73-88. valle j, da re s, henry n, fontaine t, balestrino d, latourlambert p, ghigo jm (2006) broad spectrum biofilm inhibition by a secreted bacterial polysaccharide. proc. natl acad. sci u. s.a. 103: 12558-12563. viju n, satheesh s, vincent sgp (2013) antibiofilm activity of coconut (cocos nucifera linn.) husk fibre extract, saudi j. biol. sci. 20: 85-91. viju n, anitha a, vini ss, shankar cvs, satheesh s, punitha smj (2014) antibiofilm activities of extracellular polymeric substances produced by bacterial symbionts of seaweeds. ind. j. geomar. sci. 43: 2136-2146. viju n, satheesh s, punitha smj (2016) antibiofilm and antifouling activities of extracellular polymeric substances isolated from the bacteria associated with marine gastropod turbo sp. oceanol. hydrobiol. st. 45: 11-19. viju n, satheesh s, punitha smj (2017) antifouling activities of antagonistic marine bacterium pseudomonas putida associated with an octopus. proc. natl. acad. sci., india, sect. b. biol. sci. 87: 1113-1124. vu b, chen m, crawford rj, ivanova ap (2009) bacterial extracellular polysaccharides involved in biofilm formation. molecules 14: 2535-2554. xu y, he h, schultz s, liu x, fusetani n, xiong h, xiao x, qian py (2010) potent antifouling compounds produced by marine streptomyces. bioresour. technol. 10: 1331-1336. zheng l, han x, chen h, lin w, yan lx (2005) marine bacteria associated with marine macroorganisms: the potential antimicrobial resources. ann. microbiol. 55: 119-124. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:16 utc https://www.ncbi.nlm.nih.gov/pubmed/?term=thomas%20kv%5bauthor%5d&cauthor=true&cauthor_uid=20390558 https://www.ncbi.nlm.nih.gov/pubmed/?term=brooks%20s%5bauthor%5d&cauthor=true&cauthor_uid=20390558 https://www.ncbi.nlm.nih.gov/pubmed/20390558 nova biotechnol chim (2019) 18(1): 1-9 doi: 10.2478/nbec-2019-0001  corresponding author: golovatyuk.yevgeniya@gmail.com nova biotechnologica et chimica phytotoxicity of colloidal solutions of stabilized and non-stabilized nanoparticles of essential metals and their oxides yevheniia konotop, kseniia stepanchenko, leila-anastasiia karpets, andrii zinchenko, mariia kovalenko, oleksandr smirnov, ludmila batsmanova and nataliya taran educational and scientific center, institute of biology and medicine, taras shevchenko national university of kyiv, 64/13 volodymyrska str., kyiv, 01601,ukraine article info article history: received: 18th january 2019 accepted: 14th april 2019 keywords: allium cepa (l.) root meristem mitosis chromosomal aberrations metal nanoparticles stabilized nanoparticles phytotoxicity abstract advances in nanotechnology in various fields of human activity contribute to increase of their production, improved properties and wider implementation of nanomaterials. however, increasing use may enhance their release into the environment and can lead to affecting human health. the toxicity of colloidal solutions of metal nanoparticles (cu, mn) and their oxides, obtained in the absence and presence of a stabilizer, was examined and compared with the use of the standard test system of allium cepa l.. the phytotoxicity of the experimental solutions was evaluated according to the growth response of the onion roots; the cytoand genotoxicity were estimated due to the proliferative activity of the root meristem cells. it was established that solutions of stabilized metal nanoparticles were at given concentration toxic to allium cepa l. according to the integral index of roots growth, however, were not cytotoxic. difference in the phytotoxicity of stabilized and non-stabilized metal nanoparticles and their oxides depended on their phase composition and affected root growth.  university of ss. cyril and methodius in trnava introduction advances of nanotechnology in various fields of human activity contributed to increased production and wider implementation of nanomaterials (scrinis and lyons 2007; rui et al. 2016; ditta and arshad 2016; ruttkay-nedecky et al. 2017). recent evidence suggests the efficiency of metal nanoparticles (nps) and their oxides in reducing the toxic effects of adverse stressors on crops through the activation of their protective systems, which is a good prerequisite for wider use of nanomaterials in the agriculture as fertilizers or plant protection factors (abdel latef et al. 2016; saxena et al. 2016; taran et al. 2016; konate et al. 2017). however, increased use of nanomaterials may enhance their release into the environment as well (e.g. during their synthesis, incorporation into goods, reaching wastewaters and soil etc.) and affect human health. plants, as components of ecosystems, play a significant role in accumulation and circulation of nano-sized products in nutrition chains. the potential impact of nps on plants, however, requires thorough study, since available data on their toxicity (in comparison to ionic forms or bulk material) are highly controversial (griffitt et al. 2008; ma et al. 2013; konotop et al. 2014; liu et al. 2016). the direct toxicity of metal nps and their oxides depends on the chemical composition of the raw material (for example, due to the release of toxic ions), the physical bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:46 utc mailto:golovatyuk.yevgeniya@gmail.com nova biotechnol chim (2019) 18(1): 1-9 2 table 1. characteristics of experimental colloidal solutions of nanoparticles of essential metals obtained by using the x-ray crystallography. sample size [nm] phase content lattice parameter [nm] phase mass portion [%] a b c trisodium citrate – trisodium citrate 100.00 12.817 5.625 11.484 non-stabilized mn 53 mn 97.84 4.793 4.793 15.627 64 mno 2.16 4.440 4.440 14.453 non-stabilized cu 78 cu 91.55 4.266 4.266 12.393 82 cu2o 8.45 3.617 3.617 10.724 stabilized mn – trisodium citrate 4.24 12.807 5.521 11.477 54 trisodium citrate-mn 94.76 5.745 5.745 12.836 stabilized cu – trisodium citrate 6.75 12.805 5.614 11.460 75 trisodium citrate-cu 93.25 6.836 6.836 11.957 a, b, and c – lattice parameters in three dimensions. lattice parameter refers to the physical dimension of unit cells in a crystal lattice characteristics of nps (size, shape, surface structure), and/or the ability to generate reactive oxygen species in plant tissues (navarro et al. 2008; auffan et al. 2009; horie et al. 2012; fu et al. 2014; tang et al. 2016). in addition, the toxicity of nps to plants, so called phytotoxicity, which we refer to in this article as the toxicity of nps to plants, is associated with a disturbance of nutrient uptake by roots owing to alteration of surface composition of the rhizosphere in the presence of nps (lee et al. 2012; mirzajani et al. 2013). similar effects on test objects were observed for high concentrations of nanoparticles in the nutrient medium (navarro et al. 2008; gottschalk et al. 2013). the results of some other studies are ambiguous, since the toxicity of nps does not always exceed the toxicity of bulk chemical species containing the same elements (liu et al. 2016), or depends on the metal on the basis of which the colloidal solutions were prepared. nps of essential metals, namely of cu and mn, phytotoxicity of which is researched in the present work, have already been widely used in crop production as antimicrobial agents (cu, cuo nps), or as fertilizers alternative to conventional salts (mn nps) (dimkpa et al. 2011; liu et al. 2016). it is known that the surface of nps of many metals possesses elevated chemical activity and instantly interacts with oxygen, water and organic compounds. therefore, nps in colloidal solutions require additional stabilization, which depends on the concentration of the metal phase, the chemical composition of the medium, the method of their obtaining, and the presence of complexing ligands. the lack of stabilizing agents could lead to the formation of large aggregates that are gradually oxidized (sperling and parak 2010). among stabilizers for metal nps are used e.g. ligand-complexing agents (ammonia, ethylene diamine tetraacetic acid, glycerol), surfactants (among others sodium citrate) and polymers (polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol). a common example is gold nanoparticles synthesized in an aqueous solution by reducing citrate (lin et al. 2004). the resulting nps adsorb negatively charged citrate ions on their surface and thus are stabilized due to electrostatic repulsion. the presence of stabilizing agents on the surface of nps provides them new properties, which in turn predetermine the toxicity of nanomaterials (cunningham et al. 2013; sharma et al. 2014). so, search for ways to improve the physical and chemical properties of nanomaterials is still in progress, at the same time, careful control of nanoproducts for environment safety with the use of bioassays is also needed (olkhovych et al. 2016; teplicky et al. 2018). onion, allium cepa l., belongs to the most common plant species used to assess environmental contamination. it is considered as a short-term, simple in use and sensitive test model, with reliable correlation to other test systems, including human. the macroscopic symptoms of phytotoxicity – bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:46 utc https://en.wikipedia.org/wiki/unit_cell https://en.wikipedia.org/wiki/crystal_lattice https://en.wikipedia.org/wiki/crystal_lattice nova biotechnol chim (2019) 18(1): 1-9 3 stunting of root growth, delayed development, or even death of cells/plant, represent the most sensitive test parameters. microscopic examination allows for assessing proliferative activity of root meristem cells, chromosome damage and cell division disorders, and, therefore, provides additional information on the severity of toxic effect, mechanism of genotoxicity, or potential mutagenicity of tested agents (fiskesjо 1985). therefore, the purpose of this work was to test and compare the phytotoxicity of developed colloidal solutions of essential metals nps (mn and cu) and their oxides, obtained in the absence and presence of a stabilizer, using the standard allium cepa l. test system. experimental materials colloidal solutions of stabilized and non-stabilized cu and mn nps and their oxides were developed and kindly provided by the department of technology of structural materials and materials science, national university of life and environmental sciences of ukraine, and obtained by dispersing granules of copper and manganese by pulses of electric current with amplitude 100 – 2000 a in water (lopatko 2009). concentrations of mn and cu nps in stock solutions were 3 and 4 mg.l-1, respectively. 100-fold diluted solutions were used. characteristics of the colloidal solutions of nps are given in the table 1. plant material and growth conditions the phytotoxicity of solutions of stabilized and non-stabilized cu and mn nps were studied using the standard test object allium cepa l. (fiskesjо 1985). for each experimental variant 10 equal onion bulbs, cv. chalcedony, were cultivated on experimental suspensions of nps for 5 days at 24 °c. control plants were grown on distilled water. to eliminate the effect of the stabilizer, the plants were additionally cultivated in aqueous solution of trisodium citrate in a concentration that corresponded to that in the experimental solutions of stabilized nps of essential metals, which is a 100-fold diluted stock solution (2 g.l-1) of trisodium citrate. estimation of growth response at the end of the exposure time the fresh weight, length and quantity of roots per bulb were determined. growth response was estimated fig. 1. root meristem cells of allium cepa l. in the different phases of mitosis on the 5th day of cultivation (magnification 400x). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:46 utc nova biotechnol chim (2019) 18(1): 1-9 4 according to the tolerance index (it, %) calculated as the ratio of the fresh weight of roots of experimental group to the same parameter of control group (wilkins 1978). microscopic technique onion roots were fixed in clark solution (ethanol: acetic acid = 3 : 1), macerated in 1 m hcl at the temperature 45 °c, and stained with 1 % (w/v) aqueous solution of toluidine blue. temporary oppressed slides with root’s tips were prepared and further analyzed under a light microscope (primostar, carl zeiss) at 400x magnification. cytoand genotoxicity estimation different phases of mitosis in cells of the root apical meristem were counted to calculate mitotic/phase indices, and mitosis abnormalities (fig. 1). presence of micronuclei was observed for evaluation of cytoand genotoxic effects of the experimental solutions (fiskesjo 1997). the mitotic index (mi, %) was presented as number of cells in the state of division to the total number of observed cells (3000) (eq.1): мі = × 100 %, (1) where p, m, a, t, i are quantities of cells in pro-, meta-, ana-, telo-, and interphase of mitosis, respectively. phase indices (%) were calculated as number of cells on certain mitosis phase (p, m, a, t) to the total number of divided cells. for example, prophase index (pi, %) (eq. 2): pі = × 100 % (2) statistical analysis each experiment was performed at least in triplicate. the data were subjected to analysis of variance (anova) with subsequent student's t-test or duncan's multiple range test. data are expressed as means of replicates + standard deviation, and were considered as significant at p < 0.05. fig. 2. growth response of allium cepa l. on 5th day of cultivation on colloidal solutions of non-stabilized (mn, cu) and stabilized (mn+, cu+) nanoparticles of essential metals and their oxides. results growth response the obtained results testify that the colloidal solutions of stabilized and non-stabilized nps of essential metals and their oxides do influence the growth of the roots of allium cepa l. (fig. 2). morphometric indices of onion roots under the cultivation on experimental solutions are summarized in table 2. the solution of mn nps contributed to the roots growth by 52 %, and cu nps, on the contrary, caused stunting by 49 % (fig. 3). fig. 3. index of tolerance (it, %) of allium cepa l., calculated by fresh weight of roots, on 5th day of cultivation on colloidal solutions of non-stabilized (-citrate; black columns) and stabilized (+citrate; grey columns) nanoparticles of essential metals and their oxides. data represent mean values with standard deviation of 3 replicates. means followed by the same letters were not significantly different at p < 0.05 according to the duncan’s multiple range test. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:46 utc nova biotechnol chim (2019) 18(1): 1-9 5 table 2. morphometric parameters of allium cepa l. roots under cultivation on colloidal solutions of stabilized and nonstabilized nanoparticles of essential metals and their oxides. experimental variant fresh weight of roots per 1 bulb [g] length [mm] quantity of roots per 1 bulb control h2o distilled 0.09+0.03 19.55+2.42 12.63+5.20 trisodium citrate 0.09+0.04 19.90+3.63 16.70+5.98 mn non-stabilized 0.13+0.04* 26.38+4.98** 14.50+5.42 stabilized 0.02+0.02*** 7.40+4.12*** 6.00+3.33*** cu non-stabilized 0.03+0.02*** 5.00+1.25*** 10.90+5.32 stabilized 0.006+0.004*** 3.50+1.05*** 2.51+0.83*** data represent mean values with standard deviation of 3 replicates. asterisks indicate the significance of difference at the level *р ≤ 0,05; **р ≤ 0,01; ***р ≤ 0,001 according to student’s t-test. the presence of the stabilizer in the colloidal solutions of nps suppressed strongly the roots growth by 55 and 81 % (according to the it values) for mn and cu nps, respectively. besides, only the stabilized nps limited the appearance of new roots in the experimental bulbs (table 2). it of plants grown on a solution of trisodium citrate did not differ significantly from the control values. cytoand genotoxicity to understand the causes of inhibition / stimulation of plant growth under the impact of nps of essential metals, we conducted microscopic investigation of a. cepa l. root meristem cells. effect of colloidal solutions of stabilized and nonstabilized nps on the root cells division is shown in fig. 4. the cytotoxic effect was noted for all of the experimental solutions, except for the solutions fig. 4. effect of colloidal solutions of non-stabilized (-citrate; black columns) and stabilized (+citrate; gray columns) nanoparticles of essential metals and their oxides on proliferative activity (mi, %) of root meristem cells of allium cepa l.. data represent mean values with standard deviation of 5 replicates. of trisodium citrate and mn nps. in the latter case the mi of root meristem cells did not significantly differ from the control. the most reduced proliferative activity of cells of root apical meristem (by 68 %) was observed in plants grown on solutions of non-stabilized cu nps, that correlates with tolerance indices of the same experimental variant. solutions of stabilized nps of both metals equally inhibited the division of root cells, by 18 and 14 % for mn and cu nps, respectively. proportion of mitotic phases (mainly meta-, ana and telophases) in mi value changed in a. cepa root cells that were exposed to solutions of the nonstabilized cu nps as well as to stabilized cu and mn nps (fig. 5). treatment with solutions of stabilized nps caused accumulation of cells in telophases, that led to inhibition of divisions. along with increasing number of cells in ana and telophases as result of treatment with nonstabilized cu nps, the number of cells in metaphase was reduced, that revealed crucial for proliferative activity of root meristem. as shown in the representative microscopic images (fig. 6), the majority of chromosome aberrations were recorded in anaphase for almost all experimental variants: breakes, bridges, vagrant chromosomes and spindle abnormalities. disturbed metaphase, with unorganized chromosomes on the equatorial plate, also known as c-metaphase, was observed in the root cells treated with nonstabilized mn nps (fig. 5 e, f, g). no aberrations were identified in the cells exposed to nonstabilized cu nps, probably due to the lowest number of divided cells (according to mi values). however, besides typical stages of mitosis we also bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:46 utc nova biotechnol chim (2019) 18(1): 1-9 6 observed bridges in anaphase in the control root cells. micronuclei were not detected in root cells in either of the experimental variants. discussion the nps of mn and cu for this study were chosen because these trace elements are essential for plants. they are components of active enzymes centres and participate in important processes such as photosynthesis, respiration etc. recently, mn nps were suggested as an alternative to traditional fertilizers, since their revealed positive effect on the root and shoot growth and increased the productivity of crops. moreover, they did not cause oxidative damages in plant tissues (pradhan et al. 2013; liu et al. 2016). despite the fact that cu nps are part of many commercial antimicrobial formulations, their potential phytotoxicity is still being studied. in the present research the onion root growth was dependent on the type of metal nps in the culture medium, as has been shown previously (konotop et al. 2014). similarly to liu et al. (2016) and trujillo-reyes et al. (2014), both cu and mn nps showed inhibitory and stimulating effect on root growth, respectively. it has been shown that toxicity of cu nps and its oxides is connected to oxidative damage of root tissue in arabidopsis, while the cu nps contributed much stronger to activation of oxidative stress-related genes than the corresponding cu2+ ions (tang et al. 2016). since the mitotic activity of the root meristem was not affected by solutions of non-stabilized mn nps, enhanced root growth could be assumed with involving the compensatory mechanisms, namely intensive cell elongation (table 2). on the other hand, mito-depressive effect was notable in plant cells treated with non-stabilized cu nps. according to pi values, smaller number of dividing cells in metaphase was detected along with abundant prophase stage cells, which is associated with the terminating of mitosis at the end of the prophase and delay in passing through the next cell division stages (livanos et al. 2012). to less extent, such an effect was also observed in onion root cells treated with colloidal solutions of stabilized both mn and cu nps. although separate cases of mitotic aberrations were recorded, we can not confirm the genotoxicity of studied nps solutions in researched concentrations, because the frequency of observed chromosomal disturbances was at the control level. while the genotoxic effect has already been proven for some nano-sized materials including cu nps (kumari et al. 2009; 2011; pakrashi et al. 2014; nagaonkar et al. 2015), there is scarce information concerning mn nps-induced genotoxicity. the values of it, mi and pi indicators of plants grown on the solution of trisodium citrate did not differ significantly from the control values. in light of this, the obtained data allow to speculate that stabilized nps acquire novel properties or functional qualities compared to non-stabilized ones, as has been reported previously (cunningham et al. 2013; sharma et al. 2014). it could mainly be connected with altered phase composition of tested solutions, rather than with the size of metal particles. fig. 5. phase indices (%) in allium cepa l. root meristem cells exposed to colloidal solutions of non-stabilized (mn, cu) and stabilized (mn+, cu+) nanoparticles of essential metals and their oxides. pi, mi, ai, ti – pro-, meta-, anaand telophase indices respectively. data represent mean values with standard deviation of 5 replicates. asterisks indicate the significance of difference at the level *р ≤ 0.05; **р ≤ 0.01; ***р ≤ 0.001 according to student’s t-test. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:46 utc nova biotechnol chim (2019) 18(1): 1-9 7 despite of unaffected mitotic activity of cells, there was a significant delay in root growth (it) as likely consequence of the terminated root elongation. the cell expansion can be explained by the “acid growth theory”, which postulates auxin-induced activation of plasma membrane-localized h+-atpase that acidifies the apoplast (rayle and cleland 1992). thus, an electrochemical gradient for absorbing k+ is provided, which is necessary to increase the cell turgor and further strain. recently, the auxin-dependent growth mechanism was also confirmed for root cells: high auxin concentrations suppressed, while low stimulated their elongation (barbez et al. 2017). moreover, it was reported that cuo nps influenced the metabolism of cotton plants through hormonal imbalance, namely auxins (van et al. 2016). owing to the greater correlation between surface area and mass compared to bulk metals, nps have high reactivity, can form complexes with membrane transporters and, thus, be taken up by plant (liu et al. 2016). in this way, stabilized nps with different properties compared to non-stabilized, might directly or indirectly affect elongation of root cells, as well as the emergence of new root growth points. conclusions the phytotoxicity of the colloidal solutions of essential metal nps and their oxides obtained in the presence and absence of a stabilizer was tested and compared. the solutions of stabilized mn and cu nps retarded the growth and development of allium cepa l. roots, although the proliferative activity of root apical meristem remained unaffected and genotoxicity was not confirmed. in contrast, non-stabilised cu nps caused significant mito-depressive effect. it can be assumed that phytotoxicity of colloidal solutions of nps of essential metals and their oxides depends on their properties, mainly phase composition altered by applied stabilizers; the mechanisms of their action on root growth, however, remains unclear. the wider implementation of nano-sized products in different human practices requires further research and careful control in regard of the impact on environment. acknowledgement this work was supported by the president’s of ukraine grant for competitive projects of the state fund for fundamental research (contract no. ф75/170–2018 from 20.09.2018). we would like to thank dr. m.v. karpets from national fig. 6. aberrations induced by colloidal solutions of nonstabilized and stabilized nano-particles of essential metals and their oxides in root meristem cells of allium cepa l.: a – control, b – trisodium citrate, c–h – non-stabilized mn nanoparticles, i–k – stabilized mn nanoparticles, l – stabilized cu nanoparticles. types of observed aberrations: anaphase with bridge (a, d, l); anaphase with breakes (c); spindle abnormalities in ana-phase (b, i, l); c-metaphase (e, f, g); vagrant chromosome in ana-phase (h, i, j, k). magnification 400x. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:46 utc nova biotechnol chim (2019) 18(1): 1-9 8 technical university of ukraine “igor sikorsky kyiv polytechnic institute” for the x-ray investigation of colloidal solutions of stabilized / non-stabilized cuand mn-based nps. conflict of interest the authors declare that they have no conflict of 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hazard? j. hazard. mater. 267: 255-263. van nl, ma c, shang j, rui y, liu s, xing b (2016) effects of cuo nanoparticles on insecticidal activity and phytotoxicity in conventional and transgenic cotton. chemosphere. 144: 661-670. wilkins da (1978) the measurement of tolerance to edaphic factors by means of root length. new phytol. 80: 623633. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:46 utc microsoft word partelova et al 2015_nbec_final.doc 212 nova biotechnologica et chimica 14-2 (2015) doi 10.1515/nbec-2015-0028 © university of ss. cyril and methodius in trnava removal of contaminants from aqueous solutions using hop (humulus lupulus l.) agricultural by-products denisa partelová, anna šuňovská, jana marešová, miroslav horník, martin pipíška, stanislav hostin department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic (hornik@ucm.sk) abstract: agricultural wastes can be used as an alternative to the existing sorbents for the removal of metals or synthetic dyes from contaminated liquids. in this work, the fine powdered biomass of the hop (humulus lupulus l.) variety osvald's clone 72 and variety bohemie as a sorbent for the removal of cd from aqueous solutions of cdcl2 spiked with radionuclide 109cd and synthetic dyes thioflavine t (tht) or methylene blue (mb) from single dye solutions under conditions of batch systems was used. the maximum sorption capacity q = 264 µmol cd/g (d.w.) was found in the case of the leaf biomass of hop (h. lupulus l.) variety osvald's clone 72 at the initial concentration of cdcl2 10,000 µmol/dm3, whereby the sorption capacity decreased in the order qleaves : qstems : qroots = 1.0 : 0.8 : 0.7. the sorbed amount of cd was removed from the hop biomass with the following increasing desorption efficiency of the extraction reagents: deionised h2o << 0.1 mol/dm3 hcl ≤ 0.1 mol/dm3 edta-na2. similarly as in the case of cd sorption, the kinetics of tht and mb sorption by the leaf biomass of the hop (h. lupulus l.) variety bohemie were also showed as two-phase processes. the maximum sorption of tht approx. q = 19 mg/g (d.w.) and mb approx. q = 70 mg/g (d.w.) were found within the range of the initial values of ph 4 – 7. the sorption of both dyes by the leaf biomass from single dye solutions decreased with increasing biomass concentration and on the other hand increased with increasing the initial concentrations of tht or mb. the process of tht and mb sorption was better described by the langmuir model than the freundlich model of sorption isotherm. from the obtained values of qmax, it was found that in the case of mb the dried leaf biomass showed more than 2-times higher sorption capacity (qmax = 184 mg/g; d.w.) in comparison with the value predicted for tht. obtained results suggest that dried plant biomass of hop (h. lupulus l.) as agricultural by-products can be used as a potential sorbent for both types of studied contaminants. key words: cadmium, synthetic dyes, sorption, hop, agricultural by-products, sorbents 1. introduction in recent decades, the growth of industrial activities and the increasing water usage worldwide have led to the release of various pollutants into an aquatic environment, such as heavy metals or organic compounds represented by phenols, dyes, pesticides, humic substances, detergents, and other persistent organic pollutants (abdolali et al., 2014). heavy metals, such as cadmium, often presented in industrial wastewaters are hazardous to the aquatic ecosystem and pose possible human health risk. cadmium (cd) makes its way to water bodies through the wastewaters from metal plating industries, industries of cd-ni batteries, phosphate fertilizer, mining, pigments, stabilizers and alloys (low and lee, 1991). cd is listed as one of the top toxic bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc nova biotechnologica et chimica 14-2 (2015) 213 metals, since it causes carcinogenic and renal disturbances, lung insufficiency, bone lesions, cancer, anaemia, hypertension, itaï-itaï disease and weight loss (ding et al., 2012). epa requires water suppliers to limit the concentration in drinking water to < 5 μg/dm3 for cd (epa, 2011). council directive 83/513/eec of 26 september 1983 on limit values and quality objectives for cadmium discharges (cec, 1983) determines the limit values for industrial discharges to be 0.2 mg of cd per litre of discharge. according to epa wastewater treatment manuals, cd in concentrations of 1.0 – 10.0 mg/dm3 can significantly inhibit positive biological activity of activated sludge in the wastewater treatment process. many industries, such as textile industry, used dyes to colour their products and thus produce wastewaters containing organic compounds with a strong colour, because within the dyeing processes the percentage of dyes lost in wastewaters is approx. 50 % of the dyes due to the low levels of dye-fibre fixation (mohan et al., 2007; salleh et al., 2011). large amounts of dye contaminated wastewaters are being released yearly from the industry of leather, cosmetics, pharmaceutical, and plastics, and results in an impending hazard to human health and the ecosystem (cao et al., 2014; semeraro et al., 2015). besides dyes, such effluents also contain a number of other contaminants, such as alkalis, acids, electrolytes, heavy metal ions, dissolved and other suspended solids etc. (safa and bhatti, 2011). today, there are more than 10,000 dyes commercially available. dyes have a complex aromatic molecular structure and are generally resistant to light, temperature, and oxidizers. these characteristic features make the dyes non-degradable and therefore causes bioaccumulation in living organisms, leading to severe diseases and disorders (banerjee and chattopadhyaya, 2013). dyes can affect aquatic plants because they reduce the sunlight transmission through the water as well as may impart toxicity to aquatic life and may be mutagenic, carcinogenic and may cause severe damage to human beings, such as dysfunction of the kidneys, reproductive system, liver, brain, and central nervous system (adegoke and bello, 2015). thus, the removal of heavy metals and dyes as xenobiotics from polluted waters is of great importance from an environmental and industrial point of view. the most widely used methods for removal these contaminants from wastewaters include ionexchange, chemical precipitation, reverse osmosis, evaporation, membrane filtration, adsorption, biological treatment, coagulation, flocculation, electrochemical treatment, electrodialysis, photo-catalysis, and photo-oxidation (rangabhashiyam et al., 2014; oguntimein, 2015). but major drawbacks of these technologies are long operation time, low specificity, high cost, and eco-unfriendliness. biological systems have the availability to accumulate specific metals and compounds from wastewaters. this process is called bioremediation. one type of bioremediation is phytofiltration, which uses dead tissues from plants to remove contaminants from waters (lópez et al., 2005). it is based on (bio)sorption processes. the cost of such sorption technology application can be reduced, if the sorbent is inexpensive. so, there is a constant search for alternate low-cost sorbents (ghasemi et al., 2014). the limitations which considered when choosing suitable sorbents are: the sorption and regeneration abilities, market availability, and kinetic parameters. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc 214 partelová, d. et al. sorption capacity parameter is very important for estimation of process costs. regeneration of the sorbent is also important in cyclic processes when the used sorbents are expensive (sadeek et al., 2015). natural materials that are available in large quantities, or certain waste products originated from industrial or agricultural operations may have the potential as inexpensive sorbents. agricultural wastes – plant residues – are renewable and available abundantly at no or low cost (šćiban et al., 2008). agricultural wastes can be used as an alternative to the existing conventional technologies as the sorbents for the removal of metals or xenobiotics (synthetic dyes) from aqueous solutions. several studies characterized the sorption properties of waste materials derived from plants, such as hop (lópez et al., 2005), rice (wu et al., 2016), maize (garcíarosales and colín-cruz, 2010), sunflower (oguntimein, 2015), wheat (gorgievski et al., 2013), sugar beet (aksu and i̇şoğlu, 2005), and tobacco (qi and aldrich, 2008) biomass. also, some papers suggest on the utilization of physical or chemical approaches for the modification of waste plant biomasses or techniques for production of biochar with the aim to increase their sorption capabilities (pellera et al., 2012; zafar et al., 2015; wang et al., 2016). our previous papers deal with the study of sorption processes and characterisation of sorption capabilities of lichen, moss or algae biomass to bind metals or synthetic dyes (pipíška et al., 2007; marešová et al., 2011; horník et al., 2013; šuňovská et al., 2015). the aim of this work was to evaluate the capability of fine powdered hop (humulus lupulus l.) biomass as an agricultural by-product to removal of cd or synthetic dyes thioflavine t (tht) and methylene blue (mb) from aqueous solutions. also, the effects of time, metal or dye concentration, amount of hop biomass, and ph on sorption processes were studied. for these purposes, a radionuclide 109cd with gamma-spectrometry and uv-vis spectrophotometry as detection methods and approaches were applied. 2. materials and methods 2.1 hop biomass in the experiments, the plants of hop (humulus lupulus l.) variety osvald's clone 72, genotype k-72/6/13 obtained from plant production research institute in piešťany (slovak republic) and leaf biomass of hop (h. lupulus l.) variety bohemie obtained from hop research institute co., ltd., žatec (czech republic) were used. leaves, stems, and roots of variety osvald's clone 72 and leaves in the case of variety bohemie were dried 3 days at 60 °c, ground and sieved for a particle size < 0.45 mm (variety osvald's clone 72) or between < 0.63 mm and > 0.31 mm (variety bohemie). 2.2 sorption experiments all solutions of cdcl2 (analytical grade; cas 10108-64-2; sigma-aldrich, usa) or synthetic dyes thioflavine t (tht; c.i. 49005; mr 318.86; cas 2390-54-7; fluka, usa) and methylene blue (mb; c.i. 52015; mr 319.85; cas 61-73-4; fluka, usa) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc nova biotechnologica et chimica 14-2 (2015) 215 were prepared in deionised water. in the case of cdcl2, solutions were spiked with a standardized 109cdcl2 solution. the initial ph of experimental solutions was adjusted with 1 mol/dm3 hcl or 1 mol/dm3 naoh solutions. dried biomass of hop was transferred into a 100 cm3 erlenmeyer flasks with 20 cm3 of mentioned experimental solutions containing defined initial concentrations of cdcl2, tht or mb. the exposure was carried out on a rotary incubation shaker (250 rpm) at 25 °c. in defined time intervals mixture was centrifuged (10 min at 5 000 min-1), supernatant was removed and analysed from the point of view of remaining concentration of cdcl2 through the scintillation γ-spectrometry or remaining concentration of tht or mb dyes by uv-vis spectrophotometry. from the obtained primary data, the specific adsorption q (mg of metal/dye sorbed per g of dried hop biomass) was calculated using the following equation (eq. 1): m v ccq t )( 0 −= (1) where q is the amount of metal/dye sorbed onto hop biomass (mg/g; d.w.), c0 and ct represent the initial concentration of metal/dye in solution and the concentration of metal/dye at the end of the experiment (mg/dm3), respectively, and v and m are the volume of solution (dm3) and weight of hop biomass (g; d.w.) in the experiments. in desorption experiments, the sediment of hop biomass after cd sorption was resuspended by wortexing in deionised water or 0.1 mol/dm3 edta-na2 or hcl solutions. the 109cd radioactivity leached from the hop biomass into the supernatant was determined by similar way as in the sorption experiments and the desorption efficiency deff was calculated using the following equation (eq. 2): 100* b d eff a a d = (2) where deff is the desorption efficiency (in %), ad is the 109cd radioactivity released from the hop biomass into the supernatant (in bq) and ab is the 109cd radioactivity sorbed onto the hop biomass before carrying out the desorption experiment. the sorption equilibrium data were described by sorption isotherms according to the langmuir (eq. 3; langmuir, 1918) and freundlich (eq. 4; freundlich, 1906). the non-linear forms of these mathematical models are as follows: eq eq eq cb cqb q *1 ** max + = (3) )/1(* neqeq ckq = (4) where ceq is the equilibrium concentration of the dye in the solution (mg/dm 3), qeq is the equilibrium specific sorption of dye onto the dried hop biomass (mg/g; d.w.), qmax is the maximum sorption capacity of the dried hop biomass (mg/g; d.w.), b is the langmuir equilibrium constant characterizing the affinity between the dye and the biomass of the hop (dm3/mg), k is the freundlich equilibrium constant related to sorption capacity of the hop biomass (dm3/g of biomass), n is the freundlich equilibrium constant related to intensity of the sorption (non-dimensional). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc 216 partelová, d. et al. 2.3 scintillation γ-spectrometry standardized solution of 109cd in the form of 109cdcl2 (3.94 mbq/cm 3, 50 mg/dm3 cdcl2 in 3 g/dm 3 hcl) was obtained from the czech institute of metrology (prague, czech republic). a gamma-spectrometric assembly using the well type scintillation detector nai(tl), 54bp54/2-x (scionix, netherlands) and the data processing software scintivision-32 (ortec, usa) were used for 109cd determination in the supernatants and sediment of hop biomass. a library of radionuclides was built by selecting characteristic γ-ray peaks (88.04 kev for 109cd; 661.66 kev for 137cs; 1,115.52 kev for 65zn and 1,173.24 kev for 60co) for the energy and efficiency calibration. counting time 600 s allowed obtaining data with measurement error < 2 %, which do not reflect other source of errors. 2.4 uv-vis spectrophotometry a uv-vis spectrophotometer cary 50 (varian, australia) was used for establishing calibration curves for tht and mb dyes at maximum absorbances λtht = 412 nm and λmb = 650 nm and for determination of remaining concentrations of tht or mb dyes in solutions. the influence of ph value on the analysed absorbances was taken into account. 2.5 speciation modelling the prediction of cd speciation for the synthetic wastewater as a function of ph, temperature, ionic strength, and concentrations of cations and anions was calculated using the modelling software mineql+ ver. 4.62 (environmental research software, usa). the composition of synthetic wastewater according to iaquinta et al. (2006) (ppm): 455 – cl-; 87 – so4 2-; 418 – na+ ; 78 – k+; 45 – mg2+; 138 – ca2+. 2.6 statistical analysis all analytical determinations were performed in triplicate. statistical analysis, nonlinear regression and graphical interpretation of the obtained data were carried out using programs originpro ver. 8.5 (originlab corp., usa), systat ver. 13 (systat software inc., usa), and sigmaplot ver. 12 (systat software inc., usa). 3. results and discussion 3.1 sorption of cd by hop biomass kinetics of cd sorption by dried and homogenized biomass of leaves, stems, and roots (particle size < 0.45 mm) of hop (h. lupulus l.) variety osvald's clone 72 under conditions of batch systems was showed as a two-phase process. the first, fast bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc nova biotechnologica et chimica 14-2 (2015) 217 phase can be explained as the sorption of cd2+ cations on the hop biomass surface. the second, more slowly phase lasting approx. 30 min can be attributed to the swelling of dried biomass and relating diffusion processes from the outside into the inside of sorbent particles (data not shown). the maximum sorption capacity q = 264 µmol cd/g (d.w.) was found in the case of the leaf biomass of hop at the initial concentration of cdcl2 10,000 µmol/dm 3 (table 1). generally, cadmium sorption by hop biomass increased with increasing initial cd concentration in the solution from 10 to 10,000 µmol/dm3 of cdcl2 (fig. 1). in the case of the leaf biomass, the linear dependence (r2 = 0.999) between the sorption q and the initial concentration of cdcl2 was observed. it means that neither at the highest applied initial cd concentration c0 = 10,000 µmol/dm 3 cdcl2 the leaf biomass of hop was not saturated by the studied sorbate (cd2+ ions). mentioned linear dependence was not found for the data describing the sorption of cd by stem and root biomass of hop under identical conditions (fig. 1). thus, the biomass of stem and root of hop showed a partial saturation with cd2+ cations at the highest applied initial cd concentrations. table 1. desorption of cd (%) from leaf, stem, and root biomass of hop (h. lupulus l.) variety osvald's clone 72 after 30 min by resuspending of biomass sediment originated from sorption experiments with deionised water (5.0 cm3/0.05 g; d.w.), 0.1 mol/dm3 edta-na2 (1.0 cm3/0.05; g d.w.) or 0.1 mol/dm3 hcl (1.0 cm3/0.05; g d.w.) at 25 °c. sorption experiment* desorption deff [%] biomass c0 cdcl2 [μmol/dm3] sorption q [μmol/g]; d.w. deion. h2o 0.1 mol/dm3 edta-na2 0.1 mol/dm3 hcl 10 0.30±0.01 ** 7.4 70.1 – 100 3.40±0.10 8.4 72.6 – 1 000 36.0±0.20 7.8 69.9 – leaves 10 000 264±3.00 9.1 71.0 71.5 10 0.40±0.01 6.6 70.5 – 100 3.40±0.10 7.8 74.3 – 1 000 32.2±0.40 9.5 74.0 – stems 10 000 198±1.00 19.0 73.1 71.2 10 0.30±0.01 9.4 78.0 – 100 0.60±0.01 13.4 79.5 – 1 000 34.3±0.20 16.4 82.5 – roots 10 000 190±0.01 18.7 79.2 70.0 * sorption experiments are described in fig. 1; ** ± value represents the standard deviation of the mean (n = 3). it can be expected, that the structure and material composition of particular plant organs are different. therefore, the sorption capabilities of leaves, stems, and roots biomass of hop for cd were compared. it was found that sorption capacity q for cd expressed per gram of dried biomass of hop decreased in the order qleaves : qstems : qroots = 1.0 : 0.8 : 0.7 (table 1). explanation of the differences in the sorption capabilities of particular plant organs for cd will require primarily the comparison of differences in the structures of plant tissues of humulus genera in more details. the mean value of sorption capacity for the whole hop biomass at c0 = 10,000 µmol/dm 3 cdcl2 in deionised water was q = 220 µmol/g (d.w). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc 218 partelová, d. et al. 0 2000 4000 6000 8000 10000 12000 0 50 100 150 200 250 300 leaves stems roots s or pt io n of c d [μ m ol /g ]; d. w . c 0 cdcl 2 [μmol/dm3] r2 = 0.999 fig. 1. sorption of cd (μmol/g; d.w.) by leaf –■–, stem –o– and root –∆– biomass of the hop (h. lupulus l.) variety osvald's clone 72 in dependence on initial cdcl2 concentration in deionised water, ph 5.0 after 10 min of biomass exposure at 25 °c. biomass concentration 25 g/dm3 (d.w.), fraction < 450 μm. error bars represent standard deviation of the mean (n = 3). it is generally known that the ph optimum for bivalent metal cations sorption by biomass is in the range 4 – 6, thus in the moderate acid conditions. in the strong acid conditions, the efficiency of metal cation sorption is minimal and controlled by competitive effect of h+ ions (salim et al., 2008). on the other hand, at higher ph values > 8 (cordero et al., 2004) there exists a formation of sparingly soluble hydroxides cd(oh)2 or cd(oh) 3. the ph value of sorbate solution significantly contributes to ionic equilibria determined by all components presented in the solution. in the fig. 2, the relative proportion of particular ion forms of cd in dependence on ph value of solution imitating the wastewaters predicted by the speciation modelling program mineql+ is described. in the range of ph 3 – 8, the forms of monovalent cdcl+ (52 %) and bivalent cd2+ cations dominate at minority participation of other ionic forms. over the ph 9, the proportion of cdoh+ ions increases. however, over the ph 10 dominates the proportion of cd(oh)2, which already at ph 11 represents 78 % of all cd forms existed in the model system. in the interpretation of cd sorption onto living or dead biomass as a potential sorbent, it is necessary to consider the high proportion of monovalent cation cdcl+ at physiological values of ph 6 – 8. based on this consideration, it can be concluded that an increasing clions concentration in the environment will increase the proportion of cdcl+ complexes. in practice, where inorganic sorbents are used for the removal of metals from wastewaters, the regeneration of the sorbent represents an important step in such decontamination technologies. in this step, the application of chelating agents or acids as desorption solutions is generally accepted (šuňovská et al., 2015). the efficiency of cd desorption (deff) from the hop biomass expressed in % bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc nova biotechnologica et chimica 14-2 (2015) 219 from the total amount of sorbed cd in the biomass increased in the order of the following extraction reagents: deionised water << 0.1 mol/dm3 hcl ≤ 0.1 mol/dm3 edta-na2 (table 1). desorption of cd with deionised water is more significant in the case of hop biomass originated from the sorption experiments carried out at higher initial cdcl2 concentration in the solution. this fact point out that in the hop biomass exist binding sites, which are characterized by different stability of the bond cd–biomass. it can be expected that in the case of cd sorption from diluted cdcl2 solutions (10 μmol/dm 3), cd2+ cations are binding primarily to the sites with higher stability of the bond and in the case of higher cdcl2 concentrations (10,000 μmol/dm3) to the sites with lower stability of the bond. 2 3 4 5 6 7 8 9 10 11 12 0 20 40 60 80 100 [c d] fo rm s/[ c d] to ta l*1 00 [% ] ph cd2+ cdcl+ cdcl 2 cdso 4 cdoh+ cd 2 oh3+ cd(oh) 2 cd(oh) 3 fig. 2. speciation forms of cd in dependence on ph value at the initial concentration of cdcl2 10,000 μmol/dm3 and 25 °c in the solution of inorganic salts simulating wastewaters composition according to iaquinta et al. (2006). calculated by a speciation program mineql+ ver. 4.62. 3.2 sorption of dyes by hop biomass previous experiments with the biomass of leaves, stems, and roots of hop (h. lupulus l.) variety osvald's clone 72 showed that the leaf biomass has the highest affinity and sorption capacity to cd2+ cations. therefore, in the case of the biomass of hop (h. lupulus l.) variety bohemie only the leaf biomass was studied. preliminary experiments revealed that the dried and homogenized leaf biomass (particle size < 0.63 mm and > 0.31 mm) of hop (h. lupulus l.) variety bohemie showed a higher affinity to thiazine dye – methylene blue (mb) than to benzothiazole dye – thioflavine t (tht). regarding this fact, the next experiments were carried out at different concentrations of these studied dyes. fig. 3a depicts the kinetics of tht and mb sorption q (in mg/g; d.w.) by the leaf biomass of the hop under conditions of batch, single dye systems at the initial tht concentration c0 = 40 mg/dm 3 or mb concentration c0 = 80 mg/dm 3, and at the leaf biomass concentration cb = 0.5 g/dm 3 (d.w.) and ph value 6.0. similarly, as in the case of cd sorption by dried biomass bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc 220 partelová, d. et al. of leaves, stems, and roots of hop (h. lupulus l.) variety osvald's clone 72, the tht and mb sorption were also showed as a two-phase process. in the first time of analysis (t = 10 min) of remaining dye concentration in the solution, the sorption of both dyes is characterized as a rapid process and followed by a gradual increasing amount of sorbed dye within the next 350 min of exposure up to reaching the concentration equilibrium. similar results also observed hameed (2009) in the case of mb sorption by the biomass of the grass waste. despite of the mentioned reason that the ratio of initial concentrations of dyes c0 (mb) : c0 (tht) was 2 : 1, the sorption of mb by the leaf biomass of the hop was 4-times higher than in the case of tht. this fact confirms that the dried leaf biomass of the hop showed a higher affinity to the thiazine dye mb than to the benzothiazole dye tht. in both dye molecules, the positive charge is on the quaternary nitrogen = n+ =, but on the different positions within the molecule. thus, in this case the steric effects can play an important role. 0 200 400 600 800 1000 1200 1400 1600 0 20 40 60 80 100 s or pt io n of d ye [m g/ g] ; d .w . time [min] tht mb 2 3 4 5 6 7 0 20 40 60 80 100 s or pt io n of d ye [m g/ g] ; d .w . ph tht mb fig. 3. a. kinetics of tht and mb sorption by the leaf biomass of the hop (h. lupulus l.) variety bohemie during 1 440 min of exposure in deionised water containing tht c0 = 40 mg/dm3 or mb c0 = 80 mg/dm3 at the initial value of ph 6.0 and 25 °c. b. effect of initial value of the ph on tht and mb sorption by the leaf biomass of the hop (h. lupulus l.) variety bohemie after 360 min of exposure in deionised water containing tht c0 = 40 mg/dm3 or mb c0 = 80 mg/dm3 at 25 °c. error bars represent standard deviation of the mean (n = 3). as it was mentioned, the decisive parameter in metal ions or dyes sorption is the ph value of the solution, which affects the sorption capacity of biomass mainly due to the changes in the dissociation of relevant functional groups responsible for their binding. in addition to this, the ph also affects the chemical speciation of metals as well as the chemical dissociation of dye molecules, and thus the sorption of these contaminants. from the fig. 3b, it can be seen that the maximum sorption of tht approx. q = 19 mg/g (d.w.) and mb approx. q = 70 mg/g (d.w.) were found within the range of initial values of ph 4 – 7. approx. 50 % decrease in both dyes sorption was observed at the ph 3 and the minimal sorption q ≈ 1 mg/g (d.w.) for both dyes was calculated at the initial value of ph 2. the similar dependence between the values of specific sorption (in mg/g; d.w.) and the ph value of solutions also described barka et al. (2011) for the case of mb sorption and the biomass of vascular plant scolymus hispanicus. moreover, they found that this dependence can be caused by the changes in surface charge of the dye and in functional groups a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc nova biotechnologica et chimica 14-2 (2015) 221 localized on the surface of biomass, and the value of isoelectric point of the plant biomass (pi = 5.2) as well. in the next experiment, the effect of the leaf biomass concentration on the tht and mb sorption was studied. the sorption of both dyes by the leaf biomass of the hop from single dye solutions decreased with increasing concentration of the biomass under conditions of batch systems (fig. 4a). the relationship between the sorption of dyes q and the concentration of leaf biomass cb showed a non-linear dependence. kumar and porkodi (2007) explain this dependence on the basis of their results, when in the systems with a higher concentration of biomass a negative aggregation and changes in the specific surface area (m2/g; d.w.) of biomass, as well as changes in conditions of effective mixing the biomass within the whole system were described. on the other hand, the efficiency of the removal of both dyes (in %) from the single dye solutions non-linearly increased with increasing concentration of the leaf biomass (data not shown). hameed (2009) reported that the increase of the percentage of dyes removal from the solution with increasing concentration of biomass relates with increasing the available surface binding sites responsible for dyes sorption. 0,5 1,0 1,5 2,0 2,5 0 20 40 60 80 100 s or pt io n of d ye [m g/ g] ; d .w . c b [g/dm3] tht mb 0 50 100 150 200 250 300 350 0 25 50 75 100 125 150 s or pt io n of d ye [m g/ g] ; d .w . c 0 dye [mg/dm3] tht mb r2 = 0.983 r2 = 0.931 fig. 4. a. effect of biomass amount cb (in g/dm3; d.w.) on tht and mb sorption by the leaf biomass of the hop (h. lupulus l.) variety bohemie after 360 min of exposure in deionised water containing tht c0 = 40 mg/dm3 or mb c0 = 80 mg/dm3 at the initial value of ph 6.0 and 25 °c. b. effect of initial concentration of dye c0 (in mg/dm3) on tht and mb sorption by the leaf biomass of the hop (h. lupulus l.) variety bohemie after 360 min of exposure in deionised water containing tht c0 = 20, 40, 100, 120, and 200 mg/dm3 or mb c0 = 20, 40, 80, and 320 mg/dm3 at the initial value of ph 6.0 and 25 °c. error bars represent standard deviation of the mean (n = 3). the efficiency of dyes removal as well as the sorption capacity are also strongly dependent on the concentration of sorbate – dyes in the solution. the fig. 4b depicts the dependence between the sorption of tht or mb by the leaf biomass of the hop (mg/g; d.w.) and the initial concentrations of the tht in the solution within the range 20 – 200 mg/dm3 or mb within the range 20 – 320 mg/dm3. this dependence shows a non-linear trend, whereby the sorption capacity of dried leaf biomass for both studied dyes increased with increasing the initial concentrations of tht or mb due to the higher probability of collisions between dye and biomass. however, the linear dependences within the concentration range 20 – 120 mg/dm3 for tht (r2 = 0.983) and in the case of mb within the concentration range 20 – 80 mg/dm3 (r2 = 0.931) were observed. thus, above the concentrations of tht > 120 mg/dm3 a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc 222 partelová, d. et al. and the concentrations of mb > 80 mg/dm3 the leaf biomass showed the certain degree of the saturation to sorption of both dyes. on the contrary, the efficiency of the removal of both dyes (in %) from the single dye solutions non-linearly decreased with increasing concentration of the dye in the solution (data not shown). as turabik (2008) mentioned in his work, at the lower concentrations of dyes practically all present dye molecules can interact with sufficient amount of binding sites localized on the biomass. however, in the case of higher concentrations of dyes the lower efficiency of the removal of dyes from single dye solutions is observed due to the saturation of sorption sites characterized by relevant functional groups. 0 25 50 75 100 125 150 175 200 0 25 50 75 100 125 150 q eq [m g/ g] ; d .w . c eq [mg/dm3] langmuir freundlich 0 25 50 75 100 125 150 175 200 0 25 50 75 100 125 150 q eq [m g/ g] ; d .w . c eq [mg/dm3] langmuir freundlich fig. 5. description of tht (a) and mb (b) sorption by the leaf biomass of the hop (h. lupulus l.) variety bohemie according to langmuir and freundlich isotherms. conditions of experiments are described in fig. 4b. error bars represent standard deviation of the mean (n = 3). the fact that the experiments concerning the effect of initial concentration of dyes on the sorption of dyes by the leaf biomass of the hop were carried out at constant temperature and within the time, when the concentration equilibrium was reached, allows to evaluate the obtained data by sorption isotherms. sorption isotherms according to the langmuir (eq. 3) and freundlich (eq. 4) are often used to describe the sorption data. these mathematical models provide important parameters to characterize the sorption processes of dyes or metals binding by a wide range of sorbents derived from the plant waste biomass from the point of view of the sorption mechanism, characterization of sorption surface or affinity of sorbent to sorbate, and the practical utilization of studied sorbents as well (banerjee and chattopadhyaya, 2013; oguntimein, 2015). for description of the obtained data, the linear and non-linear forms of these models can be used. however, as noted by several authors (see e.g. ncibi et al., 2009; albadarin and mangwandi, 2015), the application of mentioned models in non-linear forms is preferable than the utilization of their simplified linearized forms, mainly due to the potential changes in the distribution of errors after transforming the data to a linear form. the description of obtained data by sorption isotherms according to the langmuir and freundlich for both studied dyes in the form of the dependences between the equilibrium specific sorption qeq (in mg/g; d.w.) of dye on the leaf biomass of the hop and the equilibrium concentration of the dye ceq (in mg/dm 3) in the solution a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc nova biotechnologica et chimica 14-2 (2015) 223 is depicted in the fig. 5. in the first step, the suitability of both used mathematical models to description of obtained data was evaluated according to the comparison of determination coefficients r2. the obtained values of parameters from mathematical description of the data characterizing tht and mb sorption by the leaf biomass according to the langmuir and freundlich isotherms are mentioned in the table 2. it is evident that the process of tht and mb sorption is better described by the langmuir model (r2 = 0.972 for tht and r2 = 0.982 for mb) than according to the freundlich model (r2 = 0.913 for tht and r2 = 0.966 for mb) of sorption isotherm. similar results were obtained in our previous work also concerning the sorption of tht and mb by dried biomass of freshwater moss vesicularia dubyana (partelová, 2012). table 2. obtained values of parameters from mathematical description of the data characterizing tht and mb sorption by the leaf biomass of the hop (h. lupulus l.) variety bohemie according to langmuir and freundlich isotherms. model qmax [mg/g]; d.w. b [dm3/mg] k [dm3/g]; d.w. 1/n r2 langmuir tht mb 77.6 184 0.0129 0.0159 0.972 0.982 freundlich tht mb 3.30 7.83 0.548 0.570 0.913 0.966 from the obtained parameters of applied sorption isotherms, the parameter qmax characterizing the maximum sorption capacity (mg/g; d.w.) of the dried leaf biomass of the hop for the given sorbate – dye is the most important parameter. on the basis of this parameter, it is possible to compare the various sorbents derived from the plant waste biomass as well as to study the affinity of different sorbents to the given sorbates (dyes or metals). according to obtained values of qmax for tht and mb, it can be concluded that in the case of mb the dried leaf biomass of the hop showed more than 2-times higher sorption capacity (qmax = 184 mg/g; d.w.) in comparison with the value obtained for tht (qmax = 78 mg/g; d.w.). thus, the dried leaf biomass has more than 2-times higher affinity to mb than to tht dye. 4. conclusions experiments focused on the removal of cd from aqueous solutions of cdcl2 spiked with radionuclide 109cd and synthetic dyes thioflavine t (tht) or methylene blue (mb) from single dye solutions under conditions of batch systems showed that dried plant biomass of hop (humulus lupulus l.) as agricultural by-products can be used as a sorbent of both types of studied contaminants. the maximum sorption capacity q = 264 µmol cd/g (d.w.) was found in the case of the leaf biomass of hop (h. lupulus l.) variety osvald's clone 72 at the initial concentration of cdcl2 10,000 µmol/dm 3, whereby the sorption capacity decreased in the order qleaves : qstems : qroots = 1.0 : 0.8 : 0.7. individual experiments showed that cd sorption increased with increasing initial cd concentration in the solution. for the leaf biomass, but not in the case of stem or root biomass, the linear dependence bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc 224 partelová, d. et al. between the sorption q and the initial concentration of cdcl2 was observed. thus, the biomass of stem and root of hop showed a partial saturation with cd2+ cations at the highest applied initial cd concentrations. the sorbed amount of cd was removed from the hop biomass with the following increasing desorption efficiency (in %) of the extraction reagents: deionised water << 0.1 mol/dm3 hcl ≤ 0.1 mol/dm3 edta-na2. similarly as in the case of cd sorption by dried biomass of leaves, stems, and roots of hop (h. lupulus l.) variety osvald's clone 72, the kinetics of tht and mb sorption by the leaf biomass of the hop (h. lupulus l.) variety bohemie were also showed as a two-phase process. the maximum sorption of tht approx. q = 19 mg/g (d.w.) and mb approx. q = 70 mg/g (d.w.) were found within the range of the initial values of ph 4 – 7. approx. 50 % decrease in both dyes sorption was observed at the ph 3 and the minimal sorption q ≈ 1 mg/g (d.w.) for both dyes was calculated at the initial value of ph 2. the sorption of both dyes by the leaf biomass of the hop from single dye solutions decreased with increasing concentration of the biomass and on the other hand increased with increasing the initial concentrations of tht or mb. the process of tht and mb sorption was better described by the langmuir model than the freundlich model of sorption isotherm. from the obtained values of qmax for tht and mb, it was found that in the case of mb the dried leaf biomass of the hop showed more than 2-times higher sorption capacity (qmax = 184 mg/g; d.w.) in comparison with the value obtained for tht (qmax = 78 mg/g; d.w.). acknowledgement: this work was financially supported within the grant vega 1/0635/13 “hop as an important source of phytomedicinally active compounds”. the authors wish to thank dr. juraj faragó and dr. ivana pšenáková for providing the samples of hop. references abdolali, a., guo, w.s., ngo, h.h., chen, s.s., nguyen, n.c., tung, k.l.: typical lignocellulosic wastes and by-products for biosorption process in water and wastewater treatment: a critical review. bioresour. technol., 160, 2014, 57-66. adegoke, k.a., bello, o.s.: dye sequestration using agricultural wastes as adsorbents. water res. ind., 12, 2015, 8-24. aksu, z., i̇şoğlu, i̇.a.: removal of copper(ii) ions from aqueous solution by biosorption onto agricultural waste sugar beet pulp. process 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276-282. rangabhashiyam, s., anu, n., giri nandagopal, m.s., selvaraju, n.: relevance of isotherm models in biosorption of pollutants by agricultural byproducts. j. environ. chem. eng., 2, 2014, 398-414. sadeek, s.a., negm, n.a., hefni, h.h.h., abdel wahab, m.m.: metal adsorption by agricultural biosorbents: adsorption isotherm, kinetic and biosorbents chemical structures. int. j. biol. macromol., 81, 2015, 400-409. safa, y., bhatti, h.n.: biosorption of direct red-31 and direct orange-26 dyes by rice husk: application of factorial design analysis. chem. eng. res. des., 89, 2011, 2566-2574. salim, r., al-subu, m., dawod, e.: efficiency of removal cadmium from aqueous solutions by plant leaves and the effects of interaction of combinations of leaves on their removal efficiency. j. environ. manage., 87, 2008, 521-532. salleh, m.a.m., mahmoud, d.k., karim, w.a.w.a., idris, a.: cationic and anionic dye adsorption by agricultural solid wastes: a comprehensive review. desalination, 280, 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heavy metals from aqueous solution with tobacco dust. bioresour. technol., 99, 2008, 5595-5601. turabik, m.: adsorption of basic dyes from single and binary component systems onto bentonite: simultaneous analysis of basic red 46 and basic yellow 28 by first order derivative spectrophotometric analysis method. j. hazard. mater., 158, 2008, 52-64. zafar, m.n., aslam, i., nadeem, r., munir, s., rana, u.a., khan, s.ud.: characterization of chemically modified biosorbents from rice bran for biosorption of ni(ii). j. taiwan inst. chem. eng., 46, 2015, 82-88. wang, z., han, l., sun, k., jin, j., ro, k.s., libra, j.a., liu, x., xing, b.: sorption of four hydrophobic organic contaminants by biochars derived from maize straw, wood dust and swine manure at different pyrolytic temperatures. chemosphere, 144, 2016, 285-291. wu, y., fan, y., zhang, m., ming, z., yang, s., arkin, a., fang, p.: functionalized agricultural biomass as a low-cost adsorbent: utilization of rice straw incorporated with amine groups for the adsorption of cr(vi) and ni(ii) from single and binary systems. biochem. eng. j., 105, 2016, 27-35. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 13:49 utc nova biotechnol chim (2017) 16(2): 86-91 doi: 10.1515/nbec-2017-0012  corresponding author: sestinova@saske.sk  nova biotechnologica et chimica investigation of genotoxicity in river sediments oľga šestinová, lenka findoráková, jozef hančuľák and tomislav špaldon department of environment and hygiene in mining, institute of geotechnics, slovak academy of sciences, watsonova 45, košice, sk-040 01, slovak republic article info article history: received: 3rd march 2017 accepted: 15th november 2017 keywords: escherichia coli k12 pq37 genotoxic effect heavy metals sediments abstract the purpose of the present study was to develop a useful screening method to assess genotoxic effect of polluted bottom sediments from the water reservoir ružin no.i. the hornád and hnilec rivers drained a former mining area, have been polluted in the long-term by heavy metals (cu, as, sb, hg), which significantly contributed to environmental degradation. genotoxicity of bottom sediment was evaluated by test sos-chromopadtm 3.0 for solid samples without extraction. the mentioned test represents simple, quick and direct sediment phase toxicity testing procedure. in this test bacterial strain escherichia coli k12 pq37 was used. the results of soschromopadtm 3.0 showed that sample hornád has low potential genotoxic effect on the environment. it was determined on the basis of slight blue colouration of chromogenic paper at the point of sediment application. the sample hnilec was negative. this test allows significantly reduce the time for obtaining information about sediments genotoxicity and accept necessary security proceeding in time.  university of ss. cyril and methodius in trnava     introduction heavy metals in mine wastes can considerably influence surrounding surface waters, sediments and human health. to estimate environmental impact, heavy metal concentrations in sediments can be utilized because they are indicators of contamination and change negligibly with time. in the contaminated sediments of the water reservoir ružín no.i (eastern slovakia) there are mainly heavy metals (cu, as, sb, hg), which origin are in the geological environment of the area and are connected with the complex treatment of ore. the river hornád and hnilec dewater the area of old mining load after extraction and treatment of sulfitic ore connected with abandoned mines (rudňany, smolník and krompachy) (šestinová et al. 2015). heavy metals can affect organisms in different ways. many of them are toxic, and some of them may cause loss of genomic stability by altering the balance in chromatin metabolism. they are classified as genotoxic. genotoxic effects of heavy metals are very important as organisms health is based on genome (factori et al. 2014; marple and hasty 2004). a result of genetic damage material of an organism can thus show up for a long time, sometimes in the next generation (guecheva et al. 2001; huang and wai 2004; malachová et al. 2014). many of these metals have genotoxic effects that act directly or indirectly on organisms (mutagens). action of matter with mutagenic effects leads to damage of genetic information stored in nucleic acids. in cells non-lethal genetic changes are formed, that are of permanent character. these can be reflected into defective offspring of cell. in some cases, the process of cell bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:53 utc nova biotechnol chim (2017) 16(2): 86-91 87 replication as a result of damaged information can completely overthrow cells transformation and develop, for example, carcinogenicity. for testing genotoxicity organisms or community of organisms with specific laboratory sets such as salmonella typhimurium (e.g. chromoplate kit) or escherichia coli (e.g. sos-chromotest) are most commonly used (ghani 2010; feng et al. 2012). the genotoxicity tests have been developed for the detection of chemical mutagens and carcinogens in environmental samples. in addition to assessing the genotoxic potential of test samples they allow for direct determination of the toxic effects of wastewater and sediment samples (dutka et al. 1995; vojtková and janáková 2011; zotina et al. 2015; malachová et al. 2014). the mentioned tests are commercially available in the form of sets involving concentrated solutions, reference materials and test organisms in the form of latent eggs, which allow to detect a genotoxic material even in non-laboratory conditions. because of the rapid indicative determination of acute toxicity in monitored sediments, the sos-chromopadtm test was selected and used of the available short term indicative genotoxicity tests,. the sos-chromopad is a rapid bacterial-based colorimetric bioassay kit for the determination of toxicity of sediments, suspended sediments, soils and solid wastes. it is sensitive to a wide spectrum of toxic substances such as heavy metals, and organic and inorganic pollutants, and may be used to detect the presence of toxicants directly without solvent extraction. in the present study, the screening method, genotoxicity of sediments collected from the water reservoir ružín no. i (hornád and hnilec rivers) was used to evaluate the potential genotoxic risks on the given aquatic environment. experimental material for sediment genotoxicity testing the sediment samples were taken in two areas from the water reservoir ružín no.i, in the year 2011– 2012. the sediments from the locality hornád and hnilec branch were collected into plastic bottles by a multisampler sample device (at a depth of 50 cm). for the physicochemical and bioassays, the sample was dried at laboratory temperature, then quartered and sieved into fraction of 63 μm. the total extractable metal concentrations in the sediments were determined after mineralization with a mixture of acids (hcl/hno3/hf) in a microwave pressure digestion system (mws-3, ger). the metal concentrations in the sediments were determined by atomic absorption spectrometry (varian, aus). chns analysis was performed by an elementary analyzer (vario macro cube ger) using a thermal conductivity detector (findorakova et al. 2017). the certified reference material lgc6187 control was river sediment (elbe). the reference sample was systematically used to control the analytical precision. incubatordrier memmert 100-800 (incubation at 37°c) was used for acute genotoxicity study. the chemicals and media of the sos-chromopadtm3.0 test contained: a nutrient medium for bacterial growth, lyophilized bacteria escherichia coli k12 pq37 (non-pathogenic strain), a petri dish with blue chromogenic substrate, 4nqo (4-nitro-quinolineoxide-for positive control), disposable plastic pipette, test tubes, plastic bags for incubation and ampicillin. sample (0.1 g) treatment before determining genotoxicity was performed by the following procedures: sos-chromopadtm ver. 3.0fresh-solid. sediment samples without extraction were used. three replicates were performed for each sample set. preparation of bacterial strain escherichia coli k12 pq 37 for sediments genotoxicity testing, bacterial strain escherichia coli k 12 pq37 in test soschromopadtm was used. lyophilized bacteria escherichia coli k12 pq37 were 24 hours before each test mixed with 10 ml of culture medium and incubated in a thermostat for 16-18 hours at temperature 37°c. on the next day, the concentration of bacteria was measured by uv-vis spectrophotometer helios-υ at a wavelength of 620 nm. required bacteria concentration corresponded to the absorbance value of 0.07. the principle of the test sos-chromopadtm ver. 3.0 is that genotoxic agents cause dna damage and immediately thereafter, the cell tries to repair this damage with activation reparation system, which is called sos. the result of sos reparations affects bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:53 utc nova biotechnol chim (2017) 16(2): 86-91 88 table 1. metal concentration in the sediment from the localities in hornád, hnilec and control (average ± standard deviation). sample cu as sb hg (mg/kg) dry weight 1/2011 333±2.7 75±6.1 62±2.8 5.9±0.2 1/2012 198±3.4 39±2.7 27±3.6 6.5±0.4 2/2011 354±2.8 56±3.4 59±3.7 1.8±0.2 2/2012 359±5.1 49±4.3 32±5.7 1.9±0.5 control 82±2.7 25±1.9 14±2.5 1.6±0.3 limiting values (mg/kg) dry weight* tv 36 29 3 0.3 mpc 73 55 15 10 iv 190 55 10 (1/2011-2012 hornád and 2/2011-2012 hnilec) *norm no. 549/1998-2: tv-target value (negligible risk), mpc –maximum permissible concentration (max. tolerable risk), iv-intervention value (serious risk), control (certified reference material) table 2. results of elemental chns in sediments from hornád and hnilec. sample c h n s (%) 1/2011 40.80 7.59 9.32 0.75 1/2012 39.25 6.85 8.71 0.65 2/2011 38.93 6.46 8.14 0.63 2/2012 37.31 6.15 8.06 0.61 replacement of cell, it leads to permanent changes of the cell genetic structure, which may lead to genetically portable mutation or carcinogenic cell transformation. induction of sos functions is evaluated using the sfia gene (junior et al. 2007). working scheme of sos-chromopadtm ver. 3.0 preparation of test tubes for each sample: the bacterial suspension and sediment in the tube 1 by mixing 1 ml active bacterial culture with 0.1 g weighed portion of sediment was prepared in the shaking incubator. dilution of prepared suspension in tubes 2-5 of concentrations were: 0.5n (50%) – 0.25n (25%) – 0.125n (12.5%) – 0.0625n (6.25%) – 0.0313n (3.13%); 6 – negative control (only bacteria/0.5 ml), 7 – positive blue control (4nqo/0.050 ml). incubation of the tubes was in a thermostat during 4 hours at temperature 37°c. then 0.020 ml of a bacterial sludge in the form of dots on a petri dish with blue chromogenic substrate was applied. arrangement of dots was as follows: in the middle was positioned a positive control, a negative control and diluted samples were on the edges (7 dots on chromogenic plate). incubation of the petri dishes with active bacterial culture was in a thermostat for 20 hours at 37°c. after incubation, a wash of the sediment particulate from the surface of chromogenic paper was performed. evaluation was realized by photographic documenting (fig. 1) and visual evaluating of the intensity of blue colour dots versus positive indicator (dark blue) using a colourindex. the scale is based on 5 colour tones. white colour corresponds to colour-index 0 (nongenotoxic) and the blue colour corresponds to the colour-index 5 (the genotoxic sample). finally, the minimum effective concentration in a volume of the sample was determined. results and discussion the ph of sediment samples (1/2011-2012 and 2/2011-2012) was in range 7.25 – 7.59, indicating a slight alkaline nature. organic matter content of sediments ranged from 8.1 to 14.2 % (brázová et al. 2015; šestinová et al. 2015). table 1 summarizes the results of chemical analyses of cu, as, sb and hg in the sediments compared to limiting values for contamination with copper and arsenic according to law no. 549/1998-2 of the methodological instruction of the ministry bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:53 utc nova biotechnol chim (2017) 16(2): 86-91 89 fig. 1. the colour evaluation of the samples from hornád and hnilec at sos-chromopad plate. values indicated for the circled areas of sample application indicate the sample dilution (in %) (nc – negative control-without sediment and pc – positive control with the genotoxic standard 4nqo). of environment of the slovak republic for assessment of risks from pollution of sediments of streams and water reservoirs. the present data was compared with metal concentrations in other sediments reported for similar mining areas in krompachy, during the last decade (findoráková et al. 2017). from table 1-2 it is obvious that the sample hornád is a slightly more contaminated and has higher values of chns as the sample hnilec. sos-chromopad test has great advantage that the sediments are applied in solid state. the endpoint of the bioassay is an easy to interpret colour reaction. the results corresponding to two parallel tests of sediments are visual–colour evaluated according to a simple colour-index (fig. 1). dots on chromogenic paper enable rapid detection of genotoxicity or dna damage in sediments compared to negative and positive signal light. the basic of scores is the intensity of the blue colour of serially diluted (%) positive sample comparing with samples. principally, toxic materials interfere with recovery process and thus with synthesis of the enzyme and colour reaction. fig. 1 and table 3 show colour evaluation of the environmental samples assayed at plates sos-chromopad. from the fig. 1a it is evident that the sample hornád is non-toxic at 3.13% dilution. concentrations of responsible sediment components exceeding this dilution value bear potentially genotoxic effect(s) on the environment. in contrast, the the fig. 1b shows the results of the sediment hnilec that generated no blue colour at chromogenic paper at point of application. it confirms that the sample hnilec is negative and can be considered as nontoxic to environment according to this test. in this study we used three technical replicates of each sample for evaluation of genotoxicity (table 3). the colour reactions of the individual samples at chromogenic pad were reproducibly similar in all three repetitions. in some cases, however, the visual–colour evaluation in the soschromopad test was difficult due to appraisal colour reaction of sediments and their corresponding appraisal genotoxic effect (table 3). according to rönnpagel et al. (1995) the effects of sediment or soil matrices on the bioavailability of compounds can contribute to such difficulties to screen toxicity of solid-associated contaminants. the majority of bioassays for testing toxicity of soils and sediments have been performed on water or solvent extracts (rönnpagel et al. 1995). this study builds on our previous article, in which genotoxicity of sediment extracts (soxchlet extraction) from ebpi (can) has been described, while the sos-chromotesttm ver. 6.4 for liquid samples was used (šestinová and findoráková 2017). the two tests – soschromopad and chromotest – were performed in parallel and the results were compared. test results of sediment samples obtained using soschromotest pointed on genotoxicity (blue staining) a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:53 utc nova biotechnol chim (2017) 16(2): 86-91 90 table 3. results of visual evaluation of three parallel tests of sediments according to colour-index. colour index nc pc 1 /50 2 /25 3 /12.5 4 /6.25 5 /3.13 i. replicate hornád nc pc 50 % 25 % 12.5 % 6.25 % 3.13 % w b+++ b++ b+ b+ b w i. replicate hnilec nc pc 50 % 25 % 12.5 % 6.25 % 3.13 % w b+++ w w w w w ii. replicate hornád nc pc 50 % 25 % 12.5 % 6.25 % 3.13 % w b+++ b++ b+ b+ w w ii. replicate hnilec nc pc 50 % 25 % 12.5 % 6.25 % 3.13 % w b+++ b w w w w iii. replicate hornád nc pc 50 % 25 % 12.5 % 6.25 % 3.13 % w b+++ b+ b+ w w w iii. replicate hnilec nc pc 50 % 25 % 12.5 % 6.25 % 3.13 % w b+++ w w w w w w – white colour, b – blue colour, + intensity colour, nc – negative control, without sediment, pc – positive control (4nqo) only for the positive standard (4nqo), while the sediment samples yielded yellow coloration. this indicates to low genotoxic potential of all the extracted leachate of the studied samples. subsequently, corresponding instrumentation absorbance values of the samples were measured and the inducing activity (ic) of each extract dilution was calculated (using evp-extractable organic fraction and eop-extractable aqueous fraction). the obtained results confirmed that the epo of the samples hornád does possess a potential genotoxic effects. only a single sample dilution (12.5%) had a value of ic-1.66, exceeding the critical value of ic-1.5. nevertheless, the total ic value was closely below the critical value suggesting that neither sediment sample represents a risk in terms of genotoxicity. since no monitored metals were found in the sediment samples and effective substances could be extracted in eop, the effective compound might be a chemical substance in an organic form, with the ability to immobilize ions of toxic metals. such substances can include e.g. humic acids. it was also demonstrated by higher ic values for sample hornád in the eop. according to šestinová and findoráková (2017) the results of yield in the eop for individual metals were lower than yields in evp (especially for cu and sb in samples hornád, hnilec), on the contrary was determined their ic value in eop greater than in evp. on the basis of above mentioned results of sediment samples, we can assume for sample hornád in eop potential genotoxic effect. significant genotoxic effect has not been established for sample hnilec. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:53 utc nova biotechnol chim (2017) 16(2): 86-91 91 conclusions the ecological impact of genotoxic sediments on aquatic biota is difficult to estimate, since depends on a wide range of environmental and biological factors. a result of genetic damage material of an organism can thus show up for a long time, sometimes in the next generation. the purpose of this study was to describe sos-chromopadtm ver. 3.0 test for testing of bottom sediments from the water reservoir ružín no.i (in the area hornád and hnilec rivers) for genotoxicity directly without extraction. the results of acute genotoxicity were for sample hornád low potential genotoxic and for hnilec sample non-toxic in used sos-chromopad test. the test sos-chromopad is usefulness because is easy to perform, faster, require minimal amount of sample, environmentally friendly but is not so precise in the determination. obtained information from this study can be useful for hazard identification and risk assessment of sediment-associated contaminants. acknowledgement the work was supported by the slovak grant agency vega, grants no. 2/0079/16 and no. 2/0194/15. references brázová t, hanzelová v, miklisová d, šalamún p, vidalmartínez vm (2015) host-parasite relationships as determinants of heavy metal concentrations in perch (perca fluviatilis) and its intestinal parasite infection. ecotox. environ. safe. 122: 551-556. dutka bj, teichgraber k, lifshitz r (1995) a modified soschromotest procedure to test for genotoxicity and cytotoxicity in sediments directly without extraction. chemosphere 31: 3273-3289. environmental bio-detection products inc. available at: <http://www.ebpi-kits.com/index.php>. factori r, leles sm, nowakowski gc, rocha clsc, thomaz sm (2014) toxicity and genotoxicity of water and sediment from stream on dotted duckweed (landoltia punctate). braz. j. biol. 74: 769-778. feng s, mai bb, wei g, wang x (2012) genotoxicity of the sediments collected from pearl river in china and their polycyclic aromatichydrocarbons (pahs) and heavy metals. environ. monit. assess. 184: 5651-5661. findoráková l, šestinová o, kováčová m (2017) assessment of potential sediment contamination using screening methods (xrf, tga/ms) taking into account principles of green chemistry, eastern slovakia. environ. earth sci.76:119. ghani a (2010) toxic effects of heavy metals on plant growth and metal accumulation in maize (zea mays l). iran j. toxicol. 3: 325-334. guecheva t, henriques ja, erdtmann b (2001) genotoxic effects of copper sulphate in freshwater planarian in vivo, studied with the single-cell gel test (comet assay). mutat. res. 497: 19-27. huang s, wai owh (2004) a review of heavy metal pollution in the pearl river estuary. j. hydrodyn., ser. b 16: 367-378. junior hm, de-silvia j, arenzon a, portela cs, ferreira ic, henrique’s ja (2007) evaluation of genotoxicity and toxicity of water and sediment samples from a brazilian stream influenced by tannery industries. chemosphere 67: 1211-1217. malachová k, sezimová h, rozinek r (2014) detection of genotoxicity and toxicity of wastewater treatment plant (wtp) effluents after pretreatment with ferrate (vi). toxicol. lett. 229(10): 117-118. marple t, li h, hasty p (2004) a genotoxic screen: rapid analysis of cellular dose-response to a wide range of agents that either damage dna or alter genome maintenance pathways. mutat. res. 554: 253-266. rönnpagel k, lib w, ahlf w (1995) microbial bioassays to assess the toxicity of solid-associated contaminants. ecotox. environ. safe. 31: 99-103. šestinová o, findoráková l, hančuľák j, šestinová l (2015) study of metal mobility and phytotoxicity in bottom sediments that have influenced by former mining activities in eastern slovakia. environ. earth. sci. 74: 6017-6025. šestinová o, findoráková l (2017) assessment of eastern slovakia sediments genotoxicity and phytotoxicity using screening tests: chromotests and phytotoxkit. fresen. environ. bull. 26: 2454-2462. vojtková h, janáková i (2011) research of waste dump water mutagenicity of bacterial detection system sos chromotest. water sci. technol. 63: 2833-2837. zotina ta, trofimova ea, medvedeva my, dementyev dv, bolsunovsky ay (2015) use of aquatic plant elodea canadensis to asses toxicity and genotoxicity of yenisei river sediments. environ. toxicol. chem. 34: 23102321. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:53 utc nova biotechnol chim (2017) 16(1): 26-31 doi: 10.1515/nbec-2017-0004  corresponding author: stefan.demcak@tuke.sk nova biotechnologica et chimica utilization of poplar wood sawdust for heavy metals removal from model solutions stefan demcaka,, magdalena balintovaa, maria hurakovab, marina v. frontasyevac, inga zinicovscaiac,d and nikita yushinc a technical university of kosice, faculty of civil engineering, institute of environmental engineering, vysokoskolska 4, kosice, sk-040 01, slovak republic b slovak academy of sciences, institute of experimental physics, watsonova 47, kosice, sk-040 01, slovak republic c frank laboratory of neutron physics, joint institute for nuclear research, joliot-curie str., 6, 1419890 dubna, russia d horia hulubei national institute for r&d in physics and nuclear engineering, 30 reactorului str. mg-6, bucharest magurele, romania article info article history: received: 27th february 2017 accepted: 2nd may 2017 keywords: adsorption heavy metals poplar wood sawdust abstract some kinds of natural organic materials have a potential for removal of heavy metal ions from wastewater. it is well known that cellulosic waste materials or by-products can be used as cheap adsorbents in chemical treatment process. in this paper, poplar wood sawdust were used for removal of cu(ii), zn(ii) and fe(ii) ions from model solutions with using the static and dynamic adsorption experiments. infrared spectrometry of poplar wood sawdust confirmed the presence of the functional groups which correspond with hemicelluloses, cellulose and lignin. at static adsorption was achieved approximately of 80 % efficiency for all treated model solutions. similar efficiency of the adsorption processes was reached after 5 min at dynamic condition. the highest efficiency of cu(ii) removal (98 %) was observed after 30 min of dynamic adsorption. changes of ph values confirmed a mechanism of ion exchange on the beginning of the adsorption process.  university of ss. cyril and methodius in trnava introduction heavy metals and their compounds are excessively released into the water bodies by a wide range of industrial activities such as: metal plating, mining activities, smelting, battery manufacture, tanneries, and many other branches of industries (ngah and hanafiah 2008; saifulnizam and jaafar 2010). in comparison with organic contaminants, heavy metals are non-biodegradable and they can be accumulated in living organisms, causing various diseases and disorders. for these reasons, heavy metals must be removed from wastewater before their discharge into the aquatic environment (ahmaruzzaman 2011). nowadays, organic materials from plant wastes or by-product are often used instead of the costly conventional methods of metal ions removal from wastewater (ngah and hanafiah 2008; fu and wang 2011; nayak et al. 2017). physico-chemical processes based on adsorption on natural organic materials are cheap and effective techniques for metals removal from wastewater. according to bailey et al. (1999), low-costs organic sorbents widely abundant in nature can be used as cheap alternative to industrially produced sorbents. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:23 utc mailto:stefan.demcak@tuke.sk nova biotechnol chim (2017) 16(1): 26-31 27 wooden materials or wastes are cheap sorbent materials (ngah and hanafiah 2008; ahmaruzzaman 2011). the benefits of application of wooden by-products or wastes for wastewater treatment are determined by their high removal selectivity, good adsorption capacity and possibility of regeneration. the use of original and untreated wooden materials or wastes such as straw, tree bark, peanut skins, wood sawdust, moss and peat has been reported for pollutants removal in several studies (shukla et al. 2002; šćiban et al. 2007; asadi et al. 2008). wood sawdust, a low-costs adsorbent, is perspective for removing metal ions, some types of acid and basic dyes as well as other unwanted compounds from wastewaters (singh et al. 1993). the efficiency of the adsorption processes strongly depends on the composition of the wastewater (keränen et al. 2016). ahmad et al. (2009) showed formation of complex compounds of metal cations with wood sawdust functional groups. lignin, cellulose and hemicellulose, carbohydrates, and phenolic compounds, which contain carboxyl, hydroxyl, sulphate, phosphate, and amino groups, are the main metal binding sites (gardea-torresdey et al. 1990; crini 2006). the application of wood sawdust for removal of pollutants brings many benefits for the protection of environment and timber industry. contaminated water could be treated, and a new market would be opened for the sawdust (shukla et al. 2002). the aim of the present research was to study the sorption properties of poplar wood sawdust for copper, zinc and iron removal from model solutions. poplar sawdust was analysed by infrared spectrometry for characterization of functional groups, which can be responsible for metal binding. efficiency of heavy metals removal was analysed by colorimetric method and changes of ph values. experimental wood sample the sawdust of poplar, species of locally available wood, was sieved, and the fraction with particle size max. 2.0 mm was used for experiments. 1 g of dry poplar sawdust was weighted and used for adsorption experiments. ir spectrum of poplar sawdust was studied for characterization of present functional groups, which can be responsible for metal binding. ftir measurements of poplar sawdust were carried out on bruker alpha platinum-atr spectrometer (bruker optics, ettingen, germany). a total of 24 scans were performed on sample in the range of 4,000–400 cm−1. adsorbate solutions adsorbate solutions of cu(ii), zn(ii) and fe(ii) (initial concentration c0=10 mg.l -1) were prepared by dissolving cuso4·5h2o, znso4·7h2o and feso4·7h2o, respectively in deionised water. concentrations of appropriate ions were determined by colorimetric method (colorimeter dr890, hach lange, germany) with appropriate reagents to determine concentration of dissolved cooper, zinc, and iron. copper in the sample reacts with a reagent (bicinchoninic acid) to form a purple coloured complex in proportion to the copper concentration. zinc reacts in the sample complex with cyanide. adding cyclohexanone selectively releases zinc. the zinc then reacts with the 2-carboxy-2’-hydroxy-5’-sulfoforamazyl benzene (zincon) and forms a blue colour that is proportional to the zinc concentration. the 1,10-phenanthroline indicator reacts with iron ion in the sample to form an orange colour in proportion to the iron concentration. input ph values were also measured by ph meter inolab ph 730 (wtw, germany). adsorption studies batch adsorption experiments were carried out on static and dynamic conditions of experimental setup. in both experiments, 1 g of dry poplar sawdust was mixed with 100 ml of model solutions containing 10 mg.l-1 of copper, zinc, and iron respectively. in static conditions, the sorbent-sorbate interaction time was 24 h. to determine the contact time required for equilibrium sorption in dynamic condition the samples were removed at different time intervals 5, 10, 15, 30, 45, 60 and 120 min, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:23 utc nova biotechnol chim (2017) 16(1): 26-31 28 respectively. after the end of experiments, wood sawdust was removed by filtration through a laboratory filter paper. residual concentrations of appropriate ions were determined by colorimetric method and ph change was also measured. in both cases, the efficiency of ion removal η (in %) removal was calculated using the following equation (eq. 1): %,100 )( 0 0    c cc e (1) where c0 is the initial concentration of appropriate ions (mg.l-1) and ce equilibrium concentration of ions (mg.l-1). all adsorption experiments were carried out in triplicate under the batch conditions and results are given as arithmetic mean values. results and discussion poplar sawdust infrared spectrum the aim of the present research was to study the sorption properties of poplar wood. metal adsorption capacity is influenced strongly by the surface structures of c–o and c–oh functional groups which are present in organic materials (ricordel et al. 2001). functional groups in poplar wood sawdust were determined using ftir spectroscopy. the ir spectrum of poplar sawdust is shown in fig. 1. according to literature (kidalova et al. 2015) we can suppose that the structure of wooden sawdust is mainly formed by cellulose, hemicellulose, and lignin. strong broad oh stretching (3,650–3,000 cm-1) and c–h stretching of methyl and methylene groups (2,950– 2,800 cm-1) are present (fig. 1), possibly due to the presence of –oh functional groups or could be absorbed also from atmospheric damp. for comparison, typical for hemicelluloses is the stretching band at 1,736 cm-1 caused by presence of c=o from the acetyl groups. infrared spectra of lignin in wooden sorbent materials (zhang et al. 2015) revealed characteristic bands at 1,503 and 1,454 cm-1 (correspond to aromatic skeletal vibrations of lignin) and at 1,320 cm-1 (syringyl and guaiacyl condensed lignin). deformations detected in a range of 1,421 to 895 cm-1 wavenumber appertain to cellulose that can occur in two forms (crystalline and/or amorphous). functional groups of aromatics, carboxylic acids and alkyl halides were described at 829 cm-1 (schwanninger et al. 2004). fig.1. infrared spectrum of poplar wood sawdust. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:23 utc nova biotechnol chim (2017) 16(1): 26-31 29 adsorption study of poplar wood sawdust – static conditions results of sorption experiments under static conditions for solutions with cation concentrations 10 mg.l-1 are shown in table 1. the data suggest that poplar sawdust has a very good efficiency of removal of studied metal ions. the most efficient sorption was for copper with efficiency of 85.8 %. for zinc and iron the efficiencies were 74 % and 78 % (respectively). previously, šćiban et al. (2007) have suggested that 20 g.l-1 of poplar sawdust is efficient for removal of most of the cu(ii) ions. sorption is accompanied by changes of ph values in solutions. the ph of the solutions is an important controlling parameter in the adsorption process, since ph affects the surface charge of the adsorbent, the degree of ionization and speciation of sorbate during adsorption (rahman and islam 2009). at lower ph, the positively charged metal ion species may compete with h+ and be absorbed at the surface of the sawdust by ion exchange mechanism. at elevated, mainly neutral ph, metal cations may be absorbed by hydrogen bonding mechanism along with ion exchange. due to different properties of the metals cu(ii), zn(ii), and fe(ii), the adsorption took place at slightly different ph values (respectively). sorption of cu(ii) by poplar sawdust increases the ph of the solution. possibility of ion exchange between dissolved zn(ii) and fe(ii) metal cations and h+ from poplar sawdust was indicated by decreasing of ph. table 1. results of static sorption experiments with poplar sawdust (initial concentration of cations in solutions c0=10 mg.l -1). ion input values adsorption experiments c0 ph ce η ph [mg.l-1] [mg.l-1] [%] cu2+ 10.0 5.8 1.4 85.5 5.3 zn2+ 10.0 5.4 2.6 74.0 5.8 fe2+ 10.0 5.4 2.2 78.0 5.7 η – efficiency of ion removal, c0 – initial concentration of ions, ce – equilibrium concentration of ions. adsorption study of poplar wood sawdust – dynamic conditions sorption efficiency and changes of ph values over time are shown in fig. 2. using the poplar sawdust for absorption experiment in model solutions with dissolved ions cu(ii), zn(ii), fe(ii), the curve indicates the rapid progress of sorption. after 5 min of sorbent-sorbate interaction, approximately 80 % of metal cations were removed from the solution. fig. 2. comparison of metal sorption efficiencies η (black squares) and changes of ph values (empty triangles) over the experimental time. the residual time of experiment can be considered as a relative settled with slower changes of removal bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:23 utc nova biotechnol chim (2017) 16(1): 26-31 30 efficiency. values of cu(ii), zn(ii), and fe(ii) sorption show that ion removal might occur in twosteps staring with ion exchange and subsequent adsorption. the highest efficiency of cu(ii) removal from model solution (98 %) was obtained after 30 minutes. on the other hand, the highest efficiency of zn(ii) (88 %) and fe(ii) (85 %) removal were reached both after 45 min. changes of ph values in solutions were observed after adsorption indicating to physical and chemical processes resulting in heavy metal ions removal from aqueous solutions. as shown in fig. 2, use of poplar sawdust in dynamic conditions is connected with decrease (in case of cu2+) or increase (zn2+ and fe2+) of ph values in the tested samples. such a significant change of ph value has previously been observed at high initial concentration of dissolved heavy metals as a result of intensive ion exchange (holub et al. 2013). in all cases, changes of ph values were recorded after minutes of the adsorption. the most notable ph change was in case of fe(ii) removal (from 5.4 to 6.4). during intensive increase of ph, metal cations were absorbed by mechanism of ion exchange by hydrogen bonding, while with the decreasing metal ion concentrations the ion exchange is not so dominant and does not affect the ph (petrilakova et al. 2014). after completion of the ion exchange, the ph began to decrease gradually in all cases in our system to nearly input values (fig. 2). conclusions poplar sawdust was proved as a suitable product for removal of selected metals from solutions. the function groups of poplar sawdust were characterized by infrared spectra that confirmed the presence of hemicelluloses, cellulose and lignin. at static adsorption experiment the poplar wood sawdust exerted good capability to remove copper, zinc, and iron ions with efficiency of ~ 80 %. changes of ph at static conditions point to adsorption and ion exchange. at dynamic conditions the sorption processes for all model solutions reached 80 % efficiency already after 5 min. the highest removal efficiency of cu(ii) (98 %) was observed after 30 min, and of both zinc (88 %) and iron (85 %) after 45 min. significant changes of ph were recorded after 5 min of adsorption as a consequence of ion exchange between metal ions in model solutions and functional groups of poplar wood sawdust. the adsorption experiments showed that the poplar wood sawdust is suitable for metal removal from model acidic solutions and also provides promising perspective for use in purification of metals from wastewaters. acknowledgements this work has been supported by the slovak grant agency for science (grant no. 1/0563/15) and the cultural and education grant agency (contract no. 073tuke4/2015). references ahmad a, rafatullah m, sulaima o, ibrahim mh, chii yy, siddique bm (2009) removal of cu(ii) and pb(ii) ions from aqueous solutions by adsorption on sawdust of meranti wood. desalination 247: 636-646. ahmaruzzaman m (2011) industrial wastes as low-cost potential adsorbents for the treatment of wastewater laden with heavy metals. adv. colloid interface sci. 166: 36-59. asadi f, shariatmadari h, mirghaffari n (2008) modification of rice hull and sawdust sorptive characteristics for remove heavy metals from synthetic solutions and wastewater. j. hazard. mater. 154: 451-458. bailey se, olin tj, bricka rm, adrian dd (1999) a review of potentially low-cost sorbents for heavy metals. water res. 33: 2469-2479. crini g (2006) non-conventional low-cost adsorbents for dye removal: a review. bioresour. technol. 97: 1061-1085. fu f, wang q (2011) removal of heavy metal ions from wastewaters: a review. j. environ. manag. 92: 407-418. gardea-torresdey jl, becker-hapak mk, hosea jm, darnall dw (1990) effect of chemical modification of algal carboxyl groups on metal ion binding. environ. sci. technol. 24: 1372-1378. holub m, balintova m, pavlikova p (2013) removal of metal ions from acidic solutions using peat–a low cost sorbent. in: proceedings of the 13th international conference of environmental science and technology, athens, greece, september 5-7, 2013, 1-6. keränen a, leiviskä t, zinicovscaia i, frontasyeva mv, hormi o, tanskanen j (2016) quaternized pine sawdust in the treatment of mining wastewater. environ. technol. 37: 1390-1397. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:23 utc nova biotechnol chim (2017) 16(1): 26-31 31 kidalova l, stevulova n, terpakova e (2015) influence of water absorption on the selected properties of hemp hurds composites. p. periodica 10: 123-132. nayak a, bhushan b, gupta v, sharma p (2017): chemically activated carbon from lignocellulosic wastes for heavy metal wastewater remediation: effect of activation conditions. j. colloid interface sci. 493: 228-240. ngah ww, hanafiah makm (2008) removal of heavy metal ions from wastewater by chemically modified plant wastes as adsorbents: a review. bioresour. technol. 99: 3935-3948. petrilakova a, balintova m, holub m (2014) precipitation of heavy metals from acid mine drainage and their geochemical modeling. sspj. civil eng. 9: 79-86. rahman ms, islam mr (2009) effects of ph on isotherms modeling for cu (ii) ions adsorption using maple wood sawdust. chem. eng. j. 149: 273-280. ricordel s, taha s, cisse i, dorange g (2001) heavy metals removal by adsorption onto peanut husks carbon: characterization, kinetic study and modeling. sep. purif. technol. 24: 389-401. saifulnizam m, jaafar a (2010) reduction of fe (ii) and zn (ii) using fresh eichhornia crassipes. i: doctoral dissertation, universiti malaysia, pahang. schwanninger m, rodrigues jc, pereira h, hinterstoisser b (2004) effects of short-time vibratory ball milling on the shape of ft-ir spectra of wood and cellulose. vib. spectrosc. 36: 23-40. šćiban m, radetić b, kevrešan ž, klašnja m (2007) adsorption of heavy metals from electroplating wastewater by wood sawdust. bioresour. technol. 98: 402-409. shukla a, zhang yh, dubey p, margrave jl, shukla ss (2002) the role of sawdust in the removal of unwanted materials from water. j hazard mater 95: 137-152. singh dk, tiwari dp, saksena dn (1993) removal of lead from aqueous solutions by chemically treated used tea leaves. j environ sci eng 35: 169-177. zhang p, dong sj, ma hh, zhang bx, wang yf, hu xm (2015) fractionation of corn stover into cellulose, hemicellulose and lignin using a series of ionic liquids. ind. crops prod. 76: 688-696. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:23 utc nova biotechnol chim (2017) 16(2): 132-137 doi: 10.1515/nbec-2017-0018  corresponding author: veronika.urickova@stuba.sk  nova biotechnologica et chimica distinguishing between juniper-flavoured spirit drinks from different producers veronika uríčková, kinga dobóová and jana sádecká institute of analytical chemistry, faculty of chemical and food technology, slovak university of technology, radlinského 9, bratislava, sk-812 37, slovak republic article info article history: received: 27th march 2017 accepted: 6th december 2017 keywords: beverage chemometrics fluorescence spectroscopy abstract a simple method for classifying juniper-flavoured spirit drinks is proposed based on the ratio of fluorescence intensity values in synchronous fluorescence spectra. receiver operating curves (roc) and linear discriminant analysis (lda) were used to compute the performance of the classification. significant differences in the fluorescence intensity ratios (i316/i287 and i324/i287) observed in the spectra recorded using wavelength difference 10 nm were evaluated by roc analysis to identify cutoff values that gave ideal aucs equal to one, thus allowing for 100% correct classification of the samples according to producer criteria. lda showed that drinks of different producers could be distinguished (100% correct classification) on the basis of their differences in the fluorescence intensity ratios (i323/i287, i324/i287, i316/i287 and i325/i287). these results show that complete synchronous spectra are not required to discriminate between producers. instead of them, fluorescence intensity could be measured at selected wavelengths.  university of ss. cyril and methodius in trnava   introduction juniper-flavoured spirit drinks are spirit drinks produced by flavouring ethyl alcohol of agricultural origin with juniper (juniperus communis l. and/or juniperus oxicedrus l.) berries. the minimum alcoholic strength by volume of juniper-flavoured spirit drinks shall be 30%. for a process without fermentation juniper berries are first slightly squeezed and then prepared with 30% drinkable alcohol. after a resting period the distillation is initiated. in enterprises where juniper mashes are still fermented the production of the brandy is done in a two-step distillation. a gas chromatograph coupled with a mass spectrometer is a configuration often used in the analysis of alcoholic beverages (vichi et al. 2008). automated sequential multidimensional gc/ms is a technique capable of producing matrix-specific libraries of complex products. spectral deconvolution of gc/ms data based on these libraries provides a reliable, unambiguous means of tracking the genealogy of juniper berry content from raw materials to final products (gins) and provides a more rationale means for detecting adulterants (robbat et al. 2011). however multidimensional gc/ms method is relatively expensive, time-consuming and requires highly skilled operators. up to date, several simple and rapid methods such as ultraviolet (uv), visible (vis), infrared (ir)and fluorescence spectroscopies have been tried for determining the origin of beverages (shen et al. 2012; azcarate et al. 2013; martelovidal et al. 2013). the last technique is particularly attractive because of its high sensitivity and excellent specificity. by combining fluorescence bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:14 utc nova biotechnol chim (2017) 16(2): 132-137 133 spectroscopy and chemometric method discrimination of red wines according to grape variety (airado-rodríguez et al. 2011; saad et al. 2016; silvestri et al. 2014; yin et al. 2009), typicality (dufour et al. 2006; yin et al. 2009), manufactures (yin et al. 2009) and geographical origin (dufour et al. 2006) or reliable classification of white wines according to grape variety (azcarate et al. 2015) can be successfully achieved. furthermore, adulterations of brandy can be identified and determined by using chemometric methods even if slight fluorescent spectral variations are observed for the samples (markechová et al. 2014). the combination of fluorescence spectroscopic data with uv/vis and near ir data has improved the grouping of single-malt whiskies according to their geographic origin (mignani et al. 2012). in our previous study, we applied general discriminant analysis and support vector machine to synchronous fluorescence spectra of juniperflavoured spirit drinks from different producers, thereby obtaining 100% correct classification of juniper-flavoured spirit drinksof three producers (uríčková et al. 2015). in this paper, a simplified method for classifying juniper-flavoured spirit drinks is proposed based on the ratio of fluorescence intensity values in synchronous fluorescence spectra. receiver operating curves (roc) and linear discriminant analysis (lda) were used to compute the performance of the classification. experimental samples a total of thirty-two commercially available samples from three slovak producers (code s1–s3) were collected in 2015 – 2016. different products from the same producer and four bottles of the same product were sampled: 16 samples [klasik slovenská borovička, klasik inovecká borovička, klasik borovička jemná, borovička borec (s1)], 12 samples [trenčianska juniperus borovička, juniperus borovička, koniferum borovička (s2)] and 4 samples [borovička zlatá (s3)]. the alcoholic degree ranged within 35 – 42% ethanol. the samples were stored at room temperature and analyzed without any prior treatment. fluorescence spectroscopy the acquisition of fluorescence spectra was performed using a perkin-elmer ls 50 luminescence spectrometer equipped with a xenon lamp, a 10 mm x 10 mm x 45 mm quartz cell and fl data manager software for spectral acquisition and data processing. the slits of monochromators, scan speed, acquisition interval and integration time were set at 5 nm, 200 nm min-1, 1 nm and 0.1 s, respectively. synchronous fluorescence (sf) spectra were collected by simultaneously scanning the excitation and emission monochromators in the excitation wavelength range from 200 to 450 nm, with constant wavelength differences δλ between them. the values of δλ were varied from 10 to 100 nm, in steps of 10 nm. sf spectrum was a plot of the variations in fluorescence intensity as a function of the excitation wavelength for a fixed δλ. three spectra were recorded for each sample and the average of the three replicates was used for further analysis. fluorescence intensities were plotted as a function of the excitation wavelength. software data were exported to ascii and processed with the microsoft office excel 2010 software. statistica version 7.0 (statsoft, usa, 2004) was used for lda. univariate roc curve analysis available on metaboanalyst home page http://www.metaboanalyst.ca was used (xia and wishart 2016). results and discussion synchronous fluorescence spectra in sf, a signal is observed only when ∆λ is in accord with the interval between one excitation band and one emission band. thus, the shape and intensity of the sf spectra depend on the ∆λ value used. fig. 1 shows the averaged sf spectra of samples from the three producers recorded at ∆λ = 10 nm, 20 nm, 30 nm and 40 nm. regarding ∆λ = 10 nm (fig. 1a), sf spectrum of the s1 brands showed two overlapping bands in the wavelength range from 308 nm to 330 nm with bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:14 utc nova biotechnol chim (2017) 16(2): 132-137 134 fig. 1. averaged synchronous fluorescence spectra recorded at (a) ∆λ = 10 nm, (b) ∆λ = 20 nm, (c) ∆λ = 30 nm and (d) ∆λ = 40 nm. table 1. the ratio of fluorescence intensity (i) values for spirits of different producers. parameter producer mean standard deviation minimum median maximum i316/i287 (  10 nm) s1 1.290 0.407 0.890 1.131 2.028 s2 0.415 0.124 0.261 0.424 0.634 s3 0.176 0.006 0.170 0.175 0.183 i324/i287 (  10 nm) s1 1.197 0.347 0.879 1.024 1.920 s2 0.438 0.135 0.260 0.470 0.640 s3 0.156 0.006 0.148 0.156 0.161 i306/i282 (  20 nm) s1 1.200 0.437 0.818 1.033 1.978 s2 0.354 0.031 0.316 0.364 0.406 s3 0.209 0.005 0.203 0.210 0.214 i298/i280 (  30 nm) s1 1.839 0.615 1.378 1.545 2.953 s2 0.697 0.076 0.601 0.705 0.816 s3 0.408 0.008 0.401 0.408 0.418 maxima at 316 and 324 nm as well as two overlapping less intense bands in the wavelength range from 260 nm to 290 nm with maxima at about 274 and 281 nm. sf spectrum of the s2 brands showed a maximum at about 286 nm, a shoulder at 273 nm and less intense band in the wavelengths from 310 nm to 330 nm with slightly observable maxima at about 315 and 324 nm. sf spectrum of the s3 brand showed the highest fluorescence of all the samples with a maximum at about 287 nm and two less intense overlapping bands with maxima at 314 and 324 nm. regarding ∆λ = 20 nm (fig. 1b), broadening in spectral bands, increasing fluorescence intensity of all bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:14 utc nova biotechnol chim (2017) 16(2): 132-137 135 table 2. area under the roc curve and cut-off value for discriminating different producers using fluorescence intensity ratios.  (nm) ratio of fluorescence intensity s1 and s2 versus s3 s1 versus s2 and s3 auc cut-off auc cut-off 10 i316/i287 1 0.222 1 0.762 10 i324/i287 1 0.211 1 0.759 20 i306/i282 0.964 0.265 0.938 0.612 bands and changes in their relative intensities were observed. in addition, fluorescence maxima were blue-shifted to 262, 274, 306 and 323 nm for s1 brands; to 263, 282 and 304 nm for s2 brands or to 282, 305 and 323 nm for s3 brand. at higher ∆λ values, a further increase in fluorescence intensity together with broadening or disappearance of the bands was observed. fig. 1 suggests the potential of sf spectroscopy to differentiate between juniperflavoured spirit drinks from different producers, in particular on the basis of sf spectra recorded at small ∆λ value. a detailed analysis of the spectral features of 32 samples obtained at ∆λ = 10 nm showed that the ratio of fluorescence intensity values at 316 nm and 287 nm (i316/i287) was 1.209 ± 0.407 (mean ± sd), 0.415 ± 0.124 and 0.176 ± 0.006 for s1, s2 and s3 samples, respectively. other relevant differences in the ratio of fluorescence intensity values at 324 nm and 287 nm (i324/i287), at 306 nm and 282 nm (i306/i282) and at 298 nm and 280 nm (i298/i280) were observed in the spectra recorded using ∆λ = 10 nm, 20 nm and 30 nm, respectively (table 1). it is noticed that both fluorescence intensity ratios calculated from s1 group are always higher than those of s2 and s3 groups. receiver operator characteristic curve the main criteria (cut-off) for classifying the samples were based on receiver operator characteristic (roc) curves, which are often used to determine the cut-off point based on which subjects will be classified as either a positive or negative outcome (xia et al. 2013). univariate roc curve analysis available on metaboanalyst home page (http://www.metaboanalyst.ca) was used (xia and wishart 2016). metaboanalyst accepts a table with the sample values in rows and the feature labels in columns. the first column was a set of sample labels (s1, s2 and s3), the second column was a set of class labels (0 or 1) and next four columns was the ratio of fluorescence intensity values matrix. to find cut-off point for s1 samples, the class label was assigned a value of 0 for s1 samples and 1 for all other samples. to find cut-off point for s3 samples, the class label was assigned a value of 0 for s1 and s2 samples and 1 for s3 samples. the results for distinguishing s1 samples from s2 and s3 samples, and for separating s1 and s2 samples from s3 samples are shown in table 2. univariate roc curve analysis resulted in the areas under the curve (auc) and cut-off values (table 2) with a probability of 0.95. in general, the auc close to 0.5 means poor discrimination, whereas the auc higher than 0.9 indicates excellent separation between the two classes. regarding roc analysis of data based on spectra recorded at ∆λ = 10 nm, ideal aucs equal to one were obtained for i316/i287 ratio. the first cut-off value was 0.222, below which the sample was deemed as belonging to s3. above the second cut-off value, 0.762, the sample was deemed as belonging to s1. similar cut-off values were obtained for i324/i287 ratio (table 2) again based on ideal aucs. regarding roc analysis of data based on spectra recorded at ∆λ = 20 nm, slightly smaller aucs were obtained for i306/i282 ratio. based on cut-off values 0.265 and 0.612, one sample was incorrectly classified in both cases. roc analysis showed that i298/i280 ratio (∆λ = 30 nm) was not significant either for distinguishing s1 samples from s2 and s3 samples or for separating s1 and s2 samples from s3 samples. linear discriminant analysis (lda) in the next part, lda was applied to the ratio of fluorescence intensity values i315.5/i287, i316/i287, i316.5/i287, i317/i287, i323/i287, i324/i287 and i325/i287 based on spectra recorded at ∆λ = 10 nm. lda was bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:14 utc nova biotechnol chim (2017) 16(2): 132-137 136 table 3. stepwise discriminant analysis and raw (standardized) coefficients of discriminant functions. step variable wilks' partial p-level raw (standardized) coefficients lambda lambda root 1 root 2 1 i323/i287 0.177 0.634 0.002 37.37 (9.22) -17.09 (-4.22) 2 i324/i287 0.142 0.791 0.047 -33.71 (-8.88) 23.88 (6.29) 3 i316/i287 0.160 0.700 0.009 2.65 (0.82) -22.90 (-7.05) 4 i325/i287 0.123 0.913 0.307 -1.47 (-0.37) 19.62 (4.99) constant -3.88 -1.46 used to determine which variables most significant discriminate between three naturally occurring groups and in addition to find classification functions to predict group membership. to select the least number of variables, stepwise lda was performed and wilks’ lambda was calculated at each step. the variable with the smallest wilks’ lambda that improves classification was entered into the analysis. the results of the applied lda, according to producer criteria for each step are summarized in table 3. variables i315/i287, i316.5/i287 and i317/i287 were excluded by a stepwise method, taking into account their lower variability observed through the different producer samples. the partial wilks' lambda (the smaller the partial wilks' lambda, the greater is the contribution to the overall discrimination) indicates that variable i323/i287 contributes most, variable i316/i287 second most, variable i324/i287 third most, and variable i325/i287 contributes least to the overall discrimination. new two discriminant functions (roots), linear combinations of variables selected, were obtained to discriminate the different producers. the following two eigenvalues and canonic correlations (given in parentheses) were calculated: 4.964 (0.912) and 0.492 (0.574) explaining about 90.9 % and 100% of the total variance, respectively. raw canonical discriminant function coefficients obtained are also reported in table 3. a two-dimensional plot of discriminant functions derived from the four selected variables is shown in fig. 2. the first discriminant function (root1) mostly discriminates between s1 and the two others (s2 and s3). the second function (root2) provides some discrimination between s3 (all show negative values) and s2 (which have mostly positive values). however, the discrimination is not as clear as that provided by the root1. standardized canonical discriminant function coefficients (given in parentheses in table 3) indicate that the first discriminant function is marked mostly by variables i323/i287 and i324/i287, while the second function is weighted mostly by variables i324/i287 and i316/i287 and to a lesser extent by the other two variables. fig.2. projections of samples according to producer criteria in the space formed by the two discriminant functions. finally, the two classification functions were used for the classification of samples, with the result that 100% of samples were classified correctly according to producer criteria. the performance of the lda model was evaluated using the leaveone-out-cross-validation approach, which is the best alternative for small number of samples  less than 50 (molinaro et al. 2005). in this kind of validation, the sample set, itself, was used to validate the model. the model was repeatedly 32 times calculated leaving out a single sample and then used to predict the left-out sample. in crossvalidation step, all s1 and s3 samples were again classified correctly, however, three of s2 samples bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:14 utc nova biotechnol chim (2017) 16(2): 132-137 137 were specified as belonging to s3 group, leading to 90.6 % correct classification   conclusions significant differences in the fluorescence intensity ratios (i316/i287 and i324/i287) observed in the spectra recorded using ∆λ = 10 nm were evaluated by roc analysis to identify cut-off values that gave ideal aucs equal to one, thus allowing for 100% correct classification of the samples according to producer criteria. lda showed that drinks of different producers could be distinguished on the basis of their differences in the fluorescence intensity ratios (i323/i287, i324/i287, i316/i287 and i325/i287). all of the 32 samples that were used as input data for the analysis were also classified correctly. in addition, the results obtained by both roc and lda were similar. these results show that complete synchronous spectra are not required to discriminate between producers. instead of them, fluorescence intensity could be measured at selected wavelengths. acknowledgement this publication was supported by the competence center for smart technologies for electronics and informatics systems and services, itms 26240220072, funded by the research & development operational programme from the erdf. this work was supported by the stu grant scheme for support of excellent teams of young researchers. references airado-rodríguez d, durán-merás i, galeano-díaz t, wold j.p (2011) front-face fluorescence spectroscopy: a new tool for control in the wine industry. j. food compos. anal. 24: 257-264. azcarate sm, de araújo gomes a, alcaraz m, ugulino de araújo mc, camiña jm, goicoechea hc (2015) modeling excitation–emission fluorescence matrices with pattern recognition algorithms for classification of argentine white wines according grape variety. food chem. 184: 214-219. azcarate sm, cantarelli ma, pellerano rg, marchevsky ej, camiña jm (2013) classification of argentinean sauvignon blanc wines by uv spectroscopy and chemometric methods. j. food sci. 78: 432-436. dufour e, letort a, laguet a, lebecque a, serra jn (2006) investigation of variety, typicality and vintage of french and german wines using front-face fluorescence spectroscopy. anal. chim. acta 563: 292-299. markechová d, májek p, kleinová a, sádecká j (2014) determination of the adulterants in adulterant-brandy blends using fluorescence spectroscopy and multivariate 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[accessed: 2017, january 18]. xia j, broadhurst di, wilson m, wishart ds (2013) translational biomarker discovery in clinical metabolomics: an introductory tutorial. metabolomics 9: 280-299. xia j, wishart ds (2016) using metaboanalyst 3.0 for comprehensive metabolomics data analysis. curr. protoc. bioinformatics 55:14.10.1-14.10.91. yin ch, li h, ding ch, wang h (2009) preliminary investigation of variety, brewery and vintage of wines using three-dimensional fluorescence spectroscopy. food sci. technol. res. 15: 27-38. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:14 utc microsoft word 2_bardáčová et al nova biotechnologica et chimica 15-2 (2016) 114 doi 10.1515/nbec-2016-0012 © university of ss. cyril and methodius in trnava the activity of cell-wall modifying β-1,3glucanases in soybean grown in presence of heavy metals monika bardáčová1, marína maglovski2, zuzana gregorová2, yevheniia konotop3, miroslav horník1, jana moravčíková2, ján kraic4, daniel mihálik4, ildikó matušíková1 1 department of ecochemistry and radioecology, faculty of natural science, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic (ildiko.matusikova@ucm.sk) 2 institute of plant genetics and biotechnology slovak academy of sciences, akademická 2, p.o. box 39a, nitra, sk-950 07, slovak republic 3 educational and scientific centre “institute of biology and medicine”, taras shevchenko national university of kyiv, ukraine 4 department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic abstract: cell walls represent the first barrier that can prevent the entrance of toxic heavy metals into plants. the composition and the flexibility of the cell wall are regulated by different enzymes. the ß-1,3-glucanases control the degradation of the polysaccharide callose as a flexible regulation mechanism of cell wall permeability and/or its ability to bind metals under stress conditions. the profile and activity of ß-1,3-glucanases in the presence of heavy metals, however, has rarely been studied. here we studied these enzymes in four soybean varieties (glycine max) grown in the presence of cadmium ions. these analyses revealed three acidic and one basic enzyme isoforms in each soybean variety, but only two of the acidic isoforms in the variety moravians were substantially responsive to the presence of cd2+. since the responses of certain glucanases were detected mainly in the varieties sensitive to metal and accumulating high amounts of metals, we assume their role in the defense rather than strategic metal sequestration. key words: metal stress, glucanhydrolase, cadmium, plant defense, pr proteins 1. introduction increasing industrialization and urbanization result in emission of pollutants in the environment including toxic heavy metals. among the different heavy metals contaminating the environment, cadmium raises great concern, as it is ecotoxic and as such can heavily impact ecosystems. moreover, once taken up by plants it can enter the food chain endangering animals and humans. the cell wall is the first structure that comes in contact with heavy metals in soil. considerable amount of literature demonstrates the roles of the cell wall in heavy bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:43 utc 115 bardáčová, m. et al. metal response in sequestering heavy metals. the cell wall composition confers the ability to bind heavy metals via different functional groups (e.g., carboxyl, hydroxyl) deriving from the different polysaccharides, thus represents a both barrier and target of heavy metals. the binding and accumulation of cd in the cell wall prevents it from penetrating inside the cell (extracellular compartmentalization) where it can cause damage. on the other hand cd pollution might have severe repercussions on the development of secondary cell walls, by reducing their rigidity and robustness (popper et al., 2011). the composition and the flexibility of the cell wall are regulated by different enzymes, of which many have been shown as sensitive to metals. these include proteins involved in the oxidation and peroxidation of cell wall components (chaoui et al., 2004) and pathogenesis-related defense proteins of the glucanase family. the latter are responsible for degradation of the polysaccharide callose as one of the earliest sign of metal intoxication (piršelová et al., 2011). results indicate that the callose accumulation is not related to decreased enzymatic activity of glucanases and reinforce the hypothesis that accumulation of callose is primarily dependent on a higher rate of synthesis and/or deposition (piršelová et al., 2012; 2013). the callose-decomposing glucanases have been rarely studied in the context of heavy metal stress. to our knowledge there is available a single study on the activity of these enzymes after exposure to metal toxicity in the roots of maize and soybeans (piršelová et al., 2011). in this work we studied the activity of glucanases in a set of soybean cultivars and evaluated their potential role in metal detoxification. 2. materials and methods 2.1. plant material soybean seeds (glycine max l.), cultivars moravians, kent, gallec, cardiff were obtained from matex, s.r.o (veľké kapušany). seeds were sterilized with 0.5 % sodium hypochlorite for 5 minutes and germinated on wet filter paper in petri dishes. roots (6 – 8 mm) were replaced into petri dishes with experimental solutions of 50 mg/dm3 cd2+ in a form of cdcl2. control plant material was incubated on a distilled water. the seedlings were incubated in dark at 23 °c for 48 hours. fresh weight (fw) of seedling’s roots was measured at the end of each treatment on analytical scale (explorer pro ep 114 cm). index of tolerance (it, %) was calculated as a ratio of the weight of roots of experimental group to weight of roots of control group x 100 %. all biological determinations were performed in triplicate. 2.2. protein isolation protein extract was isolated by extraction buffer containing 20 % (v/v) glycerol, 1.5 % (w/v) polyvynilpyrrolidon, 10 % (v/v) 1 m tris-hcl (ph 8.0), 0.02 % (v/v) β-mercaptoethanol and 1 % (w/v) 100 mm phenylmetylsulfonylfluorid (sigma). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:43 utc nova biotechnologica et chimica 15-2 (2016) 116 concentration of proteins in the extract was determined according to bradford (1976) spectrophotometrically at wave lenghth 525 nm (ultrospec 1 000 uv/visiblespectropotometer, shimadzu, tokyo, japan). 2.3. spectrophotometry detection of glucanase activity overall glucanase activity in protein extracts was determined spectropfotometrically according to miller (1959). briefly, samples were mixed with 1 % (w/v) 3,5-dinitrosalicylic acid (dns) and 30 % (w/v) potassium sodium tartrate, and subsequently boiled for 5 minutes. after cooling the absorbance of samples was measured spectrophotometricaly at 540 nm (uv-1800 uv / vis (shimadzu). 2.4. in-gel detection of protein fractions with glucanase activity gel-electrophoresis separation of proteins was performed according to laemmli (1970) on the mini-protean tetra cell module (bio-rad). the 12.5 % (w/v) polyacrylamide gels contained 1 % (w/w) laminarin (sigma) as a substrate. electrophoresis was running under constant power of 18 ma in the stacking gel and 24 ma in the separation gel, for 4 h. after electrophoresis, proteins were re-natured by shaking the gels in 50 mm sodium acetate buffer (ph 5.0), 1 % (v/v) triton x-100 over night. for subsequent enzyme detection, the gels were transferred into fixing solution (7 % (v/v) acetic acid, 20 % (v/v) metanol) for 5 minutes. the fixed gels were boiled 3 – 10 minutes in a solution of 1 m naoh and 0.1 % (v/w) triphenyl tetrazolium chlorid (sigma) until red strips appeared indicating to fractions with glucanase activity. the gels were scanned by bio rad – gs-800 calibrated densitometer and stored in a 7 % (v/v) acetic acid. after detection of glucanases, the gels were stained for detection of total proteins with coomassie brilliant blue. proteins were loaded on the gels without heat treatment. molecular weights of proteins were estimated by co-electrophoresis of a protein ladder (mark 12 unstained standard, invitrogen) ranging from 2.5 to 200 kda. 2.5. statistical analysis each experiment was performed in triplicate. after verification of normal distribution and variance homogeneity the data was analysed by t test. 3. results and discussion soybean is an important staple food especially in asia, while its high nutrition value is well known. since soybean is considered as sensitive to metal toxicity and variable (genotype dependent) accumulation has been demonstrated (socha et al., 2015), we conducted a research aiming to study the soybean responses to metals bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:43 utc 117 bardáčová, m. et al. focusing on the activity of cell-wall modifying enzymes ß-1,3-glucanases. seeds of 4 soybean varieties, namely cardiff, gallec, kent a moravians, were germinated and exposed to solution with cadmium ions that has previously been shown as toxic but sub-lethal to soybean (socha et al., 2015). indeed, a strong negative impact on root growth and development was observed for each variety (p ≤ 0.01) (fig. 1). fig. 1. data on root biomass of soybean varieties grown in presence (dark columns) and absence (light columns) of cadmium ions. shown are the means ± sd (n = 4). above columns the tolerance index values are indicated for each variety. the effect of heavy metals on root biomass is considered a good indicator for sensitivity / tolerance to metal stress (piršelová et al., 2011), hence the measured values of tolerance index assigned the variety moravians as the most sensitive and the variety laurentiana the most sensitive to cadmium (fig. 1). these data are consistent with the amount of cadmium accumulated in roots: the metal uptake was the largest in the sensitive variety moravians, and relatively lower in the tolerant variety laurentian (fig. 2). fig. 2. accumulation of cd by roots of soybean genotypes. the data indicate average values from three experiments ± sd (n = 4). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:43 utc nova biotechnologica et chimica 15-2 (2016) 118 the enzymes of ß-1,3-glucanases belong to the repertoire of plant´s defense under different stresses, including metal toxicity. their total activities in plants, however, depend on many factors, including age, developmental stage and genotype (dobroviczká et al., 2013). total activity of glucanases in the roots of tested soybean varieties was variable (fig. 3). genotypic variations in the levels of enzyme have previously been confirmed for e.g. the chitinases another type of defense proteins (metwally et al., 2005). in each variety we measured significant changes in enzyme activity (p ≤ 0.05); while in the varieties cardiff and laurentian the enzymes were induced, in moravians and gallec we detected a significant reduction of their activity (fig. 3). previously piršelová et al. (2012) have described the induction of the overall activity of the glucanases of soybean (cv. korada) under the action of 500 mg/dm3 pb2+, and 300 mg/dm3 cd2+. on the other hand, inhibition of the activity was observed for 100 mg/dm3 as3+. in contrast in maize roots no significant changes were observed (piršelová et al., 2012). fig. 3. total activity of glucanase in roots of soybean cultivars under cd stress. shown are the means ± sd (n = 4). since the total enzyme activity comprises an entire set of isoforms in tissues (mészáros et al., 2013), we studied the soybean glucanases in in more detail after separation in polyacrylamide gels. in the enzyme profile of each variety we recorded the presence of at least 6 isoforms with glucanolytic activity of sizes ~ 140, 100, 70, 50, 40 and 25 kda (fig. 4a). previously piršelová et al. (2011) have detected in the variety korada only 5 isoforms of smaller sizes (65, 35, 32, 25 and 18 kda). such differences in protein size between the two studies may be due to the variety or technical reasons (different conditions of separation and isolation of proteins, the sensitivity of the detection system, etc.). polymorphism in the profile of defense proteins has rarely been described (mészáros et al., 2013). none of the detected enzyme fractions (separated based on size exerted responsivity to cadmium (data not shown). in contrast, piršelová et al. (2011) have observed a significant induction of two isoforms (32 and 35 kda) at much bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:43 utc 119 bardáčová, m. et al. higher metal doses. we further analyzed the individual glucanases separately for acidic / neutral and alkaline / neutral isoforms. these analyses revealed three acidic and one basic isoforms in each soybean variety (fig. 4b,c). two of the acidic isoforms in the variety moravians were substantially responsive to the presence of cd2+ (p < 0.05); the acid isoform a was significantly induced, while the isoform c was inhibited by the metal (fig. 4d,e). however, no such response was observable for any of the acidic isoforms in the other varieties (fig. 4d). previously a single acidic glucanase has been described in soybean (piršelová et al., 2011) that was induced by high cd concentration. in view of the differences in experimental conditions of the two studies, however, the equality of these isoforms remains uncertain. concerning the alkaline proteins we detected a single basic isoform with glucanase activity (fig. 4c), which was significantly induced by the presence of cd2+ (p < 0.05) in the varieties moravians and cardiff (fig. 4d). in earlier studies no basic isoform has been detected so far in soybean (piršelová et al., 2011). fig. 4. detection of differences between protein profiles of roots of soybean genotypes under cd stress. proteins were separated in laminarine-containing gels on sds–page (a) and native gels (b, c). band intensities of isoforms of acidic glucanases of soybean roots of 4 genotypes under cd stress (d, e, f) – shown are the means ± sd (n = 4). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:43 utc nova biotechnologica et chimica 15-2 (2016) 120 the increase but also the reduction of the activity of the individual isoforms suggests that glucanases likely play a different role in this type of stress. since the responses of certain glucanases were detected mainly in the varieties sensitive to metal and accumulating high amounts of cd2+ (fig. 1, 2, 4 c,d), we assume their role in the defense (e.g. by limiting cell wall permeability), rather than strategic metal sequestration. conversely, the minor changes of corresponding activities in the other cultivars might indicate to low importance of glucanases in this type of stress. metal tolerant varieties might utilize other components of the defense/tolerance e.g. the composition and thickness of the cell walls (yokoyama nishitani, 2004) or effective mechanisms for removal of oxidative stress (tounekti et al., 2011). in the varieties moravians and cardiff the identified glucanase isoforms might modulate the callose content in cell walls and affect their permeability and/or ability to bind metals (corrales et al., 2008). the glucanase enzymes in general regulate the callose turnover on the different types of stress (including toxic metals), or might contribute to a more specific defense against various types of metals (békésiová et al., 2008; mészáros et al., 2013; this study). for example, deposition of callose as a local and very flexible process was observed in roots cells exposed to various types of toxic metals (cd, pb and as) in soybean and corn (piršelová et al., 2012). 4. conclusions in conclusion, we identified glucanase isoforms in selected varieties of soybean that respond to presence of cadmium in growth media. studying the variety of cell wall responses to metals will allow us to identify specific mechanisms used to tolerate large doses of such pollutants. those mechanisms (including general biochemical responses or sequestering in the vacuole/cell wall) contribute to the chemical variety of cell walls and provide several possibilities for the cell walls to interfere with the toxic activity of metals. in this context, it is important to identify the natural targets of cd at the cell wall level and to figure out which of these targets can be modified to generate plants that either cannot absorb cd or can sequester cd in the cell wall thereby reducing or avoiding detrimental effects in terms of growth or productivity. acknowledgment: this work was supported by the bilateral project sk-bg-2013-0007 and by the project apvv-15-0051. we gratefully acknowledge the financial support for the stay of yg at ucm in trnava provided by slovak academic information agency saia. references corrales, i., poschenrieder, ch., barcelo, j.: boron-induced amelioration of aluminium toxicity in a monocot and a dicot species. j. plant. physiol., 165, 2008, 504-513. dobroviczká, t., piršelová, b., mészáros, p., blehová, a., libantová, j., moravčíková, j., matušíková, i.: effects of cadmium bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:43 utc 121 bardáčová, m. et al. and arsenic ions on content of photosynthetic pigments in the leaves of glycine max (l.) merrill. pak. j. bot., 45, 2013, 105-110. chaoui, a., jarrar, b., el ferjani, e.: effects of cadmium and copper on peroxidase, nadh oxidase and iaa oxidase activities in cell wall, soluble and microsomal membrane fractions of pea roots. j. plant. physiol., 161, 2004, 1225-1234. mészáros, p., rybanský, l., hauptvogel, p., kuna, r., libantová, j., moravčíková, j., piršelová, b., tirpáková, a., matušíková, i.: cultivar-specific kinetics of chitinase induction in soybean roots during exposure to arsenic. mol. biol. rep., 40, 2013, 2127-2138. metwally, a., safronova, v.i., belimov, a.a., dietz, k.j.: genotypic variation of the response to cadmium toxicity in pisum sativum. j. exp. bot., 56, 2005, 167-178. piršelová, b., mistríková, v., libantová, j., moravčíková, j., matušíková, i.: study on metal-triggered callose deposition in roots of maize and soybean. biologia, 67, 2012, 698-705. piršelová, b., matušíková, i.: callose: the plant cell wall polysaccharide with multiple biological functions. acta physiol. plant., 35, 2013, 635-644. piršelová, b., kuna, r., libantová, j., moravčíková, j., matušíková, i.: biochemical and physiological comparison of heavy metaltriggered defense responses in the monocot maize and dicot soybean roots. mol. biol. rep., 38, 2011, 3437-3446. popper, z., michel, g., hervé, c., domozych, d.s., willats, w.g., tuohy, m.g., kloareg, b., stengel, d.b.: evolution and diversity of plant cell walls: from algae to flowering plants. annu. rev. plant biol., 62, 2011, 567-590. socha, p., bernstein, n., rybanský, ľ., mészáros, p., gálusová, t., spies, n., libantová, j., moravčíková, i.: cd accumulation potential as a marker for heavy metal tolerance in soybean. isr. j. plant sci., 62, 2015, 160166. yokoyama, r., nishitani, k.: genomic basis for cell-wall diversity in plants. a comparative approach to gene families in rice and arabidopsis. plant cell physiol., 45, 2004, 1111-1121. received 12 october 2016 accepted 7 december 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:43 utc nova biotechnol chim (2020) 19(1): 30-36 doi 10.36547/nbc.v19i1.575  corresponding author: f.elhouiti@lagh-univ.dz nova biotechnologica et chimica in silico assessment of rhanterium adpressum sesquiterpenes inhibitory effect on 3 and 15-o-trichothecene acetyltransferases fatiha elhouiti, djilali tahri, mohamed ouinten and mohamed yousfi laboratory of fundamental sciences, university of amar telidji of laghouat, bp37g road of ghardaïa, laghouat 03000, algeria article info article history: received: 11th march 2020 accepted: 13th april 2020 keywords: 15-o-trichothecene acetyltransferase 3-o-trichothecene acetyltransferase docking homology modeling rhanterium adpressum abstract essential oils (eo) from leaves and flowers of rhanterium adpressum have shown to inhibit the mycelial growth and type b trichothecenes production. the four strains of fusarium culmorum and fusarium graminearum were inhibited with 0.25 μl.ml-1 of each oil. the inhibitory activity of 11 sesquiterpenes identified in these oils was here examined in silico against two key enzymes in the biosynthesis pathway of trichotecenes namely: 15-o-trichothecene acetyltransferase and 3-o-trichothecene acetyltransferase. in sesquiterpene composition, t-muurolol and α-eudesmol have the highest percentages ranging from 1.4 to 2.75 %. three-dimensional structures of these two enzymes were modeled using swiss-model with gmqe = 0.93 and qmean = -0.45 for 3-o-trichothecene acetyltransferase and gmqe = 0.93, qmean = -0.58 for 15-o-trichothecene acetyltransferase. by the results of docking, t-muurolol and α-eudesmol showed high affinity compared to 15-decalonectrin and deoxynivalenol. these molecules are all sesquiterpenes with no major conformational difference with an rmsd of 3.7 å and 3.5 å between 15-decalonectrin and α-eudesmol, t-muurolol respectively. the results of docking prove the inhibitory effect of r. adpressum eo sesquiterpenes on the enzymes of mycotoxins biosynthesis pathway of f. culmorum and f. graminearum.  university of ss. cyril and methodius in trnava introduction fusarium culmorum and fusarium graminearum are two closely related species, known by their pathogenicity and mycotoxin production of type b trichothecenes considered major determinants of their aggressiveness. it has been reported that these strains are more common on wheat grain in germany, netherlands and other countries with high levels of deoxynivalenol (don) (waalwijk et al. 2003; shah et al. 2005). two chemotypes have characterized these strains according to their production of trichothecenes: the niv chemotype, which includes isolates producing nivalenol and fusarenone x, and the don chemotype, which includes isolates producing don and acetyldeoxynivalenol (adon) (bakan et al. 2001). trichothecenes are a large group of chemical sesquiterpene epoxides therefore they all have in common a 12,13-epoxytrichothecene skeleton and characterized by a keto group in the c8 position and with its presence or absence can be distinguished type a trichothecenes and type b trichothecenes. both of f. culmorum and f. graminearum produce type b trichothecenes, including deoxynivalenol (don), nivalenol (niv) and their several acetylated derivatives. 3-acetyldeoxynivalenol (3adon) sub-chemotype was mailto:f.elhouiti@lagh-univ.dz nova biotechnol chim (2020) 19(1): 30-36 31 identified in f. culmorum while f. graminearum displayed 15-acetyl-deoxynivalenol (15adon) sub-chemotype (foroud and eudes 2009; yörük and albayrak 2012). the biosynthesis of fusarium trichothecenes has been studied in f. sporotrichioides, which produces t-2 toxin. in the biosynthetic pathway, many genes are involved such as tri 5, tri 6, tri 13, tri 7, tri 11, tri 3, tri 8 and tri 16 (garvey et al. 2009). the core structure of trichothecenes is formed from the cyclization of farnesyl pyrophosphate by trichodiene synthase, then the modifications of positions c4 and c15 are carried out by p450 monooxygenase/acetyltransferase followed by oxygenation of c8. finally, to protect itself, the fungus removes the acetyl group at c3 who added it at the beginning of the biosynthesis pathway (wagacha and muthomi 2007; yörük and albayrak 2012). in a previous study, the essential oils of the leaves and flowers of rhanterium adpressum showed remarkable effects on the mycelial growth of four strains of f. culmorum and f. graminearum (t5, bd17, inra 349 and inra 812). this effect was accompanied by a strong inhibition of type b trichothecenes production with 0.25 μl.ml-1 of each oil, adding that it has a variability in the chemical composition between the two oils that has influenced the biological activity. in this study, in silico modeling was performed to verify the inhibitory effect on the production of type b trichothecenes (sesquiterpene secondary metabolites) of sesquiterpenes present in the oil of r. adpressum on two key enzymes in mycotoxins biosynthesis in both strains: 15-o-trichothecene acetyltransferase (f. graminearum) and 3-otrichothecene acetyltransferase (f. culmorum). experimental plant material, extraction and identification collection of aerial parts of rhanterium adpressum was done in three months from each year: 2011, 2012 and 2013 in zelfana 660 km sse of algiers (32°23’46” n; 5°13’34” e). extraction of essential oils from dried aerial parts was performed with hydrodistillation using clevenger apparatus for 7 h. gc/ms analysis and identification of essential oils components were described in a previous study (elhouiti et al. 2017). homology modeling sequences of 15-o-trichothecene acetyltransferase from fusarium graminearum (pcd27416.1) and 3-o-trichothecene acetyltransferase from f. culmorum (ano39638.1) were downloaded from ncbi protein database; the number of amino acids for each protein is 517 and 511 respectively. enzymes structures were modeled with swissmodel workspace (biasini et al. 2014) based on the target-template alignment using promod3 version 1.1.0. in swiss-model, the modeling process comprises four steps, beginning from the identification of structural template(s), alignment of target sequence and template structure(s), model building and model quality evaluation where the best homology models were selected according to global model quality estimation (gmqe) and qualitative model energy analysis (qmean) statistical parameters (benkert et al. 2009; biasini et al. 2014). for flexible structure comparison, secondary structures of generated enzymes models were aligned with fatcat server (ye and godzik 2004). molecular docking structures of 11 sesquiterpenes from the essential oil of r. adpressum and structures of deoxynivalenol and 15-decalonectrin were downloaded from pubchem database and converted into pdb files with discovery studio 3.5. modeled structures of 15 and 3-otrichothecene acetyltransferase and ligands were imported to molegro virtual docker 6.0 (mvd). proper bonds, bond orders, hybridization and charges were assigned to imported molecules in preparation process. the score function used is moldock score with a resolution of 0.30 å and the coordinates of the search space are: x: 8.56, y: 32.65, z: 25.51 at 10 å. the selected search algorithm is moldock optimizer 10 runs with optimization of the energy and optimization of h-bonds at the end of the docking. the other parameters for this evolution algorithm are: population size = 50, maximum interactions = 2000, scaling factor = 0.5, cross over rate = 0.9 nova biotechnol chim (2020) 19(1): 30-36 32 table 1. percentages of sesquiterpenes in the essential oil of r. adpressum. 2011 2012 april may june april may june peak area [%] peak area [%] α-humulene 0.48 0.37 0.41 0.46 0.41 0.12 germacrene d 0.32 0.37 0.46 0.31 0.26 0.55 bicyclogermacrene 0.51 0.66 1.24 2.08 1.25 0.05 β-bisabolene 0.20 0.24 0.33 0.13 0.15 0.19 γ-cadinene 0.49 0.55 0.54 0.47 0.41 0.34 δ-cadinene 1.03 0.96 0.63 0.61 0.85 0.07 γ-selinene 0.82 0.87 0.19 0.85 0.93 0.21 nerolidol 1.07 1.06 1.19 0.96 1.25 1.68 ledol 0.79 0.49 0.54 0.50 0.51 0.11 t-muurolol 2.41 2.31 2.75 2.27 1.40 2.12 α-eudesmol 2.47 1.96 2.26 2.06 2.02 1.42 and rmsd-based for termination scheme. results and discussion sesquiterpenes in r. adpressum essential oil sesquiterpenes are less volatile than other terpenes and have great potency for stoichiometric diversity and a strong odor (buckle 2015). in the total composition of the essential oil of r. adpressum, 36 components were identified represent 77 – 82 % of the total composition of which 11 sesquiterpenes belongs to hydrocarbons and oxygenated sesquiterpenes families represent 12 – 16 % of this composition (table 1). t-muurolol and α-eudesmol have the highest percentages ranging from 1.4 to 2.75 %. important biological activities have been reported for these sesquiterpenes. these include antiinflammatory effect of α-humulene in the essential oil from cordia verbenacea (fernandes et al. 2007), fungitoxic activity of bicyclogermacrene (bohlmann and zdero 1978; silva et al. 2007), antibacterial activities, antileishmanial and antiulcerogenic of nerolidol (hada et al. 2003; arruda et al. 2005; klopell et al. 2007), antibacterial activity of β-bisabolene (nascimento et al. 2007) and antitermitic activity of t-muurolol (cheng et al. 2004). in a previous study, variability between chemical groups and in the same group was noted, which may be related to the variation of different biotope conditions (elhouiti et al. 2017). yayi-ladekan et al. (2012) observed a diurnal variation in the chemical composition of the essential oil of ocimum canum which modified the synergistic action of these constituents in the results of the antimicrobial activity. these sesquiterpenes identified in the essential oil of r. adpressum have been suggested as molecules which may be involved in mycotoxins production inhibition of the strains of f. culmorum and f. graminearum observed in the previous study (elhouiti et al. 2017). the hypothesis was that their structural analogy with trichothecenes may play an important role in inhibiting the biosynthetic enzymes of these mycotoxins such as 15-o-trichothecene acetyltransferase and 3-o-trichothecene acetyltransferase. homology modeling modeling of both structures of 15-o-trichothecene and 3-o-trichothecene acetyltransferase with promod3 1.1.0 of swiss-model gave one model for 3-o-trichothecene acetyltransferase with gmqe = 0.93 and qmean = -0.45 (86.06 % of sequence identity to 3fot.1.a sequence template) and two models for 15-o-trichothecene acetyltransferase: the first one has 85.43 % of sequence identity to 3fot.1. a sequence template and gmqe = 0.93, qmean = -0.58. the sequence identity to the sequence template (1noc.2.a) for the second model was 12.64 % with gmqe = 0.14, qmean = -6.64. in order to select the most suitable templates, the most likely structural similarity is the value at which the joint distribution is maximized, called global quality estimation score (gmqe) expressed as a number between zero and one, where higher numbers indicate higher nova biotechnol chim (2020) 19(1): 30-36 33 table 2. docking analysis of r. adpressum sesquiterpenes, deoxynivalenol and 15-decalonectrin on 3 and 15-o-trichothecene acetyltransferases. 3-o-trichothecene acetyltransferase 15-o-trichothecene acetyltransferase moldock score rerank score internal hbond le1 le3 moldock score rerank score internal hbond le1 le3 α-humulene -71.5 -57.6 6.2 0 0.4 0.6 -76.2 -30.1 6.2 0 0.4 0.6 germacrene d -90 -69.4 -5.4 0 -0.4 0.1 -78.1 -62.4 -5.2 0 -0.3 0.1 bicyclogermacrene -70.8 -34.3 0.3 0 0 0.3 -76.9 19.1 0.3 0 0 0.3 β-bisabolene -93.4 -77.1 8.3 0 0.6 0.6 -86.4 -72.9 5.7 0 0.4 0.5 γ-cadinene -79.2 -63.2 7 0 0.5 0.5 -67.8 -56.3 6.2 0 0.4 0.5 δ-cadinene -70.5 -47.9 12.7 0 0.8 0.9 -59.6 -50.2 11.6 0 0.8 0.8 γ-selinene -68.9 -60.1 13.4 0 0.9 0.6 -75.5 -63.3 13.4 0 0.9 0.6 nerolidol -103.9 -88.8 9.5 -2 0.6 0.5 -91.5 -80.2 4.3 -2.4 0.3 0.2 ledol -73.5 -60.5 -0.5 -1.9 0 0.2 -76.6 -62.4 -0.5 -1.3 0 0.2 t-muurolol -67 -59.7 14.6 -2.5 0.9 0.7 -63.3 -55.9 12.7 -0.2 0.8 0.6 α-eudesmol -76.3 -64.2 17.5 -2.5 1.1 0.8 -77.5 -69.4 14.4 -2.2 0.9 0.6 deoxynivalenol -81 -68.1 31.8 -12.3 1.5 0.7 -69.2 -0.4 37.9 -8.8 1.8 0.7 15-decalonectrin -79.4 -19.9 18.6 -5.9 0.8 0.5 -72.5 -66.4 22.2 -2.5 1 0.6 reliability (biasini et al. 2014). global model quality is assessed with the local composite scoring function qmean calculated as indicators for the overall model quality and takes values between 0 and 1 (benkert et al. 2009; biasini et al. 2014). with the structural alignment of fatcat, the two structures of 15-o-trichothecene and 3-otrichothecene acetyltransferase are significantly similar (p ≤ 0.05) and the alignment has 496 equivalence positions with an rmsd of 0.21 å. the alignment also showed no major difference with the template structure (3fot) with an rmsd of 0.22 å and 495 to 489 equivalent positions for 15-o-trichothecene and 3-o-trichothecene acetyltransferase respectively. the structures of these two enzymes belong to a conserved protein domain family (pfam 07428), which represents a conserved region of approximately 400 residues. garvey et al. (2009) mentioned that in the structure of tri 3 from f. sporotrichioides (3fot) there are two protein domains: n-terminal and c-terminal forming between them at doughnut hole where the active site is located. 15-o-trichothecene and 3-otrichothecene acetyltransferase belong to the bahd superfamily acyltransferases which have a conserved sequence catalytic motif: hx3d and an important structural motif: dfgwg (d’auria 2006). in both enzymes (15 and 3-o-trichothecene acetyltransferase), the catalytic motif is represented by: his 183, leu 184, phe 185, trp 186 and asp 187. the residues that take place of the dfgwg motif in the c-terminal are: glu 447 ser 459. molecular docking in docking analysis, the affinity of the r. adpressum sesquiterpenes was elucidated in comparison with deoxynivalenol (tri 3 produced toxin) and 15-decalonectrin which is the native tri 3 substrate. binding ability of these sesquiterpenes to enzyme active site is given in terms of moldock score and rerank score (table 2). these two scores reflect the affinity of a ligand to the active site, therefore high values indicate a strong affinity, given that rerank score in mvd provides an estimation of interaction strength (heble et al. 2016). table 2 shows the best poses for each ligand among 65 resulting poses with their internal energies. le1 represents ligand efficiency 1, which is the ratio between moldock score and the number of heavy atoms while le3 is the ligand efficiency 3 results from the division of rerank score on the number of heavy atoms. r. adpressum sesquiterpenes have a strong affinity for the active site of the two enzymes 15-otrichothecene acetyltransferase and 3-otrichothecene acetyltransferase and the oxygenated sesquiterpenes can establish hydrogen bonds with the catalytic amino acids which makes their affinities greater than the sesquiterpene hydrocarbons (table 2). t-muurolol and α-eudesmol are the best inhibitors of these two enzymes compared to the native substrate 15-decalonectrin (fig. 1) knowing that most of the amino acid interactions of the active site with the substrate are hydrophobic (garvey et al. 2009). there is a slight structural difference between these nova biotechnol chim (2020) 19(1): 30-36 34 fig. 1. t-muurolol and α-eudesmol docking poses in active site of 15-o-trichothecene acetyltransferase (on the right) and 3-o-trichothecene acetyltransferase (on the left). two compounds and 15-decalonectrin with an rmsd of 3.7 å and 3.5 å for α-eudesmol and t-muurolol respectively. the analogy of these sesquiterpenes with the substrate explains the values of docking score more or less similar (table 2) and the high affinities to catalytic active sites of the two enzymes which can make these molecules competitive inhibitors. in catalytic process, his183 may not form a hydrogen bond with the c15 hydroxyl moiety, but does form a hydrogen bond with a trapped water molecule and the residues of the conserved loop of lys 421 pro 432 are proposed to allow access to the active site. asp 187 residue of the hx3d motif is directed away from the substrate, forming a salt bridge with arg 339 and participating in α-helix capping (garvey et al. 2009). in their study, umesh et al. (2020), showed molecular details on the high affinity of rutin, carpaine, stigmasterol, β-caryophyllene, and α-eudesmol against trehalose-6-phosphate phosphatase (an enzyme involved in trehalose biosynthesis in the pathway of infection and proliferation of several pathogenic microbes) with distances to active site amino acids between 1.7 and 2.7 å. in the study of bernal and coybarrera (2014), agarofuran was shown to be a very good inhibitor for n-myristoyl transferase, a target enzyme against protozoan parasitic diseases. on the other hand, the study of ogungbe and setzer (2008), showed that the monoterpene hydrocarbons (camphene, p-cymene, limonene, myrcene, α-pinene, and β-pinene) have a weak inhibitory α-eudesmol α-eudesmol t-muurolol t-muurolol nova biotechnol chim (2020) 19(1): 30-36 35 activity against cysteine protease cruzain, an enzyme that plays an important role in the penetration and proliferation of the parasite responsible for american trypanosomiasis, while the sesquiterpene hydrocarbons (β-caryophyllene, α-vopaene, germacrene d, α-humulene and α-zingiberene) showed relatively strong inhibition. conclusions essential oils of r. adpressum have shown a significant inhibitory effect on the growth and production of type b trichothecenes of f. culmorum and f. graminearum strains. in this study, it was shown that the sesquiterpenes and especially the oxygenated sesquiterpenes identified in r. adpressum oil may be responsible for the inhibition of mycotoxins production of these strains by their high affinities to biosynthesis enzymes like 15-o-trichothecene acetyltransferase and 3-o-trichothecene acetyltransferase. the results open up new perspectives in the study of the levels and mechanisms of action of these molecules of plant origin as alternatives in the fight against phytopathogenic agents. conflict of interest the authors declare that they have no conflict of interest. references arruda dc, d'alexandri fl, katzin am, uliana sr (2005) 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173: 53-61. microsoft word 5_pukančíková et al nova biotechnologica et chimica 15-2 (2016) 142 doi 10.1515/nbec-2016-0015 © university of ss. cyril and methodius in trnava natural microflora of raw cow milk and their enzymatic spoilage potential lucia pukančíková1, sabina lipničanová1, miroslava kačániová2, daniela chmelová1, miroslav ondrejovič1 1 department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic (miroslav.ondrejovic@ucm.sk) 2 department of microbiology, faculty of biotechnology and food sciences, slovak university of agriculture in nitra, tr. a. hlinku 2, nitra, sk-949 76, slovak republic abstract: the aim of this work was to identify the main microbiota in raw cow milk from dairy farm of slovakia and to describe the selected microorganisms responsible for thermostable protease and lipase production which can affected the quality of dairy products. the main bacterial classes identifying by maldi-tof ms were gammaproteobacteria (62 %), actinobacteria (19 %) and bacilli (12 %). the dominant microbial genus of raw cow milk was pseudomonas. from milk bacteria, the strain lactococcus lactis and from the family enterobacteriaceae, namely enterococcus faecalis, hafnia alvei, citrobacter braakii and raoultella ornithinolytica were observed in raw milk. the spoilage of milk products is caused by thermostable enzymes with lipolytic and proteolytic activity. qualitative proteolytic and lipolytic activities were performed on skin milk agar and olive oil, respectively. from 16 identified microorganisms, only 8 strains (p. fragii, p. gessardii, p. lundesis, h. alvei, c. braakii, r. ornithinolytica, kocuria rhizophila and candida inconspicua) showed protease activity. quantitative protease and lipase activities were determined by casein and olive oil, respectively. the highest both activities were measured for the genus pseudomonas. while lipases produced by all isolated microbial species lose enzymatic activity at 77 °c for 30 – 40 min, almost proteases showed comparable activities during whole pasteurization experiment at selected experimental conditions (70 °c, 40 min). key words: milk, microflora, enzymes, protease, lipase, spoilage 1. introduction the milk is an important raw material for food industry. its quality can be affected by various biochemical and microbial factors such as age and health of the production animals, their feed, season and some others (vanhaecke et al., 1990). in the term of quality milk, the microbial population is very important. the microorganisms may come from the cowshed, bedding material, the seasonal feed, the teat purity and the dairy equipment (cousin, 1982; vacheyrou et al., 2011). the main part of milk microflora includes bacteria such as lactococcus, streptococcus, leuconostoc, bacillus, microbacterium, micrococcus, staphylococcus, pseudomonas, aeromonas and acinetobacter but some fungal species such as candida, kluyveromyces and pichia can be present (delavenne et al., 2011; smaržija et al., 2012; quigley et al., 2011; 2013). the present of microorganisms in the cow milk is problematic for three main aspects, namely production of microbial toxins, biofilm production and production bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc 143 pukančíková, l. et al. of undesirable enzymes. production of microbial toxins can cause some health problems, but the present of these microorganisms is monitored and contaminated milk is not used in the next processing cycle. production of biofilms allows some problematic microorganisms to survive during thermal technological operation such as pasteurization. biofilm-producing microorganisms can cause faster spoilage of milk products. the last negative aspect of present of microorganism in the milk is production of enzymes which are stable at pasteurization temperature. these enzymes can cause physical and chemical instability of milk products. the main problematic enzymes are proteolytic and lipolytic enzymes. proteolytic enzymes may lead to the defects in the final dairy products, because the protein hydrolysis causes some negative properties such as bitter flavor in the milk and gelatinization of uht milk during prolonged storage. even small amounts of proteolytic enzymes can effectively hydrolyze peptide bonds in proteins and can change the certain properties of dairy products such as physico-chemical, functional and/or sensory properties (guinot-thomas et al., 1995; celestino et al., 1997; chen et al., 2003; martins et al., 2006; teh et al., 2012). lipolytic enzymes play the important role in increasing of oxidation lability of milk lipids by hydrolysis of triacylglycerols and production of more free fatty acids which are easily oxidized (teh et al., 2011). the aim of this work was to describe the key microorganisms responsible for enzymatic spoilage of milk products by production of thermostable enzymes with lipolytic and proteolytic activities which can affect the quality of milk and milk products. 2. materials and methods 2.1. isolation and identification of bacterial strains the raw cow milk was taken from slovak dairy farm (senica, slovakia). the milk used in experiments was aseptically collected from storage tank. the collected samples were diluted and applied onto both nutrition agars, namely mpa and mrs agar. the microflora was incubated for determination of cfu for 48 hours at 6, 25 and 50 °c of psychrophiles, mesophiles and thermophiles, respectively. on the base of microand macroscopic properties of colonies, microbial isolates were collected and re-streaking on new solid media which were marked as isolates with the serial number. 2.2. preparation of samples for maldi-tof ms analysis freshly biomass of each isolate was prepared by cultivation on mpa agar for 24 hours at 30 °c, before identification of microbial isolates by maldi-tof ms. after then, one colony of microbial isolate was taken off from agar plate and solvated in 300 µl of distilled water and after intensively mixing, 900 µl of pure ethanol was added. the mixture was two-times centrifuged at 12,000 x g for 2 min. the pellet bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc nova biotechnologica et chimica 15-2 (2016) 144 of washed microbial biomass was dried at laboratory temperature and after then, 10 µl of formic acid was added. after mixing, 10 µl of acetonitrile was added and mixture was centrifuged at 12,000 x g for 2 min. the supernatant was prepared for malditof ms analysis. 2.3. identification by maldi-tof ms 1 µl of supernatant, prepared on the base of method described in the chapter 2.2., was applied on the maldi board and after drying, 1 µl of saturated solution of α-cyano-4-hydroxycinnamic acid in 0.025 % (v/v) of trifluoracetic acid in 50 % (v/v) water solution of acetonitrile was added. follows, sample was analyzed by maldi-tof ms (microflex lt/sh, bruker daltonics, germany) with flex software and the results were obtained by realtime classification software (bruker daltonics, germany). 2.4. enzyme determination 2.4.1. agar diffusion method the basic screening of ability of microbial isolates to produce of proteases and lipases was realized by agar diffusion method based on effect of enzyme on selected compounds, namely proteins and lipids forming turbidity of solid nutrition media. skim milk powder was used as substrate for proteases and olive oil as substrate for lipases. these additives were added to nutrient agar in amount of 10 % (w/v). the microbial isolates were inoculated on the cooled solid nutrient agar and incubated at 30 °c for 24 and 48 hours. the enzyme production was visualized by clarification of zone around producer colony after incubation. 2.4.2. protease production by selected microbial isolates for determination of protease production ability, microbial isolates were cultivated in 10 % (w/v) water solution of dry milk at 30 °c for 48 hours. after this time, cultivation media were centrifuged at 4,000 x g for 10 min and supernatants were used for enzyme activity determination. determination of protease activity was performed by the study of charney and tomarelli (1947) using azocasein as substrate. the enzyme reaction mixture contained 900 µl of 1 % (w/v) solution of azocasein in 5 mmol/l phosphate buffter (ph 7.4) and 100 µl of enzyme solution prepared in previous experiments. the enzyme reaction mixture was incubated at 30 °c for 15 min on the orbital shaker (150 rpm). after this time, 800 µl of 5 % (w/v) of water solution of trichloroacetic acid was added and mixture was intensively mixed on the vortex. formed precipitate was eliminated by centrifugation at 10,000 x g for 5 min. 100 µl of the clear reaction solution was pipetted into the wells of microtitration plates contained 50 µl of 0.5 mol/l sodium hydroxide. the resulted absorbance was measured at 405 nm. the protease activity was calculated on the base of proteolytic activity of standard protease isolated from bacillus subtilis (sigma aldrich, germany). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc 145 pukančíková, l. et al. 2.4.3. lipase production by selected microbial isolates for determination of lipase production ability, microbial isolates were cultivated in 10 % (w/v) water solution of dry milk at 30 °c for 48 hours. after this time, cultivation media were centrifuged at 4,000 x g for 10 min and supernatant was used for enzyme activity determination. determination of lipase activity was performed according to (benzonana and desnuelle, 1965). 100 µl of enzyme solution prepared in previous experiments was added into solution contained 5 g of olive oil and 12.5 ml of isopropanol. the enzyme reaction mixture was incubated at 30 °c for 30 min. after this time, 60 µl of standard solution of phenolphthalein was added and mixture was titrated by 0.25 mol/l potassium hydroxide to the pink color of solution stabile minimal 15 sec. the lipase activity was calculated on the base of proteolytic activity of standard lipase isolated from pseudomonas cepacia (sigma aldrich, germany). 2.5. thermal inactivation of enzymes the resistance of the enzymes to pasteurization was evaluated at 63 °c, 70 °c and 77 °c according to janness and koops (1962). the cultivation medium (100 µl) prepared by the method described in chapter 2.1. was heated in the test microtubes using a thermomixer (eppendorf, germany) at selected temperature (63 °c, 70 °c and 77 °c, respectively) during 40 min. during this time, the samples were collected and frozen immediately in liquid n2. after the measurement of the residual enzyme activity, samples were stored at -20 °c. the residual proteolytic and lipolytic activities were measured in all samples using the standard assays described in chapter 2.4.2 and 2.4.3, respectively. 2.5.1. determination of the enzyme half-life the enzyme half-life was calculated according to baur et al. (2015). the first step was calculation of inactivation rate constant (kd) by a first-order equation: tkt de e e −= 0 (1) so that: tk e e d t −=⎥ ⎦ ⎤ ⎢ ⎣ ⎡ 0 ln (2) where, kd is equal to the slope of the regression line from a plot of ln[et/e0] versus t at a particular temperature. the resulting regression lines had high correlation coefficients (r2 > 0.95), suggesting first order inactivation kinetics in the temperature range investigated. the half-life t1/2 was calculated based on equation (2), using equation (3): dk t 2ln 2/1 = (3) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc nova biotechnologica et chimica 15-2 (2016) 146 where et is the enzyme activity (u/ml) after a particular heating time, e0 is the initial enzyme activity (u/ml), t is the heating time (s), and kd is the inactivation rate constant (s-1). 2.6. statistical analysis all experiments were realized in triplicate. the obtained data were evaluated by excel (microsoft, 2010). 3. results and discussion the microbiota of raw milk is various due the possibility of contamination from cowshed, bedding material, the seasonal feed, the teat purity and the dairy equipment (glück et al., 2016). first step was determined the microbial population including psychrophilic, mesophilic and thermophilic microorganisms in raw cow milk by plate counting on selected media (mrs and mpa) (data not shown). isolated colonies showing differences in microand macro-scopic properties were re-cultivated as one-colony culture. from the whole microbial population (107 cfu/ml), the psychrophilic (4.2 × 106 cfu/ml) and mesophilic (5.1 × 106 cfu/ml) bacteria represented the major part of microorganisms of raw cow milk. the thermophilic bacteria represent only 1.3 × 102 cfu/ml. similarly, ercolini et al. (2009) found that the milk samples contain mainly mesophilic bacteria (5.0 × 103 – 6.0 × 105 cfu/ml) and psychrophilic bacteria (1.2 × 103 – 1.6 × 106 cfu/ml). after an initial screening, 30 variable microbial isolates differencing in micro/macroscopic properties were characterized and identified by maldi-tof ms. 3.1. maldi-tof ms identification of microbial isolates prepared microbial isolates were identified by maldi-tof ms. analysis was provided on the base of maldi-tof ms fingerprint of protein fraction microbial isolates. the obtained fingerprints were compared to the reference spectra of the biotyper database and their similarity was expressed as biotyper log (score). maldi-tof ms fingerprint of the isolate from raw milk was compared to reference maldi-tof ms profiles of the biotyper database. the dark grey, grey and colorless sticks in the top half colored panels indicating the peak matching (intensity and m/z value) between experimental and database reference maldi-tof ms profiles is excellent, medium and low match, respectively (table 1). the log(score) takes into account the number of matching peaks, the total number of peaks, the peak weight representing species specificity and a correlation factor related to the matching peak intensity (cherkaoui et al., 2010). the 30 isolates (6 psychrophilic, 11 mesophilic and 4 thermophilic) from raw cow milk isolated on mrs and mpa agar plates were identified by maldi-tof ms (7 isolates (27 %) with log(score) ≥ 2.3; 11 isolates (33 %) with log(score) 2.3 ≥ bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc 147 pukančíková, l. et al. and ≤ 2.0; 8 isolates (27 %) with log(score) 2.0 ≥ and ≤ 1.7. only 4 isolates were not identified for low log(score) (≤ 1.7). the 9 isolates differing in micro and macroscopic properties after cultivation on mrs and mpa plates were identified as the same species. 7 gram-positive and 9 gram-negative bacteria were identified among the isolates from milk samples. one of isolates was identified as the yeast candida inconspicua (table 1). ercolini et al. (2009) found that some milk samples do not contain gram-positive bacteria. in general, the number of gramnegative bacteria was higher than the number of gram-positive bacteria (ercolini et al., 2009). table 1. maldi-tof ms analysis of isolates from raw milk and peak matching between experimental and database reference maldi-tof ms profiles. number of isolate organism (best match) score value organism (second best match) score value 1 enterococcus faecalis 2.405 enterococcus faecalis 2.389 2 corynebacterium casei 2.016 not reliable identification 1.503 3 candida inconspicua 2.072 candida inconspicua 2.020 4 aeromonas media 1.909 aeromonas veronii 1.881 5 citrobacter braakii 1.716 not reliable identification 1.520 6 raoultella ornithinolytica 2.376 raoultella ornithinolytica 2.276 7 raoultella ornithinolytica 2.425 raoultella ornithinolytica 2.327 8 raoultella ornithinolytica 2.390 raoultella ornithinolytica 2.314 9 kytococcus sedentarius 1.921 kytococcus sedentarius 1.703 10 pseudomonas fragi 1.749 not reliable identification 1.693 11 not reliable identification 1.684 not reliable identification 1.481 12 acinetobacter johnsonii 2.035 acinetobacter johnsonii 1.842 13 raoultella ornithinolytica 2.392 raoultella ornithinolytica 2.373 14 raoultella ornithinolytica 2.353 raoultella ornithinolytica 2.258 15 lactococcus lactis 2.156 lactococcus lactis 2.111 16 not reliable identification 1.576 not reliable identification 1.573 17 sphingobacterium multivorum 1.987 not reliable identification 1.270 18 kocuria rhizophila 1.950 kocuria varians 1.837 19 pseudomonas gessardii 1.930 pseudomonas proteolytica 1.857 20 not reliable identification 1.323 not reliable identification 1.291 21 kocuria kristinae 2.103 kocuria kristinae 2.076 22 pseudomonas lundensis 2.029 not reliable identification 1.634 23 not reliable identification 1.317 not reliable identification 1.297 24 pseudomonas fragi 1.820 pseudomonas taetrolens 1.773 25 pseudomonas lundensis 2.068 pseudomonas taetrolens 1.993 26 pseudomonas fragi 2.159 pseudomonas taetrolens 1.741 27 lactococcus lactis 2.000 lactococcus lactis 1.953 28 hafnia alvei 2.092 hafnia alvei 2.083 29 micrococcus luteus 2.306 micrococcus luteus 2.277 30 hafnia alvei 2.080 hafnia alvei 1.981 legend: the dark grey (excellent match), grey (medium match) and colorless (low match) sticks in the colored panels indicated the peak matching between experimental and database reference maldi-tof ms profiles. the fig 1 shows the classification of identified microorganisms into groups of psychrophilic and mesophilic organisms presented in raw cow milk. the dominant psychrophilic microorganism in the milk sample was the bacterial genus pseudomonas (77 %) (fig. 1-a). similarly, in the culture of mesophilic organisms, the most bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc nova biotechnologica et chimica 15-2 (2016) 148 abundant bacterial microorganism was the microbial genus pseudomonas (20 %). it was observed the higher microbial diversity in the group of mesophilic microorganisms (fig. 1-b). vithanage et al. (2016) evaluated qualitative composition of microbial culture in raw cow milk by maldi-tof analysis. they found that the main bacterial classes are gammaproteobacteria, bacilli and actinobacteria. the major isolates (approximately 42 %) belongs to gammaproteobacteria (including pseudomonas), followed by bacilli (32 %) and actinobacteria (15 %). in our study, the main bacterial classes were gammaproteobacteria (62 %), actinobacteria (19 %) and bacilli (12 %). similarly to this study, the genus pseudomonas was the most abundant microbial genus in study of other authors (neubeck et al., 2015; vithanage et al., 2016) milk bacteria belong to dominant group of mesophilic microorganisms of raw cow milk. our isolate of milk bacteria was determined as lactococcus lactis. in addition to this species, marrok (2011) identified other species of milk bacteria such as lactococcus raffinolactis, leuconostoc mesanteroides, but the most numerous is lactococcus lactis. xin et al. (2017) found that bacterial composition of the raw milk is various and it affects different geographical areas. fig. 1. relative abundance of different microorganisms isolated from raw cow milk and identified by maldi-tof ms analysis. a – psychrophilic and b – mesophilic microorganisms; n.d. – not determined by maldi-tof ms analysis. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc 149 pukančíková, l. et al. from the group of undesired microorganisms, many gram-positive bacteria were determined such as streptococcus, enterococcus and staphylococcus (vithanage et al., 2016). in our samples, enterococcus faecalis, hafnia alvei, citrobacter braakii and raoultella ornithinolytica were determined. kagkli et al. (2007) suggested that the possible source of h. alvei can be the water. other undesired microorganism present in raw cow milk were clostridium (andersson et al., 1995), moraxella, listeria, corynebacterium, salmonella and escherichia (cousin, 1982; d´aoust, 1991; stark, 2000). except to bacteria, the raw cow milk can contain also yeast and fungi. in our samples, we determined the presence of the yeast candida inconspicua. similarly, quigley et al. (2013) identified some yeasts such as candida, kluyveromyces and pichia by next generation sequenation. delavenne et al. (2011) describe that in the cow milk can be present also rhodotorula, debaryomyces, geotrichum, trichosporon and cyptococcus. 3.2. identification of proteolytic and lipolytic isolates the ability of enzyme production by microbiota from milk was determined previously by several authors (hantsis-zacharov et al., 2007; teh et al., 2011; baur et al., 2015; glück et al., 2016). in total, 17 isolates were obtained from raw caw milk. the first screening of the ability of microbial isolates to produce enzymes, namely proteases and lipases, it was evaluated agar diffusion method. this method is based on the elimination of turbidity of solid media with proteins from skim milk powder or lipids from olive oil. the analysis of cultivation media was realized after cultivation of each microbial isolate at 6, 20 and 30 °c for 48 hours. the results are shown in table 2. from 17 identified species from raw cow milk, some species such as c. inconspicua, h. alvei, k. rhizophila, r. ornithinolytica (mesophiles) and c. braakii, p. fragii, p. gessardii and p. lundensis (psychrophiles) showed proteolytic activity (table 2). similarly, glück et al. (2015) found that some microorganisms isolated from cow milk produced peptidase activity such as pseudomonas, candida, microbacterium and chryseobacterium. similarly, ercolini et al. (2007) observed that the genera pseudomonas, hafnia and citrobacter were able to produce proteolytic activity on skim milk agar. lipolytic activity was not observed by this method (table 2). although, baur et al. (2016) observed that some species of pseudomonas, acinetobacter, chryseobacterium, candida, lactococcus and staphylococcus produced lipases. the growth of microbial isolates and their production of proteolytic enzymes depend on cultivation temperature. understandable, the temperature of 6 °c was suitable for growth of psychrophiles, but it was not suitable for growth of mesophiles (table 2). although, proteolytic activity was detected at 3 species from 6 identified psychrophilic bacteria from raw cow milk at 6 °c. all producers of proteolytic enzymes belong to the genus pseudomonas. the temperatures of 20 °c and 30 °c were suitable for growth of all isolated microorganism, but proteolytic activity was detected only at 8 species from 17 identified microorganisms from raw cow milk. after an initial determination of enzyme production, these 8 species were used for precise determination of their proteolytic and lipolytic activity. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc nova biotechnologica et chimica 15-2 (2016) 150 table 2. the proteolytic and lipolytic ability of identified psychrophiles and mesophiles from raw cow milk. microbial species 6 °c 20 °c 30 °c growth pa la growth pa la growth pa la p sy ch ro ph ile s acinetobacter johnsonii + + + citrobacter braakii + + + + + pseudomonas fragi + + + + + + pseudomonas gessardii + + + + + + pseudomonas lundensis + + + + + sphingobacterium multivorum + + + m es op hi le s aeromonas media + + candida inconspicua + + + corynebacterium casei + + enterococcus faecalis + + hafnia alvei + + + kocuria kristinae + + kocuria rhizophila + + + + kytococcus sedentarius + + lactococcus lactis + + micrococcus luteus + + raoultella ornithinolytica + + + legend: pa – proteolytic activity; la – lipolytic activity. 3.3. determination of proteolytic and lipolytic activity although psychrotrophic bacteria are not resistant to heat treatment of milk, their enzymes can survive the heat treatment used in dairy industry and to reduce milk quality (xin et al., 2017). they cause the coagulation and instability of milk (caldera et al., 2016). the proteolytic and lipolytic activities of microbial isolates from raw cow milk were determined on the base of their enzyme activity on their natural substrates such as azocasein and lipids (olive oil), respectively. the proteolytic activity was determined at ph 7.6 because it is known that thermophile peptidase produced by milk microbiota mainly belong to the class of metallopeptidase (ec 3.4.24.) and have an optimal ph between 6.5 and 8.0 (kohlmann et al., 1991; schokker and van boekel, 1997; koka and weimer, 2000; rajmohan et al., 2002; dufour et al., 2008). the results are showed in the table 3. table 3. proteolytic and lipolytic activity of selected microbial isolates from raw cow milk after 48 hours cultivation at 30 °c. microbial species proteolytic activity [u/ml] lipolytic activity [u/ml] candida inconspicua 30.5±2.1 0.67±0.1 kocuria rhizophila 24.9±0.2 1.50±0.2 raoultella ornithinolytica 30.6±0.8 1.33±0.2 acinetobacter johnsonii 19.9±2.1 1.17±0.4 citrobacter braakii 20.5±1.1 1.00±0.1 pseudomonas fragi 29.3±0.6 0.83±0.1 pseudomonas gessardii 70.5±4.3 0.83±0.2 pseudomonas lundensis 28.9±2.1 1.72±0.2 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc 151 pukančíková, l. et al. all of tested isolated species have proteolytic and lipolytic activity. from the literature, it is known that pseudomonas, candida and acinetobacter produce the highest both enzymatic activities (baur et al., 2015; xin et al., 2017). the genus pseudomonas can simultaneously produce proteases and lipases because the gen apra encoding metallopeptidase and the gen lipa encoding an extracellular lipase lie next to each other (woods et al., 2001; mccarthy et al., 2004; marchand et al., 2009; hasan et al., 2010; loper et al., 2012). the highest proteolytic activity was measured for the bacteria p. gessardii (70.5±4.3 u/ml) and the high lipolytic activity was observed for the bacteria p. lundesis (1.72±0.2 u/ml). 3.4. determination of spoilage enzyme thermostability the thermal inactivation of enzymes produced by microbial culture of milk is essential for obtaining of suitable dairy products (xin et al., 2017). some enzymes including lipases and proteases can withstand the thermal treatment during milk processing and then these enzymes can cause the spoilage of milk and dairy products. fig. 2. proteolytic (a) and lipolytic (b) activity of microbial isolates from raw cow milk after 40 min of pasteurization at selected temperatures (63 °c, 70 °c and 77 °c); ci – candida inconspicua; kr – kocuria rhizophila; ro – raoultella ornithinolytica; aj – acinetobacter johnsonii; cb – citrobacter braakii; pf – pseudomonas fragi; pg – pseudomonas gessardii; pl – pseudomonas lundensis. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc nova biotechnologica et chimica 15-2 (2016) 152 the thermal stability of enzymes presents in milk media fermented by our microbial isolates was determined. the analysis of enzyme thermostability during common pasteurization of cow milk was realized in the range 63 – 77 °c. the results are shown in fig. 2 which shows residual activity of proteases and lipases in milk medium after pasteurized at described temperatures (63 °c, 70 °c and 70 °c) for 40 min. the selected temperature conditions were appropriated for elimination of all studied microorganism (data not shown) but some enzymes were present and active also after pasteurization at the highest tested temperature (77 °c) for 40 min. generally, proteases were more resistant to thermal treatment as lipases. whereas lipases produced by all microbial isolates lose enzyme activity at 77 °c for 30 – 40 min, almost proteases showed the comparable activity during whole pasteurization experiment. fig. 3. the half-life of lipases produced by selected microflora of raw cow milk at described temperatures (63 – 77 °c) of pasteurization for 40 min. these data are confirmed by other authors who described that peptidases from pseudomonas spp. can withstand the heat treatments even in the uht region (110 – 160 °c) (adams et al., 1975; barach and adams, 1977; mu et al., 2009). the thermolability of lipases is best documented by half-life of these enzymes (fig. 3). 4. conclusions the dominant microbial genus of raw cow milk was pseudomonas. besides this, microbial genera aeromonas, candida, corynebacterium, enterococcus, hafnia, kocuria, kytococcus, lactococcus, micrococcus, raoultella, acinetobacter, citrobacter and sphingobacterium were presented and identified in raw milk. the main producers of spoilage enzymes were candida inconspicua, kocuria rhizophila, raoultella ornithinolytica, acinetobacter johnsonii, citrobacter braakii, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc 153 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fischer, l., hinrichs, j., scherer, s., wenning, m.: biodiversity of refrigerated raw milk microbiota and their enzymatic spoilage potential. int. j. food microbiol., 211, 2015, 57-65. quigley, l., o´sullivan, o., beresford, t.p., ross, r.p., fitzgerald, g.f., cotter, p.d.: molecular approaches to analyzing the microbial composition of raw milk and raw milk cheese. int. j. food microbiol., 150, 2011, 81-94. quigley, l., o´sullivan, o., stanton, c., beresford, t.p., ross, r.p., fitzgerald, g.f., cotter, p.d.: the complex microbiota of raw milk. fems microbiol. rev., 37, 2013, 664-698. reimmann, c., beyeler, m., latifi, a., winteler, h., foglino, m., lazdunski, a., haas, d.: the global activator gaca of pseudomonas aeruginosa pao positively controls the production of the autoinducer n-butyrylbereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc 155 pukančíková, l. et al. homoserine lactone and the formation of the virulence factors pyocynin, cyanide, and lipase. mol. microbiol., 24, 1997, 309-319. rieu, a., lemaitre, j.p., guzzo, j., piveteau, p.: interactions in dual species biofilms between listeria monocytogenes egd-e and several strains of staphylococcus aureus. int. j. food microbiol., 126, 2008, 76-82. smaržija, d., zamberlin, š., pogačić, t.: psychrotropic bacteria and milk and dairy products quality. mljekarstvo, 62, 2012, 77-95. stepanović, s., vuković, d., hola, v., di bonaventura, g., djukić, s., ćirković, i., ruzicka, f.: quantification of biofilm in microtiter plates: overview testing conditions and practical recommendations for assessment of biofilm production by staphylococci. apmis, 115, 2007, 891-899. teh, k.h., flint, s., palmer, j., lindsay, d., andrewes, p., bremer, p.: thermo-resistant enzyme-producing bacteria isolated from the internal surfaces of raw milk tankers. int. dairy j., 21, 2011, 742-747. vacheyrou, m., normand, a.c., guyot, p., cassagne, c., piarroux, r., bouton, y.: cultivable microbial communities in raw cow milk and potential transfers from stables of sixteen french farms. int. j. food microbiol., 146, 2011, 253-262. woods, r.g., burger, m., beven, c.a., beacham, i.r.: the aprx-lipa operon of pseudomonas fluorescens b52: a molecular analysis of metalloprotease and lipase production. microbiology, 147, 2001, 345-354. xin, l., meng, z., zhang, l., cui, y., han, x., yi, h.: the diversity and proteolytic properties of psychrotrophic bacteria in raw cows' milk from north china. int. dairy j., 66, 2017, 34-41. received 16 november 2016 accepted 8 december 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 14:53 utc nova biotechnol chim (2019) 18(1): 72-83 doi: 10.2478/nbec-2019-0010  corresponding author: articlesmoussaceb@yahoo.fr nova biotechnologica et chimica stabilization/solidification by hydraulic binders of metal elements from landfill leachate mira cheribet drouiche1, karim moussaceb1,, emmanuel joussein2 and jean-claude bollinger2 1 laboratoire de technologie des matériaux et de génie des procédés, faculté de technologie, université de bejaia, 06000 bejaia, algérie 2 université de limoges, pereine ea 7500 grese, université de limoges, 123 avenue albert thomas, 87060 limoges, france article info article history: received: 13th february 2019 accepted: 25th may 2019 keywords: ckd hydraulic binder leachates solidification/stabilization leaching tests tclp test abstract the objective of this work is to use stabilization/solidification (s/s) on the landfill leachates that often are heavily polluted by heavy metals and require proper treatment before discharge into the environment. the process consists of a s/s using a hydraulic binder in order to limit the solubility and mobility of the pollutants. while cement is the most used binder based on s/s values, in this study we substituted it by cement kiln dust (ckd) in two replacement ratios 25.50 and 100 %. the resulting effect on mechanical resistance and on retention of pollutants was evaluated. a metal (lead, iron and zinc) contaminated leachate from the landfill site of sidi-bouderham in algeria was mixed with an amount of cement and cement kiln dust in different proportions in order to optimize our formulations. the smooth paste was obtained and a standardized test of the test specimens was analyzed for mechanical resistance after 7 and 28 d of setting. our results show that f1p (100 % cement) and f2p (75 % cement + 25 % ckd) point on satisfactory mechanical strength and metal retention capacity. our approach suggests a promising approach for remediation of polluted sites.  university of ss. cyril and methodius in trnava introduction environmental protection has an important place in the national and international priority concern. with the emergence of new consumption, habits of populations, human health and quality of natural environments are threatened by the increasing amount of solid waste generated. to cope with this situation, political decisions have been made over the world to reduce the volume of waste to be stored: it must be kept at a minimum. in algeria, this situation poses a real problem, as it is not dealt with correctly. for this reason, a new policy of integrated solid waste management has been implemented, however its translation on the field by a sustainable environmental infrastructure program for the sound management of different types of solid waste, is still difficult to achieve as it requires improved collection and waste treatment services. in recent years, among the techniques used nowadays, solidification/stabilization (s/s) based on hydraulic binders offered multiple advantages. the s/s process stabilizes metals by sorption; lattice incorporation and precipitation during the cement hydration (cheng and bishop 1992; glasser 1997; conner and hoeffner 1998; malviya and chaudhary 2006; paria and yuet 2006; chen et al. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:15 utc mailto:articlesmoussaceb@yahoo.fr https://www.researchgate.net/institution/university_of_limoges nova biotechnol chim (2019) 18(1): 72-83 73 table 1. chemical analyses of portland cement cem i from ain el kebira (algeria). element sio2 al2o3 fe2o3 cao mgo so3 k2o na2o cem i content [%] 23.35 6.10 6.77 59.40 1.89 1.76 0.43 0.12 ckd content [%] 11.81 4.50 2.28 43.48 1.13 0.74 0.50 0.00 2009). cement matrix materials intended for immobilization vary considerably in their formulation. it is often economically and technically advantageous to add blending agents: e.g. slag and fly ash. these affect the chemical, mineralogical and microstructural constitution of the system (glasser 1997). several studies have been conducted by different researchers on s/s materials (barna et al. 1997; conner and hoeffner 1998; halim et al. 2003, 2004; mijno et al. 2004, 2007; qian et al. 2006; cyr et al. 2012; moussaceb et al. 2012, 2013; belebchouche et al. 2014, 2016). the characterization of the mechanism of leaching is very important to predict the long-term behavior of s/s materials, currently, the main tests used for characterization of leaching are: tank test and phdependence test, and a monolithic leaching test (mlt) to follow the long-term release of chemical species and pollutants (tiruta-barna et al. 2004; barna et al. 2005). this paper deals with the solidification/stabilization carried out on the leachate of the sludge from the landfill site of sidi-bouderham (algeria). on leachate and s/s materials we performed a chemical analysis (metals, sulphate, phosphate and chloride). our study contains several objectives: (1) the characterization of leachate from the landfill site to get an idea of the pollutant nature of this waste; (2) the treatment of polluted leachate in porous s/s matrices and the release of metallic species under different leaching conditions; (3) the study of the substitution effect of cement by ckd on the mechanical resistance and retention of heavy metals. experimental sample the leachate used in this study comes from the percolation of water onto the waste sewage mud from the landfill site of sidi-bouderham in algeria, classified as class ii. this leachate is then used as a liquid source for the formulation of cement pastes. the formulations investigated are based on ordinary portland cement (opc) cem i from the ain el kebira plant (algeria) mixed with cement kiln dust (ckd) from the hammam-dalaa plant (algeria). the chemical analyses of these opc and ckd are shown in the table 1. the formulations realized are based on the nf en 196-1 standard (afnor 2016). briefly, specimens is prepared by mixing cement and ckd together for 10 min in a mixer of 3 kg capacity (controls 65-l0005), in the way to obtain a homogeneous mixture. then, leachate is added and is mixed for 5 min. the ratio (eq. 1): (1) (where l (ml) refers to the amount of leachate, c (g) and p (g) is the mass of cement and ckd, respectively) was fixed in 0.32 on the way to obtain adequate hydration for a good homogeneity and a satisfactory discharge capacity; the table 2 gives the different formulations realized. finally, the cement mixtures are molded to prepare samples with dimensions of 4×4×16 cm3; three replicates were done for each. then, the molds are protected by a plastic film and they are stored in a room under a controlled temperature of (20±2 °c) for 28 d. table 2. formulation of each cement pastes realized. nomenclature cement [g] ckd [g] leachate [ml] f1p 450.0 0 145 f2p 337.5 114.5 145 f3p 225.0 225.0 145 f4p 0 450.0 145 chemical analyses and mineralogical characterization chemical analyses of dried solids were performed by x-ray fluorescence (xrf cubix panalytical).the infrared spectra of materials are performed using an ftir spectrometer (shimadzu iraffinity-1s); sample preparation was done as kbr disks. for all experimental tests (equilibrium bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:15 utc nova biotechnol chim (2019) 18(1): 72-83 74 and dynamic leaching tests), leachate solutions were previously filtered at 0.45 µm, the solution was then separated into two parts: the first was acidified at ph 2 with hno3 solution (65 %) for cation analysis, and then stored in the cold; the second was used for the determination of anions and of the soluble fraction. released metals (pb, zn and fe) were analyzed using graphite furnace atomic absorption spectrophotometry (shimadzu aa-6501f;cations (na+, k+ and ca2+) and anions (so4 2and po4 3-) were quantified by uv-visible spectroscopy (biotech engineering management co. ltd. (uk)) (rodier et al. 2009). all these analyses were performed in triplicates, and the reported values correspond to their arithmetic mean. mechanical strength the measures of compressive and flexural strength are realized after 28 d of cure onto the 4×4×16 cm3 monoliths according to nf en196-1 standard (afnor 2016), using a hydraulic press of type 65l11m2. leaching test according to the toxicity characteristic leaching procedure (tclp) the solidified/stabilized materials were assessed using the toxicity characteristic leaching procedure (tclp) as defined by u.s.epa (u.s.e.p.a. 1994). the sample specimens were crushed to reduce their particle size to less than 2.0 mm. the procedure involved shaking a 1 g crushed sample in 200 ml of 0.0992 m acetic acid + 0.0643 m naoh extraction solution (1/20 solid/liquid ratio) with a ph of 4.93±0.05, for 18 h on a rotary at about 300 rpm. the leachate was filtered through a 0.45 µm membrane filter to remove suspended solids and stored in the cold, then analyzed. acid neutralizing capacity (anc) test the purpose of this test is to determine the solubilization of chemical species as a function of ph, as well as the ability of the material to neutralize the acidic or basic solutions to which it is subjected (tiruta-barna et al. 2004; barna et al. 2005). the material was fragmented at ≤ 1 mm and immersed over a period of time in solutions containing a strong acid or base. the test itself consisted in putting in parallel various samples of the material into contact with acid or basic solutions of different concentrations. the moisture of the material is taken into account to obtain an accurate ratio l/s = 10 ml.g-1 of dry material. one of the leaching solutions was deionized water: it will allow us to obtain the natural ph of the material. the acidic solutions were prepared using nitric acid (1 m hno3) and the basic solutions with sodium hydroxide (1 m naoh). the flasks were submitted to a mechanical agitation during the time of the test at the rate of 10 turns/min. after 7 d the leachates were filtered using filter paper of 0.45 µm pore size and their ph and conductivity were measured. these leachates were then used for the determination of metals by the aas method, after being acidified to ph 2 with 65 % hno3, and for the determination of anions and the soluble fraction. pores water (pw) and maximum mobile fraction (mmf) tests this method consists in the extraction of water from the pores of the solid material. assuming that we can extract all the water by a filtration installation, the test consists by mixing finely ground material with different volumes of deionized water (tiruta-barna et al. 2004; barna et al. 2005). the material was crushed at a grain size ≤ 1 mm; solid samples were then immersed of deionized water at various l/s ratio (i.e. 200, 100, 50, 10, 5, 2 ml.g-1 of dry material) and subjected to mechanical agitation for 7 d (10 twists/min) at room temperature (23±3°c). after that, the leachates were filtered using filter paper of 0.45 µm pore size and characterized. monolithic leaching test (mlt) the leaching behavior of monolithic waste materials is part of their fundamental characterization tests. the mlt test is intended for characterizing the mass transfer mechanisms by observing the chemical elements flow released by the porous monolithic blocks when brought into bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:15 utc nova biotechnol chim (2019) 18(1): 72-83 75 contact with a fixed volume of leaching solution. the solution is renewed periodically and the dynamics of the release of some elements is determined by the physico-chemical analysis of the obtained leachate (tiruta-barna et al. 2004; barna et al. 2005). the specimens subjected to leaching were obtained by dry cutting of undried material, cleaned with compressed air, measured and weighed: their sizes were 4×4×4 cm3. the leaching solution (either deionized water or 50 g.l-1 sodium sulphate) was introduced at a ratio of liquid volume/block size (l/s) of 10 cm3.cm-2, what is enough to ensure dynamic behavior, in a hermetically closed container to prevent air penetration (carbonation from co2) and water evaporation during the experiment. for each material, all experiences were performed in duplicate. each solution where the sample is immersed was changed after 6 h, 18 h, 1 day, 2 days, 5 days, 7 days, 20 days, 28 days, giving a total of 64 days of continuous leaching. thus, we obtained 8 solutions whose physico-chemical parameters must be measured. at each renewal of the leaching solution, it was ensured that the time spent by the specimens out of the leaching was minimized. the leached solution was recovered after shaking the bottle and filtration to 0.45 µm, the filtered solid being put back into the flask for further leaching. thus, a new volume of leaching liquid was added, the flask was closed, and the new leaching sequence began. results because algerian regulations for waste disposal were recently available (official journal of the algerian democratic republic 2006) we applied their limiting values for liquid effluent discharge to assess the hazard behavior of the studied s/s wastes. leachate characterization the results of the physico-chemical characterization of leachate of the landfill site of sidi-bouderham are shown in table 3. table 3. physico-chemical characteristics obtained of leachate from of the landfill site of sidi-bouderham used in this study. element value standards ph 7.60 6.5 – 8.5 ce [ms.cm-1] 1.00 – sm [mg.l-1] 168.00 3.0 cod [mg o2.l -1] 5.00 120.0 – 130.0 pb2+ [mg.l-1] 20.00 0.5 zn2+ [mg.l-1] 40.30 3.0 fe2+ [mg.l-1] 30.23 3.0 na+ [mg.l-1] 31.83 – k+ [mg.l-1] 55.00 – cl [mg.l-1] 1,775.50 – so4 2[mg.l-1] 1,024.00 – po4 3[mg.l-1] 20.00 – the conductivity of the leachate is approximately 1 ms.cm-1, which indicates a significant presence of ionic species in solution. however, the presence of metals is effective such as pb, cu and fe, but zn is the present element (40.30 mg.l-1) which exceeds the standard value (3 mg.l-1). according to these results, the leachate can be considered as toxic and harmful; hence the need for treatment before storage. characterization of solidified/stabilized materials feasibility and chemical properties of s/s materials the chemical analyses of synthesized s/s materials are reported in the table 4. as expected, the chemical composition of the formulations is rich in sio2 and cao. moreover, the use of infrared spectroscopy (fig. 1) allows to evidence the structural characteristics of c-s-h phases (hydrated table 4. major elements of s/s materials for each formulations. element sio2 al2o3 fe2o3 cao mgo so3 k2o na2o others [%] [%] [%] [%] [%] [%] [%] [%] [%] f1p 21.36 5.32 6.46 56.55 1.40 1.53 0.41 0.12 6.85 f2p 21.32 4.79 5.39 54.40 1.70 1.31 0.44 0.11 11.17 f3p 17.80 4.84 4.53 51.68 0.97 1.10 0.49 0.11 13.20 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:15 utc nova biotechnol chim (2019) 18(1): 72-83 76 4000 3500 3000 2500 2000 1500 1000 500 10 15 20 25 30 35 a e d c b t ra n s m it ta n c e t (% ) wave number (cm -1 ) f1p 4000 3500 3000 2500 2000 1500 1000 500 10 15 20 25 30 35 40 wave number (cm -1 ) f2p e d c b a t r a n s m it ta n c e t (% ) 4000 3500 3000 2500 2000 1500 1000 500 10 20 30 40 50 60 f3p e d c b a t r a n s m it ta n c e t (% ) wave number (cm -1 ) fig. 1. infrared spectra of materials f1p, f2p and f3p, and identification of the bands observed on the ir spectrums. phases) by the presence of the si-o-ca bands near 990 cm-1 as well as formation of portlandite (characteristic band at 3,639 cm-1), carbonates (band at 1,420 cm-1) and ettringite (harmonics around 2,930 cm-1 and 2,853 cm-1 signs of the presence of clinker) during hydration period. this fact clearly explains the consolidation of the s/s materials obtained by the alkaline activation process. mechanical strength compressive and flexural strengths of solidified/stabilized materials were followed for 7 and 28 d of cure (fig. 2). in general, materials at 28 d of curing showed higher strengths than materials at 7 d of curing. this observation is common in studies and experiments dealing with concrete or cement strength reported in literature (atahan et al. 2009; zhao et al. 2013; belebchouche et al. 2014). the increase of cem 1 percentage in the formulation induces the increase of the mechanical strength (compressive and flexural) from 30 to 70 mpa in the case of the compressive one. as expected, the curing time favors such properties, which agree with the hydration process (bediako et al. 2015). the observed increased compressive strength results in this study might be due to the organic content present in the leachate, which might act as a dispersing agent, improving the dispersion of particles of cement and reducing clumping (silva and naik 2010). the effect of concrete mixing water has been widely investigated for different water sources other than ordinary freshwater. silva and naik (2010) investigated the use of partially processed sewage treatment plant water in concrete and found improvement in strength during 3 to 28 d. taha et al. (2010) have studied the use of brackish and crude produced water and found better strength even at the long curing period compared to tap water. katano et al. (2013) found that early and long-term strength for concrete mixed with high chloride content ingredients (seawater and sea sand) could be improved. al-jabri et al. (2011) reported no significant difference in strength for concrete mixed with car washing station waste water. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:15 utc nova biotechnol chim (2019) 18(1): 72-83 77 fig. 2. evolution of the mechanical strengths of solidified/stabilized formulations of leachates as a function of cement amount at 7 (red columns) and 28 (black columns) days. table 5. element concentrations after the tclp test of s/s materials. element concentration of leached elements [mg.l-1] percentage of release [%] pb2+ zn2+ fe2+ clpb2+ zn2+ fe2+ cl f1p 0.25 0.10 0.16 15.79 1.25 0.24 0.52 0.88 f2p 0.42 0.11 0.33 19.80 2.10 0.27 1.90 1.11 f3p 0.87 0.20 0.38 20.60 4.35 0.49 1.25 1.12 the addition of ckd in the formulation is not satisfactory as their influence on these mechanical properties is deleterious. several studies have been carried out to study the effect of cement substitution by ckd, bhatty (1984, 1985, 1986) studied binary, ternary and quaternary mixtures using ordinary portland cement (opc), five different ckds, two different types of fly ash (classes f and c) and slag. he observed that cements containing only ckd had reduced strength, set time and handling. the addition of fly ash to a ckd-opc system has reduced alkali content and improved strength. maslehuddin et al. (2009) evaluated the properties of cement kiln dust (ckd) blended cement concretes. the percentages of ckd were 0 %, 5 %, 10 % and 15 %, replacing cement astm c 150 type i and type v. the results showed that the compressive strength of concrete specimens decreased with the quantity of ckd. however, there was no significant difference in the compressive strength of 0 and 5 % ckd cement concretes. according to the data in fig. 1 it can be deduced that the desired strength can be obtained with 100 % of cem (f1p s/s formulation). leaching behavior of s/s materials according to tclp test to check the immobilization behavior of the solid s/s matrix against the contaminants and to determine the most efficient formulation, tclp test is performed and results are reported in the table 5. the concentrations of these elements are lower than those found on the leachate before treatment. the higher the cement dosage percentage, the lower are the concentrations. whatever the formulation of the s/s materials, the leaching values from tclp are under the regulated standards reported in table 3. transfer and leaching properties of solidified/stabilized materials according to the preceding data, it was decided to select the f1p and f2p s/s formulations (100 % and 75 % cem, respectively) for the next tests. results of acid neutralizing capacity (anc) test the solubilization of the chemical species contained in the f1p and f2p materials, depending bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:15 utc nova biotechnol chim (2019) 18(1): 72-83 78 fig. 3. solubility of chemical species in mmol.l-1 as a function of ph in the case of f1p (red circles) and f2p (blue circles) materials after anc test. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:15 utc nova biotechnol chim (2019) 18(1): 72-83 79 on the ph, is shown in figs. 3 a, b, c, d, e, f, g, h. the species can be classified into two categories: ph-independent species (phosphate) and phdependent species i.e. chloride, calcium, lead, chloride, sodium, potassium and sulphate. in the case of the ph-independent species, the solubilization of phosphates at the start of the acid attack, increases gradually (ph shift from 13 to 10) until it reaches a steady state concentration level. for the ph-dependent species, the amount of solubilized chloride is almost stable at the beginning of the acid attack (ph value shifts from 13 to 6) and an increase of dissolved chloride is observed when the ph value falls under 6. the maximum amount of clextracted is obtained for ph < 4. this can be attributed to the sorption of the chlorides within the c-s-h phases. indeed, the behavior of chloride ions may be considered as almost independent of ph. the same behavior is obtained for the sulphate that is slightly dissolved at the beginning of the acid attack (ph between 13 and 10). when the ph decreases, solubilization increases and becomes maximum when ph < 8. for zn, there is an increase of the solubilization at the beginning when ph > 10. a maximum of mobilization is achieved for 6 < ph < 8, and finally a decrease is observed for highly acidic ph this result is consistent with work of imyim (2000). fig. 4 ph and conductivity as a function of l/s ratio and solubility of chemical species as a function of l/s ratio of f1p (red circles) and f2p (blue circles) material. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:15 utc nova biotechnol chim (2019) 18(1): 72-83 80 for pb and fe, the solubilization regularly increases with the acidification of the medium. concerning k, the solubilization decreases from ph less than 9 to reach a steady-state value, the amphoteric behavior of lead accounts for the solubility minimum found between ph 8 and 10 combined with sharp solubility increases in both acidic and alkaline conditions (de windt et al. 2007). catherine (2003) found a slight increase in potassium solubilization for very alkaline (greater than 11) and acidic (less than 6). for na, it seems that the maximum values of solubilization is obtained for ph between 6 to 10 according to catherine (2003), sodium and potassium concentrations are independent of ph. pores water (pw) and maximum mobile fraction (mmf) tests the results showing the comparative evolution of ph , conductivity (fig. 4a) and the concentration of the monitored species depending on the variation of the ratio l/s for the material f1p and f2p during pw and mmf testing are recorded in the figs. 4b, c, d, e, f, j, h, i. the general evolution of the ph is an increase with decreasing l/s. the initial level of basicity is 12.3. the change in conductivity exhibits the same behavior as the ph. in fact, conductivity reflects the ionic strength of the solution then the solution of the pores of cement-based material has a somewhat high ionic strength. the maximum extracted quantities are recorded for low l/s ratios (imyim 2000; belebchouche et al. 2014). briefly, zn, fe and pb seem to have a solubility in accordance with the change in ph according to changes in l/s. the dependence of the concentration of sodium and potassium on the l/s ratio (linear with the l/s ratio in logarithmic scale) is explained by the fact that these elements come from the dissolution of highly soluble phases (imyim 2000). the chlorides behavior is like that of na with regular decreases when s/l increases. monolithic leaching test (mlt) the results of the physical parameters (ph and conductivity) of mlt tests are shown in fig. 5a and the evolution of the concentration of monitored species are shown in fig. 5b, c, d, e, f, g, h. whatever of the medium used (neutral or sulphated), the shape of curves is similar. the ph is always highly basic comprised between 11 and 12, we also observe that the ph increases over time, despite the renewal of leachates through the dissolution and diffusion of the soluble species contained in the material. the conductivity is constant along time of experiment. whatever, the elements studied, the flow decreases with time. the contents of the monitored species released into the sulphate medium are higher than those in the neutral medium; this is due to the higher ionic strength of the sulphated solution. discussion although the municipal landfill at sidi-bouderham (algeria) is dedicated to domestic household waste, it is well known that such a facility surely contains several kinds of contaminants, mainly potentially toxic metals and xenobiotic organic compounds (slack et al. 2005). among the possible treatments to immobilize such hazardous chemical species, s/s within cementitious matrixes can be a good choice; this method has been intensively applied to sewage sludge (valls and vazquez 2000; cyr et al. 2012). several studies have shown that the use of different ingredients added with cement, such as clays, zeolite, and fly ashes, allowed enhancing the performance of the s/s treatment (barth 1990; montgomery et al. 1991; adaska et al. 1998; balkan and kocasoy 2004; shi 2004; hunce et al. 2012). the studied leachate mainly contains 20 mg.l-1 pb(ii) and 1,775.50 mg.l-1 cl(-i) species, but also other potentially toxic metallic elements such as zn(ii) and cu(ii) and so4 2-. although the addition of fly ash to cement commonly improves both the mechanical and physico-chemical properties of such s/s materials (cyr et al. 2012), this is not the case in the present study with the addition of cement kiln dust ckd to ordinary portland cement opc. the tclp test on the s/s solid materials allowed to highlight the efficiency of 100 % opc addition (f1p sample) but the f2p sample with 25 % ckd can be another bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:15 utc nova biotechnol chim (2019) 18(1): 72-83 81 fig. 5. evolution of ph and conductivity as a function of time in neutral and sulphated medium, and concentration of the element flows as a function of average time (axis x; days) for the monolithic leaching test (mlt). indicated are data for neutral medium f1p (blue), sulphated medium f1p (red), neutral medium f2p (green), sulphated medium f2p (lila). good choice in spite of lower mechanical strength. according to the leaching test results, main pollutants in the leachate, pb, zn and fe were successfully solidified and approximately, 1.25 %, 0.24 % and 0.52 % of pb, zn and fe respectively released in the eluate water. for a somewhat similar case, the cementitious s/s of landfill leachate concentrate obtained after reverse osmosis has only attracted a scarce number of studies. hunce et al. (2012) added zeolite to cement, but the result was not good, they conclude that the use of zeolite as an aggregate material did not show significant differences. in the case of a zn-rich concentrate, kallel et al. (2017) have made use of clay brick waste as a substitute to coarse aggregate material in the composition of concrete mixtures; all metals but cr were effectively immobilized in such matrixes. conclusions following this study, the "s/s" stabilization/solidification method using hydraulic binders comprising artificial portland cement seems quite capable of treating contaminated leachates. it can retain metals pollutants bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:15 utc nova biotechnol chim (2019) 18(1): 72-83 82 in the leachate of the landfill. applying a series of leaching tests helps determine the leaching behavior of materials and pollutants, the effect of ph of the leachate was investigated by the nac test. ep tests and dmf provide quantities released by water leaching of the highly soluble elements such as na, k, and cl. the flow species released by the material in the mlt test inform about the transport mechanisms. the exploitation of the results allowed us to draw the following conclusions: the values of the mechanical strength of the s/s materials obtained are high. however, the resistance decreases with the increase of the amount of the substituted ckd, for the formulation f4p (100 % ckd) the resistance is lower than that recommended by the standard xp x 31-212, so it is not satisfactory for the manufacture of test pieces. it turns out that the ratio r = 0.32 is interesting. the latter has given satisfactory results by retaining almost completely the heavy metals from where the concentrations of the analyzed elements in the leachate of materials s/s return effectively within the range of algerian standards. the retention of heavy metals decreases with the increase in the amount of ckd whose release percentages of pb, zn and fe are respectively 1.25, 0.24 and 0.52 for f1p and 2.1, 0.27 and 1.09 for the formulation f2p. conflict of interest the authors declare that they have no conflict of interest. references adaska ws, tresouthick sw, west pb (1998) solidification and stabilization of wastes using portland cement, 2nd edition, portland cement association, skokie, 22 p. afnor (2016) norme française nf en 196-1 : méthodes d'essais des ciments partie 1 : détermination des résistances (septembre 2016). al-jabri ks, al-saidy ah, taha r, al-kemyani aj (2011) effect of using wastewater on the properties of high strength concrete. procedia eng. 14: 370-376. atahan hn, oktar on, tasdemir ma (2009) effects of water–cement ratio and curing time on the critical pore width of hardened cement paste. constr. build. mater. 23: 1196-1200. balkan m, kocasoy g (2004) industrial sludge solidification by using clinoptilolite. j. environ. sci. health, part a 39: 951-960. barna r, rethy z, tiruta-barna l (2005) release dynamic process identification for a cement based material in various leaching conditions. part i. influence of leaching conditions on the release amount. j. environ. manage. 74: 141-151. barna r, 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of metakaolin to stabilize sewage sludge ash and municipal solid waste incineration fly ash in cement-based materials. j. hazard. mater. 243: 193-203. de windt l, badreddine r (2007) modelling of long-term dynamic leaching tests applied to solidified/stabilized waste. waste manage. 27: 1638-1647. glasser fp (1997) fundamental aspects of cement solidification and stabilisation. j. hazard. mater. 52: 151-170. halim ce, amal r, beydoun d, scott ja, low g (2003) evaluating the applicability of a modified toxicity bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:15 utc nova biotechnol chim (2019) 18(1): 72-83 83 characteristic leaching procedure (tclp) for the classification of cementitious wastes containing lead and cadmium. j. hazard. mater. 103: 125-140. halim ce, scott ja, natawardaya h, amal r, beydoun d, low g (2004) comparison between acetic acid and landfill leachates for the leaching of pb(ii), cd(ii), as(v), and cr(vi) from cementitious wastes. 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(2009) properties of cement kiln dust concrete. constr, build. mater. 23: 2357-2361. mijno v, catalan ljj, martin f, bollinger jc (2004) compositional changes in cement-stabilized waste during leach tests – comparison of sem/edx data with predictions from geochemical speciation modelling. j. colloid. interface sci. 280: 465-477. mijno v, martin f, bollinger jc, catalan ljj (2007) stabilization/solidification process: comparison of synthetic sludge and volcanogenic massive sulphide tailings. process saf. environ. 85: 260-264. montgomery d, sollars c, perry r (1991) optimization of cement-based stabilization/solidification of organiccontaining industrial wastes using organophilic clays. waste manage. res. 9: 21-34. moussaceb k, aït-mokhtar a, merabet d (2012) influence of leaching conditions on the release kinetics of lead, chromium and nickel from solidified/stabilized cementitious materials. environ. tech. 33: 2681-2690. moussaceb k, belebchouche c, aït-mokhtar a, merabet d (2013) evaluation of the impact of ni, cr and pb contained in effluents of an industrial unit by the process of stabilization/solidification using hydraulic binders. int. j. environ. res. 7 : 485-494. paria s, yuet pk (2006) solidification-stabilization of organic and inorganic contaminants using portland cement: a literature review. environ. rev. 14: 217-255. qian g, cao y, chui p, tay j (2006) utilization of mswi fly ash for stabilization/solidification of industrial waste sludge. j. hazard. mater. 129: 274-281. rodier j, legube b, merlet n (coord.) (2009) l'analyse de l'eau (9° édition), dunod, paris. slack rj, gronow jr, voulvoulis n (2005) household hazardous waste in municipal landfills: contaminants in leachate. sci. total environ. 337: 119-137. silva m, naik tr (2010) sustainable use of resources – recycling of sewage treatment plant water in concrete. in paper presented at the second international conference on sustainable construction materials and technologies, ancona, italy, 28-30 june 2010. shi c (2004) hydraulic cement systems for stabilization/solidification. in spence rd, shi c (eds.) stabilization and solidification of hazardous, radioactive, and mixed wastes, crc press, u.s.a, p. 4977. taha r, al-harthy as, al-jabri k (2010) use of production and brackish water in concrete. paper presented at the. international engineering conference on hot arid regions (iechar), al-ahsa, kingdom of saudi arabia, 2010. tiruta-barna l, imyim a, barna r (2004) long-term prediction of the leaching behavior of pollutants from solidified wastes. advan. environ. res. 8: 697-711. u.s.-e.p.a. (1994) toxicity characteristic leaching procedure (tclp), method 1311, solid waste characterization manual sw-848, test methods for evaluating solid waste, physical/chemical methods. athens, georgia, usa. valls s, vàzquez e (2000) stabilization and solidification of sewage sludges with portland cement. cem. concr. res. 30: 1671-1678. zhao h, poon cs, ling tc (2013) properties of mortar prepared with recycled cathode ray tube funnel glass sand at different mineral admixture. constr. build. mat. 40: 951-960. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:15 utc nova biotechnol chim (2019) 18(1): 25-36 doi: 10.2478/nbec-2019-0004  corresponding author: tibor.maliar@ucm.sk nova biotechnologica et chimica biologically valuable components, antioxidant activity and proteinase inhibition activity of leaf and callus extracts of salvia sp. katarína vulganová1, tibor maliar2,, mária maliarová3, peter nemeček3, jana viskupičová4, andrea balážová5 and jozef sokol3 1 institute of physiotherapy, balneology and medical rehabilitation, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic 2 department of biotechnologies, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic 3 department chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic 4 centre of experimental medicine, institute of experimental pharmacology and toxicology, slovak academy of sciences dúbravská cesta 9, bratislava, sk-841 04, slovak republic 5 faculty of pharmacy of comenius university, kalinčiakova 8, bratislava, sk-832 32, slovak republic article info article history: received: 19th may 2019 accepted: 15th june 2019 keywords: antioxidant activity chemometrics hplc proteinase inhibition activity salvia sp. abstract sage is medicinal plant, known for its antioxidant and anti-inflammatory effects. eight extract samples were tested in this study: extract from salvia officinalis l. varieties from two different geographical localities (jaslovské bohunice and pobedim, slovakia), salvia officinalis l., variety "bicolor", salvia officinalis l., variety "purpurescens", salvia apiana, salvia divinorum, and two callus cultures of salvia sclarea l. and salvia aethiopis l. the highest values for composite parameters were observed for extract from salvia apiana. it can be concluded that prepared sage extract samples are rich on polyphenolic acids (2 950±265 g.ml-1 gaeq.) and amines (197±5.50 g.ml-1 trpeq.). hplc analysis confirmed the dominant content of rosmarinic acid in the extracts; the highest content was detected in the salvia apiana extract (1 120±15 g.ml-1). extract from salvia apiana expressed too the highest antioxidant activity (1 710 – 4 669 g.ml-1teac). similarly, the highest inhibition activity was observed for this extract on thrombin (57±3.3 %) and on other proteinases (over 80 %). spearman correlation analysis and pca analyses revealed a coherence between antioxidant activity of samples and their content of rosmarinic acid as well as inhibitory activity towards particular proteases, and revealed the significance of thiol based secondary metabolites. cluster analysis demonstrates the differences of salvia apiana extract from extracts of s. officinalis l., the group of s. divinorum extract and from callus cultures.  university of ss. cyril and methodius in trnava introduction sage is a common aromatic plant of the lamiaceae family, widely cultivated for its medicinal and culinary purposes. sage expresses various biological activities and medicinal properties such as antioxidant, antibacterial, antifungal, antiinflammatory, antispasmodic, astringent and antihidrotic (bouaziz et al. 2009; khan et al. 2011; hamidpour et al. 2014; razavi et al. 2014; bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:57 utc mailto:tibor.maliar@ucm.sk nova biotechnol chim (2019) 18(1): 25-36 26 martins et al. 2015). from the morphological point of view, the shrub of salvia apiana (the white sage) has long, thin, velvety leaves of colour ranging from shades of grey to green. small flowers of white sage can be purple or white with long anthers (adams and garcia 2005). this species can be found scattered throughout southwestern north america, with the highest abundance in southern california (borek et al. 2006). salvia divinorum, commonly known as diviner's sage, is a medicinal plant native to mexico and traditionally used by the mazatec peoples for spiritual practices due to its hallucinogenic properties. salvia sclarea l. is a spicy, aromatic plant, known as anti-inflammatory, antimicrobial, antioxidant, antispasmodic, hypoglycaemic and digestive agent (moretti et al. 1997; hammer et al. 1999; mantle et al. 2000; miliauskas et al. 2004). from herbal literature, aqueous extracts of the plant were used against various digestive disorders, as well as decoction and infusion for treating polyarthritis and acute rheumatics (peana and moretti 2002; rajagopal et al. 2013). given the ever-increasing interest in pharmaceutically important plantproduced compounds, scientists' attention is focused on their economically viable production potential. with advances in cellular, metabolic and gene engineering, plant cells and tissue cultures are now a very promising option for producing valuable phytochemicals (vanisree and tsay 2004; vanisree et al. 2004). in addition to the natural environment, plant material can grow under various conditions, for example be cultivated under specific conditions in closed containers as whole plants or their separate parts. phenolic acids and flavonoids represent the most frequent polyphenols, while soluble polyphenols can influence the positive effects of food more significantly. the polyphenolic content in sage varies with growing conditions and includes diterpenoids, flavonoids and phenolic acids and their derivatives. commonly found phenolic acids (derivatives) in sage are carnosic acid and its derivatives, rosmarinic acid (ra), methylrosmarinate (mer), caffeic acid (ca), salvianolic acid (salk), syringic acid (sa), and vanillic acid (va). it was reported, that ra derivatives from sage were more potent antioxidants in comparison to the present flavonoids, luteolin, and apigenin glycosides. earlier studies on the antioxidant activity of sage had been limited to the involvement of diterpenoid compounds. further, apigenin and its derivatives (e.g. apigenin 4'-methyl ether), scutellarein 6-methyl ether, isoscutellarein 8-methyl ether, luteolin and 6-oh-luteolin-6-methyl ether where published for s. officinalis (veličković et al. 2007). the main antioxidative effect of sage, however, has been reported to relate to the presence of carnosic acid and rosmarinic acid. the level of both compounds in the dry leaves varies widely depending on genetic factors and environmental conditions. while in general the main goal of any extraction is to maximize the mass transference of targeted compound from matrix to solvent, factors that influence the extraction of polyphenols are their stability, the method of extraction, plant matrix, ph, time, temperature, type and polarity of the solvent, and particle size (nell et al. 2008). from the medicinal point of view, there are important relationships between many pathophysiological processes in the body and proteases. trypsin (ec 3.4.21.4) acts as potential pathophysiological agent for both the acute and chronic types of pancreatitis (whitcomb 2003). thrombin (ec 3.4.21.5) plays a role in the coagulation disorder diseases, inflammation, and metastasis progression (bode 2006; scatena et al. 2007). urokinase (urokinase type plasminogen activator, upa) (ec 3.4.21.75) is an important component of the extracellular protease system specifically converting plasminogen to plasmin, participating in a number of pathophysiological processes, including extracellular matrix (ecm) lysis, thus tumour progression and metastasis of onco-transformed cells (krošlák et al. 2016). the main goal of this paper is to evaluate the antioxidant activity and proteinase inhibition activity in sage in relation to the content of biologically valuable component groups. research on inhibition activities of sage extracts or components on serine proteinases are published very rarely and it is a continuing of the similar papers in this field (fan et al. 2010). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:57 utc nova biotechnol chim (2019) 18(1): 25-36 27 experimental plant material the source of biological material for the experiments were leaves of salvia officinalis l. from two different geographical areas ‒ jaslovské bohunice and pobedim (slovakia), collected during year 2018. the sampled and dried sage leaves comprised the varieties "bicolor" and "purpurescens", further commercially purchased and dried leaves of salvia apiana and salvia divinorum. as explantate cultures in vitro are known for production of secondary metabolites (either directly or more often after elicitation), callus cultures of salvia sclarea l. and salvia aethiopis l. were also used. seeds harvested in medical plants garden of faculty of pharmacy, comenius university in bratislava (slovakia) were disinfected by the solution of 20 % (v/v) naclo (savo) for 20 min and subsequently washed three times with sterile distilled water. the seeds were placed on paper bridges in erlenmeyer flasks containing sterile distilled water. seeds germinated for 1 month under 16/8 h white light/dark period (22 000 lux) at 24 °c and relative humidity 70 %. hypocotyls acquired by separation of seedlings were under aseptic conditions transferred on paper bridges in erlenmeyer flasks with 25 ml of sterile liquid murashige-skoog growth medium (ms) supplemented either with kinetin (0.3 mg.l-1) and 1-naphthyl acetic acid (naa) (2 mg.l-1) (s. aethiopis l.) or with kinetin (0.1 mg.l-1) and 2,4-dichlorophenoxyacetic acid (1 mg.l-1) (s. sclarea l.). cultivation was performed under the conditions mentioned above. the produced calli of both salvia sp. were after 1 month of subcultivation transferred into fresh growth media enabling the formation of sufficient amount of biomass. extracts from all sage samples (dried leaves and callus cultures) were prepared by crushing followed by extraction with methanol 1 : 20 (w : v). the extraction process was carried out under reflux at 70 °c for 1.5 h. chemicals common laboratory chemicals, including dimethyl sulfoxide (dmso), methanol p.a., ethanol p.a., 2-propanol, chloroform p.a., tween 40, β-carotene type iv, sodium carbonate, sodium acetate and acetic acid were supplied by mikrochem (slovakia). 2,2-diphenyl-1-picrylhydrazyl (dpph), (±)-6-hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxylic acid (trolox), 5-(3-carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid (dtnb), phosphate buffer tablets, chromogenic substrate n-α-cbz-l-lysine thiobenzyl ester hydrochloride (z-l-lys-sbzl hydrochloride), 2,2’-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts), linoleic acid, ammonium persulfate, 2,4,6-tris(2-pyridyl)-s-triazine (tptz) and sodium dodecyl sulphate were purchased from sigmaaldrich (usa). all the enzymes used in the experiments were also supplied by sigmaaldrich. avenanthramides a, b and c were purchased from reseachem (switzerland). hplc analysis hplc separation, identification and quantification was performed using waters hplc system (waters, usa) equipped with alliance module with 2998 photodiode array (pda) detector, software empower 2 and waters symmetry c18 column (75 × 4.6 mm i.d., 3.5 μm). a gradient elution system consisted of solvent a (0.1 % formic acid in water) and b (0.1 % formic acid in methanol). a linear gradient of 5 to 100 % solvent b over 30 min at a flow rate of 1.0 mlmin-1 was used. the column temperature was set at 30 °c and the injection volume was 20 μl. detection of rosmarinic acid in extract samples was performed using diode-array detector at 325 nm. total polyphenols content determination total phenolics content (tpf) in sage extracts was determined by the method described by slinkard and singleton (1997) modified for microplate screening system. this method based on measurement of products of the reaction of phenol hydroxyl groups with the folinciocalteu reagent. the reaction mixture contained bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:57 utc nova biotechnol chim (2019) 18(1): 25-36 28 20 μl of extract/standard/ethanol (for sample/standard/blank) and 20 μl of folinciocalteu reagent. after 5 min, 200 μl of 100 g.l-1 sodium carbonate was added. after 30 min of incubation in the dark, the optical density at 760 nm (od760nm) was measured using microplate reader opsys (dynex, chantilly, virginia, usa). tpf was expressed as micrograms of gallic acid equivalent (gaeq.) per ml. total phenolic acids content determination total phenolic acids content (tpfa) in extracts of sage was determined by method described by singleton et al. (1999), reaction with arnov´s reagent modified for microplate screening system. 30 μl of extract extract/standard/ethanol as blank was mixed with 150 μl of distilled water, 30 μl of 0.5 mol.l-1 hcl, 30 μl of arnov´s reagent (100 g.l-1 sodium nitrite and 100 g.l-1 sodium molybdate in distilled water), 30 μl of 1 mol.l-1 sodium hydroxide, and 30 μl of distilled water. the optical density was measured at 490 nm (od490nm) using microplate reader opsys. tpfa was expressed as micrograms of caffeic acid equivalent (caeq,) per ml. total flavonoid content determination total flavonoids content (tfl) of sage samples was determined according to rakotoarison et al. (1997). intensity of staining solution after the reaction of flavonoids with aluminum chloride is proportional to the concentration of flavonoids. the reaction mixture consisted of the extract/standard solution/ethanol as blank (100 μl) and 20 μl of 50 g.l-1 aluminum chloride in methanol. after 30 min incubation, the optical density at 405 nm (od405nm) was measured using microplate reader opsys. tfl was expressed as micrograms of quercetin equivalent (qeq.) per ml. total thiol content determination for determination of total thiols content (tt) in the sample extracts, ellman's reagent, dtnb, was used (ellman 1959). the method is based on the reaction of sulfhydryl groups with dtnb yielding a yellow color product. briefly, 150 μl of dtnb in 0.05 mol.l-1 phosphate buffer, ph 7.0 was added to 20 μl of the extract/standard solution/ethanol as blank. od405nm was measured after 15 min of incubation using microplate reader opsys. tt was expressed as micrograms of cysteine equivalent (cyseq,) per ml. total amine content determination total amines content (ta) was determined using reaction with ninhydrin. the reaction mixture contained 20 μl of the extract/standard/ethanol as blank and 100 μl of the ninhydrin reagent (3.5 g.l-1 ninhydrin in 2-propanol). optical density at 630 nm (od630nm) was measured after 45 min of incubation at 42 °c using microplate reader opsys. ta was expressed as micrograms of tryptophan equivalent (trpeq.) per ml (kaiser et al. 1970). antioxidant activity by dpph method (dpph) the dpph radical scavenging activity of sage extracts was measured by dpph radical (•dpph) scavenging method modified for microplate screening system. a decreased absorbance of reaction mixture indicated higher free radical scavenging potency. aliquots of 25 μl of extract/standard/methanol for sample/standard/blank was mixed with 100 μl of dpph solution (3.05.10-4 mol.l-1 in methanol). after 10 min-incubation in the dark, optical density at 540 nm (od540nm) was measured using microplate reader opsys. antioxidant activity (aadpph) was expressed as micrograms of trolox equivalent (troloxeq.) per ml (brand-williams et al. 1995). antioxidant activity by abts method (abts) the 2,2'-azino-bis (3-ethylbenzothiazoline-6sulphonic acid) (abts) radical scavenging activity of sage extracts was measured by abts radical cation (abts·+) modified for microplate screening system. abts reagent was prepared by reaction of 7 mmol.l-1 abts with 2.45 mmol.l-1 ammonium persulfate in the volume of 10 ml, followed by incubation in the dark during 16 h, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:57 utc nova biotechnol chim (2019) 18(1): 25-36 29 after that was abts reagent adapted on the absorbance 0.7 at 730 nm. a decreased absorbance of reaction mixture indicated higher free radical scavenging activity. 25 μl of extract/standard/ethanol (sample/standard/blank) was mixed with 100 μl of abts reagent. after 10 min of incubation in the dark, optical density at 630 nm (od630nm) was measured using microplate reader opsys. antioxidant activity (aaabts) was expressed as micrograms of trolox equivalent (troloxeq.) per ml (ferri et al. 2013). antioxidant activity by frap method (frap) the ability of sage extracts to change the oxidation status of transition metals was measured by reduction of fe(iii)-2,4,6-tris(2-pyridyl)-striazine (tptz) complex, according to the method described by stratil et al. (2007) modified for microplate screening system. fe(iii)-tptz complex was prepared by mixing 10 ml tptz solution with 1 ml of sodium acetate buffer (ph 3.6) and 1 ml of 20 mmol.l-1 fecl3. an increased absorbance of reaction mixture indicated higher reducing ability. briefly, 165 μl of fe(iii)-tptz reagent was added to 35 μl of extract/standard/ethanol. after 5 min of incubation in the dark at 37 °c, optical density at 630 nm (od630nm) was measured using microplate reader opsys. antioxidant activity (aafrap) was expressed as micrograms of trolox equivalent (troloxeq.) per ml. antioxidant activity by reducing power method (rp) the ability to change the oxidation status of transition metals of sage extracts was measured by reduction of potassium ferricyanide, k3[fe(cn)6] complex, according to the method described by jayaprakasha et al. (2001) modified for microplate screening system. 30 μl of extract/standard/ethanol was mixed with 100 μl distilled water, 45 μl of 1 mol.l-1 hcl, 45 μl of 10 g.l-1 k3[fe(cn)6] solution, 15 μl of 10 g.l -1 sodium dodecyl sulfate solution and 15 μl of 2 g.l-1 fecl3 solution. the reaction mixture was incubated for 20 min at 50 °c, followed by measuring of optical density at 630 nm (od630nm). absorbance was measured using microplate reader opsys. an increased absorbance of reaction mixture indicated higher reducing ability. antioxidant activity (aa_rp) was expressed as micrograms of trolox equivalent (troloxeq.) per ml. proteinase inhibitory assays for determination of proteinase inhibitory activities in the extracts, photometric methods with chromogenic substrates z-lys-sbzl.hcl and dtnb were used. substrates were cleaved by trypsin (ia_ty), thrombin (ia_tr), plasmin (ia_pl) and urokinase (ia_ur) and released chromogen was detected at 405 nm. each well contained 0.6 mmol.l-1 substrate, 2.817.10-5 mmol.l-1 dmso and 20 μl of tested extract. the reaction started by the addition of the enzyme solution: 0.3 u.mg-1 of trypsin, 1.714 u.mg-1 of thrombin, 1.524 u.mg-1 of plasmin and 1.072 u.mg-1 of urokinase, each in potassium phosphate buffer (50 mmol.l-1, ph 7.0). the reaction temperature was set at 37 °c. the optical density differences were measured at 405 nm using microplate reader opsys for each sample as (eq. 1) (green and shaw 1979): δod = (od405nm in the 61-st min) – (od405nm in the 1-st min) (1) the inhibitory activity (ia) was calculated according to the following equation (eq. 2): ia [%] = [1 – (δodsample δodblank)] /(δodcontrolδodblank) x 100 (2) statistical analysis the experimental results obtained were organized into a data matrix, where the columns contained fourteen composite variables (tpc, tpac, tfc, ttc, tac, ra), five inhibitory (ia_ty, ia_tr, ia_ur, and ia_pl), four antioxidants (dpph, frap, abts and rp). in the next step, statistical methods were applied in order to evaluate the relationships between the variables studied (inhibitory activities and composites) and objects (genotypes). correlation analysis with spearmen correlation coefficient was used to reveal statistically significant correlations (α < 0.05; 0.01; bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:57 utc nova biotechnol chim (2019) 18(1): 25-36 30 0.001) between variables. the principal component analysis (pca) and cluster analysis (ca) were chosen to evaluate relationships among all the objects (genotypes) and/or for the variables studied. basic operations and calculations with data were performed by ms office 2010 and the statistical approach (pca, ca and correlation analysis) was carried out by jmp 9 software. results and discussion hplc eight extract samples were analysed by hplc on the content of selected polyphenols (rosmarinic acid, flavonoids luteolin and apigenin). rosmarinic acid, the dominant secondary metabolite, was found in all extract samples, while detection of two flavonoids – apigenin and luteolin will be a subject of the continuing study, with regard to published data (li et al. 2017; oudjedia et al. 2019). the fig. 1 represents a typical hplc chromatogram. the content of rosmarinic acid (ra) varied from 1 120±15 g.ml-1 (extract from salvia apiana) to 5.02±0.04 g.ml-1 (callus extract). the content of ra decreased in order salvia apiana > salvia officinalis l. > salvia divinorum > callus culture extracts. from results, it is evident that rosmarinic acid is typical secondary metabolite produced by differenced cells, not by callus culture cells. we can observe significant differences (p < 0.01) between particular taxon’s and varieties of sage. the particular chromatograms of eight prepared extracts are presented in fig. 1. the overlay of spectrum of rosmarinic acid (standard) and particular peak from sample 1 (extract from salvia apiana) are included. the similar content of ra was published e.g. by huafu et al. (2004), who reported that the content of rosmarinic acids in sage was 2 000 – 2 704 μg.g-1 in the aromatic herbs analysed. bandoniene et al. (2005) described that the concentration of rosmarinic acid in salvia sp. ranged from 1 000 to 930 μg.g-1, which are values comparable to our results. habán et al. (2019) described that the first time of harvesting of sage with respect to ra content is more appropriate; in the second collection period (under the same soil and climatic conditions) the plants are no longer able to produce a comparable amount of rosmarinic acid. the weather conditions of the monitored varieties statistically significantly influenced the content of ra. for the industrial application and utilization of sage as a source of rosmarinic acid it should be precisely considered and argued the selection of the taxon. fig. 1. particular chromatograms of eight sage extracts. the overlay of spectrum of rosmarinic acid as standard and subjected peak from sample 1 (extract from salvia apiana). the order of lines/extracts – 8 as follows: salvia apiana (black line), salvia officinalis l. (locality jaslovské bohunice, blue line), salvia officinalis l., variety "bicolor"(green line), salvia officinalis l., variety "purpurescens" (light blue line), salvia officinalis l. (locality pobedim, violet line), salvia divinorum brown line), callus 1: salvia sclarea (dark blue), callus 2: salvia aethiopis (dark green). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:57 utc nova biotechnol chim (2019) 18(1): 25-36 31 fig. 2. the content of composite parameters in eight prepared extract samples from native matter and callus culture of salvia sp. total phenolic content (tpc) percentage expressed as gaeq., total flavonoid content (tfl) percentage expressed as qeq., total phenolic acid content (tpca) percentage expressed as gaeq., total thiol content (ttc) percentage expressed as cyseq. and finally total amine content (tac) percentage expressed as trpeq., towards primary extract from salvia apiana. determination of component parameters all eight prepared extracts were tested on the content of composite parameters (see material and method section) as follows. results are presented in fig. 2. from results it is evident that a group of data for the measured parameters are for a particular extract sample identical and individual. the highest content of the composite parameters tested was observed for the extract from salvia apiana. relatively high content of all composite parameters was observed for four extract samples from salvia officinalis l. and on the contrary, relatively low content was observed for salvia divinorum and two extract samples from the both callus cultures. from general point of view, we can present that prepared sage extract samples are rich in polyphenolic acid (2 950±265 g.ml-1 gaeq.) and at second on amines (197.46±5.49 g.ml-1 trpeq.). from the presented results we can conclude, that the source of biologically active compounds is the dried matter of plants, but specifically for particular taxon’s. all data obtained from salvia officinalis l., salvia divinorum and from callus cultures are statistically significant on the level with p < 0.05 towards extract sample from salvia apiana. the obtained data about secondary metabolites obtained during year 2018 might differ from real time values in nature as both biotic and abiotic factors affect secondary metabolites content (murcia et al. 2017). determination of antioxidant activity parameters the primary data matrix consists of the antioxidant activities of eight extracted samples subjected to testing by all the methods used, dpph, abts, frap and rp. the results are shown in fig. 3. based on the achieved results it can be concluded that significant differences in expressed antioxidant activities among tested extract samples from native matter and callus culture of salvia sp. by all applied methods have been observed. the highest antioxidant activity was recorded by rp method (4 699±18 g.ml-1 troloxeq.) and the least antioxidant activity by the abts method (1 710±6 g.ml-1 troloxeq.). all the results reached from salvia officinalis l. salvia divinorum extracts and from callus cultures were statistically on the level with p < 0.05 towards primary extract sample from salvia apiana. callus cultures and salvia divinorum showed minimal antioxidant activity compared to native salvia apiana and salvia officinalis l. the antioxidant activity of presented extract samples correlates with content bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:57 utc nova biotechnol chim (2019) 18(1): 25-36 32 fig. 3. the antioxidant activity of eight prepared extracts from native matter and callus culture of salvia sp. determined by dpph, abts, rfap and rp methods (respectively). results as expressed as percentage of troloxeq., towards primary extract from salvia apiana. of secondary metabolites, due to fact, that secondary metabolites are major holder of antioxidant activity in plants (lee et al. 2016; pandey et al. 2018). determination of proteinase inhibition activity parameters in our work, we focused on the inhibitory activity of selected serine proteinases against four proteinases – trypsin acting as pancreatic disorder promoter, thrombin acting as promoter of diseases associated with coagulation, urokinase acting as promoter of metastatic process of onco-transformed cells and plasmin. plasmin-induced proteolysis had been described as a pathological mechanism for some diseases, e.g. cancer and certain viral diseases. the proteinases were selected as the third group of variables (secondary metabolites content, antioxidant activity parameters). as the result, the presented paper represents a continuity of several, yet published papers with identical methodology and alternate plant subject – barley (maliar et al. 2015), oat (krošlák et al. 2016) and poppy (krošlák et al. 2017), enabling comparison and discussion of obtained results. table 1. results of protease inhibition activity (ia) of eight extracts prepared from native matter and callus culture of salvia sp. on trypsin (ty), plasmin (pl), thrombin (thr) and urokinase (ur). extract sample ia ± std [%] ia_ty ia_pl ia_thr ia_ur salvia apiana 88.67±1.01 80.16±1.57 57.33±3.30 83.82±3.52 salvia officinalis l. (locality jaslovské bohunice) 55.76±3.94 80.81±1.63 59.21±4.45 64.17±4.45 salvia officinalis l., variety "bicolor" 44.79±2.94 31.17±1.36 31.88±1.33 31.74±0.92 salvia officinalis l., variety "purpurescens" 43.32±5.64 30.58±1.21 35.35±1.19 54.04±4.05 salvia officinalis l. (locality pobedim) 53.54±3.27 19.44±2.55 39.40±4.25 39.14±0.62 salvia divinorum 50.26±2.26 0 22.63±2.05 6.12±0.25 callus 1: salvia sclarea 3.80±0.12 9.00±0.26 0.21±0.01 0 callus 2: salvia aethiopis 5.04±0.51 36.74±1.32 16.84±0.87 0.93±0.04 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:57 utc nova biotechnol chim (2019) 18(1): 25-36 33 further, the selected secondary metabolites are main carriers of antioxidant activity as well as proteinase inhibition activity in the plant tissue (meijersen et al. 1991; fink et al. 2000; maliar et al. 2015; yu et al. 2018). the obtained results are presented in table 1. the highest antioxidant activity was observed for extract from salvia apiana, and ia varied from lowest on thrombin (57.33±3.30 %) up to over 80 % inhibition of other proteinases. relatively high antioxidant activity was observed for the four extract samples from salvia officinalis l., which reached values from percentile interval <20, 80>. these extracts inhibited most effectively the urokinase, however in variable manner. opposite to this, relatively low antioxidant activity (not more than 51 %, table 1) was observed for salvia divinorum and the two extract samples from both callus cultures. table 2. selected results of correlation analysis by spearmen method between all parameters (variables). statistically significant values of correlation coefficients are written with bold and indicated with asterisk. ty – trypsin; thr – thrombin; pl –plasmin; ur – urokinase; dpph, abts, frap and rp – particular antioxidant activity methods; tpf – total polyphenol content; tfl – total flavonoid content; tpfa – total polyphenolic acid content; tt – total thiol content; ta – total amines content; ra – the content of rosmarinic acid by hplc. achieved results proved significant inhibition activity of sage methanol extracts on selected proteinases. on the other hand, the measure of inhibition activity is strictly individual for particular taxon. secondary metabolites isolated in these extracts would require further research. statistical processing statistical processing was applied on the primary data matrix. it consisted of data for eight extract samples and fourteen parameters (variables). results are shown in table 2. the statistically significant values of correlation coefficients are written with bold and marked with asterisk. from the correlation analysis it is evident that several groups of parameters significantly correlate (table 1). except the known (published) logical correlation between proteinases (trypsin, thrombin and urokinase), antioxidant activity parameters (frap and rp vs. dpph and abts) and some composite parameters (tpf, tfl, tpfa and ra), we observed interesting correlation between antioxidant activity parameters (except abts) and inhibition activity on trypsin and thrombin and urokinase (respectively). this fact could be explained by properties of the polyphenolic compounds in the extract samples. these are good radical scavengers and possess relatively reactive aromatic hydroxyl groups, which serve as donors and acceptors of h-bond – key feature for inhibition mechanism in catalytic cavity of proteinase (maliar et al. 2004). similarly, correlations have broadly been published between composite parameters (tpf, tfl and tpfa) in relation to antioxidant activity by all the methods used (adomako-bonsu et al. 2017). interestingly, we found in this study a correlation between tt and inhibition activity on trypsin, thrombin and urokinase, and also an unexpected correlation between antioxidant activity (all methods) and tpf and tfl (respectively). this could indicate the presence of thio-substituted polyphenols or flavonoids in extract samples from salvia sp. parameter (variable) ia_ ty ia_ pl ia_ thr ia_ ur aa_ dpph aa_ abts aa_ frap aa_ rp tpf tfl tpfa tt ta ia_thr 0.81** 0.59 1 ia_ur 0.83** 0.64 0.95** 1 aa_dpph 0.76** 0.29 0.76* 0.71* 1 aa_abts 0.48 0.38 0.64 0.79* 0.67 1 aa_frap 0.81* 0.64 0.81* 0.83* 0.83* 0.74* 1 aa_rp 0.71* 0.57 0.76 0.83* 0.76* 0.79* 0.95** 1 tpf 0.69 0.71* 0.67 0.67 0.79* 0.60 0.91** 0.76* 1 tfl 0.91** 0.62 0.98** 0.98** 0.81* 0.71* 0.86** 0.81* 0.74* 1 tpfa 0.74* 0.83* 0.86** 0.91** 0.67 0.74* 0.93** 0.91** 0.83* 0.89** 1 tt 0.89** 0.52 0.93** 0.93** 0.88** 0.74* 0.83* 0.76* 0,76* 0.98** 0.81* 1 contents_ra 0.62 0.50 0.67 0 0.79* 0.93** 0.91** 0.91** 0.81* 0.76* 0.83* 0.79* 1 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:57 utc nova biotechnol chim (2019) 18(1): 25-36 34 noteworthy is also the correlation between the content of rosmarinic acid and inhibition activity on urokinase (only one from four tested proteinases), the ra with antioxidant activity (by all methods) and also the correlation to certain composite parameters (tpf, tpfa). surprising is a correlation of ra content with tfl and tt by this statistical method. fig. 4. two-dimensional pca from all primary data of eight evaluated extracts, four from salvia officinalis l. of different varieties and localities (green cross), salvia apiana (red circle) and extract from salvia divinorum and from the both callus culture extracts (blue diamonds). abbreviations represent metabolites or methods described in the part experimental. principal component analysis (pca) is a tool (method) that may provide more information about relation among parameters (variables). twodimensional pca containing all primary data is given in fig. 4. the position of each particular extract sample is represented by one point in the chart. concerning their location it is evident that certain similarities appear between the four samples prepared from salvia officinalis l. (green cross), three samples of salvia divinorum and the two callus culture extracts (blue diamonds), and finally the extract sample from salvia apiana presented as outlying point (red circle). from the angle of particulars vectors for each parameter it is evident the occurrence of certain relation between tpf, tpfa, ia_ur, ia_thr and ra in the first assembly and ia_ty and all four antioxidant activity parameters in the second assembly, whereas the vector for ta and ia_pl seem to be relatively outlying. this finding differs from the conclusion of correlation analysis and suggests than rosmarinic acid possesses both proteinase inhibition activity as well as antioxidant activity. this proposal is supported by the clustering analysis (ca) of the eight evaluated extract samples and the values of 14 measured parameters (fig. 5). fig. 5. cluster analysis including all primary data of eight evaluated extracts from salvia officinalis l. (green cross), salvia divinorum, both callus culture extracts (blue diamonds) and extract from salvia apiana. the results correspond to outputs of the pca analysis (fig. 4). cluster analysis moreover graphically presents the measure of similarity (or difference) among the extract samples from different sources. the extract from salvia apiana is obviously special regarding the evaluated parameters, with best antioxidant activity, proteinase inhibition activity and content of composite parameters (fig. 5). this extract was selected and assigned as a primary extract. relatively homogenous is the cluster of extracts from different plants within salvia officinalis l. the extract from salvia divinorum with two extracts from callus culture create an individual cluster with the lowest potent effects, though are of different nature and source. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:57 utc nova biotechnol chim (2019) 18(1): 25-36 35 conclusions the study revealed the inhibitory activity of sage extracts from different sources on selected proteinases, which may represent a potentially pathophysiological effector in the progression of serious human diseases. our work at first time reports that extracts of salvia sp. do possess proteinase inhibition activity, in addition to being a rich source of polyphenolic acids and amines, as well as of remarkable antioxidant activity. beside this thiols seem to be promising and active secondary metabolites in sage. inhibition properties of ra and sage extracts on selected proteinases deserve further research. acknowledgements this work was supported by the slovak research and development agency under the contracts no. apvv-160173 and apvv-17-0113. conflict of interest the authors declare that they have no conflict of interest. references adams jd, garcia c (2005) the advantages of traditional chumash healing. evid. based complement. alternat. med. 2: 19-23. adomako-bonsu ag, chan sl, pratten m, 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cui sw, guo q, ding h (2014) some physicochemical properties of sage (salvia macrosiphon) seed gum. food hydrocoll. 35: 453-462. singleton vl, orthofer r, lamuela-raventós rm (1999) analysis of total phenols and other oxidation substrates and antioxidants by means of folin–ciocalteu reagent. methods enzymol. 299: 152-178. scatena m, liaw l, giachelli cm (2007) osteopontin: a multifunctional molecule regulating chronic inflammation and vascular disease. arterioscler. thromb. vasc. biol. 27: 2302-2309. slinkard k, singleton vl (1997) total phenol analysis: automation and comparison with manual methods. am. j. enol. viticult. 28: 49-55. stratil p, klejdus b, kubá v (2007) determination of phenolic compounds and their antioxidant activity in fruits and cereals. talanta 71: 1741-1751. vanisree m, tsay hs (2004) plant cell cultures-an efficient source for the production of biologically important secondary metabolites. int. j. appl. sci. eng. 1: 29-48. vanisree m, lee cy, lo sf, nalavade sm, lin cy, tsay h (2004) studies on the production of some important secondary metabolites from medicinal plants by plant tissue culture. bot. bull. acad. sin. 45: 1-22. veličković dt, nikolova mt, ivancheva sv, stojanović jb, veljković vb (2007) extraction of flavonoids from garden (salvia officinalis l.) and glutinous (salvia glutinosa l.) sage by ultrasonic and classical maceration. j. serb. chem. soc. 72: 73-80. whitcomb dc (2003) molecular and genetic mechanisms of acute and chronic pancreatitis. int. congr. ser. 1255: 49-60. yu zj, liu s, zhou s, li h, yang f, wu l, guo l, li gb (2018) virtual target screening reveals rosmarinic acid and salvianolic acid a inhibiting metalloand serine-βlactamases. bioorg. med. chem. lett. 28:1037-1042. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:57 utc nova biotechnol chim (2018) 17(1): 1-15 doi: 10.2478/nbec-2018-0001  corresponding author: svetlana.hrouzkova@stuba.sk nova biotechnologica et chimica review present state and applications of single drop microextraction for the determination of harmful organic compounds and pollutants silvia zichová, adriana brisudová and svetlana hrouzková institute of analytical chemistry, faculty of chemical and food technology, slovak university of technology in bratislava, radlinského 9, bratislava, sk-812 37, slovak republic article info article history: received: 29th march 2018 accepted: 20th june 2018 keywords: harmful organic compounds microextraction pollutants single drop microextraction abstract single drop microextraction (sdme) nowadays earns an increasing attention by scientists due to its simplicity, low cost and the need for only common laboratory equipment. this microextraction technique combines sample cleanup and pre-concentration of analytes in one step. furthermore, a significant reduction in the amount of organic solvents needed comparing to standard lle techniques places sdme into the position of environmental friendly extraction techniques. sdme is a straightforward technique in which a micro-drop of solvent is suspended from the tip of a conventional micro-syringe and then it is in a direct contact with a sample solution in which it is immiscible or it could be suspended in the headspace above the sample. the paper overviews developments of the stateof-the-art sdme techniques for the extraction of harmful organic compound and pollutants from environmental, food and biological matrices. key extraction parameters essential for sdme performance were described and discussed.  university of ss. cyril and methodius in trnava introduction the toxicity of pesticides and pollutants and their harmful environmental effects are attracting the attention of the society and interest in the identification and quantification of such compounds in various matrices have aroused. thus, the importance is put on the development of the faster and selective analytical methodology, suitable for the determination of the low levels of these compounds. the limits of quantitation (loqs) of pesticides and pollutants are usually in mg/l or below. therefore, in order to accomplish the quantitation of these compounds in various samples, a pre-treatment step, extraction and pre-concentration step are required. generally, when conventional liquid phase extraction is used, the preconcentration of the analytes is very small or not even obtained. a lot of research efforts have been focused on the development of combined sample preparation techniques, when conventional liquid phase extraction is in the combination with a proper liquid phase microextraction technique or sorptive based microextraction technique, in order to achieve additional preconcentration of the analytes. contrary to this, in the case of the single drop microextraction (sdme) these two processes are performed in a single step (de souza pinheiro et al. 2011; andraščíková et al. 2016). currently sdme has been increasingly used in the analysis of pesticides and other environmental bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2018) 17(1): 1-15 2 pollutants in various matrices because it is simple, cheap, fast, effective and consuming only microliters of organic solvents per analysis. sdme technique is based on the principle of a distribution of analytes between a microdrop of the organic solvent and an aqueous phase or headspace phase above the solid/liquid sample. using volume of sample in a milliliter scale and microliters of organic solvent leads to a high enrichment factor obtained. the sdme procedure using conventional microsyringe has the advantage of combining manipulation and injection of the sample into a single-step extraction and thus simplify the extraction process. however, some problems such as microdrop instability leading to the dislodgement of the drop or dislocation of the drop from the needle tip, and partial solvent loss caused by the high temperature and high solubility of the drop in the sample during extraction may reduce the repeatability of the method (jeannot et al. 2010; ramos 2012). sampling repeatability requires the use of the proper syringe needle for the extraction. the use of the microdrop as a collector phase of analytes from the gaseous samples (liu and dasgupta 1995) and liquid samples (liu and dasgupta 1996) was described for the first time in the nineties of the 20th century. by the time, sdme has undergone many technical changes, and it is known in various modes. seven different modes of solvent microextraction that belong to the category of single drop microextraction are currently in use for various applications. the base classification of sdme modes is dividing into two sub-categories, two and three-phase techniques, which exist in equilibrium (table 1). all modes are based on the principle of passing the analytes from the sample directly, or through the other phase (e.g. headspace) or mediator to the final extraction solvent having a volume in microliters (ramos 2012). there are more advances to classify sdme, e.g. according to hydrodynamic features, static and dynamic modes of operation are distinguished (alexovič et al. 2016). use of the extraction solvent, which is volatile, e.g. hexane or toluene, makes the application directly compatible with gas chromatography (jeannot et al. 2010). moreover, combinations of sdme with high performance liquid chromatography (hplc) and capillary electrophoresis were reported (amde et al. 2015; garcía-vázquez et al. 2016; yohannes et al. 2016). table 1. overview of the sdme modes. sdme mode acronym two-phases techniques direct immersion di-sdme continuous flow microextraction cfme drop-to-drop microextraction ddme directly suspended droplet microextraction dsdme three-phases techniques headspace hs-sdme liquid-liquid-liquid microextraction lllme solvent-supported microextraction ssme sdme has become frequently used for the extraction of a broad scope of compounds for numerous analytical application due to the advantages as reviewed by more papers (lamboropoulou et al. 2007; jeannot et al. 2010; jain and verma 2011). in this review, the focus was given to the three main application areas of sdme for the extraction of the harmful organic compounds and pollutants in environmental, food and biological samples in the recent years. environmental samples sdme has been used to extract a wide range of organic compounds from the environmental samples, mostly several types of water and soil. overview of the applicability of sdme for environmental sample analysis is shown in the table 2. sdme was an appropriate extraction technique for liquid samples such as tap water (kaykhaii et al. 2005; lamboropoulou et al. 2007; yohannes et al. 2016), river water (kaykhaii et al. 2005; saraji and bankhshi 2005; ahmadi et al. 2006; de souza pinheiro et al. 2009; de souza pinheiro et al. 2011; soares et al. 2014; amde et al. 2015; yohannes et al. 2016), lake water (wu et al. 2008; xie et al. 2014; amde et al. 2015; yohannes et al. 2016), surface water (lopezblanco et al. 2003; lamboropoulou et al. 2007; santos et al. 2017), drinking water (lopez-blanco et al. 2003; ahmadi et al. 2006; carlos et al. 2013), effluent and influent water (amde et al. 2015), ground water (yohannes et al. 2016; santos et al. 2017), water from the farm (ahmadi et al. 2006), bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2018) 17(1): 1-15 3 sea water (tian et al. 2014) and other water samples (przyjazny and kokosa 2002). furthermore, sdme was applied for the extraction of the pesticides from the soil samples (salemi et al. 2013; williams et al. 2014; soares et al. 2015). organic pollutants, such as pesticides (organochlorine pesticides (ocps), organophosphorus pesticides (opps)), fungicides, aliphatic amines, volatile organic compounds, phenols, polyaromatic hydrocarbons (pahs), nitropahs and quinones extracted by sdme were mostly reported. generally, two sdme modes were employed, di and hs, nevertheless di was preferably used due to the chemical properties of analyte and the type of the sample. special pre-treatment was not necessary for the majority of selected applications, except addition of nacl to the sample for increasing the ionic strength by lamboropoulou et al. (2007), wu et al. (2007) and yohannes et al. (2016), adjustment of ph to 2 – 6 reported by saraji and bankhshi (2005), and filtration in the case of surface and ground water (santos et al. 2017). the volume of a sample varied in the range of 1 ml – 30 ml, the most commonly selected volume of the sample was 5 ml. the pre-treatment of the solid samples was more difficult in comparison with water samples. drying, sieving, ultrasonic extraction, sonication, centrifugation and final dilution with an appropriate solvent were used due to the matrix of the sample. the amount of sample was ranging from 2 g to 5 g (salemi et al. 2013; williams et al. 2014; soares et al. 2015). the selection of a suitable extraction solvent is a crucial step for sdme performance. according to the extracted analytes, mostly organic solvents differing in kow compatible with gc and hplc were chosen, in order to obtain the highest extraction efficiency. toluene, provided stable drop and avoided bubble formation during extraction according to vapour pressure lower than other solvents, was mostly used in direct immersion (di) mode (lamboropoulou et al. 2007; de souza pinheiro et al. 2009; de souza pinheiro et al. 2011; tian et al. 2014; santos et al. 2017). however, the bubble formation in the drop was required for the extraction of pesticides from lake water by xie et al. (2014) by exposing the liquid-gas pendant drop (cpd) to the sample. the stability of the cpd was investigated by monitoring the formation of 1 μl organic droplets (toluene, chlorobenzene) containing different volumes of air bubble in a sample solution resulting in the better stability of the drop using chlorobenzene. a formation of a liquid-gas cpd was performed using a novel extraction assembly consisting of a 10 mm long quartz capillary, a funnel-like pmma cap and a 5 ml microsyringe. the limitation of droplet instability in comparison with di-sdme was overcome, larger microdrop size and higher stirring rates could be employed, as well as liquid-gas cpd has a potential to be fully automated by using a syringe pump (xie et al. 2014). a 70to 135-fold enrichment of pesticides was obtained. in comparison to a conventional sdme, improvement of the extraction efficiency could be ascribed by the increased surface area of the microdrop. the bubble in drop (bid) was studied by williams et al. (2014) when chloroform was used as the extraction solvent. for the extraction of ocps from tap and surface water, toluene and isooctane gave similar results for the majority of target analytes, but toluene was selected due to the selectivity and no significant loss of solvent during extraction (lamboropoulou et al. 2007). the similar observation was reported for n-hexane that was selected due to its lower solubility in water, which increases the stability of the microdrop in comparison to the use of toluene. the analyte extraction was not significantly affected by the type of extraction solvent (n-hexane or toluene) (soares et al. 2014). n-hexane was suitable for the extraction of ocps and pyrethroids from drinking water (carlos et al. 2013), multiclass pesticides from water and soil (soares et al. 2014; soares et al. 2015). for the extraction of the atrazine, desethyl-atrazine, desisopropyl-atrazine from the water samples, toluene, n-hexane, and cyclohexane were investigated, however, due to their low viscosity and density, the microdrop was not stable. formation of the stable drop under the vigorous stirring and higher extraction efficiency was reached using 1-octanol as the extraction solvent (yohannes et al. 2016). solubility of the extraction solvent in water had a great effect for the extraction of 2 pesticides, α-endosulphane and β-endosulphane from water. n-hexane didn’t exhibit a good extractability of these compounds, because bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2018) 17(1): 1-15 4 s o a re s e t a l. (2 0 1 4 ) a m d e e t a l. (2 0 1 5 ) t ia n e t a l. ( 2 0 1 4 ) x ie e t a l. ( 2 0 1 4 ) c a rl o s e t a l. (2 0 1 3 ) y o h a n n e s e t a l. (2 0 1 6 ) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2018) 17(1): 1-15 5 d e s o u z a p in h e ir o e t a l. (2 0 1 1 ) d e s o u z a p in h e ir o e t a l. (2 0 1 1 ) p rz y ja z n y a n d k o k o sa ( 2 0 0 2 ) k a y k h a ii e t a l. (2 0 0 5 ) a h m a d i e t a l. (2 0 0 6 ) l o p e z -b la n k o e t a l. ( 2 0 0 3 ) s a ra ji a n d b a n k h sh i (2 0 0 5 ) l a m b o ro p o u lo u e t a l. ( 2 0 0 7 ) s a n to s e t a l. (2 0 1 7 ) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2018) 17(1): 1-15 6 w u e t a l. ( 2 0 0 7 ) s a le m i e t a l. (2 0 1 3 ) w il li a m s e t a l. (2 0 1 4 ) s o a re s e t a l. (2 0 1 5 ) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2018) 17(1): 1-15 7 it is more soluble in water in comparison to isooctane. isooctane, being solvent of low polarity, provided higher extractive efficiency and reproducibility for both analytes (lopezblanco et al. 2003). extraction of the phenols from the river water by di-sdme followed by in syringe derivatization was investigated by saraji and bankhshi (2004). several organic solvents in the broad range of polarity were investigated, resulted hexyl acetate being the most suitable for the extraction. hexyl acetate provided the selective extraction of the phenols, in comparison to other solvent, when 4­nitrophenol was not extracted. furthermore, the manipulation with hexyl acetate microdrop was acceptable without any stability problems during the extraction, however, chromatographic peak overlaps were observed (saraji and bankhshi 2004). the volatile compounds were preferably extracted by using hs mode. octanol, benzylalkohol and β-cyclodextrine were investigated as potential extraction solvents (przyjazny and kokosa 2002; kaykhaii et al. 2005; salemi et al. 2013). hssdme was found as an efficient technique for the extraction of the organic compounds such as benzene, toluene, ethylbenzene and xylenes from the water samples. gas chromatography with flame ionization detector (gc-fid) was employed. for the highest extraction efficiency under the experimental conditions, solvents with a high boiling point and low vapour pressure were investigated, so as to minimize the evaporation of the microdrop during the extraction. 1-octanol and n-hexadecane provided satisfactory extraction of the analytes. however, less impurities using n-hexadecane were interfering the analyte signal (przyjazny and kokosa 2002). the specific application of hs-sdme which was free of organic solvent was provided by wu et al. (2007), when β-cyclodextrine was used as extraction solvent for pahs from the lake water. nanofluid (nf) prepared by dispersion of zno nanoparticles in 1-hexyl-3-methylimidazoliumhexafluorophosphate was reported as an extraction solvent for three fungicides in water. a syringe tip cap (obtained from 5 ml disposable syringe) was used as a holder of the microdrop instead of the syringe needle. the microdrop was immersed on the syringe tip cap by microsyringe and after the extraction, the microdrop was placed in an eppendorf vial containing 100 μl methanol with the aim to remove nanoparticles. nowadays, nfs earn an attention due to the frequent applications in many research directions. replacing conventional solvent in liquid–liquid system by nps can cause an increase of mass transfer coefficient and increase the efficiency of extraction as the main goal (amde et al. 2015). volume of the drop is the next important factor for the efficient extraction. in general, increasing the drop volume results in the significant improvement of the extraction efficiency. however, when the microdrop volume increases further, the drop became unstable due to the gravity. the volume of the drop was in the range of 0.9 μl (ahmadi et al. 2006) and 10 μl (wu et al. 2007; amde et al. 2015). drop volume 1 μl was the most frequently applied, what can be explain by the found consensus between the volume, stability and extraction capacity of the drop (kaykhaii et al. 2005; de souza pinheiro et al. 2009; de souza pinheiro et al. 2011; williams et al. 2014; xie et al. 2014; santos et al. 2017). sampling repeatability of the microdrop requires the use of the proper syringe needle for the extraction. generally, 10 μl hamilton syringe routinely used for gc was frequently used as a holder of the microdrop during the extraction process. the needle should have a minimum dead volume, for instance using a 1 μl microsyringe, no dead volume is occurred. the modification of needle tip, e.g. cones tip, or modified microsyringe causes increasing adhesion force between needle tip and drop, thereby increasing drop stability and achieving a high stirrer speed (up to 700 rpm for the cones tip and up to 1,700 rpm for the modified microsyringe) (ahmadi et al. 2006). furthermore, improved holding of the microdrop and larger extraction solvent volumes used for the extraction were reached by attaching a 2-mm long cone that was cut off from a 200 μl pipette tip onto the needle tip of a gc microsyringe (tian et al. 2014). the similar approach was used for the modification of the hplc syringe, which was supported by a part of the pipette tip, thus the larger microdrop was obtained (wu et al. 2007). to avoid impurities, a rinse of the microsyringe before extraction was presented, mostly by the extraction solvent (przyjazny and kokosa 2002; kaykhaii et al. 2005; ahmadi et al. 2006; wu et al. 2007; bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2018) 17(1): 1-15 8 salemi et al. 2013; tian et al. 2014; yohannes et al. 2016; santos et al. 2017). sample agitation is an important factor for the reduction of the extraction time. from the film theory of convective-diffusive mass transfer, the agitation of the sample solution enhances the extraction efficiency and reduce the extraction time, since the thickness of the stagnant film around the extracting phase (i.e. nernst diffusion film) decreases with increasing stirring rate, and it results in faster extraction rate (pena-pereira et al. 2010). magnetic stirring, mechanical vibration, continuous flow and syringe plunger motion are usually used for the sample agitation to increase the amount of convective mixing or interfacial contact area for the dynamic modes of sdme. the time required to reach equilibrium in sdme dependent on the type and degree of agitation, phase volumes, interfacial contact area and equilibrium distribution constant (jeannot and cantwell 1996; jeannot et al. 2010). magnetic stirring was applied for the majority of the applications, with the velocity range from 155 rpm (soares et al. 2014) to 1,300 rpm (ahmadi et al. 2006). high velocity of agitation was used for the hs mode, when the drop is not in the direct interaction with a sample. for the extraction of the atrazine and metachlor from soil, no stirring was used and the extraction was performed under static conditions (williams et al. 2014). the positive effect of the ultrasound waves has been used in sdme for the opps extraction from the soil sample (salemi et al. 2013). for hs-sdme, the temperature is a key parameter to speed-up the rate of mass transfer of compounds from the sample to the headspace increasing the number and the amount of compounds of interest moved to the gas phase. increased temperatures lead to the decrease of the extractant-headspace phase distribution constant and this results in the decrease of sensitivity of the method. cooling of the extraction solvent, while the sample could be heated, is the promising solution of this problem. however, such an arrangement of experiment brings apparatus design complications, therefore, it is useful for ultra-trace analyses or for very volatile analytes with a low possibility to transfer to the headspace at ambient conditions (jeannot et al. 2010). extraction temperature was in the range from 17 °c (soares et al. 2014) to 60 °c (salemi et al. 2013). the increase of the ionic strength of the waterbased sample can affect the analyte transfer by the increase of the amount of salt to the aqueous phase (wang et al. 2017). the majority of the publications were reported to be without salt addition. food samples sdme extraction of the organic compounds from food samples is an important application field of sdme technique, it earns nowadays great interest proved by the published papers in recent years. overview of papers reporting the analysis of various samples, such as tea (liu et al. 2012; wu et al. 2015), wine (garbi et al. 2010; perreira dos anjos et al. 2015), vegetables (amrvrazi and tsiropoulos 2009; kin et al. 2009; wu et al. 2016) and fruits (perreira dos anjos et al. 2014; panofarias et al. 2017) are summarized in the table 3. all of them were targeted on the determination of the pesticides. no special pre-treatment was reported for liquid samples such as wine or coconut water, mainly conventional sonication, acidification, ph adjustment and centrifugation were used (garbi et al. 2010; perreira dos anjos et al. 2014; perreira dos anjos et al. 2015). on the other hand, more difficult pre-treatment was required for solid samples prior to the extraction step (amrvrazi and tsiropoulos 2009; kin et al. 2009; liu et al. 2012; wu et al. 2015; wu et al. 2016; pano-farias et al. 2017). di was the most occurred mode of sdme (amrvrazi and tsiropoulos 2009; garbi et al. 2010; perreira dos anjos et al. 2014; perreira dos anjos et al. 2015; pano-farias et al. 2017). for the extraction of 8 pesticides from cucumber and strawberries, the hs mode was selected and its application was compared to headspace solid phase microextraction (hs-spme) and solid phase extraction (spe) (kin et al. 2009). more complex apparatus was employed by wu et al. (2015, 2016) for the dynamic microwave assisted extraction (dmae) and dynamic microwave assisted extraction in combination with continuous flow microextraction (dmae-cfme) of pesticides from tea (wu et al. 2015) and cabbage, cauliflower, red cabbage and cucumber (wu et al. 2016). furthermore, dsdme using 100 μl isooctane for isolation of ocps was used to analyze samples of tea by liu et al. (2012). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2018) 17(1): 1-15 9 w u e t a l. (2 0 1 5 ) l iu e t a l. (2 0 1 2 ) pe rr e ir a d o s a n jo s e t a l. (2 0 1 5 ) g a rb i e t a l. (2 0 1 0 ) w u e t a l. (2 0 1 6 ) pe rr e ir a d o s a n jo s e t a l. (2 0 1 4 ) k in e t a l. (2 0 0 9 ) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2018) 17(1): 1-15 10 a m rv ra z i a n d t si ro p o u lo s (2 0 0 9 ) p a n o -f a ri a s e t a l. ( 2 0 1 7 ) k u m a ri e t a l. (2 0 1 6 ) p e li t a n d y e n g in (2 0 1 4 ) t a b le 2 . bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2018) 17(1): 1-15 11 for effective extraction of the pesticides from the vegetable samples and stable drop in the continuous flow by dmae-cfme, a variety of water-immiscible extraction solvents (n-hexane, cyclohexane, dichloromethane, chloroform, carbon tetrachloride, ethyl acetate, and toluene) were investigated, resulting dichloromethane and ethyl acetate as inappropriate for this mode. the major loose of these solvents was presented in the continuous sample flow arrangement. toluene exhibited the most promising results of extraction efficiency for the majority of studied compounds (wu et al. 2016). the wide scale of solvents, such as dichloromethane, chloroform, carbon tetrachloride, and chlorobenzene were examined for the extraction of the pesticides from the tea. an improved extraction chamber using as a holder of the microdrop was employed. the microdrop provided a sufficient stability in the flowing system and the microdrop transfer to analytical instruments by microsyringe after extraction was without loose of the extraction solvent. carbon tetrachloride exhibited the highest extraction efficiency for most of the analytes, also the carbon tetrachloride extract was proved to be cleaner in comparison to other solvents. chlorobenzene was not stable enough in the flow (wu et al. 2015). analyzing tea samples, cyclohexane drop immersed to the tea sample solution was not reproducible with low stability. toluene was problematic as it extracted tea pigments and further cleaning was necessary. isooctane showing the highest viscosity among studied solvents was selected as the final solvent for real-samples extraction, in addition, it formed stable microdrop (liu et al. 2012). microsyringe of the volume 10 μl was used in all of the mentioned applications and the drop of the sample was in the range of 1 μl and 10 μl. especially, the volume 100 μl was used for the dsdme mode to measure the volume of the sample. presence of the steady vortex was important for the formation of the microdrop (liu et al. 2012). to avoid carry over effect, the rinse of the microsyringe was used mostly by the extraction solvent or polar organic solvents (amrvrazi and tsiropoulos 2009; kin et al. 2009; garbi et al. 2010; perreira dos anjos et al. 2015; pano-farias et al. 2017). the highest speed of the magnetic stirrer was used for the dsdme extraction, the stirring speed above 1,100 rpm causes instability and dissolution of the solvent microdrop (liu et al. 2012). low speed of magnetic stirrer was mostly used for the di-sdme in the range of 180 – 700 rpm (amrvrazi and tsiropoulos 2009; garbi et al. 2010; perreira dos anjos et al. 2014; perreira dos anjos et al. 2015; pano-farias et al. 2017) and microwave irradiation was employed for the dmae (wu et al. 2015) and dmae-cfme (wu et al. 2016). the influence of microwave energy (100 – 350 w) on the extraction was studied. implementation of the microwave-assisted extraction (mae) prior to sdme simplify the pre-treatment process by the acceleration of the disruption of the vegetable cells under the high temperature. the high temperature improves the diffusion and mass transfer during extraction, and the dissolving capacity of extraction solvent at the same time, as well as, the target compounds dissolves faster. however, when high microwave power is used, it may cause the analyte degradation (wu et al. 2016). no salt addition was reported in the published papers for food samples analysis. satisfactory recovery values in the acceptable limit (between 70 % and 120 %) were mostly reposted. difficulties to extract some compounds were mostly explained by chemical properties of the analytes and by the interaction of the analytes with other compounds presented in the sample. low values of recovery were reported for dursban (the pesticide) with recoveries in the interval of 13.2 % – 52.4 % (perreira dos anjos et al. 2015), and for the pyrethroid insecticide λ-cyhalothrin (1.4 for grape and 1.5 for apple) (amrvrazi and tsiropoulos 2009). permethrin i and permethrin ii are compounds exhibiting higher affinity toward water phase, which is demonstrated by low partition coefficient, therefore, as a result, insufficient recovery rates were reported (39.4 % and 45.0 %, respectively). thus, it is possible to notice a difficulty of the migration of these analytes from the aqueous sample to the organic solvent. although a relatively high partition coefficient belonging to the pesticide endosulfane, a low recovery rate was reported (29.0 %), what was explained by the interaction with other matrix components comprising the coconut water. in general, extraction of the compounds with higher polarity such as ocps, compared to other classes of compounds such as opps, is more difficult bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2018) 17(1): 1-15 12 hence they promote a higher affinity for waterbased sample than for organic extractant (perreira dos anjos et al. 2014). biological samples sample preparation step is essential for the isolation of analytes from complex biological matrices and has a great influence on their reliable and accurate determination. suitable extraction technique is usually necessary because almost all biological samples are incompatible with the following chromatographic instrumentation, these samples exhibit complicated matrix for direct analysis, and components in the sample may interfere the signal acquisition. in addition, the analytes are present at concentration levels below the limit of detection of regular analytical methods (ocaña-gonzález et al. 2016). biological samples contain proteins, salts, acids, bases, and various organic components with properties close to those of searched analytes. in the recent years, applications of the sdme were aimed mostly to extraction of the several organic compounds in the biological samples such as fish (botrel et al. 2017), urine and blood of the rats (agrawal et al. 2007), human urine (gao et al. 2011; garcía-vasquéz et al. 2015), saliva (timofeeva et al. 2016) and human blood serum (shrivas and patel 2009) (table 4). the solidification of the extraction solvent in microliter scale after dispersive liquid-liquid microextraction for the extraction of drugs (ebrahimzadeh et al. 2011; ahmadi-jouibari et al. 2014; tehrani et al. 2014), medicaments (ebrahimzadeh et al. 2013; jia et al. 2013; suh et al. 2014) was also reported. a few papers were devoted to the extraction of pesticides, namely maneb (fungicide) and paraque (herbicide) in albine mice by kumari et al. (2016) and chlorpyrifos and chlorpyrifosoxon by pelit and yengin (2014). 1-undecanol, 1-dodecanol, 2-dodecanol and n-hexadecane were investigated for the extraction of maneb and paraque from albine mice. except n-hexadecane, all other solvents were found to recover comparably the same amount of targeted analytes. this maybe because all these solvents possess nearly similar affinity toward the targeted analytes. in this case, the solidification of the solvent drop was used, which offered manipulation with a microdrop without significant losing of extract. this approach was reported to be semiautomated and could be fully automated without varying from the original concept by the further modifications. finally, 1-dodecanol was chosen to be used for further experiments, due to its peculiar characteristic of being easily solidified near room temperature. (kumari et al. 2016). in the case of urine, this matrix offers several advantages as it is very easy to obtain and specimen volume is not a limitation. biomonitoring of the chlorpyrifos exposure in human urine was reported and 2-dodecanol as extraction solvent was selected due to the absence of the solvent peak presented in the same time as the analytes in the chromatogram in comparison with the other studied solvents. the extraction was performed using increased temperature, and no addition of the salt was presented. in this case, it was difficult to collect the organic phase due to the formation of smaller droplets of the solvent in the solution after addition of 0.5 g nacl to 10.0 ml of the solution (pelit and yengin 2014). conclusions sdme showed its suitability for the extraction of various type of analytes, which belongs to the wide group of harmful organic compounds and environmental pollutants from numerous types of samples. applications of sdme for real samples analysis were sorted into 3 groups according to sample nature and summarized into the form of overview tables. as it was shown by the largest group of applications, sdme has the major utilization in the area of environmental samples analysis, then followed by food samples analysis. nowadays, there is an increasing frequency of publication devoted to the sdme for biological samples analysis. sdme offered the reach of high pre-concentration, clean final extract directly suitable for analysis, fast extraction and the simplicity of the process. it was shown, that the two most common sdme modes di and hs have slightly different general fields of applicability, but they show their wide applicability for various matrices. di-sdme was approved to be suitable mostly for the extraction of the nonpolar or moderately polar volatile and semivolatile analytes from relatively clean matrices, such as bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2018) 17(1): 1-15 13 water, using appropriate water immiscible solvents, due to the direct contact of the solvent with a water sample. since volatile compounds are best preconcentrated by headspace sdme. the selection of the working parameter was also established to be critical for the reaching satisfactory recoveries of the analytes and explained by numerous examples. in conclusion, miniaturized extraction methods such as sdme represent a new approach that is currently receiving a great deal of interest of researchers in the area of sample preparation methods for analytical purposes. acknowledgements this work was supported by the slovak research and development agency under the contract no. sk-srb2016-0006. sz would like to thank for financial contribution from the stu grant scheme for support of young researchers. references agrawal k, wu fh (2007) drop-to-drop solvent microextraction coupled with gas chromatography/mass spectrometry for rapid determination of trimeprazine in urine 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poľnohospodárska knižnica | heruntergeladen 28.02.20 08:24 utc nova biotechnol chim (2019) 18(2): 133-143 doi: 10.2478/nbec-2019-0016  corresponding author: roman.tandlich@gmail.com nova biotechnologica et chimica the efficiency of a low-cost hydrogen sulphide (h2s) kit as an early warning test for assessing microbial rainwater quality and its correlation with standard indicators microorganisms mokaba shirley malema1,2, jean-marc mwenge kahinda1, akebe luther king abia3, roman tandlich4,, bongumusa m. zuma2 and eunice ubomba-jaswa5,6 1 council for scientific and industrial research, natural resources and the environment, po box 395, pretoria 0001, south africa 2 biotechnology innovation centre, rhodes university, po box 94, grahamstown, south africa 3 antimicrobial research unit, college of health sciences, university of kwazulu-natal, private bag x54001, durban 4000, south africa 4 environmental health and biotechnology research group, division of pharmaceutical chemistry, faculty of pharmacy, rhodes university, artillery road, po box 94, grahamstown, south africa 5 department of biotechnology, university of johannesburg, 37 nind street, doornfontein, 2094, gauteng, south africa 6 water research commission, lynnwood bridge office park, bloukrans building, 4 daventry street, lynnwood manor, pretoria, south africa article info article history: received: 31st october 2019 accepted: 29th november 2019 keywords: colilert® harvested rainwater hydrogen sulphide test membrane filtration water quality abstract testing microbial quality of the harvested rainwater remains a challenge in many countries. the h2s test kit is a low-cost microbiological field-based test which can be used in areas where water testing facilities are limited. this study compares its efficiency with the standard indicators microorganisms in the detection of faecal contamination of rainwater in south africa. a total of 88 rainwater samples were collected from various tanks in the eastern cape, south africa over three months in 2016. the collected samples were analysed for faecal bacterial contamination using the h2s test kit, colilert-18/quanti-tray ®/2000 and the membrane filtration technique for faecal coliforms (mft). the correspondence rate of the h2s test kit with mft was 88 %, while for the colilert® it was 76 %. the h2s test kit confirmed faecal contamination when concentrations of standards indicators microorganisms were 5 most-probable number of cells/100 cm3 or higher. overall, the best correspondence of the h2s test kit with colilert ® was observed at e. coli concentrations above 50 most-probable number of cells/100 cm3. results of the h2s test kit correlated better with mtf, while the medium used has strongly influenced the enumeration of faecal contamination. results point to strong effect of media used and revealed the need to calibrate the correspondence between the standard indicator microorganisms and the h2s test kit under local conditions for specific settings.  university of ss. cyril and methodius in trnava introduction in the year 2017, it was reported that diarrhoeal diseases were the second leading cause of death among children aged below 5 years (who 2017). although the incidences of diarrhoeal diseases bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:32 utc nova biotechnol chim (2019) 18(2): 133-143 134 mostly affect the low-income populations with poor access to safe water, sanitation, and urgent medical care, acute infectious diarrhoea is also a common cause of outpatient visits and hospital admissions in high-income regions (wazny et al. 2013). assessment of microbial quality of potable water is one of the major tools to prevent or decrease the likelihood of diarrhoeal disease outbreaks. several methods are used to detect and quantify indicators microorganisms which are indicative of the faecal contamination of potable water, i.e. escherichia coli and other faecal/thermotolerant coliforms (who 2008). if finite concentrations of these indicators microorganisms are detected in a sample of potable water, then faecal contamination is present in it and domestic use of such (untreated) potable water can result in diarrhoeal disease outbreaks. however, the enumeration of indicators microorganisms in potable water can be a complex task and requires laboratory facilities (who 2011). these laboratory facilities might not be available where the microbial water quality must be tested, as reported for the case of south africa (luyt et al. 2012). although e. coli is recommended as a standard indicator organism for this purpose, the hydrogen sulphide (h2s)-producing bacteria are an alternative faecal indicator that has been shown to correlate with e. coli levels and their detection has been developed into field test products (luyt et al. 2012; tandlich et al. 2014; murcott et al. 2015). the h2s test kit was developed to equip public health workers with a simple field test kit that can detect faecal contamination in drinking water (manja et al. 1982). a version of this method is the focus of the current study. many (developing) countries and many areas around the world lack water testing facilities, due to financial constraints and a shortage of trained personnel which further hampers their ability to ensure accurate assessments (luyt et al. 2012; wright et al. 2012). a study on the overall cost of monitoring microbial drinking water quality in sub-saharan africa indicated that financial constraints are assumed to be the main barrier to conducting water quality tests (delaire et al. 2017). conducting a microbial water quality test involves four types of expenses, namely consumables, laboratory equipment, labour (for collecting and analysing samples) and logistics such as transport and communication (hunter et al. 2009; crocker and bartram 2014; delaire et al. 2017). delaire et al. (2017) also revealed that the estimated cost of monitoring piped water supplies in sub-saharan african countries at the time of their study, based on the who recommendations, varied between 1403 usd (liberia) and 1655 672 usd (south africa) and amounted to 10931 000 usd per year for all the sub-saharan african countries. challenges relevant to this point have been reviewed for south africa (e.g. luyt et al. 2012). the h2s test kit might provide a low-cost solution in this context, which can be field-based and could be used in the household settings (delaire et al. 2017). in this way, the h2s test kit could provide an early warning about faecal contamination at the point of consumption of the tested potable water. before the current study was conducted, the same version of the h2s test kit used by the authors here had been tested on harvested rainwater in a pilot study of a community-based rainwater monitoring and treatment programme in the grahamstown/makhanda in the eastern cape province of south africa between march and june 2013 (tandlich et al. 2014). tandlich et al. (2014) collected a total of 55 samples from eight rainwater tanks during the period of the study. results indicate that the modified h2s test kit, which is a low-cost microbiological field-based test, was successfully used by non-governmental organisation volunteers to detect faecal contamination in harvested rainwater. results further revealed a 71 % rate of correspondence between the membrane-thermotolerant e. coli, which had been grown on the m-tec agar, and the h2s test kit, which was also used, in the current study. in rural and certain peri-urban areas, the use of standard indicator microorganisms for faecal contamination of water samples, i.e. colilert® for e. coli and the membrane filtration technique enumerating faecal coliforms on the m-fc agar (mft), may not be a feasible choice due to the lack of investment capacity in building laboratories (us epa 2016). further reasons could include the long distances between the sampling sites and the laboratories, where analyses are carried out (luyt et al. 2012). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:32 utc nova biotechnol chim (2019) 18(2): 133-143 135 table 1. sampling site characteristics and the description of the rainwater tanks from which the harvested rainwater was extracted. site location overhanging tree branches and birds treatment rainwater usage tank age [years] roof type s1 rhodes university yes none emergency flushing of toilets 3 tile s2 rhodes university yes none emergency drinking water source 3 galvanized s3 rhodes university yes none drinking 3 galvanized s4 rhodes university no none emergency flushing of toilets 6 asbestos s5 rhodes university yes chlorinator drinking tile s6 rhodes university no none emergency flushing of toilets 3 tile s7 kenton (coastal) no none dishwashing 8 tile s8 kenton (coastal) no none dishwashing 8 tile s9 grahamstown west no none drinking 4 galvanized s10 grahamstown west no none watering the garden 6 galvanized s11 grahamstown west no none 6 galvanized . therefore, to strengthen testing programs, capacity building must not only include laboratory development and staff training (us epa 2016). use of simple and robust methods for testing microbial water quality, which do not require laboratory facilities or the trained microbiological personnel, and yet could provide early warning with respect to microbial rainwater quality at the point of use of the rainwater, in question, i.e. household level, are needed. the h2s test kit fulfils these criteria (tandlich et al. 2014). the h2s test kit is based on detecting the presence of faecal bacteria that produce h2s (sobsey and pfaender 2002). in this test, the h2s-producing bacteria of faecal origin reduce the thiosulphate in the kit medium to h2s and hydrogen sulphide then reacts with the ferric salt (ferric ammonium citrate) to form an insoluble black ferrous sulphide precipitate (mosley and sharp 2005). members of the enterobacteriaceae group such as salmonella, citrobacter, klebsiella, and proteus can produce h2s in such a medium (sobsey and pfaender 2002). other non-enteric bacteria, typically absent in drinking water, can reduce thiosulphate into h2s under anaerobic conditions, but their growth is inhibited by the addition of 0.5 % deoxycholate to the h2s test kit medium (sobsey and pfaender 2002; tandlich et al. 2014), as well as by conducting the h2s test kit incubations under aerobic conditions. this study aims to report on the comparison of the improved and modified h2s test kit, which based on the medium of venkobachar et al. (1994) and on further improvements by tandlich et al. (2014), in the detection of faecal contamination of rainwater. furthermore, the detection of faecal contamination in rainwater using the h2s test kit and based on the two standard microorganisms that require enumeration, namely colilert® and mft, is reported. the study was also conducted in grahamstown/makhanda in the eastern cape, south africa over three months in 2016. the current paper is part of an ongoing project to establish the correspondence between the h2s test kit and the standard indicator microorganisms used in south africa and surrounding countries. examples of previous studies included that of tandlich et al. (2014), who compared the efficiency of the modified h2s test kit to that of the m-tec enumeration method. angala et al. (2019) conducted a similar study in rural and urban parts of namibia. the h2s test kit was also used to study the microbial water quality in harare, zimbabwe in 2015 (chirenda et al. 2015). however, the current study is the first one to evaluate the microbial quality of harvested rainwater and to compare the modified h2s test kit and the colilert® method. mft is used for comparison purposes as this method was applied in previous studies (tandlich et al. 2012; nhokodi et al. 2016). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:32 utc nova biotechnol chim (2019) 18(2): 133-143 136 experimental sample collection sampling was conducted weekly over a threemonth period (june-september 2016) in the eastern cape from 11 tanks situated at rhodes university, kenton-on-sea (coastal) and in homes in the grahamstown/makhanda area. the 5 l bottles used for sampling were sterilised for sampling by first washing with antibacterial soap, rinsed with tap water followed by soaking in 30 % hcl for 5 minutes and finally rinsed with sterile distilled water. sterile 5 l bottles were then used to collect rainwater samples by first rinsing the tap connected to the tanks with 70 % ethanol and letting the tap run for 30 seconds before collection. details about the sampling sites are provided in table 1.samples (total number/grand total = 88) were then transported to rhodes university’s microbiology laboratory on ice for microbial analysis within 6 h after collection. gps coordinates of the sampling sites are available from the authors upon request. the rainwater harvesting tanks were located either in public areas, where no one could have an expectation of privacy, or at private houses. sampling at private residences took place in a participatory manner, i.e. only access to the sampling sites was negotiated with the household owners and no interviews or personal information was collected at any private house sampling sites. the results of the analyses from each private house were provided to the household owners/occupants expeditiously. microbial analysis preparation of the hydrogen sulphide (h2s) test kits the h2s test kits were prepared as previously described by tandlich et al. (2012), luyt et al. (2011), tandlich et al. (2014) and angala et al. (2019). all the chemicals were purchased from merck, johannesburg, south africa). the h2s test kits sampling was performed following the instructions previously described by tandlich et al. (2014) with slight modifications of the incubation temperature. briefly, five sterile h2s test kits per sample were filled with 20 cm3 of the collected rainwater (total volume of urine jars was 40 cm3). the contents of the h2s test kits were hand-shaken for about 10 seconds and incubated at 37 °c for 72 h instead of keeping the h2s test kits in a warm place (room temperature) as described by tandlich et al. (2014). the temperature of 37 °c is inside the working range of the h2s test kit based on the medium by venkobachar et al. (1994), as shown previously for water samples in south africa by genthe and franck (1999). this temperature is feasible to be achieved in the household, e.g. incubating the h2s test kits with sampled rainwater in the kitchen oven which is set to 37 °c, based on the use of low-cost thermometer to measure the temperature. table 2. correspondence between the h2s test kit and mft and colilert ®. colilert® mft presenceb absencee presencec absencef h2s test presencea 52 0 46 6 absenced 21 15 5 31 mft presence 51 0 absence 22 15 a presence of faecal contamination indicates that all five h2s kits in a particular sample from a given sampling site on a specific sampling occasion yielded a positive signal (see fig. 1 for details); b presence of faecal contamination indicates that concentration of e. coli in the colilert® analysis of the particular sample from a given sampling site on a specific sampling occasion yielded a concentration of 1 mpn/100 cm3 or higher; c presence of faecal contamination indicates that concentration of faecal coliforms in the mft analysis of the particular sample from a given sampling site on a specific sampling occasion yielded a concentration of 1 cfus/100 cm3 or higher; d absence of faecal contamination indicates that all five h2s kits in a particular sample from a given sampling site on a specific sampling occasion yielded a negative signal (see fig. 1 for details); e absence of faecal contamination indicates that concentration of e. coli in the colilert® analysis of the particular sample from a given sampling site on a specific sampling occasion yielded a concentration below 0 mpn/100 cm3; f absence of faecal contamination indicates that concentration of faecal coliforms in the mft analysis of the particular sample from a given sampling site on a specific sampling occasion yielded a concentration below 0 cfus/100 cm3. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:32 utc nova biotechnol chim (2019) 18(2): 133-143 137 the incubated h2s test kits were checked every 12 h for any colour change. a black colour indicated the presence of h2s-producing organisms of faecal origin (positive), while no colour change indicated the absence of h2s-producing organisms of faecal origin. both positive (e. coli atcc® 25922) and negative (sterile distilled water) controls were included in each test. incubations were done using the same equipment (tandlich et al. 2014). colilert® bacteria e. coli in rainwater samples was enumerated in triplicates using the colilert18®/quanti-tray®/2000 (idexx laboratories, inc., johannesburg south africa). briefly, 100 cm3 of water sample was mixed with the colilert-18® reagent, allowed to dissolve and transferred to a quanti-tray®/2000. the trays were then sealed with a quanti-tray® sealer and incubated at 37 °c for 18 – 24 h. positive e. coli (atcc® 25922) culture (inoculated in sterile water) and negative (sterile water) controls were included. following incubation, the quanti-tray®2000 were examined under uv for fluorescent wells. the mpn/100 cm3 values were recorded according to a tabulation of 95 % confidence intervals provided by the manufacturer, i.e. the mpn concentrations are derived from a statistical distribution, which reports an mpn value of the concentration for the respective indicator microorganism. the statistical distribution provides an estimate of the likely variability of the mpn concentration through the 95 % confidence interval. in further text of this article, the most probable number per 100 cm3 of the sampled rainwater is used to report bacterial concentrations measured by colilert® and designed as mpn/100 cm3. membrane filtration technique (mft) the mft was performed in triplicates by filtering 100 cm3 of the harvested rainwater sample through a 0.45 µm sterile membrane filter (millipore, tokyo, japan). the membrane filters were placed on m-fc agar (merck, south africa) plates and incubated at 44.5±0.2 °c for 24 h. after incubation, (presumptive) faecal coliforms were counted as blue colonies. concentrations of faecal coliforms are reported as the number of colony-forming units per 100 cm3 and designated as cfus/100 cm3 in further text. statistical analysis data analysis was done using the statistical package for the social sciences (spss) ver. 16.0. the non-parametric spearman's rank correlation was used to determine if there was any correlation between the three methods for the detection of faecal contamination (colilert®, mft and h2s test). furthermore, a 2 × 2 contingency table (95 % confidence level) method (mack and hewison 1988) was used to determine sensitivity, specificity, positive predictive value, negative predictive value, false positive, false negative and accuracy. the correspondence rates (cr) were calculated as shown in eq. 1. (1) in eq. 1, a is the total number of true positive results, d is the total number of true negative results and the term grand total represents the total number of samples tested, i.e. 88. a true positive result was recorded, if the h2s test kit recorded a positive signal for faecal contamination in all five kits for particular sample from a given sampling site on individual sampling occasion and the indicator microorganism concentration of 1 mpn/100 cm3 or 1 cfus/100 cm3 or higher was measured in the same sample. a true negative result was recorded, if the h2s test kit recorded a negative faecal contamination signal in all the five kits for particular sample from a given sampling site on individual sampling occasion and the indicator microorganism concentration below 0 mpn/100 cm3 or below 0 cfus/100 cm3. these results were evaluated in line with positive and negative controls. results efficiency of the h2s test to detect faecal bacteria in harvested rainwater a total of 88 samples from 11 harvested rainwater tanks were analysed to determine the performance bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:32 utc nova biotechnol chim (2019) 18(2): 133-143 138 fig. 1. modified h2s kit on model samples indicating negative (left) and positive (right) results. these were used as a reference to evaluation of the individual kits collected throughout the study at sampling sites s1-s11 (table 1). of the h2s test kit in the detection of faecal contaminants compared to two standard methods (colilert® and mft). correspondence between the standard indicator microorganisms are shown in terms of presence and absence in table 2. fig. 1 shows the difference between a negative and a positive h2s test kit. the cr values and related parameters of comparison between the h2s test kit and the standard indicators microorganisms are shown in table 3. from data in table 3, comparison of the h2s test as the new method and mft as the standard method showed a sensitivity of 93 % and the cr value of 88 % the h2s test showed a false positive of 16 % and a false negative of 9.8 % compared to the mft. using colilert® as the standard method, the h2s test showed a sensitivity of 71 % and a specificity of 100 % to detect the presence or absence of faecal bacteria in harvested rainwater (table 3) when compared to colilert®, the h2s test had a false positive result of 0 % and a false negative of 29 %. the cr between the h2s test kit and colilert® was equal to 76 % (see table 3 for details). performance of the h2s test kit in the detection of faecal bacteria from individual rainwater tanks the h2s test detected faecal bacteria in all the tanks except at sampling site 11 (fig. 2). sampling site 11 presented low bacterial counts using colilert® compared to the other rainwater harvesting tanks throughout the sampling period. at sites s1 s2, s4 and s5, there was a similar pattern between the two methods (h2s and mft) in the detection of faecal bacteria. furthermore, the h2s test detection rate was higher in s6, s8, s9 and s10 when compared to mft. table 4 shows the measured concentrations of the standard indicator microorganisms and the h2s test kit results. fig. 2. performance of the used test types on the detection of faecal bacteria. the portion of the total samples per respective sampling site detected by colilert (black columns), membrane filtration (grey columns) and h2s test (white columns). positive negative bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:32 utc nova biotechnol chim (2019) 18(2): 133-143 139 table 3. comparison of h2s test with colilert ® and mft for performance efficiency. sensitivity1 = 100 true positive results/(true positive results + false negative results); specificity2 = 100 true negative results/(true negative results + false positive results); positive predictive value3 = 100 true positive results/(true positive results + false positive); negative predictive value4 = 100 true negative results/(true negative results + false negative); cr5 as defined in eq. 1. spearman’s correlation coefficient calculations (table 5) indicate a high degree of correlation between the results of the h2s test kit and colilert®/mft, i.e. the r values are higher than 0.60 in both cases (see below). discussion efficiency of the h2s test to detect faecal bacteria in harvested rainwater the h2s test method was assessed in this study for its efficiency in the detection of faecal bacteria in harvested rainwater. the h2s test showed a higher cr value against mft, i.e. 88 %, as compared to the colilert® method where the cr value was equal to 76 %. in a previous study by tandlich et al. (2014), the cr value of the h2s test was test in comparison to the membrane filtration method was equal to 71 %. based on the calibration in grahamstown’s surface water (tandlich et al. 2012), the cr value was estimated to be equal to 99.4 % in the five-kit test variant of the h2s test kit method used in the current study (nhokodi et al. 2016). the media used for the enumeration of the particular standard indicator microorganism will have an influence on the recovery of cells, i.e. the measured bacterial concentration. based on the cr values measured in this study, it can be seen that the h2s test kit corresponds better with mft than with the colilert® method. therefore, the cr values have to established/measured/calibrated under the local conditions and for the given source of water. results of the current study further indicate that the h2s test kit performed better, i.e. achieved higher correspondence rates with the standard indicator microorganisms, at higher bacterial concentrations above 50 mpn/100 cm3 in the rainwater samples. this observation could explain the difference in correspondence rate between colilert® and the h2s test kit. however, the h2s test kit was positive for faecal contamination, when concentrations of standards indicators microorganisms were 5 most-probable number of cells/100 cm3 or higher. therefore, the h2s test kit may be used as a compliance test to monitor the quality of harvested rainwater at the household level. it should also be emphasised that should the h2s test show a positive result, the water should not be used for any potable purposes such as drinking without treatment. the contaminated harvested rainwater could be treated at household level using treatment options such as boiling and chlorination prior to use. the modified h2s test kit used in this study was first introduced and piloted by luyt et al. (2011) and locally produced at a cost of 0.34 usd using basic laboratory chemicals and everyday household materials such as urine jars. at present, that price might have increased as the south african rand depreciated against the usd. the h2s test kit is a good early warning system that does not require laboratories or sophisticated equipment to establish whether or not water is contaminated by faecal bacteria (luyt et al. 2011). a study by wallis (1991) in thailand reported that rainwater tanks had a 20 % false positive and 41 % false negative using the h2s test compared to the mft. these results indicate that the medium used in this study performed better than previous studies in the literature, as the rate of false positives was 16 % and the rate of false negatives were at 9.8 % when h2s was compared with mft. parameter h2s vs. mft [%] h2s vs. colilert® [%] mft vs. colilert®[%] sensitivity1 93 71 70 specificity2 88 100 100 positive predictive value3 92 100 100 negative predictive value4 91 42 41 cr5 88 76 75 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:32 utc nova biotechnol chim (2019) 18(2): 133-143 140 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:32 utc nova biotechnol chim (2019) 18(2): 133-143 141 false negatives occur when standard methods indicate the presence of contamination, a positive result but the new test indicates that the water is safe, a negative result. alternatively, false positives occur when a new test indicates that a water source is contaminated, a positive result when in fact it is not. the improvement in detection ability of the h2s kit in this current study can be attributed to the modification that the kit had undergone, i.e. addition of 0.5 % deoxycholate to the h2s test kit medium. furthermore, a false positive result is less likely to lead to a risk of disease because it would result in the contaminated water not being used, subjected to additional testing or treated. however, a false negative is of great concern as it means the contaminants are not detected by the new test (sobsey and pfaender 2002) and hence users might be exposed to microbial contaminants that could make them sick. in the current study, the h2s test gave a false negative of 9.8 % with mft and 29 % when compared to the colilert® method. further development and modification of the h2s test kit might be required to align its monitoring role with the colilert® system. one possible modification could be optimisation of the bile salt(s) added to the h2s test kit medium. performance of h2s test on the detection of faecal contaminants from individual rainwater tanks analysis from the current study showed that in most cases where rainwater samples had high faecal bacterial counts, the h2s test showed a positive test as well (table 4). the observation between the colilert® and the h2s test suggests that the h2s test is more efficient when the e. coli count in the rainwater samples is ≥ 50 mpn/100 cm3. in almost all the tested samples (s1-s10) the performance of the h2s test was satisfactory and proved to be competent when compared to mft and colilert®. water samples with low faecal bacterial counts resulted in negative h2s test. this finding suggests that rainwater samples from the eastern cape province of south africa, in which low concentrations of indicator microorganisms are measured, the h2s test kit results should be approached with caution. it is advisable to validate the h2s test against standard methods, prior to distributing the test in a new setting. in addition, further part of the ongoing project, which the current study is part of, will have to focus on the environmental stress of indicator microorganisms in the harvested rainwater and the factors affecting the cultivability of the h2s test kit bacteria. knowledge of the quality of the water to be tested is very important in order to minimize false negatives, especially in cases where contamination is seldom encountered. with sufficient public education on the h2s test, households would be in charge of their own water safety. if results indicate high risk, households would be instructed to treat their water to make it fit for human consumption (matwewe et al. 2018). vasudevan and tandon (2008) conducted research on the microbial quality of rainwater from roof surfaces and performed similar tests using mpn, mft and h2s and their results were in agreement with the results obtained in this study in terms of the positive correlations observed between the 3 methods during high bacterial counts. vasudevan and tandon (2008) further stated that in some cases, h2s showed that the water was potable while the mpn method disagreed. this finding differs from the current study in that no false positives were identified between colilert® and h2s. omar and bhutada (2016) studied the bacteriological assessment of drinking water and reported that the h2s test recorded, 20 positive samples out of the 38 samples tested, while 24 samples were positive for faecal contamination with mft. the study from omar and bhutada (2016) further reported that 10.52 % of the total number of samples tested as false negatives when the h2s test was compared to mft. based on the low difference between the h2s and the mft observed in their study, the authors concluded that the h2s test was a reliable and alternative method for the detection of faecal contamination during drinking water quality surveillance and screening of large numbers of samples in a short duration in the field where laboratory facilities are limited. conclusions with an average e. coli concentration of 362 mpn/100 cm3, the h2s test can overall be bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:32 utc nova biotechnol chim (2019) 18(2): 133-143 142 considered an effective tool in the rainwater microbial quality monitoring. the h2s test was the most efficient in detection of faecal contamination, if the colilert® concentrations of e. coli were above 50 mpn/100 cm3. with the e. coli concentrations below 50 mpn/100 cm3, the h2s test kit was not always sensitive enough, nor accurate enough in detection of faecal contamination in rainwater tested. the h2s test kit results correlated well with mft in detection of faecal contamination. comparing results from this study, with those from previous studies in the eastern cape, indicates the cr, sensitivity and other parameters should be measured under local conditions, i.e. to establish the efficiency of the h2s test kit in the detection of faecal contamination of the specific source(s) of the potable water. acknowledgement this study was supported by funds from the parliamentary grant of the council for scientific and industrial research (csir project echs043) and the water research commission (wrc project k5/2593). rhodes university provided access to monitoring sites and laboratory space for sample analysis. 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(1994) assessment of bacteriological quality using a modified h2s strip test. j. water supply res. t. 43: 311-314. wallis i (1991) simple test to detect contamination of drinking water. in project report marine and freshwater research center, no 11/91. land and water resources research and development corporation, canberra, act, 12 p. wazny k, zipursky a, black r, curtis v, duggan c, guerrant r, levine m, petri jr. wa, santosham m, scharf r, sherman pm, simpson e, young m, bhutta za (2013) setting research priorities to reduce mortality and morbidity of childhood diarrhoeal disease in the next 15 years. plos med. 10: e1001446. wright ja, yang h, walker k, pedley s, elliot j, gundry sw (2012) the h2s test versus standard indicator bacteria tests for faecal contamination of water: systematic review and meta-analysis. trop. med. int. health. 17: 94-105. world health organisation (who 2008) guidelines for drinking-water quality, third edition, world health organization, geneva, switzerland. world health organisation (who 2011) guidelines for drinking water quality, fourth edition, world health organization, geneva, switzerland. world health organisation (who 2017) an update on diarrhoeal disease, world health organization, geneva, switzerland. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:32 utc nova biotechnol chim (2019) 18(2): 179-194 doi: 10.2478/nbec-2019-0020  corresponding author: mszultka@umk.pl nova biotechnologica et chimica identification of in vitro and in vivo potential metabolites of novel cardiovascular and adrenolytic drugs by liquid chromatography-mass spectrometry with the aid of experimental design malgorzata szultka-mlynska1,, katarzyna pauter1,2 and boguslaw buszewski1,2 1 department of environmental chemistry and bioanalytics, faculty of chemistry, nicolaus copernicus university, gagarina 7, 87100 torun, poland 2 centre for modern interdisciplinary technologies, nicolaus copernicus university, wilenska 4, 87100 torun, poland article info article history: received: 13th october 2019 accepted: 26th november 2019 keywords: adrenolytic drugs cardiovascular drugs high performance liquid chromatography liver microsomes mass spectrometry abstract drug metabolism in liver microsomes was studied in vitro using liquid chromatography-tandem mass spectrometry (lc-ms/ms). relevant drug was incubated with dog, human and rat liver microsomes (dlms, hlms, rlms) along with nadph, and the reaction mixture was analyzed by lc-ms/ms to obtain specific metabolic profile. grace analytical c18 column, vision ht (50 × 2 mm, 1.5 m) was implemented with acetonitrile and water (+ 5 mm ammonium acetate) in a gradient mode as the mobile phase at a flow 0.4 ml.min-1. different phase i and phase ii metabolites were detected and structurally described. the metabolism of the studied drugs occurred via oxidation, hydroxylation and oxidative deamination processes. conjugates with the glucuronic acid and sulfate were also observed as phase ii biotransformation. the central composite design (ccd) showed that factors, such as time incubation, liver microsomal enzymes concentration and nadph concentration, along with drying gas temperature, nebulizer gas pressure and capillary voltage significantly affected the final response of the method. this study describes the novel information about the chemical structure of the potential metabolites of selected biologically active compounds, which provide vital data for further pharmacokinetic and in vivo metabolism studies.  university of ss. cyril and methodius in trnava introduction in recent years there has been a growing interest in research which improves our understanding of pathological phenomena responsible for the development and progression of various illnesses. with new diagnostic tools, the background of the most complex anomalies can be discovered (li 2001; bussiere 2008). huge progress has also been made in the therapy of many illnesses, which measurably translates into the extended overall survival of the patient (wrighton et al. 1995; jia et al. 2007; rostami-hodjegan et al. 2007). furthermore, as chromatographic techniques have been developed and combined with sensitive detection methods, the use of metabolomics has increased in the recent years (goldman et al. 2004; persell 2011). this made it possible to determine and identify substances in biological samples with the view to clinical diagnosing of illnesses and their stages as biomarkers of various human diseases have been discovered (ingelman-sundberg 1999, 2004; zisaki et al. 2015). an important trend in a pharmaceutical analysis is determining metabolic profiles after administering the drug to investigate what happens to it in the organism. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc mailto:mszultka@umk.pl nova biotechnol chim (2019) 18(2): 179-194 180 currently the most popular technique in the analysis of drugs and their metabolites is high-performance liquid chromatography combined with mass spectrometry. this combination is used most often because of its very low limit of detection, good selectivity, accuracy and the precision of quantification. the information obtained can be used to identify and quantitatively determine the studied compounds (xu et al. 2007; lietz et al. 2013). the determination of metabolites requires the use of multidimensional separation techniques. therefore the lc/ms analysis of selected drugs and their metabolites is to evaluate their concentration in real time (wicha et al. 2018; zhang et al. 2018). there are drug metabolites whose structures may resemble the structure of the parent drug to a lesser or greater degree and which also show a pharmaceutical activity resembling that of the parent drug; this creates a significant problem in therapeutic drug monitoring (tdm) (kang et al. 2009). monitored therapy is a useful tool in the process of personalizing treatments with drugs of varied pharmacological activity, because drug pharmacokinetics can differ significantly from patient to patient, which results from the influence of numerous interactions, comorbidities, genetic polymorphism or patients failing to adhere to the prescribed dose schedule (guengerich 2000). the monitoring of drug concentrations in blood is widely used in selected clinical specializations, e.g. neurology, cardiology and psychiatry. the most often monitored types of drugs include antibiotics, anti-seizure medicines, anti-arrhythmic medicines, painkillers, immunosuppressant substances, anti-cancer drugs and tricyclic antidepressants (cholerton et al. 1992; nicholson et al. 1999; yan et al. 2001). the enzyme family of cytochromes p450 are membrane proteins present mostly in the microsomes of endoplasmic reticulum and in membrane structures of mitochondrion (lamb et al. 2007). microsomes are closed vesicles in the membrane of endoplasmic reticulum in liver cells (coming from mice, rats and humans) which contain a number of phase i and ii metabolic enzymes, including the monooxygenase enzymatic complex that comprises cytochrome p450 and its nadph-dependent reductase (isin et al. 2007). among the numerous microsomal fraction enzymes in liver cells, the ones that are most involved in biotransformation reactions of exogenous substances are isoenzymes of cytochrome p450. there have been discovered more than a dozen of cytochrome p450 isoenzymes that participate in the oxidation of many drugs used in medicine. each isoenzyme is coded by a different gene (degtyarenko et al. 1993). the so-called gene superfamily that codes the various isoenzymes of cytochrome p450 has been classified into families and sub-families depending on the degree of similarities in the amino acid sequence of particular isoenzymes. approximately 95 % of the currently used medicines are metabolized by this enzymes group (houston 1994; wiekers et al. 2005; sahi et al. 2010). here we report the successful application of the optimized procedure and analysis method to determine and identify the selected cardiovascular and adrenolytic drugs (aliskiren, metoprolol, mexiletine, oxprenolol, prasugrel, propafenone, propranolol and rivaroxaban) and their potential metabolites found in different microsomal liver fractions (dog, human and rat). conventional triple quadrupole mass spectrometer (ms/ms) are the most popular analytical tools due to its excellent quantitative sensitivity, stability and low cost. however, it is not suitable for drug metabolite profiling since their full-scan sensitivity is rather poor. to overcome this problem, a set of predicted precursor-product ion pairs instead of full-scan mode to search for the potential metabolites was used. to the best of our knowledge, no detailed information regarding the structure of their potential metabolites via in vitro studies has been published. transformations were investigated in the presence of nadph/udpga-p450 oxidoreductase. central composite face-centered design with fractional factorial design was used in order to evaluate the significance of selected factors. in addition, the composite design helped us to find the optimal values for each factor using the response surface method. additionally, the results obtained were compared to in vivo experiments, by analyzing plasma samples from patients differing in the level of cytochrome p450 isoenzymes after relevant cardiovascular or adrenolytic drug delivery. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 181 experimental chemicals and materials aliskiren (ali), metoprolol (met), mexiletine (mex), oxprenolol (oxp), prasugrel (pra), propafenone (prf), propranolol (pro) and rivaroxaban (riv) were obtained from sigmaaldrich (schelldorf, germany). glucose-6phosphate, β-nadp+, glucose-6-phosphate dehydrogenase and uridine 5’-diphosphoglucuronic acid triammonium salt (udpga) were obtained from sigma-aldrich (schnelldorf, germany). other chemicals of analytical quality (acetonitrile, formic acid, ammonium acetate) were purchased from merck (darmstadt, germany). microsomes derived from dog, human and rat liver cells (dlms, hlms, rlms) were used in enzymatic studies. the microsomal fractions were stored at – 80 °c and thawed immediately before each experiment. the water was obtained by means of milli-q rg apparatus by millipore (millipore intertech, bedford, ma, usa) in our laboratory. assay incubation was done in the thermomixer (eppendorf, hamburg, germany). the evaporation of samples was done on a scanvac speed vacuum concentrator (kansas city, usa). drug-free plasma samples were kindly provided by nicolaus copernicus university, collegium medicum, torun (poland) upon the permission of the bioethical commission (no. 585/2017). microsomal incubations the research on discovering the mechanism of metabolic transformations of selected bioactive compounds was carried out in model conditions with their standard solutions in the presence of microsomal enzyme fraction (coming from dog, human and rat). the incubation of the selected drugs with liver cell microsomal fraction enzymes was to be performed in appropriate reaction buffer (pbs, ph 7.4). after initial pre-incubation in a heated bath (37 °c), enzymatic reaction cofactor nadph/udpga was added to the mixture of the particular bioactive compound and microsome (enzyme). the obtained reaction mixture was subjected to further incubation at the same temperature for a specific period (15, 25 and 35 min). then the resulting solution was cooled in ice bath and an equivalent amount of cooled methyl alcohol was added to stop the reaction. this mixture was centrifuged (5 – 15 min; 5,000 rpm). the resulting supernatant was analyzed by rp-hplc with esi-ms/ms. glucuronidation was studied by the incubation of the model drug with dlms, hlms and rlms. the test and blank samples were prepared analogously to phase i metabolism samples, the exception being that 10 μl of pbs contained the udpga co-factor which mediated the relevant reaction. beside the above mentioned batches, incubations without nadph, microsomal protein, target compound, respectively, were analyzed as negative controls. high performance liquid chromatography tandem mass spectrometry instrumentation and analytical conditions the hplc system consisted of an agilent 1100 series (agilent technologies, waldbronn, germany) autosampler, binary pump, degasser and column compartment. chromatograph separation was performed on a grace analytical c18 column, vision ht (50 × 2 mm, 1.5 µm), which was protected by a hplc guard (2.1 × 5 mm, 1.8 µm) at 21 °c. the column was operated gradiently at a flow rate of 0.4 ml.min-1 with mobile phase composed of 0.1 % ammonium acetate (a) and acetonitrile (b): 0 – 2 min, 15 % b; 2 – 5 min, linear to 95 % b; 5 – 10 min, 95 % b; and 10 – 12 min, return to initial conditions. the stop time was 15 min, followed by a 3 min post-time. the injection volume was 10 µl. a 6410 triple quadrupole lc/ms (agilent technologies, waldbronn, germany) equipped with electrospray ionization (esi) source was controlled by mass hunter workstation data acquisition software and qualitative analysis software (version b.01.00). the source temperature was maintained at 290 – 350 °c, and the ionization voltage was set at 3,500 – 4,500 v. the nebulizer gas was set at 35 – 45 psi. the detection of the ions was performed in the multiple reaction monitoring (mrm) mode. optimization of the compounddependent parameters were performed by the direct infusion of a standard solution into the ion source bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 182 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 183 using a syringe pump (table 1). the studied target compounds were identified by mrm transitions of the compounds searched for using a dwell time of 100 ms and product ion scan ms/ms experiments. the ms/ms conditions were optimized as well. all the compounds were tested in both positive and negative ion modes. ms/ms parameters were optimized in a continuous flow mode using an integrated syringe pump at a flow rate 5 µl.min-1 of standard solutions. the nebulizer gas and dry gas was nitrogen, and helium (99.999 %) was used as the collision gas in the ion trap. after the best conditions for isolating the precursor on (proton adduct of the analyte) had been determined, full scan ms/ms mode was used to record product ions from the standard solution of each target compound. for each analyte, the fragmentation amplitude and isolation width were also optimized manually to increase the selectivity and sensitivity of the method and to select the most intensive and characteristic fragmentation ions for a qualitative analysis and one of the highest intensity for the quantitative analysis. application to patient samples a 100 µl volume of plasma was transferred to a disposable 2 ml centrifuge tube. subsequently, 350 µl of water and 150 µl of methanol were added, and the tube contents were then mixed gently. 900 ml of acetonitrile was added to this solution, and the sample was mixed vigorously for few seconds. after a standing time of 15 min, the mixture was centrifuged at 5,000 rpm for 5 min, and 100 µl of the clear supernatant was mixed with 900 ml of 10 % methanol. results and discussion optimization of the lc-ms/ms parameters the determination of analytes with a mass spectrometer was carried out in positive polarization (esi+) in the following modes: full table 3. analysis of variance (anova) for fit of conversion efficiency from central composite design (for pro). source of variation sum of square dfa mean square f-valueb p-value x1 6183693 1 6183693 25.60267 0.002311 x1 2 1471186 1 1471186 6.09123 0.048587 x2 1024 1 1024 0.00424 0.950191 x2 2 832855 1 832855 3.44831 0.112697 x3 2744578 1 2744578 11.36352 0.015024 x3 2 490002 1 490002 2.02878 0.204213 x1 x2 3583529 1 3583529 14.83707 0.008441 x1 x3 8270401 1 8270401 34.24238 0.001099 x2 x3 625918 1 625918 2.59152 0.158563 lack of fit 1449152 6 241525 total ss 38000000 15 a df, degrees of freedom; b test for comparing model variance with residual (error) variance. scan (fs), single ion monitoring (sim), product ion scan (pi), and multiple reaction monitoring (mrm). scanning in the full mode was performed to determine the m/z ratio value of the pseudomolecular ion (an analyte molecule with its mass enlarged by the mass of the attached proton). single ion monitoring mode was applied to allow us to select the voltage at the fragmentor (the voltage at the end of the capillary directing ions to the analyzer q1). product ion mode was to make it possible to obtain product ions for each parent ion through its fragmentation. this step was to use the previously chosen fragmentor voltage, and appropriate values of collision energy were selected for individual studied compounds. ms/ms transitions form the precursor ion to two of its main fragment ions were registered for each target compound. only one mrm transition was selected for each at least. selected mrm transitions for each analyte and the optimal instrumental conditions set for their analysis are summarized in table 1. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 184 central composite design statistica software designed 16 experiments in accordance with the ccd; the levels examined and the experimental results are listed in table 2. the analysis of variance (anova) was used to assess the significance of each factor and interaction term. the significance of each coefficient was checked using an f test and by determining the p value. the p value was used to check the significance of each coefficient, which indicated the strength of the interaction between each independent variable. anova of the quadratic regression model showed that the model was significant (p < 0.0001/0.0005) and that the lack-of-fit was not significant fig. 1. three-dimensional surface for the lc-ms analyses using box-wilson procedure obtained by plotting. response surface plots showing the effects of drying gas temperature, nebulizer gas pressure and capillary voltage on the intensity of pro. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 185 (p > 0.05), indicating that the quadratic model was valid for this study (table 3). the coefficient of determination (r2) is a measure of the global fit of the model. in our analyses, r2 was higher than 0.9860 for all the target compounds, which means that the obtained model could explain the variability in response. based on the experimental data, a second-order polynominal model was constructed by ccd; where y is the predicted response value, and x1, x2, and x3 are the coded values of drying gas temperature, nebulizer gas pressure and capillary voltage, respectively. in the optimum design process, response surface curves were not only plotted to investigate the relationship between responses and each variable and the interactions of two variables, but the optimal level of each variable was also determined for the maximum response. the typical three-dimensional response surface plots for pro are shown in fig. 1. as can be seen, drying gas temperature, nebulizer gas pressure and capillary voltage had quadratic effects on the pro response. based on the plotted surfaces, the response value reached a maximum when drying gas temperature, nebulizer gas pressure and capillary voltage was around the central points. after the results had been analyzed, the hplc-ms analysis conditions (x1 = 310 °c, x2 = 40 psi, x3 = 4,000 v for pro) were obtained. fig. 1a illustrates the effects of drying gas temperature and nebulizer gas pressure (psi) on the signal intensity during lc-ms analyses. the maximum value was obtained with the drying gas temperature located between 300 and 330 °c and nebulizer gas pressure (psi) located between 37 and 43. higher drying gas temperature and higher nebulizer gas pressure tended to result in a decrease of signal intensity. this also could be due to the fact that the decreasing intensities result from ion suppression effects in the electrospray. fig. 1b shows the effects of drying gas temperature and capillary voltage on the signal intensity during lc-ms analyses. the maximum efficiency could be obtained with 310 °c and capillary voltage equaling 4,000 v. this drying gas temperature and capillary voltage resulted in the increase in the signal intensity during lc-ms analyses. in the range of 300 – 330 °c signal intensity increased, while above the value of 330 °c no increase in the intensity was observed. a higher drying gas temperature and capillary voltage resulted in medium signal intensity, which would result in a small number of ions to the mass spectrum originating from precursor ion. fig. 1c shows the effects of nebulizer gas pressure (psi) and capillary voltage on the signal intensity during lc-ms analyses. it was observed that in the nebulizer gas pressure range of 38 – 42 psi and in capillary voltage range of 3,600 – 4,200 v signal intensity increased, while above those values no incrementation was observed. the maximum predicted value of signal intensity during lc-ms analyses was under the conditions presented in table 1. fig. 2. pareto chart of the standardized main effect for the ccd design for lc-ms method. the vertical line defines the 95 % confidence level. the plot of experimental values of efficiency of conversion versus those calculated from equation indicated a good fit, as presented in fig. 2 (for pro). as result of the full factorial design, pareto chart was drawn for each studied cardiovascular drug in order to visualize the estimated effects of the main variables and their interactions. pareto chart gives a graphical presentation for these effects and it allows looking at both the magnitude and the importance of an effect. in the above mentioned pareto charts, the bars (variables) that graphically overpass the significance line exert a statistically significant influence on the results obtained. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 186 in vitro incubation of selected cardiovascular and adrenolytic drugs with mammalian liver microsomes there are a large number of components in the hlm, among which many compounds may elute in the same time with the target compounds during the analysis. in order to solve this problem, we performed using a mass hunter metabolite id software; peaks present in the analyte were evaluated in relation to control samples by comparing their retention time in postacquisition analyses, ms spectra and peak area. in order to identify the potential metabolites of selected cardiovascular and adrenolytic drugs, their possible structures were assumed according to the known common metabolic pathways and the structures of parent drugs. then the total ion chromatogram (tic) of the liver microsomal solution after incubation with a relevant drug was compared with that of blank liver microsomal solution (without nadph) to find the possible metabolites. finally, the potential metabolites were identified by comparing their lc-ms and ms2 spectra with those of target compounds. in the case of riv 6 metabolites were found and identified in all in vitro incubations. metabolite structures were first elucidated by liquid chromatography-mass spectrometry with a detailed analysis of the changes in molecular mass and interpretation of the fragmentation patterns, compared with those of the parent drug. the product ion spectra of metabolites m1, m2, m3, m5 and m6 exhibited losses of 18 amu corresponding to h2o molecule, 46 amu (hcooh), 58 amu (hoccoh) and 72 amu (co2+co), indicating that metabolism occurred at the morpholinone moiety. the hydroxylated metabolite m4 showed a product ion spectrum that was different from that for the other hydroxylated metabolites, namely m1, m2 and m3. in the case of m4, fragmentation at the oxazolidinone moiety indicated that the hydroxylation had taken place at the oxazolidinone ring. hydroxylation occurred at the morpholinone moiety, leading to metabolites m1, m2 and m3 and at the oxazolidinone moiety, leading to metabolite 4. in the case of m5 and m6, combinations of morpholinone and oxazolidinone hydroxylation were also identified. among all oxidative reactions, the hydroxylation leading to m1 was the major primary pathway identified in incubations with liver microsomes across all the species. in the case of pra 4 metabolites were found and identified in all in vitro incubations. one of them was characterized for thiolactone metabolite m1 (in the literature named as r-95913). then thiolactone metabolite was oxidatively metabolized by hepatic cytochrome p450 to the compound m2 containing a thiol group (in the literature named as r-138727), which was further metabolized to its s-methyl analogue m3 (in the literature named as r-106583). the analytes were analyzed by an lc-ms/ms in the multiple reaction monitoring mode using the respective [m+h]+ ions, m/z 332 – 109 for m1, m/z 498 – 206 for m2, m/z 364 – 316 for m3 and m/z 390 – 318 for m4. in the case of ali 4 metabolites were found and identified in all in vitro incubations. the mass spectra of metabolites m1 and m2 showed molecular ions at m/z 538, indicating that they are demethylated metabolites. o-demethylation was assigned to the region of fragment c in the two possible positions for methoxy groups. the mass spectrum of metabolite m3 showed a molecular ion at m/z 480. o-dealkylation was assigned to the region of fragment c, indicating oxidation of ali, with loss of the propoxy side chain. hence, compound m3 could be the methoxy phenol derivative. metabolite m4 was identified as a glucuronic acid conjugate of m3. in the case of pro 7 metabolites were found and identified in all in vitro incubations. this molecule is interesting because of the availability of numerous oxidation sites present on the structure. hence, the first step was to identify hydroxylated metabolites. the presence of monohydroxylated (m/z at 276) and dihydroxylated propranolol (m/z at 292) metabolites was observed. the abundance of m/z 260, the parent ion, was low, demonstrating that the drug was extensively metabolized. during the study also the presence of hydroxypropranolol glucuronide (m/z 452) and dihydroxypropranolol glucuronide (m/z 468) was observed. in the case of oxp 2 metabolites were found and identified in all in vitro incubations. the proposed microsomal transformation bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 187 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 188 of oxprenolol (m/z 224) include n-dealkylation and ring-hydroxylation (o gain) as well as the combination of these two pathways. during the study also the presence of hydroxyoxprenolol glucuronide (m/z 571) was observed. in the case of met 2 metabolites were found and identified in all in vitro incubations. the proposed microsomal conversion of metoprolol include benzylic hydroxylation or aromatic hydroxylations (m/z 284) and demethylation (m/z 254). in the case of mex 2 metabolites were found and identified in all in vitro incubations. the proposed microsomal conversion of mexiletine include aliphatic (m/z 178) and aromatic (m/z 196) hydroxylation. in the case of prf 2 metabolites were found and identified in all in vitro incubations. for propafenone (m/z 342) with a known metabolism, the only product characterized for phase i that was observed and identified was single o gain at various positions. in other words, the only metabolic pathways that was mimicked with the use of microsomal fraction was the hydroxylation (m/z 358). during the study also the presence of hydroxypropafenone glucuronide (m/z 647) was observed. to elucidate the structures of the unknown metabolites, we studied the fragmentation of the [m+h]+ ions of met and its metabolites using lc/esi/ms/ms experiments. major metabolites were compared with the retention time of the commercial reference standards. the postulated major metabolic pathways of incubated cardiovascular and adrenolytic drugs with dlms, hlms and rlms is presented in table 4. these pathways show that the hydroxyl moiety, carbon-carbon double bond, and carbonoxygen double bond are the metabolic sites in vitro. the hydroxyl groups is the primary metabolic sites with further formation through reduction and oxidation to generate dehydrated and hydrogenated metabolites, which is similar to the investigated in vivo metabolism in real biological samples. the production of potential metabolites of target compounds was all increased with the prolongation of the incubation time. among them, the demethylated metabolites were the major metabolites, while the hydroxylated were the minor metabolites; glucuronidated constituted the fewest of target compounds. based on the above discussion, the selected chromatograms of ali in 3 kinds of liver is presented in fig. 3. this may be due to the different genotype and activity of hepatic metabolic enzymes in different species. the detailed proposed fragmentation for all potential metabolites is presented in table 5. fig. 3. hplc-chromatograms of ali potential metabolites (m1 – m4) in three types liver microsomes: (a) dlms, (b) hlms and (c) rlms. all the postulated phase i and ii metabolic reactions are typical of processes catalyzed a b c bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 189 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 190 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 191 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 192 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 193 by isoenzymes of the cytochrome p450 group. it may be concluded that the mammalian liver microsomes having enzymatic activity may be very useful in the study of the metabolism of cardiovascular and adrenolytic drugs. analysis of biological samples from patients in order to examine the proposed metabolic pathways of target compounds, the data coming from the suggested method with the use of liver microsomes fractions were studied in detail and were compared with the results obtained from biological samples originating from patients receiving cardiovascular and adrenolytic drugs. the presence of drug and its potential metabolites in samples from the patients were additionally confirmed by ms/ms spectra. the abundance of relevant parent ion, was low, demonstrating that the drug was extensively metabolized. applying the developed lc-ms/ms method in plasma samples were identified: metoprolol and its two metabolites o-demethylated metoprolol (m/z = 254) and hydroxylated metoprolol (m/z = 284); propranolol and hydroxylated propranolol (m/z = 276) was observed; mexiletine and hydroxylated mexiletine (m/z = 196); oxprenolol n-dealkylated oxprenolol (m/z = 224) and propafenone and hydroxylated propafenone (m/z = 358), respectively. conclusions in this study, we applied lc-ms/ms to identify phase i and phase ii metabolites of selected cardiovascular and adrenolytic drugs as a model compounds detected after in vitro incubation of the studied drug with dog, human and rat liver microsomes fraction. to the best of our knowledge, no detailed information regarding the structure of their potential metabolites via in vitro studies has been published. the results obtained show the similarity among analyzed mammalian species. the main biotransformation route of the studied compounds was identified as reduction, oxidation, dehydroxylation and glucuronic acid conjugation. as a result, the presented approach with in vitro and in vivo studies can be helpful in prognosing the influence of the selected drugs and their metabolites from different therapeutic groups on the activity of metabolizing enzymes; it may also be used in planning a targeted therapy. acknowledgments the work was financially supported by the national science centre in the frame of the project sonata 12, no. 2016/23/d/st4/00305 (2017-2020). conflict of interest the authors declare that they have no conflict of interest. references bussiere jl (2008) species selection consideration for preclinical toxicology studies for biotherapeutics. expert opin. drug metab. toxicol. 4: 871-877. cholerton s, daly ak, idle jr (1992) the role of individual human cytochromes p450 in drug metabolism and clinical response. trends pharmacol. sci. 13: 434-439. degtyarenko kn, archakov ai (1993) molecular evolution of p450 superfamily and p450-containing monooxygenase systems. febs lett. 11: 1-8. goldman dp, joyce gf, escarce jj, pace je, solomon md, laouri m, landsman pb, teutsch sm (2004) pharmacy benefits and the use of drugs by the chronically ill. jama 291: 2344-2350. guengerich fp (2000) common and uncommon cytochrome p450 reactions related to metabolism and chemical toxicity. chem. res. toxicol. 14: 611-650. houston jb (1994) utility of in vitro drug metabolism data in 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today 6: 357-366. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc nova biotechnol chim (2019) 18(2): 179-194 194 lietz cb, gemperline e, lingjun l (2013) qualitative and quantitative mass spectrometry imaging of drugs and metabolites. adv. drug delivery rev. 65: 1074-1085. nicholson jk, lindon jc, holmes e (1999) 'metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological nmr spectroscopic data. xenobiotica 29: 1181-1189. persell sd (2011) prevalence of resistant hypertension in the united states, 2003-2008. hypertension 57: 10761080. rostami-hodjegan a, tucker gt (2007) simulation and prediction of in vivo drug metabolism in human populations from in vitro data. nat. rev. drug discov. 6: 140-148. sahi j, grepper s, smith c (2010) hepatocytes as a tool in drug metabolism, transport and safety evaluations in drug discovery. curr. drug discov. technol. 7: 188-198. wicha sg, kloft ch (2018) quantitative systems pharmacology in model-informed drug development and therapeutic use. curr. opin. sys. biol. 10: 19-25. wienkers lc, heath tg (2005) predicting in vivo drug interactions from in vitro drug discovery data. nat. rev. drug discov. 4: 825-833. wrighton sa, ring bj, vandenbranden m (1995) the use of in vitro metabolism techniques in the planning and interpretation of drug safety studies. toxicol. pathol. 23: 199-208. xu rn, fan l, rieser mj, el-shourbagy ta (2007) recent advances in high-throughput quantitative bioanalysis by lc-ms/ms. j. pharm. biomed. anal. 44: 342-355. yan z, caldwell gw (2001) metabolism profiling, and cytochrome p450 inhibition & induction in drug discovery. curr. topics med. chem. 1: 403-425. zhang z, tang w (2018) drug metabolism in drug discovery and development. acta pharm. sin. b 8: 721-732. zisaki a, miskovic l, hatzimanikatis v (2015) antihypertensive drugs metabolism: an update to pharmacokinetic profiles and computational approaches. curr. pharm. des. 21: 806-822. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:44 utc https://www.scopus.com/record/display.uri?eid=2-s2.0-85047524773&origin=resultslist&sort=plf-f&src=s&st1=wicha&st2=s&nlo=1&nlr=20&nls=count-f&sid=fda0d1b9af0be7556af2fafa08a160bf&sot=anl&sdt=aut&sl=40&s=au-id%28%22wicha%2c+sebastian+g.%22+56114980500%29&relpos=2&citecnt=0&searchterm= https://www.scopus.com/record/display.uri?eid=2-s2.0-85047524773&origin=resultslist&sort=plf-f&src=s&st1=wicha&st2=s&nlo=1&nlr=20&nls=count-f&sid=fda0d1b9af0be7556af2fafa08a160bf&sot=anl&sdt=aut&sl=40&s=au-id%28%22wicha%2c+sebastian+g.%22+56114980500%29&relpos=2&citecnt=0&searchterm= https://www.scopus.com/record/display.uri?eid=2-s2.0-85047524773&origin=resultslist&sort=plf-f&src=s&st1=wicha&st2=s&nlo=1&nlr=20&nls=count-f&sid=fda0d1b9af0be7556af2fafa08a160bf&sot=anl&sdt=aut&sl=40&s=au-id%28%22wicha%2c+sebastian+g.%22+56114980500%29&relpos=2&citecnt=0&searchterm= microsoft word 8_miklovicbaranboca nova biotechnologica et chimica 15-2 (2016) 182 doi 10.1515/nbec-2016-0018 © university of ss. cyril and methodius in trnava thieno[3,2-c]pyridine complex of ni(ii) with unusual magnetic properties jozef miklovič1, peter baran2, roman boča1 1 department of chemistry, faculty of natural science, university of ss. cyril and methodius in trnava, trnava, sk-917 01, slovak republic (roman.boca@ucm.sk) 2 department of chemistry, juniata college, huntington, pa 16652, usa abstract: thieno[3,2-c]pyridine (thpy) has been prepared in a free form and embodied into the [ni(thpy)2(h2o)2(ac)2] complex as a ligand. the x-ray structure shows a molecular structure of the complex with ni−o(ac) = 2.059, ni−oh2 = 2.078, and ni−n(thpy) = 2.124 ǻ. electronically the complex behaves like a compressed tetragonal bipyramid. the molecular units are linked into a complex system of hydrogen bonds. two units show a π−π stacking of the aromatic rings (3.8 ǻ). there are planes of tetragons formed of the nickel atom with the in-plane ni...ni separation of 7.74 ǻ and the inter-plane ni...ni contacts at a = 9.65 ǻ. the effective magnetic moment shows a gradual decrease on cooling from the room temperature and an abrupt drop below 20 k typical for the zero-field splitting of s = 1 systems. above the room temperature the effective magnetic moment shows anomalies – a decrease and then an increase. key words: ni(ii) complex, x-ray structure analysis, magnetic data, zero-field splitting 1. introduction at the present, one of the dynamically developed field of heterocyclic chemistry is represented by the synthesis and characterization of new systems that can form coordination compounds with transition metals. structural and spectral studies of complexes containing n-heterocyclic ligands are of a considerable interest from the standpoint of coordination and bioinorganic chemistry. especially quinoline isosteres (furopyridines, thienopyridines) in which the benzene ring is replaced by furan or thiophene ring are components of many biologically active compounds. new pharmacophores with potential antipsychotic activity posses the thieno[3,2-c] and furo[3,2-c]pyridine ring system (new et al., 1989). therefore, efficient synthetic methods for these types of heterocycles are of a great interest. the synthesis of the furo[3,2-c]pyridines and pyrrolo[2′,3′,4,5]furo[3,2-c]pyridines has already been reported (krutošíková et al., 1994; bencková et al., 1995; krutošíková et al., 1996; juristová et al., 2007). the above heterocycles can be readily coordinated to metal centres through the n-donor atom. the coordination chemistry of them has already been outlined (miklovič et al., 2004; baran et al., 2005). however, systematic studies need much more effort, and in addition to the structural features also the spectral and magnetic data would be interesting. some data along this direction has also been published (boča et al., 2008; titiš et al., 2010). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:02 utc 183 miklovič, j. et al. 2. experimental section the ligand thieno[3,2-c]pyridine (thpy) has been prepared by a procedure described elsewhere (eloy et al., 1970). nickel(ii) acetate tetrahydrate (0.249 g, 1 mmol) has been dissolved in 4 cm3 of a dry ethanol and on stirring a solution containing thieno[3,2-c]pyridine (0.405 g, 3 mmol) in 3 cm3 of dry ethanol has been added. after 30 min of stirring, the volume has been reduced to a half using the vacuum rotational evaporator. on cooling a precipitate was separated, washed with 1 cm3 of cold ethanol and dried. anal. calcd for c18h20n2nio6s2 (hereafter 1): c, 44.7; h, 4.17; n, 5.80; s, 13.3. found: c, 44.9; h, 4.10; n, 5.60; s, 13.3 %. single crystals suitable for the x-ray analysis have been obtained by crystallization from the diethylether/tetrahydrofuran mixture (1 : 1). ir spectra were measured on magna-ftir-750 spectrometer (nicolet) in kbr pellets in the 4 000 – 400 cm−1 region. carboxyl stretching frequencies can indicate a coordination mode of acetate anion. following a tentative assignment for 1 (νa(coo) = 1 596, νs(coo) = 1 336 cm −1), the difference in observed stretching modes δ = 260 cm−1 indicates a unidentate coordination mode for the acetate anions, what is consistent with findings from a crystallographic studies. electronic spectra were measured in the nujol mull on specord-200 (analytical jena). they show three principal spin-allowed d-d bands with peaks at the positions of 9 730 (3t2g ← 3a2g), 15 940 ( 3t1g(f) ← 3a2g), and 26 560 cm −1 (3t1g(p) ← 3a2g), respectively. however, there exist arms at 13 880 and 22 500 cm−1, which confirm a symmetry reduction to the tetragonal and/or rhombic pattern. diffraction data for 1 were collected on a three-circle-platform diffractometer equipped with a ccd detector (bruker, smart 1κ). the frame data were acquired with the smart software at 300 k using mo kα radiation. the structure was solved by direct method (shelx-90) and refined by least-squares method on f2 (shelxtl-93, incorporated shelxtl v5.1). hydrogen atoms were geometrically positioned and left riding on their parent atoms during structure refinement. the structural data for 1: empirical formula, c18h20n2nio6s2; formula mass, 483.19; colour, pale turquoise; crystal system, monoclinic; space group, p2(1)/c; a = 9.6513(15) å; b = 12.7271(19) å; c = 8.8086(13); α = γ= 90 deg; β = 110.143(2) deg; v = 1 015.8(3) å3; t = 298(2) k; z = 2; λ = 0.71073 å; ρ(calcd) = 1.580 g/cm3; μ = 1.199 mm−1; hkl ranges, (−12, 12), (−16, 7), (−11, 10); f(000) = 500; no. of data collected, 6067; r(f) [i > 2σ(i)], 0.0485 (0.0332); rw(f 2) (all data), 0.0852; largest peak and hole/e å−3, 0.311/ −0.329; data / restraints / parameters, 2296 / 0 / 134. deposit number in the cambridge crystallographic data centre – ccdc no 292402. the squid magnetometer (quantum design, mpms-xl7) was used for measurements of the magnetic data: temperature dependence of the magnetic susceptibility, and field dependence of the magnetization. the magnetic susceptibility taken at b = 0.1 t has been corrected for the underlying diamagnetism and converted to the effective magnetic moment. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:02 utc nova biotechnologica et chimica 15-2 (2016) 184 3. results and discussion single crystal x-ray structural analysis confirms that the complex under study possesses a molecular structure. two thpy ligands, two aqua ligands, and two acetato ligands form the {nin2o2o’2} chromophore with an approximate rhombohedral coordination. the ortep drawing of the x-ray determination is shown in fig. 1. the metal-ligand distances are nio(ac) = 2.059, nioh2 = 2.078, and nin(thpy) = 2.124 ǻ. thus the complex approximately forms an elongated bipyramid. an average ni-o distance in a set of [ni(h2o)6] 2+ complexes adopts a value of d (nio) = 2.055 å whereas d (nin) = 2.145 å is an average value in [ni(nh3)6] 2+ complexes. therefore, the electronic properties of the {nin2o4} chromophore need be evaluated taking into account a relaxation of bond lengths relative to the complexes with homogeneous ligand sphere. the displacement parameters are ( )i i id dδ = − and they define the (electronic) distortion parameters: str ( ) / 2z x yd δ δ δ= − + , str ( ) / 2x ye δ δ= − (1) in the light of this correction to the heterogeneous donor set, i.e. δx,y(ni-o) = +1.35 pm and δz(ni-n) = −2.10 pm, the axial and rhombic distortion parameters of the complex 1 become dstr = −3.45 pm, estr = 0.95 pm and e/|d| = 0.275. contrary to pure geometrical aspects, the complex behaves electronically like a compressed tetragonal bipyramid. however, another choice of axes yields δx,y(ni-o,n) = −1.70 pm and δz(ni-oh2) = +2.30 pm, dstr = +4.00 pm, estr = 1.25 pm and e/|d| = 0.312. to this end it must be stated that the sign of the d-parameter cannot be safely fixed owing to the critically high rhombicity. fig. 1. ortep drawing of 1 with atom numbering (50 % probability ellipsoids). a similar architecture has been found in an analogous complex, [ni(fupy)2(h2o)2(ac)2] hereafter 2, with fupy = furo[3,2-c]pyridine, where ni−o(ac) = 2.052, ni−oh2 = 2.073, and ni−n(fupy) = 2.129 ǻ yield dstr = −2.35 pm. in the [ni(iqu)2(h2o)2(ac)2] monomeric units, 3, containing the iso-quinoline ligands bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:02 utc 185 miklovič, j. et al. (iqu), the distances inside the chromophore are ni−o(ac) = 2.061, ni−oh2 = 2.089, and ni−n(iqu) = 2.115 å, so that dstr = −5.00 pm (ivaniková et al., 2006). in both cases the pure geometric aspects refer to the elongated tetragonal bipyramid while the complexes electronically behave like a compressed tetragonal bipyramid. fig. 2. mercury view to the π−π stacking of aromatic rings in 1. fig. 3. the system of hydrogen bonds in 1 forming a 20-membered ring. view along the a-direction. a detailed inspection to the crystal structure of 1 confirms that a complex system of the closed contacts is formed (fig. 2, fig. 3). two thpy molecules of the adjacent bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:02 utc nova biotechnologica et chimica 15-2 (2016) 186 centers exhibit a π−π stacking at the distance 3.84 – 3.88 ǻ and the n...s contacts 3.91 ǻ. these contacts propagate a chain. four units inside the unit cell ale linked by hydrogen bonds which form a 20-membered ring involving 4 ni atoms. these four ni atoms exhibit a rhombus that is propagated along the b-c plane with the shorter ni...ni contact 7.74 ǻ. such planes are stacked each above another with the interplane ni...ni contact c = 9.65 ǻ. temperature evolution of effective magnetic moment for 1 is rather complex (fig. 4). on cooling from the room temperature it shows a gradual decrease until 25 k (region ii) owing to some temperature-independent paramagnetism. a drop of μeff below 25 k (region i) evidences a zero-field splitting typical for s = 1 systems (boča et al., 2008; titiš et al., 2010). near room temperature the μeff starts to decrease (region iii) and above 350 k it increases (region iv). t/k 0 50 100 150 200 250 300 350 400 μ e ff/ μ b 0 1 2 3 4 b/t 0 1 2 3 4 5 6 7 m m ol /( n a μ b ) 0 1 2 t = 2.0 k b = 0.1 t 0 10 20 30 40 50 2.8 2.9 3.0 3.1 3.2 3.3 i ii ii iii iv 200 250 300 350 400 χ m ol /( 10 -6 m 3 m ol -1 ) 0.04 0.06 0.08 200 250 300 350 400 [χ m ol /( 10 -6 m 3 m ol -1 )] -1 10 15 20 25 t = 4.6 k fig. 4. the magnetic functions for 1. lines – fitted. the magnetization data were taken at t = 2.0 k and 4.2 k not evidencing a saturation at b = 7 t. the magnetization per formula unit is lower than mmol/na = 2.0 μb expected for s = 1 systems. this again confirms that some zero-field splitting applies. there is no evidence for the existence of a remnant magnetization when the direction of the magnetic field is reversed. the magnetic susceptibility data was treated by the spin-hamiltonian of the general form: 2 2 2 2 2 2 , 1 b ˆ ˆ ˆˆ ( / 3) ( ) ˆ ˆ ˆ( sin cos sin sin cos ) k l z x y m x k l x y k l y z k z h d s s e s s b g s g s g sμ ϑ ϕ ϑ ϕ ϑ − − − = − + − + + + r h h h (2) where the zeeman (last) term needs averaging over a number of grid points (k, l) distributed uniformly over one hemisphere. the eigenvalues for the given “working” magnetic field enter the partition function; its derivative determines the magnetization and magnetic susceptibility (boča, 1999; 2012). moreover, the magnetic susceptibility has been corrected for the constant term χtip (temperature-independent bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:02 utc 187 miklovič, j. et al. paramagnetism plus the uncertain portion of the diamagnetic susceptibility) that reflects the linear increase of the effective magnetic moment at ambient temperature. also a molecular-field correction (zj) has been accounted for by the formula: zfs zfs tip/[1 ( ) ]zjχ χ χ χ= − + (3) as a result of the fitting procedure applied for susceptibility below 280 k the following set of magnetic parameters has been determined: gav = 2.254, d/hc = 2.59 cm−1, e/hc = 0.56 cm−1, χtip = 6.0 × 10 –9 m3 mol–1, and (zj)/hc = –0.012 cm−1; the discrepancy factor on susceptibility was r(χ) = 0.004. however, the same quality of the fit is obtained with gav = 2.257, d/hc = –2.50 cm −1, e/hc = 0.63 cm−1, χtip = 6.3 × 10–9 m3 mol–1, and (zj)/hc ~ 0 (table 1). table 1. structural and magnetic parameters for related complexes [ni(base)2(h2o)2(ac)2]. averaged bond lengths d (ni−o) = 2.055 å and d (ni−n) = 2.145 å. 1 (thpy) 2 (fupy) 3 (iqu) r(ni-ac)/ǻ 2.059 2.0735 2.0614 r(ni-oh2)/ǻ 2.078 2.0519 2.0886 r(ni-n)/ǻ 2.124 2.1295 2.1153 dstr/pm –3.45 (+4.00) –2.2 –5.0 estr/pm 0.95 (1.25) 1.0 1.4 gx 2.147 2.094 gy 2.149 2.104 gz 2.257 (2.254) 2.179 2.139 d/cm-1 –2.50 (2.59) –5.0 –5.3 e/cm-1 0.63 (0.56) 0.5 1.2 the detected g-value adopts a value typical for ni(ii) complexes (boča et al., 2008; titiš et al., 2010). the d-parameter for rhombic zero-field splitting systems with {nin2o4} chromophore is of the order of −5 cm −1. namely, in analogous complexes the following set of magnetic parameters has been detected: d/hc = −5.0 cm−1 in [ni(fupy)2(h2o)2(ac)2], and d/hc = −5.3 cm −1 in [ni(iqu)2(h2o)2(ac)2] (ivaniková et al., 2006). much higher value of the tip in the present case needs to be viewed as an effective parameter that accounts for the inter centre interactions due to the hydrogen-bond network and the π-π interaction between the aromatic rings. the molecular-field interaction of an antiferromagnetic nature is effective at the lowest temperature window. the magnetic behaviour above the room temperature (regions iii and iv) stays unmodelled so far. most probably a phase transition applies. the present structural and magnetic data can be utilized in enrichment of the magnetostructural d-correlations and also more advanced statistical methods can be applied for them (titiš et al., 2010; augustín et al., 2015). 4. conclusions it has been demonstrated that the complex [ni(thpy)2(h2o)2(ac)2] possesses molecular structure with the {nin2o2o’2} chromophore. the bond lengths alone show geometry of the elongated tetragonal bipyramid. however, after correction to the averaged ni-o and ni-n distances the coordination polyhedron matches bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:02 utc nova biotechnologica et chimica 15-2 (2016) 188 a compressed tetragonal bipyramid with a large rhombicity. magnetic susceptibility data confirms the s = 1 state and the magnetic response at low temperature is influenced by the zero-field splitting. the data fitting yields d/hc = –2.5 cm–1; however also d/hc = +2.6 cm–1 is a good solution so that the sign of the d-parameter cannot be fixed reliably owing to a critically high rhombicity. there is a magnetic anomaly near and above the room temperature. acknowledgements: slovak grant agencies (vega 1/0534/16, vega 1/0522/14 and apvv-14-0078) are acknowledged for the financial support. references augustín, p., boča, r.: magnetostructural relationships for fe(iii) spin crossover complexes. nova biotechnol. chim., 14, 2015, 96-103. baran, p., boča, m., boča, r., krutošíková, a., miklovič, j., pelikán, p., titiš, j.: structural characterization, spectral and magnetic properties of nickel(ii) complexes with furopyridine derivatives and isothiocyanate. polyhedron, 24, 2005, 1510-1516. bencková, m., krutošíková, a.: synthesis of pyrrolo[2´,3´:4,5]furo[3,2c]pyridines. monatsh. chem., 126, 1995, 753-758. boča, r.: theoretical foundations of molecular magnetism, elsevier, amsterdam, 1999. boča, r.: zero-field splitting in metal complexes. coord. chem. rev., 248, 2004, 873 p. boča, r., titiš, j.: magnetostructural d-correlation for zero-field splitting in nickel(ii) complexes. in: cartere, t.w., verley, k.s. (eds.), coordination chemistry research progress, nova science publishers, new york, 2008, 247-304. boča, r.: a handbook of magnetochemical formulae, elsevier, amsterdam, 2012, 991 p. eloy, f., derickere, a.: synthèse des thiénopyridines. bull. soc. chim. belg., 79, 1970, 301-311. ivaniková, r., boča, r., dlháň, ľ., fuess, h., mašlejová, a., mrázová, v., svoboda, i., titiš, j.: heteroleptic nickel(ii) complexes formed of n-donor bases, carboxylic acids, and water: magnetostructural correlations. polyhedron, 25, 2006, 3261-3268. juristová, n., štefanovová, e., ďurčeková, t., prónayová, n., gatial, a., krutošíková, a.: synthesis and reactions of [1]benzofuro[3,2c]pyridine. nova biotechnol., 7, 2007, 107-114. krutošíková, a., dandárová, m.: substituted vinyl azides in synthesis of furo [3, 2-b: 4, 5-b'] dipyrroles and pyrrolo [2', 3': 4, 5] furo [3, 2-c] pyridines. heterocycles, 37, 1994, 1695-1700. krutošíková, a., sleziak, r: synthesis of 2-arylfuro[3,2-c]pyridines and their derivatives. collect. czech. chem. commun., 61, 1996, 1627-1636. miklovič, j., krutošíková, a., baran, p.: two furopyridine complexes of nickel(ii) isothiocyanate. acta cryst., c60, 2004, m227-m230. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:02 utc 189 miklovič, j. et al. new, j.s., christopher, w.l., yewich, j.p., butler, r.f., schlemmer jr. r. f., van der maelen, c.p., cipolline, j.a.: the thieno[3,2-c]pyridine and furo[3,2-c]pyridine rings: new pharmacophores with potential antipsychotic activity. j. med. chem., 32, 1989, 1147-1156. titiš, j., boča, r.: magnetostructural d-correlation in ni(ii) complexes – reinvestigation of the zero-field splitting. inorg. chem., 49, 2010, 3971-3973. received 16 november 2016 accepted 6 december 2016 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 15:02 utc microsoft word duresova et al 2015_nbec_final.doc 176 nova biotechnologica et chimica 14-2 (2015) doi 10.1515/nbec-2015-0025 © university of ss. cyril and methodius in trnava comparison of cd and zn accumulation in tissues of different vascular plants: a radiometric study zuzana dürešová1, anna šuňovská1, miroslav horník1, martin pipíška1, marcela gubišová2, jozef gubiš2, juraj lesný1 1department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic (hornik@ucm.sk) 2plant production research institute, national agricultural and food centre, bratislavská cesta 122, piešťany, sk-921 68, slovak republic (gubisova@vurv.sk) abstract: the aim of the present work was to compare the accumulation and translocation of cd and zn in plants of tobacco (nicotiana tabacum l.), celery (apium graveolens l.), maize (zea mays l.), giant reed (arundo donax l.), and alpine pennycress (noccaea caerulescens l.) under conditions of short-term hydroponic experiments using nutrient solutions spiked with radionuclides 109cd or 65zn, and direct gammaspectrometry. it was found that the time-course of metals accumulation in studied plants was not different in terms of target metal, but it was significantly different on the level of plant species. the highest values of cd accumulation showed plants of giant reed, whereby the accumulation decreased in the order: giant reed > tobacco > alpine pennycress >> maize and celery. on the basis of concentration ratios (cr) [me]shoot / [me]root calculation for both metals, it was found that cd and zn were in prevailing part accumulated in the root tissues and only partially accumulated in the shoots, where the amount of accumulated cd and zn increased from the oldest developed leaves to the youngest developed leaves. the cr values corresponding to these facts were calculated in the range 0.06 – 0.27 for cd and for zn 0.06 – 0.48.in terms of plant species, the cr values obtained for cd decreased in the order: maize > celery > tobacco and giant reed > alpine pennycress. the similarity between studied objects – individual plant species on the basis of the obtained variables defining cd or zn accumulation at different conditions of the experiments as well as the relationships between obtained variables and conditions of the experiments were subjected to multivariate analysis method – cluster analysis (ca). according to the findings and this analysis, it can be expected that plants of tobacco and giant reed will dispose with similar characteristics as plants of alpine pennycress, which are classified as zn/cd hyperaccumulators, in terms of cd or zn accumulation and other positive parameters for their utilization in phytoremediation processes and techniques. key words: cadmium, zinc, plants, accumulation, translocation, speciation modelling, cluster analysis 1. introduction heavy metals are well-known risk elements for the environment as non-degradable and persistent contaminants, which can be harmful for humans and the environment. the pollution of the environment with heavy metals predominantly comes from anthropogenic sources. despite the fact that hg, pb, and cd emissions from the nonferrous metal industry decreased in europe over the last 50 years, the increasing industrial activities in other continents (asia, africa) could significantly affect the global emissions of metals (ettler, 2015). the biota may require some of these bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:05 utc nova biotechnologica et chimica 14-2 (2015) 177 elements considered to be essential (e.g. iron, zinc, copper or molybdenum) in trace quantities, but at higher concentrations they may become toxic. however, metals like cadmium, lead or aluminium are non-essential and toxic even at very low levels (iannone et al., 2015). cadmium (cd) represents one of the most dangerous of the trace elements not only because of its significant occurrence in the environment together with zinc (zn) as its chemical analogue, but also because of its negative accumulation, impact on living organisms, and being the fourth most toxic element to vascular plants as well. according to the agency for toxic substances and disease registry cd is actually ranked at the 7th place within the priority list of hazardous substances based on their frequency of occurrence in the environment, toxicity and adverse potential to affect human health (atsdr, 2013). first, second, and third places in the mentioned priority list belong to arsenic (as), lead (pb), and mercury (hg), respectively. cadmium is also considered as a priority pollutant by the european community, within the water framework directive (ec, 2001) and is released into the environment mainly by power stations, heating systems, metal working industries, waste incinerators, urban traffic, cement factories, and as a by-product of phosphate fertilisers (hawrylak-nowak et al., 2014). the free ionic form cd2+ is the most bioavailable and consequently more toxic for organisms and plants, but it can complex with oxides and organic compounds, and it is not soluble above ph 7.5. the content of cd in crop plants varies in a wide range from 5 up to 400 μg/kg of dried weight (kabata-pendias and mukherjee, 2007). even at low concentrations, cd is toxic for most plants, while at concentrations greater than 5 – 10 mg/kg of leaf dry weight it leads to plant death (white and brown, 2010). cd is easily taken up by plant roots and transported to aerial parts, thus entering into the food chain causing health problems in animals and humans (bernard, 2008). cd-induced toxicity in plants can be described by: growth reduction, chlorosis and necrosis of leaves, wilting, redbrown colouration of leaf margins or veins (gill et al., 2013). moreover, cd can disturb the water balance, mineral nutrition, photosynthesis, respiration as well as general plant development (wójcik and tukiendorf, 2005). however, the biochemical and physiological basis of cd phytotoxicity is not always clear (hawrylak-nowak et al., 2015). cadmium, like other heavy metals, can accept a pair of electrons from a coordinate covalent bond, react with –sgroups, –oh groups, amino groups, and carboxylic acid termini, and thus affect sulfhydryl groups and n atoms in proteins causing their inactivation (lambers et al., 2008). soil-to-plant transfer of cd is the major exposure pathway for humans with regards to soil contamination (liang et al., 2013). food chain contamination is the main source of cd entry to human, especially non-smoking general population, and is the main constraint for food safety and agricultural land quality (atafar et al., 2010). excessive dietary intake of cd is accumulated in the body throughout life, and can lead to kidney malfunction and other diseases (mclaughlin et al., 1999). besides the ability to take up essential elements, plants are able to absorb and accumulate other metals, even those of a toxic character or with unidentified metabolic functions (hawrylak-nowak et al., 2014). toxic trace heavy metals, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:05 utc 178 dürešová, z. et al. such as cd, have chemical properties similar to essential micronutrients, such as zn, and are generally transported in plants by the same transporters as those essential micronutrients (tavarez et al., 2015). these mechanisms responsible for toxic metals uptake, translocation and accumulation in plant tissues represent serious risks related to their entering into the food chain. on the other hand, these mechanisms can be utilized for removal of heavy metals and radionuclides from contaminated environment, thus for remediation of contaminated environment by plants. phytoremediation, the use of plant systems to remove the contaminants from the soils and water has recently attracted a great deal of attention as an alternative means of soil decontamination, since it is a cost-effective, environment-friendly approach, applicable to large areas (bauddh et al., 2015). the capability of the plants to uptake contaminants, survive in contaminated soil and the low bioavailability of the contaminants in the soil are some of the limiting factors that influence phytoremediation efficiency (chirakkara and reddy, 2015). the plenty of plant species (including agricultural crops, grasses, shrubs, water plants, alpine plants a.o.) were subjected to characterization their potential for phytoremediation techniques. some of them showed an ability to tolerate and accumulate high concentrations of heavy metals and therefore were defined as hyperaccumulators. in order to fully utilize the contaminated sites and to overcome the disadvantage of phytoremediation, a new strategy of combining phytoremediation with energy crops cultivation, with a view to achieving low cost decontamination of soil through the production of biofuels (bauddh et al., 2015; pandey et al., 2016). a further current area of interest, at least for those metal ions essential for the human diet, also lies in the prospect of transferring the trait to crop plants as a way of combatting dietary microelements deficiencies (visioli and marmiroli, 2013). our previous works studied the accumulation of zn, co, cd or cs from nutrient solutions by roots of hydroponically cultivated plants of sunflower (helianthus annuus l.), tobacco (nicotiana tabacum l.), celery (apium graveolens l.), lettuce (lactuca sativa l.), and giant reed (arundo donax l.) plants (horník et al., 2005; horník et al., 2007; horník et al., 2008; horník et al., 2009; guldanová et al., 2010; šuňovská et al., 2012; dürešová et al., 2014). the aim of this work was to compare the uptake, translocation, and accumulation of heavy metals cd and zn in various plants including tobacco (n. tabacum l.), celery (a. graveolens l.), maize (zea mays l.), giant reed (arundo donax l.), and alpine pennycress (noccaea caerulescens l.) under conditions of short-term hydroponic experiments. a quantitative description of cd and zn accumulation was carried out using nutrient solutions spiked with radionuclides 109cd or 65zn, and direct gamma-spectrometry. the selection of the chosen plants included a typical model plant (tobacco), root crop (celery), agricultural crop (maize), potential energy crop (giant reed), and alpine plant (alpine pennycress) classified as a hyperaccumulator of heavy metals. in addition, the similarity between individual plant species on the basis of obtained variables defining the studied metals accumulation in plants at different conditions of the experiments, and the relationships between obtained variables and conditions of the experiments, were evaluated applying multivariate analysis method – cluster analysis (ca). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:05 utc nova biotechnologica et chimica 14-2 (2015) 179 2. materials and methods 2.1 plant material seeds of tobacco (nicotiana tabacum l.) were obtained from plant production research institute in piešťany (slovak republic). seeds of celery (apium graveolens var. rapaceum mill.) and maize (zea mays convar. saccharata koern.) were used as commercially available seeds from the company semo, inc. (czech republic). seeds of alpine pennycress (noccaea caerulescens l.) were purchased from b&t world seeds (france). all mentioned seeds were germinated and grown in pots filled with granulated perlite as an inert carrier and watered with 25 % or 50 % strength nutrient medium according to hoagland (1920). the composition of the full strength hoagland medium (100 % hm) was (in mg/dm3) mgso4.7h2o – 370; kno3 – 404; cacl2 – 444; nah2po4.2h2o – 292; na2hpo4.12h2o – 46.5; feso4.7h2o – 17.9; nano3 – 340; nh4cl – 214; nh4no3 – 160; h3bo3 – 8.5; na2moo4.2h2o – 0.06; mnso4.5h2o – 5.0; znso4.7h2o – 0.66; cuso4.5h2o – 0.8. table 1. conditions of the experiments evaluating the accumulation of cd and zn in plants of tobacco (n. tabacum l.), celery (a. graveolens l.), maize (z. mays l.), giant reed (a. donax l.), and alpine pennycress (n. caerulescens l.). plant metal conditions of the experiments cd 25 % hm; ph 6.0; 10 μmol/dm 3 cdcl2; 40.5 kbq/dm 3 109cdcl2; 12h/12h day/night (2 000 lx); 60 % relative humidity; 22±2 °c tobacco zn 25 % hm; ph 6.0; 10 μmol/dm 3 zncl2; 36.4 kbq/dm 3 65zncl2; 12h/12h day/night (2 000 lx); 60 % relative humidity; 22±2 °c cd 50 % hm; ph 5.5; 10 μmol/dm 3 cdcl2; 45.0 kbq/dm 3 109cdcl2; 12h/12h day/night (2 000 lx); 60 % relative humidity; 22±2 °c celery zn 25 % hm; ph 5.5; 10 μmol/dm 3 zncl2; 50.0 kbq/dm 3 65zncl2; 12h/12h day/night (2 000 lx); 60 % relative humidity; 22±2 °c cd 50 % hm; ph 5.5; 10 μmol/dm 3 cdcl2; 45.0 kbq/dm 3 109cdcl2; 12h/12h day/night (2 000 lx); 60 % relative humidity; 22±2 °c maize zn 50 % hm; ph 5.5; 5.9 μmol/dm 3 zncl2; 49.0 kbq/dm 3 65zncl2; 12h/12h day/night (2 000 lx); 60 % relative humidity; 22±2 °c cd 25 % hm; ph 6.0; 10 μmol/dm 3 cdcl2; 95.5 kbq/dm 3 109cdcl2; 12h/12h day/night (4 000 lx); 60 % relative humidity; 25/22 °c giant reed zn 25 % hm; ph 6.0; 1 μmol/dm 3 zncl2; 74.4 kbq/dm 3 65zncl2; 12h/12h day/night (4 000 lx); 60 % relative humidity; 25/22 °c cd 25 % hm; ph 6.0; 10 μmol/dm 3 cdcl2; 168 kbq/dm 3 109cdcl2; 16h/8h day/night (11 450 lx); 60 – 80 % relative humidity; 28/15 °c * alpine pennycress zn 25 % hm; ph 6.0; 10 μmol/dm 3 zncl2; 164 kbq/dm 3 65zncl2; 16h/8h day/night (11 450 lx); 60 – 80 % relative humidity; 28/15 °c * * conditions provided by a plant growth chamber (kbwf 720, binder, germany). plants of giant reed (arundo donax l. var. versicolor) were obtained by vegetative propagation under in vitro conditions. shoots development from lateral buds was induced in an ms medium (murashige and skoog, 1962) with an addition of 2– bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:05 utc 180 dürešová, z. et al. 4 mg/dm3 of bap (6-benzylaminopurine) as a cytokinin-type growth regulator. for rooting of regenerated shoots and in vitro storage of giant reed plants, ms medium without an addition of growth regulators was used. subsequently, giant reed plants were transferred and pre-cultivated in a 25 % hm. after 4 – 8 weeks of plant pre-cultivation, seedlings were gently removed from perlite and roots were washed free of any adhering perlite granules by deionized water and used in the accumulation experiments. 2.2 accumulation experiments and conditions of the experiments plants from the pre-cultivation phase were transferred into erlenmeyer flasks (100 – 250 cm3) with a cover to protect plant roots against lights and cultivated for 8 days in a 50 – 150 cm3 (according to plant) hoagland medium (hm) containing cd (in the form of cdcl2) or zn (in the form of zncl2 or znso4) and spiked with 109cdcl2 or 65zncl2. the conditions of the individual experiments are detailed in the table 1. in time intervals, samples of nutrient solution were taken and 109cd or 65zn radioactivities were measured by gamma-spectrometry. at the end of the experiments, plants were harvested, roots were carefully rinsed in deionized water and incorporated radioactivity in roots, stems or leaves was measured. individual parts of plant were then oven dried (at 60 °c for 24 hours) and dry weights were determined. 2.3 radiometric analysis all nutrient solutions used in the experiments were spiked with 109cdcl2 and 65zncl2 radioactive solutions obtained from the czech metrological institute (czech republic). the radiometric analysis in terms of volume or specific radioactivity of 109cd and 65zn in the samples of nutrient solutions or individual parts of plant was realized using a well type nai(tl) scintillation gamma-spectrometer 54bp54/2-x or 76bp76/3 (scionix, the netherlands or envinet, czech republic) and the data processing software scintivision-32 (ortec, usa). for energy and efficiency calibration, a library of radionuclides was built by selecting characteristic γ-ray peaks for 109cd (eγ = 88.04 kev), 137cs (eγ = 661.66 kev) or 65zn (eγ = 1115.52 kev), and for the calibration, standard solutions of 109cdcl2, 137cscl or 65zncl2 with known radioactivity including the half-life of radionuclides ( 109cd: t1/2 = 462.6 d; 137cs: t1/2 = 11 019 d, and 65zn: t1/2 = 243.9 d) were used. 2.4 speciation modelling the qualitative and quantitative proportion of the ion forms of cd and zn in nutrient solutions were calculated by the software mineql+ ver. 4.62 (environmental research software, usa). this software employs chemical equilibrium models to calculate metals speciation and solubility equilibria in the laboratory or natural aqueous systems under a variety of conditions including bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:05 utc nova biotechnologica et chimica 14-2 (2015) 181 a gas phase with constant partial pressure, ph, temperature, ionic strength, and concentrations of cations and anions. 2.5 statistical analysis all analytical determinations were performed in triplicate. the statistical significance of differences in calculated values of cd or zn accumulation in plant tissues were evaluated by a multiple range test to ascertain differences between individual groups. the level of significance p was 0.05 in all cases. to evaluate the similarity degree between the objects – individual plant species on the basis of obtained variables and conditions of the experiments as well as to evaluate the relationships between the obtained variables and conditions of the experiments, the cluster analysis (ca) was applied. parameters describing the obtained variables, such as cd or zn accumulation in plants (in μmol/g; fresh wt.) or concentration ratio (cr) defined as the ratio of the concentration of cd or zn accumulated in shoots to concentration of cd or zn accumulated in roots ([me]shoot / [me]root), and the conditions of the experiments, such as the initial concentrations of cd2+ or zn2+ ions (in μmol/dm3) predicted by the speciation modelling, the ph value of the nutrient solutions, the strength of hoagland medium (25 % or 50 % hm) or conditions of the plant cultivation (temperature, relative humidity, day/night photoperiod or intensity of illumination) were utilized in this analysis. statistical analysis, evaluation, and graphical interpretation of the obtained data were carried out using programs originpro ver. 8.5 (originlab corp., usa), statgraphics centurion ver. 15 (statpoint technologies inc., usa), systat ver. 13 (systat software inc., usa), and sigmaplot ver. 12 (systat software inc., usa). 3. results and discussion fig. 1 depicts the kinetics of cd and zn accumulation in plants of tobacco (n. tabacum l.), celery (a. graveolens l.), maize (z. mays l.), giant reed (a. donax l.), and alpine pennycress (n. caerulescens l.) during 8 days of plant cultivation under hydroponic conditions. the conditions of the experiments were partly different (see table 1) in terms of the initial concentrations of cd (in the form of cdcl2) and zn (in the form of zncl2 or znso4), the ph of nutrient solutions and the strength of hoagland medium (hm) as well as from the point of view of conditions of plant cultivation (temperature, relative humidity, day/night photoperiod or intensity of illumination). to carry out a calculation of free cd2+ and zn2+ ionic forms proportion and concentration in nutrient solutions as the most bioavailable metal forms for plants, the program mineql+ was used. however, this prediction has to be considered with caution, because the modelling program does not include the participation of the plant. therefore, it can be used for the evaluation of initial conditions of the experiments (without plants). according to in silico speciation analysis by mentioned equilibrium modelling software, it was found that cd present in a 25 % or 50 % hm representing the model bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:05 utc 182 dürešová, z. et al. of soil solution can occur in 15 different inorganic chemical forms within the range of ph 3 – 12. in the case of zn, 12 different inorganic chemical forms were calculated under the same conditions. at the ph 5.5 or 6.0, 25 % or 50 % hm, and initial concentration of cdcl2 10 μmol/dm 3, which were included in the experiments, the proportion of cd2+ form from the total amount of cd was in the range 71.7 % to 77.1 %. in the case of zn and the given initial conditions, the proportion of zn2+ form represented 92.2 % – 94.8 %. 0 1 2 3 4 5 6 7 8 0,00 0,05 0,10 0,15 0,20 0,25 time [day] c d ac cu m ul at io n [μ m ol /g ]; fr es h w t. tobacco celery maize giant reed alpine pennycress 0 1 2 3 4 5 6 7 8 0,00 0,05 0,10 0,15 0,20 0,25 time [day] z n ac cu m ul at io n [μ m ol /g ]; fr es h w t. tobacco celery maize giant reed alpine pennycress fig. 1. kinetics of cd (a) and zn (b) accumulation in plants of tobacco (n. tabacum l.), celery (a. graveolens l.), maize (z. mays l.), giant reed (a. donax l.), and alpine pennycress (n. caerulescens l.) during 8 days of plant cultivation under hydroponic conditions. conditions are detailed in table 1. data are presented as arithmetic means of triplicate experiment. from the fig. 1, it is evident that the curves describing the cd accumulation (fig. 1a) in tobacco, celery, maize, giant reed, and alpine pennycress plants show a relatively similar trend with the curves obtained for zn (fig. 1b). this result corresponds with the fact that cd and zn are mutual physico-chemical analogues as well as that cd is transported within the plants via the zn pathways, but the mechanisms responsible for cellular zn tolerance cannot detoxify cd effectively (zhao et al., 2006). however, there were significant differences between plant species in time-course of cd and zn accumulation and in the obtained values of accumulation (μmol/g; fresh wt.). the most different trend of the curve describing the cd and zn accumulation showed plants of giant reed, when the two-phase process given by a slow cd or zn accumulation within the first 3 days of plant cultivation followed by exponential increase of accumulation within the next 5 days was observed. at the other studied plant species, the typical curves with gradual increasing the cd or zn accumulation in plants were found. the phytotoxic symptoms at used cd concentrations were not observed in either case. the fig. 2 compares the differences in the obtained values of cd and zn accumulation in individual plant species on the basis of multiple range test at the level of p < 0.05. it was found that the highest value of cd accumulation showed plants of giant reed and the accumulation decreased in the order: giant reed > tobacco > alpine pennycress >> maize and celery. quezadahinojosa et al. (2015) also found the variability in cd contents among the six different perennial plant species growing in a wooded pasture of the swiss jura mountains, where the soils are geogenically enriched in cd. in the case of zn, when a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:05 utc nova biotechnologica et chimica 14-2 (2015) 183 the different initial concentrations of zn (see table 1) were used, the order of plants for zn accumulation was: tobacco and alpine pennycress > celery > maize >> giant reed. it should be mentioned that the nutrient solution for giant reed contained a 10-fold lower concentration of zn in comparison with plants of tobacco and alpine pennycress. from these results, it can be concluded that tobacco and giant reed showed a similar cd accumulation efficiency as plants of alpine pennycress, which are classified as zn/cd hyperaccumulators. in the case of celery and maize, as typical agricultural crops, the cd and zn accumulation reached significantly lower levels. 0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 12 1 c b ab m et al a cc um ul at io n [μ m ol /g ]; fr es h w t. cd zn alpine pennycress giant reedmaizecelerytobacco a c 1 2 3 fig. 2. comparison of cd (a) and zn (b) accumulation in plants of tobacco (n. tabacum l.), celery (a. graveolens l.), maize (z. mays l.), giant reed (a. donax l.), and alpine pennycress (n. caerulescens l.) after 8 days of plant cultivation under hydroponic conditions. conditions are detailed in table 1. mean values from triplicate experiments with the same letter (for cd) or number (for zn) above columns are not significantly different at the p < 0.05 level based on multiple range test. in terms of phytoremediation technologies, it is important to mention that toxic metals or radionuclides have to be accumulated mainly in above-ground parts of plants. in general, significant differences in distribution of cd and zn within shoots and roots of studied plant species and higher percentage distribution of cd into the shoots from the total accumulated amount of cd in comparison with the data obtained for zn were found as well (fig. 3). the highest proportion of translocated cd into shoots was observed in plants of tobacco, celery and alpine pennycress, whereby the distribution of cd for account of shoots decreased in the order: tobacco, celery and alpine pennycress > maize > giant reed. in the case of zn, when the different initial concentrations of zn (see table 1) were used, the percentage distribution into shoots decreased in the order: giant reed >> alpine pennycress > celery > tobacco > maize. within the shoots, the direct gammaspectrometric determinations revealed that the amount of accumulated cd and zn (in specific radioactivities bq/g of dry wt. or bq/cm2) increased from the oldest developed leaves to the youngest developed leaves (data not shown). this result was observed in all studied plant species. zhang bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:05 utc 184 dürešová, z. et al. et al. (2015) reported that cd accumulation within the plants of two castor cultivars (ricinus communis l.) decreased in the order: root > stem > young leaf > old leaf. 0 20 40 60 80 100 zn a lp in e pe nn yc re ss g ia nt r ee d m ai ze c el er y 323 23 21 3 233 2 c abb a ab c b aa d is tr ib ut io n of m e [% ] root shoot a 1 t ob ac co a lp in e pe nn yc re ss g ia nt r ee d m ai ze c el er y t ob ac co cd fig. 3. distribution of cd and zn in roots and shoots of tobacco (n. tabacum l.), celery (a. graveolens l.), maize (z. mays l.), giant reed (a. donax l.), and alpine pennycress (n. caerulescens l.) after 8 days of plant cultivation under hydroponic conditions. conditions are detailed in table 1. mean values from triplicate experiments with the same letter (for cd) or number (for zn) above columns are not significantly different at the p < 0.05 level based on multiple range test. also, for the evaluation of cd and zn mobility in conductive tissues of studied plants in the term of their translocation efficiency it was established a non-dimensional concentration ratio (cr) which represents the ratio of metal concentration in the above-ground part of plants [me]shoot (µmol/g; dry wt.) to metal concentration in roots [me]root (µmol/g; dry wt.). from the data mentioned in table 2, it is evident that on the basis of these concentration ratios [me]shoot / [me]root cd and zn were in prevailing part accumulated in the root tissues and only partially accumulated in shoots. in general, the obtained values of cr were very similar for both target metals cd and zn. this result is in agreement with the above mentioned fact that cd and zn are mutual physico-chemical analogues and that cd is transported via the zn pathway as well. some present differences in the obtained cr values between cd and zn were probably caused by lower concentrations of zn in nutrient solutions. the cr values were calculated in the range 0.06 – 0.27 for cd and for zn 0.06 – 0.48. similar concentration ratios found połeć-pawlak et al. (2005) for plants of arabidopsis thaliana cultivated under hydroponic conditions during 14 days in hm containing 25 or 50 μmol/dm3 of cd2+. from the point of view of plant species, the cr values obtained for cd decreased in the order: maize > celery > tobacco and giant reed bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:05 utc nova biotechnologica et chimica 14-2 (2015) 185 > alpine pennycress. these results suggest that plants of celery and maize with a lower ability to accumulate cd and zn from nutrient solutions showed higher values of cr. on the other side, plants, such as tobacco, giant reed and alpine pennycress, with a higher ability to accumulate cd and zn from nutrient solutions showed lower values of cr. this fact can be related to ability of these plants to bind and detoxicate of metals, such as cd and zn, in root cells and cellular compartments (e.g. vacuoles). thus, in this case the root system acts as a protective barrier to further metals movement into the above-ground parts of plants. this character results in a high tolerance to metals and metals accumulation in plants or in given plant tissues. salt et al. (1999) reported that zn was mostly complexed to histidine in roots, transported as zn2+ in the xylem sap, and complexed to organic acids in leaves. it can be expected that mentioned processes are also involved in cd accumulation. some works reported that the application of chelating agents, such as ethylenediaminetetraacetate (edta) or citric acid (ca), can increase not only the concentration of metals in soil solutions, but also the translocation of metals within the plants (horník et al. 2008; horník et al. 2009; guldanová et al., 2012; ehsan et al., 2014). table 2. comparison of the values of non-dimensional concentration ratio (cr) calculated for cd and zn after 8 days cultivation of plants of tobacco (n. tabacum l.), celery (a. graveolens l.), maize (z. mays l.), giant reed (a. donax l.), and alpine pennycress (n. caerulescens l.) under hydroponic conditions. conditions are detailed in table 1. mean values from triplicate experiments with the same letter (for cd) or number (for zn) above columns are not significantly different at the p < 0.05 level based on multiple range test. cr ([me]shoot / [me]root) ± s.d. plant cd zn tobacco 0.12±0.02c 0.06±0.014 celery 0.16±0.02b 0.14±0.013 maize 0.27±0.03a 0.18±0.022; * giant reed 0.11±0.01c 0.48±0.201; ** alpine pennycress 0.06±0.01d 0.06±0.014 * initial concentration of zn 5.9 μmol/dm3 in a nutrient solution; ** initial concentration of zn 1.0 μmol/dm3 in a nutrient solution. the realized experiments were deliberately designed for different initial conditions of the experiments and conditions of the plant cultivation (see table 1). this fact allows to evaluate the relationships between variables obtained at the end of experiments and these conditions as well as to find the similarity between objects – individual plant species. for these purposes, the method of multivariate analysis – cluster analysis (ca) was applied and parameters describing the obtained variables, such as cd or zn accumulation in plants (in μmol/g; fresh wt.) and concentration ratio (cr) defined as the ratio of the concentration of cd or zn accumulated in shoots to concentration of cd or zn accumulated in roots ([me]shoot / [me]root) and the conditions of the experiments, such as the initial concentrations of cd2+ or zn2+ ions (c0 cd 2+ or c0 zn 2+; in μmol/dm3) predicted by the speciation modelling, the ph value of nutrient solutions, the strength of hoagland medium (25 % or 50 % hm) or conditions of the plant cultivation (temperature, relative humidity, day/night photoperiod or intensity of illumination) were utilized in this analysis. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:05 utc 186 dürešová, z. et al. 0 4 8 12 16 20 24 d is ta nc e t ob ac co c el er y m ai ze g ia nt r ee d ne p en ny cr es s 0 4 8 12 16 20 24 d is ta nc e c 0 c d fr ee ac cu m ul at io n c d h m c d t r c on di tio nsph 0 4 8 12 16 20 24 d is ta nc e t ob ac co c el er y m ai ze g ia nt r ee d pe nn yc re ss 0 4 8 12 16 20 24 d is ta nc e c 0 z n fr ee c on di tio ns ph cc um ul at io n z n h m z n t r fig. 4. dendrogram of cluster analysis including squared euclidean distance and ward’s method as the aggregation criterion for the evaluation of the similarity degree between objects – individual plant species on basis of obtained variables and conditions of the experiments (a, c) as well as the relationships between obtained variables and conditions of the experiments (b, d) for cd (a, b) and zn (c, d). conditions of the experiments are detailed in table 1. from ca including squared euclidean distance and ward’s method as the aggregation criterion for the evaluation of the similarity degree between objects represented by studied plant species, it was found that in the case of cd plants of tobacco, giant reed, and alpine pennycress, these formed separate cluster (fig. 4a, no. 1). it means that these plants showed similar characteristics from the point of view of cd accumulation and relationships between conditions of the experiments and obtained data describing cd accumulation. thus, it can be expected that plants t ob ac co g ia nt re ed a lp in e p en ny cr es s c el er y m ai ze d is ta nc e d is ta nc e d is ta nc e d is ta nc e t ob ac co g ia nt re ed a lp in e pe nn yc re ss c el er y m ai ze c 0 c d2 + ph c d ac cu m ul at io n c d c r c 0 z n2 + ph z n ac cu m ul at io n c on di tio ns of c ul tiv at io n % h m z n c r a b c d % h m c on di tio ns of c ul tiv at io n 1 2 1 2 1 2 1 2 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:05 utc nova biotechnologica et chimica 14-2 (2015) 187 of tobacco and giant reed will dispose with similar characteristics as plants of alpine pennycress, which are defined as zn/cd hyperaccumulators, in terms of cd accumulation and other positive parameters for their utilization in phytoremediation processes and techniques. liu et al. (2016) also classified the plants of tobacco (n. tabacum l.) as hyperaccumulators of cd on the basis of realized experiments under hydroponic conditions and characterized the effect of conditions of the experiments (chemical inhibitors, temperature or illumination) on cd accumulation. on the other side, plants of celery and maize as agricultural crops are significantly different from this group, because they formed a second separate cluster (fig. 4a, no. 2). within the evaluation of relationships between the conditions of the experiments and obtained values for cd accumulation (cd accumulation and cr for cd), the correlations between c0 cd 2+, ph and cd accumulation (fig. 4b, no. 1) as well as between the strength of hm (representing the different concentrations of macroand microelements and ionic strength) and cr for cd (fig. 4b, no. 2) were found. from these results, it can be concluded that cd accumulation through the root system of studied plants is strongly dependent on initial concentration of free cd2+ ions and the value of ph. on the other hand, the concentration ratio cr describing the cd translocation from roots-to-shoots is primarily affected by the strength of hm corresponding with concentrations of macroand microelements in nutrient (soil) solutions. in the case of zn, on the basis of the similarity degree, two separate clusters were also formed. the first cluster was represented by plants of tobacco and alpine pennycress (fig. 4c, no. 1) and the second cluster formed plants of celery, maize, and giant reed (fig. 4c, no. 2). similarly, as in the case of cd, the two logical correlations between c0 zn 2+ and zn accumulation (fig. 4d, no. 1) and between the strength of hm and cr for zn (fig. 4d, no. 2) were found. 4. conclusions the obtained results showed that the time-course of cd accumulation in plants of tobacco, celery, maize, giant reed, and alpine pennycress cultivated under conditions of short-term hydroponic experiments had a relatively similar trend with curves obtained for zn. this result supports the fact that cd and zn are mutual physico-chemical analogues as well as that cd is transported within the plants via the zn pathways. on the other side, a significant difference in time-course of cd and zn accumulation and in the obtained values of accumulation (μmol/g; fresh wt.) between plant species existed as well. the differences in the obtained values of cd and zn accumulation in individual plant species were evaluated by multiple range test at the level of p < 0.05. the highest values of cd accumulation showed plants of giant reed and the accumulation decreased in the order: giant reed > tobacco > alpine pennycress >> maize and celery. from the point of view of cd and zn distribution within the plants, the highest proportion of translocated cd into shoots was observed in plants of tobacco, celery, and alpine pennycress. a similar result was also found in the case of zn. within the shoots, the amount of accumulated cd and zn increased from the oldest developed leaves to the youngest developed leaves. on the basis of concentration bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 17.01.20 10:05 utc 188 dürešová, z. et al. ratios (cr) [me]shoot / [me]root calculation for both metals, it was found that cd and zn were in prevailing part accumulated in the root tissues and only partially accumulated in shoots. the cr values corresponding to these facts were calculated in the range 0.06 – 0.27 for cd and for zn 0.06 – 0.48. from the point of view of plant species, the cr values obtained for cd decreased in the order: maize > celery > tobacco and giant reed > alpine pennycress. this result suggests that plants of tobacco, giant reed, and alpine pennycress showed the ability to bind and detoxicate of metals, such as cd and zn, in root cells and cellular compartments in larger extent than studied agricultural crops (maize and celery). the similarity between studied objects – individual plant species on the basis of obtained variables and conditions of the experiments as well as the relationships between obtained variables and conditions of the experiments were subjected to multivariate analysis method – cluster analysis (ca). according to this analysis, it can be expected that plants of tobacco and giant reed will dispose with similar characteristics as plants of alpine pennycress, which are defined as zn/cd hyperaccumulators, in terms of cd accumulation and other positive parameters for their utilization in phytoremediation processes and techniques. also, it was found that cd and zn accumulation through the root system of studied plants is strongly dependent on initial concentration of free me2+ ions and the value of ph. on the other hand, the concentration ratios cr describing the cd or zn translocation from roots-toshoots are primarily affected by concentrations of macroand microelements in the nutrient (soil) solutions. acknowledgement: this work was supported by the slovak research and development agency under the contract no. apvv-0380-12. references atafar, z., mesdaghinia, a.r., nouri, j., homaee, m., yunesian, m., ahmadimoghaddam, m., mahvi, a.h.: effect of fertilizer application on soil heavy metal concentration. environ. monit. assess., 160, 2010, 83-89. atsdr (agency for toxic substances and disease registry): priority list of hazardous substances, u.s. department of health and human services, atlanta, ga, usa, 2013. available from: <www.atsdr.cdc.gov>. bauddh, k., singh, k., singh, b., singh, r.p.: ricinus communis: a robust plant for bio-energy and phytoremediation of toxic metals from contaminated soil. ecol. eng., 84, 2015, 640-652. bernard, a.: cadmium & its adverse effects on human health. indian j. med. res., 128, 2008, 557-564. chirakkara, r.a., reddy, k.r.: biomass and chemical amendments for enhanced phytoremediation of mixed contaminated soils. ecol. eng., 85, 2015, 265-274. clemens, s., aarts, m.g.m., thomine, s., 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crystal structure and magnetism of [cu(cyclam)ni(ncs)4(h2o)2]n ivana kočanováa, juraj kuchára, martin orendáčb, roman bočac and juraj černáka, a department of inorganic chemistry, institute of chemistry, faculty of sciences, p. j. šafárik university in košice, košice, sk-041 54, slovak republic b institute of physics, faculty of science, p. j. šafárik university in košice, košice, sk-041 54, slovak republic c department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic article info article history: received: 23rd january 2017 accepted: 26th may 2017 keywords: copper nickel crystal structure chain-like structure magnetic properties abstract [cu(cyclam)ni(ncs)4(h2o)2]n (1) (cyclam = 1,4,8,11-tetraazacyclodecane) exhibits bent 1d crystal structure in which paramagnetic cu(ii) and ni(ii) atoms are linked by bridging 2-ncs ligands. the cu(ii) atom exhibit tetragonally elongated hexacoordination in the 4+2 form with one tetradentate macrocyclic cyclam ligands placed in the equatorial plane while the axial positions are occupied by s atoms from bridging ncsligands. the ni(ii) atom in nin4o2 donor set is deformed octahedrally coordinated by four isothiocyanato ligands among which two in trans positions are bridging in nature; additional aqua ligands occupy the remaining two positions in trans arrangement. weak hydrogen bonding interactions of the o-h···s type links the formed chains into 3d supramolecular structure. the magnetism of 1 is dominated by a sizable single-ion anisotropy dni/hc = +7.49 cm -1 along with a weak exchange interaction of the ferromagnetic nature.  university of ss. cyril and methodius in trnava introduction heterobimetallic compounds represent enhanced interest in magnetic studies as in some case their magnetic properties cannot be viewed as a simple addition of the magnetic properties of the two magnetically active centers. in the case of cu-ni heterobimetallic compounds (černák et al. 2012a) the ones exhibiting chain-like structures and containing both cu and ni central atoms paramagnetic (p-p type) can be viewed as model compounds for observation of the phenomenon predicted by furusaki et al. (1994), namely, that in some cases such systems may behave as ferromagnets despite the presence of antiferomagnetic interactions between the present metallic centers with half (s = 1/2, cu(ii)) and integer (s = 1, ni(ii)) spins. the literature data shows that p-p type cu-ni compounds are numerous (černák et al. 2012a). on the other hand, the number of cu-ni compounds with onedimensional (1d) crystal structures is limited. as examples we can mention [ni(cyclam)cu(h2o)2(2,4-pydc)2]n·8h2o (cyclam = 1,4,8,11-tetraazacyclodecane, 2,4-pydc = pyridine2,4-dicarboxylato) (huang et al. 2013), [cu(en)2(μ2-h2o)2ni(ac)4]·4h2o (en = ethane-1,2diamine, ac = acetato) (nesterova et al. 2010) or {[cu(en)2] [ni(c5o5)2(h2o)2]}n (c5o5 = 4,5dihydroxy-cyclopentanetrionate(2-)) (wang et al. 2009). previously, we have synthesized and structurally characterized several 1d cu-ni bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:46 utc mailto:juraj.cernak@upjs.sk nova biotechnol chim (2017) 16(1): 68-75 69 compounds with diamagnetic [ni(cn)4] 2bridging anion (p-d type), e.g. cu(cyclam)ni(cn)4 (černák et al. 2010) or cu(bapen)ni(cn)4 (bapen = n,n'bis(3-aminopropyl)-1,2-diaminoethane) (černák et al. 2012b). with the aim to prepare cu-ni compounds of the p-p type with 1d structure we have replaced in our synthetic procedure the diamagnetic [ni(cn)4] 2building block by paramagnetic [ni(ncs)4(h2o)2] 2one; this complex anion was already used, e.g. in {culα[ni(ncs)4(h2o)2]} (lα = 5,12-dimetyl1,4,8,11-tetraazacyclotetradecane-4,11-diene). (bieńko et al. 2007). as the results of our experiments we have isolated novel title complex [cu(cyclam)ni(ncs)4(h2o)2]n (1) and here we report its crystal structure, spectroscopic characterization along with its magnetic properties. experimental materials nickel(ii) dichloride hexahydrate nicl2·6h2o, potassium thiocyanate kscn, copper(ii) perchlorate hexahydrate cu(clo4)2·6h2o and cyclam (1,4,8,11-tetraazacyclodecane) were purchased from commercial sources and used as received. syntheses of [cu(cyclam)ni(ncs)4(h2o)2]n (1) in syntheses 15 cm3 of aqueous-ethanolic solution (2:1, vv) of nicl2·6h2o (0.07 g, 0.3 mmol) were slowly successively under stirring added 0.12 g of kscn (1.2 mmol) dissolved in 10 cm3 of water, and 20 cm3 of aqueous solution formed of cu(clo4)2·6h2o (0.11 g, 0.3 mmol) and cyclam (0.06 g, 0.3 mmol). the formed violet solution was filtered and left aside for crystallization. within two weeks light pink needles separated which were collected by filtration and dried on air. yield: 20 %. anal. calc. for c14 h28 cu n8 ni o2 s4 (mr = 590.93) (%): c, 28.46; h, 4.78; n, 18.96; cu, 10.75; ni, 9.93. found: c, 28.50; h, 4.69; n, 18.89; cu, 10.53; ni, 9.80. ir (cm–1; s = strong, m = medium, w = weak, v = very, sh = shoulder, b = broad): 3544 msh; 3477 ssh; 3397 vs; 3243 m; 3215 w; 3167 s; 3151 w; 2954 m; 2918 wsh; 2875 w; 2113 vs; 2098 vs; 1652 w; 1614 m; 1455 m; 1422 m; 1386 w; 1317 w; 1295 m; 1235 w; 1120 m; 1093 s; 1064 m; 1024 s; 987 s; 882 m; 806 w; 779 m; 654 m; 517 w; 474 m; 434 m. physical measurements elemental analyses (c, h, n) were performed on a chnos elemental analyzer (vario micro). the metal contents were determined by complexometry (cu) and gravimetry (ni). infrared spectra were recorded on a ft-ir avatar 330 thermo-nicolet instrument using kbr pellets technique (1:200) in the range of 4000 – 400 cm–1. the magnetic susceptibility of sample 1 (56 mg) was measured using the squid magnetometer (mpms, quantum design) at b = 0.1 t and the background contribution arising from the gelcap and straw was subtracted from the experimental data. raw data was corrected for underlying diamagnetism and transformed to the effective magnetic moment. the magnetization data was taken at t = 2.0 k until b = 5.0 t. x–ray crystallography x–ray experiments were carried out on a four– circle –axis xcalibur2 diffractometer equipped with a ccd detector sapphire2 (oxford diffraction). the crysalis software package (oxford diffraction 2003) was used for data collection and reduction. analytical absorption corrections using crystal faces were applied (clark and reid 1995). the crystal structure of 1 was solved by direct methods and further refined using the shelxs–97 and shelxl–97 program (sheldrick 2015), respectively, incorporated in the wingx program package (farrugia 1999). hydrogen atoms in the cyclam ligand were placed in the calculated positions and allowed to ride on the parent atoms with isotropic thermal parameters tied with the parent atoms (u(h) = 1.2u(ch2), u(h) = 1.2u(n)). water hydrogen atoms in 1 were located with the program calc–oh (nardelli 1999) and their isotropic thermal parameters were tied with the parent oxygen atoms (u(h) = 1.5u(o)). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:46 utc nova biotechnol chim (2017) 16(1): 68-75 70 table 1. crystal data and structure refinement for 1. empirical formula c14 h28 cu n8 ni o2 s4 formula weight 590.93 temperature [k]291(2) wavelength [å] 0.71073 å crystal system triclinic space group p-1 unit cell dimensions [å, ] a= 7.4927(5) b=8.0603(6) c=11.1747(8) α=106.318(7) =100.658(6) γ=105.429(8) volume [å3] 599.25(8) z 1 density (calculated) [g.cm-3] 1.638 absorption coefficient [mm-1] 2.049 crystal dimensions [mm3] 0.1x0.2x0.5  range for data collection [] 2.80 to 25.00 index ranges –8  h  8, –9  k  9, -13  l  13 reflections coll./ obs. 2116/1638 absorption correction analytical tmin/tmax 0.565/0.882 goodness-of-fit on f2 (all/obs.) 0.997/0.998 final r indices [i>2(i)] r1 = 0.0505, wr2 = 0.1299 r indices (all data) r1 = 0.0639, wr2 = 0.1355 largest diff. peak and hole [e å–3] 1.07 and –0.74 the structural figures were drawn using the diamond software (brandenburg 2008). crystal data and final parameters of the structure refinement are summarized in table 1, while selected geometric parameters are given in table 2. possible hydrogen bonds are gathered in table 3. results and discussion synthesis and spectroscopic characterization from the aqueous-ethanol systems cu2+ – cyclam – ni2+ – ncs– the complex [cu(cyclam)ni(ncs)4(h2o)2]n (1) was isolated and chemically characterized. the choice of the cyclam as blocking ligand coordinated to the cu(ii) atom, in the lack of another chelating ligand, was motivated by significantly higher stability of the [cu(cyclam]2+ complex (logk= 27.2) with respect to the analogous ni(ii) one (logk= 22.2) (king 2005). similar mild conditions were used for preparation of analogous compound {culα[ni(ncs)4(h2o)2]} with (lα = 5,12-dimethyl-1,4,8,11tetraazacyclotetradecane-4,11-diene) (bieńko et al. 2007). the most characteristic absorption band in the ir spectrum of 1 (fig. 1) is the very strong split one with close peak positions at 2113 and 2098 cm–1 due to the presence of the n-bonded ncsligand (nakamoto 1997; chandra et al. 2008). in analogous {culα[ni(ncs)4(h2o)2]} with two different structural functions of ncs– ligands (terminal and bridging) the values of 2121 and 2088 cm-1 were reported (bieńko et al. 2007). the presence of cyclam ligand is indicated by several observed absorption bands. as characteristic absorption bands we can mention those arising from n─h stretching vibrations, which were observed in the regions of 3244─3228 cm–1, in line with the literature data 3265─3180 cm–1 (nakamoto 1997) as well as those arising from stretching vibrations of the methylene groups observed in the region 2954─2875 cm–1. the presence of aqua ligands manifest itself by broad and strong absorption band at 3397 cm–1 with two shoulders at 3544 and 3477 cm–1 which were ascribed to asymmetric and symmetric ν(oh) stretching vibrations; the corresponding absorption band due to δ(h2o) vibrations was located at 1652 cm–1. further absorption bands are gathered in the experimental part. fig. 1. ir spectrum of 1. crystal structure the crystal structure of compound [cu(cyclam)ni(ncs)4(h2o)2]n consists of quasilinear chains exhibiting composition [-cu(cyclam)bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:46 utc nova biotechnol chim (2017) 16(1): 68-75 71 µ-scn-ni(ncs)2(h2o)2-µ-ncs-]n with transpositions of the bridging ligand in the respective coordination polyhedra (fig. 2). analogous chainlike structure was reported for [cu(en)2][mn(ncs)4(h2o)2] (kou et al. 1998). the cu(ii) atom in the title complex exhibitstetragonally elongated hexacoordination with elongated axial bonds as the consequence of the jahn-teller effect. in the equatorial plane there are nitrogen atoms from the cyclam ligand situated, while bond lengths of cu-n are 2.014(4) (4×) å (table 2) and are comparable with the average value of (2.0205(3)) å found in {culα[ni(ncs)4(h2o)2]} (lα = 5,12-dimethyl-1,4,8,11-tetraazacyclo tetradecane-4,11-diene) (bieńko et al. 2007). the axial positions are occupied by sulfur atoms from bridging thiocyanato ligands, bond length of cu-s is 3.027(2) å. the observed value is slightly shorter as the reported values of 3.071(1) and 3.065(3) å for {cu(en)2[ni(en)(scn)3]2}n (shen et al. 2001) and [cu(en)2][mn(ncs)4(h2o)2] (kou et al. 1998), respectively. the ni(ii) central atom (-1 site) in [ni(ncs)4(h2o)2] 2anionis deformed octahedrally coordinated. the equatorial plane is occupied by four atoms of nitrogen from ncs ligands, while in the axial ones are placed two aqua ligands (donor set is o2n4) (fig. 2). two transpositioned ncsligands link ni(ii) and cu(ii) central atoms generating those chain-like structure (fig. 2). ni-n bond lengths are from the range from 2.050(4) to 2.069(4) å, while the ni-o bond (2.123(3) å, 2x) is somewhat longer; the experimental values well correspond to those observed in the similar compound {culα[ni(ncs)4(h2o)2]} (bieńko et al. 2007). as to the other geometric parameters it should be noted that the formed chains are substantially bent on the sulfur atom with the value of the angle (n2)c2-s2-cu1 97.10(2) (table 2) which support the assumption about weak coordination of the bridging scnligand to the cu(ii) central atom. nevertheless, such bent coordination is not uncommon as almost the same value 97.109(2)˚ was reported for {cu(en)2[ni(en)(scn)3]2}n (shen et al. 2001). fig. 2. view of the chain-like structure of [cu(cyclam)ni(ncs)4(h2o)2]n. the thermal ellipsoids are drawn on 30 % probability level. dashed line represents the weaker cu-s coordination bond. symmetry codes: i: 1-x, -y, -z; ii: 2-x, -y, 1-z ; iii: -1+x, y, -1+z; iv: 1+x, y, 1+z. the formed chains [-cu(cyclam)-µ-scnni(ncs)2(h2o)2-µ-ncs-]n are interconnected by hydrogen bonding interactions of the o-h∙∙∙s type formed by aqua ligands and sulfur atoms from thiocyanato ligands (fig. 3, table 3). the 2dhydrogen bonding network can be described as formed of two topologically different fused rings r1 and r2 with descriptors 24 (8)r and 2 2 (12)r , respectively (fig. 3); for the nomenclature of the descriptors see (bernstein et al. 1995). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:46 utc nova biotechnol chim (2017) 16(1): 68-75 72 the geometric parameters associated with o-h∙∙∙s hydrogen bonding interactions (table 3) are comparable with those observed in {cul[ni(ncs)4(h2o)2]} (l = l = n-dl-5,12dimethyl-1,4,8,11-tetraazacyclotetradeca-4,11diene) (bieńko et al. 2007). table 2. selected geometric parameters [å, ] for 1. cu1-n4 2.011(4) cu1-n3 2.012(4) ni1-n1 2.047(4) ni1-n2 2.066(4) ni1-o1 2.120(3) s2-c2 1.643(5) s1-c1 1.638(5) c1-n1 1.158(5) c2-n2 1.146(6) n4-cu1-n3 85.39(18) n1-ni1-n2 88.91(15) n1-ni1-o1 89.66(15) n2-ni1-o1 88.61(17) c1-n1-ni1 165.4(4) c2-n2-ni1 169.9(4) n1-c1-s1 179.5(4) n2-c2-s2 178.4(4) c2-s2-cu1 97.08(18) magnetic study the effective magnetic moment at the room temperature for 1 adopts a value of eff = 3.62 b (fig. 4). such a value can be reconstructed by applying the high-temperature formula 2 2 1/ 2 eff ni ni ni cu cu cu [ ( 1) ( 1)]g s s g s s     . using the constituent spins sni = 1 and scu = 1/2, an estimate for the uniform g-factor is gav = 2.18. cooling down to about 15 k does not cause visible changes of the effective magnetic moment, however, below 15 k its sharp decrease is detected; at 1.8 k eff = 2.55 b. the observed behavior indicates a strong magnetic anisotropy characterized by the axial zero-field splitting parameter dni. there is no indication for a pronounced exchange coupling and if this is the case, it has to be of the ferromagnetic nature. for heteroleptic complexes with trans{nio2n4} chromophore the formula for estimation of the structural distortion parameter dstr based on bond distances around the ni(ii) central atom was elaborated elsewhere (ivaniková et al. 2006). the calculated value of dstr = 15.4 pm for 1 suggests a presence of sizable and positive value of d>> 0. analogous strongly elongated bipyramid was found in heteroleptic complexes [ni(im)4(ac)2] (dstr = 15.5 pm; im = imidazole) (titiš et al. 2007) and [ni(dmeiz)4(h2o)2]cl2 (dcal = 10.67 using casscf calculations; dmeiz = 1,2-dimethylimidazole) (singh et al. 2014). the magnetization per formula unit at t = 2.0 and b = 5 t adopts a value of m1 = mmol/nab = 2.62; this value is far from the spin-only estimate 1 ni ni cu cu m g s g s  ~ 3.27 so that also this data confirms a presence of the considerable dni. the magnetic functions have been fitted simultaneously by minimizing the error functional ( ) ( )f r r m  consisting of the relative errors for the susceptibility and magnetization, respectively. the exchange hamiltonian of the form: 2 2 2 2 ni cu ni ni, ni 1 b ni ni, ni, ni, 1 b cu cu, cu, cu, ˆˆ ( ) ( / 3) ( cos sin cos sin sin ) ( cos sin cos sin sin ) kl z z k x k l y k l z k x k l y k l h j s s d s s bg s s s bg s s s                            has been used where the first term refers to the isotropic exchange, the second is the singleion anisotropy of the ni-center, and the remaining terms represent the zeeman interaction for a set of orientations of the field along grids {k, l} distributed uniformly over one hemisphere (hudák et al. 2013). this form secures a correct powder average: the eigenvalues are inserted into the partition function zkl from which the magnetization mkl and susceptibility kl are calculated, and then averaged. the fitting procedure converged to the following set of magnetic parameters: j/hc = +0.15 cm-1, gni = 2.284 (gcu = 2.1, fixed), dni/hc = +7.49 cm -1, and the temperature-independent magnetism tim = – 1.9 × 10-9 m3 mol-1. the quality of the fit is excellent, as the discrepancy factors are r() = 0.038 and r(m) = 0.012. to this end, a sizable and positive (easy plane) magnetic anisotropy has been confirmed along with a weak exchange coupling of the ferromagnetic nature. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:46 utc nova biotechnol chim (2017) 16(1): 68-75 73 table 3. possible hydrogen bonding interactions [å, º] in 1. d-h...a d(d-h) d(h∙∙∙a) d(d∙∙∙a) <(dha) o1-h2o1∙∙∙s1vi 0.85 2.52 3.336(4) 160 o1-h1o1∙∙∙s1v 0.85 2.64 3.447(4) 159 symmetry codes: v: x, -1+y, z; vi: -x, -y, -z. fig. 3. view on the supramolecular structure formed by hydrogen bonding interactions in [cu(cyclam)ni(ncs)4(h2o)2]n. r1 and r2 are hydrogen bonded rings with descriptors 24 (8)r and 2 2 (12)r , respectively. only selected atoms are shown for clarity. symmetry codes: i: 1-x, -y, -z; v: x, -1+y, z; vi: -x, -y, -z. the model could be extended by considering more centers in exchange interaction within the chain and/or plane. however, the magnetic centers are rather far each other and thus such kind of interaction would be visible only at very low temperature. although the crystal structure itself suggests that 1 might be considered as a new representative of alternating chain with alternation of half (s =1/2) and an integer (s = 1) spins, yet more detailed study in the milikelvin temperature range is necessary to elucidate this conjecture. the study involving specific heat and magnetization measurements would clarify the role of hydrogen bonds as potential exchange bridges as well as the effect of single–ion anisotropy of ni(ii) ions. conclusions from the aqueous-ethanol system cu(ii) – cyclam – ni(ii) – ncsa novel chain-like compound [cu(cyclam)ni(ncs)4(h2o)2]n (1)was isolated and characterized. results of x-ray structure analysis have shown that its crystal structure is formed of infinite chains in which the paramagnetic cu(ii) and ni(ii) atoms are linked by bridging 2-ncs groups; the chains are further interlinked by hydrogen bonding interactions of the o-h∙∙∙s type yielding a 2d supramolecular system. investigation of magnetic properties revealed the presence of a weak ferromagnetic interaction and a sizable single–ion anisotropy of the ni(ii) atom as predicted by the calculated value of structural distortion parameter dstr. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:46 utc nova biotechnol chim (2017) 16(1): 68-75 74 t/k 0 50 100 150 200 250 300  e ff / b 0 1 2 3 4 b/t 0 1 2 3 4 5 m m o l/( n a  b ) 0 1 2 3 0 10 20 30 40  m o l/( 1 0 -6 m 3 m o l-1 ) 0 5 t = 2.0 k b = 0.1 t fig. 4. magnetic functions for 1. left – effective magnetic moment (inset: molar magnetic susceptibility); right – magnetization per formula unit. lines – fitted. acknowledgements this work was supported by the slovak grant agencies vega 1/0063/17 and apvv-14-0078. we thank student michal hegedüs for help in synthetic experiments. references bernstein j, davis re, shimoni l, chang n-l (1995) patterns in hydrogen bonding: functionality and graph set analysis in crystals. angew. chem. int. ed. eng. 34: 1555-1573. bieńko a, kłak j, mroziński j, domagała s, korybutdaszkiewicz b, woźniak k (2007) magnetism and crystal structures of (cumnii)-mn-ii and (cuniii)ni-ii ordered bimetallic chains. polyhedron 26: 50305038. brandenburg k (2008) diamond. crystal impact (version 3.1f) gbr, bonn, germany. černák j, kuchár j, stolárová m, kajňaková m, vavra m, potočňák i, falvello lr, tomás m (2010) preparation, spectroscopic and magnetic characterization of cu(cyclam)m(cn)4 complexes exhibiting onedimensional crystal structures (cyclam=1,4,8,11tetraazacyclotetradecane, m = ni, pd, pt). trans. met. chem. 35: 737-744. černák j, kočanová i, orendáč m (2012a) copper-nickel heterobimetallic compounds. comm. inorg. chem. 33: 2-54. černák j, stolárová m, čižmár e, tomás m, falvello lr (2012b) cu(bapen)m(cn)4·h2o complexes exhibiting chain-like structures (bapen = n,n '-bis(3-aminopropyl)1,2-diaminoethane, m = ni, pd): preparations, crystal structures, spectroscopic and magnetic properties. trans. met. chem. 37: 321-329. chandra s, pundir m (2008) spectroscopic characterization of chromium(iii), manganese(ii) and nickel(ii) complexes with a nitrogen donor tetradentate, 12-membered azamacrocyclic ligand. spectrochim. acta a69: 1-7. clark rc, reid js (1995) the analytical calculation of absorption in multifaceted crystals. acta crystallogr. a51: 887-897. farrugia lj (2012) wingx and ortep for windows: an update. j. appl. crystallogr. 45: 849-854. furusaki a, sigrist m, lee pa, tanaka k, nagaosa n (1994) random exchange heisenberg chain for classical and quantum spins. phys. rev. lett. 73: 2622-2625. huang s-l, zhang l, lin y-j, jin g-x (2013) discrepant gas adsorption in isostructural heterometallic coordination polymers: strong dependence of metal identity. cryst. eng. comm.15:78-85. hudák j, boča r, moncoľ j, titiš j (2013) magnetism of dinuclear benzoato cobalt(ii) complexes modeled bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:46 utc nova biotechnol chim (2017) 16(1): 68-75 75 by a general bilinear exchange. inorg. chim. acta 394: 401-409. ivaniková r, boča r, dlháň ľ, fuess h, mašlejová a, mrázová v, svoboda i, titiš j (2006) heteroleptic nickel(ii) complexes formed from n-donor bases, carboxylic acids and water: magneto structural correlations. polyhedron 25: 3261-3268. king br (2005) encyclopedia of inorganic chemistry, wiley, university of georgia, 3393 p. kou h-z, liao d-z, cheng p, jiang z-h, yan s-p, wang gl, yao x-k (1998) a new one-dimensional thiocyanatobridged bimetallic compound [cu(en)2mn(ncs)4(h2o)2]n. synthesis, crystal structure, and magnetic properties can. j. chem. 76: 1102-1107. nakamoto k (1997) infrared and raman spectra of inorganic and coordination compounds, part b: applications in coordination, organometallic, and bioinorganic chemistry, wiley, new york, usa. nardelli m (1999) modeling hydroxyl and water h atoms. j. appl. cryst. 32: 563-571. nesterova ov, petrusenko sr, nesterov ds, kokozay vn, skelton bw, jezierska j, linert w, ozarowski a (2010) a (cuniii)-ni-ii complex with ethylenediamine: crystal structure and ferromagnetic behaviour of an aquabridged heterometallic chain containing ambidentate ni(oac)4 2blocks. eur. j. inorg. chem. 22: 3529-3535. oxford diffraction (2003) crysalis red and crysalis ccd software (ver. 1.171.1), oxford diffraction ltd., abingdon, oxfordshire, england. sheldrick gm (2015) shelxt – integrated space-group and crystal-structure determination. acta crystallogr. c71: 3-8. shen l, xu y-z (2001) structure and magnetic properties of a novel two-dimensional thiocyanato-bridged heterometallic polymer {cu(en)2[ni(en)(scn)3]2}n. j. chem. soc., dalton trans. 23: 3413-3414. singh sk, gupta t, badkur p, rajaraman g (2014) magnetic anisotropy of mononuclear ni-ii complexes: on the importance of structural diversity and the structural distortions. chem. eur. j. 20: 1030510313. titiš j, boča r, dlháň ľ, ďurčeková t, fuess h, ivaniková r, mrázová v, papánková b, svoboda i (2007) magneto structural correlations in heteroleptic nickel(ii) complexes. polyhedron 26: 1523-1530. wang c-c, ke m-j, tsai c-h, chen i-h, lin s-i, lin t-y, wu l-m, lee g-h, sheu h-s, fedorov ve (2009) [m(c5o5)2(h2o)n] 2as a building block for hetero and homo-bimetallic coordination polymers: from 1d chains to 3d supramolecular architectures. cryst. growth des. 9: 1013-1019. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:46 utc nova biotechnol chim (2019) 18(2): 102-114 doi: 10.2478/nbec-2019-0013 corresponding author: hezekiahfatoki@gmail.com, dmsanni@futa.edu.ng nova biotechnologica et chimica physicochemical properties, kinetics and thermodynamic studies of polyphenol oxidase from sorghum (sorghum bicolor (l.) moench) for potential use in industry toluwase hezekiah fatoki and david morakinyo sanni enzyme biotechnology unit, department of biochemistry, federal university of technology akure, pmb 704 akure, ondo state, nigeria article info article history: received: 27th july 2019 accepted: 10th november 2019 keywords: kinetics physicochemical properties polyphenol oxidase sorghum bicolor thermodynamics abstract advances polyphenol oxidase (ppo) from sorghum bicolor (white and yellow varieties) grains were investigated for optimum processing condition. the partially purified enzyme was obtained from two varieties of sorghum bicolor by step-wise separation through ion-exchange and size-exclusion chromatography. the final purification gave a yield of 7.33 % and 12.3 % for ppo from white and yellow sorghum respectively. the ppo has vmax and km of 2.66 u.ml -1 and 19.72 mm for white sorghum, 1.33 u.ml-1 and 12.92 mm for yellow sorghum. the optimal ph of ppo activity was found at ph 4 and ph 7 for white and ph 4 and ph 8 for yellow sorghum. the pka 7.4 and 8.7 were obtained for ppo from white sorghum, and pka 5.4, 7.4 and 8.5 for yellow sorghum. the ppo residual activity were above 70 % at 5 hours of incubation within the neutral ph ranges for white sorghum, while those of yellow sorghum were below 40 %. the optimum temperature of 40 ºc and 30 ºc for white and yellow sorghum ppo respectively. the average value of enthalpy (δh), entropy (δs) and gibbs free energy (δg) obtained at 20 min of incubation and temperature 50 – 80 °c were respectively 49.03 kj.mol-1, 129.52 j.mol-1.k-1, and 92.81 kj.mol-1 for white sorghum ppo, and 90.1 kj.mol-1, 9.29 j.mol-1.k-1, and 93.37 kj.mol-1 for yellow sorghum ppo. zn2+, fe2+ and ascorbic acid inhibited ppo while cu2+, na+ and k+ activated the enzyme. the results suggest the processing parameters for controlling ppo in potential industrial application of white and yellow sorghum grains. university of ss. cyril and methodius in trnava introduction sorghum (sorghum bicolor (l.) moench) is indigenous to africa and is a member of the grass family poaceae and has high morphological variations (deu and hamon 1994; hariprasanna and patil 2015). sorghum is ranked as the fifth most important grain in terms of production, preceded by wheat, rice, maize and barley (sanni and fatoki 2017a). nigeria is the third largest world producer after the united states and india (faostat 2011), accounting for about 71 % of the total sorghum production in west africa which could be of different varieties, with two best known species which are sorghum vulgare and sorghum bicolor (l.) moench (ogbonna 2011). sorghum is a dietary staple for about 500 million people in over 30 countries of the semi arid tropics (dahlberg et al. 2011). sorghum find its application in human food, such as bread, malt drinks and beer; livestock feed; and renewable energy source such as bioethanol (dahlberg et al. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:25 utc mailto:hezekiahfatoki@gmail.com mailto:dmsanni@futa.edu.ng nova biotechnol chim (2019) 18(2): 102-114 103 2011). it is a source of nutrients and bioactive compounds, especially 3-deoxyanthocyanidins, tannins and polycosanols, which beneficially modulate, in vitro and in vivo, the metabolic markers for varieties of diseases. (sanni and fatoki 2017a). polyphenol oxidases (ppo; 1, 2-benzenediol: oxygen oxidoreductase; ec 1.10.3.1) are copper containing oxidoreductases that catalyze the hydroxylation and oxidation of phenolic compounds in the presence of molecular oxygen. they are widely found in plants, animals and microorganisms (yoruk and marshall 2003). the three types of polyphenol oxidases which were classified according to their ability to oxidizes different types of phenolic compounds are catecholase (1,2-benzenediol: oxygen oxidoreductase; e. 1.10.3.1), laccase (ec 1.10.3.2) and tyrosinase (monophenol monooxygenase; ec 1.14.18.1) (sanchez-ferrer et al. 1995; erhan 2003; mayer 2006). ppo cause oxidative browning reactions in many foods of plant origin, which result into deterioration in food quality by changing nutritional and organoleptic properties (martinez and whitaker 1995), but ppo has its advantage in the processing of tea and malt. the ppo has been characterized either partially or fully, from different plant sources which include sweet potato (manohan and wai 2012) and yams (sanni and fatoki 2017b). also, ppo of most grains of industrial application has been studied which include wheat (interesse et al. 1980; kihara et al. 2005; naqvi et al. 2013), barley (huynh and jerumanis 1977). the potential industrial application of sorghum in africa include large scale production of beverages (such as malt, pito/dolo, burukutu, kunu etc.), breads (such as kisra, injera, sourdough etc.) and porridges. although polyphenol oxidase content has been screened in grains of from fifty different sorghum varieties (dicko et al. 2002) as well as the activity of ppo during malting of sorghum (sanni and fatoki 2017a). the understanding of thermodynamics, kinetics activities and physicochemical properties of purified ppo of sorghum will enhance the processing and production of sorghum-based products. reduction in heating time and optimization of heating temperatures to minimize damage of nutrients and sensory components are part of the objectives of the current food processing technologies (sant’anna et al. 2012). the activity and thermostability of enzymes are important issues often considered when assessing the economic feasibility of enzyme-based industrial processes. high stability is generally considered an economic advantage for a desirable enzyme because of reduced enzyme consumption (souza et al. 2015) whereas low thermostability favors the inhibition of undesirable enzyme in an industrial process. the thermodynamic and kinetic studies can provide valuable information about the thermostability of enzymes at the operating temperature. hence, this present work focused on the investigation of substrate specificity, kinetics parameters, effect of ph, ph stability, effect of ph on kinetics parameter, optimum temperature, thermal stability, and effect of salts on the activities of polyphenol oxidase from two varieties of sorghum bicolor grains (fig. 1) toward advancement of industrial utilization. fig. 1. photograph of studied sorghum samples (a) white sorghum (b) yellow sorghum. a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:25 utc nova biotechnol chim (2019) 18(2): 102-114 104 experimental sample collection fresh sorghum (white and yellow) was purchased from oba market in akure, nigeria and dried at room temperature. the sample was identified and authenticated at the department of crop, soil and pest management (csp) of the federal university of technology, akure, nigeria. preparation enzyme crude extract one hundred and fifty grams (150 g) of sorghum grains were weighed and homogenized with 450 ml of chilled phosphate buffer (25 mm, ph 6.8) containing 0.5 % triton x-100 and 0.01 % ascorbic acid, for 5 min and filtered through cheese cloth. the filtrate was centrifuged at 6,000 rpm at 4 ºc for 30 min. the supernatant was used as crude extract for further analysis. assay for polyphenol oxidase activity ppo enzyme activity was determined with a spectrophotometer by measuring an increase in absorbance at 420 nm at room temperature by using catechol as substrate (eq. 1). the reaction mixture consisted 0.1 ml freshly prepared enzyme extract and 2.9 ml of 20 mm catechol in 20 mm phosphate buffer ph 6.8 while the blank contained the buffer and the substrate. one unit (u) of ppo activity was defined as the amount of the enzyme that increased the absorbance by 0.001 per minute under the conditions of the assay (sanni 2016) (eq. 1). 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝐼𝑛𝑐𝑟𝑒𝑚𝑒𝑛𝑡𝑎𝑙 × 𝑇𝑖𝑚𝑒 × 𝑉𝑜𝑙𝑢𝑚𝑒 activity (u.ml-1) of polyphenol oxidase, incremental is 0.001, time (min) of assay is 10 and the volume (ml) of assay is 3 (eq. 2). 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 𝐴𝑐𝑖𝑣𝑖𝑡𝑦 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 specific activity (u.mg-1) of polyphenol oxidase, activity (u.ml-1) and protein concentration (mg.ml-1). determination of protein concentration protein concentration was measured according to the method of bradford (1976) using bovine serum albumin (bsa) as standard. purification of enzyme ammonium sulphate precipitation the crude extract obtained from each species was brought to 30 – 80 % ammonium sulphate (nh4)2so4 saturation by slowly dissolving the precise amount of the solid salt evaluated by encorbio ammonium sulphate calculator using magnetic stirrer. after the mixture stood for 2 h, the precipitate was separated by centrifugation at 6,000 rpm for 30 min at 4 ºc. the precipitate was dissolved in 15 ml of 20 mm phosphate buffer (ph 6.8) and dialyzed against the same buffer at 4 ºc for 72 h, with three changes of buffer. the dialyzed samples (partial extract) were further centrifuge at 14,000 rpm, and the resulting supernatant were used as the enzymes source in further experiments. purification on deae-sephacel chromatography the certain amount of pre-swollen deae-sephacel stored in ethanol was poured into a clean 500 ml beaker and stirred with addition of distilled water to form a slurry. this was carefully loaded into column (2.5 × 20 cm) and washed with 500 ml of distilled water and later equilibrated with about 1 l phosphate buffer (20 mm, ph 6.8). fifteen milliliters (15 ml) of dialyzed sample was loaded onto the column and washed with the same buffer at the flow rate of 18 ml.h-1 until the absorbance of the fractions at 280 nm was nearly zero. the bound protein was eluted with linear gradient of 0.0 – 0.5 m nacl in 20 mm phosphate buffer ph 6.8 and collected in fractions of 5 ml. the absorbance of the fractions was measured at 280 nm and assayed for ppo activity. the fractions with ppo activity were pooled and concentrated by dialyzing against 4 m sucrose. (1) (2) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:25 utc nova biotechnol chim (2019) 18(2): 102-114 105 purification on sephadex g-100 fifteen grams of sephadex g-100 was soaked and allowed to swell in 20 mm phosphate buffer ph 6.8 for 72 h at room temperature. this was poured carefully into the column. (2.5 × 70 cm) and allowed to packed very well without space. it was later equilibrated with 1 l of 20 mm phosphate buffer ph 6.8. 15 ml of the concentrated pooled fraction from the deae-sephacel step was carefully applied to the column. the proteins were eluted using the same buffer and 5 ml fractions were collected at a flow rate of 12.0 ml.h-1. the absorbance of the fractions collected was measured at 280 nm and were assayed for ppo activity while those that contained ppo were pooled and used for further analysis. physicochemical properties, kinetics and thermodynamic of ppo substrate specificity of ppo substrate specificity was determined using 2 different substrates; l-dopa and catechol. the assay for ppo was carried out using 20 mm of l-dopa at 475 nm, as well as 20 mm of catechol at 420 nm, as earlier described. determination of kinetics parameter the catechol concentrations (30, 25, 20, 15, 10 and 5 mm) were prepared in 20 mm phosphate buffer (ph 6.8). ppo activity was assayed as according to standard assay procedure earlier described. kinetics parameter; km and vmax were evaluated using lineweaver-burk plot. effect of ph on ppo activity effect of ph on polyphenol oxidase activity was determined by incubating 0.1 ml of purified ppo with 0.9 ml of 20 mm catechol prepared in each buffer ph range 2.0 – 9.0 using 20 mm glycine/hcl (ph 2.0 – 3.0), 20 mm sodium acetate buffer (ph 4.0 – 5.0), 20 mm phosphate buffer (ph 6.0 – 7.0) and 20 mm tris/hcl buffer (ph 8.0 – 9.0) at room temperature. the enzymes activity was determined at 420 nm. effect of ph on ppo stability the effect of ph on stability was determined by incubating 0.2 ml enzyme solution in 1.8 ml of the buffer solution between ph range 4.0 – 9.0, using 20 mm na-acetate buffer (ph 4.0 – 5.0), 20 mm phosphate buffer (ph 6.0 – 7.0) and 20 mm tris/hcl buffer (ph 8.0 – 9.0) at room temperature. the residual polyphenol oxidase activity was determined according to the standard assay procedure. effect of ph on kinetics parameter effect of ph on kinetics parameters was determined according to the method of kolawole et al. (2005). michaelis-menten constant (km) and maximum reaction velocity (vmax) were determined using catechol as substrates. substrate concentrations (30, 25, 20, 15, 10 and 5 mm) was prepared in different buffer ph between 4 – 9, using 20 mm na-acetate buffer (ph 4.0 – 5.0), 20 mm phosphate buffer (ph 6.0 – 7.0) and 20 mm trishcl buffer (ph 8.0 – 9.0); polyphenol oxidase activity was assayed according to standard assay procedure earlier described. data were plotted according to the method of lineweaver-burk. the vmax/km value was evaluated and plotted against the ph. the apparent pka of ionized amino acids was obtained from the peak of the plot of vmax/km against ph. effect of temperature on ppo activity effect of temperature on ppo activity was carried out between 30 °c to 90 °c at 10 °c interval. the assay mixtures consisting of 0.2 ml of the enzyme and 1.8 ml of 20 mm catechol were incubated at each temperature for 10 min, and the absorbance was read at 420 nm according to the standard assay procedure earlier described. effect of temperature on ppo stability thermal stability of the ppo was determined by incubating purified enzyme at different temperatures range of 30 – 90 ºc for 60 min. at 10 min interval the residual activity was determined according standard assay procedure. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:25 utc nova biotechnol chim (2019) 18(2): 102-114 106 thermal inactivation was evaluated in the temperature range of 50 – 80 °c after 20 min of enzyme incubation (manohan and wai 2012), and the residual ppo activity (a) was determined according to the standard procedure described earlier. the data obtained were used to evaluated activation energy (ea), enthalpy (δh), entropy (δs) and gibbs free energy (δg) using equation 1 to 11. in the thermodynamic study, the percentage residual ppo activity is calculated by comparison to the initial enzyme activity (a0). the rate constants k for first-order inactivation was determined from the slopes of the inactivation time courses according to the following equation (eq. 3). log ( a a0 ) = − ( k 2.303 ) 𝑡 where a0 is the initial enzyme activity (before heating where time t is zero) and a is the activity after heating for time t. a useful indication of the rate of a first-order chemical reaction is the half-life t1/2, of a substance, the time it takes for its concentration to fall to half the initial value. the time for [a] to decrease from [a0] to ½[a0] in a first-order reaction. the half-life of the enzyme (t1/2) calculated according to the following equation (eq. 4). t1/2 = (ln 2) k the main point to observe about this result is that, for a first-order reaction, the half-life of a reactant is independent on its initial concentration. in addition, decimal reduction time (d-value) is defined as the time required to pre-incubate the enzyme at a given temperature to maintain 10 % residual activity or reduce the initial activity by 90 % (sant’anna et al. 2012; manohan and wai 2012; wong and lee 2014), and was estimated from the relationship between k and d according to the eq. 5. 𝐷 = ln10 𝑘 the z-value is the temperature increase required for one-log10 reduction (90 % decreases) in d-value or the temperature needed to vary d-value one log unit. it was determined from a plot of log d versus temperature (ºc). the slope of the graph is equal to -1/z (sant’anna et al. 2012; wong and lee 2014). the temperature of treatment and the rate constant (k) in a denaturation process was related according to the arrhenius equation (eq. 6). 𝑘 = 𝐴e − 𝐸𝑎 𝑅𝑇 equation 2.4 can be transformed as in eq. 7 ln 𝑘 = ln 𝐴 – 𝐸𝑎 𝑇 −𝑅 ln𝑘 = −𝑅 ln𝐴 + 𝐸𝑎 𝑇 where r is the universal gas constant (8.314 j.mol-1.k-1), k is the reaction rate constant value, a is the arrhenius constant, ea is the activation energy (energy required for the inactivation to occur), and t is the absolute temperature in kelvin. slopes were calculated by linear equation (y = mx + c). the energy of activation of denaturation (ea) was calculated from the slopes of these arrhenius plots, (-r ln k) values versus reciprocal of absolute temperatures (1/t) according to eq. 8, and the ordinate intercept corresponds to –r ln a. the values of the activation energy (ea) and arrhenius constant (a) allowed the determination of different thermodynamic parameters such as variations in enthalpy (δh; eq. 9), entropy (δs; eq. 10) and gibbs free energy (δg; eq. 11), respectively (sant’anna et al. 2012), according to the following expressions. ∆𝐻∗ = 𝐸𝑎 − 𝑅𝑇 ∆𝑆∗ = 𝑅 (ln𝐴 – ln ( 𝐾𝐵 ℎ𝑝 ) – ln 𝑇) ∆𝐺 ∗ = ∆𝐻∗ − t∆𝑆∗ where kb is the boltzmann constant (1.38˟10-23j.k-1), hp is the planck constant (6.626˟10-34 j.s), and t is the absolute temperature (k). effect of salts and ascorbic acid the effect of salts such as copper sulphate, zinc sulphate, iron sulphate, sodium chloride, potassium chloride and ascorbic acid, at three different concentrations (10, 30 and 50 mm) on enzyme activity were examined. the salts and ascorbic acid were each prepared in 20 mm catechol. the assay (3) (4) (5) (6) (7) (8) (9) (11) (10) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:25 utc nova biotechnol chim (2019) 18(2): 102-114 107 table 1. purification of ppo from white and yellow sorghum. samples purification steps volume protein total protein activity total activity specific activity yield fold [ml] [mg.ml -1] [mg] [u.ml-1] [u] [u.mg-1] [%] white crude 345.00 4.47 1,540.25 2.24 773.84 0.50 100.00 1.00 ammonium sulphate precipitation 33.00 66.07 2,180.34 5.24 172.92 0.08 22.35 0.16 ion-exchange chromatography on deae-sephacel 80.00 6.70 535.68 2.60 164.40 0.31 21.24 0.61 gel filtration on sephadex g-100 45.00 1.60 47.70 1.26 56.70 1.19 7.33 2.37 yellow crude 325.00 8.93 2,901.93 2.56 832.00 0.29 100.00 1.00 ammonium sulphate precipitation 35.00 59.22 2,072.81 5.30 185.50 0.09 22.30 0.31 ion-exchange chromatography on deae-sephacel 100.00 3.75 339.40 1.40 140.00 0.41 16.83 1.44 gel filtration on sephadex g-100 55.00 0.06 2.97 1.86 102.30 34.44 12.30 120.00 was done at room temperature as earlier described above. the percentage effect (inhibition or activation) was calculated (eq. 12). 𝐸𝑓𝑓𝑒𝑐𝑡 [%] = [ 𝐴𝑡−𝐴0 𝐴0 ] × 100 where a0 is initial ppo activity (without salt/additive); at is ppo activity with salt/additive, where (-) is inhibition, (+) is activation at a baseline of zero (0). data analysis all experiments were performed in triplicates and average of the data sets were processed using microsoft excel 2010. results and discussion the results of ppo activity obtained from two varieties of sorghum bicolor in this project, confirmed the occurrence and distribution of ppo in sorghum. reports by other authors have shown the presence of ppo in both the leaves and the grain of sorghum (luthra et al. 1988; gowda et al. 1989; dicko et al. 2006). the crude activity of ppo was found to be high in malted and nonmalted yellow sorghum grains than in white counterpart (sanni and fatoki 2017a). purification parameter of the ppo the polyphenol oxidase activity of white sorghum was found to be 2.234 u.ml-1 and 2.055 u.ml-1 for crude and ammonium sulphate precipitation samples respectively while that of yellow sorghum was 2.560 u.ml-1 and 5.300 u.ml-1 for crude and ammonium sulphate precipitate samples respectively (table 1). the elution profile of both varieties showed the same pattern. two activity peaks were obtained from deae-sephacel chromatography, one in flow through fractions and the other in the gradient elution fractions. overall, the polyphenol oxidase (ppo) activities of yellow sorghum were found to be slightly higher than that ppo of the white sorghum. the purification of ppo from many plants to homogeneity has remained difficult possibly due to the high phenolic content and irreversible binding of phenolics to ppo during purification steps (mishra and gautam 2016). in this study, there was an obvious difference in the purification results obtained from two varieties of sorghum bicolor. these variations in fold purification and yield could be due to differences in the concentration and types of phenolics affecting their overall binding to the ppo protein (yoruk and marshall 2003). two peaks were obtained from ion exchange profile of ppo activity in both sorghum varieties, (12) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:25 utc nova biotechnol chim (2019) 18(2): 102-114 108 fig. 2. lineweaver-burk plot for ppo from white (round) and yellow (square). and it may possibly be an indication that the ppo consist of two isoforms. polyphenol oxidases isolated from barley, steeped barley, and green malt were resolved into two active forms on sephadex g-200 column (huynh and jerumanis 1977). the two isoform of ppo obtained for each varieties of sorghum grains in this study, align with the evidence of three isoforms of crude ppo found in the sorghum leaves (dicko et al. 2006). substrate specificity of ppo ppo has higher affinity using catechol than l-dopa in both samples while yellow sorghum showed more activity than white sorghum in both substrates. the ppo from both white and yellow sorghum in this study showed greater specificity for catechol than l-dopa and this indicates di-phenolase activity. mishra and gautam (2016) have reported the substrate specificity of ppo to be in the order of 4-methyl catechol > tertbutylcatechol > dihydrocaffeic acid > pyrocatechol > l-dopa, d-dopa > caffeic acid > chlorogenic acid > pyrogallol. moreover, dicko et al. (2006) observed difference in mono-phenolase and o-diphenolase activities of sorghum varieties and found that sorghum ppos are more active fig. 3. effect of ph on ppo from white (round) and yellow (square). the result was computed relative to the alkaline optimum ph expressed as 100 % for each ppo respectively with o-diphenols than with monophenols, and corroborated other findings from most plant ppos (martinez and whitaker 1995). previous study has shown that germination decreases the o-diphenolase activity and slightly increases the monophenolase activity of ppo, while the zymography revealed that germination does not induce new ppo isoenzymes in sorghum grain (dicko et al. 2006). since the activities of ppo can be kinetically controlled, during possessing the modulation of these activities will have interesting applications in the food and chemical industries (dubey et al. 1998; espin et al. 2001). determination of kinetics parameters the kinetics parameters obtained using lineweaverburk plot were vmax and km are 2.66 u.ml -1 and 19.72 mm for white sorghum ppo and 1.33 u.ml-1 and 12.92 mm for yellow sorghum ppo respectively using catechol substrate as shown in fig. 2. the vmax and km showed that ppo from white sorghum have much better capacity and more affinity for the substrate used than that of yellow sorghum. km is a measure of the amount of enzyme that is bound in any form whatsoever to the substrate. the results showed that the rate of polyphenol oxidase activity bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:25 utc nova biotechnol chim (2019) 18(2): 102-114 109 fig. 4. effect of ph on stability of ppo from (a) white sorghum and (b) yellow sorghum. the result was expressed as percent relative activity to the activity at 0 hour (100 %). in white sorghum was better than that of yellow sorghum. effect of ph on ppo activity the two varieties of sorghum bicolor ppo exhibited two optimal ph of activity each. optimal ph of 4 and 7 were obtained for white sorghum ppo while that of yellow sorghum were found to be ph 4 and ph 8 as shown in fig. 3. ppo from white sorghum showed high enzymatic activity of 93 % and 89 % at alkaline ph of 8.0 and 9.0, while that of yellow revealed a sharp reduction in activity after the optimal at ph 9.0. however, white sorghum ppo revealed a high percent relative activity of 85.95 % at ph 4.0 while yellow sorghum ppo revealed a lower percent relative activity of 44.62 % at ph 4.0. these results indicate the possible occurrence of two ppo isoforms in both varieties of sorghum bicolor, having different ph for optimal activity. however, the optimum ph of both varieties of sorghum was similar to the ph optima of partially purified ppo from two species of eggplants that have optimum activity at ph 4.0 – 4.5 and 7 (solanum depressum), as well as ph 4 and 8 (solanum gilo) for the oxidation of catechol (sanni 2016). the optimum ph depends on the amino acid components of active site, while the isoelectric point depends of the full enzyme primary and tertiary structure (fatoki 2016). effect of ph on ppo stability the ppo residual activity were found to be relatively stable above 50 % at 2 h of incubation in all the ph ranges for white sorghum while those of yellow sorghum were below 50 % as shown in fig. 4. white ppo stability were found in both acidic region (ph 5 and 6) with percent relative activity of 80 % and 90 % as well as in alkaline region (ph 8 and 9) with relative activity of 75 % and 76 % respectively after 5 h of incubation. the white sorghum ppo was found to be relatively stable at 5 hours of incubation retaining over 70 % of its initial activity within the neutral ph while that of yellow sorghum were below 40 %. this showed that ppo from white sorghum is highly stable within the neutral ph than that yellow sorghum. this may be an indication of possible difference in the amino acid residues composition at the active site of the ppo from these two varieties of sorghum. effect of ph on kinetics parameters the dependencies of ph on kinetics parameters showed that activity of ppo from white sorghum was optimal at ph 8 independent of the substrate concentrations as shown in fig. 5 while ppo from yellow sorghum was optimal at ph 6 – 7 independent of the substrate concentration. the slight difference observed in the ppo activity bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:25 utc nova biotechnol chim (2019) 18(2): 102-114 110 fig. 5. effect of ph and substrate concentration on activity of ppo from (a) white sorghum and (b) yellow sorghum. may be due to difference in variety of sorghum. at concentration 25 mm and 30 mm, the optimum activity was in close range of ph 7 – 8. lineweaverburk plot was used to evaluate the kinetics parameter at various ph for both sorghum. varieties (data not shown) from which vmax and km were obtained. the plot of vmax/km against ph gave pka 7.4 and 8.7 for ppo from white sorghum and pka 5.4, 7.4 and 8.5 for ppo from yellow sorghum (data not shown). the effect of ph on the kinetic parameter showed that ph dependencies of ppo activity at independence of the substrate concentrations, better provide direct useful insight for the evaluation of the pka of an enzyme. though a pka value does not represent the microscopic ionization of a particular group but is a combination of this value and various equilibrium constants between different conformational states of the molecule. these results suggest the occurrence of cysteine (thiol group) and histidine (imidazole group) at active site of sorghum (sorghum bicolor) polyphenol oxidase. kolawole et al. (2005) earlier predicted the occurrence of cysteine and histidine at the active site of fonio millet seed (digitaria exilis) beta-amylase. effect of temperature on ppo activity the optimum temperature of ppo from both white and yellow sorghum were found at 40 ºc and 30 ºc respectively. the ppo from both sorghum bicolor varieties exhibited a similar pattern of gradual reduction in enzymatic activity after the optimal temperature were obtained. however, ppo from white sorghum revealed a higher percentage activity of 90 and 100 % compare to 100 and 94 % obtained for yellow sorghum ppo at 30 and 40 °c respectively as shown in fig. 6. the optimum temperature of 40 ºc and 30 ºc obtained for ppo activity in white and yellow sorghum was followed by a decrease in relative enzyme activity as the temperature increase above the optimum value. this result probably favored increase activity of ppo during germination process in beverage production. sanni and fatoki (2017a) reported increase in ppo activity in germinating sorghum in the malting floor during the early stage of brewing process while kilning and mashing at temperature of about 120 ºc will ensured total loss of ppo activity and favors the late stage of brewing process. the decrease in activity at high temperature was possibly owing to the thermal denaturation of ppo. effect of temperature on ppo stability and dynamics the result showed that ppo from both white and yellow sorghum varieties were most stable at 30 – 40 ºc as the ppo showed over 60 % residual bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:25 utc nova biotechnol chim (2019) 18(2): 102-114 111 fig. 6. effect of temperature on ppo from white (round) and yellow (square). the result was computed relative to the optimum temperature expressed as 100 % for each ppo respectively activity were observed between 30 – 50 ºc after 60 min of incubation in white and yellow sorghum. the ppo from white sorghum showed 37 % and 0 % relative activity after 60 min of incubation at 60 ºc and 70 – 90 ºc respectively, while over 20 % relative activity was observed after 60 min of incubation at 60 – 80 ºc for yellow sorghum. the result showed that ppo from yellow sorghum is thermally stable than ppo of white sorghum. the moderate thermal stability of yellow sorghum ppo will favor the development of flavors and color during the curing in malt production but will be a detrimental in confectionary processes if not adequately control at onset with another parameter such as additives. the inactivation constant (kinact) increased from 5.8 × 10-3 to 39.5 × 10-3 min-1 for white sorghum ppo, and 0.9 × 103 to 27.5 × 103 min-1 for yellow sorghum ppo, as the temperature increases from 50 – 80 ºc respectively. it was observed that half-life (t1/2) of the ppo from both sorghum varieties, decreases as temperature increases. d-value is the temperature sensitivity parameter and z-value is the temperature increase needed for a 90 % reduction in d-value. these results of kinetic study showed that the thermal inactivation of sorghum’s ppo using catechol as substrate followed first-order kinetics however, the inactivation constant (kinact or k-value) of ppo from white sorghum was linear fig. 7. arrhenius plot for ppo from white (round) and yellow (square) sorghum between 50 – 80 ºc at 20 min of incubation. the slope equals activation energy (ea). while that of yellow sorghum was hyperbolic or non-linear. d-value and k-values decreased and increased, respectively with increasing temperature in both varieties, indicating faster polyphenol oxidase inactivation at higher temperatures. manohan and wai (2012) described denaturation of sweet potato ppo as a first-order reaction with k values between 0.0075 and 0.0657 min-1. the half-life (t1/2) value of sorghum ppos in this study was between 117.46 to 17.54 min for white sorghum ppo and 770.0 to 25.20 min for yellow sorghum ppo. z-value is the temperature increase needed to vary d-value one log unit. the z-value of 36.9 °c and 20.58 °c, obtained for white and yellow sorghum ppo respectively. in general, high z-values mean more sensitivity to the duration of heat treatment and lower z-values mean more sensitivity to increase in temperature (barrett et al., 1999). differences in the kinetics of heat activation of the two varieties of sorghum ppos may result from differences in their composition which is reflective of their variety or the agronomic and climatic conditions under which they were grown (chutintrasri and noomhorm 2006). the arrhenius plot for the two varieties of sorghum ppo were shown in fig. 7. the results thus suggest that white and yellow ppo are relatively thermo bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:25 utc nova biotechnol chim (2019) 18(2): 102-114 112 table 2. thermodynamics parameter of ppo from white and yellow sorghum after 20 minutes of incubation. sorghum temp. kinact t1/2 d z temp. ea lna δh# δs# δg# sample [ºc] [10-3 min-1] [min] [min] [ºc] [k] [kj.mol-1] [kj.mol-1] [j.mol-1.k-1] [kj.mol-1] ppo white 50 5.90 117.46 390.27 323 49.16 -129.15 90.87 60 7.70 90.00 299.04 333 49.07 -129.40 92.16 36.90 51.84 14.00 70 13.10 52.90 175.77 343 48.99 -129.65 93.46 80 39.50 17.54 58.29 353 48.91 -129.89 94.76 yellow 50 0.90 770.00 2,558.43 323 90.30 -8.92 93.18 60 14.70 47.14 156.64 333 90.22 -9.17 93.45 20.58 92.99 28.46 70 37.10 18.68 62.06 343 90.14 -9.42 93.37 80 27.50 25.20 83.73 353 90.05 -9.66 93.46 stable enzymes with the ea of 51.84 kj.mol-1 and 92.99 kj.mol-1, respectively, at 20 min of incubation. high activation energy reflects a greater sensitivity of the sorghum ppos to temperature change as indicated for ppo from other sources (weemaes et al. 1998; chutintrasri and noomhorm 2006). this suggests that the denaturation process requires a high energy input for the enzyme-substrate complex to initiate denaturation probably due to a possible compact structure of enzymes and the strength of the thiol groups (–sh) or disulfide bond (–s–s–) at the active site (björck 1992). thermal inactivation of ppo from sorghum varieties in this study gave an average value of δh, δs and δg of 49.03 kj.mol-1, 129.52 j.mol-1.k-1, and 92.81 kj.mol-1 for white sorghum ppo, and 90.1 kj.mol-1, 9.29 j.mol-1.k-1, and 93.37 kjmol-1 for yellow sorghum ppo respectively at 20 min of incubation and temperature of 50 – 80 °c as shown in the table 2. the high values of change in enthalpy obtained for the different treatment temperatures revealed that the enzyme undergoes a considerable change in conformation during denaturation. positive values of δh of the ppo indicate the endothermic nature of the oxidation reactions. the negative values of entropy indicate that there are no significant processes of aggregation respectively, otherwise the values of entropy would be negative (anema and mckenna 1996). the gibbs free energy (δg) is a measure of the spontaneity of the inactivation processes. the free energy of the ppo of both varieties of sorghum increases slightly with increasing temperature, and the result of this study showed gibbs free energy obtained for ppo from both sorghum varieties were the same and positive, which showed that the reactions or inactivation processes of ppo were not spontaneous which could be due to presence of disulphide and imidazole linkage at the active sites (dietler and lerch 1982). effect of salts and ascorbic acid on ppo activity the effect of salts and additives on the ppo activity at three different concentrations from the two sorghum varieties is given in table 3. ascorbic acid, zn2+ and fe2+ have increased inhibitory effect on ppo activity from both white and yellow sorghum varieties in concentration dependent manner (from 10 to 50 mm). similar activation effect was observed for cu2+. highest activation effect was obtained at 30 mm for na+ and 50 mm for k+ in both varieties. this result corroborates the evidence of full activity of yam polyphenol oxidases (ppo1 and ppo2) in the presence of na+ and cu2+ (yapi et al. 2014). the involvement of copper as a prosthetic group in ppo is essential for enzyme activity since the active site of the enzyme consists of two copper atoms each in the ligand field with three conserved histidine residues. copper-chelating reagents inactivate enzyme, while activity can be restored by the addition of excess copper (yoruk and marshall 2003). previously, strong inhibitory effect of ascorbic acid has been reported for ppo from sweet potato (manohan and wai 2012), cassava (wong and lee 2014), eggplant (sanni 2016), and dioscorea (sanni and fatoki 2017b). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:25 utc nova biotechnol chim (2019) 18(2): 102-114 113 table 3. effects of salts and ascorbic acid on activity of ppo from white and yellow sorghum. salt/additive effect on ppo activity [%] white sorghum yellow sorghum 10 mm 30 mm 50 mm 10 mm 30 mm 50 mm znso4 -39.65 -47.72 -61.35 9.90 14.99 -14.99 cuso4 34.08 38.58 54.53 29.99 34.93 44.98 feso4 -81.63 -83.65 -95.90 -41.75 -58.25 -83.25 nacl 0.00 69.44 20.43 100.00 250.00 41.75 kcl 59.22 65.34 70.50 25.00 41.75 125.00 ascorbic acid -51.01 -42.87 -46.91 -58.25 -25.00 -33.25 (-) inhibition, (+) activation. zero was taken as the baseline value for the control (ppo activity in the absence of salt or ascorbic acid). conclusions polyphenol oxidase (ppo) was successfully extracted from the grains of white and yellow varieties of sorghum (sorghum bicolor (l) moench). the results on the enzyme properties obtained in this study provide a good starting point for processing and potential use of sorghum grains for large scale production of edible foods, such as beverages (such as malt, pito/dolo, burukutu, kunu etc.), bread (such as kisra, injera, sourdough etc.), pharmaceutical excipient, and others. operating at the optimal parameters can inhibit browning activity of ppo and 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pretreatment method of beech and poplar wood and wheat straw in 2g biofuel production processing andrej pažitný1,2,, albert russ1, štefan boháček1, štefan šutý2 and vladimír ihnát3 1pulp and paper research institute, jsc, dúbravská cesta 14, bratislava sk841 04, slovak republic 2institute of polymer materials, faculty of chemical and food technology, slovak university of technology, radlinského 9, bratislava sk-812 37, slovak republic 3slovak forest products research institute, dúbravská cesta 14, bratislava sk-841 04, slovak republic article info article history: received: 29th april 2020 accepted: 1st june 2020 keywords: beech wood cellulose accessibility enzymatic hydrolysis poplar wood steam explosion pretreatment wheat straw abstract monosaccharides such as glucose, xylose and arabinose are the main monomer units of which cellulose and hemicelluloses are composed. the cellulose and hemicelluloses content in many biomass species makes them suitable for 2g bioethanol production. today, when 1g bioethanol production is closely monitored due to its enormous consumption of food raw materials such as wheat or corn grains, larger companies are gradually moving to pilot operations of 2g bioethanol production. however, cellulose and hemicelluloses contained in biomass are only very slightly accessible to enzymes used in 2g bioethanol production. therefore pretreatment methods such as steam explosion are very suitable to use for fractionation of cell structure. in this paper, we tested the cellulose accessibility. we compared the cellulose accessibility of wheat straw particles with wooden particles obtained from beech and poplar. particle size was less than 0.7 mm. we identified the optimal conditions of steam explosion pretreatment at reaction temperature of 200 °c for wheat straw, poplar and beech wood particles. the main indicator of accessibility was concentration of monomers obtained from enzymatic hydrolysis. the concentration of monomer was determined by high performance liquid chromatography. the experimental results showed different accessibility measure for each type of biomass species.  university of ss. cyril and methodius in trnava introduction hexose and pentose sugars are suitable for bioethanol production, however, they are predominantly chemical components of starch, cellulose and hemicelluloses. in general, lignocellulosic biomass rich in cellulose and hemicelluloses include raw materials with worse cellulose accessibility due to their recalcitrant nature and complex structure (stankovská et al. 2018). the composition of different lignocellulosic materials may vary considerably between each other (jørgensen et al. 2007; joshi et al. 2011; bertero et al. 2012; hazuchová et al. 2017; kucharska et al. 2018) and this fact is justified by the genetic variability and diversity among different sources (prasad et al. 2007; iqbal et al. 2013; shahzadi et al. 2014). some lignocellulosic materials are less suitable for 2g bioethanol production. the disadvantages of materials which are less suitable or unsuitable for 2g bioethanol production include low availability of plants or crops in nature, presence of waxes and resins, low cellulose content, high cellulose crystallinity, mailto:pazitny@vupc.sk nova biotechnol chim (2020) 19(1): 89-97 90 high lignin and ash content, structure of lignin, presence of acetylated hemicelluloses and lignincarbohydrate complexes, high degree of polymerisation, low pore volume and low specific surface area of cellulose (thian hong 2013). although specific pretreatment methods can cause favourable changes in lignocellulosic material structure, less suitable or unsuitable materials such as softwood (pine, spruce and fir) without using more energy-intensive thermo-hydromechanical fractionation are not useful for largescale 2g bioethanol production. the potentially suitable lignocellulosic materials for utilization in 2g bioethanol production include beech wood, poplar wood, and also wheat straw which is an agricultural waste and much of this waste remains unused. the reasons for selection of the raw materials are good geographic and time availability, high holocellulose content and low prices whereas these raw materials are mostly waste or redundant materials. however, cellulose and hemicelluloses are poorly accessible in these materials and pretreatment processes are needed to be applied to obtain significantly enhancement of cellulose accessibility for the following production processes. one of the industrially utilized processes, also used in 1g bioethanol production, is enzymatic hydrolysis (eh). this process is necessary to use as a control process for cellulose accessibility level determination since it gives information about hydrolysate compositions which can vary significantly depending on biomass species and enzymatic hydrolysis conditions (gigac et al. 2017; pažitný et al. 2019a). as published, enzymatic hydrolysis gives a relatively good response for cellulose accessibility when the compositions of original and pre-treated samples are compared and it is also a major financial item in bioethanol production process due to high enzyme cost (pažitný et al. 2019b). optimal pretreatment conditions for each raw material can result in structural recalcitrance reduction and enhanced cellulose accessibility (pažitný 2019). various suitable thermo-mechanical or thermohydro-mechanical pretreatment methods were published by several authors while thermochemical pretreatments tend to generate soluble sugars, degradation products of sugars and lignin, various other soluble components, and organic acids (larsson et al. 1999; weil et al. 2002; kumar and wyman 2008, 2009; stankovská et al. 2018; xiong et al. 2020). these methods are physicochemical pretreatment methods and they include steam explosion (stex), ammonia fiber explosion (afex) and liquid hot water (lhw) pretreatment. stex seems to be the most suitable and universal physicochemical pretreatment method because lhw and afex have limited efficiency in wood biomass processing (wyman et al. 2005). in case of afex, ammonia regeneration is required as well. thus, the most perspective thermo-hydro-mechanical pretreatment seems to be the steam explosion procedure and its continuous alternative steam extrusion with the use of a low alkali concentration or without the use of any chemicals (thian hong 2013; brezániová et al. 2016; gigac et al. 2017; lopez et al. 2019). steam explosion is a promising pretreatment method for disintegration of the compact structure of hardwood such as beech or poplar wood and wheat straw and thrifty to hydrolysable components (russ et al. 2016; olguin et al. 2018; pažitný et al. 2019a). also many grass species such as miscanthus x giganteus, panicum virgatum (switchgrass), phalaris arundinacea l. (reed canary grass) with high cellulose content belong to the plant species suitable for their conversion to 2g bioethanol (pažitný et al. 2013; pidlisnyuk et al. 2016). some authors also applied the mentioned pretreatment method to miscanthus x giganteus (menardo et al. 2013). miscanthus is a genus of about 20 species of perennial grasses native to eastern asia, northern india and africa (heaton et al. 2010). menardo's research team found that the intensity of pretreatment strongly affects the reduction of hemicelluloses at the level by about 15 % – 98 % between temperature ranging from 180 °c and 200 °c, however, the cellulose degradation is not affected by steam explosion pretreatment. exotic grass species such as miscanthus are cultivated solely for experimental purposes in slovakia but they may be a good starting point for experimental design of pretreatment processes. the aim of our work was to optimize the pretreatment method for three waste materials based on biomass available in slovakia. beech wood, poplar wood and wheat straw particles were nova biotechnol chim (2020) 19(1): 89-97 91 subjected to the steam explosion pretreatment at various temperatures and the obtained hydrolysates were determined by high performance liquid chromatography (hplc). the concentration of monosaccharides in hydrolysates showed the cellulose accessibility level for individual biomass materials. experimental lignocellulosic materials lignocellulosic materials as sources of holocellulose for the steam explosion pretreatment were beech wood (fagus sylvatica l.) obtained from bukóza píla jsc (hencovce, slovakia), poplar wood (populus alba l.) obtained from municipal waste deposited on fedinova street (bratislava, slovakia) and wheat straw of winter wheat (triticum aestivum l.) of variety evina grown in the bratislava self-governing region (senec, slovakia). mechanical pretreatment of lignocellulosic materials lignocellulosic materials were pre-treated mechanically by dry milling in brabender mill (brabender®, gmbh & co. kg, germany). a finer fraction was obtained by a bottom sieve and a 0.7 mm mesh screen was finally used for the laboratory testing. the pre-treated biomass particles were impregnated with fresh water so that the final moisture content of the samples during steam explosion was at least 85 % (w/w). water content in which the sample was soaked prior to steam explosion pretreatment was calculated to be 15 % (w/w) of the dry biomass. the samples were soaked in water at 20 °c for at least 30 min prior to steam explosion pretreatment. steam explosion pre-treatment the prepared materials were subjected to steam explosion at different temperatures (180 °c; 200 °c; 220 °c, respectively) with corresponding pressures (12.4 bar; 15.4 bar; 29.2 bar, respectively) and residence time of 10 minutes. before each pretreatment, a sample of particles (100 g o.d.) was impregnated in water (5 l) for 30 min. the pretreatment was carried out in a 2 l stainless steel batch reactor (amar equipments pvt. ltd., india), in which the impregnated particles were loaded at the top and heated to the required temperature. when the pre-set residence time concluded (10 min), the steam-treated biomass was released from reactor by rapid depressurization of steam explosion reactor vessel. after the steam explosion pretreatment, the pretreated materials were labelled as steam explosion pre-treated (stexp) samples and they were subjected to cellulose accessibility control by enzymatic hydrolysis in contrast with non-pretreated (np) materials as the original samples of biomass. cellulose accessibility control by enzymatic hydrolysis enzymatic hydrolysis of the non-pre-treated and pre-treated biomass particles with cellic ctec3 at a dose of 15 % (w/w) (g cellic ctec3 / 100 g cellulose) was carried out at 50 °c, ph = 5.0 for 72 hours and 12.5 % (w/w) of the samples. the ph was adjusted continuously during the process using 0.1 n sulphuric acid or 0.1 n sodium hydroxide. the hydrolysate samples were sampled after 24, 48 and 72 hours, respectively. determination of monosaccharides in hydrolysates actual presence and concentration of monosaccharides and inhibitors was determined using the procedure of national renewable energy laboratory (sluiter et al. 2008). monosaccharides (glucose, xylose and arabinose) and generated inhibitors (formic acid, acetic acid and residual inhibitors) were determined in hydrolysates by hplc method with rezex roa (organic acid) h+ column. the hplc system was supplied by chromservis sk (chromservis sk ltd., slovak republic). refractive index detector ri 101 shodex was a part of the hplc system. it was used for detecting and quantitative analysis of monosaccharides (glu, xyl and ara, respectively) and inhibitors. hydrolysates were injected into the hplc system through smartline nova biotechnol chim (2020) 19(1): 89-97 92 table 1. availability of stexp samples for enzymatic hydrolysis testing of cellulose accessibility. lignocellulosic material temperature [°c] 180 200 220 wheat straw available available available poplar wood unavailable available unavailable beech wood available available available universal mounting bracket for manual injection valves. each sample was analysed three times. the mobile phase passing through column was 0.005 n sulphuric acid at a flow of 0.5 ml.min-1 and temperature set to 30 °c. chromatography data processing was performed by the software clarity version 5.3.0.180 (dataapex ltd., czech republic) and concentration of individual monomer and inhibition products present in hydrolysates were calculated by external standards of monitored monomers and inhibitors. results and discussion hplc chromatograms of stexp samples of biomass the aim of this work was to study the chemical compositions of hydrolysates obtained after enzymatic hydrolysis of np and stexp samples of wheat straw, poplar and beech wood particles. the resulting monomers and their concentration in hydrolysates are affected strongly by the original chemical composition of biomass particles and the pretreatment temperature (russ et al. 2016). additionally, removing hemicelluloses at high temperatures can effectively increase cellulose digestibility (zhao et al. 2012) and enhanced cellulose accessibility for enzymes provides high concentration of monomers due to dramatic changes in biomass morphology after pretreatment aimed at enhancing the reactivity of biomass (podgorbunskikh et al. 2019). the resulting monomers – glucose (glu), xylose (xyl) and arabinose (ara) were determined using hplc combined with external standard methods. these methods are used to perform an accurate qualitative and quantitative analysis of monitored monomers and also formic acid, acetic acid and residual inhibitors in lignocellulosic hydrolysates (llano et al. 2017). according to several scientific papers (kačík et al. 2012; zhao et al. 2012; kidalová et al. 2015; demčák et al. 2017; demčák et al. 2019; ibrahim et al. 2020) it can be predicted that chemical structure of wheat straw, poplar and beech wood particles is mainly composed from cellulose, hemicelluloses and lignin. those monomers were generated just from holocellulose made of cellulose and all of the hemicelluloses during the action of enzymes. table 1 shows the available stexp samples pre-treated at different temperatures. the samples were subsequently hydrolyzed. the obtained hydrolysates had different concentration of monomers based on the specific chemical composition and porous structure of np and stexp samples of wheat straw, poplar and beech wood particles with various holocellulose contents (table 2). moreover, the base bulk density of raw materials mildly affects the glu and xyl yields of enzymatic hydrolysis and this physical property is different in case of the selected lignocellulosic materials (vasco-correa and shah 2019). the bulk density extent can be an advantage when considering transport and logistics of lignocellulosic material of higher density exhibiting a more compact structure. hplc analysis showed that the monomers in hydrolysates had a different elution time (table 3). the elution time did not depend table 2. the content of chemical components in selected lignocellulosic materials (aruss et al. 2016; bkačík et al. 2012; cibrahim et al. 2020). lignocellulosic material lignin [%] cellulose [%] holocellulose [%] awheat straw 17.25 46.50 73.10 bpoplar wood 17.49 – 19.04 43.44 – 45.07 80.61 – 81.89 cbeech wood 24.30 43.00 65.70 nova biotechnol chim (2020) 19(1): 89-97 93 on monomer concentration, however, it depended on set-up and conditions of hplc method. fig. 1 demonstrates three hplc chromatograms from hydrolyzates of stexp samples pre-treated at different temperatures (180 °c; 200 °c; 220 °c). the hydrolysates were sampled after 72 hours. all the chromatograms show the strong signal of glu monomer (elution time of 11.80 minutes) and the weaker signals of c5 sugars such as xyl (elution time of 12.60 minutes) and ara (elution time of 13.85 minutes). significant reduction of c5 sugars can be seen in case of beech and wheat straw hydrolysates obtained from stexp at a lower temperature. the lignocellulosic biomass decomposition mechanism during thermo-hydromechanical refining processes is known. the rate of this decomposition is higher for hemicelluloses, much lower for cellulose and the lowest for lignin (sandberg et al. 2013; pažitný 2019). this explanation can be projected into the fact that the lower concentrations of c5 sugars correspond to lower peaks with equal elution time and they belong to the pretreatments at higher temperatures. table 3. elution time of analysed sugar monomers in hydrolysates. analysed component elution time [min] glucose 11.80 ± 0.01 xylose 12.60 ± 0.02 arabinose 13.85 ± 0.02 production and determination of monosaccharides the average values for concentrations of monosaccharides determined by the hplc method depend on enzymatic hydrolysis (eh) time and also on the steam explosion temperature (fig. 2 – 4). the qualitative hplc method revealed the monomers of both cellulose and hemicelluloses. the hydrolysates from wheat straw (fig. 2), poplar particles (fig. 3) and beech particles (fig. 4) contained monomers glu and xyl. in addition of these monomers, however, the enzymatic hydrolysates obtained from poplar particles also contained monomer ara (fig. 3). various compositions of enzymatic hydrolysates and their fig. 1. hplc chromatograms from hydrolyzates after 72 h of enzymatic hydrolysis of stexp samples pre-treated at different temperatures (a – wheat straw particles, 180 °c, 200 °c, 220 °c; b – poplar particles, 200 °c; c – beech particles, 180 °c, 200 °c, 220 °c). a b c nova biotechnol chim (2020) 19(1): 89-97 94 0 10 20 30 40 50 60 70 80 90 100 c o n c e n tr a ti o n o f m o n o sa c c h a r id e s (g .l -1 ) eh 24 h eh 48 h eh 72 h without stex stex 180 °c stex 200 °c stex 220 °c individual monomers are caused by different representation of natural compounds. the highest total monosaccharide (glu+xyl) concentration of 91.3 g.l-1 was obtained after enzymatic hydrolysis of wheat straw particles pre-treated by steam explosion at the temperature of 220 °c (hydrolysis time of 72 hours, stex temperature of 220 °c, green column in fig. 2). slightly lower concentration of analysed monosaccharides was found in the enzymatic hydrolysate of poplar particles pre-treated by steam explosion at the temperature of 200 °c after enzymatic hydrolysis for 72 hours (90.0 g.l-1, fig. 3). generally, concentrations of monosaccharides were mildly higher in case of wheat straw due to higher cellulose content made accessible at higher temperature while hemicelluloses were decomposed. it is also relevant that original cellulose content in wheat straw is also mildly higher than in wood materials (table 2). production and determination of inhibitors the main inhibitor for each hydrolysed type of particles was acetic acid which acted as a slight hydrolysis inhibitor (yang et al. 2011). concentration of acetic acid ranged from 1.7 g.l-1 however, the correlation of concentrations related to pretreatment temperatures was not significant. high concentration of acetic acid causes the process inhibition but its specific concentration may be useful in specific pretreatment to coproduce xylooligosaccharides and fermentable sugars (lai et al. 2019). formic acid concentration was the highest in wheat straw hydrolysates (2.0 g.l-1), much lower concentration was in hydrolysates 0 10 20 30 40 50 60 70 80 90 100 c o n c e n tr a ti o n o f m o n o sa c c h a r id e s (g .l -1 ) eh 24 h eh 48 h eh 72 h stex 200 °c without stex fig. 2. influence of steam explosion (stex) temperature on concentrations of monosaccharides (glu; xyl; glu+xyl) in hydrolysates obtained during eh of wheat straw particles. fig. 3. influence of steam explosion (stex) at 200 °c on concentrations of monosaccharides (glu; xyl; ara; glu+xyl+ara) in hydrolysates obtained during eh of poplar particles. nova biotechnol chim (2020) 19(1): 89-97 95 0 10 20 30 40 50 60 70 80 90 c o n c e n tr a ti o n o f m o n o sa c c h a r id e s (g .l -1 ) eh 24 h eh 48 h eh 72 h without stex stex 180 °c stex 200 °c stex 220 °c of poplar particles (0.0 g.l-1 – 1.2 g.l-1) and the lowest concentration in hydrolysates of beech particles (0.0 g.l-1 – 0.3 g.l-1). hplc method also identified other inhibitors such as 5-hydroxymethylfurfural and furfural, however, their concentration was not significant. of all the inhibitors, formic acid is the strongest inhibitor to cellulases due to complete inactivation of enzymes (cantarella et al. 2004; panagiotou and olsson 2007; yang et al. 2011). formic acid concentration generating in enzymatic hydrolysis affects selection of suitable raw material for 2g bioethanol production. formic acid had low concentration in all samples taken from mixtures during enzymatic hydrolysis. according to our results wheat straw is a less suitable raw material than wooden materials, especially when pre-treated at high temperatures as formic acid concentration mildly increases with steam explosion temperature. 2g bioethanol production from selected biomass species the obtained results showed that wheat straw, poplar and beech particles are biomass resources suitable for 2g bioethanol production. however wheat straw is a more seasonal biomass and vice versa mentioned wooden materials are better storable due to lower biodegradability when compared to wheat straw. combination of all studied biomass resources and their mixing before thermo-hydro-mechanical pretreatment in 2g bioethanol production require further research. however, 2g bioethanol based on the studied biomass resources is not only suitable for use in biofuels but also as the main component in disinfection materials inactivating viruses including coronaviruses and other pathogens (zhao and gao 2020). conclusions pretreatments of selected raw materials based on different temperature showed a significant impact of steam explosion temperature. in two biomass pretreatment cases – wheat straw and beech particles that were pre-treated at three different temperatures increase of concentration of monosaccharides was found with enhancing temperature. the highest total concentration of detected monosaccharides (91.3 g.l-1) was obtained by enzymatic hydrolysis of wheat straw particles pre-treated by steam explosion at the temperature of 220 °c and slightly lower in case of hydrolysed poplar particles pre-treated by steam explosion at the temperature of 200 °c (90.0 g.l-1). both hydrolysates were sampled after hydrolysis time of 72 hours. it is important to be careful when using wheat straw as a raw material for 2g bioethanol production due to higher concentration of formic acid generated at higher temperature of steam explosion pretreatment (2.0 g.l-1) because this compound is the strongest inhibitor of cellulases by complete inactivation of enzymes. based on the obtained results it may be considerably useful to combine the selected raw fig. 4. i influence of steam explosion (stex) temperature on concentrations of monosaccharides (glu; xyl; glu+xyl) in hydrolysates obtained during eh of beech particles. nova biotechnol chim (2020) 19(1): 89-97 96 materials before thermo-hydro-mechanical pretreatment in 2g bioethanol production. the starting point for pretreatment of selected biomass resources appears to be the optimum steam explosion temperature at 200 °c. acknowledgement this work was supported by the slovak research and development agency under the contract no. apvv-18-0240. conflict of interest the authors declare that they have no conflict of interest. references bertero m, puente gdl, sedran u (2012) fuels from biooils: bio-oil production from different residual sources, characterization and thermal conditioning. fuel 95: 263271. brezániová z, fišerová m, stankovská m, gigac j, opálená e, puškelová j (2016) effect of 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surgery. br. j. oral. maxillofac. surg. 58: 250-253. zhao x, zhang l, liu d (2012) biomass recalcitrance. part i: the chemical compositions and physical structures affecting the enzymatic hydrolysis of lignocellulose. biofuels, bioprod. bioref. 6: 465-482. nova biotechnol chim (2018) 17(1): 66-73 doi: 10.2478/nbec-2018-0007  corresponding author: akmal84a@gmail.com nova biotechnologica et chimica chemical composition and biological activity of seed oil of amaranth varieties soyibjon s. bozorov, nodir sh. berdiev, uchkun j. ishimov, shukhratjon s. olimjonov, jamolitdin f. ziyavitdinov, akmal m. asrorov and shavkat i. salikhov institute of bioorganic chemistry of academy of sciences of uzbekistan, laboratory of proteins and peptides chemistry, 83 m.ulughbek str., 100125, tashkent, uzbekistan article info article history: received: 4th may2018 accepted: 1st august 2018 keywords: amaranth oil fatty acids refractometric detection reversed-phase hplc squalene triglycerides abstract the work is devoted to study of seed oil composition of amaranth varieties: kharkov, lera, andijan and helios, acclimatized in uzbekistan. we demonstrated the possibility of using reversed-phase hplc using a refractometric detector, which allows simultaneous determination of squalene and triacylglycerides in plant seeds and determining the authenticity of amaranth oils. established seed oiliness ranged from 6.39 to 7.81 % of the initial mass. amaranth oil samples contained quite large amount of unsaturated fatty acids 72.72 – 73.28 %, 1.17 % of which is omega-3alpha-linolenic acid. the squalene content in the seeds ranged from 0.35 % to 0.55 %. it was established that the squalene content in oils obtained by extraction is greater than the one obtained by cold pressing. in the triacylglyceride composition of the investigated cold-pressed and extracted oils, no significant differences were found.  university of ss. cyril and methodius in trnava introduction amaranth oil is a rich source of unsaturated fatty acids and thus is widely used as antioxidant means. antioxidative and related properties of amaranth oil are often linked with squalene, which is acyclic polyunsaturated hydrocarbon of triterpene nature (c30h50) containing 12 double bonds. in a purified form, squalene is a colourless, almost tasteless, transparent liquid with no significant odour. the name squalene came from the latin squalus a shark, the liver of which is a rich source of this compound. in addition to shark liver, squalene is found in olive and amaranth oils, as well as in oils from wheat germ and rice bran (bondioli et al. 1993; makeev et al. 1999; jeleznov et al. 2009). squalene is an intermediate in biosynthesis of cholesterol, although 10 % of it is converted to cholesterol (govind rao and acaya 1968). besides, squalene is a part of the secretion mass of sebaceous glands of human skin (up to 12 – 14 %), due to which it is easily absorbed and penetrates into the body. moreover, it accelerates the penetration of substances dissolved in it. in human body, squalene is synthesized as a result of complex biochemical reactions leading to the formation of vital substances, such as coenzyme q10, cholesterol, bile acids, vitamin d, sex and other steroid hormones. when the formation processes of these biologically active substances are violated, metabolic disorders leading to the development of atherosclerosis, cardiovascular and other diseases are in progress (berg et al. 2002; kuntz and kuntz 2008). in recent literature, recommendations on the use of squalene in medicine, cosmetology, gerantology bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:26 utc nova biotechnol chim (2018) 17(1): 66-73 67 have widely been presented (kamimura et al. 1992; chernenko et al. 1997; tokhtaboev 1999; chernenko and glushenkova 1998; abdurakhimov and makhmudov 2014). squalene hasa unique ability to saturate cells with oxygen. allegedly, this is due to the fact that squalene as an unsaturated compound is unstable, and for the saturation of double bonds and transition to a more stable states it needs 12 hydrogen atoms, the best source of which are water molecules. authors consider that squalene easily reacts with water, resulting in the formation of molecular oxygen, which saturates all cells (jeleznov et al. 2009). most squalene is contained in amaranth seed oil (up to 8 %), which makes it possible to recommend the plant to produce squalene as another alternative to shark liver. squalene accumulates in olive oil, in rice bran, in germs of wheat seeds during their germination and its significance for the plant metabolism is not clear yet. it is assumed that the seeds of these plants are deficient in oxygen upon germination, and squalene, as an antihypoxic compound, performs a protective function. like other antioxidant triterpenes, squalene is involved in antioxidative processes. well-known antioxidants, such as vitamins e and a, beta-carotene are terpenes, or contain isoprene groups characteristic for terpenes (huang et al. 2009). the ability of amaranth seeds, rich in squalene, to maintain high germination for dozens of years, possibly, is due to its extraordinary antioxidant properties. oil of most wild plants and cultivars are among very valuable medicines used for a long time in medical practice. amaranth oil which is obtained from the plant seeds has a very special place among organic vegetable oils. its oil content reaches 6.3 – 8.0%, which is 1.5-times greater than the oil content of rice and wheat seeds (jeleznov et al. 2009). the dark-orange coloured liquid has specific taste, without bitterness. the fatty acids composition of amaranth oil obtained from the seeds of various species differed slightly. the sum of saturated fatty acids is 24.58 – 24.84 % with dominance of palmitic acid (20.34 – 20.68 %). linoleic (43.0 – 43.21 %) and oleic acids (22.57 – 22.39 %) are predominant among unsaturated fatty acids which makes up 66.75 – 66.85 % of oil. seeds of some varieties contain elaidic acid (1.07 – 1.15 %), a trans-isomer of oleic acid, which is very rare in nature. in addition, amaranth oil contains phospholipids (up to 10 %), tocopherols (2 %) and phytosterols (up to 2 %) that possess medicinal effects on the animal and human organism (govind rao and acaya 1968). scientists of uzbekistan have been conducting research on the adaptation of various varieties of plants of the genus amaranthus in different climatic zones of uzbekistan and obtaining cold pressed oils from amaranth seeds over recent years (chernenko et al. 1997; chernenko and glushenkova 1998; tokhtaboev 1999; abdurakhimov and maxmudov 2014). the aim of the work is investigating the fatty acid and triacylglyceride composition and determining the quantity of squalene in amaranth seeds acclimatized in uzbekistan. the seeds of amaranth varieties amaranthus hypochondriacus – kharkov, lera and amaranthus cruentus – andijan and helios, grown in andijan region, were used in this work. experimental isolation of squalene from seeds the sample of the seeds (0.20 – 0.35 g) was ground in a porcelain mortar with 0.2 – 0.3 g of quartz sand sieved through a hole with diameter of 0.25 mm to the visual homogeneity of the powder. the resulting powder was transferred to a 10 ml flask and insisted in acetone by periodical stirring for 30 min. after settling the main part of the solid phase, the solution was filtered through a tfe-fg-3 teflon filter in a filter cartridge and chromatographed in 2 μl portions. determination of triacylglyceride composition and quantity of squalene in amaranth oil crude oil from the seeds of amaranth was extracted by two methods: cold pressing and extracting with organic solvents in soxhlet apparatus with subsequent removal of the solvent and drying. in order to determine triacylglycerides, 100 μl of the testing oil samples were dissolved in 900 μl of absolute acetone. the resulting solutions were bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:26 utc nova biotechnol chim (2018) 17(1): 66-73 68 centrifuged at 6,000 rpm. the clear supernatant is chromatographed by 2 μl in hplc. chromatography a liquid chromatograph agilent technologies 1260 with a refractometer was used. chromatographic column 4.6 x 250 mm, agilent eclipse xdb c18, 5 μm was included. for the preparation of mobile phases acetone (imp., reahim) and acetonitrile (sigma) were used. chromatograms were registered and processed using the open lab program (agilent technologies). mobile phase: 25 ml of acetonitrile is added to a 250 ml volumetric flask and the solution was brought to the mark with acetone, stirring. feed rate: 1 ml/min., to calibrate the response of the detector, squalene solutions were prepared in the eluent in concentrations of 0.075 – 0.200 mg/ml. squalene (97 %, alfa aestar, lancaster) was used as a standard sample. obtained solutions were chromatographed, as well as the test samples. gc-ms chromatography hp 9010 column (50 m capillary) was used with a stationary phase based on peg. chromatography conditions were: injector: 180 °c; detector 250 °c; the program for the column thermostat was: the beginning of 100 °c (0.5 min), by 10 °c in a minute until 230 °c, delay at 230 °c for 5 min. helium at the rate of 1.7 ml/min was used as carrier gas. parameters of the massselective detector were: scan mode from 40 to 1,000 amu; ionization by electron impact 70 eb. the used mass spectral libraries were the database/w9n11.l and database/rtlpest3.l. fatty acids composition investigation on the fatty acid composition of amaranth oil was carried in accordance with state standards 30418-96 (p 51483-99 2017). the method was based on conversion of fatty acids’ triglycerides into methyl (ethyl) ester of fatty acids with subsequent gc-analysis. the method is applicable in the range of 0.1 – 100 % of mass portions of fatty acids. for the conversion of triglycerides of fatty acids into their methyl esters, 0.2 ml of each tested oils sample is dissolved in 2 ml of diethyl ether. then 0.1 ml of 10 % koh solution in methanol is added to the samples and incubated at 100 °c for 3 min. further 0.2 ml of hexane and 1 ml of water were gradually added to the samples. after phases were separated, organic phase was analysed in a volume of 2 μl in gc-ms. antiatherosclerotic property experimental studies on antiatherosclerotic effect of amaranth oil were carried on 40 white mongrel male rats with an initial body weight of 250 – 300 g. a tween model of hyperlipidemia was used by intraperitoneal administration of a single detergent tween-80 at a dose of 200 mg/kg. animals under hyperlipidemia treatment and prophylactic regime were administered orally 5 ml/kg of amaranth oil and an aqueous solution of latitude in a dose of 200 mg/kg once a day. the duration of the experiment was 5 days. on the sixth day, blood was taken from the iliac artery, the material was frozen until the day of the biochemical analysis. the following lipid profile indices were studied: total lipids, triglycerides, lowdensity lipoproteins, very low-density lipoproteins, atherogenic coefficient. to assess the antioxidant properties, the level of lipid peroxidation products in plasma was measured. determination of malonedialdehyde malonedialdehyde levels in samples were determined by using molar extinction coefficient (ishimov et al. 2016). table 1. oiliness and squalene percentage of amaranth seeds (m±m; n = 5). cultivar kharkov lera andijan helios oiliness [%] 7.81±0.17 7.55±0.23 6.39±0.06 7.68±0.16 squalene [%] 0.55 ±0.02 0.48±0.03 0.35±0.02 0.44±0.02 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:26 utc nova biotechnol chim (2018) 17(1): 66-73 69 fig. 1. hplc analysis of amaranth oil, determination of quantity of squalene and triacylglyceride compositions (the numbers and the names of the identified substances are given in table 2). ± means indicate relative standard deviation. results and discussion the oiliness of the investigated amaranth varieties was in the range of 6.39 – 7.81 % of the initial mass of the seeds. the highest oil content was determined in the variety kharkov (7.81±0.17 %), and the lowest is in andijan (6.39±0.06 %). the oil contents of lera and helios varieties were 7.55±0.23 % and 7.68±0.16 %, respectively (table 1). exact methods for the quantification of squalene in amaranth seeds have been developed and processed in products (sulpice and ferezou 1984; brunner et al. 1991; lehmann 1996; grieveson et al. 1997; hagiwara et al. 1998; bruni 2001; spanggord et al. 2002; spanggord et al. 2006; grigoriadoua et al. 2007). in this work we used simultaneous quantitative determination of squalene and triacylglycerides in one cycle of chromatographic separation. the advantage of the method is simplicity of sample preparation and availability of chemical reagents. the essence of the proposed method is separation table 2. squalene content and triacylglyceride composition of amaranth seed oils (rsd≤4 %; n=5). amaranthus hypochondriacus amaranthus cruentus kharkov lera andijan helios c/p ext. c/p ext. c/p ext. c/p ext. squalene [%] 6.79 7.12 6.05 6.87 5.29 5.88 5.53 6.25 triacyl-glycerides quantity [molar %] l3 4.96 4.37 5.90 5.47 7.22 7.68 5.42 5.88 l 2о 12.98 11.85 13.60 12.90 12.69 12.15 11.94 13.06 l 2p 15.68 13.87 16.00 15.61 15.26 15.39 13.34 15.15 lps 0.35 0.09 0.13 0.11 0.17 0.15 0.12 0.09 lо2 11.63 12.88 10.79 11.93 11.62 11.36 12.09 11.77 l2с+ lоp 15.58 19.09 16.60 19.54 18.12 17.72 18.12 16.10 lp2 4.60 7.61 5.10 8.45 7.91 7.96 7.35 4.72 о3 8.09 8.98 7.47 6.05 6.91 7.50 8.16 9.95 lоs 11.00 10.86 12.66 9.49 11.20 9.74 11.62 13.17 о2p 3.99 4.07 5.30 4.12 4.48 4.45 5.08 5.57 о2s 3.99 1.70 1.83 2.03 1.85 2.21 1.71 1.64 оp2 2.76 3.28 3.77 3.08 2.07 2.73 2.90 2.16 оps 0.38 1.34 1.37 1.23 0.50 0.95 2.15 0.74 designations: c/p – cold pressed oil; ext. – extracted oil; l – linoleic acid; o – oleic acid; p – palmitic acid; s – stearic acid; where lp2 stands for two acyls of linoleic acid and one acyl of palmitic acid. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:26 utc nova biotechnol chim (2018) 17(1): 66-73 70 of amaranth oil components in a chromatographic column by polarity and their registration by refractometric detector. a typical chromatogram of amaranth oil, obtained from kharkov variety, is shown in fig. 1 and corresponding results for the oiliness and squalene content are given in table 1. the seed oiliness degree of these four amaranth varieties, ranging from 6.39 to 7.81 % (table 1), can be considered as high oil content. the highest oiliness degree among 104 amaranth genotypes has been established to be 8.7 %, whereas the lowest compiled 1.9 % (he and corke 2003). our results revealed that the content of squalene in the seeds of the amaranth varieties ranged from 0.35 to 0.55 % of the initial mass of the seeds or 5.48 – 7.81 % of the oil (table 1). the higher content of squalene, like oil content, was determined in kharkov variety (0.55±0.02 %), and the lowest in andijan (0.35±0.02 %). there were no significant differences in varieties lera and helios with squalene contents 0.48±0.03 % and 0.44±0.02 %, respectively. in comparison to squalene content in amaranth oil of up to 104 genotypes (1.04 to 7.3 %), seed oils of these four amaranth varieties can be expected as a rich squalene source (he and corke 2003). hplc results, obtained by the analysis of the contents of squalene and triacylglycerides in amaranth seed oils, are represented in table 2. the content of squalene in the cold pressed seed oils of the investigated amaranth varieties was in the range from 5.29 to 6.79 %. the highest percentage was found in varieties kharkov (6.79 %) and lera (6.05 %). in andijan (5.29 %) and helios (5.53 %) the squalene content was much lower. these data are also confirmed in experiments to determine the amount of squalene in extracted oils: kharkov – 7.12 %, lera – 6.87 %, andijan – 5.88 % and helios – 6.25 %. besides, obtained results indicate insignificant differences among triacylglyceride compositions in cold pressed and extracted oils of the investigated amaranth oils (table 2). the sum of saturated fatty acids made up 26.72 – 27.28 % with the dominance of palmitic acid from 22.6 % to 23.19 %; unsaturated fatty acids compiled 72.72 – 73.28 % with a predominance of linoleic acids (40.16 – 47.31 %) and oleic acids (24.67 – 31.31 %). three high intensity peaks in fig. 2 demonstrate palmitic, oleic and linoleic acids. in the oils of the studied amaranth varieties, omega-3-alpha-linolenic acid was found in the range of 1.17% – 1.65 %. earlier has been established a positive correlation between amaranth seed oil and oleic acid contents (hlinková et al. 2013). similar results were found in our work since kharkov and helios varieties had higher oiliness degree and oleic acid content. besides, the negative correlation between oleic acid and linoleic acid quantities corresponds to results in 10 amaranth samples of a. cruentus and a. hypochondriacus (hlinková et al. 2013). molar ratios of predominant unsaturated oleic (c 18:1) and linoleic (c 18:2) acids correspond to previous results in 5 amaranthus accessions fig. 2. gc – chromatogram of fatty acids of amaranth oil. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:26 utc nova biotechnol chim (2018) 17(1): 66-73 71 table 3. chemical compositions of fatty acids of amaranth oil of investigated varieties (n = 5). ± means indicate relative standard deviation representing two species (oleic acid compiled 22.8 – 31.5 % and linoleic acid 39.4 – 49.1 %, respectively) (table 3). the results obtained with the predominant saturated palmitic acid differed from 21.4 to 22.2 %, which correspond to previous studies (jahaniaval et al. 2000). comparative review of fatty acid compositions of amaranthus species demonstrate that palmitic (c 16:0), oleic (c 18:1) and linoleic (c 18:2) acids have been established as predominant in several studies (gamel et al. 2006; venskutonis and kraujalis 2013). in our work the molar ratio of saturated and unsaturated fatty acids was established ~ 27:73 among the four amaranth seed oils. comparable results (23.7:76.3 and 22:78, respectively) were demonstrated in a. hypochondriacus and a. cruentus (el gendy et al. 2018). high percentage of squalene and unsaturated fatty acids in amaranth oil makes it possible for use in medicine. therefore, we studied the effects of amaranth oil on antioxidative system in rats. coronary heart disease and atherosclerosis are on the first places by morbidity and mortality in economically developed countries. the role of diet therapy in the prevention and treatment of atherosclerosis is significant. from this point of view, modifying the fat composition of the diet, in particular reducing the content of saturated fatty acids and increasing unsaturated fatty acids, exerts a pronounced influence. analyses on the antiatherosclerotic effect of oil from seeds of amaranth plants (helios variety), acclimatized in uzbekistan, on the tween model of hyperlipidemia was carried. the results showed, that tween model in rats was accompanied by a number of metabolic changes (table 4), among which significant changes in the plasma lipoprotein spectrum: the relative content of fractions of plasma lipoproteins of very low density increased by 1.9-times and low density lipoproteins 2.6-times. there was a decreasing level of high-density lipoproteins, that 1.9-times lower degree was observed in experimental animals as tween was introduced. total cholesterol values increased by 68 % and triglycerides by 77 %. all these changes led to more than 5-times greater coefficient of atherogenicity, increasing from 0.793±0.12 (outcome) to 4.47±0.30 (control). thus, feeding with amaranth oil (helios variety) significantly reduced the concentration of atherogenic lipid profile of the blood plasma in animals, in particular the total cholesterol for 27.0 %, triglycerides 9 % and low-density lipoproteins 38 %. high-density lipoproteins in basal conditions are considered as antiinflammatory factor, capable of destroying oxidized lipids forming an inflammatory response. amaranth oil authentically increased the level of anti-atherogenic high-density lipoproteins cholesterol by 64 % compared to control. all these changes led to decrease in the coefficient of atherogenicity, respectively 2.4and 1.9-times, which dropped from 4.47±0.30 (control) to 1.9±0.14. simultaneously, by processes of increasing lipid peroxidation and as a result of interaction with the antioxidative system, a significant decrease in antioxidant activity occurs. results show, in rats under the tween model, activation of lipo-oxidation processes occurred comparing to indicators in intact animals. thus, the concentration of malonedialdehyde increased by 51 %, and conjugated dienes by 26 %. as intensity of lipid peroxidation lowered, antioxidant and catalase activities degrees decreased by 39 %. these results correspond to studies on patients, which demonstrated dosedependent reduction of high-density lipoproteins, reversely proportional to squalene concentration, compiled 3.33 % in amaranth oil composition. while the reductions of low-density lipoproteins were fluctuating, the concentration of very lowdensity lipoproteins increased (martirosyan et al. 2007). on the 5th day of the treatment with quantity [molar %] [±0.05 %] fatty acids kharkov lera andijan helios с 14 : 0 myristic 0.27 0.28 0.27 0.24 с 16 : 0 palmitic 23.19 22.60 22.27 22.80 с 18 : 0 stearic 3.16 3.57 3.59 2.99 с 18 : 1 oleic 31.31 24.67 24.52 28.80 с 18 : 2 linoleic 40.16 47.01 47.31 42.83 с 18 : 3 linolenic 1.23 1.17 1.26 1.65 с 20 : 0 arachidic 0.66 0.69 0.78 0.69 saturated 27.28 27.14 26.91 26.72 unsaturated 72.72 72.86 73.00 73.28 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:26 utc nova biotechnol chim (2018) 17(1): 66-73 72 table 4. effects of amaranth oil of helios variety on lipids’ profile of rats under hyperlipidemia tween model (м±m; n = 10). groups, doses total cholesterol [mg/100 ml] triglycerides [mg/100 ml] high-density lipoproteins [mg/100 ml] very low-density lipoproteins. [mg/100 ml] low-density lipoproteins [mg/100 ml] atherogenicity coefficient intact animal 52.0±0.3 60.0±0.6 31.0±0.5 11.0±0.3 12.0±0.4 0.793±0.12 control (hlp+н2о) 87.5±0.3 106.0±0.4 16.0±0.5 21.2±0.5 31.5±0.3 4.470±0.30 hlp+amaranth oil, 5 ml/kg 64.0±0.3* 80.0±0.6* 26.7±0.5* 17.5±0.3* 19.5±0.4* 1.900±0.14* *hlp – hyperlipidemia condition amaranth oil, a significant reduction in the level of peroxidation was observed; since the mda level decreased aby 20 % (from 4.03±0.25 μm/ml.min. to 3.25±0.19 μm/ml. simultaneously, conjugated dienes decreased by 19 % (from 7.17±0.18 units to 5.80±0.16). previously, decreased levels of mda were also observed in patients with heart diseases that consumed 6 – 12 g of amaranth oil possessing 3.33 % squalene (martirosyan et al. 2007). obtained results enable us to conclude that the amaranth oil have a pronounced atherogenic effect, and can be applied in the form of biologically active food additives. conclusions in this work we studied chemical compositions of amaranth seed oil, acclimatized in uzbekistan and its oiliness degree. we established that amaranth oil contains amounts of unsaturated fatty acids and squalene comparable with amaranth oils obtained in other regions. our results showed that its biological activity resemble lowering the level of lipoproteins rats in agreement with previous reports. thus, seed oil of acclimatized amaranth variety studied in this work can be considered as a source of biologically active substances. a detailed study of the biochemical composition of amaranth seeds of various species and products of their processing, in order to obtain biologically active substances and food additives, can serve as basis for further research. acknowledgments the work was supported by innovative project of academy of sciences of uzbekistan i-2016-5-16/3 introduction of technology for complex processing of amaranth plants to produce feed for livestock, and also oil cake and flour for the needs of the pharmaceutical food industry. we express our sincere thanks to researchers of andijan state university for providing with amaranth seeds of the investigated varieties and cold-pressed oils. references abdurakhimov sa, makhmudov ra (2014) analyzing and technical regulation of vegetable oils extracted by extraction method according to international standards. scientific and technical journal "standard" of uzstandart agency. 2: 28-30. berg jm, tymoczko jl, stryer l. 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99-100. chernenko tv, khodzhaeva ma, glushenkova ai, turakhozhaev mt (1997) composition of the lipids and carbohydrates of the seeds of amaranthus caudatus. chem. nat. sci. 33: 624-626. el gendy ang, tavarini s, conte giuseppe, pistelli l, hendawy sf, omer ea, angelini lg (2018) yield and qualitative characterisation of seeds of amaranthus hypochondriacus l. and amaranthus cruentus l. grown in central italy. ital. j. agron. 13: 63-73. gamel th, mesallam as, damir aa, shekib la, linssen jp (2007) characterization of amaranth seed oils. j. food lipids 14: 323-334. he hp, corke h (2003) oil and squalene in amaranthus grain and leaf. j. agric. food chem. 51: 7913-7920. ishimov uj, abdullayeva gt, ziyavitdinov jf, asrorov am, ergashev na, asrarov mi (2016) the effects of isolated fractions of red pepper capsicum annuum l. on the mitochondrial permeability transition pore and lipid peroxidation. j. microbiol. biotech. food sci. 5: 259-262. jahaniaval f, kakuda y, marcone mf (2000) fatty acid 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and prospects for their use. 1999. moscow (russia), pushchino, 100-103. martirosyan dm, miroshnichenko la, kulakova sn, pogojeva av, zoloedov vi (2007) amaranth oil application for coronary heart disease and hypertension. lipids health dis. 6: 1-12. spanggord rj, sun m, lim p, ellis wy (2006) enhancement of an analytical method for the determination of squalene in anthrax vaccine adsorbed formulations. j. pharm. biomed. anal. 42: 494-499. spanggord rj, wu b, sun m, lim p, ellis wj (2002) development and application of an analytical method for the determination of squalene in formulations of anthrax vaccine adsorbed. j. pharm. biomed. anal. 29: 183-193. state standards of russian federation p 51483-99 (2017) vegetable oils and animal fats. determination by gas chromatography of constituent contents of methyl esters of total fatty acid content, 763 p. sulpice jc, ferezou j (1984) squalene isolation by hplc and quantitative comparison by hplc and glc. lipids 19: 631-635. tokhtaboev nx (1999) prospects for complex processing of amaranth plant (amaranthus hybridus l.) as a food source. problems of flora and fauna of fergana valley and their rational use. “regional conference” materials. 1999. andijan (uzbekistan), 132. venskutonis pr, kraujalis p (2013) nutritional components of amaranth seeds and vegetables: a review on composition, properties, and uses. compr. rev. food sci. f. 12: 381-412. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:26 utc https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=venskutonis%2c+petras+r https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=kraujalis%2c+paulius nova biotechnol chim (2017) 16(1): 1-11 doi: 10.1515/nbec-2017-0001  corresponding author: jamrichova.daniela@gmail.com, lenka.tisakova@ucm.sk nova biotechnologica et chimica how to approach heterogeneous protein expression for biotechnological use: an overview daniela jamrichová, lenka tišáková, veronika jarábková and andrej godány department of biology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic article info article history: received: 8th june 2017 accepted: 26th june 2017 keywords: escherichia coli gene expression protein purification heterologous abstract production of recombinant proteins in escherichia coli expression systems has shown many advantages, as well as disadvantages, especially for biotechnological and other down-stream applications. the choice of an appropriate vector depends on the gene, to be cloned for purification procedures and other analyses. selection of a suitable production strain plays an important role in the preparation of recombinant proteins. the main criteria for the selection of the host organism are the properties of the recombinant produced protein, its subsequent use and the total amount desired. the most common problems in eukaryotic gene expression and recombinant proteins purification are, for instance, post-translational modifications, formation of disulphide bonds, or inclusion bodies. obtaining a purified protein is a key step enabling further characterization of its role in the biological system. moreover, methods of protein purification have been developed in parallel with the discovery of proteins and the need for their studies and applications. after protein purification, and also between the individual purification steps, it is necessary to test protein stability under different conditions over time. shortly, all the essential points have been briefly discussed, which could be encountered during production and purification of a recombinant protein of interest, especially from eukaryotic source and expressed heterogeneously in prokaryotic production system.  university of ss. cyril and methodius in trnava introduction over years, the production of recombinant proteins in the escherichia coli expression system has shown many advantages, as well as disadvantages. there is the evidence of such recombinant protein evolution reflecting in a wide range of commercially available production escherichia coli strains with modified properties for the heterogeneous production of eukaryotic proteins under various conditions. the major advantage of an expression system with e. coli as a host organism is time, technical and financial effectivity. e. coli cells can be propagated under common laboratory conditions in a simple culture medium with selected ions, salts and carbon sources. in the exponential phase, its generation time is 20 min, in a complex culture medium containing vitamins, salts, microelements, amino acids and glucose, at an intensive passage at 37 °c (sezonov et al. 2007). it is doubtless that the production of recombinant proteins in various microbial systems (especially those of eukaryotic origin), has revolutionized the whole biochemical and biotechnological overview of the basic approach how to obtain high yield of protein bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:22 utc mailto:jamrichova.daniela@gmail.com nova biotechnol chim (2017) 16(1): 1-11 2 in a decent purity. many scientific studies focused on application output of proteins have encountered obstacles with heterogeneous expression of eukaryotic genes in prokaryotic systems, because of miscellaneous reasons. this paper could serve as brief overview that pays attention to crucial steps and potential hinders in production of eukaryotic recombinant proteins in heterogeneous e. coli, methods of isolation, purification and detection of the (overexpressed) recombinant proteins, especially (fig. 1). gene design when planning production of recombinant protein, it is inevitable to take into consideration several steps in preparation of the gene cloned. principally, before starting the experimental part, it is important to apply bioinformatics approaches to evaluate the usability/suitability of the gene of interest for the planned downstream procedures. various in silico analyses could be conducted by online programs and tools, such as restriction digestion analysis of the gene, primer design for amplification of the gene, primer melting temperature calculation, verification the open reading frames (orfs). it should be emphasized that e. coli codon usage evaluation is one of the key factors for heterogeneous expression, so it must be performed prior to gene design. nowadays, several commercial companies provide codon usage optimization as one of their bonus services along with gene synthesis in a selected type of vector. in the case of using a synthetic gene for recombinant protein production, much time could be saved in cloning procedures and the real starting point in the laboratory is the expression part. last but not least, when thinking about fusion protein preparation, it is good to evaluate, whether to design a (synthetic) gene with or without a stop codon. finally, hasty decision while designing a gene, cloning the gene or directly ordering a synthetic one, could rapidly decrease the rate of getting relevant results and increase the sources needed to reassess or repeat the procedures. fig. 1. overview of the steps (in blocks) for heterogeneous protein production with all the essential steps involved – obstacles during protein production (bars above blocks) and their consequences (bars below the corresponding blocks). vector selection the choice of an appropriate vector for successful down-stream procedures depends on the gene to be cloned. all expression vectors must contain three important signal sequences including promoter, terminator and ribosome binding site (rbs). currently, the most common expression vectors are designed as a combination of replicon, promoter, rbs, selection marker, multicloning site (mcs), affinity tag to facilitate purification, and sometimes a fusion protein to increase protein solubility. there is a wide range of commercially available expression vectors, so the selection of a suitable bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:22 utc nova biotechnol chim (2017) 16(1): 1-11 3 vector for a specific gene of interest to be cloned must be not only complex, but also substantial. therefore, attention should be paid to the needs of the (individual) gene to be cloned and expressed, when searching for the most suitable expression vector. the promoter is a cardinal part of the expression vector, as a promotor sequence regulates the initial stage of gene expression, rna polymerase attachment, and controls the rate of mrna synthesis. there are a number of vectors on the market with different types of promoters, while the most commonly used promoter types are listed in table 1. escherichia coli expression systems for cloning under laboratory conditions and in industrial scale-up production technologies, non-pathogenic e. coli strains have become the starting point for the developing new strains with features optimized for gene cloning or recombinant protein expression and production. the fundamental task in the heterologous protein production in bacterial expression systems is the correct folding of the produced protein. since the e. coli production strains lack the systems necessary to handle more complex eukaryotic proteins or to further modify them (e.g. glycosylation), special strains have been developed, e.g. origami that supports the formation of disulphide bridges in the cytoplasm (derman et al. 1993) or rosetta (novagen, usa) with modified trna codes providing easier expression of eukaryotic genes in the prokaryotic systems (guillaume et al. 2006; nakamura et al. 2008). selection of a suitable production strain plays an important role even for following downstream procedures. the main criteria for the selection of the host organism are the properties of the recombinant protein produced, its subsequent use and the total amount. inappropriate choice of the production strain may cause the recombinant protein to be inactive, insoluble, degraded, or can only be produced in limited amounts. therefore, e. coli strains have been developed that lack nucleases and proteases, to prevent degradation of the recombinant dna and the produced protein. a variety of e. coli strains with various desired properties have been developed by mutations of specific genes (baneyx 1999; yokoyama 2003; klock et al. 2005; terpe 2006). the most common e. coli strains include bl21 (derived from the b strain) and derivatives thereof. bl21 strains are non-pathogenic, capable of growth in simple culture media, allowing higher gene expression under the t7 bacteriophage promoter integrated into the bacterial chromosome of the bacterium (studier and moffatt 1986). nowadays, there are many biotechnologically and therapeutically used proteins produced in e. coli cells (chrastilová et al. 2007). selection of a host strain when using e. coli as a production organism for expression of recombinant proteins, some drawbacks could be encountered, directly derived from its inherent properties, especially due to its prokaryotic nature. there are some typical and commonly occurring problems in expressing recombinant genes and producing recombinant proteins. post-translational modifications protein post-translational modifications (phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis) increase the functional diversity of the proteome by the covalent addition of functional groups or proteins, proteolytic cleavage of regulatory subunits or degradation of entire proteins. these modifications generally influence almost all aspects of normal cell biology and pathogenesis, therefore, identifying and understanding ptms it is critical to identify and understand them, when focusing on gene expression and protein production. there are no posttranslational modifications occurring during the protein production in e. coli, such as n-glycosylation, c-glycosylation, acetylation, phosphorylation, which might be indispensable for proper functions of eukaryotic proteins (stevens 2000; yin et al. 2007). this problem can be addressed by using evolutionary higher systems, for example, yeasts, or mammalian cells. from another point of view, lack of post-translational modifications can also be bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:22 utc nova biotechnol chim (2017) 16(1): 1-11 4 beneficial, when protein production in nonglycosylated form is more suitable for subsequent applications, for instance, protein crystallisation. formation of disulphide bonds to ensure catalysis of protein disulphide bonds (dsb) formation, there have been evolved elaborate dsb systems in escherichia coli (inaba 2009). disulphide bonds are important for protein structure and functionality but are only formed in the periplasm under catalysis of the dsb system (andersen et al. 1997; bardwell 1994). therefore, if disulphide bond formation is required, the recombinant protein should be directed to the periplasmic space by means of a cleavable table 1. digest of the most commonly used promoters in e. coli expression vectors. signal peptide (e.g. pelb) (chung et al. 1998; le-calvez et al. 1996). such a signal is used by w3110 production strains as well as by some vectors, for example the pet26b (makrides 1996; matthey et al. 1999). the lower abundance of proteins in the periplasm facilitates the recombinant protein purification (choi and lee 2004). however, only small yields of the final proteins are usually obtained in the periplasm, which is marked as the major drawback of this type of production. a more convenient approach is the use of specific, commercially available strains, for example origami strain (novagen). these strains involve mutated genes encoding for thioredoxine reductase (trxb) and glutathione reductase (gor) in the dsb system, allowing cytoplasmic cysteine reduction by thioredoxin and glutathione redoxine (bessette et al. 1999). if further formation of disulphide bonds is needed, the gene for thioredoxin can by fused with the gene of interest and subsequently expressed in the trxb / gor e. coli strain (lavallie et al. 1993). if the protein does not get the correct tertiary structure (folding), it is usually insoluble and creates a so-called inclusion bodies within the bacterial host (chatterjee and esposito 2006). formation of inclusion bodies proteins in inclusion bodies are inactive, aggregated and insoluble. they usually require denaturation and subsequent renaturation, promotor type features references lac inducer: iptg. lac promoters and their lacuv5 derivatives are weak promoters therefore not often used for recombinant proteins production. müller-hill et al. (1968), calos (1978), deuschle et al. (1986), makoff and oxer (1991) trp inducer: indolyl-3-acrylic acid. it is used to produce majority of protein. hallewell and emtage (1980), tacon et al. (1983), yansura (1990) tac inducer: iptg. a hybrid type of promoter prepared by joining (-35) trp and (-10) lac promoter regions. tac is 10 fold stronger than lacuv5 promoter. de boer et al. (1983) λpl inducer: heat shock. a strong type of promotor with wide use in industrial biotechnology at higher fermentation temperatures. menart et al. (2003), dodd et al. (2005), valdezcruz et al. (2010) t7 inducer: lactose or iptg. basal expression is controlled by laciq as well as coexpression of t7 lysozyme. moffatt and studier (1987), baneyx (1999), graumann and permstaller (2006), stano and patel (2004) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:22 utc nova biotechnol chim (2017) 16(1): 1-11 5 re-folding, which complicates protein production and reduces yields (graslund et al. 2008). on the other hand, if there is no problem with protein renaturation, the inclusion bodies can be easily separated by centrifugation since they contain only small amounts of contaminants, and ultimately, the higher purity of the recombinant protein (baneyx and mujacic 2004; cabrita and bottomley 2004). formation of inclusion bodies can be suppressed by slowing expression of the (trans)gene. this can be achieved in different ways e.g. by reducing the cultivation temperature, using a weaker promoter, application of lower concentrations of inducer. slowing down the formation of the protein increases the chance for its proper folding. presence of lipopolysaccharides another obstacle in the production of proteins in e. coli may be the presence of lipopolysaccharides which constitute a part of the cell wall of gram-negative bacteria. these lipopolysaccharides have pyrogenic effects and cause complications in the purification of end products. therefore, de-pyrogenation is needed (terpe 2006; yin et al. 2007), despite the reduced final yield of the pure protein and possible adverse effect on protein stability and native conformation (petsch and anspach 2000). toxicity of the recombinant proteins toxic (recombinant) proteins cause death of the host cells or complication during their cultivation. since the toxicity for the given expression strain can be specific, the strain can be replaced by a more compatible one for protein production. another option is the use of a highly regulated expression system, where the expression of the gene of interest is regulated by an inducible promoter and transcription terminator. controlled protein production can result from keeping the plasmid copy number low, while protein toxicity can by modified by engineering the recombinant protein sequence (saida 2007). nucleotide sequence of the gene of a foreign origin the eukaryotic gene cloned may contain introns. since e. coli lacks the necessary splicing mechanism for eukaryotic mrna, production of active protein poses a serious problem in e. coli. this can be overcome by using complementary dna (cdna) prepared from mature mrna or using a synthetic gene (le hir et al. 2003). purification of recombinant proteins obtaining a purified protein from expression systems is often a key step enabling its characterization and clarification of its role in biological systems. purification of recombinant proteins represents a series of procedures that lead to the obtaining of a single protein species from complex bacterial lysates. methods of protein purification have been developed in parallel with the discovery of proteins and the need for their further study. until the beginning of the 20th century, filtration, precipitation and crystallization were mainly used to isolate proteins. the breakthrough occurred in 1903 when cvet separated plant pigments in a calcium carbonate column and subsequently, in 1906, introduced the concept of chromatography for this method. nearly two decades later, in 1924, svedberg proved that proteins can be separated by analytical ultracentrifugation (philo 1997). in the course of several years, other methods of protein separation were discovered, namely electrophoresis, affinity chromatography (ac) and ion exchange chromatography. since the mid20th century, there has been an intensive development of various types of chromatographic matrices. initially, starch or modified dextran (sephadex) were only used to allow the separation of proteins by size (porath and flodin 1959). to obtain a protein of even a higher purity, other matrices such as polyacrylamide (raymond and weintraub 1959), methyl acrylate (curtain 1963) or agarose (hjrtén 1964) have been developed that have several advantages over the starch gels like higher stability or easier handling. on the other hand, some disadvantages could emerge, e.g. toxicity of the acrylamide, which comes from a nature of this substance. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:22 utc nova biotechnol chim (2017) 16(1): 1-11 6 protein and peptide tags to facilitate detection and purification of the recombinant protein, protein or peptide tags (labels) have been implemented. these tags represent exogenous sequences with high affinities to specific biological or chemical ligands (bornhorst and falke 2000; arnau 2006a). affinity labelling allows for simple detection of proteins with low immunogenicity or no antibodies available (brizzard 2008). protein tags have been proven very suitable in protein purification where the labelled protein can be purified directly from the cell lysate or supernatant (lichty 2005). affinity tags may be located at the n or c-terminus of the recombinant protein (justesen et al. 2009), for some specific applications it is possible to place the tag even on both ends. the use of tags can improve the biochemical properties and increase the yield of the produced protein, simplify renaturation conditions, increase the solubility of the produced protein, or prevent its proteolysis. however, the use of affinity tags may also have adverse impacts on labelled proteins since, for example, can alter the conformation of the labelled protein, inhibit enzyme activity, table 2. the most commonly used stabilizing agents for protein storage. class examples value / usage buffers phosphate buffer, tris-hcl, hepes, hepes – naoh, etc. stability salts kcl, nacl, (nh4)2so4 stability ionic detergents sodium deoxycholate, sodium cholate, ctab, sds stability and increase protein solubility non-ionic detergents triton x-100, nonidet p-40, tween 20, brij 35, etc. cryoprotectants glycerol, saccharose storage and stability chelating agents citric acid, salicylic acid, edta, egta stability reducing agents β-mercaptoethanol (bme), dithiotreitol (dtt), tris(2-carboxyethyl)phosphine (tcep) stability, prevents formation of disulphide bonds ligands, cofactors mg2+, atp phosphate stability protease inhibitors pmsf (phenylmethylsulfonyl fluoride), tpck (tosyl phenylalanyl chloromethyl ketone), tlck (tosyl-l-lysyl-chloromethane hydrochloride) prevents protein degradation cause toxicity, alter biological function and reduce the protein yield (arnau 2006b). therefore, when choosing an affinity tag, several inevitable points have to be considered to achieve the desired biochemical features of the protein of interest. these include the bacterial production strain used, the conditions of purification, the desired purity and the final amount of protein. chromatographic methods for protein purification presently, various types of liquid chromatography are used to purify recombinant proteins. these differ in the principle of separation of the purified protein and include affinity-, ion exchange-, gel permeation-, or hydrophobic chromatography. due to the large number of available methods bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:22 utc nova biotechnol chim (2017) 16(1): 1-11 7 and various chromatographic matrices it is necessary to select the purification method prior to the construction of the recombinant molecule. purification methods based on protein-peptide fusion or on a fusion protein with high affinity for a particular ligand enable using a simple affinity chromatography. other protein purification methods involve various interactions, such as antibody–antigen, enzyme–substrate, or protein– metal ion. to facilitate purification, modern expression vectors contain specific sequences enabling better detection and/or subsequent purification of expressed proteins, or polypeptide sequences that affect protein solubility. the flag was the first tag and represents a hydrophilic sequence of eight amino acids (dykddddk) recognized by a monoclonal antibody. this antibody allows detection of the fusion protein and its purification by immobilization on a suitable chromatographic matrix (hopp et al. 1988). currently a histidine tag (his-tag, his6-tag) consisting of 6-12 residues is routinely used for protein purification (janknecht et al. 1991; lichty 2005). the high-affinity binding of the imidazole group of histidine to a transition metal like ni2+, co2+, cu2+, fe2+ or zn2+is the basis of a method known as immobilized metal affinity chromatography (imac) that has become one of the basic purification procedures for the isolation of recombinant proteins. the metallic ions are immobilized on an agarose or sepharose matrix by chelation with imidodiacetic acid (ida), nitrilotriacetic acid (nta) or tris (carboxymethyl) ethylenediamine (ted) (porath et al. 1975; hochuli et al. 1987). such a matrix is used for purification of his-tagged proteins under both native and denaturation conditions to yield up to ten mg of protein per 1 ml of a matrix. the protein can be eluted from the matrix with buffer containing imidazole (usually 150– 300 mm), edta (eluting with metal ions) or by lowering the buffer ph to values in the range 2.5–7.5. the procedure itself and selection of the most suitable metal ion should be optimized separately for each individual protein based on its physicochemical properties (bolanos-garcia and davies 2006). noteworthy, washing out the metal from the matrix can cause insufficient purity of the protein. this can be avoided using a so called pentadentate (ted) matrix, which binds the metal ions very tightly and thus prevents their leaching, leaving only one free spot for the protein to bind. if affinity chromatography fails to provide the product of the desired purity, other chromatographic methods should be used in subsequent steps, e.g. ion exchange chromatography that separates the molecules by their charge. the stationary phase consists of matrices from dextran derivatives (sephadex) or agarose (sepharose) with alkaline functional groups – anionic exchangers, or with acidic functional groups – cationic exchangers. ion exchange chromatography has been frequently used not only for protein purification but also for its concentrating. another option for protein purification is a gel permeation chromatography method, also called a molecular sieve, which separates proteins based on their molecular size and also could be used for desalination of protein samples. monitoring the concentration and purity of proteins determination of the purity of a recombinant protein is of key importance for its subsequent use. however, a direct method for quantification of the purity of the protein sample is missing. the available procedures always start assessing the amount of individual types of impurities such as of nucleic acids, carbohydrates, lipids, isoenzymes, inactive enzymes. for this operation, the proper type of chemical/physical method should be chosen. the protein purity value is dependent on the type of test method, as well as its sensitivity (detection limit). therefore, several factors are taken into account when deciding for a particular approach, in particular, the amount of protein available (yield and concentration), the nature of the impurities, and the type of solution, in which the protein is re-suspended. the number of analytical methods for monitoring the purity of proteins include colorimetry, electrophoresis, mass spectrometry, dynamic light scattering and enzyme analysis (rhodes et al. 2009). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:22 utc nova biotechnol chim (2017) 16(1): 1-11 8 storage of purified proteins in terms of maintaining the protein quality required for further procedures, storage of purified proteins is crucial. storage conditions depend on the type of protein, therefore there is no general rule and procedure available. importantly, protein samples should not be exposed to extreme changes in ph or temperature, and other factors that could potentially lead to protein denaturation. long-term storage of the protein solution at room temperature also requires the use of antibacterial and antimycotic agents to avoid biological contamination and destruction of the protein sample. during and after protein purification it is necessary to test the stability of the protein under different conditions, simplest on small aliquots of samples, e.g. at room temperature, at 4–6 °c, at lower temperatures –20 °c, and for its enzymatic activity and concentration over time. several storage conditions may have an adverse effect on protein stability and activity. an illustrative example is storage at –20°c in 50 % glycerol, which ensures protein stability, however, when followed by purification the glycerol has to be removed from the sample (e.g. by dialysis) to avoid dropping the protein amount and activity. protein storage is enhanced by rapid freezing without the use of a cryoprotectants (such as glycerol, ethylene glycol, sucrose), however in this case protein freezing and defrosting can cause protein denaturation, aggregation and precipitation (cao et al. 2003). proteins can be unstable and lose their biological activity as a result of various physical and chemical procedures, too, even at low temperatures (carpenter et al. 2002; simpson 2010). therefore, when choosing the proper protein storage conditions, it is important to consider what the purified protein will be used for (determination of specific enzymatic activity, protein crystallization, and interaction studies etc.). protein stabilizers or serum albumins (e.g. bsa) can be added to the protein to extend the shelf life. the presence of albumin in a sample is not an obstacle when the protein activity is tested in the following steps, but can interfere in structural studies. in contrast, the addition of substances that prevent from the growth of microorganisms is recommended, as they reduce the freezing point, or preserve the protein in the solution. when adding such substances, it is necessary to perform protein stability studies over time. it should be mentioned that there has been no universal stabilizing solution developed for storage of all the various protein samples yet. it is also necessary to be cautious about optimal conditions for the specific protein sample that can vary during storage (tab. 2) since storage may alter intermolecular interactions. further, optimal protein storage conditions may not be necessarily the same as the conditions for determining the protein activity. moreover, additives used to ensure protein stability might act as inhibitors of enzymes. that is the reason why interfering substances should be taken into consideration and must be removed or sufficiently diluted before the use of the protein. currently the market offer comprises many commercial kits with a wide variety of specific solutions to test protein and storage stability under different conditions. conclusions in this paper, all the steps and factors have been summarized and briefly discussed which could be encountered during production and purification of a recombinant protein of interest, especially from eukaryotic source, and expressed heterogeneously in prokaryotic production system. it is inevitable to mention that despite the simplicity and overall effectiveness of e. coli production system in combination with various commercially available expression vectors, the final amount and purity of a recombinant protein is insufficient and, many times, contaminated, insoluble or inactive. in many cases, when a soluble protein is inactive or not crystallized after all the separation and purification steps, there is an urgent need to 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what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for forein genes. j. biotechnol. 127: 335-347. yokoyama s (2003) protein expression systems for structural genomics and proteomics. curr. opin. chem. biol. 7: 39-43. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 27.02.20 14:22 utc nova biotechnol chim (2020) 19(1): 52-60 doi 10.36547/nbc.v19i1.577  corresponding author: jose.isagani.janairo@dlsu.edu.ph nova biotechnologica et chimica a sequence-dependent classification algorithm for crohn’s disease – causing nod2 protein mutations jose isagani b. janairo1, and marianne linley l. sy-janairo2 1biology department, de la salle university, 2401 taft avenue, manila 0922, philippines 2institute of digestive and liver diseases, st. luke’s medical center – global city, rizal drive, taguig 1634, philippines article info article history: received: 10th december 2019 accepted: 3rd march 2020 keywords: artificial neural networks inflammatory bowel disease machine learning personalized medicine abbreviations: ann artificial neural network cd crohn’s disease dcm disease-causing mutation nod2 nucleotide-binding oligomerization domain-containing protein 2 npv negative predictive value ppv positive predictive value rf random forest svm support vector machine abstract certain nod2 protein mutations have been associated with the onset of the inflammatory bowel disease, crohn’s disease (cd). nod2 is involved in the inflammatory response of the gut to the microbial community, wherein its functional impairment through mutations may lead to cd progression. considering the significant role that nod2 plays in cd pathogenesis, predicting whether a specific type of nod2 mutation is the cause of cd can greatly aid the accuracy of the disease diagnosis. hence, a novel sequence-based classification algorithm built on artificial neural network (ann) is herein presented that can predict whether a specific nod2 mutation can cause cd or not. the nod2 mutant types and their association with cd were taken from literature, and the calculated sequence-order coupling numbers were used as the classification predictors. the formulated ann classifier exhibited satisfactory predictive ability, with 82.4 % accuracy, 62.5 % sensitivity, 100 % specificity, 100 % positive predictive value, and 75 % negative predictive value. the presented ann classifier provides a proof-of-concept that predicting the onset of cd from nod2 protein variant is possible.  university of ss. cyril and methodius in trnava introduction crohn’s disease (cd) is characterized by chronic transmural inflammation of the gastrointestinal tract. pathogenesis of cd is multifactorial, wherein one of the key drivers of the disease involves mutations in the nod2 protein (yamamoto and ma 2009). nod2 is encoded by the card15 gene in the human chromosome 16 (strober and watanabe 2011). this protein plays a critical role in microbe / pathogen sensing, wherein the leucine-rich region of nod2 binds to the muramyl dipeptide (mdp) of the bacterial cell wall. once activated by mdp, nod2 initiates downstream signaling events relevant to the host immune response. thus, nod2 mutations may lead to the impaired regulation and response of the host to bacterial interactions, which increases the risk to unusual ileal inflammation (sidiq et al. 2016). various nod2 mutations have been associated with cd susceptibility, wherein the missense mutations and frameshift mutation appear to be the most common type of mutations associated with cd progression (cuthbert et al. 2002; hampe et al. 2002; economou et al. 2004). other less frequent mutations are also linked with cd pathogenesis, while other nod2 mutations do not lead to cd (lesage et al. 2002). clearly, possible connections between the properties of nod2 protein mutants and cd susceptibility exist, but remain to be uncovered. thus, this study aims to use machine learning to formulate a predictive model that can classify nod2 mutations as disease-causing or non-disease-causing based on mailto:jose.isagani.janairo@dlsu.edu.ph nova biotechnol chim (2020) 19(1): 52-60 53 protein numerical representations. artificial neural network (ann) is a powerful machine learning technique that can uncover non-obvious patterns or associations from datasets of various characteristics. ann has been widely used in medical diagnosis, particularly in cancer classification and prediction (khan et al. 2001), tuberculosis (er et al. 2010), among others. having the ability to predict whether a specific nod2 mutation maybe associated with cd can greatly improve disease detection and therapy. in addition, this ability becomes even more valuable after considering that nod2 mutation type influences the response of the patient towards a particular treatment (niess et al. 2012). early detection of the disease is one of the main challenges in inflammatory bowel disease, such as cd (flamant and roblin 2018). thus, such predictive model can potentially help improve disease diagnosis, as well as lay the groundwork for the greater adoption of personalized medicine for the management of cd. experimental data mining nod2 protein disease-causing mutants (dcm), and non-disease-causing mutants (ndcm) were taken from (lesage et al. 2002). the list contains 30 dcms, and 13 non-dcms, both which are mostly point mutations. additional nod2 mutant variants were taken from clinvar (https://www.ncbi.nlm.nih.gov/clinvar/), a database that shows the relationship between genetic variants and phenotypes (landrum et al. 2014). the archive was searched using the search string [nod2 and (("crohn disease") and (benign or pathogenic))]. the search yielded 76 results, but after removing duplications, silent mutations, and inconclusive medical assignment for each variant, 16 nod2 mutants were added to the dataset. the resulting 59 nod2 protein variants (table 1) were then numerically represented using the 30 sequence-order coupling number (socn) based from schneider-wrede descriptors (schneider and wrede 1994), calculated using protrweb (xiao et al. 2015). this web server for calculating protein descriptors require the protein sequence as the input. the canonical sequence of human nod2 was taken from www.uniprot.org (uniprot id: q9hc29), which also served as the basis of the mutant sequences. the functional impact of the mutations on the nod2 protein was also assessed using the provean protein tools as implemented in the provean web server version 1.1.3 (choi et al. 2012) (http://provean.jcvi.org/seq_submit.php). the input in the provean web server is the wild type protein sequence, the position of the mutation, and the amino acid substitution. the utilized classification threshold was the default value of -2.5. from the provided information, the web server will then determine if the submitted mutation for analysis is either deleterious or neutral. the full dataset, which contains the 59 nod variants and the corresponding 30 socns is available in the supporting information. statistical analysis all statistical analyses were carried out using tibco statistica version 13.4.0.14. statistical difference was probed between dcm and ndcm nod2 protein variants through anova using the 30 socn. the same descriptor set was also utilized to segregate the 43 nod2 protein variants through a two-cluster solution using k-means clustering. after the exploratory data analysis, the dataset was then used to create various machine learning classification models. dcm / ndcm served as the categorical response variable, and the calculated sequence-order coupling numbers served as the continuous descriptors. for the artificial neural network (ann) based classification model, a feed-forward multilayer perceptron architecture was adopted, wherein sum of squares was the error function, the hidden unit activation function used was tanh, and identity was the output unit. bootstrap subsampling was employed, wherein 10 subsamples were gathered in which 50 % was dedicated for training, 30 % for testing, and 20 % for validation. for support vector machine (svm) classification, the radial basis function (rbf) kernel was utilized, leading to the automatic selection of the best gamma and c parameters. the gamma and c parameters are involved in the definition of the hyperplanes which leads to the separation and classification of the cases. 75 % https://www.ncbi.nlm.nih.gov/clinvar/ http://www.uniprot.org/ http://provean.jcvi.org/seq_submit.php nova biotechnol chim (2020) 19(1): 52-60 54 table 1. association of nod2 mutations with cd progression as reported in lesage et al. (2002) and information from the clinvar dat. dcm refers to disease-causing mutation, while ndcm means non-disease-causing mutation. nod2 variant association with cd reference provean score functional impact of mutation based on provean prediction r138q dcm lesage et al. 2002 -2.120 neutral a140t dcm lesage et al. 2002 -0.464 neutral w157r dcm lesage et al. 2002 1.142 neutral t189m dcm lesage et al. 2002 -0.494 neutral r235c dcm lesage et al. 2002 -2.779 deleterious l248r dcm lesage et al. 2002 -4.286 deleterious p268s ndcm lesage et al. 2002 -0.614 neutral n289s dcm lesage et al. 2002 -2.417 neutral d291n dcm lesage et al. 2002 -2.097 neutral t294s ndcm lesage et al. 2002 -3.033 deleterious a301v ndcm lesage et al. 2002 -3.631 deleterious r311w dcm lesage et al. 2002 -4.602 deleterious l348v ndcm lesage et al. 2002 -2.291 neutral h352r ndcm lesage et al. 2002 -4.166 deleterious r373c dcm lesage et al. 2002 -2.984 deleterious n414s dcm lesage et al. 2002 -1.796 neutral s431l dcm lesage et al. 2002 -2.000 neutral a432v ndcm lesage et al. 2002 -1.104 neutral e441k dcm lesage et al. 2002 0.090 neutral 558dellg dcm lesage et al. 2002 -11.499 deleterious a612t dcm lesage et al. 2002 -3.608 deleterious a612v ndcm lesage et al. 2002 -3.645 deleterious r684w dcm lesage et al. 2002 -3.092 deleterious r702w dcm lesage et al. 2002 -3.285 deleterious r703c dcm lesage et al. 2002 -3.313 deleterious r713c dcm lesage et al. 2002 -2.838 deleterious a725g ndcm lesage et al. 2002 -1.275 neutral a755v ndcm lesage et al. 2002 -3.070 deleterious a758v ndcm lesage et al. 2002 -0.953 neutral e778k dcm lesage et al. 2002 -2.579 deleterious v793m dcm lesage et al. 2002 -0.804 neutral e843k dcm lesage et al. 2002 0.482 neutral n853s dcm lesage et al. 2002 -4.637 deleterious m863v dcm lesage et al. 2002 -0.070 neutral a885t dcm lesage et al. 2002 -1.407 neutral g908r dcm lesage et al. 2002 -5.822 deleterious a918d dcm lesage et al. 2002 -4.932 deleterious g924d dcm lesage et al. 2002 0.149 neutral v955i ndcm lesage et al. 2002 -0.435 neutral v972i ndcm lesage et al. 2002 -0.633 neutral g978e dcm lesage et al. 2002 -1.646 neutral 1007fs dcm lesage et al. 2002 n.d. n.d. a292v ndcm clinvar id 319441 -2.391 neutral a612s ndcm clinvar id 319452 -2.850 deleterious a849v ndcm clinvar id 97855 -3.122 deleterious d154n ndcm clinvar id 319426 -0.914 neutral g1032s ndcm clinvar id 319475 0.737 neutral l682f ndcm clinvar id 319457 -3.467 deleterious q902k ndcm clinvar id 319471 -1.023 neutral r391h ndcm clinvar id 319442 -0.414 neutral r471c ndcm clinvar id 319446 -2.087 neutral r708h ndcm clinvar id 319459 -1.413 neutral r716h ndcm clinvar id 319460 -2.092 neutral r791q ndcm clinvar id 97850 -0.251 neutral t245m ndcm clinvar id 319434 -0.886 neutral v92i ndcm clinvar id 319425 -0.275 neutral v162i ndcm clinvar id 319427 0.486 neutral v955i ndcm clinvar id 97869 -0.435 neutral nova biotechnol chim (2020) 19(1): 52-60 55 of the data set was used for training, while the remaining 25 % served as the test set, and the results were validated by applying a 10-fold cross validation. for random forest classification, the random test data proportion was set to 0.30, and 0.5 for the subsample proportion. the stopping parameters were set as follows: minimum n cases =5, maximum n cases = 10, minimum n child in node = 5, maximum n of nodes = 100. for the boosted trees regression, the learning rate was set to 0.1, with the following conditions: number of additive terms = 200, random test data proportion = 0.30, subsample proportion = 0.4. the stopping parameters were set as follows: minimum n of cases = 5, maximum n of levels = 10, minimum n in child node = 1, maximum n of nodes = 3. the predictive performances of the constructed models were evaluated using the diagnostic indices of accuracy, sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv). these indices are calculated as follows eq. 1 – 5 (trevethan 2017): (1) (2) (3) (4) (5) the values used for the calculation were taken from the results of the validation sets of the ann-based classification models, and test sets for the svm and tree-based classification models. results variations between dcm and ndcm nod2 mutants based on the location, and nature of the mutations were observed. most of the dcms were located at the leucine-rich region (lrr) of nod2, while ndcms occurred mostly fig. 1. frequency of mutations based on the domain location within the nod2 protein. blue columns represent diseasecausing mutation and gray columns represent non-diseasecausing mutations. designation of mutation type is based on lesage et al. 2002, and clinvar. at the non-domain part of the protein (fig. 1). for the nature of the mutations, conservative mutations accounted for 71 % of the ndcms and only 3 % for the dcms. most ndcms involved mutations to an aliphatic residue, while the dcms exhibited a scattered type of mutations (fig. 2). fig. 2. frequency of mutations based on the nature of the mutant amino acid. blue columns represent disease-causing mutation and gray columns represent non-disease-causing mutations. designation of mutation type is based on lesage et al. 2002, and clinvar. table 2. confusion matrix for the comparison between the pathogenicity and functional impact of the nod2 mutations. deleterious neutral dcm 14 15 ndcm 8 20 nova biotechnol chim (2020) 19(1): 52-60 56 table 3. model architecture and performance of artificial neural network – based classification algorithms. model descriptor / selection basis network structure algorithm prediction accuracy diagnostic performance a 1.30 multilayer perceptron bfgs 6 training = 70.6 % sensitivity = 40 % full model input layer: 30 testing = 52.9 % specificity = 17 % hidden layer: 22 validation = 27.3 % ppv = 28.6 % output layer: 2 npv = 25 % b 1.10 multilayer perceptron bfgs 4 training = 47.1 % sensitivity = 60 % segmented input layer: 10 testing = 58.8 % specificity = 67 % hidden layer: 8 validation = 63.6 % ppv = 60 % output layer: 2 npv = 66.7 % c 11.20 multilayer perceptron bfgs 12 training = 77.8 % sensitivity = 50 % segmented input layer: 10 testing = 52.9 % specificity = 60 % hidden layer: 8 validation = 54.5 % ppv = 60 % output layer: 2 npv = 50 % d 21-30 multilayer perceptron bfgs 16 training = 80 % sensitivity = 62.5 % segmented input layer: 10 testing = 82.4 % specificity = 100 % hidden layer: 8 validation = 45.5 % ppv = 100 % output layer: 2 npv = 75 % the functional impact of the mutations on nod2 was also assessed using provean. as seen in table 2, a deleterious mutation weakly associates with nod2 pathogenicity since it only accounts for 48 % of disease-causing mutations. this is in contrast to mutations with neutral functional impact, which account for 71 % of nondisease-causing mutations. the difference between dcm and ndcm nod2 mutants based on the 30 socn was initially probed table 4. model architecture and performance of svm – based classification algorithms. model descriptor / selection basis prediction accuracy diagnostic performance gamma capacity support vectors e 1.30 training = 52.3 % sensitivity = 0 % 0.033 1.000 44 full model test = 46.7 % specificity = 0 % (44 bounded) overall = 50.5 % ppv = 0 % validation = 40.9 % npv = 0 % f 1.10 training = 52.3 % sensitivity = 0 % 0.100 1.000 44 segmented test = 46.7 % specificity = 0 % (44 bounded) overall = 50.5 % ppv = 0 % validation = 40.9 % npv = 0 % g 11.20 training = 52.3 % sensitivity = 0 % 0.100 1.000 44 segmented test = 46.7 % specificity = 0 % (44 bounded) overall = 50.5 % ppv = 0 % validation = 40.9 % npv = 0 % h segmented test = 46.7 % specificity = 0 % (44 bounded) overall = 50.5 % ppv = 0 % validation = 40.9 % npv = 0 % nova biotechnol chim (2020) 19(1): 52-60 57 table 5. model architecture and performance of random forest – based classification algorithms. model descriptor / selection basis prediction accuracy diagnostic performance i 1.30 training = 85 % sensitivity = 37.5 % full model test = 58 % specificity = 73 % overall = 76 % ppv = 50 % npv = 61.5 % j 1.10 training = 65.7 % sensitivity = 50 % segmented test = 62.5 % specificity = 71 % overall = 64.4 % ppv = 55.6 % npv = 66.7 % k 11.20 training = 77.1 % sensitivity = 30 % segmented test = 41.7 % specificity = 50 % overall = 62.7 % ppv = 30 % npv = 50 % l 21-30 training = 74.2 % sensitivity = 30 % segmented test = 41.7 % specificity = 50 % overall = 62.7 % ppv = 30 % npv = 50 % through one way-anova and k-means clustering. as anticipated, anova yielded a non-significant difference (p > 0.05) between the two classes of nod2 mutants, owing to the small variations in their respective socns. a similar case was observed for the two-cluster solution created through k-means clustering. the first cluster only contained the truncated nod2, the 1007fs, while the second cluster contained the remaining 42 nod variants. evidently, these two statistical methods were unable to discriminate dcm from ndcm nod2 variants based on the 30 socns. several binary classification models using the socns as the predictors were then formulated utilizing various machine learning algorithms, which include ann (table 3), svm (table 4), random forest (table 5), and boosted trees (table 6). the ann-based classification algorithm and boosted trees yielded better predictive models compared with those produced using svm and random forest. optimization of the selection of the descriptors was done to improve the performance of the classification models, and was conducted systematically through the systematic segmentation of the 30 socns. comparing tables 3-6, models d, m, and p yielded the reliable classification models as demonstrated by its satisfactory prediction accuracy for the training, test, and validation sets. however, model d was deemed as the best predictive model since it required only 10 predictors, and it exhibited a good balance in accuracy and diagnostic performance. the diagnostic indices of sensitivity, specificity, ppv, and npv were used to further probe the performance of the constructed models. thus, the overall performance of model d is satisfactory, based on the algorithm accuracy, which indicates how often the classifier is correct. moreover, the sensitivity and specificity of the model are also satisfactory. sensitivity demonstrates the ability of the model for positive classification, while specificity for negative identification (wong and lim 2011). ppv and npv demonstrate how many were indeed true positives and true negatives from the ratings given by the classifier (trevethan 2017). discussion understanding the relationship between nod2 mutant type, and cd susceptibility is of paramount importance, considering the pivotal role of nod2 in cd pathogenesis. however, such connection remains to be fully understood. as what has been previously demonstrated from a study of 612 nova biotechnol chim (2020) 19(1): 52-60 58 table 6. model architecture and performance of boosted trees – based classification algorithms. model descriptor / selection basis prediction accuracy diagnostic performance m 1.30 training = 90.2 % sensitivity = 88.9 % full model test = 72.2% specificity = 56 % overall = 84.7 % ppv = 66.7 % npv = 83.3 % n 1.10 training = 73.2 % sensitivity = 55.6 % segmented test = 66.7 % specificity = 78 % overall = 71.2 % ppv = 71.4 % npv = 63.6 % o 11.20 training = 95.1 % sensitivity = 55.6 % segmented test = 55.6 % specificity = 56 % overall = 83.1 % ppv = 55.6 % npv = 55.6 % p 21-30 training = 82.9 % sensitivity = 77.8 % segmented test = 77.8 % specificity = 78 % overall = 81.4 % ppv = 77.8 % npv = 77.8 % european cd patients, nod2 mutations can either be disease-causing, or non-disease-causing (lesage et al. 2002). several studies have identified that certain nod2 mutations, such as the frameshift mutation 1007fs, as markers or indicators of cd susceptibility (ogura et al. 2001). it is believed that the frameshift mutation leads to a truncated nod2 in the leucine-rich region (lrr), thereby impairing its function. consequently, most known cd dcms occur at the lrr (fig. 1). apart from impairing the ligand-binding function of the protein, mutations at the lrr may also lead to a destabilized protein structure resulting to a loss of function for nod2 (maekawa et al. 2016). however, analysis on the functional impact of the mutations revealed that less than half of analyzed dcms have deleterious functional impact. this suggests that mutational pathogenicity observes multiple possible mechanisms apart from impairment brought by the mutation. aside from the location of the mutation, a striking difference between dcm and ndcm is the nature of the mutation. it was observed that the known dcm are mostly non-conservative (3 % conservative mutations), as opposed to ndcm that are 71 % conservative. while several diseases are also caused by conservative mutations, it is possible that the non-conservative mutations may have greater impact on the nod2 protein. the nonconservative mutations may significantly alter the microenvironment in which the mutation is located owing to change in property of the amino acid substitution. on the other hand, the conservative non-disease-causing mutations may have little effect on the nod2 loss-of-function since most conservative ndcm involve aliphatic to aliphatic substitutions (fig. 2). these findings therefore present a viable opportunity to deploy statistical methodologies in order to uncover associations between nod2 mutant type and cd progression. however, creating predictive models of protein mutants based on the primary structure is challenging due to the minute variations introduced by the point mutations. sequence – order coupling numbers are therefore ideal descriptors to be used, since this numerical representation of proteins reflects the sequence-order effect. for example, the nod2 mutants a432v and a612v have identical amino acid composition, but the substitution is located at different positions. this positional difference is adeptly captured by this class of descriptors since these two mutants have different values for the 30 socns. in addition, socn can fully describe the observed differences between dcm nova biotechnol chim (2020) 19(1): 52-60 59 and ndcm with respect to the location and nature of the mutation. this frequently used protein descriptor class is based on distance matrices derived from amino acids, their sequence-order, and physicochemical properties (schneider and wrede 1994; chou 2000). the 30 different socn represents the rank of the socn. for example, the first socn describes the coupling of adjacent residues, the second socn describes the coupling of between all second most contiguous residues, so forth. thus, this protein descriptor class can potentially reveal hidden association between nod2 mutation effect and cd susceptibility. the presented ann classifier provides a proof-ofconcept that predicting the onset of cd from nod2 protein variant is possible. the presented classification model (model d) is reliable after considering that the other models exhibited overfitting as characterized by the high training set accuracy but extremely low test accuracy. in addition, the other models were unable to classify ndcm nod2 variants, as demonstrated by the low scores obtained for specificity and npv. out of the 16 classification models created, only model d demonstrated a satisfactory accuracy for the training and test sets, in addition to respectable scores for the diagnostic indices. statistical endeavors that aimed to enhance cd detection involved the formulation of machine learning classification algorithms based on endoscopic data (mossotto et al. 2017), multivariate analysis of magnetic resonance spectroscopic data of gastrointestinal tissues (bezabeh et al. 2001), neuro-fuzzy classifier based on multitudes of clinical data (ahmed et al. 2017), and serological, genetic, and inflammatory markers-dependent random forest classifier (plevy et al. 2013). recently, an svm classification model that can categorize individuals into healthy or cd patients based on exome variations was reported (wang et al. 2019). the svm classifier used over 10,000 genes for the classification, including the nod2 gene. in the present study, the focus of the classifier is to categorize whether mutations are disease-causing or not, based on variations in the nod2 protein. thus, the present study has therefore demonstrated a new way that is solely dependent on the sequence of the nod2 protein which can potentially enhance detection and diagnostics. while the created algorithm is presently constrained by availability of data for training and validation, it is expected that the model will improve its predictive ability as more mutation types are incorporated in the system. it should also be taken into consideration the relationship between population predisposition, nod2 mutations, and cd progression. for example, nod2 mutations were absent in japanese cd patients (yamazaki et al. 2002). thus, the present utility of the algorithm may be restricted to the population group from which the data was taken. conclusion differences between nod2 crohn’s diseasecausing mutations and non-disease-causing mutations were observed. the variations were related to the location and nature of the mutations. based from these, a comprehensive statistical analyses were conducted which demonstrated the possibility of predicting the association of nod2 mutations with cd susceptibility. the ann model exhibited satisfactory capability to predict whether a specific nod2 mutation is associated with the onset of cd, based on sequence-order coupling numbers. the presented classifier sets itself apart from previously reported algorithms by using the primary structure of nod2 as the predictor. the formulated predictive model is potentially useful for the enhanced diagnosis and understanding of crohn’s disease. conflict of 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numerical representation schemes of protein sequences. bioinformatics 31: 1857-1859. yamamoto s, ma x (2009) role of nod2 in the development of crohn’s disease. microbes infect. 11: 912-918. yamazaki k, takazoe m, tanaka t, kazumori t, nakamura y (2002) absence of mutation in the nod2/card15 gene among 483 japanese patients with crohn’s disease. j. hum. genet. 47: 469-472. nova biotechnol chim (2018) 17(1): 38-47 doi: 10.2478/nbec-2018-0004  corresponding author: alzbeta.marcek.chorvatova@ucm.sk nova biotechnologica et chimica preparation of metal nanoparticles by femtosecond laser ablation tibor teplicky1,2, dusan chorvat2, miroslav michalka2 and alzbeta marcek chorvatova1,2, 1faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovakia 2international laser centre, ilkovicova 3, bratislava, sk-84104, slovakia article info article history: received: 30th april 2018 accepted: 1st july 2018 keywords: chlorella sp. cobalt colloids laser ablation nanoparticles nickel zinc abstract nanoparticles (nps) proved to have numerous applications in various fields, including biomedicine and environmental sciences. in this work, we designed and created an apparatus for fabrication of metal nps directly in liquids initiated by femtosecond laser pulses. the laser parameters leading to ~10 j/pulse energy and 0.1 gw peak power resulted in predominantly spherical particles with the sizes varying from <10 nm to ~100 nm in diameter. nps generated from cobalt and zinc targets were smaller in order of magnitude compared to that of nickel. the fabricated nps were characterized by scanning electron microscopy, while spectroscopic properties were investigated by absorption spectroscopy and spectrally resolved fluorescence imaging. we also tested the possible interaction of the created nps with living algae for their potential use for environmental research. employing such ultrashort laser opens route to provide on-demand production of np's in-situ at even factory environment.  university of ss. cyril and methodius in trnava introduction nanotechnology is proving to be a powerful tool in modern technologies. in this study we target a production of nanoparticles (np's), which features many application in medicine, cosmetics, environmental remediation, biomedical devices, sensors, renewable energy sources and semiconductors in recent years (kim et al. 2007; gupta et al. 2011; langer et al. 2015). nps possess unique properties due to its small sizes and composition. composition of nps can be pure, consisting only of one element, or can consist of more complex structures and compounds (e.g. quantum dots, etc.). nowadays, most widely commercially used nps are silver nps used mostly for antimicrobial use (bondarenko et al. 2013). on the other hand metal oxides nps of zno proved to have also antimicrobial properties and are used as additive in cosmetics for scattering of uv light (serpone et al. 2007; padmavathy et al. 2008). we also demonstrated its interaction with living cells (teplicky et al. 2015). cuo nps found its application in electronics in semiconductors or in heat transfer of nanofluids (ebrahimniabajestan et al. 2011) and sensors (li et al. 2008). there are many possible ways to synthesize nps, which can be categorized into following groups: chemical, physical, photochemical and biological. each process has its advantages and drawbacks. it includes fabrication complexity of synthesis, particle sizes and size distribution, or quantity and quality of nps. main drawback is the cost of production. chemical synthesis of nps in solution usually employs metal precursor, reducing agent and stabilizing agents. study of kim et al. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:33 utc nova biotechnol chim (2018) 17(1): 38-47 39 (2006) described the synthesis of spherical agnps. in this study, the authors showed the ability to control the size of nps by adjusting the temperature to around 100 °c, as well as injection rate using the polyol process and a modified precursor injection technique. chen et al. (2007) published chemical synthesis of ag nps in a simple oleylamine-liquid paraffin system. in contrast to chemical synthesis, the physical approach to np's synthesis is based on decomposition (evaporation)-condensation process. it usually utilizes the input energy in form of the heat or the electric arcs to produce nps. advantage of the physical approach are in large amounts of the nps produced in the form of dust. drawbacks include the high energy consumption and longer periods of the time for thermal stability and condensation of sufficient amount of nps. lee and kang (2004) developed a technique for fabrication of powder ag nps by thermal decomposition. photochemical synthesis approach provides clean alternative to the previous methods, utilizing photoinduced fabrication of np's from pure target, simple manipulation and control over parameters of generated nps. the main advantage of this method is the ability to produce nps ranging from metallic to non-metallic precursors in various transparent media e.g. glasses, polymers, including water, without the need of any chemical add-ons. ultrashort laser pulses offer both high laser intensity and precise laser-induced breakdown threshold (liu et al. 1997) suitable for micromachining and laser ablation (arboleda et al. 2015). while the method has been described two decades ago, it was limited to high-energy physics laboratories and its wider application was possible only after commercial availability of amplified femtosecond (fs) laser systems. in this regard, amplification of fs pulses in ytterbiumdoped fiber (limpert et al. 2002) has provided a route for high-energy, high-repetition rate fs sources. high energy fs pulses focused tightly with microscope objective on a surface delivers vast amount of energy to a targeted area without influencing its surroundings (hwang et al. 2004). it is able to evaporate material of any kind within the volume that is limited only to the focal point. evaporated material escapes the targeted area and forms dust or colloid of nano/micro-particles. an interesting option is a controlled mobility of generated np's, applicable for surface patterning (menéndez-manjón et al. 2009). mechanisms of interaction of ultra-intense laser pulses with solids have been extensively studied under various conditions (perez and lewis 2002; pereira et al. 2004; amoruso et al. 2007). in this regard, mechanisms of np formation using fs laser ablation significantly differ from the case of using other laser sources. ablation of materials with ultrashort pulses in solvents has a very limited heat-affected volume, thus does not alter the liquid and is a suitable route for np synthesis directly in solvents without any chemical modification. it was described that efficiency of laser plasma generation and subsequent formation of nps (povarnitsyn et al. 2007; noël et al. 2007) are dependent on many parameters such as laser pulse length, energy, wavelength, interaction with subsequent pulses or metal target properties (eliezer et al. 2004; noël and hermann 2009; rouleau et al. 2017). the most important pre-requisites for fast, efficient and stable np generation in laboratory conditions by fs lasers are i) medium-energy pulses with high enough peak power, but avoiding induction of highly nonlinear processes, ii) high repetition frequency that allow reasonable speed of np generation and iii) fast mechanical/optical xyz scanning of the target, to avoid the interaction of subsequent pulses with the previously irradiated volume and to keep the focus of the beam at optimal position. important issue is also to select the correct laser wavelength in order to avoid direct photochemical interaction with already synthesized np's. in this work we describe design and implementation of experimental apparatus for fabrication of metal np's directly from metal targets in solvents using amplified fs laser pulses. for the reasons given above, our aim was to create an instrument based on fs laser pulses with medium energy and high-repetition rate, combined with fast xyz micropositioner. experimental experimental setup scheme of the experimental setup for direct laser ablation of metals in liquids is shown at fig. 1. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:33 utc nova biotechnol chim (2018) 17(1): 38-47 40 metal discs from pure cobalt, nickel and zinc, held in a glass petri dish, were covered by deionised water or by ethanol. in order to minimize the absorption of already fabricated nps, the laser focal point was moving constantly over the surface. no further preparation for the np material was carried out. fig. 1. scheme of the laser ablation of metal discs in liquids. spirit ultrafast ytterbium amplified laser (highq, spectraphysics, newport) was used for the ablation of metal surfaces. the laser parameters were following: 300 fs pulse width, 1,040 nm wavelength; 4 w mean power operated at 100 khz which leads to ~10 j/pulse energy and 0.1 gw peak power. laser light was focused at metal discs surface by plan n 10x/0.25 uis2 objective (olympus). movement of the beam over the surface was realized by motorized xyz micropositioner (walter uhl technische mikroskopie gmbh & co.) using joystick interface (fig. 2). materials metal discs of pure elements (goodfellow cambridge ltd.) were used as a substrates for fabrication of nps: cobalt – co (99.9 %), ø 25 mm, thickness 1.0 mm; zinc –m zn (99.99 %), ø 25 mm, thickness 3.0 mm; nickel – ni (99.99 %), ø 25 mm, thickness 3.2 mm. scanning electron microscopy (sem) field-emission scanning electron microscope (leo 1550, carl zeiss, germany) was used to characterize the size and structural properties of fabricated nps. for imaging by in-lens detection of secondary electrons we have used voltage of 5 – 15 kv and magnification of 10,000 – 200,000x. samples for electron microscopy were prepared by drying of np solution on pure silicon wafer substrate. elemental composition of the np's was checked and approved by edx analysis. absorption spectroscopy (as) absorption spectra were measured by dual-beam cary 50 bio (varian) spectrophotometer within 300 – 800 nm range. quartz cuvettes with 2 mm beam path were used. spectra were obtained from the colloidal solutions dissolved to concentration with <0.2 od at 800 nm. due to different concentration of samples, all spectra were further normalized to absorbance values measured at 800 nm in each solution. confocal fluorescence microscopy (clsm) confocal laser scanning fluorescence microscopy was done by lsm510 meta scanhead on axiovert 200m microscope stand (both carl zeiss, germany). zeiss plan-apochromat 20x/0.75 lens was used to visualize the surface of silicon substrates with dried nps. zeiss c-apochromat 40x/1.2w lens was used to visualize the interaction of np colloids with algae. for excitation we used 450-nm diode laser line, for detection we utilized two channels: first with band-pass filter 435 – 485 nm for reflected light, and long-pass filter lp 505 nm for fluorescence. spectral imaging was done with 450 nm laser excitation and meta spectral detector within 515 – 665 nm range. live cell imaging was done using band-pass filters of 500 – 550 nm, 650 – 710 nm and transmission channel. living cells chlorella sp. was obtained from the faculty of natural sciences, university of ss. cyril and methodius in trnava collection of the green algae, as described previously (teplicky et al. 2017). the green algae of genus chlorella sp. were previously isolated from the main drinking water supply. green algae were cultivated in hoagland cultivation medium (hoagland 1920). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:33 utc nova biotechnol chim (2018) 17(1): 38-47 41 results fabrication of metal nanoparticles with the aim to fabricate nps in various solvents, metal disc plates were covered by selected solvent and laser focus was set to the plate surface. focus of the laser beam was manually moved over the surface for about 1 – 2 min. irradiation of the plate by fs laser pulses caused ablation of the surface which was followed by creation of np colloid by dispersion of np's in the solvent by brownian motion. process of np generation could be observed visually by the change of the color of the solvent, which locally turned from transparent to grey/black. the effectivity of np generation was dependent on the selected material and solvent, e.g. for cobalt the np's generation in ethanol was several times less (and slower) than that of water. further preparation of np's involved drying on silicon wafer. no other treatment was necessary for sem or clsm imaging. size and shape of nanoparticles to semi-quantitatively characterize the shape and size of the nps generated by fs laser beam, dried samples were imaged by sem microscopy (fig. 3). we found that the np geometry in all cases was predominantly spherical with sizes varying from <10 nm to ~100 nm in diameter. np's generated from cobalt and zinc targets were smaller in order of magnitude compared to that of nickel. ethanol solution exhibited smaller efficiency of the np generation and somewhat larger degree of clustering during drying process. we also observed larger distribution of np size in the case of cobalt. spectroscopic properties of nanoparticles absorption spectra of the nps show strong influence of the solvent on their photo-chemical behavior. the absorption spectra in ethanol (fig. 4a) show mostly non-specific diffraction effect with the exception of zinc, with the distinct peak at 340 nm. absorption of zno nps was previously described in distilled water in uv region above 200 nm, with a smaller peak between 350 – 380 nm (singh and gopal 2007). in strong contrast to the results in ethanol, absorption spectra in water (fig. 4b) revealed very pronounced resonance peaks at 300 – 450 nm for zinc and cobalt, while the absorption of nickel nps remain low and non-specific. note the logarithmic scale of the fig. 4b due to extremely high absorption fig. 2. photograph of the experimental setup for nps generation by direct laser ablation: l) laser, b) beam delivery optics, o) objective, s) sample, m) micropositioner. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:33 utc nova biotechnol chim (2018) 17(1): 38-47 42 coefficient of the zinc in the uv region. others also demonstrated dependence of the np absorption spectra on the used solvents (li et al. 2015) and observed results were interpreted in terms of dielectric constant, hydrogen bonding, etc. (khan et al. 2011). in our work, water was chosen as a solvent comparable to the environment in the cytosol of living cell and organisms. however, a strong dependence of the spectra on the solvents indicates that, in the future, other solvents, including glycerol or dimethylsulfoxide need to be tested to insure comparability with true environmental conditions in cells. based on the interesting absorption characteristics of the zinc np's, we performed confocal fluorescence microscopy of dried zinc nps (fig. 5). the result shows pronounced fluorescence in the regions where the zinc nps form clusters. indeed, the resolution of optical microscopy does not allow to resolve individual np such as in the case of sem microscopy, however the information about np fluorescence is an interesting finding. with the aim to gain more insight into the potential origin of detected fluorescence we performed spectrally resolved imaging of the zn sample. the emission spectra show distinct green fluorescence of dried zinc np's peaking at 520 nm, compared to the non-specific background signal from silicon substrate. using zno nps, multiple emission maxima, including one at 527 nm, were previously described (estrada-urbina et al. 2018) and fluorescence of zno and its nanocomposits was demonstrated between 450 and 520 nm (li et al. 2015). as a result, various zno nps are efficiently applied for multicolor bioimaging (kang et al. 2017). in the light of these data, our results indicate a possible oxidation of recorded zn nps. interaction of nanoparticles with living algae to demonstrate the potential use of the generated nps for environmental research, we added the np colloids to living algae chlorella sp. algae were incubated with np for 60 h at room temperature. fig. 3. sem image of nps from various targets and solvents. a) cobalt in water, b) cobalt in ethanol, c) zinc in water, d) zinc in ethanol, e) nickel in water, f) nickel in ethanol. scale bar: 20 nm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:33 utc nova biotechnol chim (2018) 17(1): 38-47 43 fig. 4. absorption spectra of nps created in ethanol (a) and water (b). we previously demonstrated that the exposure of the algae to toxic substances, namely sodium hypochlorite, lead to complete abolishment of the red fluorescence with maximum at 680 nm (related to chlorophylls) and significant increase in the green fluorescence of unknown origins (marcek chorvatova et al. 2018). here, we recorded the interaction of np with algae (fig. 6) at both, the red spectral channel (650 – 710 nm) and the green spectral channel (500 – 550 nm). we observed that cobalt nps have no visible effect on the endogenous fluorescence of algae (fig. 6b) when compared to control conditions (fig. 6a). on the other hand, zn nps provoked clear clustering of cells (fig. 6c) and lead to decrease of both, the green and the red fluorescence finally, nickel nps created dark deposits (shown at fig. 6d as arrows), from which some could be found also inside the algae (data not illustrated), suggesting the capacity of the algae to ingest the zn nps. algae are well known to play a role as primary producers in most aquatic ecosystems, estimating the effects of heavy metals on the growth and photosynthesis of algae can significantly impact the overall ecological risk assessment of heavy metals. some heavy metals including zinc and nickel are essential for growth at very low concentrations but toxic at higher levels (reed and gadd 1990). the toxic effect of nickel ions on photosynthetic electron transport chain was demonstrated (elsheekh 1993). in addition, in agreement with our observations, nickel nps were shown to penetrate the cells and cause deterioration fig. 5. reflectance (a), fluorescence (b) and composite (c) image of zn nps dried from water solution at si substrate. scale bar: 10 µm. 300 400 500 600 700 800 1,0 1,5 2,0 2,5 r e l. a b s o rp ti o n [ a .u .] wavelength [nm] cobalt zinc nickel 300 400 500 600 700 800 1 10 100 r e l. a b s o rp ti o n [ a .u .] wavelength [nm] cobalt zinc nickel a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:33 utc nova biotechnol chim (2018) 17(1): 38-47 44 of several cellular characteristics (oukarroum et al. 2017). concerning cobalt, constituent of vitamins, its higher concentrations were inhibitory to o2 uptake by the algae (el-sheekh et al. 2003). zn serves as cofactor for various enzymatic reactions, as well as dna transcription (miazek et al. 2015). despite this fact, toxic effects of zno nps were also largely studied and showed greatest inhibition of growth and chlorophyll content in algae when compared to other nps (ko et al. 2018). our observations of the red fluorescence decrease are in agreement with lowering of the chlorophyll content and clustering of cells can also be a result of the np toxicity. however, the precise mechanism underlying interaction of living algae with nps needs to further elucidated in the future. discussion in this study, we described a newly developed apparatus for photochemical generation of nps from various metal targets using focused fs nir beam. we proved successful generation of np colloids in pure solvents by this method using sem microscopy. size of the generated np's was within the range of 10 – 100 nm. for future fabrication of np colloids, a flow-cell design of the reaction chamber will be needed to assure that the already generated np will be not irradiated by the focused fs beam, which can lead to cluster formation and further photochemical reaction induced by the focused light. this effect might be responsible for broader size distribution in some fig. 6. chlorella spirulis algae incubated with np colloids. rows: a) control, b) cobalt, c) zinc, d) nickel. columns: left – green autofluorescence, middle – red autofluorescence, right – transmission image. scale bar: 20 m. arrows: nanoparticle deposits. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:33 utc nova biotechnol chim (2018) 17(1): 38-47 45 of our samples (e.g. ni, or co+ etoh). based on previous experience with gold nps, we also avoided the use of 520 nm excitation due to spectroscopic overlap of the laser wavelength and possible extinction peaks of the colloid. it is known that an absorption band of zinc np colloid at uv wavelenth region is due to surface plasmon resonance (spr) at zno nps. spr frequencies depend on the environment, inter-particle distance, size, shape and composition of the np's (amekura et al. 2007). for example, zn nps dispersed in silica exhibit two optical extinction peaks around ~250 nm and ~1,050 nm both of which were ascribed to surface plasmon resonances. our results confirm the presence of spr for both zinc and cobalt. very strong spr signal from zn np's was observed in water at 350 nm, somewhat weaker extinction peak of co np's was observed in water at 390 nm (fig. 4). ethanol colloids show in comparison only a weak spr from zinc. interestingly, the deposited np's on si substrate excited by 450 nm laser show also fluorescence with emission maximum around 520 nm (fig. 5 and 6). this fluorescence peak has been reported previously in published studies, however the origin of fluorescence is not fully understood (monticone et al. 1998; guo et al. 2000). the ability to generate fluorescent signal opens a new prospect for metal np sensing, which should be investigated further in more details. production of heavy metals and metals in general caused higher occurrence of metals in the environment. in order to understand the mechanisms behind the toxicity of these substances, the physico-chemical properties of the metal particles and their interaction with living cells should be thoroughly analyzed in relevant test environments. we demonstrated viable route of simple np production by photochemical method, applicable to virtually all combination of solvents and targets. one of the advantages of this method is a very small absolute volume of metals needed for successful np generation, which is an interesting option for rare or very toxic samples. by our setup the np's can be also generated in-situ in the presence of cells in the same solvent (e.g. physiological solution) without the need for any subsequent chemical treatment. we also evaluated the interaction of the created nps with living algae and showed patterns of different behavior of the algae in their presence. nevertheless, more profound study is needed to precisely determine the effect of the nps on living organisms. conclusions we successfully designed and created an apparatus for direct synthesis of nps from various metal targets by fs laser ablation in liquids. the size range of the generated np was approximately 10 – 100 nm. nps were characterized by uv-vis absorption spectroscopy, sem and clsm imaging with spectral detection. we also performed first evaluation of their possible interaction with living cells. presented work is the first step towards an on-demand synthesis of metal np used as models of environmental pollution in various fields of biotechnology and environmental sciences. acknowledgements this work was supported by the slovak research and development agency under contract number apvv 140858, grant vega 2/0123/18 and by eu-h2020 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micro and nano-structured surfaces. in periasamy a, konig k, so ptc (eds.), multiphoton microscopy in the biomedical sciences xv, book series 9329: 93290d-01 to 93290d-10. teplicky t, danisova m, valica m, chorvat d jr, marcek chorvatova a (2017) fluorescence properties of chlorella sp algae. adv. electr. electron. eng. 15: 362-367. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:33 utc nova biotechnol chim (2017) 16(1): 48-53 doi: 10.1515/nbec-2017-0007  corresponding author: simona.kvasnova@umb.sk nova biotechnologica et chimica zinc bioaccumulation by microbial consortium isolated from nickel smelter sludge disposal site simona kvasnováa,, ľudmila hamarováb and peter pristašc a department of biology and ecology, matej bel university, tajovského 40, banská bystrica, sk-914 01, slovak republic b institute of biology and ecology, pavol jozef šafárik university in košice, šrobárová 2, košice, sk040 01, slovak republic c institute of animal physiology, slovak academy of science, šoltésovej4-6, košice, sk040 01, slovak republic article info article history: received: 20th february 2017 accepted: 11th may 2017 keywords: bacteria consortium heavy metals bioremediation zinc bioaccumulation arthrobacter abstract heavy metal pollution is one of the most important environmental issues of today. bioremediation by microorganisms is one of technologies extensively used for pollution treatment. in this study, we investigated the heavy metal resistance and zinc bioaccumulation by microbial consortium isolated from nickel sludge disposal site near sereď (slovakia). the composition of consortium was analyzed based on maldi-tof ms of cultivable bacteria and we have shown that the consortium was dominated by bacteria of genus arthrobacter. while consortium showed very good growth in the zinc presence, it was able to remove only 15 % of zinc from liquid media. selected members of consortia have shown lower growth rates in the zinc presence but selected isolates have shown much higher bioaccumulation abilities compared to whole consortium (up to 90 % of zinc removal for nh1 strain). bioremediation is frequently accelerated through injection of native microbiota into a contaminated area. based on data obtained in this study, we can conclude that careful selection of native microbiota could lead to the identification of bacteria with increased bioaccumulation abilities.  university of ss. cyril and methodius in trnava introduction elevated concentrations of heavy metals are introduced into the environment through metalliferous mining, metal smelting, metallurgy industry activities, and waste disposal sites. this global environmental problem has developed over the past few decades due to the rapid increase in industrialization and urbanization (dixit et al. 2015). some heavy metals are essential elements for the existence of living organisms because they perform several functions in biological systems. these heavy metals such as ca, co, cr, cu, fe, k, na, ni, and zn are essential nutrients; some of heavy metals are relatively harmless but can be toxic in larger amounts or certain forms. essential metals function as catalysts for biochemical reactions are stabilizers of protein structures and bacterial cell walls and serve in maintaining osmotic balance. many other metals have no biological role (e.g. ag, al, cd, au, pb, and hg) are non-essential and potentially toxic to living organisms, especially microorganisms (hussein et al. 2005). heavy metals toxicity leads to decreases in biodiversity and productivity and thereby, results changes in structure and function of ecosystems (mayor et al. 2013). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:35 utc mailto:simona.kvasnova@umb.sk nova biotechnol chim (2017) 16(1): 48-53 49 heavy metals polluted environments are however a unique source of poly-extremotolerant microorganisms for modern biotechnology and bioremediation applications (pristas et al. 2015). the available physicochemical methods of metal removal are not only expensive but also produce secondary sludge and are, therefore, not ecofriendly. hence, the need to remove hazardous trace metals with eco-friendly method is acute (singh et al. 2012). the opposite of these physicochemical strategies is bioremediation. this process use microbes to transform pollutants in environment into less toxic substances or immobilization in order to reduce their bioavailability. bioremediation gained more attention recently to clean up the polluted environment and ensures a safe and economical alternative to commonly used physicochemical methods (guo et al. 2010). the activity of microorganisms may lead to adsorption and precipitation of potentially toxic elements by biosorption or bioaccumulation, but also to increase their solubility or formation of volatile compounds with mechanisms such as biovolatilization or bioleaching (boriová et al. 2015). it is well known that certain species, such as pseudomonas sp., escherichia sp., bacillus sp., acinetobacter sp., and enterobacter sp. can effectively remove metals in pure cultures, but multispecies consortia can better withstand extreme conditions often encountered with industrial waste like spikes of ph or high metal concentration. they provide a microenvironment, which could be very beneficial for metal precipitation. the positive interaction between constituent species may also facilitate the survival of sensitive strains. in view of these facts, the applicability of consortia in bioremediation processes, offer promising solution for combating heavy-metal pollution in the environment (carpio et al. 2016; malik, 2004). this work investigates influence of heavy metals on bacterial growth, metabolic activity and compares zinc bioaccumulation by pure cultures and microbial consortium isolated from heavy metals contaminated nickel sludge disposal site. experimental sample collection, isolation, growth, and identification of bacteria sludge samples were obtained from sludge waste disposal site of nickel smelter near sereď (slovakia). samples from the surface and 10 cm depth at three different locations were aseptically collected, transferred to the laboratory and stored in the cold (4 °c) prior to microbiological analysis. to the 0.5 g of solid sample 10 ml of sterile pbs solution was added and after 20 min of intensive mixing 0.5 ml aliquot was transferred to the liquid diluted lb medium (lb medium (becton dickinson, usa) diluted 1 : 3 with distilled water) and cultivated aerobically at room temperature. after 24 h of cultivation the bacterial culture was transferred to the fresh diluted lb medium and after another 24 h of cultivation used in subsequent analyses. this mixed bacterial culture is referred as consortium in further. to analyse the composition of consortium, aliquots of consortium was spread on non-selective agar medium (tsa – tryptone soya agar, oxoid, usa) and cultivated under aerobic conditions at laboratory temperature for 48 h. the individual colonies were selected on the basis of cell and colony morphology and used for further analyses. after repeated cultivation and control for the purity, the isolates were typed and identified by the maldi tof ms approach (kopcakova et al. 2014). the impact of heavy metals on metabolic activity of consortium for evaluation of heavy metal influence on metabolic activity of consortium we have used biolog ecoplates™, which contain 31 different carbon sources and a blank in triplicate (insam, 1997). each well of the biolog ecoplate™ was inoculated with 100 μl of inoculum (overnight consortium culture diluted 1 : 50 in phosphate buffered saline, thermo fisher scientific, usa) and incubated at 25 °c. filter sterilized nicl2 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:35 utc nova biotechnol chim (2017) 16(1): 48-53 50 and cocl2 were added at appropriate concentrations. absorbance readings were taken periodically (after 24, 48, and 144 h) at 590 nm with a microplate reader (synergy 2, biotek, usa). the metabolic response (mr) of consortium was expressed as average well optical density of all carbon sources (mr=σ odi/31; gryta et al. 2014). bioaccumulation of zinc the bioaccumulation of zinc was tested in liquid diluted lb medium. fresh overnight culture of consortium or selected isolates was inoculated (1 : 100) into diluted lb medium supplemented with 10 mm zncl2 and cultivated for 18 h at room temperature. following cultivation, the cell biomass was removed by centrifugation at 3 000 g, the wet weight of biomass was measured and residual zinc concentration in the spent liquid medium was determined spectrophotometrically using dithizone method (ahmed and alam, 2003). all experiments were done at least in duplicates. results and discussion heavy metals pollution is one of the most serious environmental issues, nowadays. heavy metals significantly impact soil bacterial community due to their toxic effect. to transform pollutants into less toxic substances or immobilization in order to reduce their bioavailability, bioremediation by microorganisms is frequently used (guo et al. 2010). in several countries e.g. in usa, 12 % of the in situ projects and 11 % of the ex situ projects have used bioremediation (us epa, 2010). the activity of microorganisms led to adsorption and precipitation of potentially toxic elements by mechanism biosorption or bioaccumulation. bioremediation is frequently accelerated through injection of native microorganisms into a contaminated area. the aim of our study was to analyse the native microbiota of nickel sludge disposal site near sereď. this landfill belongs to the most serious ecological burdens in slovakia and is composed of nickel sludge with extremely high heavy metal content: fe (50–80 %), cr2o3 (2.5–3.5 %), sio2 (6–8 %), al2o3 (6–8 %), cao (2.5–3.5 %), ni (0.17 %), p2o3 (0.6 %) (michaeli et al. 2012). to the best our knowledge of the microbiota of the sludge was never analyzed. recently, remenár et al. (2014) analysed cultivable bacteria from nickel and cobalt contaminated agricultural soils adjacent to the landfill. in our experiment, the bacterial consortium was isolated from the mixed sludge samples. the consortium showed relatively good growth in the presence of several heavy metals (data not shown). to test influence of nickel and cobalt on metabolic activity of obtained consortium biolog ecoplates™ were used. both metals showed strong inhibition of metabolic activity of consortia (fig. 1). at both concentrations tested, cobalt exerted much stronger inhibition effect on consortium compared to nickel. there are no general rules on the relative toxicity of nickel and cobalt because published data are often contradictory, even for similar type of microbial systems (gikas 2008). several authors however, reported stronger inhibitory effect of cobalt over nickel. cobalt is more potent inhibitor than nickel of the growth of escherichia coli and pseudomonas putida (wu et al. 1994). schmidt and schlegel (1989) who studied the effects of ni(ii) and co(ii) on several bacteria isolated from metal contaminated soils, have also reported that bacterial resistance to co(ii) is lower compared to ni(ii) resistance. arthrobacter butzleri has been reported to be more sensitive to ni(ii) than to co(ii) (otth et al. 2005). fig. 1. effect of heavy metals to metabolic activity of isolated consortium – expressed as average well colour optical density in each microplate. microbial consortium obtained was tested for the heavy metal bioaccumulation using zinc bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:35 utc nova biotechnol chim (2017) 16(1): 48-53 51 as a model. upon cultivation in liquid zinc supplemented medium, the consortium was able to remove of about 15 % of zinc and bioaccumulation of zinc reached 0.18 mg/g of wet biomass weight (fig. 2). observed bioaccumulation abilities of isolated consortium are not very high and several authors reported much higher accumulation rates. singh et al. (2012) reported that microbial consortium isolated from coal could efficiently remove over 80 % of metals, such as ni, zn, cd, cu and cr, while the removal of pb was nearly 45 %. according to carpio et al. (2016) microbial consortium isolated from the contaminated water and sediment could remove more than 50 % of zinc from medium. bacterial consortia isolated from contaminated environments are usually genetically complex and consist of multiple bacterial taxa (carpio et al. 2016) of different physiological and bioaccumulation properties. fig. 2. comparison of zinc removal from medium by bacterial strains and consortium. the composition of isolated consortium was analysed using cultivation approach and subsequent identification of isolates by maldi-tof ms analysis. the analysis showed that consortium is composed by gram-positive bacteria only and it is dominated by actinobacteria of arthrobacter genus (fig. 3). among 22 randomly selected isolates at least 16 were affiliated to this genus. carpio et al. (2016) reported predominance of proteobacteria in microbial consortium isolated from the contaminated water and sediment. remenár et al. (2014), who analysed microbiota of soils near nickel sludge in sereď, reported the dominance of actinobacteria in these soils. according their research in nickel-contaminated soils dominate genus streptomyces (18.1 %) followed by arthrobacter (15.2 %), rubrobacter (5.7 %), and glycomyces (4.8 %) genera. the occurrence of arthrobacter genus in heavy metal contaminated soil is not surprising and it was reported by several authors (alvarez et al. 2017; henne et al. 2009). the phylum actinobacteria and genus arthrobacter constitute some of the main and most diverse phyla within the domain bacteria. these taxa exhibit diverse physiological and metabolic properties, such as production of extracellular enzymes and the formation of a wide variety of secondary metabolites. this versatility in secondary metabolite production makes them important tools for biotechnological applications such as bioremediation (ludwig et al. 2012). the environmental prevalence of arthrobacter may be due to its ability to survive long periods under stressful conditions, such as temperature shifts, oxygen radicals, and toxic chemicals, including heavy metals. arthrobacter sp. is one of the most important genus in relation to tolerance to heavy metals and its potential use in bioremediation (alvarez et al. 2017). based on these data several isolates (nh3, nh5, nh8, and nh9) identified as arthrobacter spp. by maldi tof analysis. no reliable identification was obtained for nh1 isolate nevertheless the isolate was identified as arthrobacter spp. by 16s rrna sequencing (data not shown). the obtained results showed that growth in the presence of zinc and bioaccumulation abilities vary greatly among isolates. all isolates showed lower growth rates compared to consortium (fig. 2) and no clear correlation was observed between growth rates and zinc bioaccumulation. while for the most isolates bioaccumulation ability was lower compared to consortium, nh1 isolate was able to eliminate the most of zinc from medium, despite the slowest growth rate. nh1 isolate is able to take up to 95 % of zinc (0.5 mg of zinc per 1 g of wet cell biomass) and therefore it is the most appropriate isolate for bioremediation use (fig. 2). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:35 utc nova biotechnol chim (2017) 16(1): 48-53 52 fig. 3. maldi tof based similarity cluster analysis of isolates from indigenous microbiota of nickel sludge disposal site near sered. the maldi tof based identification is shown next to the clades. it has been suggested that stimulating the growth of indigenous microorganisms with metal bioaccumulation capacity may be a useful strategy for immobilizing metals in soils and preventing contamination of groundwater (valentine et al. 1996). several studies focused on microbial metal bioaccumulation involving multi or single-component systems of pure cultures. the advantages of the individual components lies in the providing information on metal binding characteristics of individual organisms, but cannot be directly applied to the behaviour of metals in heterogeneous systems. studies focused on the behaviour of multi-component systems, like a consortium of microorganisms, represent an approach, even if still simplified, closer to the real situations in which bacteria are found in natural systems (ledin, 2000; alvarez et al. 2017). these advantages mainly consist of synergistic interactions among members of the consortium. our data suggest that analysis of contaminated environments could lead to the identification of bacteria with increased bioaccumulation abilities. conclusions indigenous microbiota of nickel sludge disposal site near sereď have been found to be dominated by arthrobacter spp. the microbiota was analysed from the point of metal bioaccumulation capabilities and while the highest growth rates were observed for mixed bacterial consortium, the highest zinc bioaccumulation rates were observed for individual strain. despite extreme conditions, heavy metals contaminated sites could be source of autochthonous microflora, which could be used to concentrate, remove and recover metals from contaminated sites and thereby enhance the efficiency of treatment processes. acknowledgements this work was in part supported by vega 1/0229/17 and vvgs-2016-275 grants. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:35 utc nova biotechnol chim (2017) 16(1): 48-53 53 references ahmed mj, alam m (2003) a rapid spectrophotometric method for the determination of mercury in environmental, biological, soil and plant samples using diphenylthiocarbazone. spectroscopy 17: 45-52. alvarez a, saez jm, costa jsd, colin vl, fuentes, ms, cuozzo sa, amoroso mj (2017) actinobacteria: current research and perspectives for bioremediation of pesticides and heavy metals. chemosphere 166: 4162. boriová k, urík m, matus p (2015) biosorption, bioaccumulation, biovolatilization of potentially 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perspect. 102: 297-300. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:35 utc nova biotechnol chim (2020) 19(1): 116-123 doi 10.36547/nbc.v19i1.586  corresponding author: r.tandlich@ru.ac.za; roman.tandlich@tul.cz; roman.tandlich@gmail.com nova biotechnologica et chimica citizen science based monitoring of microbial water quality at a single household in a south african local municipality during the covid19 lockdown roman tandlich1,2,3, 1faculty of pharmacy, rhodes university, artillery road, p.o. box 94, grahamstown/makhanda 6140, south africa, tel: 00-27-46-603-8825, fax: 00-27-46-603-7506 2 faculty of health sciences, technical university of liberec, studentská 2, 461 17 liberec, czech republic 3regional director for africa, the international emergency management society (tiems), rue deux eglises 39, 1000 brussels, belgium article info article history: received: 5th june 2020 accepted: 26th june 2020 keywords: drinking/potable water hygiene h2s test kit makana local municipality pandemic abstract personal hygiene and access to potable/drinking water, which is safe for human consumption, are critical to containing the covid19 pandemic. the principle of citizen science, with limited use of laboratory resources, was applied to monitoring of microbial quality of the municipal water supply in makana local municipality in the eastern cape province, south africa. samples were taken just before and during 30 days of the strictest phase of the covid19 nation-wide lockdown in south africa. the h2s test kit was used as the basis for the microbial testing, while a cell phone app was used for the temperature monitoring at the author’s house. during the study, the ambient temperature ranged from 17 to 29 °c, with decreases below 18 °c occurring on three out of 12 sampling occasions. thus the results of the h2s test kit might have been slightly influenced by the fluctuations of the ambient temperature. on 8 sampling occasions between 1 and 4 h2s test kits were positive for faecal contamination. three samples or 25 % were free of faecal contamination. one sample had all five h2s test kits were positive for faecal contamination. results of statistical testing indicated that potable/drinking water from the municipal supply in makana local municipality was probably faecally contaminated at the author’s household on an intermittent basis. ongoing monitoring of microbial drinking water quality is necessary and continuing at the sampled location.  university of ss. cyril and methodius in trnava introduction the 2017 united kingdom risk register of civil emergencies indicated that there was a very high chance of a pandemic influenza occurring in the next few years (uk government 2017). those predictions came true when the covid19 disease outbreak started in late december 2019 (tandlich et al. 2020). results of a recent genetic study indicate that the sars-cov-2 virus (the causative agent of covid19) originated most likely in bats in parts of asia and was subsequently transferred into pangolins where it mutated (andersen et al. 2020). the mutated virus then got transferred humans and mutated again; and started causing mild to severe (respiratory) disease in humans (andersen et al. 2020). the sars-cov-2 virus has been shown possess phylogenetic similarities to the causative agent of the severe acute respiratory syndrome (sars), i.e. the sars mailto:r.tandlich@ru.ac.za mailto:roman.tandlich@tul.cz mailto:roman.tandlich@gmail.com nova biotechnol chim (2020) 19(1): 116-123 119 cov virus (wang et al. 2020). the previous sars outbreak(s) did not reach the same global proportions as the sars-cov-2 virus pandemic. therefore, even though sars-cov-2 virus is not an influenza virus, the scope and impact of the covid19 pandemic is analogical to the pandemic influenza, which had been predicted as imminent to occur (uk government 2017). on 30th january 2020, the world health organisation (who) declared the covid19 a health emergency of international concern (who 2020a). the covid 19 pandemic is caused by an infectious disease agent, i.e. a virus, and can therefore be classified as a first-generation disaster (bulíkova et al. 2011). as of 25th june 2020, there have been 9,296,202 confirmed cases of covid19, including 479,133 deaths worldwide (who 2020b). as the morbidities and mortalities keep climbing on a daily basis, the primary health outcomes of the disasters have been devastating globally and the human cost of the covid19 pandemic has been staggering. currently there is no vaccine against the covid19, which has been approved and is being used for the vaccination of the global population. therefore mitigation, mainly non-pharmaceutical interventions and public health strategies, are used to contain and to limit the spread of covid19. personal hygiene forms a core part of those interventions and strategies to contain the sars-cov-2 virus. access to sufficient volumes of potable water, which is microbially safe for human consumption and domestic uses is critical for the maintenance of hygiene and thus prevention of the spread of covid19 (hyde 2020). armitage and nellums (2020) stated that access to water and sanitation is a challenge in many places on earth, and population in low-income settlements in urban areas could be particularly vulnerable. neal (2020) stated that a re-alignment of the priorities and functioning of the water sector, as well as uses of water in general, are a must. parts of south africa have been experiencing water quality problems since at least 2011 (luyt et al. 2011; tandlich et al. 2014; malema et al. 2019) and drought since at least 2015 (monyela 2017). previous studies by the author and his collaborators indicate that municipal potable/drinking water has suffered from faecal contamination in makana local municipality in the eastern cape province of south africa (luyt et al. 2011). on 15th march 2020, a national state of disaster was declared in south africa (south african government gazette 2020a). from 27th march 2020, the entire territory of south africa was placed under lockdown, i.e. movement of the population was limited to staying at home and only leaving personal dwellings/households to perform very limited activities (south african government gazette 2020b). examples include purchase of groceries and or visiting a doctor’s surgery (south african government gazette 2020b). provision of drinking/potable water to the households, i.e. the taps not running dry would have been the most important task for the south african government at local, provincial and national level. for this, the national department of water and sanitation has been rolling out a programme for the installation of water storage facilities across the territory of south africa (all africa 2020). access to drinking/potable water is critical to wash hands and to maintain personal hygiene. at the same time, the microbial safety of the provided drinking water is also of the essence during the covid19 pandemic. this is mainly to prevent the outbreak of diarrhoeal diseases which can cause cascading effects of the covid19 pandemic. citizen science is a concept where the laymen/persons who have not received a formal scientific training perform data collection on topics of scientific interest/research (e.g. angala et al. 2019). the author has worked and collaborated with other researchers over the past decade on the development, usage and benchmarking of the h2s test kit against standard indicator microorganism test for microbial water quality (luyt et al. 2011; tandlich et al. 2014; malema et al. 2019). the h2s test kit is a viable tool to perform testing for faecal contamination of drinking water in areas where formal laboratory access is logistically a challenge (luyt et al. 2011; tandlich et al. 2014; malema et al. 2019). the correspondence rates, along with the rates false positive and false negative results, in the analysis of rainwater was recently published on by malema et al. (2019). use of the kit in citizen science monitoring was also conducted previously (e.g. angala et al. 2019; nqowana 2019). it is assumed in further text of this 117 nova biotechnol chim (2020) 19(1): 116-123 118 study that there is similarity in the monitoring performance of the h2s test kit and the standard indicator mcroorganisms in the detection of faecal contamination in the makana municipal drinking water and the rainwater in the eastern cape province of south africa (malema et al. 2019). the author reports the results of a one-location monitoring of microbial water quality just before and during the strictest phase of the covid19 nationwide lockdown in south africa. data was collected in isolated settings without or with limited access to a formal laboratory, and using the tools of citizen science. the author is a formallytrained scientist, but the tools used here are those that would be available to the citizen scientists, with limited equipment and laboratory assistance. experimental temperature calibration and measurement of the ambient temperature during the study the first task in the project was to devise a lowcost, reliable and laboratory-independent method to monitor the ambient temperature during the h2s test kit incubations. during the water testing, the ambient temperature was monitored in the lounge, bathroom (sampling site) and the cupboard used to store the h2s test kit samples at the author’s house. this was done using the thermometer app (trajkovski labs, www.trajkovski.net). that measurement was done to establish whether the ambient/incubation temperature for the h2s test kits was inside the recommended interval of 18 – 25(37) °c (tandlich et al. 2012). the thermometer app was downloaded onto the author’s cell phone from google play store (alphabet inc., mountain view, usa). the temperature values, which were measured using the app during the study, are designated as tapp in further text of this article. the tapp readings were obtained by placing the cell phone with the active thermometer app onto the same surface in the respective room in the house or in the cupboard with the h2s test kit samples (see below), until a constant temperature reading was achieved. the average time for achieving a constant tapp value was equal to 2 – 5 min, if the ambient temperatures ranged from 17 to 25 ºc. the tapp readings stabilised after about 15 min, around the minimum and maximum temperatures measured in the study (table 1). the tapp readings were calibrated against the temperature readings taken suing a portable, but accurate thermometer (designated as treal in further text). the accurate thermometer was purchased from rs components (product number: rs stock no. 798-9133, midrand, south africa). this accurate thermometer was calibrated against the mercury-based thermometers in the author’s laboratory in the faculty of phamacy at rhodes university. the two types of thermometers were found to consistently provide the same readings of air temperatures. therefore the accurate thermometer was used in the study for the tapp vs. treal calibration. the tapp vs. treal calibration was done by taking readings with both methods at the author’s house on 10 sampling days during the study (table 1). if the temperature readings from the app are to be reliable, then there must be a correlation between the treal values and the tapp values. this correlation was investigated using the spearman’s correlation coefficient (rho) using the social sciences online calculator (https://www.socscistatistics.com/tests/spearman/de fault2.aspx). the correlation was also investigated using the pearson correlation coefficient (r) using the same programme (see https://www.socscistatistics.com/tests/pearson/defa ult2.aspx for details ). the rho and r values indicated a strong and linear correlation bewteen tapp and treal. thus an equation was derived using linear regression to convert tapp into the treal for the temperature monitoring during the microbial water quality sampling (microsoft excel 2013; microsoft south africa, johannesburg, south africa). the linear regression-based equation is shown in eq. 3, the tapp readings were taken throughout each sampling day and converted into the treal (see below). microbial water quality monitoring and volume calibration the microbial water quality monitoring was based on the use of the h2s test kit and a cell-phone app. all consumables were purchased from the same http://www.trajkovski.net/ https://www.socscistatistics.com/tests/spearman/default2.aspx https://www.socscistatistics.com/tests/spearman/default2.aspx https://www.socscistatistics.com/tests/pearson/default2.aspx https://www.socscistatistics.com/tests/pearson/default2.aspx nova biotechnol chim (2020) 19(1): 116-123 119 suppliers and the h2s test kits were prepared using the general methodology of luyt et al. (2011) and malema et al. (2019). those activities had been carried out in a microbiological laboratory in the faculty of pharmacy of rhodes university, makana local municipality before the lockdown began. minor changes in the logistics of the sampling were required, as compared to luyt et al. (2011) and malema et al. (2019). that was the result of the nationwide lockdown. sampling was performed on the makana-supplied municipal drinking/potable water in one sampling location, i.e. a bathroom at the author’s house. that sampling took place from 26th march 2020 (day 0 of lockdown) until 30 days into the lockdown (number of days increasing accordingly to the length into the lockdown period, e.g. 27th march 2020 was day 1 and so on). all samples were taken in the said bathroom at the author’s house from a sink tap/tap in a hand-wash basin. the house is located in kingswood, which is a middle-class to upper-middle class suburb in the grahamstown/makhanda area of makana local municipality. during a particular sampling, the outside of the h2s test kits and the sampled bathroom tap were surface-sterilised using baby wet wipes (purchased before the onset of the lockdown crazy store, grahamstown/makhanda, south africa). the single bathroom tap was opened for 10 seconds and one h2s test kit was filled to half volume with the sampled drinking water. the h2s test kit was then closed and hand-shaken for 10 seconds. the process was repeated with 5 h2s test kit during a single sampling. therefore a sample in further text represents one set of five h2s test kits taken on a single sampling occasion/day. the single-sample volume of potable water was measured by taking readings from the house’s municipal water meter right before and right after the individual sampling was completed. the municipal water meter at the author’s house was not calibrated by makana local municipality since 2015. therefore a citizen-science based calibration of the water meter was performed by the author after the lockdown level decreased, i.e. after the 1st june 2020. this was done with the minimum necessary use of laboratory resources. for the calibration, 3 five-litre plastic bottles were taken from the author’s house. the water meter reading was taken from the municipal water meter and it was recorded. then a single of those bottles filled up to the brim in the bathroom at the author’s house. right after the individual water bottle was filled to the brim, another reading was taken from the municipal water meter. the sampling with 5 litre bottles was repeated three times in total. all three bottles were then transported into the laboratory at rhodes university and weighed using a micro t7e balance (premier scale services, johannesburg, south africa). the weight of each 5 litre bottle, when filled with the sampled municipal drinking water was recorded. the water was then emptied out of the bottles and the inside of the bottles was dried. all of the five-liter bottles were weighed again and volume of each 5 litre bottle was calculated using eq. 1. (1) in eq. 1, mwater is the weight of the five-litre bottle filled with sampled water in kg. at the same time, mbotttle is the weight of the empty five-litre bottle in kg and ϱ is the density of water at the average treal in the author’s house and the laboratory at the faculty of pharmacy. the ϱ value was calculated as 998.4305 kg.m-3. after the particular sampling, the five h2s test kits were kept at room temperature in dark. colour change from brown to black was monitored for 72 hours, as mentioned in tandlich et al. (2014) and malema et al. (2019). a negative h2s test kit was recorded and was considered negative for faecal contamination, if the liquid colour inside it remained brown after 72 hours of incubation. on the other hand, an h2s test kit was recorded as positive for faecal contamination, if the liquid inside it turned black after 72 hours of incubation. results of the water samples were evaluated in the following way. each municipal water sample was assigned a score (x) of 0, 1 or 2. the score 0 was assigned to a sample, if all five h2s test kits were negative for the faecal contamination on a particular sampling occasion (nhokodi et al. 2016; malema et al. 2019). the sample was assigned a score of 1, if 1-4 h2s test kits were positive for the faecal contamination (nhokodi et al. 2016; malema et al. 2019). finally, the sample was assigned a score of 2, if all five h2s test kits were positive for the faecal contamination (tandlich et al. 2014; nhokodi et al. 2016; malema nova biotechnol chim (2020) 19(1): 116-123 118 et al. 2019). then the average h2s test kit score (xavg) was calculated using eq. 2. (2) in eq. 2, xi stands for the score in a particular one of the 12 samples taken at the author’s house during the study. if the potable water, supplied by makana local municipality to the auhtor’s house, was free of faecal contamination and microbially safe for human consumption, then the xavg value was not statistically significantly different from 0 at 5 % level of significance. if the potable water, supplied by makana local municipality to the author’s house, was not free of faecal contamination and microbially safe for human consumption based on the h2s test kit results, then the xavg value would be statistically and significantly different and higher than 0 at 5 % level of significance. the x data statistical testing was tested for normality using the kolmorogovsmirnov test at 5 % level of significance (see https://www.socscistatistics.com/tests/kolmogorov/ default.aspx for details). periodically throughout the study, blank samples were taken by pouring the oasis water was into 5 sterile h2s test kit. the oasis water is the reverseosmosis-treated water from oasis (grahamstown/makhanda, south africa). based on preliminary results which were obtained before the current study had begun, the oasis water was found to be free of faecal contamination. this was the case at the point of purchase and during storage of up to 7 – 9 weeks (data not shown). during the study, the blanks were prepared by pouring 20 ml of oasis water into five sterile h2s test kits using. blank sampling, processing and incubations were conducted a similar procedure, as stated for the samples above. all blanks were negative for faecal contamination throughout the study. therefore results of the study shown in table 2 were not influenced by the sampling process, sample processing or incubations during the study. in addition to blanks being processed, the surface of the sampled sink tap was tested periodically for the presence of potential faecal contamination. twenty ml of oasis water was poured into a sterile h2s test kit and the content was hand-shaken with the kit lid on. after the liquid turned brown inside the kit, a sterile cotton swab was submerged into the liquid. that moistened swab was used to swipe the faucet of the sampled sink. the process was repeated for the neck of the tap and the pipe leading into the sink. the cotton part of each swab was then cut with sterile scissors (sterilised with 70 % isopropanol hand sanitiser, buco, grahamstown/makhanda, south africa). twenty ml of oasis water was added and the kit was closed with the lid. contents were hand-shaken and the h2s test kits were incubated in the same fashion as samples or blanks. all of the swab kits were all found to be negative for faecal contamination after 72 hours of incubation. therefore, the surface sterilisation with the baby wet wipes was efficient in prevention of crosscontamination of the h2s test kits from the bathroom sink tap at the author’s house (the samples tap). based on this observation and the negative blanks, any faecal contamination detected in the potable municipal water at the author’s house originated from the distribution system or lack of treatment by makana local municipality. results and discussion temperature calibration and measurement of the ambient temperature during the study the values of tapp and the treal are shown in table 1. usage of the spearman’s correlation to describe the relationship between the independent and the dependent variable implies that there is a the relationship between the two, but this is not necessarily linear. the rho value for the tapp and the treal was equal to 0.9908 and the value was statistically significant on the 5 % level of significance (p-value = 0). therefore there was a general correlation between the tapp and the treal. the pearson correlation coefficient value (r) for the tapp and the treal was equal to 0.9902 and the value was statistically significant on the 5 % level of significance (p-value < 0.00001). the relationship between the two variables was linear, based on the r value. the final equation from the linear regression analysis can be seen in eq. 3. treal = 0.7803 + 0.8298 × tapp; r 2 = 0.9822 (3) temperature readings from the study and related to the microbial samples incubations can be found 120 https://www.socscistatistics.com/tests/kolmogorov/ nova biotechnol chim (2020) 19(1): 116-123 121 table 1. results of temperature calibration between the readings from the trajkovski lab app (tapp) and the accurate thermometer (treal). tapp (°c) treal (°c) 14 12 17 15 18 15 29 25 23 20 21 19 21 18 24 20 25 23 36 30 in table 2. as it can be seen, the treal ranged from 17 to 29 °c. the minimum value of treal was observed on days 13, 21 and 30 of the strictest phase of the national lockdown in south africa. this is outside of the interval from 18 to 25(37) °c which was previously reported as the optimum incubation temperature (tandlich et al. 2012). results of the h2s test kit might have been influenced by the fluctuations of the ambient temperature at the author’s house. however, this influence was likely minimal from a practical point of view, as the majority of the temperature readings were inside the optimum ambient temperature interval. on the other hand, the temperatures on days 13, 21 and 30 also icnreased above 17 °c for parts of the respective day. microbial water quality monitoring and volume calibration the average sampled volume of municipal potable water per single sampling was equal to (5.5 ± 0.2) table 2. results of the microbial water quality in makana local municipality from 26th march 2020 (day 0) and the lockdown days (day 1 to day 30 in the lockdown). sampling day treal (°c) xi (dimensionless) 0 20 – 22 1 1 21 – 24 1 2 25 – 29 0 3 22 1 5 22 – 23 1 7 23 1 10 22 – 23 0 13 17 – 21 1 17 21 0 21 17 – 22 1 24 23 – 27 2 30 17 – 21 1 litres. during the calibration of the water meter, the average volume of sampled water, based on the water meter reading, was on average also equal to (5.5 ± 0.2) litres. the volume of sampled water based on the weighing is equal to (5.1 ± 0.1) litres. the calculator for the social sciences, used in other test in this paper did not allow for the calculation due to low number of replicates. therefore the mann-whitney test at 5 % level of significance was performed using the statology calculator (see https://www.statology.org/mannwhitney-u-test-calculator/ for details). the test criteria were as follows: the u statistic = 11; p-value = 0.4895 for the two-tailed test. therefore, the volume readings based on the water meter and the weighing method/citizen-science calibration were not signifcantly different at 5 % level of significance. the x values for all 12 samples are shown in table 2. eight out of 12 samples (67 %) had a score of 1, i.e. on 8 sampling occasions between 1 and 4 h2s test kits were positive for faecal contamination. three of the twelve samples or 25 % were free of faecal contamination, i.e. had a x value of 0. one sample had a score of 2 and this occurred on day 24, when all five h2s test kits were positive for faecal contamination. microbial water quality for the makana municipal water supply was comparable just before the strictest lockdown phase and during its duration. compared to the results of nhokodi et al. (2016), the current study had a lower proportion of the municipal water samples which are free of faecal contamination, i.e. 25 % vs. 72 %. the x data statistical testing was tested for normality using the kolmorogov-smirnov test at 5 % level of significance. data was found not to be significantly different from a normal distribution (d-statistic = 0.36849, p-value = 0.05723). next, the t-test at 5 % level of significance was run to check whether xavg was statistically significantly different from 0. the xavg value was equal to (0.83 ± 0.57) and it was statistically and significantly different, as well as higher, than 0 (t statistic = 5, p-value = 0.0002-0.0004, at: https://www.socscistatistics.com/tests/tsinglesampl e/default2.aspx). that means the average x value (or level of faecal contamination of the tested drinking water) in makana local municipality was https://www.statology.org/mann-whitney-u-test-calculator/ https://www.statology.org/mann-whitney-u-test-calculator/ https://www.socscistatistics.com/tests/tsinglesample/default2.aspx https://www.socscistatistics.com/tests/tsinglesample/default2.aspx nova biotechnol chim (2020) 19(1): 116-123 122 higher than 0, i.e. there is significant indication that potable water in makana local municipality was microbially contaminated during days 0 – 30 of the strictest phase of the national lockdown in south africa. this, however, only applies to the author’s house. next, the t-test at 5 % level of significance was rerun to check whether xavg was statistically significantly different from 1. the xavg value was not statistically significantly different from 1 (t statistic = -1; p-value = 0.3388). therefore the municipal supply of drinking water at the kingswood suburb in grahamstown/makhanda was intermittently contaminated with faecal contamination during the 0 – 30 days of the strictest phase of the national lockdown in south africa. there were no occurence of diarrhoeal diseases at the author’s house during the sampled period. however, it has to be stated that the municipal water supply was not used for drinking. the domestic uses of the municipal drinking water were greywater production and cooking. results of the current study indicate that microbial water quality could have occurred in makana local municipality during the lockdown. these findings should, however, be treated with caution, as only one sampling location could be sampled. that was the result of taking place during the strictest phase of the covid19 lockdown in south africa, i.e. the limited movement of the population. further sampling is currently ongoing and it began once the limitations on movement have been eased across south africa, i.e. after 1st june 2020. ongoing sampling will be exnpanded from the middle-class and upper-middle-class neighbourhood to include the communal taps that are used to supply in low-income settlements around makana local municipality. these will be an extension of the networks and working relationship with stakeholders which have already been established (nqowana 2019). citizen science in water resource management has been studied by various authors in south africa (e.g. graham and taylor 2018). building of community of practice focused on various aspects of the water resource management and the use of web platforms have been utilised (graham and taylor 2018). many citizen-science water quality monitoring programmes have been ongoing around south africa and the world. in makana local municipality, a web-based tools to report problems with water service delivery had been developed some time ago (mobisam 2020). these and similar strategies have been instrumental in the improvement of tools for improved water management in south africa, empowerment of the population in water-related matters and in increasing some capacity in the water sector in the govenrment sphere around the country. collaboration between rhodes university and the technical university of the liberec, both of which the author is currently associated with, has been underway since 2019. it will be focused on the bench-marking the citizen science approach from this study against the best practices from the european union. in addition, the citizen science approach and relevant best practices will be benchmarked for the disaster risk and emergency management space through the the international emergency management society (tiems). this is an ongoing engagement of tiems on covid19 (tandlich et al. 2020). conclusions data on microbial quality of municipal water supply during the lockdowns are necessary for maintenance of hygiene measures and to curb the covid19 spread. in addition, microbial quality of drinking water supplies that are available to the south african population must be maintained in compliance with the regulatory requirements. this is necessary to prevent the development of cascading disasters impacts during the covid19 lockdowns. prevention of the cascading effects of disasters is critical to prevention of drainage human and other resources from the public health covid19 campaigns. preliminary data from this study provide a snapshot of possible problems with microbial water quality in the makana local municipality in the eastern cape. further research is ongoing. acknowledgement the author would like to express his gratitude to the rhodes university sandisa imbewu fund for providing funding for the consumables h2s test kits. neither of the draft nor the final version of the current study have undergone any kind of review by either any of the organisations that the author is affiliated with. therefore, no endorsement of the study by either of these institutions should be implied. nova biotechnol chim (2020) 19(1): 116-123 121 conflict of interest the author declares that they have no conflict of interest. references all africa (2020) south africa: water and sanitation provides water tanks and hygiene essentials to kwazulu-natal residents amid 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(in press). world health organisation (who 2020a). statement on the second meeting of the international health regulations (2005-2020) emergency committee regarding the outbreak of novel coronavirus (2019-ncov). world health organisation (who 2020b). coronavirus disease (covid-19) situation dashboard. 123 https://allafrica.com/stories/202006240245.html https://mobisam.net/ https://www.tandfonline.com/doi/full/10.1080/02508060.2020.1773648 nova biotechnol chim (2021) 20(2): e716 doi: 10.36547/nbc.716 1 nova biotechnologica et chimica incorporation of disaccharides and dimethyl sulfoxide into alginate beads increases post-thaw viability of immobilized saccharomyces boulardii yeast igor vysekantsev, iryna buriak, valentyna martsenyuk, tetiana gurina, evgeniya pushkova institute for problems of cryobiology and cryomedicine of the national academy of sciences of ukraine, 23, pereyaslavska str., kharkiv 61016, ukraine  corresponding author: buriakiryna@gmail.com article info article history: received: 22nd october 2020 accepted: 21st april 2021 keywords: cryopreservation cryoprotectants gel immobilization saccharomyces boulardii abstract gel-immobilized microorganisms are increasingly used in microbiological industries. currently, the problem of developing technologies for long-term storage of microorganisms immobilized in gel carriers remains urgent. low-temperature storage is the most effective method of preserving microorganisms. the viability of immobilized cells is affected by cryoprotective properties of gel matrix as well as cooling regimes. therefore, the effects of incorporation of cryoprotective agents in alginate gel and cooling regimes on the viability of immobilized saccharomyces boulardii cells after cryopreservation were studied. incorporation of non-permeable cryoprotectants (sucrose, lactose, and trehalose) and permeable one dimethyl sulfoxide (dmso) in alginate gel beads promoted an increase in the viability of immobilized cells after freeze-thawing. the highest viability rates of the gelimmobilized cells were observed in the alginate gel beads incorporating combinations of dmso (5 – 10 % v/v) and one of the disaccharides (10 – 20 % w/v). in all experiments, slow cooling provided significantly higher viability if compared to the rapid immersion of the samples into liquid nitrogen.  university of ss. cyril and methodius in trnava introduction microorganisms immobilized in gels are widely used in medicine, veterinary medicine, pharmaceuticals, food industry, animal feed production and biotechnology for environmental restoration. gels of polysaccharides (alginate, chitosan, carrageenan) are most often applied to immobilize probiotic products (shori 2017). bioreactors based on immobilized microorganisms in various industries have the number of technological and economic advantages (gbassi and vandamme 2012; martín et al. 2015; żur et al. 2016; shori 2017). the problem of storing immobilized microorganisms both at the stages of biotechnological production and in the form of commercial products is urgent. techniques for preserving gel-immobilized microorganisms are under development. cryopreservation and lyophilization are the most effective ways of preserving microorganisms of various taxonomic groups (she and petti 2015). it is logical that low temperatures and lyophilization can also be used to store the microorganisms immobilized in gel beads. previously the results of such studies have been reported (kearney et al. 1990; tsen et al. 2007; solanki et al. 2013; cagol et al. 2018). mailto:buriakiryna@gmail.com nova biotechnol chim (2021) 20(2): e716 2 cryopreserved samples are usually stored in liquid nitrogen (-196 °с) or in liquid nitrogen vapors at temperatures below -150 °с. during the cryopreservation, microorganisms are exposed to a number of damaging physicochemical factors at the cooling and warming stages, the severity of those is associated with water crystallization – recrystallization. it is possible to minimize the damaging effect of these factors by adjusting the cooling rates and introducing various cryoprotective agents (cpas) into the preservation medium (mazur et al. 1970; gordienko and pushkar' 1994; pegg 2007). investigation of the effect of the composition of preserving media and cooling rates on the viability of s. boulardii cells when freezing cell suspensions to -196 °c has shown that the highest viability was provided by cooling at a rate of 1 °c.min-1, at which the initial number of viable cells was observed (vysekantsev et al. 2012). with an increase in the cooling rate up to 5 – 40 °c.min-1, the number of viable cells decreased and was minimal after rapid cooling by immersing the samples into liquid nitrogen. the addition of sucrose as a cpa to the preserving medium contributed to a significant rise in cell viability in the samples frozen at a rate of 5 – 20 °c.min-1 (vysekantsev et al. 2012). it was also shown that freezing s. boulardii and escherichia coli cells to -196 °c in solutions of polysaccharides sodium alginate, carrageenan and agar also increased the cell viability compared to cell suspensions in physiological saline (vysekantsev et al. 2015). during the immobilization by ionotropic gelation, the beads (microspheres) acquire a rigid shell due to the cross-linking of sodium alginate molecules with divalent cations (ca2+) (woodward 1988). the manifestation of physicochemical factors during the cryopreservation of cells in alginate beads and in polysaccharide solutions will differ. this can affect the survival of probiotic microorganisms immobilized in the beads and, therefore, the therapeutic effect of their use. the probiotic strain of the yeast s. boulardii is widely exploited in medical practice and the food industry to treat and prevent the dysbiosis of various origins (mcfarland 2010; moré and swidsinski 2015). the immobilization of s. boulardii in gel beads and the development of methods for long-term storage of such products may hold promise for the pharmaceutical and food industries. the research was aimed to develop the composition of gel carriers and experimentally substantiate cooling regimes that will ensure a high viability rate of immobilized s. boulardii cells during the cryopreservation. experimental research object the s. boulardii cncm i-745 were isolated from the commercial product enterol® (biocodex, france). they were grown in wort agar plates with sugar content 8 ° according to balling scale (afanasʼyeva 1976) at 30 °c for 48 h. the cells were harvested and suspended in physiological saline (0.9 % w/v). the yeast concentration in the suspension was 1 × 108 cells.ml-1. immobilization to immobilize yeast cells, three series of alginate carriers incorporating cpas were prepared. in the first series, 1 % (w/v) sodium alginate gel incorporated one of the disaccharides: sucrose, trehalose or lactose. the concentration of disaccharides in the gel made 10 % and 20 % (w/v). in the second series, the 1 % sodium alginate gel contained dimethyl sulfoxide (dmso) at concentrations of 5 %, 10 %, and 15 % (v/v). in the third series, a 1 % sodium alginate gel contained various combinations of each of the disaccharides with dmso: 10 % or 20 % disaccharide + 5 %, 10 %, or 15 % dmso (table 1). all solutions were tyndalized (poliak et al. 2008). gel beads with the cells immobilized in them were obtained by ionotropic gelation (tsen et al. 2007). the bead diameter was 2000 ± 100 μm. the stabilization of the beads in the 0.2 m cacl2 solution lasted 20 min. before freezing, the samples were kept at 0 °c in melting ice. freezing the beads (by 100 pieces) were placed into 2.0 ml cryovials (nunc, usa). the samples were frozen in two ways: 1) rapid freezing down to -196 °c by nova biotechnol chim (2021) 20(2): e716 3 immersion into liquid nitrogen; 2) slow cooling at a rate of 1 °с.min-1 up to -40 °с in the programmable freezer (cryoson, germany) followed by immersion into liquid nitrogen. frozen samples were thawed after 10 days in a water bath at 37 °c for 10 min. viability assessment the beads were dissolved in 4 % edta (w/v) in a water bath at 30 °c. the viability of yeast cells was assessed by the plate koch’s method by colony formation on wort agar (lusta and fikhte 1990). the number of macrocolonies formed on the agar surface was recorded. each macrocolony was formed after the reproduction of one viable cell, i.e. the colony-forming unit (cfu). this easy method makes it possible to assess the integral characteristics of the viability of microbial cells. cell viability in beads was assessed before and after cryopreservation. in this case, the viability of the yeast before freezing served as a control. the result of assessing viability after freezing was presented as % of the control (eq. 1): (1) statistical analysis for statistical processing of experimental data, the averages x̅ and standard deviations sx̄ were determined. the results were presented as x̅ ± sx̄. to determine the statistical significance of differences between the comparison groups, student's t-test was used. the significance level was 0.05. the data obtained were statistically processed using the ms excel software. each series of experiments was performed six times (n = 6). results and discussion effect of cooling regimes on the viability of yeast cells immobilized in gel beads without cpas it was found that after rapid cooling, 14.69 % of yeast cells remained viable in gel beads without cpas. after slow cooling, 56.48 % of viable cells remained. the findings indicate that the cooling regimes affect the viability of s. boulardii yeast cells immobilized in alginate gel beads during the cryopreservation. higher viability of the immobilized cells was provided by slow cooling (1 °с.min-1). these results were similar to the data obtained in studies on suspensions of free cells s. cerevisiae (mazur 1961) and s. boulardii (vysekantsev et al. 2012). similar results were obtained with rapid and slow freezing of s. boulardii cells embedded in various gels, in particular sodium alginate without beads formation (vysekantsev et al. 2015). this dependence of the viability of yeast cells on the cooling rate was consistent with the main provisions of the twofactor and multifactorial theories of cell cryoinjury (mazur et al. 1970; pegg 2007). under slow cooling a more thermodynamically stable structure is known to be formed in samples to be frozen. this eliminates mechanical damage to cells during the freeze-warming. the alginate hydrogel itself exhibits cryoprotective properties due to its three-dimensional structure inhibiting the growth of ice crystals (brockbank and ga 1988; zhang et al. 2018), as well as due to hydrogen bonding with intracellular water molecules (brockbank and ga 1988) and the formation of a highly viscous intercellular environment, which acts as a mechanical barrier and protects cells against extracellular ice crystals (ponomareva et al. 2018). since the hydrogels contain mainly bound water (rehm 2009), during freeze-thawing in alginate gels, inhibition of crystallization-recrystallization occurs in comparison with aqueous solutions. additionally, the cooling rate also affects the dynamics and severity of these processes. viability of s. boulardii cells immobilized in gel beads incorporating dmso after rapid and slow cooling since the viability rates of cells immobilized in beads of 1 % (w/v) alginate gel after freezing were not high enough, in subsequent experiments the influence of cryoprotective substances with different mechanisms of protective action (permeable and impermeable) (fuller 2004) on the viability of immobilized cells was studied. dmso   %100%  freezingbeforecellsviableofnumber freezingaftercellsviableofnumber viability nova biotechnol chim (2021) 20(2): e716 2 fig. 1. viability of s. boulardii cells immobilized in alginate gel beads incorporating dmso (a); sucrose (b); lactose (c) and trehalose (d). ■ – rapid cooling; □ – slow cooling. note: a – significant differences as compared to the control (number of viable cells prior to freezing), p<0.05; b – significant differences between viability after rapid and slow cooling. was used as a permeable cpa. this cpa is most widely used in cryopreservation of eukaryotic cells (awan et al. 2020). it was found that the addition of 10 % or 15 % dmso to the composition of the alginate gel led to increasing of the number of viable immobilized yeast cells after rapid and slow freezing compared to the gel with no additives. cell viability after slow cooling of the beads significantly exceeded the viability of rapidly cooled cells. thus, after rapid cooling of the gel beads containing 10 % and 15 % dmso, the cell viability was 22.43 % and 28.67 %, and after slow cooling that made 72.54 % and 84.75 %, respectively (fig. 1a). in alginate gel beads incorporating 5 % dmso, a significant increase in cell viability (69.48 %) was observed after slow cooling. after rapid cooling of the beads incorporating 5 % dmso, cell viability did not increase. dmso has the ability to bind water that reduces the free water content in extracellular and intracellular spaces. the presence of dmso inside a cell reduces the probability of intracellular crystallization. due to this, a mechanical injury to cells was reduced owing to structural rearrangements of ice during freeze-thawing, and cell viability was increased. 0 10 20 30 40 50 60 70 80 90 100 5 10 15 v ia b il it y [ % ] dmso concentration [%] a,b a a,b a a a,b 0 10 20 30 40 50 60 70 80 90 100 10 20 v ia b il it y [ % ] sucrose concentration [%] a a a,b a,b a b 0 10 20 30 40 50 60 70 80 90 100 10 20 v ia b il it y [ % ] lactose concentration [%] a a a,b a,b 0 10 20 30 40 50 60 70 80 90 100 10 20 v ia b il it y [ % ] trehalose concentration [%] a a,b a,b a c d 4 nova biotechnol chim (2021) 20(2): e716 5 viability of s. boulardii cells immobilized in gel beads incorporating disaccharides after rapid and slow cooling the effect of incorporation of impermeable cpas, i.e. disaccharides to the alginate gel beads on the post-cryopreservation viability of immobilized yeast cells was studied. after rapid cooling to -196 °с in gel beads with 10 or 20 % sucrose, the viability was 35.1 and 30.9 % of cells, respectively (fig. 1b). after slow cooling, the beads incorporating 10 % sucrose retained 76.0 % of viable cells, and in the ones with incorporation of 20 % sucrose viability was kept at 78.2 % of the cells. in beads of alginate gel incorporating 10 or 20 % lactose, the viability of 22.43 and 28.67 % of cells, respectively, was observed after rapid cooling (fig. 1c). after slow cooling, 70.7 and 84.5 % of viable cells remained, respectively. the viability of yeast cells in gel beads with the addition of 10 and 20 % trehalose after rapid cooling was 25.0 and 31.3 % (fig.1d), and after slow cooling this was 67.3 and 74.1 %. the results obtained showed that the addition of sucrose, lactose, and trehalose disaccharides to the alginate gel contributed to significant increasing in the viability of immobilized s. boulardii cells after freezing to -196 °c. in all the samples, the number of viable yeast cells after slow cooling significantly exceeded the number of viable cells in rapidly cooled samples. impermeable cpas, i.e. disaccharides used in the experiments due to hydration reduce the amount of free extracellular water, and as osmotic agents they diminish the content of intracellular water (fuller 2004). this leads to a rise in the viscosity of solutions and to a decrease in cryodamage to cells during the formation of ice crystals. this assumption is supported by the studies of other authors, who demonstrated that the addition of trehalose and β-cyclodextrin to alginate beads reduced the content of free water and decreased its activity (calvo and santagapita 2016). during freezing, the content of freezable water as well as its activity decreased with increasing sucrose concentration in the sodium alginate solution (pongsawatmanit et al. 1999). viability of s. boulardii cells immobilized in gel beads incorporating the combinations of one of disaccharides and dmso after rapid and slow cooling the effect of incorporating combinations of a permeable cpa dmso and one of the disaccharides in alginate gel on viability of immobilized s. boulardii yeast cells after rapid and slow cooling was studied. after rapid cooling in gel beads incorporating combinations of 10 % sucrose and 5 %, 10 % or 15 % dmso, remained 27.22 %; 38.31 %; 26.52 % of viable cells (table 1), and in gel beads incorporating the combinations of 20 % sucrose and 5 %, 10 % or 15 % dmso the viability made 48.25 %; 38.7 %; 26.01 %, accordingly. after slow cooling in gel beads with the addition of combinations of 10, 20 % sucrose and 5, 10 % dmso, the number of viable cells did not differ from the control (before freezing). in samples of gel beads with the addition of combinations of 10 %, 20 % sucrose and 15 % dmso, cell viability was 84.22 % and 48.84 %. the viability of yeast cells immobilized in gel beads incorporating 10 % lactose combined with 5 %, 10 %, 15 % dmso after rapid cooling was 21.07 %; 30.33 %; 23.18 %, and in gel beads incorporating the combinations of 20 % lactose and 5 %, 10 %, 15 % dmso the viability was 35.93 %; 27.59 %; 14.47 % (table 1). after slow cooling in the gel beads with the addition of 10 % lactose and 5 %, 10 %, 15 % dmso, remained 80.07 %; 79.67 %; 63.04 % viable cells. in gel beads incorporating 20 % lactose and 5 %, 10 %, 15 % dmso after slow cooling the viability was 87.01 %; 81.28 %; 64.00 %. the number of viable yeast cells immobilized in gel beads incorporating the combinations of 10 % trehalose and 5 %, 10 %, 15 % dmso after rapid cooling was 29.49 %; 47.54 %; 30.41 %, and in gel beads with the addition of 20 % trehalose and 5, 10, 15 % dmso – 25.52; 44.2; 36.71 % (table 1). after slow cooling, the viability of cells immobilized in gel beads incorporated 10 % trehalose combined with 5 %, 10 %, 15 % dmso was 80.1 %; 87.43 %; 64.19 %. in the gel beads containing 20 % trehalose and 5 %, 10 %, 15 % dmso the viability made 74.9 %; 89.36 %; 83.25 %. slow cooling in samples of beads with the nova biotechnol chim (2021) 20(2): e716 6 addition of 5 % or 10 % dmso in combination with one of the disaccharides increases the viability rate of the cells in comparison with the samples containing one cpa. in this case, the combined protective effect of permeable and impermeable cryoprotective substance was observed table 1. viability of s. boulardii yeast immobilized in alginate gel incorporating the combinations of dmso and disaccharides after freezing to -196 °c. dmso [%] disaccharides viability [% from control] disaccharide type concentration [%] rapid cooling slow cooling x̅ ± sx̄ confidence level x̅ ± sx̄ confidence level 5 sucrose 10 27.22 ± 2.64 a, b, e 92.41 ± 3.04 b, e 20 48.25 ± 2.85 a, b, e 95.42 ± 2.82 b, e lactose 10 21.07 ± 2.16 a, b, e 80.07 ± 3.10 a, b, e 20 35.93 ± 2.84 a, b, e 87.01 ± 3.18 a, b, e trehalose 10 29.498 ± 2.10 a, b, e 80.1 ± 3.21 a, b, e 20 25.52 ± 2.58 a, b, e 74.9 ± 2.76 a, b, e 10 sucrose 10 38.31 ± 2.93 a, b, c, e 93.29 ± 2.55 b, e 20 38.7 ± 2.74 a, b, c, e 93.71 ± 2.90 b, e lactose 10 30.33 ± 22.41 a, b, c, e 79.67 ± 3.35 a, b, e 20 27.59 ± 2.70 a, b, c, e 81.28 ± 3.10 a, b, e trehalose 10 47.54 ± 2.16 a, b, c, e 87.43 ± 2.82 a, b, e 20 44.2 ± 3.05 a, b, c, e 89.36 ± 2.75 a, b, e 15 sucrose 10 26.52 ± 2.77 a, b, d, e 84.22 ± 3.80 a, b, d, e 20 26.01 ± 2.69 a, b, d, e 48.84 ± 2.77 a, b, d, e lactose 10 23.18 ± 2.14 a, b, d, e 63.04 ± 3.10 a, b, d 20 14.47 ± 3.17 a, b, d, e 64.00 ± 3.42 a, b, d trehalose 10 30.41 ± 3.25 a, b, d, e 64.19 ± 2.75 a, b, d, e 20 36.71 ± 2.90 a, b, d, e 83.25 ± 3.15 a, b, d, e 1 % alginate gel without additives 14.69 ± 0.35 a, b 56.48 ± 2.30 a, b x̅ – average, sx̄ – standard deviation, a – confidence level <0.05 between x̅ values before and after freezing down to -196 °c, b – confidence level <0.05 between x̅ values after slow and rapid freezing, c – confidence level <0.05 between x̅ values at dmso concentration of 5 % and 10 % and corresponding disaccharide concentrations (10 % and 20 %), d – confidence level <0.05 between x̅ values at dmso concentrations of 10 % and 15 % and corresponding disaccharide concentrations (10 % and 20 %), e – confidence level <0.05 between x̅ values in 1 % alginate gel samples without additives and in the samples with cryoprotectants. (pakhomova et al. 2013). obtained results indicate that the viability of cells immobilized in gel beads containing the combination of disaccharide with dmso does not exceed the viability rate of yeast in beads during rapid cooling in comparison with beads containing only one cryoprotective agent. when immersed into liquid nitrogen, the protective mechanisms of both impermeable and permeable cryoprotective substances do not have time to be fully implemented. conclusions slow cooling (1 °с.min-1) provides a higher viability of s. boulardii cells immobilized in alginate gel beads both without additives and with cryoprotective substances. incorporation of nova biotechnol chim (2021) 20(2): e716 7 impermeable cpas (sucrose, lactose, trehalose) and a permeable one (dmso) as well as the combinations of these disaccharides with dmso in the alginate gel increases the viability of s. boulardii cells immobilized in the gel after freezing to -196 °c. the viability of s. boulardii cells immobilized in alginate gels incorporating the combinations of 5 – 10 % dmso and one of the disaccharides at a concentration of 10 – 20 %, after slow freezing, exceeds the cell viability rates in gel samples with the addition of one cpa (dmso or disaccharide). the maximum viability of the immobilized cells after slow cooling was observed in gel beads incorporating 5 – 10 % dmso combined with 10 – 20 % sucrose or 10 % dmso combined with 20 % trehalose. taking into account recommendations of the food and agriculture organization of the united nations and the world gastroenterological organization, regarding the composition of various commercial probiotic and symbiotic products, and our results, the incorporation of disaccharides into the gel carriers for immobilization can be proposed. this is relevant for technologies for long-term storage of immobilized probiotic microorganisms. for freezing low cooling rates are preferable to be used. acknowledgement this research was funded by the national 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microbiol. 53: 215-219. vysekantsev i, artuyants a, buriak i (2015) preservation rate of microorganisms after freezing down to -196 °c in noncovalent gels. j. tissue sci. eng. 6: 88. vysekantsev ip, babinets om, martsenyuk vf, gurina tm (2012) comparative study of cryopreservation regimens on free and immobilized cells of saccharomyces boulardii probiotic. probl. cryobiol. 22: 21-29. woodward j (1985) immobilized cells and enzymes: a practical approach, irl press, oxford, uk, 177 p. zhang c, zhou y, zhang l, wu l, chen y, xie d (2018) hydrogel cryopreservation system: an effective method for cell storage. int. j. mol. sci. 19: 3330. żur j, wojcieszyńska d, guzik u (2016) metabolic responses of bacterial cells to immobilization. molecules. 21: 958. nova biotechnol chim (2019) 18(1): 59-65 doi: 10.2478/nbec-2019-0008  corresponding author: r.tandlich@ru.ac.za nova biotechnologica et chimica effect of fly ash-lime treatment on the acute toxicity of greywater towards daphnia magna sinoyolo nondlazi1, nosiphiwe ngqwala1, bongumusa m. zuma2, paul k. mensah3 and roman tandlich1, 1 environmental health and biotechnology research group, division of pharmaceutical chemistry, faculty of pharmacy, rhodes university, grahamstown 6140, south africa 2 lugaju innovations, water sciences unit, 3-33 philip frame rd, frame office park, chiselhurst, east london/buffalo city metropolitan municipality 5247, south africa 3 institute for water research, rhodes university, grahamstown 6140, south africa article info article history: received: 31st december 2018 accepted: 12th may 2019 keywords: daphnia magna mixed effluent acute toxicity small-scale irrigation abstract acute toxicity of raw and treated greywater towards daphnia magna was assessed in this study. treatment was performed with exposure of greywater to the fly-lime mixture after 48 h of exposure, 100 % mortality of d. magna was recorded when testing the following volumetric fractions of the raw greywater streams in the tested liquid medium (%; v/v): 10 % for kitchen greywater, 5 – 10 % for bathroom greywater and 1.25 – 10 % for laundry greywater. after greywater treatment with the fly-ash-lime mixture with ph adjustment to 7.0, 80 % of neonates of d. magna survived after exposure to treated laundry greywater in all dilutions at 48 h. at the same time, 100 % of neonates survived exposure to treated bathroom and kitchen greywater at all volumetric fractions. therefore greywater had acute toxicity to d. magna, i.e. greywater treatment was required before its discharge or reuse. values of the pearson’s correlation coefficient between the chemical components of the raw greywater and treated greywater and the survival of d. magna indicated a lack of statistically significant correlation at 5 % level of significance (p-value > 0.05 in all cases), i.e. the survival of d. magna was independent of the concentration of chemical constituents in greywater samples tested. further studies will have to be conducted on the chronic toxicity of the greywater effluent after treatment with the fly-lime mixture. experiments from this study will have to be re-run for the fully scaled-up version of the fly-lime mixture-based greywater treatment systems.  university of ss. cyril and methodius in trnava introduction greywater accounts for a large volume of the drinking water that is consumed daily in a household (nondlazi et al. 2017). composition of greywater undergoes fluctuations and it can be considered a complex waste (chemical and biological components; abed and scholz 2015). if greywater is to be reused, it must be classified as an effluent and its potential toxicity to the environment must be carefully evaluated. toxicity can be evaluated by measuring concentrations of the following parameters: chemical oxygen demand (cod), heavy metals, organic pollutants and faecal coliforms (hernandez leal et al. 2012). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:09 utc mailto:r.tandlich@ru.ac.za nova biotechnol chim (2019) 18(1): 59-65 60 however, measurement of at least some of these parameters is time-consuming and expensive; and it is fair to say that it is almost impossible to achieve complete (chemical) characterisation of effluents with complex chemical composition (complex effluents; villegas-navarro et al. 1999; li et al. 2009). greywater would be an example of such a complex effluent and its toxicity must be characterised using holistic approaches. biomonitoring is an important tool used to evaluate environmental changes based on biological responses (oertel and salánki 2003). daphnia spphas long been considered and used as a standard organisms in water ecotoxicology (adema 1978), in part due to its sensitivity and toxicity profile which is compatible with the toxicity profiles of higher organisms (martins et al. 2007). in addition, this organism is highly responsive to exposure towards toxic components of effluents, its parthenogenesis and other factors result in quick reproduction of the crustacean; and d. magna is also easy to cultivate (in a laboratory; sakai 2001; gopi et al. 2012). nondlazi et al. (2017) reported on the efficiency of a pilot-scale version of the flyash-lime-filter tower (flft) in greywater treatment. this system can be classified as a sanitation technology and thus toxicity testing with d. magna can be used to characterise the flft effluent before discharge (tyagi et al. 2007). here the authors seek to perform the acute toxicity testing of the simulated flft effluent. experimental consumables concentrations of the following chemical parameters were determined in greywater samples: ammonium (nh4 +), chloride (cl-), phosphate (po4 3-), nitrate (no3 -), and chemical oxygen demand (cod) as outlined by nondlazi et al., 2017). the ph value was measured using the hanna comb ph meter (hannah instruments, south africa). the turbidity values were measured using the lutron tu-2016 portable turbidity meter (the instrument group, south africa). mechanical shaking was done using the ts-520d orbital shaker (already enterprise inc., taiwan). all masses were measured using a pioneer™ pa214 analytical balance purchased from ohaus corporation (usa). hydrochloric acid (hcl) 32 % (w/v) was purchased from merck (pty.) ltd. (south africa). the daphtoxkit f ™ magna was purchased from toxsolutions (south africa – an authorized distributor of microbiotests in south africa). the daphtoxkit f ™ magna contents were used as purchased from the supplier. fly ash and lime were procured as outlined earlier (nondlazi et al. 2017). greywater sampling the greywater samples were collected in 1 dm3 schott bottle, which were sterilised as outlined by nondlazi et al. (2017). the individual greywater streams were collected separately, i.e. as laundry greywater, kitchen greywater and bathroom greywater. all schott bottles were filled to the brim with the respective streams of greywater, the bottles were capped and stored at 4 °c until analysis/use was performed. the following operations were then performed according to the standard operating procedure developed by the manufacturer (microbiotests 2015): preparation of the standard freshwater solution according to iso 6341, the hatching of the dormant eggs of d. magna (ephippia) and the range-finding test. the number of dead or immobilised neonates was recorded after 24 h and 48 h. volumetric fractions and greywater concentrations are considered synonymous in the text of this article. toxicity testing to assess the treated greywater toxicity, treatment of the sampled greywater with the fly-ash/lime layer of the flft was simulated (nondlazi et al. 2017). for this, 10 g of fly ash/lime was weighed out using a pioneer™ pa214 analytical balance and transferred into a 100 cm3 erlenmeyer flask. next, 30 cm3 of greywater was added into the erlenmeyer flask and the ph of the liquid phase/suspension was adjusted to 7.0 using 5 m hcl. treatment was performed by shaking of the erlenmeyer flasks on the mechanical orbital shaker at 150 rpm at 25 °c for 96 h. the ph values of all suspensions of greywater and the fly-ash-lime mixture were monitored every 24 h and re-adjusted bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:09 utc nova biotechnol chim (2019) 18(1): 59-65 61 to 7.0 using 5 m hcl as needed. after 96 h, the samples were removed from the shaker and left to stand at room temperature for 30 min to settle the particulate matter. then the supernatant was transferred to volumetric flask, and relevant dilutions were performed. the experiments were conducted in 2016 and all d. magna were procured from a commercial source. therefore no wild crustaceans were captured, harmed or sacrificed during conducting of this study. next, the definitive and acute toxicity test was performed with d. magna according to the manufacturer’s instruction (microbiotests 2015). statistical evaluation of the measured data paleontological statistics software for education (past) version 2.17c was used to conduct the statistical analysis of the data. the mortality and immobility data of the greywater samples were compared to the control by means of the mannwhitney test at 5 % level of significance. further statistical analysis was done to determine the correlation between the concentration of the chemical constituents of the greywater and the survival of daphnia magna. this was done by means of pearson’s correlation coefficient calculator (stangroom 2016). the correlation coefficients were calculated to indicate, whether the raw and/or treated greywater characteristics are likely important causes for the observed toxicity levels towards d. magna. results and discussion physicochemical analyses results results of physicochemical analyses for the raw greywater and the treated greywater are shown in table 1 and 2. the physicochemical constituents of the raw greywater fell inside the following intervals: ph < 6.83±0.06, 7.13±0.06 >; turbidity < 51.3±0.6, 85±2 > ntu; cod < 571±22, 658±20 > mg.dm-3; no3 < 18±0.6, 20±1> mg.dm-3; po4 3< 0.54±0.03, 0.84±0.03 > mg.dm-3, cl< 8.94±0.07, 9.9±0.3 > mg.dm-3 and nh4 + < 44.7±0.5, 62.1±0.6 > mg.dm-3. table 1. characteristics of raw greywater.* parameter bgw kgw lgw ph 6.94±0.07 7.13±0.06 6.83±0.06 turbidity 51.3±0.6 68.7±0.6 5.0±2.0 [ntu] cod 571±22 658±20 616±31 [mg.dm-3] no3 18.00±0.60 20.0±1.0 19.7±1.0 [mg.dm-3] po430.77±0.04 0.54±0.03 0.84±0.03 [mg.dm-3] cl8.9±0.1 9.9±0.3 9.7±0.3 [mg.dm-3] nh4+ 44.7±0.5 55.2±0.7 62.1±0.6 [mg.dm-3] *bgw: bathroom greywater, kgw: kitchen greywater, lgw: laundry greywater. values of the physicochemical parameters of the treated greywater samples (table 2) fell inside the following intervals: ph < 7.02±0.04, 7.05±0.07 >; turbidity < 21±0.0, 25±0.0 > ntu; cod < 543±13, 552±16 > mg.dm-3; no3 < 17.8±0.2, 19.3±0.7 > mg.dm-3; po4 3< 0.08±0.01, 0.60±0.02 > mg.dm-3; cl< 6.35±0.09, 9.9±0.2 > mg.dm-3 and nh4 + < 22.35±0.38, 22.73±0.38 > mg.dm-3. there was no statistical difference between chemical parameter of the untreated/raw and treated greywater in all the different types of greywater. the p-values ranged from 0.076 to 0.081 for the laundry, kitchen and laundry greywater for cod, nh4 +, cl-, po4 and no3 -. table 2. characteristics of treated greywater.* parameters bgw kgw lgw ph 7.03±0.05 7.05±0.07 7.02±0.04 turbidity [ntu] 21±0 21±0 25±0 cod 552±16 552±13 543±13 [mg.dm-3] no3 17.8±0.2 19.3±0.7 17.9±0.7 [mg.dm-3] po4 30.08±0.01 0.09±0.01 0.60±0.02 [mg.dm-3] cl6.35±0.09 9.90±0.20 7.51±0.77 [mg.dm-3] nh4 + 22.73±0.38 22.57±0.38 22.35±0.38 [mg.dm-3] *bgw: bathroom greywater, kgw: kitchen greywater and lgw: laundry greywater. acute toxicity test with d. magna it has been shown in literature that the survival rates of d. magna reached maximum values, when bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:09 utc nova biotechnol chim (2019) 18(1): 59-65 62 the crustacean is exposed to the ph values from 7.00 to 8.33 (ghazy et al. 2011). because contact with the fly ash-lime particles led to an increase in the ph the greywater to around 13.1 (zuma and tandlich 2017), the ph values of the treated greywater was adjusted to around 7.00 to guarantee optimum conditions for the d. magna survival. the survival of d. magna that was exposed to the control (standard freshwater solution) was 100 % (n = 4), i.e. the viability of the test organism without any exposure to the (diluted) greywater was likely optimum under the conditions of the acute toxicity test. results of the toxicity testing are shown in fig. 1 and 2. after 48 h of exposure to the individual raw greywater streams, complete mortality of d. magna was observed at the following greywater or concentrations of each stream: 10 % for raw kitchen, 5 – 10 % for raw bathroom and 1.25 to 10 % for laundry greywater. laundry detergents contain surfactants, bleaching agents and additives (misra et al. 2010). hernandez leal et al. (2012) suggests that the toxic effect of greywater towards d. magna was caused by the anionic surfactant concentrations above 80 mg.l-1. this could have been the case in the present study. the survival of d. magna was significantly affected (p-values = 0.0124) by exposure to raw laundry greywater as a 100 % mortality is observed from the 1.25 – 10 % concentration, in comparison with the control (fig. 1c). the treated laundry greywater showed a slight but significant difference against the control with an average of 22 % mortality from the 2.5 – 10 % concentration after 48-hour exposure (p-value = 0.0131) (fig. 2a). there was a significantly lower survival rate of d. magna in the raw bathroom greywater (for all measured concentrations) (fig. 1) when compared to the survival in the treated sample (p = 0.0147) (fig. 2). a 100 % mortality was observed in the 5 and 10 % concentrations, which is significantly lower when compared to the 0 % mortality in the treated samples at the same greywater concentrations. the exposure to raw kitchen greywater resulted in a significantly lower survival rate of the neonates (fig. 1b), when compared to the survival rate of d. magna exposed to the treated kitchen greywater (p = 0.0131) (fig. 2b). in the treated greywater a 100 % survival was observed in all the greywater concentration greywater concentration greywater concentration fig. 1. survival of the d. magna reported as percentage of the total number of individual crustaceans after 48 h exposure to raw greywater (a ‒ bathroom, b ‒ kitchen and c ‒ laundry) at different concentrations (n = 5). a b c bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:09 utc nova biotechnol chim (2019) 18(1): 59-65 63 . five concentrations, and for the raw greywater 0 %; 1 % and 4 % survival was observed for the 10 %; 5 % and 2.5 % greywater concentrations respectively. the exposure to mixed greywater was done only for the treated greywater, and a slight but significant difference (p = 0.01312) in the survival of the d. magna neonates when compared to that of the control samples. these results suggest that the raw greywater would require dilution before reuse or disposal, as this would ensure that the raw greywater is not toxic to the environment. a 1:1 dilution ration is usually recommended for greywater intended for reuse (mzini and winter 2015). an 80 % survival of the neonates was observed at the 10 % greywater concentration. a 0 % mortality was observed for the d. magna neonates exposed to the treated bathroom and kitchen greywater at all concentrations. the increasing concentrations of nitrate found in surface water and groundwater are becoming a concern, yet little information has been published on toxicity of nitrate to common organisms used for toxicity testing (scott and crunkilton 2000). nitrate naturally enters groundwater and surface water via runoff from decomposition of vegetation, natural geological deposits, soil nitrogen, and atmospheric deposition (scott and crunkilton 2000). the acute toxicity of nitrate to several species of fish generally falls between 100 and 1,000 mg.l-1 no3-n (tomasso and carmichael 1986). the survival rate of d. magna starts to decrease when the concentration of nitrates in the water is 462 mg.l-1 no3-n (scott and crunkilton 2000). in this study the nitrate concentration was calculated to be in the range 17.8±0.2 – 20±1 mg.l-1 which would not be toxic to the d. magna. in this study, concentration of phosphate ions were obtained to be in the ranges of 0.08±0.01 – 0.84±0.03 mg.dm-3. chloride is abundant in natural waters and is an important constituent in biological functions (elphick et al. 2011). it facilitates a variety of ion-exchange mechanisms though trans-membrane chloride channels and it forms salts with each of the major cations (na, k, ca, and mg), but is highly soluble and exists primarily in the environment as a dissociated monovalent anion (elphick et al. 2011). the toxicity of chloride in aquatic environments is a result of its tendency to occur at elevated concentrations in effluents greywater concentration greywater concentration greywater concentration fig. 2. survival of d. magna after 48 h exposure to greywater leached with fly ash/lime (a ‒ laundry, b ‒ kitchen and c ‒ bathroom greywater) at different concentrations (n = 5). a b c bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:09 utc nova biotechnol chim (2019) 18(1): 59-65 64 from industrial operations (van voast 2003). an acute toxicity of d. magna study conducted by elphick et al. (2011) shows that at concentration levels of 0.2 to 1.0 mg.l-1 of chloride the survival rate of d. magna is within the ranges of 62.5 – 75 % (n = 10 daphnids). the chloride concentration of the greywater samples used in this study was obtained to be in the range of 6.35±0.09 – 9.9±0.3 mg.dm-3, which can still encourage the 62.5 – 75 % survival and growth rate of the d. magna in the sample. further statistical analysis was done to determine the correlation between the concentration of the chemical constituents of raw greywater and the survival of d. magna in raw greywater. this was done by means of pearson’s correlation (r) and the respective p-values (stangroom 2016). the correlation between the po4 3concentration and the survival of d. magna was characterised by the r value of -0.3259, but the negative correlation was not statistically significant at 5 % level of significance as the p-value = 0.3920. the r for nh4 + and the survival of d. magna was obtained and found to be -0.2537, the r value for cod and the survival of d. magna was equal to -0.0695 and finally the r value for the correlation between the concentration of chlorides and the survival of d. magna was fund to stand at 0.0363. all these correlations were not statistically significant at 5 % level of significance as the p-value were higher than 0.05 in all cases. there was also no statistically significant correlation between the no3 concentration and the survival of d. magna in raw greywater, as indicated by the following values r = 0.2042 with the p-value of 0.598. therefore, the concentrations of the individual components of raw greywater did not influence the survival/mortality of d. magna in the acutetoxicity test performed in this study. please add the following sentence: it is possible that the cause for the toxicity of raw greywater to d. magna might have been a result of the cumulative toxic effect of the greywater components measured. the correlation between the treated greywater concentrations of po4 3and the survival of the d. magna had the respective r value of 0.2775, but that correlation was not statistically significant as the respective p-value was equal to 0.3820. similar trends were observed for the other chemical components of greywater. the r for cland the survival of d. magna in the treated greywater after 48 h was equal to -0.2252 and the p-value > 0.05. for no3 and the survival of d. magna in the treated greywater, the r value was 0.2289 and the p-value was again higher than 0.05. finally, the ammonium ions and the survival of d. magna in the treated greywater had the r value of 0.3206 and the p-value > 0.05, while the r value for the survival of d. magna and the cod concentration was equal to 0.0253 and the respective p-value of 0.938. these results indicate that there was no statistically significant relationship between the concentration of the chemical constituents and the survival rate of the d. magna in treated greywater. conclusions raw greywater in this study was toxic to d. magna in the acute toxicity test. leaching greywater with fly ash/lime resulted in a significant decrease in the turbidity and other physicochemical constituents of the treated greywater after 96 h. the treatment also significantly reduced the toxicity of greywater, as it was observed in the survival rate of the d. magna. toxicity testing, as performed in this study, can be an effective tool to assess the effluent quality from various greywater streams. further studies will have to be conducted on the chronic toxicity of the flft effluent and on the applicability of this approach in fully scaled up flft. conflict of interest the authors declare that they have no conflict of interest. references abed ns, scholz m (2015) chemical simulation of greywater. environ. tech. 37: 1631-1646. adema dmm (1978) daphnia magna as a test animal in acute and chronic toxicity tests. hydrobiologia 59: 125-134. elphick jr, bergh kd, bailey hc (2011) chronic toxicity of chloride to freshwater species: effects of hardness and implications for water quality guidelines. environ. toxicol. chem. 30: 239-246. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:09 utc nova biotechnol chim (2019) 18(1): 59-65 65 ghazy mme, habashy mm, mohammady ey (2011) effects of ph on survival, growth and reproduction rates of the crustacean, daphnia magna. aust. j. basic appl. sci. 5: 1-10. gopi ra, ayyappan s, chandrasehar g, krishna v, goparaju a (2012) effect of potassium dichromate on the survival and reproduction of daphnia magna. bull. environ. pharmacol. life sci. 1: 89-94. hernandez ll, soeter am, kools sae, kraak mhs, parsons jr, temmink h, zeeman g, buisman cjn (2012) ecotoxicological assessment of greywater treatment systems with daphnia magma and chironomus riparius. water res.. 46: 1038-1044. li fy, wichmann k, otterpohl r (2009) review of the technological approaches for grey water treatment and reuses. sci. total environ. 407: 3439-3449. martins j, teles, lo, vasconcelos v (2007) assays with daphnia magna and danio rerio as alert systems in aquatic toxicology. environ. internat. 33: 414-425. microbiotests (2015) daphtoxkit f magna: crustacean toxicity screening test for freshwater (standard perating procedure). available at: http://www.microbiotests.be/toxkit-microbiotests/testprotocols/ (website accessed on march 31st, 2019). misra rk, patel jh, baxi vr (2010) reuse potential of laundry greywater for irrigation based on growth, water and nutrient use of tomato. j. hydrol. 386: 95-102. mzini ll, winter k (2015) analysis of grey-water used for irrigating vegetables and possible effects on soils in the vicinity of umtata dam, eastern cape. water sa 41: 115-120. nondlazi s, ngqwala np, zuma bm, tandlich r (2017) investigating the viability and performance of the pilot scale fly ash/lime filter tower for onsite greywater treatment. desalin. water treat. 91: 349-364. oertel n, salánki j (2003) biomonitoring and bioindicators in aquatic ecosystems. in ambasht rs, ambasht nk, modern trends in applied aquatic ecology, springer, boston, usa, p. 219-246. sakai m (2001) chronic toxicity test with daphnia magna for examination of river water quality. j. environ. sci. health b 36: 67-74. scott g, crunkilton rl (2000) acute and chronic toxicity of nitrate to fathead minnows (pimephales promelas), ceriodaphnia dubia, and daphnia magna. environ. toxic. chem.19: 2918-2922. stangroom j. (2016) social statistics, pearson correlation coefficient calculator (2016) available at: http://www.socscistatistics.com/tests/pearson/default2.a spx (website accessed on: december 25th, 2016). tomasso jr, carmichael gj (1986) acute toxicity of ammonia, nitrite, and nitrate to the guadalupe bass, micropterus treculi, bull. environ. contam. toxicol. 36: 866-870. tyagi vk, chopra ak, durgapal nc, arvid k (2007) evaluation of daphnia magna as an indicator of toxicity and treatment efficiency of municipal sewage treatment plant. j. appl. sci. environ. mgt. 11: 61-67. van voast wa (2003) geochemical signature of formation waters associated with coal-bed methane. aapg. bull. 87: 667-676. villegas-navarro a; romero gonzalez mc, rosas lopez e (1999) evaluation of daphnia magna as an indicator of toxicity and treatment efficacy of textile wastewater. environ internat. 25: 619-624. zuma bm, tandlich r (2017) modifications and monitoring of the laboratory scale greywater filter tower treatment system (chapter 6). in tandlich r (ed.) novel approaches to rainwater harvesting and sanitation in developing countries, nova science publishers, new york, usa. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:09 utc http://www.microbiotests.be/toxkit-microbiotests/test-protocols/ http://www.microbiotests.be/toxkit-microbiotests/test-protocols/ http://www.socscistatistics.com/tests/pearson/default2.aspx http://www.socscistatistics.com/tests/pearson/default2.aspx nova biotechnol chim (2018) 17(2): 125-131 doi: 10.2478/nbec-2018-0013  corresponding author: peter.pristas@upjs.sk nova biotechnologica et chimica genetic variability in acidithiobacillus spp. – a working horse of environmental biotechnologies peter pristas1,, jana kiskova1, ivana timkova1, lenka malinicova1, alena luptakova2, maria kusnierova2 and jana sedlakova-kadukova1 1department of microbiology, institute of biology and ecology, faculty of science, pavol jozsef šafarik university in košice, šrobárova 2, košice, 041 54, slovakia 2institute of geotechnics, slovak academy of sciences, watsonova 45, košice, 043 53, slovakia article info article history: received: 16th october 2018 accepted: 21st december 2018 keywords: acidithiobacillus complex species environmental biotechnology genetic variability phylogeography abstract the genus acidithiobacillus comprises 7 species of gram-negative obligatory acidophilic chemolithotrophic bacteria that derive energy mainly from the oxidation of reduced sulphur compounds. four of the species also catalyse the dissimilatory oxidation of ferrous iron while three (a. thiooxidans, a. albertensis, and a. caldus) do not. bacteria from the genus acidithiobacillus are often associated with mineral biotechnologies (biomining) and acid mine drainage. while acceleration of mineral solubilisation is a positive aspect in environmental biotechnologies, it is undesirable in acid mine drainage with strong negative ecological impact and there is profound interest in genetics and genomics of these bacteria. representatives of acidithiobacillus genus occur world-wide, however there are limited data on acidithiobacillus spp. variability from slovakia. in our work the variability of acidithiobacillus spp., from slovakia was analysed and the presence of a ferrooxidans was detected. in addition, for the first time we report here on the occurrence of a. albertensis as well. comparative analyses confirmed pronounced genetic and genomic diversity within the genus, especially within a. ferrooxidans and a. thioxidans complexes. based on data presented, several acidithiobacillus species could be considered as a complex species and the description of several new species is very probable in the near future.  university of ss. cyril and methodius in trnava introduction the genus acidithiobacillus comprises a group of gram-negative obligatory acidophilic chemolithotrophic bacteria that derive energy mainly from the oxidation of reduced sulphur compounds. the members of the genus are motile by one or more flagella and comprise both mesophiles and moderate thermophiles (kelly and wood 2000). acidithiobacilli use various sulphur compounds as electron donor and therefore, are often found in metal sulphide deposits all over the world (karavaiko et al. 2003), fresh waters associated with sulphide deposits (gonzalez-toril et al. 2003) and seawater (kamimura et al. 2003). the final electron acceptor is usually oxygen (kelly and wood 2000) however, fe3+ and s0 were reported to be alternative electron acceptors for some species (ohmura et al. 2002). the ability to fix co2 makes acidithiobacilli independent of an organic carbon source and allows them to thrive in mine waste ecosystems where the organic carbon concentration is extremely low (dold et al. 2005). the acidithiobacillus genus was formed when the former thiobacillus genus was split into the genera bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:21 utc nova biotechnol chim (2018) 17(2): 125-131 126 acidithiobacillus, halothiobacillus, and thermithiobacillus (kelly and wood 2000). the type species of the genus, a. thiooxidans was described nearly 100 years ago as thiobacillus thiooxydans (waksmann and joffe 1922) and up to now 7 species within the genus were validly described. all species are autotrophs capable of growth utilising inorganic sulphur compounds as sole energy substrate. four of the species (a. ferriphilus, a. ferrivorans, a. ferrooxidans, and a. ferridurans) also catalyse the dissimilatory oxidation of ferrous iron while three (a. thiooxidans, a. albertensis, and a. caldus) do not. representatives of acidithiobacillus genus occur world-wide in a diverse range of natural (acid rock drainage, sulphur springs, etc.) and industrial settings (ore concentrates, leaching solutions of the mining industry, etc.), with varying physicochemical characteristics. bacteria from the genus acidithiobacillus are often associated with mineral biotechnologies (biomining) and acid mine drainage. biomining utilises these bacteria for recovery of metals from sulphidic low grade ores and concentrates. acid mine drainage results in acidification and metal contamination of soil and water emanating from the dissolution of metal sulphides from deposits and mine waste storage (johnson 2014). acidophilic microorganisms play a central role in these processes by catalysing aerobic oxidation of sulphides. while acceleration of mineral solubilisation is a positive aspect in mineral biotechnologies, it is undesirable in acid mine drainage with strong negative ecological impact and there is profound interest in genetics and genomics of these bacteria. dna based analyses have already revealed pronounced genetic diversity within acidithiobacillus genus, especially within a. ferrooxidans complex. the diversity probably could be not attributed to the geographic distribution of acidithiobacilli as practically all known species were detected worldwide (nunez et al. 2017). more than 50 % of all acidithiobacillus isolates characterised at 16s sequence level can be mapped to asia, followed by europe (20 %), south (9 %), and north america (7 %), and africa and oceania (less than 2 % each). from the slovakia territory two isolates were characterised, both identified as a. ferrooxidans (nunez et al. 2017). the aim of our study was to understand the variability within acidithiobacilli in slovakia and worldwide based on genetic and genomic analyses. experimental molecular identification of acidithiobacillus spp. isolates from the slovakia molecular identification of acidithiobacillus spp. isolated recently from smolnik (east slovakia) acid mine drainage water – lc isolate (luptakova et al. 2012) and a. albertensis ts isolate from hodrušahámre (central slovakia) gold mine (pristas et al.; manuscript in preparation) were performed using widely accepted 16s based methodology (yarza et al. 2014). the sequences obtained were deposited into genbank database under accession numbers mh740926 and mh796351 for lc and ts isolate, respectively. the sequences were compared against database of 16s sequences of type strains of bacteria and archaea using blastn analysis (altschul et al. 1990). phylogenetic placement of isolates was confirmed by multiple sequence comparison against 528 type strains of acidithiobacillus spp. downloaded from rdp database (available at https://rdp.cme.msu.edu/) using the neighborjoining method implemented in mega 7 software (kumar et al. 2016) with 500 bootstrap replications. analysis of genetic variability within acidithiobacillus genus to analyse genetic variability in acidithiobacillus genus 16s rrna sequences of lc and ts isolates obtained in this study were added to the pre-aligned 16s acidithiobacillus spp. sequences and analysed as described above. for genome comparison, available genome sequences of acidithiobacillus spp. were downloaded from the genbank database (https://www.ncbi.nlm.nih.gov/genome). final dataset was composed of 8 sequences gca_001931655 for a. albertensis, gca_000214095.3 for a. ferrivorans, gca_000175575.2 for a. caldus, gca_000227215.2 for a. thiooxidans, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:21 utc https://rdp.cme.msu.edu/ https://www.ncbi.nlm.nih.gov/genome nova biotechnol chim (2018) 17(2): 125-131 127 gca_000021485 for a. ferrooxidans, gca_000179815.2 for acidithiobacillus sp. ggi221, gca_002341825.1 for acidithiobacillus sp. uba2486, and gca_002733395.1 for acidithiobacillus sp. norp59. for each pair of sequences ani value (average nucleotide identity) was calculated using eztaxon server (https://www.ezbiocloud.net/tools/ani; yoon et al. 2017). based on ani values obtained a similarity dendrogram was constructed using upgma algorithm implemented in mega 7. for functional genome comparisons the rast server pipeline (rapid annotation by subsystem technology) available at https://rast.theseed.org/ was used (overbeek et al. 2014). results and discussion two recently isolated acidithiobacillus spp. isolates were identified using 16s rrna sequencing followed by blastn analysis of sequences obtained. the 16s rrna sequence of lc isolate from smolnik acid mine drainage water showed the highest similarity (99.7 %) to the 16s rrna sequences of type species of a. ferrooxidans strain atcc 23270. the 16s rrna sequence of ts isolate from hodruša-hámre gold mine has shown the highest similarity (99.7 %) to the a. albertensis dsm 14366 sequence. multiple sequence alignments confirmed identification of both isolates resulted from blastn analysis (fig.1). the lc isolate sequence grouped with a. ferrooxidans sequence, while this of ts isolate grouped with a. albertensis sequence. the tree indicates that the ability to obtain energy from the dissimilatory oxidation of ferrous iron is probably of old evolutionary origin as all four species able to utilise ferrous ions (a. ferriphilus, a. ferrivorans, a. ferrooxidans, and a. ferridurans) formed well supported group phylogenetically distant from the species unable to utilize ferrous ions. the analysis indicates that a. caldus is the most distant species of acidithiobacillus genus, forming a separate branch non-related to the other members of the genus (fig. 1). up to now, up to 120 acidithiobacillus spp. isolates or environmental 16s rrna sequences were identified from the european territory, most of them identified as a. ferrooxidans and a. thiooxidans and the occurrence of a. ferrooxidans related bacteria have already been reported from slovakia as well (nunez et al. 2017). however, no a. albertensis related sequences have been reported neither from the slovak nor european territory yet. a. albertensis species was described in 1983 (bryant et al. 1983) and it is not studied very frequently. a. albertensis related isolates were reported up to now mainly from china and canada (nunez et al. 2017), while our observation is the first report on a. albertensis from europe. biotechnology and environmental protection interest led to the characterization of multiple acidithiobacillus spp. isolates mainly from industrial environments. most of the isolates were identified as a. ferrooxidans or a. thiooxidans as for many years these two species were the only validly described species of the acidithiobacillus . fig. 1. unrooted phylogenetic tree documenting phylogenetic placement of acidithiobacillus spp. from slovakia (a. ferrooxidans lc and a. albertensis ts; indicated with asterisks) inferred using the neighbor-joining method. the bar represents genetic distance shown in number of base substitutions per site. numbers at nodes are bootstrap values after 500 repetitions. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:21 utc https://www.ezbiocloud.net/tools/ani https://rast.theseed.org/ nova biotechnol chim (2018) 17(2): 125-131 128 genus (jones et al. 2016). in the recent years great diversity within the genus was recognised and several new species of acidithiobacilli were described (hallberg et al. 2010; hedrich and johnson 2013; falagan and johnson 2016). to understand genetic variability in acidithiobacillus genus all available 16s rrna sequence of acidithiobacillus-related bacteria were downloaded from rdp database (cole et al. 2014) and compared using phylogenetic approach. the resulted phylogenetic tree (fig. 2) clearly documents extraordinary variability within acidithiobacillus genus. while the recently described species e.g. a. ferrivorans and a. ferridurans form monophyletic, well supported clades in the tree, other species are spread over the tree and could be found in multiple branches, indicating genetic variability within the genus. the diversity within acidithiobacillus genus have already been reported (karavaiko et al. 2003; ni et al. 2008) and based on our data the existence of at least 20 species could be proposed solely on 16s rrna analysis. recent advances in genomics allowed us to better understand the common and distinctive features some of acidithiobacillus species isolated from similar habitats and provided data to understand potential adaptation mechanisms of these bacteria to the harsh environments. complete or draft genome sequences of several acidithiobacillus species have become available in the recent few years (cárdenas et al. 2016). comparative genomic analyses indicate that genomes of acidithiobacilli are relatively similar. the genome sizes vary from 2.78 to 3.78 mbp, g + c content vary from 52.5 to 67.5 %, and numbers of predicted genes vary from 2648 to 4007. in all these parameters a. caldus is the most distant species, showing the highest g + c content, the smallest genome size, and the smallest number of genes (data not shown). based on the functional comparative genomic analyses of acidithiobacillus species, it was shown that different metabolic pathways might allow these microorganisms to acquire energy, carbon sources, and nitrogen sources from acidic environments, ensuring their survival and proliferation in these environments (fig. 3). using rast server pipeline (overbeek et al. 2014) very similar numbers of genes were found e.g. those of involved in rna metabolism or cell wall and capsule subsystems in all acidithiobacillus species analysed. the species, however, differ significantly in number of genes classified to the nitrogen or phosphorus metabolism, and protein metabolism, as well as stress response (fig. 3). the comparisons indicate that non-motile species of the genus (a. ferrooxidans and a. thioxidans) completely lack the genes for motility and chemotaxis. the phenotypic diversity observed within the genus is thus a consequence of genetic and genomic diversity. to evaluate the genomic diversity in more detail, fig. 2. unrooted phylogenetic tree documenting phylogenetic diversity within 584 available 16s rrna sequences of acidithiobacillus spp. the tree was inferred using the neighbor-joining method. the placement of validly described species is shown. the bar represents genetic distance shown in number of base substitutions per site. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:21 utc nova biotechnol chim (2018) 17(2): 125-131 129 fig. 3. comparative genomics of selected acidithiobacillus species. number of genes classified to the selected subsystems as identified by rast server is shown. average nucleotide identity (ani) values were calculated for every pair of genomic sequences and compared. ani was proposed recently as a means to compare genetic relatedness among prokaryotic strains. the method compares nucleotide-level genomic similarity between the coding regions of two genomes and is considered the best measure of genetic relatedness among bacterial strains (figueras et al. 2014; jain et al. 2018). ani values in range from 0.993 to 0.640 were observed among analysed set of sequences. based on ani values relatedness tree of acidithiobacillus spp. was constructed (fig. 4). the tree shows congruent topology with 16s rrna based tree (fig. 1). the ferrous iron-oxidising species form a separate group distant from the species that are unable to utilize ferrous ions, and (like in 16s rrna based tree) a. caldus is the most divergent species of acidithiobacillus genus related distantly only to any other species. the tree also documents diversity within the genus when the yet uncharacterised isolates uba2486 and norp59 have produced significantly low ani values and therefore could be assigned as a new species within acidithiobacillus genus. similarity values higher than 0.95 are necessary to place the isolates to the same species (figueras et al. 2014). the first species of the acidithiobacillus genus was fig. 4. upgma (unweighted pair group method with arithmetic mean) generated dendrogram showing relatedness of acidithiobacillus sp. based on ani (average nucleotide identity) values. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:21 utc nova biotechnol chim (2018) 17(2): 125-131 130 described already 100 years ago (waksmann and joffe 1922) and up to now 7 species of acidithiobacilli were validly described, most of them in the recent 20 years. however, application of modern genetic and genomic methods indicates that variability within the genus is highly underestimated and several species, especially a. ferrooxidans and a. thioxidans, have to be considered a complex species and the description of multiple new species of acidithiobacilli is very probable in the near future. conclusions our data indicate that genetic variability of acidithiobacillus genus is highly underestimated. several species, especially a. ferrooxidans and a. thioxidans, have to be considered a complex species and the description of multiple new species of acidithiobacilli is very probable in the near future. from the slovakia territory a. ferrooxidans species is reported predominantly, but for the first time we report here on the occurrence of a. albertensis as well. acknowledgement the work was financially supported by research agency of the ministry of education, science, research and sport of slovak republic under the vega project 1/0229/17. references altschul sf, gish w, miller w, myers ew, lipman dj (1990) basic local alignment search tool. j. mol. biol. 215: 403-410. bryant rd, mcgroarty km, costerton jw, laishley ej (1983) isolation and characterization of a new acidophilic thiobacillus species (t. albertis). can. j. microbiol. 29: 1159-1170. cárdenas jp, quatrini r, holmes ds (2016) progress in acidophile genomics. in quatrini r, johnson db (eds.), acidophiles: life in extremely acidic environments, caister academic press, uk, pp. 179198. cole jr, wang q, fish ja, chai b, mcgarrell dm, sun y, brown ct, porras-alfaro a, kuske cr, tiedje tm (2014). ribosomal database project: data 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compounds. extremophiles 7: 95-99. karavaiko gi, turova tp, kondraeva tf, lysenko am, kolganova tv, ageeva sn, muntyan ln, pivovarova ta (2003) phylogenetic heterogeneity of the species acidithiobacillus ferrooxidans. int. j. syst. evol. microbiol. 53: 113-119. kelly dp, wood ap (2000) reclassification of some species of thiobacillus to the newly designated genera acidithiobacillus gen. nov., halothiobacillus gen. nov. and thermithiobacillus gen. nov. int. j. syst. evol. microbiol. 50: 511-516. kumar s, stecher g, tamura k (2016) mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. mol. biol. evol. 33: 1870-1874. luptakova a, prascakova m, kotulicova i (2012) occurrence of acidithiobacillus ferrooxidans bacteria in sulfide mineral deposits of slovak republic. chem. eng. trans. 28: 31-36. ni yq, he ky, bao, jt, yang y, wan ds, li hy (2008) genomic and phenotypic heterogeneity of acidithiobacillus spp. strains isolated from diverse habitats in china fems microbiol. let. 64: 248-259. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:21 utc nova biotechnol chim (2018) 17(2): 125-131 131 nunez h, moya-beltran a, covarrubias pc, issotta f, cardenas jp, gonzalez m, atavales j, acuna lg, johnson db, quatrini r (2017) molecular systematics of the genus acidithiobacillus: insights into the phylogenetic structure and diversification of the taxon. front. microbiol. 8: 30. ohmura n, sasaki k, matsumoto n, saiki h (2002). anaerobic respiration using fe3+, s0 and h2 in the chemolithotrophic bacterium acidithiobacillus ferrooxidans. j. bacteriol. 184: 2081-2087. overbeek r, olson r, pusch gd, olsen gj, davis jj, disz t, edwards ra, gerdes s, parrello b, shukla m, vonstein v, wattam ar, xia f, stevens r (2014) the seed and the rapid annotation of microbial genomes using subsystems technology (rast). nucleic acids res. 42 (database issue): d206-d214. waksman sa, joffe js (1922) thiobacillus thiooxidans, a new sulfur-oxidizing organism isolated from the soil. j. bacteriol. 7: 239-256. yarza p, yilmaz p, pruesse, e, glöckner fo, ludwig w, schleifer kh, whitman wb, euzeby j, amann r, rossello-mora r (2014) uniting the classification of cultured and uncultured bacteria and archaea using 16s rrna gene sequences. nat. rev. microbiol. 12: 635-645. yoon sh, ha sm, lim jm, kwon sj, chun j (2017). a large-scale evaluation of algorithms to calculate average nucleotide identity. antonie leeuwenhoek 110: 1281-1286. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:21 utc nova biotechnol chim (2018) 17(1): 95-102 doi: 10.2478/nbec-2018-0010  corresponding author: katarina.hrobonova@stuba.sk nova biotechnologica et chimica hplc separation and determination of dicoumarol and other simple coumarins in sweet clover katarína hroboňová1,, jana sádecká1 and jozef čižmárik2 1institute of analytical chemistry, faculty of chemical and food technology, slovak university of technology in bratislava, radlinského 9, bratislava, sk-812 37, slovak republic 2comenius university, faculty of pharmacy, department of pharmaceutical chemistry, odbojárov 10, bratislava, sk-812 37, slovak republic article info article history: received: 15th march 2018 accepted: 16th may 2018 keywords: hplc-dad coumarins sweet clover synchronous fluorescence abstract dicoumarol is a mycotoxin, that acts as a blood anticoagulant, is formed during the microbial action of molds and fungi in spoiled hay or silage containing highcoumarin plant. a hplc-dad method for determination of coumarins, including dicoumarol, coumarin, and 4-hydroxycoumarin was developed. methanol and acetic acid were used as mobile phase with gradient elution. the simultaneous separation was performed using c18 type of stationary phase. the recoveries were 88.6 – 92.6 %, 91.8 – 95.0 %, and 89.7 – 94.1 % (evaluated for three concentration levels) for dicoumarol, coumarin, and 4-hydroxycoumarin respectively. the parameters of system suitability (repeatability of retention times and peak areas) were determined for evaluation of the method. the method showed a good linearity in the concentration range 0.7 – 100 µg.ml-1 for dicoumarol, 0.05 – 100 µg.ml-1 for coumarin and 4-hydroxycoumarin with correlation coefficients higher than 0.9885. extracts of sweet clover herb, hay, and spoiled hay were subjected to hplcdad analysis. the most abundant compound in sweet clover herb and hay extracts was coumarin. in spoiled sweet clover hay extract the 4-hydroxycoumarin was detected in addition. the formation of 4-hydroxycoumarin was also observed in the synchronous fluorescence spectra recorded at the wavelength difference of 90 nm (difference between emission and excitation wavelength).  university of ss. cyril and methodius in trnava introduction sweet clover (melilotus albus or melilotus officinalis l.) is widespread plant in central europe. besides of vitamin c, allantoin, tannins, mineral salts and flavonoids it contains coumarin compounds. during the microbial action of molds and fungi (penicillium jenseni, penicillium nigricans) in spoiled hay or silage containing highcoumarin plant, the dicoumarol is formed (4-hydroxycoumarin is the intermediate of dicoumarol production from coumarin (poulton et al. 1980). dicoumarol, a symmetrical biscoumarin (3,3´-methylenebis(4hydroxycoumarin; fig. 1), is potentially toxic and as potent vitamin k antagonist and anticoagulant may cause animal death due to internal or external hemorrhaging (stahmann et al. 1941; smith et al. 1965; casper et al. 1983). its discovery led to the development of another modern anticoagulant drug, warfarin (4-hydroxy-3-(3-oxo-1 phenylbutyl)-coumarin). the european committee prohibited the presence of dicoumarol in cosmetic products and food (regulation (ec) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:01 utc nova biotechnol chim (2018) 17(1): 95-102 96 no 1334/2008); eu regulation (ec) no 1223/2009). fig. 1. chemical structures of coumarin, 4-hydroxycoumarin, and dicoumarol (respectively). several analytical techniques are available for determination of dicoumarol, coumarin, and 4-hydroxycoumarin such as colorimetry (emery et al. 1970), fluorescence spectrometry (marcolan et al. 2011; poláček et al. 2015), and liquid chromatography including thin layer chromatography (bellis et al. 1967). the literature provides several studies involving the use of hplc with uv, ms and fluorescence detectors for determination of individual coumarin, 4-hydroxycoumarin, and dicoumarol or in group with other phenolic compounds. only a few papers deal with simultaneous separation and determination of selected compounds in sweet clover. table 1 summarizes some studies using hplc for the determination of investigated coumarins in different matrices. techniques, such as ultrasonic liquid extraction for plant and cosmetics samples (muir et al. 1992; ikeda et al. 2009; xi et al. 2010) or accelerated solvent extraction for food sample (vieriková et al. 2009) were applied for sample pretreatment. the aim of the present work was to develop a rapid and sensitive hplc method for simultaneous determination dicoumarol, coumarin, and 4-hydroxycoumarin in sweet clover herb and hay. experimental chemicals and sample the standards of dicoumarol (98 %), 4-hydroxycoumarin (98 %), and coumarin (99 %), were purchased from sigma-aldrich (st. louis, usa). methanol (hplc grade) and acetic acid (99 %, an analytical grade) were purchased from merck (darmstadt, germany). the deionized water (resistivity of 18.2 mω/cm) was obtained from a aquamax ultra (series 370) water purification system. the sample of sweet clover (melilotus officinalis l.; aerial parts) herb was purchased from slovakia. the herb was dried to constant weight during 4 hour at 30 °c and powdered. the fresh herb was incubated without access to light and air at 23 °c for 14 and 21 days. preparation of standard solutions the stock solutions of dicoumarol, coumarin, and 4-hydroxycoumarin were prepared by dissolving of standards in methanol (concentration of analyte 1 mg.ml-1) and stored at 8 °c. the working standard solutions and mixed standard working solution (concentration of each analyte 100 µg.ml-1) were prepared by appropriate dilution of stock solutions with methanol. the solutions were filtered through a 0.45 µm nylon membrane filter. solutions of dicoumarol were prepared daily. solutions of 4-hydroxycoumarin and coumarin were stable during a week. sample preparation an accurately weighted portion of sample (approximately 0.1 g) was mixed with 20 ml of methanol and extracted under stirring on mechanical shaker for 60 min at 23 °c. the mixture was centrifuged for 10 min (4,000 rpm). the supernatant was used for hplc analysis. hplc analysis hplc system (agilent technologies, series 1200) consisted of a binary pump, an autosampler, a column oven, a diode array detector, and a fluorescence detector. data were acquired and controlled by the agilent chemstation chromatographic data system software. separations of analytes were performed in reverse phase mode using a symmetry c18 (150 mm x 3.9 mm i.d., 5 µm particle size; column i), symmetry c18 (75 mm x 4.6 mm i.d., 3.5 µm particle size; column ii), xbridge c18 (50 mm x 4.6 mm i.d., 3.5 µm particle size; column iii) (waters, usa) column. the isocratic and gradient elutions were performed using the mobile phases consisted of methanol with the addition 0.3 % acetic acid (a) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:01 utc nova biotechnol chim (2018) 17(1): 95-102 97 table 1. hplc methods for separation and determination of coumarin, 4-hydroxycoumarin, and dicoumarol. stationary phase mobile phase detection lod linear range sample reference coumarin c18 acetonitrile/0.1 % formic acid gradient elution ms – 0.026-13.5 g.ml-1 green tea with sweet-herbaceous odour yang et al. (2009) c18 acetonitrile/0.1% formic acid (50/50 v/v) dad 0.02 g.kg-1 0.05-500 g.kg-1 food, biscuits vieriková et al., (2009) 4-hydroxycoumarin c18 methanol/phosphate buffer ph 3 (65/35 v/v) uv (286 nm) fl (372/290 nm) 0.0121 g.ml-1 0.0125 g.ml-1 – plant sample (morinda citrifolia) ikeda et al. (2009) c18 methanol/acetonitrile/ phosphate buffer ph 4 gradient elution uv (280 nm) 0.0200 g.ml-1 0.07-4 g.ml-1 pharmareutical preparative vijayakumar et al. (2009) dicoumarol phenyl methanol/acetonitrile/ water gradient elution dad 0.2 g.ml-1 0.5-60 g.ml-1 cosmetics xi et al. (2010) c18 methanol/acetate buffer ph 6 (25/75 v/v) uv (303 nm) 2 g.ml-1 – sweet clover hay, silage muir et al. (1992) dad – diode array detection; fl – fluorescence detection; lod – limit of detection; ms – mass spectrometry; uv – ultraviolet detection. and 0.3 % aqueous solution of acetic acid (b). the gradient profiles and flow rates used are summarised in table 2. the injection volume was 10 µl, and column temperature was maintained table 2. the gradient profiles and flow rates for hplc separation of target coumarins on tested columns. column flow rate [ml.min-1] time [min] a [%] b [%] column i 0.8 0 7 10 11 65 40 0 0 35 60 100 100 column ii 1.6 0 2.1 2.7 4.0 65 40 0 0 35 60 100 100 column iii 1.6 0 2.1 2.7 4.0 65 40 0 0 35 60 100 100 a – 0.3 % acetic acid; b – methanol containing 0.3 % acetic acid at 23 °c. the diode array detector was operated in the wavelength range of 190 – 400 nm and chromatograms were obtained at 300 nm for dicoumarol and 280 nm for other compounds under study. the fluorescence detector was operated in the emission wavelength range of 270 – 500 nm and chromatograms were obtained at excitation wavelength 300 nm and emission wavelength 390 nm for detection of dicoumarol. synchronous fluorescence analysis lumina fluorescence spectrometer (thermo scientific) equipped with a 150 watt ozone-free xenon lamp was were used. excitation and emission slits were both set at 5.0 nm. scan speed was 200 nm/min. pmt voltage was set at 500 v. the samples were placed in a 1 cm quartz cell. method validation for the validation of the hplc-dad method, parameters such as linearity, limits of detection (lod), limits of quantification (loq), precision, and accuracy were evaluated. the calibration curves of analytes were constructed after injection of standard solutions and obtained by plotting a graph of mean peak area versus corresponding concentration of analyte (seven concentration bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:01 utc nova biotechnol chim (2018) 17(1): 95-102 98 levels, three replicate injections of each solution). the lods and loqs values were determined empirically by the injection of the series of diluted solutions of the analytes and were calculated as a signal to noise ratio of 3 and 10, respectively. the recovery tests were performed in sample of dried sweet clover spiked at three concentration levels of dicoumarol, coumarin, and 4-hydroxycoumarin. spiked sample was held for 1 hour before extraction. spiked and unspiked samples were treated by the same procedure. seven independent extractions of each sample were carried out. intra-day and inter-day precisions of the method were evaluated for seven preparations of sample within one day and three days, making triplicate injections under the working conditions and (expressed as rsd %). results and discussion hplc separation reversed phase separation mode is most commonly used for hplc separation of mixtures of coumarin derivatives including of dicoumarol as documented table 1. the work was focused on development of hplc method for separation and determination of three of comarins, coumarin, 4-hydroxycoumarin, and dicoumarol. coumarin is precursor of dicoumarol and 4-hydroxycoumarin is a probable intermediate compound. (smith et al. 1938) the 4-hydroxycoumarin is converted into dicoumarol in the presence of formaldehyde (stahmann et al. 1941). at the beginning of the study, a suitable analytical column, which would offer sufficient separation for quantification of analytes was estimated. three reversed-phase c18 type analytical columns, symmetry c18, and xbridge c18 with different particle size and column length, were tested. the mobile phases consist of methanol and acetic acid at different ratios (fig. 2) were investigated for isocratic elution. the effectiveness of the separation was evaluated using standard solution containing coumarin, 4-hydroxycoumarin, and dicoumarol. the baseline separations of analytes were achieved, but the retention factors of dicoumarol were significantly higher in comparison to retention factors of coumarin and 4-hydroxycoumarin (higher than 7 for all tested stationary and mobile phases), which lead to longer analysis times. gradient elution profiles shown in table 2 were investigated, which resulted in effective separation of target compounds in analysis time lower than 15 min. 0 2 4 6 8 10 12 14 16 a/b (5 0/50) column i a/b (6 0/40) a/b (5 0/50) column ii a/b (6 0/40) a/b (5 0/50) column iii a/b (6 0/40) k coumarin 4-hydroxycoumarin dicoumarol fig. 2. effect of mobile phase composition on retention factor of coumarins obtained for three analytical columns and isocratic elution with mobile phase a/b. a – 0.3 % acetic acid; b – methanol; flow rate 0.8 ml.min-1; dad detection. the complete separation of analytes was achieved (resolution values higher than 2) using all tested columns as is shown in fig. 3. the chromatographic characteristics for tested columns are summarised in table 3. the repeatability of the retention times and areas (determined for standard solutions at concentration level of analytes 10 µg.ml-1) were lower than 1.1 % for all analytes and columns under study. all tested stationary phases were suitable for separation of coumarin, 4-hydroxycoumarin, and dicoumarol. the satisfactory resolution and symmetrical peaks of analytes were obtained. while the symmetry c18 type of column is stable under ph range 2 – 8, the xbridge c18 stationary phase is very resistant to harsh conditions (ph 1 – 12) resulted from bridged ethylene hybrid technology. the results indicated, that fast separation using xbridge c18 was achieved, but this column was characterized by a lower column efficiency (greater hetp values) in comparison to other columns under study. therefore, the symmetry c18 type column was chosen as suitable column for next analyses of real samples. the decrease of particle size and column length and use of higher flow rate (column ii) resulted in decrease of analysis time (~2.5 times in comparison with column i) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:01 utc nova biotechnol chim (2018) 17(1): 95-102 99 a column i coumarin b column ii 4-hydroxycoumarin c column iii dicoumarol fig. 3. chromatographic separations of coumarin (1), 4-hydroxycoumarin (2), and dicoumarol (3) on three analytical columns and uv spectra of compounds under study. mobile phase composition and flow rate as table 2, dad detection (chromatograms at 280 nm). and finally decrease of solvent consumption. coumarins allow uv absorbance and some of them fluorescence too (hroboňová et al. 2013). spectrophotometric detection of coumarins was realised by dad detector operated in the interval from 190 to 400 nm where the absorption maxima of target compounds are presented (280 nm for coumarin and 4-hydroxycoumarin, 300 nm for dicoumarol; fig. 2). the maximum excitation and emission wavelength for fluorescence detection of dicoumarol were selected from excitation and emission spectra. as a result, the 300/390 nm (excitation/emission) wavelength pair was chosen for purpose of this study. validation of the method the analytical figures of merit were determined under optimized separation and detection conditions. parameters including lod, loq, linear range, precision and accuracy were calculated to demonstrated the validation of the developed hplc-dad method (listed in table 4). the calibration curves of the three analytes were table 3. retention times (tr), resolution (rs), symmetry factor (as), and height equivalent of theoretical plate (hept) for hplc separation of coumarins on tested analytical columns. a mobile phase and gradient profiles as in table 2. tr [min] rs as hept [µm] column i coumarin 6.47 7.5 0.81 11.0 4-hydroxycoumarin 8.7 10.6 0.81 6.5 dicoumarol 9.86 0.87 1.7 column ii coumarin 2.36 5.9 0.88 11.1 4-hydroxycoumarin 3.1 6.1 0.85 5.4 dicoumarol 3.53 0.87 2.2 column iii coumarin 1.59 4.7 1.7 25.0 4-hydroxycoumarin 2.22 10.3 1.8 9.3 dicoumarol 3.29 0.92 2.3 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:01 utc nova biotechnol chim (2018) 17(1): 95-102 100 table 4. linearity and sensitivity of the hplc-dad method. parameter coumarin 4-hydroxycoumarin dicoumarol linear range [µg.ml-1] 0.05-100 0.05-100 0.7-100 regression equation y = 70165c + 3.9 y = 139610c + 11.4 y = 22050c + 38.8 r2 0.9983 0.9896 0.9885 lod [µg.ml-1] 0.01 0.02 0.2 loq [µg.ml-1] 0.05 0.05 0.7 created after the injection of a mixed standard solution. results showed good linear relationship for each analyte (coefficient of determination in the range 0.9885 – 0.9983). the lods of the method were calculated as a signal to noise ratio of 3 using the injection of series of more diluted standard solutions of analytes. the loqs were evaluated using signal to noise ratio of 10. the lods and loqs of coumarin and its derivatives were in the range 0.01 – 0.2 µg.ml-1 and 0.05 – 0.7 µg.ml-1, respectively. since the lods and loqs of dicoumarol obtained for fluorescence detection were higher (2.1 and 6.5 µg.ml-1, respectively) dad detector was used in plant samples analyses. recovery tests of method were performed for dried sample spiked with dicoumarol levels at three concentration levels. the recovery values of dicoumarol, coumarin, and 4-hydroxycoumarin were higher than 85 %. as documented table 5, good repeatability of the recovery was achieved (rsds less than 4 % for all spiked concentration levels). the intra-day and inter-day precisions of developed method, determined for spiked sample were lower than 5 % and 9 %, respectively. analysis of real plant samples in order to show reliability and applicability of the developed method, the extracts of sweet clover samples were analysed. samples of herb, dried plant (aerial parts), and herb incubated for 14 and 21 days, were used in this study. sample extracts were prepared in methanol. comparison of retention times from hplc and uv spectra of sample peaks and standards were used for characterization of coumarins in extracts. analyses of dried plant and herb extracts confirmed the absence of dicoumarol and 4-hydroxycoumarin in tested samples (fig 4a, b). the content of coumarin in dried plant sample was 3.3 – 0.1 mg.g-1. the 4-hydroxycoumarin was formed in the fresh herb incubated for 14 and 21 days (fig. 4c). the formation of 4-hydroxycoumarin was also observed in the synchronous fluorescence spectra recorded at the wavelength difference (difference between emission and excitation wavelength) of 90 nm (fig. 5), using the recently developed method (poláček et al. 2015). fig. 5. synchronous fluorescence spectra of fresh herb, dried plant, fresh herb after incubation and 4-hydroxycoumarin. table 5. recovery values of extraction method for spiked dried sweet clover sample. coumarin 4-hydroxycoumarin dicoumarol recovery [%] rsd [%] recovery [%] rsd [%] recovery [%] rsd [%] c1 91.8 2.2 89.7 2.1 92.6 2.5 c2 95.0 3.0 94.1 2.6 92.5 3.1 c3 93.2 2.6 90.8 2.4 88.6 2.6 n = 7; c1 – 0.2 mg.g-1 of coumarin; 0.2 mg.g-1 of 4-hydroxycoumarin; 1.0 mg.ml-1 of dicoumarol; c2 – 1.0 mg.g-1 of coumarin; 1.0 mg.g-1 of 4-hydroxycoumarin; 2.0 mg.g-1 of dicoumarol; c3 – 2.0 mg.g-1 of coumarin; 2.0 mg.g-1 of 4-hydroxycoumarin; 10.0 mg.g-1 of dicoumarol. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:01 utc nova biotechnol chim (2018) 17(1): 95-102 101 fig. 4. chromatograms of extracts of meliloti herba fresh herb (a), dried plant (b), and herb after incubation (c). stationary phase: column i, mobile phase composition and flow rate as table 2, dad detection (chromatograms at 280 nm); 1coumarin, 24-hydroxycoumarin, 3dicoumarol. conclusions a simple reversed-phase hplc-dad method including stationary phase of c18 type and methanol-acetic acid as mobile phase with gradient elution was developed for simultaneous determination of coumarin, 4-hydroxycoumarin, and dicoumarol in extracts of sweet clover samples. the separation of coumarins by hplc was rapid, efficient and reproducible, and the method presented acceptable results for sensitivity, linearity, precision, and accuracy. the lod obtained by the proposed method are comparable to other methods with spectrophotometric detection. moreover, one should note that wider linear concentration range of each compound under study was achieved. the method has potential to be used in medical plant and related products analysis, since currently there is an interest on characterization and determination of biologically active compounds in natural samples. acknowledgement this publication was supported by the competence center for smart technologies for electronics and informatics systems and services, itms 26240220072, funded by the research & development operational programme from the erdf and funded by the slovak research a meliloti herba b meliloti herba, hay c meliloti herba herb incubated for 14 (a) and 21 (b) days bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:01 utc nova biotechnol chim (2018) 17(1): 95-102 102 and development agency under the bilateral project sk-srb-2016-0006. references bellis dm, spring ms, stoker jr (1967) the biosynthesis of dicoumarol. biochem. j. 103: 202-206. casper hhme, alstad ad, monson sb, johnson lj (1983) dicoumarol levels in sweet clover toxic to cattle. amet. assoc. vet. lab. diagnosticians, 25th annu. proc., 4148. emery ae, gear jr (1970) determination of dicoumarol in spoiled sweet clover. can. j. plant sci. 50: 39-45. eu regulation (ec) no 1223/2009 of the european parliament and of the council of 30 november 2009, on cosmetic products (oj l 342, 22.12.2009, p. 59). official journal of the european union l 342: 59. hroboňová k, lehotay j, čižmárik j, sádecká j (2013) comparison hplc and fluorescence spectrometry methods for determination of coumarinn derivatives in propolis. j. liq. chromatogr. relat. technol. 36: 486503. ikeda r, wada m, nishigaki t, nakashima k (2009) quantitation of coumarin derivatives in noni (morinda citrifilia) and their contribution of guenching effect on reactive oxygen species. food chem. 113: 11691172. marcolan m, martins pa, pedrosa va, rodrigues rh, de oliveira hpm, codognoto l (2011) spectrofluorimetric determination of coumarin in commertial tablets. j. fluorescenc. 24: 733-738. muir ad, goplen bp (1992) quantitative reversed-phase hplc analysis of dicoumarol in sweet clover hay and silage samples. j. agric. food chem. 40: 820-823. poláček r, májek p, hroboňová k, sádecká j (2015) fluorescence spectrometry as a tool for determinationof coumarins by multivariate calibration. j. fluoresc. 25: 297-303. poulton je, mcree de, conn ee (1980) intracellular localisation of two enzymes involved in coumarin biosynthesis in melilotus alba. plant physiol. 65: 171175. regulation (ec) no 1334/2008 of the european parliament and of the council of 16 december 2008, on flavouring and certain food ingredients with flavouring properties for use in and foods and amending council regulation (eec) no 160/91, regulation (ec) no 2232/96 and (ce) no 110/2008 and directive 2000/12/ec. official journal of the european union l 354/34: 15. smith wk, brink ra (1938) relation of bitter ness to the toxic principle in sweet clover. j. agric. res. 57: 145-154. smith wk, gorz hj (1965) sweet clover improvement. adv. agron. 17: 163-231. stahmann ma, huebner cf, link kp (1941) studies on hemorrhagic sweet clover disease. v. identification and synthesis of the hemorrhagic agent. j. biol. chem. 138: 513-527. vieriková m, germuška r, lehotay j (2009) determination of coumarin on food using ultraperformance liquid chromatography-electrospraytandem mass spectrometry. j. liq. chromatogr. rel. technol. 32: 95-105. vijayakumar eks, dhore dm, kumar m (2009) hplc method for simultaneous determination of impurities and degradation products in zynisamide. indian j. pharm sci. 71: 521-526. xi h, ma q, wang c, bai h, liu x, wang y (2010) simultaneous determination of 17 coumarins in cosmetics by high performance liquid chromatography. j. instrum. anal. 29: 1168-1172. yang z, kinoshita t, tanida a, sayama h, morita a, watanabe n (2009) analysis of coumarin and its glycosidically bound precursor in japanese green tea having sweet-herbaceous odour. food chem. 114: 289-294. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:01 utc nova biotechnol chim (2018) 17(2): 150-159 doi: 10.2478/nbec-2018-0016  corresponding author: dalidakso@gmail.com nova biotechnologica et chimica synthesis of ternary bioactive glass derived aerogel and xerogel: study of their structure and bioactivity dalila ksouri1, hafit khireddine1, ali aksas1, tiago valente2, fatima bir1, nadir slimani2, belén cabal3, ramón torrecillas3 and josé domingos santos2 1laboratoire de génie de l’environnement (lge), faculté de technologie, université de bejaia, bejaia, 06000, algerie 2department of materials, faculty of engineering, university of porto (feup), portugal 3nanomaterials and nanotechnology research center (cinn), csic university of oviedo (uo), avda de la vega 4-6, el entrego 33940, san martín del rey aurelio, spain article info article history: received: 7th june 2018 accepted: 12th november 2018 keywords: aerogel bioactive glass bioactivity sol-gel 63s abstract in this work ternary bioactive glasses with the molar composition 63 % sio2, 28 % cao, and 9 % p2o5 have been prepared via sol-gel processing route leading to xerogel or aerogel glasses, depending on the drying conditions. two types of drying methods were used: atmospheric pressure drying (evaporative), to produce xerogels, and supercritical fluids drying, to obtain aerogels. both dried gels were subjected to heat-treatment at three different temperatures: 400, 600 and 800 ºc in order to the removal of synthesis byproducts and structural modifications. the resulting materials were characterized by x-ray diffraction (xrd), fourier transforms infrared spectroscopy (ftir), scanning electron microscopy (sem), thermal gravimetric analysis (tga) and differential thermal analysis (dta), and by in vitro bioactivity tests in simulated body fluid. the influence of the drying and the sintering temperature of their structure, morphology, and bioactivity of the final products were evaluated. the results show a good bioactivity of xerogel and aerogel bioactive glass powders with the formation of an apatite layer after one day of immersion in sbf solution for aerogel bioactive glass powders and a particle size less than 10 nm. an apatite layer formed after 3 days in the case of xerogel bioactive glass powders and a particle size around 100 nm.  university of ss. cyril and methodius in trnava introduction the bioactive glasses constitute an important group of biomaterials that have wide application in medical and dental fields (mehdipour et al. 2012; al-noaman et al. 2012). in fact, when bioactive glasses are in contact with body fluid or tissue, they generate a series of physical and chemical reactions leading to the formation of hydroxyapatite surface layer (zhitomirsky et al. 2009; bellucci et al. 2011). certain compositions of bioactive glasses containing sio2-cao-p2o5 can bond to both soft and hard tissue without any intervening fibrous layer. the method of elaboration of the bioactive glasses is greatly influencing the results of the bioactivity and biocompatibility. sol-gel technique is the most widely used for the synthesis of bioactive glasses (li et al. 2013). the sol-gel process involves hydrolysis, polymerization, gelation, drying, and a dehydration process. gels are usually classified as aerogels and xerogels. when the liquid from the gel is evaporated at room temperature, the solid gels left behind are called xerogels. whereas, when bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:30 utc mailto:dalidakso@gmail.com nova biotechnol chim (2018) 17(2): 150-159 151 the liquid from the gel is extracted at the supercritical state of liquid, these materials are called aerogels. the removal of solvent above its critical point occurs with no capillary pressure because there are no liquid-vapor interfaces. thus in the aerogel process, there is a greatly reduced driving force for shrinkage (catauro et al. 2015). the conditions of drying the gel and sintering temperature in the sol-gel method are also an important factor affecting the structure, morphology, and bioactivity of the bioactive glasses. the bioactivity responses depend upon the physical and chemical characteristics of the materials and particularly upon its chemical composition, crystallinity, particle size, surface structure, microstructure and surface roughness (deligianni et al. 2001; valerio et al. 2004). there has been increased interest in fabricating sol-gel derived nanoscale bioactive glasses. nanoscale particulate bioactive glasses have shown advantages over conventional (micrometer-sized) bioactive glasses due to their large surface area and enhanced solubility as well as reactivity coupled with the possibility to induce nanotopographic surface features in composite materials (eroltaygun 2013). in this work, ternary bioactive glasses with the molar composition 63 % sio2, 28 % cao, and 9 % p2o5, known as 63s bioactive glass, have been prepared via sol-gel processing route leading to xerogel or aerogel glasses, depending on the drying conditions. in the literature, there are many articles about 63s obtained by atmospheric pressure drying (catauro et al. 2015), but none by supercritical drying, at least for the author’s knowledge. this paper compares the structure, morphology and in vitro bioactivity of identical chemical composition bioactive glasses produced by the sol-gel process at the supercritical condition of ethanol (aerogel) and by the most used synthesis route to obtain bioactive glasses (xerogel). the influence of the calcination temperatures was also evaluated. experimental preparation of bioactive glass powders the composition of studied bioactive glass belongs to the ternary system sio2-cao-p2o5 with 63s composition: 63 sio2 28 cao 9 p2o5 (mol %). glass powders were synthesized through a sol-gel process. xerogel bioactive glasses (labelled as xg) were prepared in a similar way as previously described by other authors (doostmohamadi et al. 2011). briefly, tetraethylorthosilicate (teos), which was selected as a silica precursor for the sol, was added to ethanol as an alcoholic media and the mixture was stirred for 30 min. distilled water was added to the solution and allowed to mix until the solution became clear with h2o : teos molar ratio of 4 : 1. after 30 min, triethyl phosphate (tep) was added to the stirring solution and after another 20 min, calcium nitrate tetrahydrate was added to the mixture. hydrochloric acid (hcl, 2n) was used as a catalyst and the final solution was stirred for one hour additional. to obtain a xerogel bioactive glass powder (xg), the solution was heated at 60 °c for 10 h and dried at 130 °c for 20 h. another identical solution was autoclaved at supercritical conditions of ethanol solvent (pressure of 63 bars, a temperature of 243 °c) to obtain an aerogel bioactive glass (labelled as ag). the dried gel powders (xerogel and aerogel) were crushed and calcined at different temperatures: 400, 600 and 800 ºc, for 2 h with a heating rate of 5 °c/min, then the furnace was naturally cooled down. the obtained glasses were labelled as xg 400 and ag 400 when the calcination takes place at 400 °c, xg 600 and ag 600 when it was at 600 °c, xg 800 and ag 800 at 800 °c. characterization of bioactive glasses thermal behaviour of these bioactive glasses was performed with a thermogravimetric-differential thermal analyzer (ta instruments, q600) in a flowing air atmosphere at a heating rate of 5 ºc/min from the ambient temperature to 1,000 °c. x-ray diffraction (xrd) analysis of the bioactive glasses was conducted on a bruker d8 discover diffractometer using cuk α radiation (λ = 1,54060 å) working at 40 kv and 40 ma in a step-scanning mode from 10° to 80°, at a scan speed of 0.04 °/s. the morphology of the obtained bioactive glasses was studied by scanning electron microscopy (sem) nova nano sem 200 equipped with the silicon sutw-sapphire edax detector, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:30 utc nova biotechnol chim (2018) 17(2): 150-159 152 fig. 1. xrd pattern of bioactive glass powders: a– xg 400, b– xg 600, c– xg 800, d– ag 400, e– ag 600 and f– ag 800. resolution: 125.01. the microstructure of the surface of the samples in vitro tests was performed using a high resolution (schottky) environmental scanning electron microscope with x-ray microanalysis and electron backscattered diffraction analysis: quanta 400 feg esem / edax genesis x4m. samples were coated with au/pd thin film, by sputtering, using the spi module sputter coater equipment. glasses were also characterized using transmission electron microscopy (tem) (jeol-2000ex). in vitro bioactivity the evaluation of in vitro bioactivity of the bioactive glass powders prepared by xerogel and aerogel at different calcined temperature was performed using sbf solution prepared according to kokubo protocol (kokubo 1990). in vitro bioactivity tests were used to evaluate the formation of an apatite layer on the surface of the bioactive glass pellets, considered as an indicator of in vivo bioactivity (leonor et al. 2002). to study the bioactivity, the bioactive glass powders were pressed into pellets of 15 mm in diameter and 2 mm in thickness and were soaked in simulated body fluid (sbf) solution prepared according to the protocol developed by kokubo (1990) at 37 °c and ph = 7.4 for 1, 3 and 7 days with a surface area to volume ratio of 0.1 cm-1 (zhao et al. 2005). the sbf solution of soaking samples was refreshed every two days. after being soaked, the pellets were extracted from the solution rinsed with deionized water then dried in air at room temperature. the pellets without soaking in sbf are labelled as zero-day (0 d). the formation of an apatite layer on the surface of the pellets was investigated by x-ray diffraction (xrd) for phase analysis, scanning electron microscopy (sem) for morphology and energy dispersive spectroscopy (eds) for elemental analysis. results and discussion characterization of the bioactive glass powders bioactive glass powders were characterized by different analysis methods in order to evaluate the drying treatment and the influence of calcination temperature on the structure, morphology, and bioactivity of bioactive glass powders. the phase formation behaviour of the bioactive glass powders derived from xerogel and aerogel at different calcination temperatures was investigated by xrd and the results patterns are shown in fig. 1. the powders calcined at 400 °c and 600 °c for both xerogel and aerogel bioactive glass powders take an amorphous state indicating an intern disorder and glassy nature of these powders (saboori et al. 2009; radev et al. 2012). the heat treatment at 600 ºc resulted in some crystallite growth. while, the powders calcined at 800 °c illustrated a crystalline structure referred to the wollastonite (casio3) (rainer et al. 2008), bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:30 utc nova biotechnol chim (2018) 17(2): 150-159 153 fig. 2. ftir spectra of bioactive glass powders: a– xg 400, b– xg 600, c– xg 800, d– ag 400, e– ag 600 and f– ag 800. this specified that the crystallization process depending on the heat treatment conditions (elbatal et al. 2003). the formation of bioactive glass powders was further confirmed by ftir spectral analysis illustrated in fig. 2. all the samples showed an adsorption band at 1,036–1,082 cm-1 assigned to the si-o-si asymmetric bond stretching vibrations, the bond in the 785 – 790 cm-1 region is attributed to the symmetric si-o-si stretching vibrations (lucas-girot et al. 2011; jiang et al. 2011) and the absorption around 453 ‒ 463 cm-1 is due to the vibrational mode of the bending of si-o-si (ma et al. 2011) indicating that all the samples are essentially composed of si-o-si network. these bands are narrower in the case of aerogel bioactive glass, and become larger with the increase of calcination temperature especially for xerogel fig. 3. tg/ dta curves of prepared bioactive glass powders: a– xg 400, b– xg 600, c– xg 800, d– ag 400, e– ag 600 and f– ag 800. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:30 utc nova biotechnol chim (2018) 17(2): 150-159 154 bioactive glass. ftir spectra involve the band around 956 cm-1 attributed to the stretching vibrations of si-oh bonds (tarasyuk et al. 2006), the band at 1,396 cm-1 is assigned to the vibrations of po2 and/or no3 groups (qian et al. 2009), this band disappear in xerogel bioactive glass powders calcined at 600 °c and 800 °c but remains in the aerogel bioactive glass powders which can be explained by the transformation of po2 to the oxide p2o5 and/or the degradation of the nitrate no3 after calcination up to 600 °c in the case of the xerogel bioactive powder however, the remaining of the band at 1,396 cm-1 in the aerogel bioactive glass powders can be explained by their porous structure that contains small pores with a nanometric scale, which may imprison the po2 and/or no3 that are not reacted during the sol-gel process. the band at 2,372 cm-1 is attributed to the adsorption of co2 by the atmospheric (hajiali et al. 2010). the weak inflection at 1,620 – 1,653 cm-1 and the broad band centred at 3,438 – 3,455 cm-1 are assigned to o–h band of adsorbed water and structural hydroxyl group respectively (mehdipour and afshar 2012). the samples were characterized by thermo-gravimetric and differential thermal analysis (tg/dta). the fig. 3 a-f shows tg/dta traces of the developed bioactive glass powders. the results indicated that the weight loss decreases with increasing calcination temperature. overall, fig. 4. sem micrographs of bioactive glass powders: a– xg 400, b– xg 600, c– xg 800, d– ag 400, e– ag 600 and f– ag 800. a d b e c f fig. 5. tem micrographs of prepared bioactive glass powders: a– xg 400, b– xg 600, c– xg 800, d– ag 400, e– ag 600 and f– ag 800. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:30 utc nova biotechnol chim (2018) 17(2): 150-159 155 xerogel bioactive glass powder calcined at 400 °c exhibits a greater weight loss with 23 % following by aerogel bioactive glass powder calcined at 400 °c with 15 %. the xerogel and aerogel bioactive glass powders calcined at 600 °c shows the same weight loss with 11 %. whereas, the xerogel bioactive glass powder calcined at 800 °c shows a slight difference comparing to aerogel bioactive glass powder calcined at 800 °c with 6 % and 4 %, respectively. the first mass loss stage occurs around 100 °c to 150 °c can be associated with the endothermic evaporation of the adsorbed water on the surface of the bioactive glass powders. a second weight loss occurs at 350 °c to 420 °c is due to the elimination of the remaining organic groups and the residual nitrates used during the sol-gel process (riti et al. 2015). in addition, a third weight loss in the range of 550 °c to 660 °c is probably attributed to the polycondensation of residuals alkoxides (cestari 2016), these loss weights can be explained by the glass transition temperature of bioactive glass particles (gönen et al. 2016). a fourth weight loss at 690 °c and 850 °c is observed in the case of xerogel and aerogel bioactive glass powders calcined at 800 °c respectively can be attributed also to the structural transformation (charoensuk et al. 2013). the different temperature of the fourth weight loss for the xerogel and aerogel bioactive glass powders could be attributed to their different particle size after the processes. the exothermic final process fig. 6. xrd pattern of bioglasses before and after soaking in sbf solution for 1, 3 and 7 days: a– xg 400, b– xg 600, c– xg 800, d– ag 400, e– ag 600 and f– ag 800. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:30 utc nova biotechnol chim (2018) 17(2): 150-159 156 fig. 7. sem morphologies of bioactive glass pellets after soaking in sbf solution for 7 days: a– xg 400, b– xg 600, c– xg 800, d– ag 400, e– ag 600 and f– ag 800. occurs at approximately 850 ºc and is related to the onset of crystallization. thermal processing drives off the –oh groups, causing further formation of o–si–o bonds. the nitrates were also removed during stabilization by thermal processing. the drying conditions have an important effect on the porosity. sem micrographs (fig. 4) show that the obtained aerogel bioactive glass powders are less dense and more porous in comparison to xerogel bioactive glass powders. aerogels are mostly constituted of agglomerate spherical particles with a spongy structure. whereas xerogel bioactive glass powders are constituted of blocks, the apparition of white particles on the surface can be assigned to a crystal phase. the values of particle size obtained from the results of image analysis of tem micrographs also confirmed the sem results. xerogel bioactive glass powders are composed of uniform particles readily identified by tem (fig. 5 a-c). the micrographs show a size particle between 20 nm to 100 nm with inhomogeneity of forms and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:30 utc nova biotechnol chim (2018) 17(2): 150-159 157 shapes. on the contrary, the aerogel network can be described as an assembly of clusters. these are formed by the aggregation of small homogeneous spherical particles with a size less than 10 nm. the tem micrographs revealed that size of the bioactive glass powders increases with increasing of calcination temperature. this is can be explained by the structural contraction and densification during the sol-gel process of the developed bioactive glass powders and weight loss because of the polycondensation of the residual alkoxide (cestari 2016). in vitro assays the fig. 6 shows the xrd patterns of bioactive glass pellets prepared from xerogel and aerogel before and after soaking in sbf solution for 1, 3 and 7 days. it can be seen (fig. 6), the apparition of two diffraction peaks at 26 and 32 ° (2θ) corresponding to (002) and (211) reflections of an apatite phase (nayak et al. 2010; ma et al. 2011). these peaks are observed after one day in the case of aerogel bioactive glass pellets (fig. 6 d-f) and after 3 days for xerogel bioactive glass pellets (fig. 6 a-c). these results indicate that an apatite crystal was deposited on the surface of the pallets. however, the diffraction peaks are less defined in xerogel bioactive glass pellets that may be due to a lower thickness of the calcium phosphate layer. the elemental analysis of bioactive glass pellets before and after soaking in sbf for 1, 3 and 7 days was confirmed by energy dispersive x-ray analysis (eds) technique. the results (table 1) showed a remarkable increase in the intensity of calcium and phosphorus elements and reduction of the silica in all the pellets from the first day of immersion in sbf solution. the decrease of silica in xerogel and aerogel bioactive glass pellets calcined at 400 °c and 800 °c after 1 day of immersion showed a slight difference compared to bioactive glass pellets without immersion 0 day. after 7 days of immersion, we noticed the almost total disappearance of element si and the appearance only of elements ca and p which improve that the surfaces of the pallets are covered by an apatite layer. excellent bioactive and resorbent properties of xerogel 63s bioactive glasses have been previously reported. table 1. the quantitative eds analysis spectrum of bioglasses before and after soaking in sbf solution for 1, 3 and 7 days. samples /at [%] c o si p ca xg 400 5.74 61.01 21.04 2.68 9.52 xg 400, 1d 5.05 60.91 14.04 7.23 12.76 xg 400, 3d 7.43 57.26 0.47 12.07 22.76 xg 400, 7d 6.10 55.87 0.46 13.11 24.43 xg 600 8.67 58.59 20.20 2.61 9.93 xg 600, 1d 5.90 61.71 6.65 10.00 15.74 xg 600, 3d 5.53 57.71 0.89 12.17 23.70 xg 600, 7d 5.76 58.64 0.57 12.66 22.36 xg 800 5.41 58.12 23.52 2.94 10.02 xg 800, 1d 15.62 50.50 12.20 7.76 13.93 xg 800, 3d 14.61 50.55 4.66 10.68 19.51 xg 800, 7d 11.92 53.03 2.18 12.39 20.48 ag 400 3.99 60.98 23.76 4.11 7.16 ag 400, 1d 5.62 59.00 17.64 6.80 10.94 ag 400, 3d 9.18 57.08 4.72 11.21 17.82 ag 400,7d 8.11 58.25 1.53 12.45 19.66 ag 600 4.23 59.48 24.96 4.08 7.24 ag 600, 1d 12.91 52.63 7.57 10.69 16.20 ag 600, 3d 10.99 55.39 1.74 13.02 18.88 ag 600, 7d 16.09 51.83 1.10 12.61 18.37 ag 800 5.09 59.66 24.10 3.16 8.00 ag 800, 1d 5.36 57.43 14.57 8.75 13.89 ag 800, 3d 5.99 56.99 6.91 11.63 18.49 ag 800, 7d 6.00 56.74 2.84 12.70 21.72 the formation of bone-like apatite layer during bioactivity experiments was also evaluated by examining the change of surface morphology during the incubation in sbf solution. the fig. 7 showed the surface morphology of samples before and after soaking in sbf for 1, 3 and 7 days. the results showed that the surface morphology changed with incubation periods. after 1 day soaking in sbf compared to 0 days as control, the micrographs revealed the formation of individual spherical apatite grain in the case of xerogel bioactive glass pellets and a fully covered surface with an extensive calcium phosphate precipitate with tiny flak on the shape for aerogel bioactive glass pellets. with increasing incubation periods, the formation of apatite grains increase too, after 3 days soaking the surface of xerogel bioactive glass pellets was fully covered by an apatite layer, and this layer was composed of numerous spherical particles. on the other hand, the more growing of the apatite layer for aerogel bioactive glass pellets. however, the density of the precipitate (apatite layer) increased significantly after 7 days of immersion. the ability of aerogel bioactive glass to promote rapid formation of apatite layer after only one day of soaking can be explained by faster bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:30 utc nova biotechnol chim (2018) 17(2): 150-159 158 interchange between ca2+ ions of aerogel bioactive glass and the h3o + from sbf that can give rise to the formation of si–oh groups on the surface of the pellets that induces the apatite nucleation (petil et al. 2011). in addition, the rate of apatite formation depends on differences in the texture and morphology of xerogel and aerogel bioactive glass that improve the apatite deposition in sbf is extensive in rougher regions and sparse in flatter regions (pereira et al. 1995; petil et al. 2011). the formation of an apatite layer on the surface of the pellets is also more important when the temperature is increased. it found that the aerogel bioactive glass powder has a nanoparticle size and the formation of an apatite layer on the surface is faster and it is designated to compensate an organ or tissue deficient in a pathology, trauma or aging tissues (el-kady et al. 2010; hajiali et al. 2010). conclusions in this study, ternary bioactive glass powders were successfully synthesized via the sol-gel route from two types of dried gel: xerogel and aerogel. the results obtained show that the texture of elaborated bioactive glass powders is strongly influenced by the drying conditions of the gels and the calcination temperature. it found that the aerogel bioactive glass powders are characterized by less dense and spongy structure with a particles size less than 10 nm and an apatite layer is obtained after only one day of immersion in sbf solution. however, the xerogel bioactive glass powders presented a dense structure with a particle size around 100 nm and a surface covered with an apatite layer after three days of immersion in sbf solution. the bioactive glass powders calcined at 800 °c presented a good properties and in vivo tests will 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ar, zhitomirsky i (2009) electrophorotic deposition of bioactive glass/polymer composite coating with and without ha nanoparticle inclusions for biomedical applications. j. mater. process. technol. 209: 1853-1860. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:30 utc 404 not found nova biotechnol chim (2017) 16(2): 92-98 doi: 10.1515/nbec-2017-0013  corresponding author: asimonovicova@fns.uniba.sk  nova biotechnologica et chimica responses of aspergillus niger to selected environmental factors alexandra šimonovičová1,, sanja nosalj1, alžbeta takáčová2, tomáš mackuľak3, karol jesenák4 and slavomír čerňanský2 1 department of soil science, faculty of natural sciences comenius university, ilkovičova 6, bratislava, sk-842 15, slovak republic 2 department of environmental ecology, faculty of natural sciences comenius university, ilkovičova 6, bratislava, sk-842 15, slovak republic 3 department of environmental engineering, institute of chemical and environmental engineering, faculty of chemical and food technology, slovak university of technology, bratislava, sk-812 37, slovak republic 4 department of inorganic chemistry, faculty of natural sciences comenius university, ilkovičova 6, bratislava, sk-842 15, slovak republic article info article history: received: 15th february 2017 accepted: 1st november 2017 keywords: aspergillus niger environmental factors enzymes growth organic acids pelletization abstract four wild type strains of a. niger were collected from soil and stream sediments representing environments with variable level of as, sb, al, fe, cd, cu, and zn contamination. banská štiavnica-šobov (s), pezinok-kolársky vrch (p) and slovinky (sl) represent contaminated localities. locality gabčíkovo (g) was as a control site. the influence of toxic elements in these substrates on fungal growth, colony size, enzymatic activity, production of organic acids and their pelletization in water suspensions with montmorillonite was studied. the aim of our study was to find out how the wild type strains from (contaminated) environment will behave in different model solutions. we also wanted to add some new information in this area of study, because that there is some gap in the available knowledge.  university of ss. cyril and methodius in trnava     introduction aspergillus niger strains belong to the most widespread microscopic filamentous fungi in the environment (nováková et al. 2012; šimonovičová 2013). this strain is very often used to study variable processes, such as heavy metal accumulation or bioleaching (gan et al. 2016; peťková et al. 2013; xu et al. 2014), production of organic acids (hu et al. 2016) or different enzymes (akhter et al. 2011; mrudula and murugammal 2011). environmental factors affecting the fungus growth include type of soil and its properties such as ph, contents of organic matter and heavy metals and potentially toxic elements; all these represent primary indicators with very rapid influence on microorganisms (šimonovičová 2014). physiological properties of microorganisms changed in dependence on these factors. environmental pollution strongly affected not only biochemical, but also macroand micromorphological characteristics of a. niger strains (šimonovičová et al. 2013). four wild type strains of a. niger were collected from soil and stream sediments representing environments with different of contamination. the influence of toxic elements in these substrates on fungal growth, colony size, enzymatic activity, production of organic acids and their pelletization in water suspensions with montmorillonite was studied. the aim of our study was to find out how the wild type strains from (contaminated) environment will behave in bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:56 utc nova biotechnol chim (2017) 16(2): 92-98 93 different model solutions. we also wanted to add some new information in this area of study, because that there is some gap in the available knowledge. fungal pellets have many advantages of easy harvest, low fermentation broth viscosity and high yield of proteins (zhang and zhang 2016). it was discovered, that mycelial pellets are effective as a biomass carrier for the immobilization of bacteria for the degradation of target pollutants (zhang et al. 2011), for degradation of lignin in water and bioremediaton of soil contaminated with pentachlorophenol pcb (rubilar et al. 2009). experimental microscopic fungi four wide types of a. niger strains were isolated from terrestrial substrates and stream sediment from šobov, pezinok, gabčíkovo and slovinky study sites in slovakia (fig. 1). the sampling was done from surface horizons at the 10-20 cm depths. the samples were transported and stored in pvc sacks at 4 °c in the dark. prior to chemical analyses, samples were air-dried at room temperature, homogenized, and then passed through a 2-mm sieve, then stored in a field-moist condition at 4 °c in the dark. all procedures were carried out according to čurlík and šurina (1998), šimanský (2011) and hrivňáková et al. (2011). total organic carbon content was measured by dichromate oxidation (nelson and sommers 1996). the isolation of fungal strains was realized using the dilution plating method (dilution of 10-4 cfu) from 10 g of substrate. dilutions were plated on sabouraud maltose agar (sab) (himedia, mumbai, india) for isolation. studied strains were isolated from mixed culture of soil microscopic filamentous fungi. pure cultures of all strains were cultivated in an incubator at 25 °c for 5-7 days on sab and identified according to micromorphological features and pcr analyses (šimonovičová et al. 2013; jesenák et al. 2015). growth and colony size of a. niger wild type strains the growth and the size of colonies of all studied a. niger wild type strains were observed after cultivation on sab agar (sabouraud dextrose agar, it means mycological peptone, dextrose and agar – himedia,mumbai, india) in a temperaturecontrolled oven at 25 °c for 5-7 days in three replicated runs. influence of the original substrates from šobov, pezinok, gabčíkovo and slovinky sites was studied by adjustment of ph values of sab agar to ph 3-5-7 and 9. fig. 1. study of polluted sites šobov, pezinok, slovinky and gabčíkovo. the last mentioned locality was the control site.  enzymatic activity of a. niger wild type strains enzymatic activity of the strains was studied using diagnostic culture media specific for each individual enzyme as follows: cellulase activity (ce) on congored medium, esterase activity (ea) on tween 80 medium, lipase activity (la) on spirit blue medium and protease activity (pa) on gelatine p3 medium. productivity of enzyme is visible as so-called "halo" effect (kraková et al. 2012; šimonovičová and čerňanský 2016) which is a creating zone or a ring of certain diameter around the fungal colony. production of organic acids of a. niger wild type strains pre-treatment of samples. the samples were stabilised at -4 °c. the samples were defrosted in water bath at 20 °c before hplc analysis. then, all samples were homogenised and filtered through regenerated cellulose membrane filters with pore size 0.45 μm. hplc analysis. analysis of the samples was realized using a hplc device with a pda detector (younglin 9100). the mobile phase used to analysis included methanol and water; their bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:56 utc nova biotechnol chim (2017) 16(2): 92-98 94 table 1. characteristics of the soils from the natural sites where the fungal strains were isolated. strain locality ph substrate an s banská štiavnica-šobov 3.12 ultra acidic dystric cambisol (contaminated and eroded without vegetation); % cox 0. 49; al 727 – 506 mg/kg an p pezinok 5.3 strongly acidic stream sediment; % cox 7.2; as 363 mg/kg; sb 93 mg/kg; fe 82.8 mg/kg an g gabčíkovo 7.7 slightly alkaline eutric fluvisol; % cox 6.3, without any contamination, vegetation is salici-popuetum an sl slovinky 8.5 strongly alkaline technosol without vegetation; % cox 0.3 – 0.8; as 305 – 511 mg/kg; cd 8.6 – 13.4 mg/kg; cu 7 372 – 9 227 mg/kg; pb 2 964 – 8 078 mg/kg; zn 24 786 – 47 291 mg/kg concentration during analysis was changed from initial ratio 10:90 up to 90:10. analysis of the samples was carried out at 25 °c. the column (gracesmart, rp 18, 150 mm length, od 4.6 mm) for selective separation of analytes was used. the flow rate was 1 ml.min-1 and the wave length of pda detector ranged from 222, 210, 200 to 235 nm (mackuľak et al. 2011). pelletization of a. niger wild type strains the fungal pellets were prepared in a 60 ml sab medium with ph adjusted to 3, 5, 7 and 9 (respectively) enriched with a 10 ml suspension of conidia from each strain and 1 g of montmorillonite. controls at each adjusted ph value were without montmorillonite. cultivation was carried out using a shaker unimax 2010 (heidolph, germany) at 135 rpm. then, the pellets were carefully washed with large amount of distilled water and stored in petri dishes where number and size of the pellets were measured. microstructure of the pellets was recorded using a digital stereomicroscope leica dmd 108. results and discussion microscopic fungi in spite of the fact, that aspergillus niger belongs to cosmopolitan strains of soil filamentous fungi (domsch et al. 2007; klich 2002), it has not been isolated from all soil types in slovakia (šimonovičová 2013). on the other hand this strain was very frequent-to dominant in contaminated soils and substrates (šimonovičová et al. 2016). this strain belongs to metal tolerant fungi (anahid et al. 2011; iram et al., 2009, 2013). four wild type strains of a. niger were used in this study that originate from soils and stream sediments from various localities in slovakia (fig. 1, table 1). fig. 2. colony size of four a. niger wild type strains depending on ph of the growth substrate. the first strain an-s was isolated from dystric cambisol (contaminated and eroded) on the locality šobov, near banská štiavnica. this ultra-acidic soil lacks vegetation and also is very poor on organic material. from stream sediment on the locality pezinok-kolársky vrch (pezinok) was isolated the second strain an-p. the substrate on this site is strongly acidic and rich on organic material, but contaminated with very high amount of as, sb and fe (table 1). from eutric fluvisol, a slightly alkaline substrate rich on organic material from the locality gabčíkovo, was isolated the third strain – an-g. this strain was taken as a control strain, because the locality lacks any contamination. the strongly alkaline technosol without vegetation on the locality slovinky was the source of the fourth strain an-sl. the corresponding content of organic bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:56 utc nova biotechnol chim (2017) 16(2): 92-98 95 material is minimal, and the soil is contaminated with several toxic elements including as, cd, cu, pb and zn (table 1). table 2. production of organic acids (mg/l) by the studied a. niger wild type strains. sensitivity mg/l 0.095 0.1 0.1 strain formic acid acetic acid oxalic acid an-s 0.108 0.143 0.173 an-p 0.144 0.1 0.175 an-g 0.238 0.1 0.228 an-sl 0.164 0.11 0.138 growth and colony size of a. niger wild type strains average sizes of colonies growing on the cultivation media with different ph were as follows: 2.37 cm at ph 3, 2.42 cm at ph 5 and 2.32 cm at ph 7 (fig. 2). the largest colonies were always recorded under conditions most similar to their original environment, i.e. an-s formed colonies of 2.8 cm at ph 3, an-p of 3.7 cm at ph 5, an-g of 4 cm at ph 7 and an-sl of 2.8 cm at ph 9. an exception was the strain an-s1 isolated from strongly alkaline substrate that formed colonies of quite similar size at different ph (2.2 cm at ph 3, 2 cm at ph 5 and 1.9 cm at ph 7) (fig. 2). even though microscopic filamentous fungi occur in soils and other terrestrial substrates of various ph, it is obvious that they prefer acidic or neutral environmental conditions (chen et al. 2013; this study). enzymatic activity of a. niger wild type strains relatively slow growth of all a. niger strains were recorded on the substrate assigned for esterase activity assessment and very low growth, almost negligible, was recorded on the substrates for proteases and cellulases. the growth of all a. niger strains was very good on the substrate for lipases. the so-called "halo" effect was recorded for all strains. the obtained results confirm a higher lipase activity for the an-g strain compared with the other strains studied. inhibition of metabolism as well as of enzymatic activity can be an expression of the quality of the original environment from which the fungal strains were isolated. the lowest metabolism and enzymatic inhibition was recorded for the strain an-g isolated from unpolluted environment. however, the real influence of the metal(loid) contaminants and other soil properties such as (low) carbon content, humidity etc. on metabolism of the other studied strains (an-p, an-s, an-sl) must be further studied. fig. 3. significantly higher number of pellets with the lower diameter (compared to pellets without clay minerals) as a consequence of the montmorillonite presence using a. niger strains isolated from šobov at ph 3 (a), pezinok at ph 7 (b), gabčíkovo at ph 7 (c) and from slovinky at ph 9 (d). production of organic acids of a. niger wild type strains according to production of organic acids by a. niger strains (table 2) the results of hplc analysis suggests the ability of all the strains to decompose organic pollutants in contaminated soil samples. such decomposition products can include lower acids, notably oxalic-, formicand acetic acids. the abundance of individual acids (their ratio) in the samples varied, likely due to enzymatic activity of the particular a. niger strain. the highest concentrations of organic acids, especially of oxalic acid, were recorded in the pre-treated sample an-g of the control strain from gabčíkovo as a probable consequence of the highest lipase activity of the microorganism. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:56 utc nova biotechnol chim (2017) 16(2): 92-98 96 fig. 4. filaments of pellets in the control sample an-g (a) and changing the shape of the pellets in the presence of montmorillonite (b). partial intermediates of decomposition of organic acids during bioleaching gradually separated organic acids due to disruption of the equilibrium in term of lysis of the cells of a. niger strains. due to disruption of the equilibrium, the complete degradation of organic acids e.g. to acetone, methanol, aldehyde etc. was reached (amiri et al. 2012). the a. niger strains effectively degrade pollutants to simple acids with no significant negative ecological impacts to the environment. subsequently, such produced substances can be utilized by other microorganisms as a source of carbon or energy. these processes can stimulate decomposition procedures of resistant types of pollutants occurred in landfills confirmed by seepages (xu et al. 2014). metabolites of microscopic filamentous fungi such as organic acids and amino acids affect ph of the medium. acidification of the medium supports the mobility of metals, especially the transport mechanism of metal from the environment into the fungal cell and backwards (gadd 2004). pelletization of a. niger wild type strains one of the factors influencing the growth of pellets is the presence of inorganic powders. these are, for example, clay minerals, which are a regular part of soils and sediments. in our case, we studied the influence of clay mineral montmorillonite on the growth of pellets. we find out that in the presence of montmorillonite there was significantly higher number of pellets; however, the diameter of the pellets was lower (fig. 3a-d) throughout the growing period. this claiming is valid for each strain tested. this phenomenon can be explained by several reasons. most probably higher amount of nucleation sites which are constituted by microscopic particles of clay minerals is responsible for increasing of the number of pellets coupled with spherical inhibition of fungal growth expressed in the diameter changes. another possible reason is a relative increase in the length of fungal filaments in pellets (fig. 4a-b). according to control experiments, the largest pellets were produced by the an-s strain at ph 3 (2.67 mm) and ph 9 (3.04 mm), followed by the an-p at ph 5 (5.28 mm) and ph 7 (4.7 mm), the an-g at ph 3 (4.39 mm) and ph 5 (3.65 mm) and an-sl at ph 9 (5.35 mm), (fig. 5a-d). fig. 5. pellets of different sizes in the control samples. a. niger strains isolated from the localities šobov at ph 9 (a), pezinok at ph 7 (b), gabčíkovo at ph 7 (c) and from slovinky at ph 5 (d) with a diameter 5.35 mm. according to fomina and gadd (2002), the changes in size of the pellets, shape and length of their mycelia of cladosporium cladosporioides and humicola grisea was also confirmed. all these changes can be explained by a significant increase in the number of inorganic particles in the culture medium. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:56 utc nova biotechnol chim (2017) 16(2): 92-98 97 conclusions the obtained results confirm the effects of environmental factors such as ph values of the substrate, % cox and contents of potentially toxic elements on physiological properties of aspergillus niger strains. influenced were especially the growth of colonies and the metabolism. lipase activity in the strain an-g was higher compared with the other studied strains. pollutants such as as, sb, al, cd, cu, pb and zn in all substrates except for those from the gabčíkovo site inhibited the fungal metabolism as well as enzymatic activity. this effect was probably even enhanced by extremely low content of organic carbon in the samples from the šobov and slovinky sites. on the other hand, higher content of organic carbon in the substrate does not increase enzymatic activity in case of the samples an-p and an-g. the production of pellets was significantly reduced in the presence of montmorillonite. in this case, all strains produced a lot of pellets with small sizes. the mechanisms behind smaller pellet formation are unknown but might represent a fungal adaptation to presence of toxic compounds. also, it can be assumed application of fungal pellets in remediation of landfills, where the contents of metal(loid)s are higher, especially during bioleaching of mining wastes with low contents of organic carbon. acknowledgement this work was supported by slovak national grant agency vega 1/0482/15. references akhter n, morshed ma, uddin a, begum f, sultan t, azad ak (2011) production of pectinase by aspergillus niger cultured in solid state media. int. j. biosci. 1: 33-42. amiri f, mousavi sm, yaghmaei s, barati m (2012) bioleaching kinetics of a spent refinery catalyst using aspergillus niger at optimal conditions. biochem. eng. j. 67: 208-217. anahid s, yaghmaei s, ghobadinejad z (2011) heavy metal tolerance of fungi. sci. iran. c 18: 502-508. chen h, mothapo nv, shi w (2013) soil moisture and ph control relative contributions of fungi and bacteria to no2 production. microb. ecol. 69: 180-191. čurlík j, šurina b (1998) príručka terénneho prieskumu a mapovania pôd. výskumný ústav pôdnej úrodnosti, bratislava, 136 pp. domsch kh, gams w, anderson th (2007) compendium of soil fungi. second edition, taxonomically revised by w. gams. ihw-verlag eching, 672 pp. fomina ma, gadd mg (2002) influence of clay minerals on the morphology of fungal pellets. mycol. res. 106: 107117. gadd gm (2004) microbial influence on metal mobility and application for bioremediation. geoderma 122: 109-119. gan m, song z, zhu j, liu x (2016) efficient bioleaching of heavy metals from contaminated sediment in batch method coupled with the waste assistance of 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ash by aspergillus niger precipitation of metallic salt crystals and morphological alteration of the fungus. biotechnol. rep. 3: 8-14. zhang j, zhang j (2016) the filamentous fungal pellet and forces driving its formation. crit. rev. biotechnol. 36: 1066-1077. zhang s, li a, cui d, yang j, ma f (2011) performance of enhanced biological sbr process for aniline treatment by mycelial pellet as biomass carrier. bioresour. technol. 102: 4360-4365. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 07:56 utc nova biotechnol chim (2021) 20(2): e652 doi: 10.36547/nbc.652 1 nova biotechnologica et chimica optimization of callus induction with enhancing production of phenolic compounds production and antioxidants activity in callus cultures of carob (ceratonia siliqua l.) assia lozzi1, , rachid mentag2, driss alami-halimi1, rabha abdelwahd2, abdelhadi abousalim1 1department of plant production, protection and biotechnologies, institute of agronomy and veterinary medicine hassan ii, rabat-instituts 6654, morocco 2biotechnology unit, regional center of agricultural research, national institute of agricultural research, rabat 415, morocco  corresponding author: assia.lozzi@gmail.com article info article history: received: 12th august 2020 accepted: 16th august 2021 keywords: antioxidant activity callus cultures ceratonia siliqua condensed tannin flavonoids polyphenols abstract carob (ceratonia siliqua l.) is an important mediterranean plant species with worldwide commercial and medicinal uses. the establishment of a callus culture protocol as an alternative system to produce polyphenols of chemical and pharmaceutical interest was made in the present study for the first time in carob. explant type and the light regime are two important factors that influence morphogenic responses and biochemical production. maximal callus induction (100 %) and biomass accumulation were obtained in cotyledon explants under both tested light regimes (16-h photoperiod and darkness). however, leaf-derived callus produced higher amounts of polyphenols (tpc) and flavonoids (tfc) but a lower amount of total condensed tannins (tct) as compared to cotyledon-derived callus. the light treatment has significantly increased tct content but decreased the antioxidant activity in carob callus cultures. strong and positive correlations were obtained between tpc, tfc, and the antioxidant activities with correlation coefficients in range 0.68 – 0.98. the obtained results indicated that calli of c. siliqua have the potential for enhanced production of phenolic compounds with antioxidant activity that is favored by cultivation under dark condition.  university of ss. cyril and methodius in trnava introduction ceratonia siliqua l., commonly known as ‘carob’, is an important xerophytic plant belonging to the fabaceae family. due to its important economic and ecologic values, it has drawn much attention in recent years. the interest in carob as an inexpensive source of several products has been increasing over the last few years. carob seeds are being used for carob bean gum extraction. carob pods can also be exploited for the production of sugar syrups (fidan et al. 2020), cocoa substitutes (akdeniz et al. 2021), bioethanols (germec et al. 2020), and secondary metabolites (kyratzis et al. 2021). carob pods and leaves are being in use since olden time to cure digestive disorders, asthma, pharyngitis, bronchitis, and anemia (rtibi et al. 2017; sargin and büyükcengiz 2019). furthermore, mailto:assia.lozzi@gmail.com nova biotechnol chim (2021) 20(2): e652 2 many studies have been conducted on several carob-tree parts for their bioactive components, including total phenolics which exhibit potent cytotoxic, anticancer, anti-inflammatory, hepatoprotective, nephroprotective, antidiabetic, sedative, and antioxidative properties (benchikh et al. 2014; custódio et al. 2015; aboura et al. 2017; ghanemi et al. 2017; othmen et al. 2020; gurel et al. 2021). the major phenolic compounds in this plant are phenolic acids, flavonoids, and tannins (stavrou et al. 2018). however, phenolic contents in carob field-grown plants are highly variable due to genetics and environmental factors (custódio et al. 2007; el hajaji et al. 2011; korkmaz et al. 2020). in vitro culture techniques emerge as efficient biotechnological tools to produce commercially important medicinal compounds such as polyphenols, under aseptic and controlled conditions. these techniques would allow continuous, economical, viable, and sustainable production of pure bioactive compounds, regardless of the climatic and geographic conditions, during a shorter and more flexible production cycle (dias et al. 2016; gai et al. 2020). different factors affect phenolic production in in vitro plant cultures, such as explants type and light conditions, although responses vary with the class of secondary metabolites and among species (estell et al. 2016; farhadi et al. 2017; shehzad et al. 2021). it must be noted that while studies on callus culture of carob have been reported (ksia et al. 2008; lozzi et al. 2015; 2018; 2019; zouari and el mtili 2020), the antioxidant properties of their phenolic compounds have not been investigated. to our knowledge, this study represents the first attempt to demonstrate that the callus culture of carob is a reliable tool for polyphenols production. the objectives of the present work were to: (i) optimize methods for producing vigorous carob callus by monitoring the effects of explant type and light conditions, (ii) determine the content of the total phenolics, flavonoids, and condensed tannins and (iii) evaluate the antioxidant activity in in vitro calli to elucidate the medicinal potency of in vitro cultures of this plant. experimental plant materials and callus culture mature seeds were collected from selected c. siliqua trees growing in beni mellal, morocco, treated with concentrated sulfuric acid (98 %) for 60 min, washed and then immersed for 48 h in sterile distilled water. cotyledons were aseptically excised from seeds and segmented into portions of 4 – 6 mm in length. leaf, hypocotyl, epicotyls, and root segments were cut from two-month-old seedlings, germinated on the carob culture medium (lac) that we have developed and successfully applied in our previous research on carob micropropagation (lozzi et al. 2019). all explants (cotyledons, leaves, hypocotyls, epicotyls, and roots) were inoculated on the lac medium supplemented with 5 µm 2,4dichlorophenoxyacetic acid (2,4-d), 3 % (w/v) sucrose and 0.7 % (w/v) agar. the cultures were maintained at 26 ± 2 °c, either in darkness or under a 16-h photoperiod (60 μmol.m–2.s–1) for five weeks. extraction callus cultures of known fresh weight were dried in a hot air oven at 60 °c to constant weight. each finely ground dried sample (0.1 g) was mixed with 70 % (v/v) acetone (40 ml). the mixtures were then centrifuged at 4,000 rpm for 20 min. the supernatant was collected and maintained at 4 °c until used. total phenolic content (tpc) total phenolic content of the calli extracts was determined spectrometrically according to the folin–ciocalteu assay (singleton and rossi 1965) using the gallic acid as standard. the calli extract (0.1 ml) was mixed with 1.4 ml of deionized water and 0.1 ml of folin-ciocalteu reagent. after 6 min of incubation, 0.5 ml of na2co3 (20 %) was added and the reaction mixture was allowed to incubate in the dark for 30 min. absorbance was measured at 750 nm and results were expressed as mg gallic acid equivalents (gae) per gram of dry weight of the sample. nova biotechnol chim (2021) 20(2): e652 3 total flavonoid content (tfc) total flavonoid content was evaluated in the extracts using the aluminum chloride (alcl3) assay (lamaison et al. 1990). 1 ml of alcl3·6h2o (2 %) was added to 1 ml of acetonic extract and the absorbance was read 10 min later at 430 nm. quercetine was used to make the calibration curve, and the results were expressed as mg of quercetine equivalents (qe) per g dry matter in extracts. total condensed tannin (tct) condensed tannins were analyzed using the vanillin–hcl method (broadhurst and jones 1978). 0.1 ml of calli extract was added to 3 ml of vanillin (4 %) and 1.5 ml of highly concentrated hcl. the absorbance was measured at 500 nm after standing for 20 min in a dark room. the results were expressed as mg of catechin equivalent (ce) per g of dry weight. dpph radical-scavenging assay the free radicals scavenging activity of the in vitro callus of carob was evaluated spectrophotometrically using the 2,2-diphenyl-1picryl hydrazyl (dpph) method (brand-williams et al. 1995). briefly, 1 ml of 0.1 mm dpph solution (in ethanol) was added to 0.5 ml of calli extract diluted in 1.4 ml of ethanol. the mixtures were vortexed and incubated for 30 min in darkness. the absorbance was measured at 515 nm and the ascorbic acid was used as a standard. the radical scavenging activity was calculated according to the following formula (eq. 1): (1) where a0 is the absorbance of the negative control (ethanol) and a1 is the absorbance of the reaction mixture containing the sample extract. ferric-reducing power (frap) the reducing power of the extract was evaluated spectrophotometrically using the assay of oyaizu (1986). 0.22 ml of the extract was added to 0.25 ml of phosphate buffer (0.2 m, ph 6.6) and 0.25 ml of potassium ferricyanide (1 %). the mixture was then incubated at 50 °c for 20 min. afterward, 0.25 ml of 10 % trichloroacetic acid was added, and the mixture was mixed with 1 ml of distilled water and 0.25 ml of iron trichloride (0.1 %). the absorbance was read at 700 nm. the increased absorbance of the reaction mixture meant increased reducing power of the calli extract. statistical analysis all extractions and assays were carried out in triplicate. the results were expressed as mean ± standard deviation. the experimental data were processed with the analysis of variance (anova) followed by duncan’s multiple range test (dmrt) at 5 % to determine significant differences among the mean values. pearson’s correlation test was used to identify the correlation between total phenolics and antioxidant activity. results and discussion effect of explant type and light conditions on induction the percentage of callus induction and the dry weights (dw) of calli were significantly (p<0.001) affected by explant type, confirming the results of previous works (gourguillon et al. 2018; rameshkumar et al. 2018; wu et al. 2020). a successful callus formation was achieved in both cotyledon and leaf explants (fig. 1a, b). however, the cotyledon explants showed 100 % callus induction and a much better callus dry weight than leaf explants (table 1). attempts to induce callus from hypocotyls and epicotyls were completely ineffective (fig. 1c, d). the root explants exhibited poor callus formation, turned brown and died within 5 weeks (fig. 1e). the difference of response between the explants could be related to the difference in endogenous hormone level and the competence of cells to respond to the external signals (jiménez and bangerth 2001a, 2001b; wu et al. 2020; saeedpour et al. 2021). besides of the explant type, light is another factor that may influence competence by modifying the concentration of endogenous hormone in explant tissues (jiménez and bangerth 2001a; su et al. nova biotechnol chim (2021) 20(2): e652 2 2014; vinterhalter et al. 2020). callus development of c. siliqua in response to light conditions is missing in the cited literature. in the present investigation, callus growth (dw) on cotyledon explants was similar under both tested light regimes (16-h photoperiod and darkness). the maximum dry weight (53.9 ± 4.9 mg.l-1) being achieved in darkness. note that the present result is considerably higher than our previous studies (lozzi et al. 2015, 2018) on callus induction from callus cotyledons using other basal culture media: ms (murashige and skoog 1962), b5 (gamborg et al. 1968), wpm (lloyd and mccown 1980) and dkw (driver and kuniyuki 1984), indicating that lac medium is more suitable for callus induction from carob cotyledons. in the case of leaf explants, the biomass accumulation (dw) in callus formed in the dark was significantly higher than that in the light. previous studies revealed a close relationship between light regimes and callus biomass accumulation (ju et al. 2014; shehzad et al. 2021) which indicate the need for optimal light conditions for optimum biomass accumulation. table 1. comparative response of callus induction in ceratonia siliqua from mature cotyledons and leaves in photoperiod and darkness. type of explant light conditions callus induction [%] callus dw [mg.l-1] mature cotyledons 16-h photoperiod 100.0a 53.9 ± 4.9a darkness 100.0a 52.5 ± 7.0a leaves 16-h photoperiod 96.3a 30.8 ± 5.0b darkness 73.6b 24.4 ± 1.1c significance of two-way anova type of explant (a) *** *** light conditions (b) ** * a×b ** ns values are mean ± se. mean values within a column followed by the same letter are not significantly different by duncan’s multiple range test (5 %). dw – dry weight; ∗, ∗∗, ∗∗∗ – significance at p≤0.05, p≤0.01, and p≤0.001, respectively (two-way anova). fig. 1. callus induction from different explants of ceratonia siliqua l. a – mature cultyledons; b – leaf explants; c – hypocotyl explants; d – epicotyls explants; e – root explants. arrows indicate callus tissues. 4 nova biotechnol chim (2021) 20(2): e652 5 effects of explant type and light conditions on antioxidant compounds the total phenolic contents of the extracts are shown in fig. 2. in contrast to the finding on callus induction and biomass accumulation, the tpc, tfc, and tct contents in leaf derived calli extracts were significantly higher when compared to cotyledon-derived calli extracts. leaf-derived callus produced the highest amounts of phenolic compounds (21.4 ± 1.4 mg gae.g−1 in darkness and 20.6 ± 0.9 in light) which were about three-fold higher than those of cotyledon callus. similarly, the leaf-derived calli produced a higher amount of flavonoids than those of cotyledon explant (7.5 ± 0.9 mg.qe.g−1 in darkness and 7.0 ± 0.7 in light). among calli derived from cotyledon explants, regardless of the light conditions, the level of tpc was not significantly different (p<0.05). similar behavior was observed among calli derived from leaf explants, indicating that the light regime tested did not influence the accumulation of these compounds. similar results were documented by fazal et al. (2016) in callus culture of prunella vulgaris where tpc production was not influenced by light conditions. opposite results were however, obtained with calli from tecoma stans and cnidium officinale when illumination induced a strong accumulation of polyphenols (rameshkumar et al. 2018; adil et al. 2019). in contrast, the tfc level was significantly (p<0.01) influenced by the light regime. the total flavonoid content was higher in darkness (3.8 ± 0.2 mg.qe.g−1 in cotyledon-derived callus and 5.3 ± 0.3 in leaf-derived callus) than in periodic light. in contrast to the finding on tpc and tfc accumulation in carob calli extracts, the cotyledon-derived callus accumulated higher amounts of tct, which were about two-fold higher than those of leaf-derived callus; the difference being significant (p<0.001). furthermore, callus grown under periodic light enhanced the accumulation of tct (1.7 ± 0.2 mg.ce.g−1 in cotyledon-derived callus and 0.84 ± 0.07 in leafderived callus) as compared to dark incubation. similarly, (castro et al. 2016) found a positive response of light on the tct in byrsonima verbascifolia callus cultures, whereas favre et al. (1991) found no effect in quercus callus. fig. 2. effects of explant type and light conditions on antioxidant compounds in callus cultures of carob (ceratonia siliqua l.). a – total phenolic content; b – total flavonoid content; c – total condensed tannin. mean values within a column followed by the same letter are not significantly different by duncan’s multiple range test (5 %). the levels detected in the present study were found to be higher than those reported in different aerial parts of moroccan carob, which ranged from 0.45 to 2.64 and from 15.8 to 18.08 mg.gae.g−1 of phenolic content in intact carob leaves and pulp (hajaji et al. 2010) and from 0.25 to 1.02 mg.ce.g−1 of condensed tannins in carob pulp (el bouzdoudi et al. 2016). while in the aceton extract a b c nova biotechnol chim (2021) 20(2): e652 2 of carob seed peel, total phenolics, flavonoids, and condensed tannins were found to be 0.16 µg.gae.g−1, 0.5 µg.qe.g−1 and 0.5 µg.ce.g−1, respectively (lakkab et al. 2019). other studies on algerian, tunisian, portuguese, and cypriot carob showed different levels of total phenolic content that ranged from 1.8 to 307 and 6 to 336 mg.gae.g−1 of leaves and pods (papagiannopoulos et al. 2004; custódio et al. 2009; ortega et al. 2009; meziani et al. 2015; correia et al. 2018; ayache et al. 2020; kyriacou et al. 2021).these authors attributed the differences not only on genetic, geographical, environmental, and cultural conditions, but also on technological factors such as the extraction methodologies and the used solvent system. effects of explant type and light conditions on antioxidant activity total antioxidant capacity may vary depending on the assay used (tabart et al. 2009). therefore, using more than one method is recommended to evaluate this property (tabart et al. 2014). in the present study, the antioxidant activities of carob calli extracts were determinate using two widely known assays, based on electron transfer reaction, the dpph free radical-scavenging activity (dpph) and ferric-reducing power (frap). however, the two methods clearly showed that carob calli extracts contain considerable antiradical and antioxidant activities (table 2). dpph assay showed that all extracts were able to reduce the purple-coloured radical dpph into pale, yellowcoloured dpph-h. free radical scavenging activity in different treatments ranged from 60.92 ± 4.98 to 88.82 ± 0.10 %, which levels were the highest in leaf-derived callus followed by cotyledon-derived callus. cultivation in the light or darkness had no apparent effect on scavenging activity in leafderived callus. in contrast, light treatment induced a significant decrease of the scavenging activity in cotyledon-derived callus. similar behavior was observed for the frap test that measures the ability of the extract to reduce fe3+ to fe2+ and may serve as a significant indicator of its antioxidant potential (govindan and muthukrishnan 2013). these findings are in line with the results reported by ali and abbasi (2014) in the cell culture of artemisia absinthium, where dark-grown cultures showed maximum tpc, tfc, and antioxidant activity compared to light treatments. weremczukjeżyna et al. (2013) also reported that the hairy roots of dracocephalum moldavica cultivated in the darkness showed the highest antioxidant activity compared to those cultivated under photoperiod. very few studies about the effect of light and dark on secondary metabolites accumulation and the antioxidant activity in callus tissue culture are reported. the increased antioxidant activity under darkness in the present study could be attributed to the stressed callus culture conditions. indeed, there is increasing evidence that some light conditions such as uv, high-intensity light, and continuous dark may act as a stressful factor requiring a change in the oxidative balance in plants which produce a large amount of scavenging enzymes and antioxidant molecules as an answer to the increased generation of reactive oxygen species (ros), and thereby minimise potential oxidative damage (gogorcena et al. 1997; close and mcarthur 2002; ali et al. 2005; pashkovskiy et al. 2018). dark stress was reported to induce structural and oxidative damage in other fabaceae species such as soybean and phaseolus vulgaris (gogorcena et al. 1997; wang et al. 2021). our results are in good agreement with a previous report considering the antioxidant activity in intact carob leaves, pulp, and seeds (hajaji et al. 2010; lakkab et al. 2019; ayache et al. 2020). the dpph scavenging activity reached 61.17 %, 80.9 % and 79.4 % respectively. this antioxidant activity may be attributed to the levels of phenolic content, flavonoids, and condensed tannin. based on the correlation coefficient values calculated for the total phenolic components and the antioxidant assays, it could be suggested that both the scavenging activity and the reducing power of the analyzed extracts are strongly and positively correlated with the phenolic (r2 = 0.84 and 0.98, respectively) and flavonoid accumulation (r2 = 0.71 and 0.68, respectively) (table 3). these results agree with the earlier reports on carob pulp extract (benchikh et al. 2014; benchikh and louailèche 2014). in contrast, significant (p<0.01) negative relationships were observed of tct tannins with tpc, tfc, dpph, and frap, with correlation coefficients ranging between -0.86 and -0.73. furthermore, the frap was significantly 6 nova biotechnol chim (2021) 20(2): e652 5 p<0.01) correlated with dpph (r2 = 0.88) which rises the hypothesis that compounds which express reducing activity meet the criteria for exhibiting antiradical effect. table 2. effects of explant type and light conditions on antioxidant compounds in callus cultures of carob (ceratonia siliqua l.). values are mean ± se. mean values within a column followed by the same letter are not significantly different by duncan’s multiple range test (5m%). fw – fresh weight; dw – dry weight; od – optical density; gae – gallic acid equivalents; qe – quercetine equivalents; ce – catechin equivalent; ns – nonsignificant; ∗, ∗∗, ∗∗∗ – significant at p≤0.05, p≤0.01, and p≤0.001, respectively (two-way anova). table 3. pearson’s correlation coefficients of different biochemical parameters of carob calli extracts. tpc tfc tct dpph tpc tfc 0.78** tct -0.73** -0.80** dpph 0.84** 0.71** -0.86** frap 0.98** 0.68* -0.78** 0.88** * correlation is significant at the 0.05 level, ** correlation is significant at the 0.01 level. conclusion the present study demonstrates, for the first time, the potential of callus cultures of ceratonia siliqua as a new source of phenolic compounds which possesses an important antioxidant activity. leaves-derived calli were found to produce significantly higher levels of phenolics and showed higher antioxidant activity than the cotyledonderived callus. however, cotyledon-derived callus accumulated more tct. the light treatment has significantly increased total condensed tannins content, but significantly decreased the antioxidant activity in callus cultures. moreover, a positive correlation between antioxidant activity and total phenolics and flavonoids contents were found in carob callus cultures. further works on improving protocols for in vitro cultivation of carob and characterization of individual groups of bioactive components responsible for the biological activity are needed. conflict of interest the authors declare that they have no conflict of interest. references aboura i, nani a, belarbi m, murtaza b, fluckiger a, dumont a, benammar c, tounsi ms, ghiringhelli f, rialland m, khan na, hichami a (2017) protective effects of polyphenol-rich infusions from carob (ceratonia siliqua) leaves and cladodes of opuntia ficus-indica against inflammation associated with diet-induced obesity and dss-induced colitis in swiss mice. biomed. pharmacother. 96: 1022-1035. adil m, ren x, jeong br (2019) light elicited growth, antioxidant enzymes activities and production of medicinal compounds in callus culture of cnidium officinale makino. j. photochem. photobiol. b: biology 196: 111509. akdeniz e, yakişik e, pirouzian hr, akkin s, turan b, tipigil e, toker o, ozcan o (2021) carob powder as cocoa substitute in milk and dark compound chocolate formulation. j. food sci. technol. 233: 1-9. ali m, abbasi bh (2014) light-induced fluctuations in biomass accumulation, secondary metabolites production and antioxidant activity in cell suspension cultures of artemisia absinthium l. j. photochem. photobiol. b: biol. 140: 223-227. ali mb, hahn e-j, paek k-y (2005) effects of light intensities on antioxidant enzymes and malondialdehyde content during short-term acclimatization on micropropagated phalaenopsis plantlet. environ. exp. bot. 54: 109-120. type of explant light conditions dpph [%] frap [od] mature cotyledons 16-h photoperiod 60.92 ± 4.98c 0.29 ± 0.04c darkness 76.60 ± 7.14b 0.38 ± 0.01b leaves 16-h photoperiod 86.93 ± 1.91a 0.65 ± 0.02a darkness 88.82 ± 0.10a 0.61 ± 0.01a significance of two-way anova type of explants (a) *** *** light conditions (b) ** ns a×b * ** 7 nova biotechnol chim (2021) 20(2): e652 8 ayache s ben, saafi eb, emhemmed f, flamini g, achour l, muller cm (2020) biological activities of aqueous extracts from carob plant (ceratonia siliqua l.) by antioxidant, analgesic and proapoptotic properties evaluation. molecules. 25: 3120. benchikh y, louailèche h (2014) effects of extraction conditions on the recovery of phenolic compounds and in vitro antioxidant activity of carob (ceratonia siliqua l.) pulp. acta bot. gall. 161: 175-181. benchikh y, louaileche h, george b, merlin a (2014) changes in bioactive phytochemical content and in vitro antioxidant activity of carob (ceratonia siliqua l.) as influenced by fruit ripening. ind. crops prod. 60: 298-303. brand-williams w, cuvelier m-e, berset c (1995) use of a free radical method to evaluate antioxidant activity. lwtfood sci. technol. 28: 25-30. broadhurst rb, jones wt (1978) analysis of condensed tannins using acidified vanillin. j. sci. food. agric. 29: 788-794. castro ahf, braga k de q, de sousa fm, coimbra mć, changas rcr (2016) callus induction and bioactive phenolic compounds production from byrsonima verbascifolia (l.) dc. 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onalbaeva et al. 2016). among them very promising are biotechnological methods using bioleaching as this method overcome the operational and technical problems associated with lithium recovery. traditionally acidophilic bacteria are widely applied, however, their usage is mostly oriented on metal recovery from sulphidic ores or metal-bearing waste materials, but their utilisation in aluminosilicates is limited (sedláková-kaduková et al. 2017). the most abundant lithium containing rocks are pegmatites with various li minerals (zhang et al. 2018). the most interesting li mineral for industry is spodumene due to its high li content, large deposits and low content of other impurities mailto:jana.sedlakova@upjs.sk nova biotechnol chim (2020) 19(2): 175-182 176 (metals). other minerals are petalite, lepidolite amblygonite, zinnwaldite and eucryptite. lepidolite is the most abundant li containing mineral, however, containing less li than spodumene (tadesse et al. 2019). the mineral composition of lepidolite and its aluminosilicate lattice was the reason why heterotrophic fungi have been selected for li bioleaching from this mineral. heterotrophic fungi have a great potential to degrade aluminosilicates resulting in metal extraction from these ores because in nature, fungi significantly contribute to the silicate bearing rocks (including mica, and iron and manganese bearing rocks) weathering (gadd 2010). they use two, often, synergistic actions to degrade mineral substrates, namely biomechanical and biochemical processes. however, the microbial activity is considered to have much more significant impact on the degradation of these substrates than mechanical actions. in biomechanical weathering process fungal hyphae may directly penetrate into intact mineral material or by producing extracellular mucilaginous substances they can indirectly deteriorate the mineral firstly by acidic and metal-chelating metabolites excreted to mucilaginous slime but also by forming a mechanical pressure to the mineral by shrinking and swelling of biofilms (gadd 2007). biochemical action involves several mechanisms, such as acidolysis, complexolysis, reductive mobilization and the function of mycelium to bound the soluble metal species (fomina et al. 2005). silicate biodegradation may be also accelerated by carbonic acid attack due to the co2 released during fungal respiration (sterflinger 2000). among the most bioleaching active fungal species been isolated and used in metal bioleaching belong species from the aspergillus genus, with the most efficient aspergillus niger (šimonovičová et al. 2012). it was found that a. niger can degrade dunite, serpentine, olivine, muscovite, feldspar, kaolin, spodumene and nepheline (gadd 2007), however its ability to deteriorate lepidolite was not previously reported. according to our previous experience as well as published studies dealing with the utilization of aspergillus for metal bioleaching (dursun et al. 2003; littera et al. 2011; marcinčáková et al. 2014) we decided to study not just the lithium bioleaching but also consequent lithium bioaccumulation in the aspergillus biomass. therefore, this work was aimed to investigate the ability of microscopic fungus a. niger to solubilize and subsequently accumulate li from lepidolite. for this purpose, not just changes in ph, lithium concertation in media but also concentration of li accumulated in biomass were determined experimentally. besides, biomechanical process of lepidolite degradation resulting in li dissolution was examined using xrd and sem analyses. experimental ore samples the raw lepidolite sample was obtained from prof. rowson (university of birmingham, uk). the sample origin is in the mine beauvoir (france). the mineralogical composition of lepidolite used in experiments is in table 1. microorganisms the fresh culture of aspergillus niger obtained from dpt. of soil science, faculty of natural sciences in bratislava, slovakia was maintained at 4 °c on a solid sabourad dextrose agar (himedia laboratories) slant. stock cultures were subcultured every month. bioleaching experiment based on our former research (marcinčáková et al. 2014) we conducted the pre-cultivation phase for 8 d. obtained biomass was further used in the experiment. bioleaching media were composed of 5 g.l-1 glucose and 0.5 g.l-1 (nh4)2so4. both, medium and mineral were sterilised by autoclaving for table 1. mineralogical composition of lepidolite [%]. sio2 al2o3 k2o li2o fe2o3 tio2 cao mgo na2o 51.0 26.0 7.75 3.79 0.50 0.03 0.05 0.05 0.38 nova biotechnol chim (2020) 19(2): 175-182 177 20 min at 120 °c. initial ph value was adjusted to 5.1. 2 g of crushed mineral and 5 ml of cultivated conidia (biomass) were added into 200 ml of media. the experiment was carried out under 21 °c without shaking. at day 4, 11, 18, 25, 33 and 41 samples for the measurement of li content in medium were withdrawn. after collection, the samples were filtered through the 0.45 µm-pore-size membrane filter. at the end of the experiments biomass was separated from the medium and li content was analysed separately in the bioleaching residue, medium and biomass to calculate li recovery efficiency. the experiment was conducted in duplicate. to study the effect of glucose on bioleaching, assays were conducted in the medium with different variants of glucose concentrations in media, namely 5, 10 and 20 g.l-1. biomass and residue processing after the experiments at the end of experiment the biomass was removed and washed three times with distilled water. the bioleaching residue was obtained after the filtering the rest of the medium and washed with distilled water. air-drying of the biomass and residue samples for 24 h was used prior mineralisation in oven for 4 h at 500 °c. to determine accumulated lithium the biomass was digested by the 2 m hcl. the amount of biomass was calculated per 1 l of media. analytical and characterisation methods li concentration was measured by atomic absorption spectrophotometer (perkin elmer 3100). the initial sample and final leaching residues were also mounted with silver paste on aluminium stubs, then coated with 300 – 400 a au/pd in a sputtering unit and finally examined in a jeol scanning electron microscope. mineral composition before and after the bioleaching process was determined by a diffractometer bruker d2 phaser (bruker axs, gmbh, germany) in bragg-brentano geometry (configuration theta2theta), cukα radiation. results and discussion rapid decrease of ph value in bioleaching media due to an organic acid production was observed within first 7 d (fig. 1) followed by the slow ph decrease phase up to 42th d. acid concentration and biomass dry weight increases (fig. 1) indicated the active growth phase of aspergillus niger (aung and ting 2005; amiri et al. 2011). the beginning of the slow ph decrease in media indicated the end of the active growth phase of the fungus (observed after 10 d of incubation), and has been associated with the accumulation of secondary metabolites and beginning of the stationary phase (qu et al. 2013). a small increase in ph (from 5.2 to 5.6) was observed at the end of the growth phase in a control medium as a result of the carbon deficit essential fig. 1. relationship between the changes of ph and a. niger biomass production during li bioleaching from lepidolite (a – exponential growth phase, b – reduced growth phase and c – stationary phase). glucose concentration 5 g.l-1, ph 5.1, temperature 21 °c. data represent mean ± error (n = 3). nova biotechnol chim (2020) 19(2): 175-182 178 for microbial growth and indicated no production of organic acids. in contrast, significant drop of ph values has been associated with accumulation of organic acids in the media as the result of high metabolic activity of the a. niger cells. according to various authors (rezza et al. 2001; aung and ting 2005; amiri et al. 2011; gadd et al. 2014; bahaloo-horeh et al. 2016) malic, citric, gluconic and oxalic acids were produced by the microscopic fungus a. niger. the predominant organic acid secreted by a. niger in glucose medium is citric acid (anjum et al. 2010). at the beginning of the incubation at ph 5.1, however, gluconic acid was probably produced since glucose oxidase is the most active at ph of 4.5 – 6.5 and inactive at ph levels below 3 (ruijter et al. 2002; wu and ting 2006). the citric acid production by a. niger starts at a ph below 3 and proceeds at ph 2 (amiri et al. 2011). the ph decrease below 3 was observed after the 14th d of the incubation. according to amiri et al. (2011) the lower ph values resulted in decreasing the gluconic acid production and increasing of the citric acid concentration. oxalic acid biosynthesis under optimal production conditions occurs in the ph range of 5 – 8 (gadd et al. 2014; bahaloo-horeh et al. 2016). the malic acid does not play as important role in bioleaching as citric and gluconic acids because it is produced in lower amounts and under both anaerobic and aerobic conditions it is easily degraded (bahaloo-horeh and mousavi 2017). since ph of the bioleaching medium was between 5 – 2, citric acid was probably the most important bioleaching agent in the process. the pka values of three carboxylic functional groups of citric acid were found at ph 3.1, 4.7 and 6.4 (max et al. 2010). on the basis of dissociation constant values at ph = 2.8 it can be assumed that only one carboxylic group of citric acid was dissociated resulting in the formation of lithium citrate (ilgar et al. 1993). the reaction can be expressed as follows: lithium was found in the solution after 25th d of incubation for the first time, its concentration was 27 μg.l-1 (fig. 2). subsequently, rapid increase in lithium concentration was observed. however, part of the solubilised lithium was accumulated into the biomass that was confirmed by the biomass analysis. it is possible that simultaneous lithium bioaccumulation could significantly contribute to li solubilisation from mineral since rapid decrease of li+ cations concentration caused by their accumulation in the mycelium shifted the equilibrium and resulted in the increased rate of the li dissolution. at the end of the experiment li amount in biomass was 110 μg.g-1 of dry weight. a total of 9.5 mg of li was recovered from 1 kg of lepidolite. the production of organic acids as the main metal leaching agents is significantly affected by the amount of sugars in the medium (castro et al. 2000). the latter authors found that higher glucose concentration in media leads to higher citric acid fig. 2. changes of li concentration in media during li bioleaching by a. niger. glucose concentration 5 mg.l-1, ph 5.1, temperature 21 °c. data represent mean ± error (n = 3). nova biotechnol chim (2020) 19(2): 175-182 179 fig. 3. final recovery of li from lepidolite by a. niger bioleaching under various glucose concentrations. initial ph 5.1, temperature 21 °c. data represent mean ± error (n = 3). production. previous studies (wu and ting 2006; bahaloo-horeh and mousavi 2017) showed that the most important organic acid in bioleaching by a. niger is citric acid, while ilgar et al. (1993) even recognized citric acid as responsible for lithium solubilisation from β-spodumene. to optimize the bioleaching efficiency, we studied the influence of various glucose concentration in medium to enhance the lithium bioleaching efficiency from lepidolite. low-nutrient medium with initial glucose concentration 5, 10, and 20 g.l-1 was used for the experiment. no significant changes in media ph were observed among experimental variants. fast ph decrease was recorded in all three media from 5.1 to 2.6 up to 19th d of the bioleaching with no changes till the end of experiment. however, higher glucose concentration in media significantly affected the amount of a. niger biomass produced in media. the highest amount of biomass (1,080 mg.l-1) was found in medium with highest glucose concentration. favourable effect of higher saccharide concentration on a. niger growth and biomass production was confirmed by various authors (castro et al. 2000; wu and ting 2004; anjum et al. 2010; qu et al. 2013). here, higher fungal growth resulted in higher li recovery. the highest lithium recovery 11.5 mg.kg-1 of lepidolite (summarised from li bioleaching into solution and li bioaccumulation into biomass) was found at glucose concentration 20 mg.l-1, while only 9.5 mg of li was recovered from kg of lepidolite at the lowest glucose concentration in media (5 mg.l-1) (fig. 3). the direct connection between the metal recovery percentage and fungal growth was also observed by amiri et al. (2011) in their study of metal dissolution from spent catalysts by a. niger. the authors suggested that higher bioleaching efficiency was caused by higher metabolite production. sem analysis showed high interpenetration activity of a. niger into the lepidolite (fig. 4). it confirms that biomechanical process was also a part of li bioleaching from lepidolite. however, according to literature (gadd 2010) biomechanical processes are considered less important in mineral weathering in comparison with biochemical processes. the micro-morfological changes support the observation that fungal carboxylic acids, e.g. citric and oxalic acids, with strong chelating properties disrupted the surface of the mineral due to chemical corrosive action and resulted in the li mobilization to the solution confirming complexolysis as main fig. 4. attachment of a. niger hyphae to lepidolite surface observed with sem. hyphae growing on the surface of lepidolite particles (a) and produced spores attached to the surface (b). a b nova biotechnol chim (2020) 19(2): 175-182 180 mechanisms of fungal bioleaching. the changes of the mineral caused by the fungal leaching were also studied by comparing the mineral phases prior to and after the bioleaching through xrd analysis (fig. 5) (sedláková-kaduková et al. 2020 – supplementary information). prior to bioleaching, the crystalline phase of polythionite, muscovite and kaolinite were clearly detected. after the bioleaching the phase changes with a dominance of muscovite were observed (fig. 5). appearance of new silicate phase of sio2 was detected. the formation of silicate phases was observed also during the aluminosilicate phlogopite bioleaching by bacteria acidithiobacillus ferrooxidans (tariq et al. 1993). in the bioweathering of both silicate and aluminosilicate minerals the breakup of si–o–si (siloxane) as well as al–o bonds or cation removal from the silicate crystal lattice can result in the collapse of the silicate lattice structure (gadd 2010) contributing to li release into the solution phase. conclusions microscopic fungus aspergillus niger can solubilize li from lepidolite through two main mechanisms – biochemical leaching and biomechanical deterioration. the biochemical process involved complexolysis by mostly citric fig. 5. x-ray diffraction of original ore (a) and bioleached residue (b) (sedláková-kaduková et al. 2020 – supplementary information). nova biotechnol chim (2020) 19(2): 175-182 181 acid and consequent bioaccumulation of lithium inside the fungal biomass. contribution of biomechanical process was confirmed by sem analyses. hyphal penetration along the crystal boundaries together with the erosion of mineral surface by organic acids was observed. the results also displayed significant differences in li bioleaching efficiency when various glucose concentrations were used. the highest efficiency was found at the highest (20 g.l-1) glucose concentration. these findings indicate that a. niger could be effective for li bioleaching from hardrock lithium ore lepidolite. acknowledgements the authors thank to dr. n. rowson from university of birmingham for the supply of the lepidolite used in this work. the work was fully supported by a grant from the slovak national grant agency under the vega project 1/0229/17. no potential conflict of interest was reported by the authors. conflict of interest the authors declare that they have no conflict of interest. references amiri f, mousavi sm, yaghmaei s (2011) enhancement of bioleaching of a spent ni/mo hydroprocessing catalyst by penicillium simplicissimum. sep. purif. technol. 80: 566-576. anjum f, bhatti hn, asgher m, shahid m (2010) leaching of metal ions from black shale by organic acids produced by aspergillus niger. appl. clay sci. 47: 356-361. aung kmm, ting yp (2005) bioleaching of spent fluid catalytic cracking catalyst using aspergillus niger. j. biotechnol. 116: 159-170. bahaloo-horeh n, mousavi sm (2017) enhanced recovery of 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corresponding author: cyril.rajnak@ucm.sk  nova biotechnologica et chimica diamagnetic cobalt(iii)tris(o-ethylxanthate) and nickel(ii)bis(o-ethylxanthate) filip varga1, ján titiš1, cyril rajnák1,, ján moncoľ2 and roman boča1 1 department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnva, nám. j. herdu 2, trnava, sk-917 01, slovak republic 2 institute of inorganic chemistry, fchpt, slovak university of technology, bratislava, sk-812 37, slovak republic article info article history: received: 11th october 2017 accepted: 16th november 2017 keywords: ab initio calculations co(iii) complex magnetism ni(ii) complex o-ethyl xanthate uv-vis spectra abstract diamagnetic [co(xanth)3] and [ni(xanth)2] complexes have been prepared by reaction of co(ii) and ni(ii) salts with potassium o-ethyl xanthate (kxanth). the isolated co(iii) and ni(ii) complexes have been characterized by single-crystal x-ray crystallography, uv-vis and ir spectroscopy, computational methods, and magnetic measurements.  university of ss. cyril and methodius in trnava       introduction transition metal complexes containing chelating ligands are the subjects of extensive research for a long time due to the broad range of their structural, spectral and magnetic properties (gerloch and constable 1994). in the past few decades, such coordination compounds have attracted increased attention of chemists because of their potential applications in various fields as chemical sensors, nonlinear optical materials and/or molecular magnetic materials (noro et al. 2009). over the past ten years, an extraordinary interest on single-molecule magnets (smms) has been registered. single-molecule magnets, attractive for high-density information storage and quantum computing, are metal complexes, that beside other interesting quantum phenomena, exhibit slow relaxation of the magnetization. the earlier type of smms was based on high-spin polynuclear complexes, such as a dodecanuclear plaquette-type complex known as mn12ac. in course of time a plethora 3d-metal complexes were identified as smms, including simple mononuclear systems containing mn(iii) (grigoropoulos et al. 2013), fe(iii) (mossin et al. 2012), fe(ii) (feng et al. 2013), fe(i) (samuel et al. 2014), co(ii) (rajnák et al. 2017), ni(ii) (lomjansky et al. 2017), ni(i) (lin et al. 2016) and cu(ii) (boča et al. 2017) central atoms (frost et al. 2016). among these systems, the s-donor ligands combined with the cobalt(ii) centres attract a considerable attention because of the pronounced magnetic anisotropy that supports the smm behaviour (zadrozny and long 2011). however, co(ii) can be readily oxidized to co(iii) in course of the complexation reaction on air giving rise the diamagnetic complex. tetracoordinate ni(ii) complexes in their nearly tetrahedral geometry are also promising smms. some ligands prefer the square planar arrangement around the ni(ii) central atom and these complexes are diamagnetic as well. some relationships between the structure and magnetic properties are compared in table 1. herein we are bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:17 utc nova biotechnol chim (2017) 16(2): 138-146 139 table 1. comparison of structure-magnetism relationships for related compounds. complex chromophore magnetic property reference (ph4p)2[co(sph)4] tetrahedral smm zadrozny and long 2011 [co(pph3)2cl2] tetrahedral smm yang et al. 2013 [co(pph3)2br2] tetrahedral smm boča et al. 2014 [co(pph3)2i2] tetrahedral smm saber and dunbar 2014 [co(pph3)2(scn)2] tetrahedral smm rajnák et al. 2016 [ni(pph3)2cl2] tetrahedral paramagnet, no smm lomjanský et al. 2016 [ni(pph3)2(ncs)2] planar diamagnet, no smm rajnák, unpublished reporting about synthesis, characterization and structural properties of the [co(xanth)3] and [ni(xanth)2] complexes with chelating s-donor ligand which were found to be diamagnetic. experimental chemicals and handling all inorganic and organic reactants of reagent grade quality were purchased and used as received (sigma-aldrich). the solvents ethanol and acetonitrile were used without further purification. synthesis of [co(xanth)3] (1) the compound 1 has been prepared as follows. 0.16 g (1 mmol) of potassium o-ethyl xanthate, was dissolved in ethanol (10 cm3) under an intense stirring. 0.064 g of cobalt(ii) chloride was also dissolved in 10 cm3 of ethanol under an intense stirring (the molar ratio of xanth: cobalt(ii) chloride = 2:1). the two solutions were mixed and the final solution was refluxed for 4 hours. the filtrate was left for the duration of 5 days for a spontaneous evaporation. deep dark green crystals were obtained by slow evaporation of the solution at room temperature. yield: 0.085 g anal. calc. for 1, c9h15coo3s6 (m = 422.57 g mol-1): c, 25.58; h, 3.58; s, 45.53. found: c, 25.88; h, 3.56; s, 46.02. selected ir bands (kbr) /cm-1: 2976(w), 2484(w), 1898(w), 1861(w), 1721(w), 1464(w), 1444(m), 1433(m), 1387(m), 1365(s), 1273(w), 1233(s), 1143(w), 1116(s), 1055(w), 1030(s), 1000(s), 861(m), 810(w), 657(w), 548(w), 438(s). uv/vis (nujol) νmax/103 cm-1 (relat. absorb.): 15.8 (0.435), 20.7 (0.526). synthesis of [ni(xanth)2] (2) the compound 2 was prepared as follows. a 100 cm3 round bottom flask was charged with potassium ethyl xanthogenate (0.150 g, 0.94 mmol) and acetonitrile (20 cm3) and slight heated. nicl2·6h2o (0.111 g, 0.47 mmol) was added once. yellowish solution changed colour immediately to green. reaction was heated with an oil bath for four hours at 80°c. in process of cooling a mixture changed colour to brown. after 3 days dark red crystalline compound was filtered off and dried in vacuum. yield: 0.026 g. anal. calc. for 1, c6h10nio2s4 (m = 301.10 g mol-1): c, 23.93; h, 3.35; s, 42.60. found: c, 24.14; h, 3.05; s, 41.13. selected ir bands (kbr) /cm-1: 2982(w), 2935(w), 2891(w), 2503(w), 1464(m), 1429(w), 1388(w), 1366(s), 1325(w), 1244(s), 1144(w), 1113(s), 1058(w), 1020(s), 996(s), 855(m), 808(w), 662(w), 554(w), 435(s) (s – strong, m – medium, w-weak); s-c=s (554 cm-1), c=s (1058 – 1144 cm-1), c-o-c (855 cm-1), νch3 (2935 cm-1), νch2 (2891 cm-1) a δc-h (1325 – 1464 cm-1). uv/vis (nujol) νmax/103 cm-1 (relat. absorb.): ~15.0 (0.191), 20.7 (0.459), 23.8 (0.513). physical measurements elemental analyses were carried out by flash 2000 chns apparatus (thermo scientific). the ir spectra were measured on atr holder with highly effective diamond crystal in the region of 4000  400 cm−1 by nicolet 5700 spectrometer (thermo electron) with dtgs/kbr detector. the solid sample for ft-ir measurements was not dried prior to its using and was used as freshly synthesized. absorption uv-vis spectra for solid sample (nujol mull) were measured by specord. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:17 utc nova biotechnol chim (2017) 16(2): 138-146 140 table 2. crystal data and structure refinement for 1, 2. empirical formula c9h15coo3s6 c6h10nio2s4 formula weight (g.mol-1) 422.50 301.09 crystal system trigonal orthorhombic space group r–3 pbca t (k) 100(1) 100(1) a (å) 14.8164(14) 7.4174(8) b (å) 14.8164(14) 7.1003(6) c (å) 12.8790(12) 20.760(2) α (°) 90 90 β (°) 90 90 γ (°) 120 90 v (å3) 2 448.5(5) 1 093.36(19) ρcalc (g.cm−3) 1.719 1.829 z 6 4 radiation type cu kα cu kα µ (mm−1) 1.54186 1.54186 f(000) 1 296 616 abs. coefficient 15.426 9.439 r1[f2 > 2σ(f2)], wr2(f2) 0.0328, 0.0755 0.0476, 0.1220 δρmax, δρmin (e å−3) 0.45, -0.59 1.45, -0.45 ccdc no. 1569076 1569077 250 plus (analytica jena) with the dad detector in the range of 9 000 – 50 000 cm-1. magnetic data was taken with the squid apparatus (quantum design, mpms-xl7) in the rso mode of the detection. the detected magnetic moment of the specimen has been converted to the molar magnetic susceptibility mol and the dimensionless product function t/c0 with the reduced curie constant c0 = na0b2/kb containing the fundamental physical constants in their usual meaning. x-ray crystal structure determination data collection and cell refinement of 1, 2 were made by stoe stadivari diffractometer using pilatus3r 300k hpad detector and microfocused source xenocs fox3d with cukα at 100k. corrections to lorentz, polarization and multi-scan absorption effects were applied. the structure was solved by charge-flipping or direct methods and refined anisotropically by common least-squares methods. the programs superflip (palatinus and chapuis 2007), shelxt (sheldrick 2015), shelxl (ver. 2016/6) (sheldrick 2015) and olex2 (dolomanov et al. 2009) have been used for structure determination, refinement and drawing. the hydrogen atoms were refined with fixed distances from the parent carbon atoms. crystal data on 1, 2 are presented in table 2. results and discussion structural data the compound 1, [co(xanth)3], possesses a molecular structure. the central co(iii) atom is hexacoordinate by three bidentate xanth ligands (fig. 1). the co-s distances are almost identical (2.268 – 2.282 å, see table 3). as expected, the s-co-s angles are acute (41 – 51 deg). the compound 1, [co(xanth)3], crystallizes in trigonal system in space group r–3. the cobalt atom lies on 3-fold axis and is coordinated in distorted octahedron. the coordination octahedron around cobalt atom of 1 is formed by six sulfur atoms [co1–s1 = 2.2693(7) å and co1–s2 = 2.2743(8) å (table 3)] of three bidentate carbamate group of xanth. the compound 2, [ni(xanth)2] crystallize in orthorhombic system in space group pbca (table 2) and it possesses a molecular structure with the planar chromophore {niiis4} – see fig. 2. the nickel atom lies in the centre of symmetry and is bonded by four sulfur atoms [ni1–s distance in region 2.21– 2.22 å (table 4)] of two bidentate carbamate groups of xanth square-planar coordination. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:17 utc nova biotechnol chim (2017) 16(2): 138-146 141 fig. 1. molecular and crystal structure of complex [co(xanth)3], 1. table 3. selected bond lengths (å) and angles (°) in complex 1. co1–s1 2.2693(7) co1–s2 2.2743(8) co1–s1i 2.2693(7) co1–s2i 2.2743(8) co1–s1ii 2.2693(7) co1–s2ii 2.2744(8) s1–co1–s1i 94.18(3) s1–co1–s1ii 94.19(3) s1i–co1–s1ii 94.19(3) s1ii–co1–s2i 166.63(2) s1–co1–s2 76.73(2) s1i–co1–s2 166.63(2) s1–co1–s2ii 166.63(2) s1i–co1–s2ii 96.22(2) s1i–co1–s2i 76.73(2) s1–co1–s2i 96.22(2) s1ii–co1–s2ii 76.73(2) s1ii–co1–s2 96.22(2) s2i–co1–s2 94.31(3) s2i–co1–s2ii 94.31(3) s2–co1–s2ii 94.31(3) symmetry codes: (i) 1-y, x-y, z; (ii) 1+y-x, 1-x, z fig. 2. molecular and crystal structure of the complex [ni(xanth)2], 2. electronic spectra the solid state electronic spectra of 1 and 2 measured in nujol mull are presented in fig. 3. these complexes exhibit a similar spectral profile with a number of electronic transitions that have varying peak intensity. the first visible transitions are slightly intense and located in the range of 15 000 – 25 000 cm. this part of the spectrum is almost identical for both complexes. in this range bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:17 utc nova biotechnol chim (2017) 16(2): 138-146 142 we can identify two transitions at ~15 000 and 20 000 cm. moreover, in the spectrum of the ni(ii) complex another peak is visible at ~24 000 cm. for the co(iii) complex this transition is missing, however, there is a broad asymmetric band in this location with a maximum at 28 000 cm. more intense and clearly identifiable peaks are located in the range of 30 000 – 50 000 cm. table 4. selected bond lengths (å) and angles (°) in complex 2. ni1–s1 2.2182(8) ni1–s1iii 2.2183(8) ni1–s2 2.2201(7) ni1–s2iii 2.2201(7) s1–ni1–s1iii 180.0 s1–ni1–s2 79.43(3) s1iii–ni1–s2iii 79.43(3) s1iii–ni1–s2 100.57(3) s1–ni1–s2iii 100.57(3) s2–ni1–s2iii 180.0 symmetry codes: (iii) 1-x, -y, 1-z; (iv) -x, 1-y, -z the hexacoordinate co(iii) complexes in the strong crystal field possess the electronic ground term 1a1g that is well separated from its excited counterparts. a modelling using the generalized crystal-field theory (gcft) with the crystal field poles f4 = 20 000 cm-1 (10dq = 33 333 cm-1) lifts the first spin-allowed d-d transition to e1 = 26 000 cm-1 (1a1g  1t1g) and the second one to e2 = 28 000 cm-1 (1a1g  1t2g). the squareplanar ni(ii) complexes in the strong crystal field also possess the electronic ground term 1a1g. a modelling by the gcft with the crystal field poles f2 = 20 000 and f4 = 10 000 cm-1 gave the lowest spin-allowed excitation energies at e1 = 14 900 cm-1 (1a1g  1a2g) and e2 = 21 300 cm-1 (1a1g  1b1g). ab initio wavenumber/cm1 10000 15000 20000 25000 30000 35000 40000 45000 50000 in te n si ty /a .u . 0.0 0.5 1.0 1.5 ni(ii) co(iii) kxanth fig. 3. solid state electronic spectra for complexes 1 and 2. spectrum of the kxanth is added for comparison. calculations based on multireference casscf method improved by nevpt2 approach have been performed to verify the crystal-field. theory results. to this end, the active space extended by the second d-shell (n electrons, where n = 6 for co(iii) and 8 for ni(ii), were correlated in 10 orbitals) and def2-tzvp basis set were used within these calculations. averaging of 10 triplet and 15 singlet states of ni(ii) and 5 quintet, 45 triplet and 50 singlet states of co(iii) was utilized. the results obtained for the experimental geometry of complexes are collected in table 5. it can be seen, the agreement between the gcft and ab initio results is good (table 6). thus, estimation of the crystal field poles in the previous analysis and the conclusions that follow may be considered correct. comparison of the calculated results with the experimental spectra also confirms this claim. table 5. assignment of transitions in 1 and 2. 1 2 experimental ab initio experimental ab initio 11000 w 13500 3t1g 2800 – 4300 16000 m 18100 5t2g 13500 sh 13500 3eg 20500 m 19400 3t2g 16000 m 16700, 17800, 28000 s 21500 1t1g 20600 m 18200 31500 s 32800 1t2g 23700 m 23400 3b2g, 23400 35000 s 34400 3t2g 31500 s 36500 s 36500 3t1g 39500 s 36000 w – weak, m – medium, s – strong, sh – shoulder bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:17 utc nova biotechnol chim (2017) 16(2): 138-146 143 table 6. calculated transition energies below 50 000 cm-1 for 1 and 2a. 1, [co(xanth)3] 2, [ni(xanth)2] gcft casscf-nevpt2 gcft casscf-nevpt2 term energy mult. energy term energy mult. energy 1a1g 0 1 0 1a1g 0 1 0 5t2g 3t1g 3t2g 1t1g 1t2g 3t2g 3t1g 3eg 3t1g 3t2g 5eg 1t2g 15095 15095 15095 16003 16003 16003 16045 16045 16045 26046 26046 26046 28160 28160 28160 39708 39708 39708 39738 39738 39738 43430 43430 45803 45803 45803 46315 46315 46315 48429 48429 49973 49973 49973 3 3 3 5 5 5 3 3 3 1 1 1 1 1 1 3 3 3 3 3 3 5 5 3 3 3 3 3 3 3 3 1 1 1 1 1 1 13433 13471 13487 18097 18126 18158 19305 19454 19461 21442 21459 21479 32393 32891 32907 34281 34386 34406 36540 36548 36602 39661 39671 41659 41664 41710 41740 41742 41852 42923 42941 44395 44445 44597 45531 45586 47628 3b1g 3a2g 3eg 1a2g 3b2g 1b1g 1eg 3eg 1a1g 1b2g 3a2g 1eg 3eg 4179 4554 7588 7588 14900 20846 21277 23443 23443 23970 23970 31317 37520 38958 41225 41225 45180 45180 3 3 3 3 1 1 1 3 1 3 3 3 3 1 3 1 1 1 2770 2967 4288 13502 16677 17813 18257 23399 23438 24332 25556 26762 36081 37096 37965 40728 40795 41733 a gcft calculations in an idealized octahedral (square planar) geometry; ab initio casscf calculations in the experimental geometry. magnetic data the measured magnetic susceptibility consists of the inherent temperature-dependent paramagnetic signal arising from the uncompensated spin and/or orbital angular momentum para, temperatureindependent paramagnetism tip arising from the close lying excited states, the underlying diamagnetism dia and the signal of the sample holder h (usually diamagnetic). eventually some traces of the dioxygen  or other paramagnetic impurities pi might be also present so that  = para +tip +dia +h +pi                  (eq. 1)  on heating, the molar magnetic susceptibility of 1 decreases and passing through the zero it becomes almost constant (fig. 4). the inherent paramagnetism of a hexacoordinate co(iii) complex possessing the ground electronic term 1a1g is thought to be absent, para ~ 0. the lowtemperature data refer to the traces of dioxygen that is present in the powder sample and obeys the curie law. also, the feature round 50 k is typical for the solidus-solidus phase transition of o2(s). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:17 utc nova biotechnol chim (2017) 16(2): 138-146 144 t/k 0 50 100 150 200 250 300  m o l/1 0 -9 m 3 m o l-1 -5 0 5 10 15 t/k 0 50 100 150 200 250 300  t / c 0 -0.2 0.0 0.2 0.4b = 0.1 t o2   fig. 4. magnetic data for 1. left – molar magnetic susceptibility (si units), right – dimensionless product function.  this signal escapes at the room temperature when (300 k) = -2.5 × 10-9 m3 mol-1 (si units) consisting only  = tip +dia +h. the underlying diamagnetism can be calculated by means of the pascal constants, however also a simple estimate using the molar mass mr can be applied such as dia [10-12 m3 mol-1] = -5mr [g mol-1]; this amounts to dia = -2.1 × 10-9 m3 mol-1. as the excited states of the same multiplicity are far away the ground state the temperatureindependent paramagnetic term will be small. it can be hand-calculated by the spin-hamiltonian formalism that offers the formula 2 tip a 0 b2 ( ) / 3xx yy zzn         (eq. 2) containing the -tensor components (restricted to the first excitation energy) 2 0 1 1 0 ˆ a aa l e e       (eq. 3) the matrix element of the angular momentum operator for an octahedral co(iii) system is t/k 0 50 100 150 200 250 300  m o l/1 0 -9 m 3 m o l-1 -5 0 5 10 15 t/k 0 50 100 150 200 250 300  t / c 0 -0.2 0.0 0.2 0.4b = 0.1 t z x, y fig. 5. magnetic data for 2. left – molar magnetic susceptibility (si units), right – dimensionless product function. lines – calculated with f2 = 20 000 and f4 = 10 000 cm-1. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:17 utc nova biotechnol chim (2017) 16(2): 138-146 145 1 1 1 , , 1g ˆa t 2x y zl  and using the first singlet excitation energy e/hc = 21 000 cm1 one gets tip = +2.4  109 m3 mol1. the computer evaluation using the generalized crystal field theory yields tip = +3.3  109 m3 mol1. the molar susceptibility for 2 at low temperature starts to decay on heating (fig. 5), however it reaches almost constant value at the room temperature of mol = 6.7 × 10-9 m3 mol-1 (si units). this behaviour clearly deviates from the curie law. the product function develops according to a straight line and this feature is a fingerprint of a considerable temperature-independent paramagnetism tip. small anomaly around 50 k is attributed to the solidus-solidus phase transition of dioxygen traces which are present in the powder sample. using the crystal field poles f2 = 20 000 and f4 = 10 000 cm-1, the gcft calculations for a quadro-ni(ii) gave tip = +3.1  109 m3 mol1. conclusions two novel low-spin co(iii) and ni(ii) complexes have been prepared and characterized by spectroscopic methods. x-ray structure analysis has shown that the complexes 1 and 2 possess with {cos6} and {nis4} chromophore, respectively. the study of bond angles and distances has revealed coordination polyhedron in octahedral (1) and square planar (2) arrangements. magnetic studies confirmed a premise on diamagnetic behaviour of both complexes. our primary goal was to prepare paramagnetic compounds of co(ii) and ni(ii). however, all attempts to prepare such compounds using the xanth ligand were unsuccessful. acknowledgement slovak grant agencies (apvv-14-0078, apvv-16-0039, vega 1/0919/17, vega 1/0534/16) are acknowledged for the financial support. this article was also created with the support of the mšvvaš of the slovak republic within the research and development operation programme for the project university science park of stu bratislava (itms project no. 26240220084) cofounded by the european regional development fund.. references boča r, rajnák c, titiš j, valigura d, (2017) field supported slow magnetic relaxation in a mononuclear cu(ii) complex. inorg. chem. 56(3): 1478-1482. dolomanov o, bourhis lj, gildea rj, howard jak, puschmann h, (2009) olex2: a complete structure solution, refinement and analysis program j. appl. crystallogr. 42: 339-341. feng x, mathoniere c, jeon i, rouzieres m, ozarowski a, aubrey ml, gonzalez mi, clerac r, long jr (2013) tristability in a light-actuated single-molecule magnet. j. am. chem. soc. 135: 15880-15884. frost jm, harriman klm, murugesu m (2016) the rise of 3-d single-ion magnets in molecular magnetism: towards materials from molecules? chem. sci. 7: 24702491. gerloch m, constable ec (1994) transition metal 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knižnica | heruntergeladen 28.02.20 08:17 utc nova biotechnol chim (2017) 16(2): 138-146 146 samuel pp, mondal kc, amin sk n, roesky hw, carl e, neufeld r, stalke d, demeshko s, meyer f, ungur l, chibotaru lf, christian j, ramachandran v, van tol j, dalal ns (2014) electronic structure and slow magnetic relaxation of low-coordinate cyclic alkyl(amino) carbene stabilized iron(i) complexes. j. am. chem. soc. 136: 11964-11971. sheldrick gm (2015) shelxt – integrated space-group and crystal-structure determination acta crystallogr. a71: 38. sheldrick gm (2015) crystal structure refinement with shelxl. acta crystallogr. c71: 3-8. yang f, zhou q, zhang y, zeng g, li g, shi z, wang b, feng s (2013) inspiration from old molecules: fieldinduced slow magnetic relaxation in three air-stable tetrahedral cobalt(ii) compounds. chem. commun. 49: 5289-5291. zadrozny jm, long jr (2011) slow magnetic relaxation at zero field in the tetrahedral complex [co(sph)4]2–. j. am. chem. soc. 133: 20732-20734. zadrozny jm, telser j, long jr (2013) slow magnetic relaxation in the tetrahedral cobalt(ii) complexes [co(eph)4]2– (e = o, s, se). polyhedron 64: 209-217. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:17 utc nova biotechnol chim (2017) 16(2): 113-123 doi: 10.1515/nbec-2017-0016  corresponding author: miroslav.ondrejovic@ucm.sk  nova biotechnologica et chimica the optimization of propagation medium for the increase of laccase production by the white-rot fungus pleurotus ostreatus miroslava hazuchová, daniela chmelová and miroslav ondrejovič department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic article info article history: received: 14th july 2017 accepted: 12th november 2017 keywords: cultivation laccase lignocellulose propagation response surface methodology white-rot fungus abstract the lignocellulolytic enzymes are routinely produced by submerged fermentation using lignocellulosic material, but for more effective production, it would be suitable to precede the production phase on the lignocellulose by propagation phase in the nutrition medium suitable for growth of the fungi. therefore, the aim of this study was to increase the laccase production by the white-rot fungus pleurotus ostreatus by two-step cultivation strategy. in the first step, propagation medium was optimized for the maximal biomass growth, the second step included the laccase production by produced fungal biomass in media with the selected lignocellulosic material (pine sawdust, alfalfa steam and corn straw). from our experiments, parameters such as glucose concentration, yeast extract concentration and ph of propagation medium were selected as key factors affecting growth of p. ostreatus. the optimal conditions of propagation medium for maximal fungal growth determined by response surface methodology were: glucose concentration 102.68 g/l, yeast extract concentration 43.65 g/l and ph of propagation medium 7.24. these values were experimentally verified and used statistical model of biomass production prediction was appropriate adjusted. thus prepared fungal biomass produced in the media with lignocellulose approximately 9-16 times higher concentrations of the laccase in 3 times shorter time than the fungal biomass without propagation phase in optimized propagation medium.  university of ss. cyril and methodius in trnava       introduction laccases (benzenediol: oxygen oxidoreductase; ec 1.10.3.2) are multi-copper oxidases that require only molecular oxygen for oxidation of wide range of substrates. therefore, these enzymes are potentially used for different industrial applications including agro-food, chemical or pharmaceutical industries (rodríguez-delgado et al. 2015; legerská et al. 2016). the main problem of laccase applications for the commercial purposes is the high costs of their production. the key factors for laccase production are a selection of the suitable producer, medium composition and cultivation conditions. laccases are produced by various organisms such as bacteria, filamentous fungi, insects or higher plants. the most significant producers are white-rot fungi (bhattacharya et al. 2011). from a group of white-rot fungi, pleurotus ostreatus appears to be a usable microbial producer. several studies have been reported on high laccase production by this fungal strain (bhattacharya et al. 2011; el-batal et al. 2015; ergun and urek 2017). laccases produced by p. ostreatus exhibit significant differences in their properties. molecular weights bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:08 utc nova biotechnol chim (2017) 16(2): 113-123 114 of produced isoenzymes varied from 46-86 kda and ph and temperature optima varied from 2.0 to 8.0 and from 25 to 65 °c depending on used substrate, respectively (baldrian 2006). the composition of cultivation medium (mainly carbon and nitrogen sources) and cultivation conditions (ph, temperature, aeration, agitation etc.) are important factors for successful production of laccases. laccase production can be increased by the addition of various aromatic compounds. arantes et al. (2011) found that the addition of lignocellulosic material to the medium enhanced lignolytic enzyme production in some white-rot fungi. several authors have proved that higher laccase production was measured in the media contained lignocellulosic materials such as wheat straw, corn, coffee husk, cedar sawdust or wheat bran than this in the media without lignocellulose (singh et al. 2013; gonzáles et al. 2013; kneževic et al. 2013). the advantages of the selection of lignocellulosic material as carbon source for laccase production are: low cost of raw material, the availability and content of potential laccase inductors in selected material. moreover, suitable cultivation conditions need to set up for optimal laccase production. except of some physical factors (ph, temperature), the important factor for the laccase production is type of cultivation. laccases can be produced by batch, fed-batch or continuous cultivation. for higher effectivity, it can be used a multi-step approach (michelin et al. 2018). in first steps, the fungal biomass grows in propagation medium under favourable conditions resulting in the greatest fungal growth, followed by the second step in which are produced laccases in production medium (chmelová and ondrejovič 2013). this method is more appropriate used for the production of secondary metabolites, including laccases because the conditions for their production are often different than these for biomass growth. the aim of this study was to increase laccase production by the white-rot fungus p. ostreatus by the two-step cultivation strategy, while in the first step propagation medium was optimized by response surface methodology (rsm) for the maximum fungal growth and the second step was adapted for laccase production in media with selected lignocellulosic biomass. experimental microorganism pleurotus ostreatus dsm 1833 was purchased from leibniz-institute dsmz (german collection of microorganisms and cell cultures, germany). this strain was maintained on malt agar (biolife, italy) at 4 °c. for all experiments, the suspension of fungal mycelium was prepared by plaque scraping (1 cm2) of culture from agar plate using microbiological loop and mixing in sterile deionized water (10 ml). the inoculum was used for the inoculation of media in ratio 1 : 10 (v/v). lignocellulosic materials lignocellulosic materials used as the carbon source for production media were pine sawdust (pinus nigra; soft wood), alfalfa steam (medicago sativa; forage) and corn straw (zea mays; agricultural waste). these materials were extracted in methanol by soxhlet extractor during 12 hours for removal of extractive compounds, then were dried at 100 °c to constant weight and were homogenized to the particle size ≤ 1.0 mm. for determination of the composition of each selected lignocellulosic material, 1 g of this material was mixed with 2.5 mol/l of naoh in ratio of 1 : 20 (w/v). this mixture was incubated at the laboratory temperature for 16 hours on rotary shaker (150 rpm) and after this time, the mixture was centrifuged (10 min, 4 000 rpm). hemicelluloses were precipitated from the supernatant by addition of ethanol in ratio 1 : 4 (w/v). the mixture was incubated for 24 hours at 4 °c, centrifuged (10 min, 4 000 rpm) and decanted. precipitates were dried at 100 °c to the constant weight (hemicellulose content). the residue of lignocellulosic material was washed to neutral reaction by deionized water and dried. dry precipitate (0.5 g) was mixed with 72 % (v/v) sulphuric acid in ratio of 1 : 10 (w/v) and the mixture was incubated for 2.5 hours at the laboratory temperature. then, deionized water was added to the mixture to final volume of 100 ml and after this, it was incubated for 1 hour at 100 °c. the mixture was filtered through filtrate paper from glass fibres. in the filtrate neutralized by solid bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:08 utc nova biotechnol chim (2017) 16(2): 113-123 115 table 1. levels of the factors tested in response surface methodology. factor coded levels -1.682 -1 0 1 1.682 glucose concentration (g/l) 74.75 100 137.5 175 200.25 yeast extract concentration (g/l) 24.9 35 50 65 75.1 ph 5.3 6.0 7.0 8.0 8.7 nahco3, reducing saccharide content was measured by dns method. solid residues were washed by warm deionized water and dried to the constant mass (klason lignin content). propagation the influence of different carbon sources (glucose, xylose, lactose, fructose, saccharose, cellulose, lignin or xylan) in the concentration of 10 g/l with ammonium sulphate as nitrogen source (2 g/l) in phosphate buffer (ph 7.0) and the influence of different nitrogen sources (ammonium sulphate, yeast extract, peptone, tryptone, albumin, casein or potassium nitrate) in the concentration of 2 g/l with glucose as carbon source (10 g/l) in phosphate buffer (ph 7.0) on fungal growth were tested. cultures were incubated at 30 °c on a rotary shaker (150 rpm) for 14 days. the same cultivation conditions were used for the determination of effect of suitable concentration of carbon and nitrogen sources on growth of the fungal biomass. glucose and yeast extract concentrations were tested in concentration range from 2 – 200 g/l and 1 – 100 g/l, respectively. effects of initial ph and temperature of the cultivation on fungal growth were evaluated in the medium contained glucose and yeast extract as carbon and nitrogen sources in concentration 10 g/l and 2 g/l, respectively, for 14 days. the ph values (4.0 – 9.0) were modified by 1 mol/l hcl or 1 mol/l naoh. tested temperatures varied in range of 15 – 37 °c. rsm was used for the optimization of three selected factors: glucose concentration, yeast extract concentration and ph values for enhancing of biomass growth of p. ostreatus. the three independent variables were investigated at five different levels (-1.682, -1, 0, 1, 1.682) (table 1).   dry biomass [g/l] of p. ostreatus was fitted using a second-order polynomial equation (equation 1) and a multiple regression of the data was carried out for obtained an empiric model related to the factors. where xi are independent variables causing the y response and bi are regression coefficients describing dependences between measured properties and coded values of observed factors. production biomass of p. ostreatus produced in the optimized propagation medium after 5× re-washed by sterile deionized water (two step cultivation strategy) as well as biomass from slant agar used for the storage of fungi (one step cultivation) were used for laccase production in the medium with lignocellulosic biomass (pine sawdust, alfalfa steam or corn straw). the composition of production medium is shown in table 2. the laccase production by the white-rot fungus p. ostreatus was evaluated in liquid production medium containing lignocellulosic material for 14 days at 30 °c. table 2. the composition of production medium (sánchez and viniegra-gonzáles 1996). component concentration (g/l) lignocellulosic material 10 yeast extract 2 k2hpo4 0.5 mgso4.7 h2o 0.5 feso4 0.02 nacl 0.01 znso4 0.02 mnso4 0.02 cuso4 0.025 determination of glucose concentration by dns method the sample (0.1 ml) was pipetted to 0.8 ml dns reagent (3,5-dinitrosalicylic acid) (miller 1959). (eq. 1) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:08 utc nova biotechnol chim (2017) 16(2): 113-123 116 after thorough mixing, the mixture was boiled in water bath for 5 min and cooled to the laboratory temperature. after 10 min 8.0 ml of deionized water was added to the reaction mixture and mixed. absorbance was measured at 540 nm (microplate reader, biotek el 800) and glucose concentration was evaluated from calibration curve of glucose. determination of enzymatic activities laccase activity was determined by oxidation of abts (2,2'-azino-bis(3-ethylbenzothiazoline-6sulphonic acid) (shin et al. 1987). the assay mixture contained 150 µl of 50 mmol/l phosphate buffer (ph 5.0) with 1 mmol/l abts and 50 µl of enzyme extracts. the oxidation of abts was monitored by measuring of absorbance at 450 nm. activity of laccase was expressed in unit [u] as the amount of enzymes able to oxidation of 1μmol of abts per minute. cellulase activity from the production medium (0.5 ml) was determined with α-cellulose (50 mg) as substrate in 0.1 mol/l mcilvain buffer (ph 4.8) with 0.1 % (w/v) sodium azide (1 ml). the mixture was cultivated for 24 hours at 30 °c and 150 rpm. after this time, the mixture was centrifuged (4 000 rpm, 10 min) and cellulase activity was determined at 540 nm as the amount of reducing saccharides releasing during reaction with α-cellulose. native-page the laccase isolation procedure was performed according to the modified method by chefetz et al. (1998). the supernatant from the production medium was concentrated (vivaspin1® 30 kda) and applied to native-page. native-page was performed with 5 % (w/v) stacking gel and 12 % (w/v) separation gel using a vertical gel electrophoretic system (5 w, 180 v, 20 ma, 30 min) (bio-rad) (laemmli 1970). gel from fig. 1. the effect of carbon source (a), nitrogen source (b), initial ph of the propagation medium (c) and cultivation temperature (d) on the production of dry biomass of the white-rot fungus pleurotus ostreatus. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:08 utc nova biotechnol chim (2017) 16(2): 113-123 117 native-page was stained with phosphate buffer (0.1 mol/l; ph 5.0) with 1 mmol/l of abts (for laccase detection); 1 mmol/l abts and 10 µmol/l h2o2 (for laccase and lignin peroxidase detection) or 1 mmol/l abts, 10 µmol/l h2o2 and 1 mmol/l mn2+ ions (for laccase, lignin peroxidase and manganese peroxidase detection). statistical analysis all experiments were carried out in triplicate. data are presented at the mean with the standard deviation. statgraphics plus 5.1 (statpoint technologies, inc. usa) was used to evaluate regression equations in the optimization by rsm. results and discussion the most common method for the production of laccase and other lignocellulolytic enzymes is based on the cultivation of appropriate microbial producer, such as white-rot fungus, on the lignocellulosic material such as wood, straw or hay (gonzáles et al. 2013; kneževic et al. 2013; chmelová and ondrejovič 2016). the results of this method are not suitable for the commercial production of target enzymes. the fermentation production of lignocellulolytic enzymes can be improved by the simple propagation of microbial producer within two-step cultivation process. during the first step of cultivation, conditions favouring the microbial growth are generated, while during the second step conditions beneficial for laccase production are established. propagation selection of optimization range the propagation medium must comply for the growth of biomass and therefore, it is necessary its optimization. in order to optimize fungal growth, we first assessed the effect of different carbon and nitrogen sources on p. ostreatus growth. similarly, cultivation conditions, such as ph and temperature of the cultivation, are able to affect the biomass growth. the results are showed in fig. 1. from the results (fig. 1), the most suitable carbon source was glucose (0.73 ± 0.14 g/l) and yeast extract as nitrogen source (5.12 ± 0.45 g/l) for the biomass growth. lihua et al. (2009) observed that glucose was the best substrate for the fungal biomass growth. p. ostreatus was able to utilize pentoses (fructose, xylose) and hexose (glucose) (fig. 1a). the polysaccharide xylan was also suitable substrate for biomass production (0.55 ± 0.09 g/l). the lowest production of biomass was noted in the media with lactose (0.08 ± 0.08 g/l). from organic nitrogen sources, namely yeast extract, peptone, tryptone, albumin and casein, the most suitable substrate was yeast extract (fig. 1b) for biomass growth. inorganic nitrogen sources were not appropriate for the effective biomass growth. organic sources can provide proteins and amino acids resulting to more abundant growth of the fungus (das et al. 2016). el-batal et al. (2015) similarly found that organic nitrogen sources were better alternative for the biomass growth than inorganic nitrogen sources. for the selection of cultivation conditions, the biomass production in the propagation medium was comparable within ph range 6.0-9.0 (fig. 1c). bettin et al. (2011) observed that suitable ph value for biomass growth was 7.4-7.5. kalmis et al. (2008) suggested that optimum ph for biomass growth is at between 6.5 and 7.0. temperatures of 15 °c and 37 °c were not suitable for the biomass production. the most suitable temperature for cultivation of p. ostreatus appears 30 °c (fig. 1d). similar to our results, bellettini et al. (2016) described the optimal temperatures for pleurotus spp. growth in the range of 25-30 °c. from the previous results, it is evident that the most suitable carbon and nitrogen sources for the growth of p. ostreatus glucose and yeast extract, respectively. in the next step, it was necessary to find the effects of glucose and yeast extract concentrations of fungal biomass growth. results are showed in fig. 2. it was observed that 100 g/l of glucose concentration and 50 g/l of yeast extract concentration stimulated fungal growth. gem et al. (2008) found that the increase of yeast extract concentration had the positive effect on the biomass production. wang et al. (2005) found the best conditions for the biomass production were: glucose concentration of 40 g/l and corn steep liquor concentration of 20 g/l. it seems that high concentrations of glucose (200 g/l) and yeast bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:08 utc nova biotechnol chim (2017) 16(2): 113-123 118 extract (75 – 100 g/l) had the inhibition effect on the biomass growth (fig. 2). optimization of propagation medium in this work, 17 experiments were carried out according to the rsm (table 3). based on the results from fig. 1 and fig. 2, independent variables were glucose concentration, yeast extract concentration and initial ph of propagation medium. dependent variable was dry biomass. the rsm was used for the evaluation of relationships between variables (myer and montgomery 2001). table 3 shows the design and the results of experiments carried out by rsm. the second-order polynomial model (equation 1) was used to evaluate the results of optimization. the high value of the coefficient of determination (r2 = 0.899) indicates that only 10.1 % of total variation was not explained by the model. the fitted response for the above regression model is shown in fig. 3. the fungal biomass growth increased with higher glucose and yeast extract concentration. thereafter, the fungal growth decreased as the ph decreased (ph < 7.2). table 3. the experimental matrix for the optimization of dry biomass production (g/l) with coded levels of independent variables. exp. glucose concentration (g/l) yeast extract concentration (g/l) ph dry biomass (g/l) 1 100 (-1) 35 (-1) 6.0 (-1) 73.5 2 100 (-1) 65 (1) 8.0 (1) 82.9 3 137.5 (0) 50 (0) 7.0 (0) 113 4 175 (1) 65 (1) 6.0 (-1) 44.4 5 175 (1) 35 (-1) 8.0 (1) 46.0 6 100 (-1) 65 (1) 6.0 (-1) 20.1 7 175 (1) 65 (1) 8.0 (1) 33.9 8 137.5 (0) 50 (0) 7.0 (0) 85.6 9 100 (-1) 35 (-1) 8.0 (1) 89.5 10 175 (1) 35 (-1) 6.0 (-1) 62.9 11 137.5 (0) 50 (0) 7.0 (0) 118.5 12 137.5 (0) 24.9 (-1.682) 7.0 (0) 82.4 13 200.25 (1.682) 50 (0) 7.0 (0) 116.3 14 74.7505 (-1.682) 50 (0) 7.0 (0) 80.1 15 137.5 (0) 75.1 (1.682) 7.0 (0) 49.1 16 137.5 (0) 50 (0) 5.3 (-1.682) 18.6 17 137.5 (0) 50 (0) 8.7 (1.682) 27.6 fig. 2. the effect of glucose (a) and yeast extract concentration (b) on production of dry biomass of the white-rot fungus pleurotus ostreatus at 30 °c and ph 5.0 for 14 days. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:08 utc nova biotechnol chim (2017) 16(2): 113-123 119 fig. 3. 3d surface graphs showing the effect of yeast extract concentration, glucose concentration and initial ph value on the growth of p. ostreatus expressed as dry biomass (g/l) for 14 days at 30 °c. the optimal values of selected variables were chosen to the evaluation of variable interactions and to determine the optimal level of each variable for the maximal response. according to the studies of rsm, the maximum biomass growth produced by p. ostreatus can be reached at optimal conditions, namely glucose concentration 102.68 g/l, yeast extract concentration 43.65 g/l and ph 7.24 for predicted dry biomass recovery of 109 g/l. these conditions were experimentally verified and predicted value of dry biomass (109 g/l) coincided with experimentally measured values (99.5 g/l). production of lignocellulolytic enzymes chemical composition of lignocelluloses lignocellulose belongs to natural substrates for growth of white-rot fungi in the environment. it proved to be a suitable substrate for the fungal growth and attractive feedstock for the laccase production. the composition of lignocellulosic material directly affect the efficiency of laccase production (levin et al. 2008; elisashivili et al. 2009) and therefore, selected lignocellulosic materials were analysed for cellulose, table 4. the content of cellulose, hemicelluloses and lignin in selected lignocellulosic materials. lignocellulosic material cellulose content (%) hemicellulose content (%) lignin content (%) pine sawdust (softwood) 30.1 ± 4.6 22.4 ± 3.8 23.7 ± 1.4 alfalfa steam (forage) 27.0 ± 2.0 48.4 ± 1.6 16.7 ± 0.7 corn straw (agricultural waste) 41.7 ± 2.0 52.3 ± 0.3 5.0 ± 0.0 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:08 utc nova biotechnol chim (2017) 16(2): 113-123 120    fig. 4. the production of laccase (a, c) and cellulases (b, d) by the white-rot fungus pleurotus ostreatus in the production medium with selected lignocellulosic material at 30 °c, ph 7.0 during 14 days (a, b – the production of lignocellulolytic enzymes by the one-step cultivation; c, d – the production of lignocellulolytic enzymes by the two-step cultivation strategy).     hemicelluloses and lignin content (table 4). the highest content of cellulose and hemicelluloses were measured at corn straw (41.7 and 52.3 %, respectively). the lowest amount of lignin was observed in corn straw (5.0 %). pointer et al. (2014) determined the comparable amount of selected components of lignocellulose in straw corn, specifically 38.8 % of cellulose, 44.4 % of hemicelluloses and 11.9 % of lignin. alfalfa steam is composed from 27.0 % of cellulose, 48.4 % of hemicelluloses and 16.7 % of lignin. bidlack and buxton (1992) determined similar content of lignin in alfalfa steam (17.4 %). from selected material, the highest content of lignin was determined in pine sawdust (23.7 %). in general, similar to our results, sjöström (1993) determined that cellulose, hemicelluloses and lignin contents varied among selected softwood among 33-42 %, 22-40 % and 27-32 %, respectively. production of laccase the effect of propagation step on increasing of the laccase production by the white-rot fungus p. ostreatus was tested in the media contained selected lignocellulosic material, namely pine sawdust, alfalfa steam or corn straw (two-step cultivation strategy). for the comparison, media with lignocellulosic biomass was directly bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:08 utc nova biotechnol chim (2017) 16(2): 113-123 121 inoculated from slant agar with p. ostreatus culture (one-step cultivation) (fig. 4). the results from laccase production in the media with lignocellulose without pre-cultivation of biomass in the propagation medium are shown in fig. 4a. the highest laccase activity was measured in the production medium with corn straw (0.44 u/ml) and the lowest was observed in the medium with pine sawdust (0.12 u/ml). the laccase production was noted at 6th day of cultivation in the media with all tested lignocellulosic materials. the greatest cellulase activity (fig. 4b) was measured at 7th day of cultivation in the medium with alfalfa stems (19.1 u/l). higher cellulase activities were observed in the production medium with corn straw (16.3 u/l) and the lowest cellulase activity was measured in the media with pine sawdust (5.3 u/l). the results in fig. 4 also demonstrate that the composition of lignocellulosic material leads to changes of the enzyme production. the laccase production seems to be stimulated by lower lignin content in lignocellulosic material (fig. 4a). specifically, the lowest lignin content was determined in corn straw, following by alfalfa steam and pine sawdust (table 4). similarly, cellulase production was higher in the cultivation medium containing lignocellulosic material with lower lignin content (fig. 4b) than that in the media with higher lignin content. these findings were observed in works of other authors (sun et al. 2004; elisashvili and kachlishvili 2009). in media with produced fungal biomass from the optimized propagation medium, the laccase activity was the highest at 4th day of cultivation (fig. 4c). the maximal laccase activity was observed in the medium with corn straw (3.80 u/ml). the laccase activity in the production medium with alfalfa steam and pine sawdust was approximately two times lower (1.93 and 1.76 u/ml, respectively) than those in the media with corn straw. widiatuti et al. (2008) reached the maximal laccase activity in first week of the cultivation. in the comparison with the one-step of cultivation (fig. 4a), laccase activities were greater 9-16 times by the two-step cultivation strategy. moreover, the slightly increase of laccase production was also noted in 14th day of the cultivation. the use of different substrates for the cultivation of white-rot fungi can affect the secretion of different lignolytic enzymes. it is obvious, that the most suitable substrate for laccase production is corn straw. this material shows the lowest content of lignin (table 4) although, lignin is considered as laccase inductor. cellulase activities (fig. 4d) detected in all tested media under the two-step cultivation strategy were similar to the one-step of cultivation (fig. 4b). cellulases are probably constitutively produced during the cultivation. p. ostreatus is a white-rot fungus which can produce lignolytic enzymes, namely manganese peroxidases (mnp) and laccases (das et al. 2016) which can oxidize abts used as the substrate. therefore, the presence of these enzymes in the media was determined by native-page. results are shown in fig. 5. fig. 5. the presence of lignolytic enzymes in the production medium. lane 1 – abts (presence of laccases), lane 2 – abts and h2o2 (presence of laccases and lignin peroxidases) and lane 3 – abts, h2o2 and mn2+ (presence of laccases, lignin peroxidases and manganese peroxidases). from native-page (fig. 5), only two laccase isoforms were detected (fig. 5, lanes 1-3). lane 1 in fig. 5 shows two bands of laccases stained by abts solution. lane 2 of page gel stained with abts and hydrogen peroxide for lignin peroxidase (lip) presence shows the same bands corensponding only the presence of laccase isoforms. similarly, lane 3 stained with the solution of abts, hydrogen peroxide and mn2+ ions also shows two laccase isoforms without the presence of other bands. therefore, it can be written that p. ostreatus cultivated at the described conditions produces only two isoforms of laccase. lane 1 lane 2 lane 3 lac1 lac2 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:08 utc nova biotechnol chim (2017) 16(2): 113-123 122 similar to our results, muñoz et al. (1997) did not detect lip and mnp activities during the cultivation of p. eryngii. conclusions the special attention has been devoted the laccase production from cheap sources, such as agricultural wastes or agro-industrial residues. the suitable technique of laccase production by the white-rot fungus p. ostreatus seems to be the two-step cultivation strategy. in the first step, it is advisable to cultivate of fungal producer at the optimal propagation conditions. the rsm prediction shows that the optimal propagation conditions were: glucose concentration 102.68 g/l, yeast extract concentration 43.65 g/l and ph 7.24. in the second step, p. ostreatus produced laccases with 9-16 times higher activities than laccases produced by the one-step cultivation directly on the lignocellulose material without the previous propagation step. moreover, the cultivation time of laccase production by the two-step cultivation strategy was three times shorter than this by onestep cultivation of p. ostreatus. p. ostreatus under the selected cultivation conditions produces two isoforms of laccases without the presence of other lignolytic enzymes. acknowledgement this work was supported by the projects fppv-17-2017 and apvv-16-0088. references arantes v, silva em, milagres amf (2011) optimal recovery process conditions for manganese-peroxidase obtained by solid-state fermentation of eucalyptus residue using lentinula edodes. biomass bioenergy 35: 4040-4044. baldrian p (2006) fungal laccases-occurrence and properties. fems microbiol. 30: 215-242. bellettini mb, fiorda fa, maieves ha, teixeira gl, ávila s, hornung ps, júnior am, ribani rh (2016) factors affecting mushroom pleurotus spp. saudi j. biol. sci., in press. bettin f, osório da rosa l, montanari q, calloni r, gaio ta, malvessi e, moura da silveira m, dillon ajp (2011) growth kinetics, production, and characterization of extracellular laccases from pleurotus sajor-caju ps2001. process biochem. 46: 758-764. bhattacharya ss, 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krupodorova 1 , victor yu. barshteyn 1,, olena v. pokas 2 1 institute of food biotechnology and genomics of the national academy of sciences of ukraine, osipovskogo str. 2a, kyiv 04123, ukraine 2 institute of epidemiology and infectious diseases of the national academy of medical sciences of ukraine, m. amosova srt. 5, kyiv 03038, ukraine  corresponding author: barmash14@gmail.com article info article history: received: 18 th november 2020 accepted: 21 st april 2021 keywords: dual-culture plate assay macromycetes saccharomycetaceae species interactions abstract the trend to search novel natural antifungal compounds has recently been increasing. interspecific interactions between 30 macromycetes species and fungal pathogens (issatchenkia orientalis, candida albicans strains) have been evaluated using dual culture plate assay. interaction reactions between studied fungi were different: deadlock after mycelia contact or at a distance, overgrowth without initial deadlock, partial or complete replacement after initial deadlock with contact. domination of replacement (78.7 %) of pathogenic fungi by macromycetes was established. complete replacement was almost twice more frequent (30.7 %) than partial replacement (19.3 %). these results clearly indicate effectiveness of macromycetes against pathogenic fungi via contact antagonism. strong antagonistic activity with high antagonistic index (ai) was established by xylotrophic species ganoderma applanatum (ai = 22.5), lentinula edodes (ai = 21.0), flammulina velutipes, irpiciporus litschaueri and pleurotus ostreatus (ai = 20.5), leaf litter decay fungus crinipellis schevczenkoi (ai = 21), and soil saprotroph lyophyllum shimeji (ai = 21).  university of ss. cyril and methodius in trnava introduction one of the major and global health care threat in dermatology, gynecology, and pediatrics continue to represent fungal infections. the most common fungal pathogen is candida albicans that is the predominant cause of candidiasis. such fungal infections can be subdivided into major groups: cutaneous (skin and its appendages), mucosal (oropharyngeal, esophageal, and vulvovaginal) and systemic (bloodstream infections, i.e., candidemia and other forms of invasive candidiasis) (papon et al. 2013). aids and cancer patients as well as in transplant individuals are at high risk of candidiasis due to the immunosuppression. in addition, frequently isolated non-albicans candida species like issatchenkia orientalis (formerly candida krusei) have shown gradual emergence as a cause of invasive candidiasis (samaranayake and samaranayake 1994; samaranayake et al. 2014) and also onychomycosis in patients with chronic mucocutaneous infections (hossain and ghannoum 2001). also of concern is the continued emergence of new facts of resistance to the existing commercial drugs. mailto:barmash14@gmail.com nova biotechnol chim (2021) 20(1): e760 2 at the same time, poor effect of some drugs and high possibility of candidiasis recurrence, as well as the toxicity of drugs of modern drug therapy, based on ketoconazole, emphasize the search for new effective antifungal agents with low bayside effect. it is worth considering that interspecific fungal antagonism can be a powerful emerging tool in the development of mycoproducts (or biotechproducts) against pathogenic fungi. the encouraging results of studies of the antifungal activity of macromycetes against most pathogenic fungi have contributed to the growing interest in this problem. numerous experiments have shown inhibition of the growth of candida sp. under the influence of various species of basidiomycetes: lentinula edodes (hirasawa et al. 1999; hearst et al. 2009), agrocybe perfecta, climacodon pulcherrimus, oudemansiella canarii, pycnoporus sanguineus (rosa et al. 2003), oudemansiella spp. (vahidi and namjoyan 2004), laetiporus sulphureus (turkoglu et al. 2007; petrović et al. 2014), coprinus cinereus (ndyetabura et al. 2010), agaricus brunnescens, lactarius vellereus (current name: lactifluus vellereus), terfezia boudieri (dogǎn et al. 2013), amanita muscaria, a. phalloides, lactarius spp., l. densifolius, l. gymnocarpoides (current name: lactifluus gymnocarpoides), russula kivuensis (chelela et al. 2014), cantharellus cibarius (popova 2015), agaricus bisporus, trametes gibbosa (waithaka et al. 2017), morchella esculenta, verpa bohemica (shameem et al. 2017), entoloma speculum (kodiyalmath and krishnappa 2018), pleurotus ostreatus (gutef et al. 2020). these examples show clearly the significant antifungal potential of fungi and testify the relevance and prospects of such investigations. altogether, the knowledge and understanding of different mechanisms of biological, chemical, physical action, directly caused by the species interaction of fungi, reveals alternative ways of their use as natural biotechnological tools for ecological bioremediation, biologically control the pathogens of agricultural crops or forests and as biological helpers in healing serious diseases (petre 2015). the aim of our study was to investigate the interspecific interactions between macromycetes species and saccharomycetaceae fungi in dualculture. experimental fungal species thirty macrofungi from different ecophysiological (wood decaying, saprotrophic, entomophilic, and leaf litter decaying) and taxonomic groups were screened (table 1). all the mushroom species used in this study were kindly supplied by the mushroom culture collection (ibk) of the m.g. kholodny institute of botany of the national academy of sciences of ukraine (bisko et al. 2016). stock cultures were maintained on beerwort agar-agar slants at 4 ºc. the issatchenkia orientalis kudryavtsev 301, candida albicans (c.p. robin) berkhout 17/138 and clinical strains of c. albicans 311, 315, 319 were from the culture collection of microorganisms of the institute of epidemiology and infectious diseases of the national academy of medical sciences of ukraine. strains c. albicans 311, 315, 319 were isolated from people with different infections with undergone long-term antibiotics therapy and show resistance to antibiotic drugs (mainly to streptomycin, penicillin, oxacillin, ampicillin, some of them to amoxicillin and others). stock cultures were maintained on potatodextrose-agar (pda) slants at 4 ºc. all fungi were transferred from stored cultures to pda petri dishes and cultured at 26 ± 1 ºc to obtain mycelial colonies. mycelial plugs 8-mm were cut from at the mycelial active growth stage using a sterile borer and used as inoculum in dual culture. dual-culture experiments macromycetes were screened for their ability to suppress the mycelial growth of saccharomycetaceae fungi in vitro dual-culture plate assays on pda by the method described by badalyan et al. (2002; 2004). the antagonistic ability of each fungal organism was determined using a rating scale for the 3 main types of reactions (a, b, c) and 4 sub-types (ca1, cb1, ca2, and cb2). types a and b were deadlock (mutual inhibition, in which nova biotechnol chim (2021) 20(1): e760 3 neither organism was able to overgrow the other) at mycelial contact (a), or at distance (b); type c replacement, overgrowth without initial deadlock. the intermediate sub-types scored were: ca1 partial and ca2 complete replacement after initial deadlock with mycelial contact; cb1 partial, and cb2 complete replacement after initial deadlock at a distance. the following score was assigned to each type or sub-type of reaction: a = 1; b = 2; c = 3; ca1 = 3.5; cb1 = 4; ca2 = 4.5; cb2 = 5. the antagonism index (ai) was then calculated for each fungal species using the following formula (eq. 1): ai=a(nx1)+b(nx2)+c(nx3)+ca1(nx3.5)+cb1(nx4) +ca2(nx4.5)+cb2(nx5) (1) where n = frequency of each type or sub-type of reaction (badalyan et al. 2002; 2004). saccharomycetaceae together with the macromycetes were incubated in the thermostat at 26 ºc. the growth of fungi was monitored daily for a month. the morphological changes (coloration of mycelia and agar, visualization of the demarcation line, producing of exudate drops, formation of dense zones of mycelium, fruiting body primordia etc.) in interacted colonies were noted. the type of interaction was recorded on the 30 th day of the experiment. all experiments were independently conducted in three replicates. results and discussion the interaction between fungi is quite broadly, engaging and important field of study. of particular interest is the cause-effect relationship of biological and biochemical bases for immune and defense reactions of competing species. different concepts have been used to characterize the macromycetes interaction with other fungi in co-culture (bruce and highley 1991; badalyan et al. 2002; 2004; barinova et al. 2008; owaid 2017; pasaylyuk 2017). to our opinion, the determination of antagonistic ability according to the method of badalyan et al. (2002; 2004) using a rating scale and calculation of an antagonistic index is more appropriated for the screening and evaluation of the antifungal potential of macromycetes in solid medium. the interactions between all tested fungi were established. the results obtained at the process of growth in dual cultures are shown in table 1 and some examples are illustrated in fig. 1. performed screening allowed easy to determine the most active fungus species with high antagonistic index (ai) from different ecological groups: xylotrophic species g. applanatum (ai = 22.5), l. edodes (ai = 21.0), f. velutipes, i. litschaueri and p. ostreatus (ai = 20.5), leaf litter decay fungus c. schevczenkoi (ai = 21), and soil saprotroph l. shimeji (ai = 21). previous experiments also showed that xylotrophic fungi p. ostreatus, f. velutipes, ganoderma sp. had the strongest competitive effect against mycoparasitic fungi (clonostachys rosea, trichoderma harzianum, t. pseudokoningii, and t. viride) (badalyan et al. 2004). moreover, it was found that p. ostreatus possessed strongest antagonistic activity against cereal pathogenic fungi (gaeumannomyces graminis var. tritici (current name: gaeumannomy ces tritici), bipolaris sorokiniana, fusarium culmorum, rhizoctonia cerealis (current name: ceratobasidium cereale) (badalyan et al. 2002). studied by us fungi produced a different variety of interaction reactions: deadlock after mycelial contact or at a distance, overgrowth without initial deadlock, partial or complete replacement after initial deadlock with contact (fig. 2). however, domination of replacement (78.7 %) of pathogenic fungi by macromycetes was established. furthermore, complete replacement was almost twice more frequent (30.7 %) than partial replacement (19.3 %). these results clearly indicate effectiveness of macromycetes against pathogenic fungi via contact antagonism. this tendency is in line with observation of antagonistic activity of 17 species xylotrophic basidiomycotina against pathogenic fungi (bipolaris sorokiniana, fusarium culmorum, gaeumannomyces graminis var tritici, rhizoctonia cerealis) (badalyan et al. 2002). probably different mechanisms affect species interactions. generally, any type of antagonism (at mycelial or hyphal level) or parasitism is caused by production of different compounds such as ferments, toxins, antimycotic substances that are capable of volatility as well as able to the diffusion (woodward and boddy 2008). it is important to nova biotechnol chim (2021) 20(1): e760 2 remark that response of species interaction in certain model experimental conditions can change table 1. the interactions of studied fungi and their antagonism index (ai). fungi species strain i. orientalis 301 c. albicans strains ai 17/ 138 311 315 319 auriporia aurea (peck) ryvarden 5048 c c ca1 c a 13.5 coprinus comatus (o.f. müll.) pers. 137 ca2 ca2 ca1* ca1 ca1* 19.5 cordyceps militaris (l.) fr. 1862 ca1 ca2 ca1* ca1 ca1* 18.5 crinipellis schevczenkoi bukhalo 31 c ca2 ca2 ca2 ca2 21.0 cyclocybe aegerita (v. brig.) vizzini 1853 c c ca1 c c 15.5 flammulina velutipes (curtis) singer 1878 ca2 ca2 ca2* ca1 ca1* 20.5 fomes fomentarius (l.) fr. 355 ca2 ca2 ca2 ca2 a* 19.0 fomitopsis betulina (bull.) b.k. cui, m.l. han &y.c. dai 327 ca2 c ca2 ca2 a* 1 7.5 fomitopsis pinicola (sw.) p. karst. 1523 c c c* c c 15.0 ganoderma applanatum (pers.) pat. 1701 ca2 ca2 ca2* ca2 ca2 22.5 ganoderma lucidum (curtis) p. karst. 1900 c c c* c c 15.0 grifola frondosa (dicks.) gray 976 a b ca2* a ca2 13.0 hericium erinaceus (bull.) pers. 970 a a b* a ca1 8.5 hypsizygus marmoreus (peck) h.e. bigelow 2006 ca1 a a* ca1 ca1* 12.5 inonotus obliquus (fr.) pilát. 1877 c c ca1 c a 13.5 irpiciporus litschaueri (lohwag) zmitr. 5312 ca2 ca2 ca1 ca1 ca2 20.5 laetiporus sulphureus (bull.) murrill 352 c c c* c c* 15.0 lentinula edodes (berk.) pegler 502 c ca2 ca2* ca2 ca2* 21.0 lepista luscina (fr.) singer 64 a a a* a a* 5.0 lyophyllum shimeji (kawam.) hongo 1662 ca2 ca2 c* ca2 ca2 21.0 morchella esculenta (l.) pers. 1953 a b a a* a 6.0 ophiocordyceps sinensis (berk.) g.h. sung, j.m. sung, hywel-jones & spatafora 1928 ca1 ca1 ca2 ca1 ca1 18.5 oxyporus obducens (pers.) donk 5085 c c c c c 15.0 phellinus igniarius (l.) quél. 1578 a ca2 ca1 ca1 a 13.5 pleurotus djamor (rumph. ex fr.) boedijn 1526 b ca2 ca1* ca2 ca2 19.0 pleurotus eryngii (dc.) quél. 2015 ca2 ca2 ca1* c ca1 19.0 pleurotus ostreatus (jacq.) p. kumm. 551 ca2 ca2 ca1* ca2 ca1* 20.5 schizophyllum commune fr. 1768 c ca2 c a c* 14.5 trametes versicolor (l.) lloyd 353 c c c c c 15.0 xanthoporia radiata (sowerby) ţura, zmitr., wasser, raats & nevo 2454 a a a* a* a* 5.0 legend: a – deadlock after mycelial contact, b – deadlock at a distance, c – overgrowth without initial deadlock, ca1 – partial replacement after initial deadlock with contact, ca2 – complete replacement after initial deadlock with contact, * – the presence of polymorphism of c. albicans. in natural habitat due to the effecting any antagonistic or parasitism mechanisms that provide full functioning of ecosystem (bruce and highley 2001). generally, c. albicans 17/138 and i. orientalis were more sensitive to all investigated fungi than c. albicans clinical isolates. current studies may provide the basis to determine the correlation between the interaction of macromycetes and the degree of candida strains virulence. during the experiments the polymorphism (formation pseudohyphae or/and hyphal form) of c. albicans clinical isolates has been established (fig. 3): c. albicans 311 in co-cultivation with 17 fungi (entomophilous c. militaris, soil saprotrophic like c. comatus, l. luscina, l. shimeji and xylotrophic f. velutipes, f. pinicola, g. applanatum, g. lucidum, g. frondosa, h. marmoreus, h. erinaceus, l. sulphureus, l. edodes, p. djamor, p. eryngii, p. ostreatus, x. radiate), c. albicans 315 ̶ with 2 fungi (soil saprotroph m. esculentaand xylotroph x. radiata), c. albicans 319 ̶ with 12 fungi (entomophilous c. militaris, soil saprotrophic c. comatus, l. luscina, and xylotrophic f. velutipes, 4 nova biotechnol chim (2021) 20(1): e760 5 f. betulina, f. fomentarius, h. marmoreus, l. sulphureus, l. edodes, p. ostreatus, s. commune, x. radiate). thus, all three clinical isolates of c. albicans fig. 1. illustration of competitive interactions between mycelia of studied fungi: a – deadlock at mycelial contact of m. esculenta and c. albicans 315; b – deadlock at distance, interaction of p. djamor and i. orientalis 301; c – overgrowth without initial deadlock, interaction of f. betulina and c. albicans 17/138; d – partial replacement after initial deadlock with contact of p. eryngii and c. albicans 319; e, f – complete replacement after initial deadlock with contact of i. litschaueri and c. albicans 17/138, front and reverse sides of petri dishes, respectively. note: right pathogenic fungus, left macromycete fungus. showed polymorphism in the case of co-cultivation only with x. radiata. two clinical isolates c. albicans 311 and 319 showed this feature with growth in dual culture with c. militaris, c. comatus, l. luscina, f. velutipes, h. marmoreus, l. sulphureus, l. edodes, p. ostreatus, x. radiata. it is a b c d e f fig. 2. frequency of type (a – deadlock after mycelial contact, b – deadlock at a distance, c – overgrowth without initial deadlock) and subtype (ca1 – partial replacement or ca2 – complete replacement after initial deadlock with contact) interactions between colonies of studied fungi, expressed as percentage of the total number (450) of pairings tested. nova biotechnol chim (2021) 20(1): e760 6 known that the basic function of hypha formation of opportunistic pathogens like candida spp. is to invade the substrate they are adhered to. however, probably, in this case, this may be a response to a change the conditions in medium, it can become more extreme in particular, a sharp change in ph as fig. 3. illustration of polymorphism of c. albicans clinical isolates (right) in co-cultivation with macromycetes (left): h. erinaceus and c. albicans 311(a), x. radiata and c. albicans 315 (b), f. fomentarius and c. albicans 319 (c). a reply of the synthesis of metabolites by antagonist. the competition or antagonism experienced during co-cultivation led to a change in the morphological characteristics of fungal colonies (fig. 4). these changes can be mediated by the up-regulation of genes involved in antagonism (iakovlev et al. 2004). the formation of a mycelial ridge of g. frondosa upon contact with a colony of i. orientalis has been established (fig. 4a). during deadlock reaction localized aerial mycelia of f. betulina was produced at the interface between confronted fungi (fig. 4b). the presence of central stria was observed in case of co-cultivation g. applanatum with i. orientalis (fig. 4c). after 20 days of growth, observed the appearance of fungal metabolites on f. fomentarius colony in the form of exudate of dark brown drops (fig. 4d) and on p. ostreatus and l. shimeji colonies in the form of shining small golden drops of liquid (fig. 4e, 4f). the production of analogical golden secondary metabolites was also found on edges of colonies l. edodes in dual culture with verticillium sp. and pythim sp. (owaid 2017). fungus f. fomentarius is distinguished by its ability to form primordia in culture. however, it should be noted that this process took place more intensively in the area of c. albicans location (fig. 4d). fruit-body primordia were also reported only in pairings of lentinus tigrinus with bipolaris sorokiniana (badalyan et al. 2002). interaction of c. comatus with c. albicans 311 resulted in the formation of brown barrages in the antithetic zone line of interaction with the pathogen, сolony morphology and color changes in the mycelia and reversume of xylotrophic fungus colony (fig. 4g, 4h, 4j, 4k). similar finding with brown barrages has already been reported as a resistance of l. edodes strain ufla-le5 to trichoderma sp. (santos 2019). changes in colony color and morphology were also observed in cocultivation of c. comatus with c. albicans 319 (fig. 4g, 4i, 4j, 4l). it is probably that diffusion of metabolites of candida albicans strains stimulated laccase activity (oxidation of polyphenols) of c. comatus and the producing of brown pigments in c. comatus hyphae by activating some general cellular mechanisms of stress resistance. it should be noted that the presence of laccase was previously established by us in 21 fungi species (krupodorova et al. 2014), but only co-cultivation of c. comatus and c. albicans strains 311, 319 allowed us to reveal it. thus, co-cultivation in our case can be considered as a biotechnological tool that allows increasing the production of c. comatus laccase. obtained from c. comatus mycelia (strain jt-01) laccase was shown previously as antipathogenic protein that possessed antiproliferative and hiv-1 reverse transcriptase inhibitory activities (zhao et al. 2014). the enhanced activity of lignin-modifying enzymes (particular laccase, manganese peroxidase, phenol b c a nova biotechnol chim (2021) 20(1): e760 7 fig. 4. illustration the changes in the morphological characteristics of fungal colonies: the formation of a mycelial ridge of g. frondosa upon contact with a colony of i. orientalis 301 (a); localized aerial mycelia of f. betulina at the interface between confronted fungi c. albicans 319 (b); the presence of central stria in case of co-cultivation g. applanatum with i. orientalis 301 (c); primordia formation of f. fomentarius in dual culture with c. albicans 17/138 (d); producing in form shining small golden drops of liquid on colonies of p. ostreatus by co-cultivation c. albicans 311 (e) and of l. shimeji by co-cultivation with c. albicans 311 (f); formation of brown barrages in the antithetic zone line of interaction with the pathogen, сolony morphology and color changes in the mycelia of c. comatus (h) and reversume of xylotrophic fungus colony (k) in dual culture with c. albicans 311; changes in front colony colour and morphology (i) and reversume (l) of c. comatus in cocultivation with c. albicans 319. g and j front and reversume of c. comatus colonies in mono culture, respectively (control). note: right – pathogenic fungus, left – macromycete fungus. a b c d e f h i k l j g nova biotechnol chim (2021) 20(1): e760 8 oxidase and peroxidase activities) in fungal coculture and the important role of these enzymes in the antagonistic interaction between species pointed out by different researchers (li 1981; 1983; bruce and highley 1991; nia 1992; freitag and morrel 1992; rayner et al. 1994; savoie et al. 1998; hatvani et al. 2002; badalyan et al. 2002; 2004; uzar et al. 2017). in addition, dark zones can be due to melanin production. it can function as a physical barrier, preventing access by cell walldegrading enzymes of other organisms (bull 1970), and also it can be an important part of the protective response against mycelial invasion that allows to adapt to environmental stress or to attack of antagonists (lee et al. 2008). generally, exudates producing as well as color changes should be considered a consequence of the fact, that сo-cultivation lead to a significantly enhanced production of constitutively present compounds and/or to an accumulation of cryptic compounds that are not detected in axenic cultures of the producing strain (marmann et al. 2014) and there are could be responsible for the antifungal or/and fungistatic activity. conclusion the study touched on the antagonistic activity is one of the integral stages into the understanding fungal-fungal competition, physiological as well as biochemical processes of their interaction. presented research may provide valuable insights into interspecific fungal interactions. co-cultivation led to a change in the morphological characteristics of some macromycete fungal colonies such as the formation of a mycelial ridge, aerial mycelia, barrages, drops of exudate, changed the colony color, and polymorphism of c. albicans clinical isolates. obtained results indicate the considerable antifungal potential of studied 30 macromycetes against issatchenkia orientalis and candida albicans strains. studied fungi produced a different variety of interaction reactions: a deadlock after mycelial contact (18.7 %), b deadlock at a distance (2.6 %), c overgrowth without initial deadlock (28.7 %), and subtypes ca1 partial replacement (19.3 %) or ca2 complete replacement after initial deadlock with contact (30.7 %). domination of replacement (78.7 %) of pathogenic fungi by macromycetes was established. the complete replacement was almost twice more frequent (30.7 %) than partial replacement (19.3 %). these results clearly indicate the effectiveness of macromycetes against pathogenic fungi via contact antagonism. performed screening allows to determining the most active mushrooms with high antagonistic index (ai): xylotrophic species g. applanatum (ai = 22.5), l. edodes (ai = 21.0), f. velutipes, i. litschaueri and p. ostreatus (ai = 20.5), leaf litter decay fungus c. schevczenkoi (ai = 21), and soil saprotroph l. shimeji (ai = 21). these fungi are promising species for further studies to investigate and isolate potential antifungal metabolites with strong action. acknowledgement we thank to prof. nina a. bisko (m.g. kholodny institute of botany of the national academy of sciences of ukraine) for providing of fungi from the culture collection of mushrooms (ibk). this research was funded by the national academy of sciences of ukraine (state registration number 0118u003812) for the institute of food biotechnology and genomics of the national academy of sciences of ukraine and by the national academy of medical sciences of ukraine (state registration number 0119u002041) for institute of epidemiology and infectious diseases of the national academy of medical sciences of ukraine. conflict of interest the authors declare 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(basidiomycetes). iran j. pharm. res. 2: 115-117. waithaka pn, gathuru em, githaiga bm, onkoba km (2017) antimicrobial activity of mushroom (agaricus bisporus) and fungal (trametes gibbosa) extracts from mushrooms and fungi of egerton main campus. njoro kenya. j. biomed. sci. 6: 1-6. woodward s, boddy l (2008) interactions between saprotrophic fungi. british mycological society symposia. 28: 125-141. zhao s, rong c-b, kong c, liu y, xu f, miao q-j, wang sx, wang h-x, zhang g-q (2014) a novel laccase with potent antiproliferative and hiv-1 reverse transcriptase inhibitory activities from mycelia of mushroom coprinus comatus. biomed res. int. 2014: 417461. 10 nova biotechnol chim (2019) 18(1): 10-17 doi: 10.2478/nbec-2019-0002  corresponding author: sestinova@saske.sk nova biotechnologica et chimica earthworms as useful bioindicator of soils contamination around košice city, slovakia oľga šestinová, jozef hančuľák and tomislav špaldon department of environment and hygiene in mining, institute of geotechnics, slovak academy of sciences, watsonova 45, sk-040 01 kosice, slovak republic article info article history: received: 27th march 2019 accepted: 14th july 2019 keywords: bioaccumulation factor earthworms heavy metals industrialized soils toxic risk abstract this study was conducted to investigate heavy metals bioaccumulation in industrialized soils in surrounding of košice city (slovakia), using earthworms. in the present research, we used ecotoxicity tests with dendrobaena veneta (7 and 28-day bioassays) to infer about potential toxic risks to the agricultural (a) and permanent grass vegetation (pgv) of soils around the plant u. s. steel košice. the total fe, cu, zn, pb, cd, cr and as contents and eco-toxicological tests of industrialized soils from the košice area were performed for 12 sampling sites in years 2016 ‒ 2017. an influence of the sampling sites distance from the largest steel producer plant on the total concentrations of heavy metals was determined for fe, cd, cr and as. it was found that earthworms (dendrobaena veneta) in some cases caused a decrease of metals concentration in contaminated soils, the largest metal concentration differences were recorded in the samples pgv (4) u. s. steelplant-main gate. the results of the bioaccumulation factors of heavy metals in d. veneta (bafs/7-28 d) are < 1 for the studied metals order in the sequence: cr < fe < pb < cu < as and > 1 for zn > cd.  university of ss. cyril and methodius in trnava introduction urban environmental quality is vital to be investigated as large parts of human populations live in cities. however, continuous urbanization and industrialization in urban areas pose a great threat to environment as well as human. soils are susceptible to anthropogenic contamination with metallic trace elements that generate risks to ecosystems and human health. in industrialized areas urban soils are highly modified and intensively managed. košice, the city in eastern slovakia, is exposed to typical urban contamination sources such as road traffic, municipal sphere, and small industrial sources. furthermore, being the largest steel producer in central europe, it is long-term environmentally loaded by the iron and steel works that represent the largest source of (metal) contamination in slovakia (hančuľák et al. 2015). the transport of heavy metals in soil is the result of processes between soil and metal components, which include processes of physical, chemical, and biological nature (violante et al. 2008). their monitoring and studying should be complex to provide relevant picture on their effects and impacts. the combination of chemical measurements with the calculation of the bioaccumulation factor (baf) and person matrix correlations (r) can be a useful tool in risk assessment. earthworms by virtue of their habitation participate in the formation of soil structure and regulate dynamics of soil organic matter, therefore (directly or indirectly) can modulate the transfer of inorganic and organic bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:51 utc mailto:sestinova@saske.sk nova biotechnol chim (2019) 18(1): 10-17 11 fig. 1. the study area and sampling localities. chemicals (toxicants) in soils. for this reason they are considered as useful biological indicator of several metals in soils (suthara et al. 2008). furthermore, they relatively efficiently accumulate certain essential and non-essential metals thus also can affect soil processes and local biodiversity directly by transferring metals to other organisms (morgan et al. 2001). bioaccumulation can be defined as the uptake of toxicants by living cells. the toxicant can be transported into the cell across the cell membrane and accumulate intracellularly. the accumulated metals are detoxified through cell metabolic cycle (coelho et al. 2018). earthworms have already been used in several studies on environmental contamination by heavy metal, and usually were considered a good bioindicator for soil pollutants. their biological activities can also influence the accumulation and/or biodegradation of inorganic contaminants including heavy metals (lingxiangyu et al. 2010). many authors have shown that bioassays provide a general indication of metal toxicity in soils cycle (das et al. 2011; yu et al. 2012; xu et al. 2014; findoráková et al. 2017; maity et al. 2018). these tests can be successfully used to evaluate potential toxicity and to assess the environmental risks of heavy metals. the aim of this study was evaluation of the impact of anthropogenic heavy metals in urban soils from the košice area, using bioassays on earthworms, and by means of the bioaccumulation factor and person matrix correlations. experimental soil sampling and analysis košice city has about 239 000 inhabitants and is located the košice basin in the river valley hornád in the eastern part of slovakia (europe). in the period of 2016 – 2017, 12 soil samples were collected in the košice suburban, in surrounding of u. s. steel košice, which is located about 10 km south-westwards from the centre of the city (fig. 1). annual raw steel production capability is 4.5 million metric tons. the technology of steel production has a significant impact on the urban environment. furthermore, the agricultural and industrial activities contribute to contamination of the soil in this region. the seven sampling sites were localised on area of the u. s. steel košice. permanent grass vegetation soils (pgvs) were sampled on following localities (fig. 1): 1 ‒ perín, 2 ‒ u. s. steel-slag heap, 3 ‒ gomboš (veľká ida), 4 ‒ u. s. steel-plant (main gate), 5 ‒ pereš-south, 6 ‒ u. s. steel-north-west and 7 ‒ u. s. steel north-eastern. the five samples of agricultural soils (as) were collected in localities (fig. 1): 1 ‒ perín, 2 ‒ u. s. steel-slag heap, 3 ‒ gomboš (veľká ida), 4 ‒ u. s. steel-plant west and 5 ‒ pereš-south. the soils were sampled at a depth of 20 ‒ 40 cm into plastic bags. soil samples were homogenized, dried at room temperature (25 °c) and sieved through 2-mm sieve. for analysis of soils were used the mixed samples of total weight of 5 kg. the concentrations of the heavy metals (fe, cu, zn, pb, cd, cr and as) in soil samples were bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:51 utc nova biotechnol chim (2019) 18(1): 10-17 12 measured using spectro xepos x-ray fluorescence spectrometer model xepos 3 (range of elements: na(11) – u(92), scattering targets: mo, co, al2o3, pd, hopg-crystal, x-ray lamp (type vf50): pd with be window, resolution: 145 kev on line kα mn). all the analysed samples were conducted in triplicate and the data were expressed to soil dry weight unit. the used control reference soil (crm tm52, microbiotests, belgium) contained: 85 % (w/w) quartz, 10 % (w/w) kaolin, 5 % (w/w) peat and calcium carbonate (ph of 6±0.5) (oecd soil). the certified river sediment lgc6187 was used for validation of data. all data were analysed using spss ver. 9.0 statistical software. statistical correlations at the levels p < 0.05 and p < 0.01 were considered significant. soil physico-chemical parameters were determined (ph value in h2o and kcl solutions and oxidation–reduction potential (eh) (iso/dis 10390), organic matter (om) (stn en 12879). biological assay with d. veneta earthworms (dendrobaena veneta) are often used as bio-monitoring organisms for assessment of soil pollution, because they are widespread in their terrestrial distribution and have a capacity to accumulate and concentrate large quantities of inorganic and organic pollutants. the experimental design was based on oecd guideline 317/2010 for the testing of chemicals related to environmental fate, and mortality. adult earthworms were purchased from a local supplier, and were allowed to acclimatize for one week in the experimental conditions. ten earthworms (also c.w. – control worms) were added to each plastic box (9 cm x 9 cm x 3 cm) with 100 g of dry soil sample. as reference, c.w. was added to 100 g of crm soil. then distilled water was added to the boxes to obtain 30 % (w/w) moisture of soil. the boxes with soils were kept at laboratory temperature for 7 and 28 d (respectively). the mortality of earthworms in soils (replicated tests a – d) was determined and compared with the data from control reference soil. the earthworms from individual boxes were collected and lyophilized at -50 °c and 50 pa (íl shin-bio base, the netherlands). the concentrations of heavy metals in the d. veneta tissue were determined after mineralization with a mixture of acids hno3/hf/h3bo3 (5:2:20) in the microwave system mws33 (germany) by atomic absorption spectrometry (aasvariant, australia). bioaccumulation of heavy metals in d. veneta tissues bioaccumulation factor for earthworms (bafs) were calculated according to oecd (2010): bafs = (metal) earthworm / (metal) soil. the relationship between mortality of earthworms (ande absolute number of dead earthworms) and total metal concentrations were evaluated by pearson matrix correlation. as control, earthworms fed in non-contaminated (crm) soil were used. table 1. soil physico-chemical parameters considered in this study. soil type locality ph ph eh om [kcl] [h2o] [mv] [%] pgv 1 perín 6.88 6.81 462 10.3 2 u. s. steel-slag heap 7.18 7.13 472 7.90 3 gomboš 7.45 7.55 443 6.60 4 u. s. steel-plantmain gate 7.83 7.72 391 5.39 5 pereš-south 5.67 6.10 534 4.80 6 u. s. steel-north-west 6.34 6.89 476 6.50 7 u. s. steelnorth-eastern 7.22 7.38 369 7.74 as 1 perín 6.20 7.20 490 4.65 2 u. s. steel-slag heap 6.99 7.74 441 4.34 3 gomboš 7.23 7.95 522 4.94 4 u. s. steel-plant west 7.47 7.80 422 4.11 5 pereš-south 5.43 6.68 457 4.36 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:51 utc nova biotechnol chim (2019) 18(1): 10-17 13 table 2. metal concentration (average ± sd) in soil (dw) at locality u. s. steel košice. locality fe cu zn pb cd cr as [%] [mg.kg-1] [mg.kg-1] [mg.kg-1] [mg.kg-1] [mg.kg-1] [mg.kg-1] permanent grass vegetation soils 1 3.3±2.1 27.1±3.9 88.7±4.3 28.9±0.9 2.0±1.1 91.7±4.1 10.6±1.8 2 3.4±2.7 27.2±2.7 157±6 50.1±2.3 1.6±0.8 85.8±3.4 13.4±2.9 3 2.5±1.8 22.5±2.8 65.9±6.9 29.9±1.5 1.9±1.2 68.8±2.8 12.5±2.1 4 10.5±3.8 64.9±6.1 1,084±11 379±14 21.4±2.9 278±14 94±13 5 2.5±1.5 24.7±2.2 64.4±5.7 30.5±1.4 3.3±1.4 106±9 9.9±2.3 6 2.9±1.7 30.8±4.3 86.5±4.6 36.8±2.2 1.2±0.1 248±11 10.8±1.8 7 3.6±2.9 28.0±3.0 95.5±6.2 46.3±2.9 2.7±0.8 264±12 12.7±1.1 agricultural soils 1 2.5±2.0 30.5±2.6 60.2±4.7 26.3±1.2 2.1±0.7 61.6±3.1 7.3±0.9 2 3.5±2.6 28.4±3.2 83.8±6.3 30.4±1.7 9.1±2.3 55.3±2.9 15.9±4.5 3 2.6±1.4 54.0±5.8 67.9±5.1 32.9±2.3 1.8±0.6 160±10 10.8±3.1 4 3.6±1.9 32.3±4.6 121±9 69.3±5.8 4.4±1.2 69.1±4.6 34.8±8.8 5 2.7±1.8 29.9±2.7 62.4±3.9 29.5±2.7 6.9±2.1 83.3±6.2 9.0±5.6 crm 2.4±0.9 83.6±1.5 439±12 77.2±3.3 2.7±1.3 84.0±5.3 24.0±3.4 low no. 220/2004-2 [mg.kg-1dw] limit 70 200 115 1 90 results and discussion earthworms play important role in soil ecotoxicological risk assessment (spurgeon et al. 2003; dai et al. 2004) as the metal content in earthworm tissues is partly dependent on the metal content of soils. this is determined by several factors including physio-chemical edaphic interactions and factors such as ph, concentration of different cations and of organic matter (c : n ratio) and cation-exchange-capacity (kizilkaya 2005). the soil types examined here were of silty-clay texture from various areas to keep representativeness, and their quality was established with reference to law (220/2004, no.2, slovak republic). table 1 summarizes the results of basic physico-chemical parameters of the permanent grass vegetation soils and agricultural soils (pgv and as, respectively) sampled from the košice area close to u. s. steel košice (fig. 1). the soil samples (both types) presented a slightly alkaline reaction, and ph/kcl and also ph/h2o were in the range from 6.34 to 7.83 (table 1). exceptions were the pgv and a-soils at locality pereš-south (5) that were slightly acidic (5.43 – 6.68). organic matter (om) content of the pgv-soils ranged between 4.8 to 10.7 %, in contrast to the a-soils that were relatively low (4.11 to 4.94 %). the results summarized in table 2 indicate to significant contamination with cadmium, chromium and arsenic comparing to the limits defined by the slovak law no. 220/2004-2 about the quality of the soil. the highest concentrations of studied metals were measured in vicinity of sites located south of the ironworks. at the main gate of the u. s. steel-plant (locality 4) high fe, zn, pb, cd, cr and as concentrations were detected for pgv soils (table 2). as for the a-soils were determined the highest values of metals from the areas: u. s. steel-plant west (locality 4) ‒ mainly for as, and gomboš (locality 3) for cr (table 2). the lowest average concentration of heavy metals in both the soil types were found in localities distant from metallurgical industry, as are perešsouth (locality 5) and perín (locality 1) (table 2). bioaccumulation factors (bafs) are used in the risk assessment to estimate trophic transfer of contaminants such as metals from soil and can be helpful to the prediction of risks associated with this transfer. baf can be derived from laboratory studies through the determination of steady-state concentrations or kinetic estimation methods. the baf is considered as the equilibrium ratio between the metal concentration in any organ of an organism and its concentration in the ambient media (soil). in this study, bioaccumulation tests were performed in the collected industrialized soils to determine uptake of fe, cu, zn, pb, cd, cr and as by earthworms d. veneta. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:51 utc nova biotechnol chim (2019) 18(1): 10-17 14 table 3. metal concentration in soil (dw) of u. s. steel košice after 7-days bioassay (average ± sd). locality fe cu zn pb cd cr as [%] [mg.kg-1] [mg.kg-1] [mg.kg-1] [mg.kg-1] [mg.kg-1] [mg.kg-1] permanent grass vegetation soils (pgvs) 1 3.1±1.7 15.3±3.5 49.5±4.2 27.9±1.1 1.9±0.8 90.3±3.9 7.8±0.6 2 2.9±1.8 22.6±2.2 106±4 48.7±2.2 1.5±0.7 87.8±2.9 9.1±1.9 3 2.0±1.9 16.4±1.9 61.2±2.9 28.1±0.6 1.9±0.6 66.9±2.4 8.7±1.3 4 9.1±2.9 55.7±7.9 1,022±13 358±12.6 19.5±1.7 258±12 67.3±7.8 5 1.9±1.1 19.9±2.1 48.7±5.1 29.9±1.5 3.1±1.1 106±7 8.4±1.7 6 2.3±0.8 23.1±3.4 74.6±6.3 35.4±1.9 1.1±0.1 248±11 9.8±2.3 7 2.8±1.5 26.2±2.7 92.0±8.4 46.2±3.0 2.8±0.9 264±13 10.6±2.8 agricultural soils (as) 1 2.3±0.5 18.9±1.6 55.8±3.6 25.3±0.6 1.9±0.9 60.1±2.8 6.1±0.9 2 3.1±1.3 20.5±1.4 79.8±4.1 29.1±1.2 8.7±2.2 52.9±3.0 11.8±1.4 3 2.5±0.5 50.3±4.3 67.1±2.3 32.0±1.5 1.5±1.3 152±12 8.4±2.1 4 3.8±1.4 31.1±2.6 110±10 64.3±3.6 4.1±2.6 64.3±4.5 30.3±2.8 5 2.3±1.2 25.8±1.9 62.0±4.1 24.7±1.3 5.9±3.7 82.8±6.3 7.5±0.7 crm 2.1±2.0 80.5±6.3 421±12 76.1±9.3 2.6±0.7 84.0±8.8 21.5±1.6 c.w. 0.05±0.8 4.2±1.3 49.8±2.4 1.7±1.9 0.5±0.4 1.0±0.1 0.7±1.4 c.w./7d. 0.06±0.6 4.1±1.2 49.2±1.6 1.7±0.8 0.4±0.8 0.9±0.7 0.6±0.9 crm (control reference soil); c.w. – control worms; c.w./7d. – control worms after 7 d. effect of metals on mortality of d. veneta after periods of 1 – 7 and 1 – 28 d of exposure (respectively) at the end of the tests (replicates a-d) showed that the earthworms’ mortality was influenced by industrialized soils already after 7 d of exposure (fig. 2). on the other hand, earthworms in some cases caused significant decrease of metals concentration in the soils (table 3). the largest metal concentration differences were recorded in the pgv soil samples at u. s. steel-plant-main gate (locality 4) (as = 67.3 mg.kg-1, cr = 259 mg.kg-1, cd = 19.5 mg.kg-1, pb = 368 mg.kg-1, zn = 1,022 mg.kg-1 and fe = 9.1 mg.kg-1), and table 4. metal concentration in soil (dw) of u. s. steel košice after 28-days bioassay (average ± sd). locality fe cu zn pb cd cr as [%] [%] [mg.kg-1] [mg.kg-1] [mg.kg-1] [mg.kg-1] [mg.kg-1] permanent grass vegetation soils (pgvs) 1 3.2±1.5 18.1±2.9 62.1±3.5 28.1±1.8 2.0±0.9 90.9±2.3 9.1±0.8 2 2.9±1.1 23.5±3.1 126±6 49.3±4.4 1.5±1.0 87.8±2.9 12.7±3.2 3 2.1±0.8 19.7±2.7 64.8±3.2 28.9±1.5 1.9±0.5 67.5±3.4 10.2±5.3 4 9.6±1.9 56.1±5.8 1,066±10 369±14 20.7±8.4 270±10 82.1±8.8 5 1.9±0.6 21.9±2.6 59.2±3.7 31.1±1.9 3.2±1.2 106±6 8.9±2.5 6 2.3±0.9 25.7±2.9 80.9±6.0 35.9±2.5 1.2±0.6 248±9 9.9±2.9 7 2.8±1.3 27.1±1.7 94.5±9.1 46.8±2.8 2.8±1.1 265±11 11.2±4.1 agricultural soils (as) 1 2.3±0.9 19.5±1.3 59.3±4.1 26.0±0.9 2.0±0.7 61.4±1.3 7.1±1.2 2 3.1±1.1 22.4±0.9 82.9±6.2 29.1±1.2 9.1±2.1 54.1±3.3 13.4±5.1 3 2.5±0.8 52.7±2.8 66.4±2.8 31.7±2.5 1.7±1.8 159±9.5 9.1±2.8 4 3.8±0.9 31.8±4.1 119±8 67.2±4.2 4.2±2.5 66.1±3.9 33.1±6.1 5 2.3±1.0 27.4±2.7 61.9±3.8 29.1±2.3 6.4±2.1 82.9±5.7 8.7±1.4 crm 2.1±1.7 80.8±5.6 424±11 77.0±10 2.7±0.5 84.6±5.8 23.4±2.5 c. w 0.05±0.8 4.2±1.3 49.8±2.4 1.7±1.9 0.5±0.4 1.0±0.1 0.7±1.4 c.w/28 0.06±0.6 4.1±1.2 49.2±1.6 1.7±0.8 0.4±0.8 0.9±0.7 0.7±0.9 low no. 220/2004-2 (mg/kg dw) limit 70 200 115 1 90 30 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:51 utc nova biotechnol chim (2019) 18(1): 10-17 15 fig. 2. effect of metals on mortality of d. veneta (ande) after periods of 1 – 7 (chequered columns) and 1 – 28 (filled columns) days of exposure at the end of the tests (average values: n/1-7 =2 and n/1-28 = 3). in the samples of a-soils at localities (4) u. s. steel-plant west as = 30.3 mg.kg-1 and (3) gomboš cr = 152 mg.kg-1, 7 d after earthworms exposure (table 3). the results show possible to use of earthworms in the remediation of industrialized soils. after a longer period of 28 d of exposure (table 4) the earthworms’ mortality was much higher than after 7 d. the metal concentration values recorded were higher in the corresponding soil samples, (both pgvs and as), at simultaneous decrease of metal concentrations in the earthworms. further research is need about this topic, it can be hypothesized that chemical extractions like naoac would be useful to predict the potential bioaccumulation of certain metals considering the matrix physico-chemical properties. the values of the bafs/7-28d are < 1 for the studied metals and order in the following sequence: cr < fe < pb < cu < as, while are > 1 for zn > cd (tables 5 and 6). it is noteworthy that the baf values in the agricultural soils are greater on day 28 when compared to values on day 7 (tables 5 and 6). concentrations of zn and cd in the earthworm tissues were higher than that of the values cr < fe < pb < as < cu, indicating that these elements could be better bio-accumulated and migrated from the studied soils to the earthworm tissues. fig. 3. correlation between metal concentrations and mortality of earthworms after 7 d (a) and 28 d (b) exposure to soils from localities with permanent grass vegetation (pgv 1 – 7) and agricultural soils (as 1 – 5) (crm-6). a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:51 utc nova biotechnol chim (2019) 18(1): 10-17 16 table 5. bioaccumulation factors for metals in earthworm tissues fed in as (localities 1 ‒ 5) and pgv (localities 1 ‒ 7) (dw) in surrounding of košice after 7 d bioassays. locality fe cu zn pb cd cr as permanent grass vegetation soils (pgvs) 1 0.06 0.86 1.35 – 0.95 0.04 0.41 2 0.06 0.29 0.66 – 1.56 0.04 0.25 3 0.22 0.41 1.37 – 0.79 0.11 0.57 4 0.07 0.15 0.12 0.03 0.19 – 2.14 5 0.16 0.82 2.33 0.01 0.36 0.03 0.26 6 0.14 0.27 1.16 0,01 0.92 – 0.22 7 0.16 0.25 1.9 0,02 0.56 0.02 0.24 agricultural soils (as) 1 0.13 0.23 1.41 – 0.47 0.02 0.21 2 0.11 0.27 1.7 – 0.22 0.03 0.13 3 0.14 0.26 1.33 – 0.44 – 0.11 4 0.08 0.21 0.79 – 1.65 – 0.19 5 0.09 0.24 1.63 0.08 0.21 – 0.01 crm 0.03 0.05 0.18 0.01 0.07 – – previously, efficient regulation of zn uptake level has been demonstrated in some organisms by mechanism of either increased exclusion or decreased uptake from environmental media (heikens et al. 2001; demuynck et al. 2007; leitgib et al. 2007; tang et al. 2013). according to coelho et al. (2018) zn is probably the most likely metal that often limits earthworm populations. the study indicates that earthworms have efficient potential for bioaccumulation of metals in their tissues which can be used as an ecological indicator of soil contaminations. pearson matrix correlation coefficients can point on relation between toxicity to organisms and metal concentration in the soils (shuai et al. 2019). correlation between mortality of earthworms (ande) after 7 and 28 d exposure to metals in soils (respectively) were calculated (fig. 3a, b). the results show the possible positive correlation between mortalities of earthworms and fe concentrations after 7 d (r = 0.25 to 0.80 at individual localities). lack of correlation was identified for concentration of zn, as and pb, and the lowest values were detected in case of cd and cr after 7 d bioassays (fig. 3a). according to the correlation coefficients, the metal contamination level in the soils followed the sequence: of pgvs at localities (5) > (1) > (4) > (3) > (6) > (7) > (2) and as at localities (5) > (4) > (2) > (1) > (3). table 6. bioaccumulation factors for metals in earthworm tissues fed in as (localities 1 ‒ 5) and pgv (localities 1 ‒ 7) (dw) in surrounding of košice after 28 d bioassays. locality fe cu zn pb cd cr as permanent grass vegetation soils (pgvs) 1 0.02 0.61 1.53 0.06 0.77 0.03 0.58 2 0.03 0.45 0.75 0.05 1.20 0.03 0.37 3 0.03 0.42 1.37 0.05 0.98 0.04 0.68 4 0.00 0.19 0.15 0.01 0.10 0.03 0.10 5 0.09 0.46 3.3 0.09 0,29 0.04 0.82 6 0.04 0.35 1.19 0.08 0.65 0.02 0.57 7 – 0.30 1.6 0.03 0.30 0.01 0.30 agricultural soils (as) dw 1 0.15 0.49 1.42 0.08 1.10 0.09 1.11 2 0.09 0.39 1.9 0.10 0.29 0.12 0.75 3 0.14 0.17 1.32 0.07 0.51 0.04 0.90 4 0.07 0.27 0.82 0.08 1.90 0.10 0.36 5 0.12 0.33 1.64 0.09 0.34 0.07 0.80 crm 0.05 0.07 0.20 0.01 0.42 0.04 0.25 after 28 d exposure of earthworms to metals in soil samples (fig. 3b) we revealed possible positive correlation between earthworms mortality and metal concentrations in the order cd > cr > fe > zn > cu. the high concentrations of metals have no significant effect on the mortality of earthworms; our bioassays results exhibited high earthworms tolerance to contaminated soils probably because of distinct detoxification mechanisms developed. conclusions local industry located in the region influences the quality of the environment, including soils. the košice area, in addition to typical urban contamination sources is long-term environmental loaded by the iron and steel. the total concentrations of selected metals as well as ecotoxicological impact of industrialized soils from košice area were determined and revealed significant contamination with fe, cd, cr and as. for certain metals, significant concentration differences in time were recorded in bioassays for the soils sampled from both permanent grass vegetation (locality at u. s. steel-plant-main gate) and agricultural fields (u. s. steel-plant west and gomboš) suggesting that earthworms can remove part of polluting metal from samples. the bafs indicated positive correlation between bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:51 utc nova biotechnol chim (2019) 18(1): 10-17 17 toxicity and concentration of metals for zn > cd. correlation has been found between mortality and certain metals as well. the obtained results may provide a baseline data for soil (contamination) condition and information about ecosystem in urban regions subjected to emission of industrial contamination sources. acknowledgement the work was supported by the slovak grant agency for the vega project no. 2/0165/19. conflict of interest the authors declare that they have no conflict of interest. references coelho c, foret c, bazin c, leduc l, hammada m, inácio m, bedell jp (2018) bioavailability and bioaccumulation of heavy metals of several soils and sediments (from industrialized urban areas) for eisenia fetida. sci. total. environ. 635: 1317-1330. dai j, becquer t, rouiller jh, reversat g, bernhard-reversat f, nahmani j, lavelle p (2004) heavy metal accumulation by two earthworm species and its relationship to total and dtpa-extractable metals in soils. soil biol. biochem. 36, 91-98. das ks, chakrapani gj (2011) assessment of trace metal toxicity in soils of raniganj coalfield, india. environ. monit. assess. 177: 63-71. demuynck s, grumiaux f, mottier v, schikorski d, lemière s, leprête a (2007) cd/zn exposure interactions on metallothione in response in eisenia fetida (annelida, oligochaeta). comp. biochem. physiol. c. 145: 658-668. findoráková l, šestinová, o, kováčová m (2017) assessment of potential sediment contamination using screening methods (xrf, tga/ms) taking into account principles of green chemistry, eastern slovakia. environ. earth sci. 76: 119. hančuľák j, kurbel t, špaldon t, šestinová o, findoráková l, fedorová e (2015) development of atmospheric deposition on selected elements in the area of košice. in 19th conference on environment and mineral processing, ostrava, technical university of ostrava, czech republic, p. 135-140. heikens a, peijnenburg wjgm, hendriks aj (2001) bioaccumulation of heavy metals in terrestrial invertebrates. environ. pollut. 127: 335-341. kızılkaya r (2005) the role of different organic wastes on zinc bioaccumulation by earthworm lumbricus terrestris l. (oligochaeta) in successive zn added soil. ecol. eng. 25: 322-331. law no.220/2004, supp. 2, on the protection and use of agricultural land and on the amendment to act no. 245/2003 coll. on integrated prevention and control of environmental pollution, slovak republic. lingxiangyu l, zhenlan x, jianyang w, guangming t (2010) bioaccumulation of heavy metals in the earthworm eisenia fetida in relation to bioavailable metal concentrations in pig manure. bioresour. technol. 101: 3430-3436. leitgib l, kalman j, gruiz k (2007) comparison of bioassays by testing whole soil and their water extract from contaminated sites. chemosphere 66: 42-434. maity s, poráčová j, dey p, vašková j, vaško l, sedlák v, mydlárová blaščáková m (2018) antioxidant responses in the earthworm aporrectodea caliginosa of eastern slovakia: application of principal component analysis as a tool to identify metal contaminated areas. environ. monit. assess. 190: 21. morgan e, richards spg, morgan aj (2001) stable strontium accumulation by earthworms: a paradigm for radiostrontium interactions with its cation analogue, calcium. enviorn. toxicol. chem. 20: 1236-1243. oecd, test no. 317: bioaccumulation in terrestrial oligochaetes, organization for economic cooperation and development, 30 p. spurgeon dj, weels jm, van gestel cam (2003) a summary of eleven years progress in earthworm ecotoxicology. pedobiologia 47: 558-606. shuai f, chaoyang w, yuan x, lanhai l, daishe w (2019) heavy metals uptake and transport by native wild plants: implications for phytoremediation and restoration. environ. earth sci. 78: 103. suthara s, singh s, dhawanc s (2008) earthworms as bioindicator of metals (zn, fe, mn, cu, pb and cd) in soils: is metal bioaccumulation affected by their ecological category? ecol. eng. 32: 99-107. tang q, liu gj, zhang h, sun ry (2013) distribution of environmentally sensitive elements in residential soils near a coal-fired power plant: potential risks to ecology and children’s health. chemosphere 93: 2473-2479. vioalante a, cozzolino v, perelomov l, caporale ag, pigna m (2010) mobility and bioavailability of heavy metals and metalloids in soil environments. j. soil. sci. plant nutr. 10: 268-292. xu, p, wang, y, zhang, y, li, j, wang, h (2014) toxicity and bioaccumulation of ethofumesate enantiomers in earthworm eisenia fetida. chemosphere 112: 163169. yu d, li j, zhang y, wang h, guo b, zheng l (2012) enantioseletive bioaccumulation of tebuconazole in earthworm eisenia fetida. j. environ. sci. 24: 21982204. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:51 utc nova biotechnol chim (2019) 18(1): 37-43 doi: 10.2478/nbec-2019-0005  corresponding author: katarina.hrobonova@stuba.sk nova biotechnologica et chimica application of solid-phase extraction for isolation of coumarins from wine samples katarína hroboňová, andrea špačková and martina ondáková institute of analytical chemistry, faculty of chemical and food technology, slovak university of technology in bratislava, radlinského 9, sk-812 37 bratislava, slovak republic article info article history: received: 10th may 2019 accepted: 20th june 2019 keywords: hplc simple coumarins solid-phase extraction wine abstract coumarins can be as a result wine storage and aging in wood drums and they can affect organoleptic characteristics of wine. the aim of this work was to determine the content of coumarins in wine samples originated from slovak tokaj wine region. the hplc method with high specificity, accuracy, precision, and recovery was proposed. spe sorbents of c18 type, styrene-divinylbenzene copolymer and molecularly imprinted polymers were compared for extraction of six coumarins, coumarin, aesculin, scoparone, scopoletin, 4-methylumbelliferone, and herniarin. higher recoveries (above 89 %; except aesculin – recoveries higher from 68 %, rsds less than 6 %) were obtained with selective polymeric sorbent laboratory prepared by molecularly imprinted technology. the results showed that content of coumarins in wine samples are in ng.ml-1 concentration levels and depend on the age and wine puttony.  university of ss. cyril and methodius in trnava introduction coumarins, a class of compounds that contain a 1,2-benzopyrone skeleton, occur as secondary metabolites mainly originated from shikimic acid pathway. these compounds are widely distributed in the seeds, roots and leaves of many plants of apiaceae, rutaceae, asteraceae and fabaceae families. the source of coumarins (glycosylated or aglycone forms) in wine could be wood of drums in which it is matured. wood releases the coumarins and other compounds (e.g. phenolic acids) into the wine, which contribute to wine taste (e.g. glycosides are bitter, aglycones are acidic). the content of coumarins in wood changes through the degradation of the wood lignin during the heat treatment of the barrel and also depends on the type of the wood (ribéreau-gayon et al. 2006; de rosso et al. 2009; canas 2017). the main coumarins in wine are aesculetin (6,7-dihydroxycoumarin), aesculin (6,7-dihydroxy-coumarin-6glucoside), scopoletin (7-methoxy-6-hydroxycoumarin), and scopolin (scopoletin-7-glucoside). in some wines, 4-hydroxycoumarin and umbelliferone (7-hydroxycoumarin) were identified (liberatore et al. 2010; canas 2017). coumarins can be used for evaluation of wine, as a possible markers of storage of wines in wood barrels. in addition, the coumarins exhibit a wide range of biological effects, e.g. anti-inflammatory, antithrombotic, antimicrobial, antifungal, antiviral (including anti-hiv), anticonvulsant, antioxidant, and antitumor (kubrak et al. 2017). wine is very complex matrices, often components of very low concentrations are of interest (concentrations of ng.ml-1 for coumarins; salagoity-auguste et al. 1987). therefore, it is necessary to use sample preparation and analytical bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:01 utc mailto:katarina.hrobonova@stuba.sk nova biotechnol chim (2019) 18(1): 37-43 38 fig. 1. names, structural formulas and pka values of simple coumarins. methods for determination of coumarins that are characterised by high selectivity and sensitivity. sample preparation is an essential part of chemical analyses. the main goal of sample preparation for subsequent hplc analysis is removal of matrix interferences and to obtain sample aliquot or final analytes extract in solvent which is compatible with the hplc mobile phase used. sometimes, depending on detection sensitivity, pre-concentration of target analytes is needed. the most widely and routinely used sample preparation technique (clean-up and preconcentration) for the extraction of analytes from a complex matrix (including wine samples) is solid-phase extraction (spe). this technique is rapid, easy to perform, can be automated, and low amount of solvents is handled (compared with liquid-liquid extraction). a broad range of sorbents based on electrostatic, hydrophobic or polar interactions exists for spe. these traditional spe sorbents are widely available, well characterized and have high binding capacity, but only a few types (e.g. c18 type) are used in the majority of spe sample preparations, and they are the solvents in wash and/or elution steps which are varied according to the applications. sometimes their selectivity is limited and the target analyte often co-elutes with interfering compounds from matrix. there are several publications, which present applications of different sample preparation techniques in the wine analysis (lorrain et al. 2013), mainly for extraction of selected phenolic compounds. research in solid-phase extraction techniques is focused on development of sorption materials with increasing selectivity for target analytes. a novel type of spe sorbets are molecularly imprinted polymers (mips) ‒ synthetic tailor-made materials with a pre-defined selectivity for a target analyte or structurally related compounds based on specific interactions between analyte and binding sites on mip. this technology enables to reduce the presence of interferences during the extraction procedure and obtain higher recoveries even at low concentration levels. mips are obtained by polymerising functional and cross-linking monomers around a template molecule, which lead to a highly cross-linked three-dimensional network polymer. after polymerisation, the template molecule is extracted and binding sites having their shape, size, and functionalities complementary to the target analyte are established. the resulting imprinted polymers are stable, robust, and resistant to a wide range of ph, solvents, and temperature. the mip solid-phase extraction (mispe) was applied in wine analysis for determination of quercetin (molinelli et al. 2002), ochratoxin a (giovannoli et al. 2014), phthalates (barcielaalonso et al. 2017), organophosphorous herbicides (sanagi et al. 2011), catechin (büyüktuncel et al. 2018), umbelliferone (machyňáková et al 2017a). in this paper the content of coumarins in wine samples were determined. different spe sorbents, c18 bonded silica, styrene-divinylbenzene 7-hydroxycoumarin (umbelliferone) pka = 8.01 7-methoxycoumarin (herniarin) coumarin 7-methoxy-6hydroxycoumarin (scopoletin) 6,7-dimethoxycoumarin (scoparone) pka = 7.13 6,7-dihydroxycoumarin-6-βd-glucoside (aesculin) pka = 8.12 4-methyl-7-hydroxycoumarin (4-methylumbelliferone) pka = 7.80 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:01 utc nova biotechnol chim (2019) 18(1): 37-43 39 copolymers, and mip based sorbents have been tested and compared for extraction of simple coumarins (fig. 1) the most important natural coumarins. hplc method was used for determination of the extracted analytes. experimental chemicals and samples the standards of 7-hydroxycoumarin (99 %), 7-methoxycoumarin (98 %), 4-methyl-7-hydroxycoumarin (98 %), 6,7-dimethoxycoumarin (98 %), 7-methoxy-6-hydroxycoumarin (98 %), 6,7dihydroxycoumarin-6-β-d-glucoside (98 %), and coumarin (99 %) used for purpose of this study were purchased from sigma-aldrich (st. louis, usa). solvents for spe extraction and preparation of hplc mobile phases, methanol and ethanol of hplc gradient grade and acetic acid (99 %) were purchased from merck (germany). hplc grade water was prepared by aquamax ultra (series 370) water purification system. the tokaj wine (sample i: puttony 3 (2002), sample ii: puttony 4 (2004), sample iii: puttony 5 (2002), sample iv: puttony 6 (2003) was a product of slovak winery. preparation of standard solutions stock standard solutions of each compound (aesculin, scopoletin, coumarin, scoparone, 4-methylumbelliferone, herniarin,) were prepared in methanol/1 % acetic acid (20/80; v/v) to reach concentration of 0.1 mg.ml-1 and stored in freeze at –20 °c. the mixed working solutions were prepared by diluting the stock solutions obtaining lower concentrations. sample preparation the spe cartridge (c18 hydra (i), hr-x (ii), hrp (iii) (all cartridges purchased from chromabond, macherey-nagel, germany), affinimip phenolic (iv) (purchased from affinisep, france) and cartridge with 0.1 g of laboratory prepared mip-umbelliferone adsorbent) was preconditioned with 3 ml of methanol and subsequently with 3 ml of 12 % ethanol. the appropriate volume of sample (5 ml) was passed through cartridge followed by washing with 3 ml of water. finally, the analytes were eluted with 1 ml of methanol/acetic acid (9/1; v/v). extract was filtered through a 0.45 µm nylon membrane filter and used for the injection into hplc. the extraction procedure was repeated three times for each kind of tested sorbent material. for solidphase extraction the vacuum manifold was used. hplc analysis a hplc agilent technologies (series 1200) consisting of a binary pump, an autosampler, a column oven, a diode array detector, and fluorescence detector was used. hplc analyses were performed on a kinetex c18 (100 mm x 4.6 mm i.d., 5 m particle size) analytical column. the column was kept at 23 °c. an injection volume was 20 µl. the mobile phase consisted of methanol/acetic acid (99/1; v/v) (a) and 1 % aqueous solution of acetic acid (b) was mixed by the gradient program as follows, 0 ‒ 12 min linear gradient of a from 20 to 45 %, 12 ‒ 12.5 min linear gradient of a from 45 to 100 %, 12.5 ‒ 14.5 min 100 % of a, 14.5 ‒ 15 min a reverse gradient of a from 100 to 20 %, and 15 ‒ 19 min 20 % a. the flow rate was 1.0 ml min-1. the diode array detector was operated in the wavelength range of 190 ‒ 400 nm and chromatograms were recorded at 280 nm for coumarin and 330 nm for aesculin, scopoletin, scoparone, 4-methylumbelliferone, herniarin. fluorescence detector was set at the wavelengths 330 nm (ex) and 450 nm (em) and fluorescence spectra were scanned in the wavelength range of 340 ‒ 500 nm. results and discussion different types of spe sorbents were selected for purpose of this study, i) alkyl – bonded silica, ii) styrene-divinylbenzene copolymeric sorbents, and iii) selective mip based polymeric sorbents. in this work, all selected cartridges and laboratory prepared mip-umbelliferone cartridge were used in the same spe procedure with optimal extraction conditions included conditioning with methanol and 12 % ethanol, washing with water, and analyte eluting with methanol/acetic acid (9/1; v/v). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:01 utc nova biotechnol chim (2019) 18(1): 37-43 40 fig. 2. recovery values of coumarins on different spe sorbents. efficiencies of spe sorbents evaluated by recovery values were calculated and compared (fig. 2). coumarins under study represent less polar type of compounds and from this reason the reversed phase approach was selected for spe. reversed phase mode involves a polar or moderately polar sample matrix and a nonpolar adsorbent. octadecyl silica type of sorbent (i) is most frequently used for isolation of less polar compounds. the retention of analytes from polar matrices is due to the van der waals forces between the carbon-hydrogen bonds in the analyte and the functional groups on the silica surface. to elute a compound from adsorbent, a nonpolar solvent is used (the disruption of the forces analyte-packing adsorbent). the hydrophobic (ii) and high porous (iii) polymeric polystyrene-divinylbenzene sorbents were also used for purpose of the study. the advantage of these adsorbents is higher resistance in whole ph range, which leads to applicability of the spe methods for acidified samples to. they are suitable for extraction of compounds from complex, food, pharmaceutical or biological samples (ii) and from water samples (iii). the efficiency values of spe for sorbents i and ii are approximately the same and higher than 89 %, except aesculin, where sorption recovery was significantly lower (less than 64 %). the lover recoveries were obtained for some of compounds for adsorbent iii. the best extraction performance of adsorbent ii, in comparison with i and iii adsorbents, is related to aromatic functional groups and hydrophobic moieties of the sorbent which produce - and hydrophobic interactions for retention of analytes. mip sorbents, on contrary to the traditional spe sorbents (e.g. silica-based sorbents) are characterised by higher selectivity and specificity table 1. parameters of the mispe-hplc methoda. compound tr [min] regression equation concentration range lod loq aesculin 2.51 y = 131.1x – 3.3 3.0 – 100 1.0 3.0 r2 = 0.987 ng.ml-1 ng.ml-1 ng.ml-1 scopoletin 6.50 y = 1219.7x + 13.2 3.0 – 100 1.0 3.0 r2 = 0.998 ng.ml-1 ng.ml-1 ng.ml-1 coumarin 8.38 y = 3650.8x + 5.3 0.5 – 100 0.2 0.5 r2 = 0.995 µg.ml-1 µg.ml-1 µg.ml-1 scoparone 8.98 y = 82.8x + 1.3 10.0 – 100 3.0 10.0 r2 = 0.998 ng.ml-1 ng.ml-1 ng.ml-1 4-methylumbelliferone 9.45 y = 306.3x + 6.2 0.5 – 100 0.2 0.5 r2 = 0.997 ng.ml-1 ng.ml-1 ng.ml-1 herniarin 11.30 y = 25.5x – 0.2 12.0 – 100 4.0 12.0 r 2 = 0.998 ng.ml-1 ng.ml-1 ng.ml-1 a coumarin ‒ parameters for uv spectrophotometric detection; other coumarins – parameters for fl detection. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:01 utc nova biotechnol chim (2019) 18(1): 37-43 41 table 2. accuracy and precision of the mispe-hplc method a. compound recovery (%) ± sd c1 c2 c3 aesculin 68.7±0.8 70.9±0.9 72.1±0.9 scopoletin 91.4±3.2 94.7±3.8 96.0±3.3 coumarin 90.0±3.1 89.8±2.6 89.0±2.9 scoparone 94.9±1.1 91.3±1.1 95.4±1.4 4-methylumbelliferone 91.3±4.5 95.3±5.1 92.3±4.5 herniarin 95.2±2.3 97.3±2.3 92.3±2.0 a coumarin ‒ c1 1.0 µg.ml-1, c2 5.0 µg.ml-1, c3 50.0 µg.ml-1; other coumarins ‒ c1 0.01 µg.ml-1, c2 0.05 µg.ml-1, c3 0.1 µg.ml-1; n = 6. to target analyte, leading to effective elimination of matrix interferences. mip selectivity is related fig. 3. chromatograms of coumarins mixture (a; line a ‒ uv spectrophotometric detection, b ‒ fluorescence detection), wine sample iii extract without (b) and after (c) mispe extraction. the peaks indicate for aesculin ‒ 1, scopoletin ‒ 2, coumarin ‒ 3, scoparone ‒ 4, 4-methylumbelliferone ‒ 5, herniarin ‒ 6. to recognition cavities in polymeric matrices which are complementary to the target molecule in size, shape, and arrangement of the functional groups. two types of mips were tested for purpose of this study, mip selective for phenolic compounds (iv) and coumarins (v). although the sorbent iv was designed for phenolic compounds, the recovery values (higher than 88 %) indicated, that it was suitable for isolation of investigated coumarins. the lowest recovery of aesculin (about 50 %) results from incompatibility of mip imprinted cavity with shape and functionality of analyte. the laboratory synthesised mip (v) by imprinting of umbelliferone molecule was selective for template and also for other structural analoges (fig. 1) (machyňáková et al. 2017b). this sorbent shows complete retention of all investigated coumarins with highest values of recovery, 92 ‒ 98 %, compared with traditional and polymeric sorbents i-iv. the advantage of mip based sorbents is alongside increased selectivity also reusability. sorbent can be reused up to fifty times without losing extraction capacity. the real sample of wines were selected in order to demonstrate the applicability for the mispe method. parameters including lod, loq, linear range, correlation coefficient, precision and accuracy were examined to validate the developed mispe method coupled with hplc-dad/fld. by using hplc method, acceptable asymmetry (as ~ 0.9), good peaks resolution (rs  2.1), and analysis time lower than 15 min were obtained (fig. 3a). the rsds repeatability of retention times varied from 0.2 to 0.5 %. lods and loqs (table 1) were calculated using 3 sa and 10 sa criteria (sa is the standard deviation of the intercept of calibration curve). the calibration curves were linear in working ranges from loq to 100 µg.ml-1 a b c bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:01 utc nova biotechnol chim (2019) 18(1): 37-43 42 for coumarin detected with uv detection and in the loq – 100 ng.ml-1 concentration range for other coumarins detected with fluorescence detector. the precision and accuracy (expressed as the recovery) of the developed method was determined for spiked sample at three concentration levels of each analyte (table 2). the intra-day precision expressed as rsd (%) ranged from 1.2 to 5.4 %. the obtained recoveries ranged from 68.7 to 96.0 %. two of coumarins, scopoletin and 4-methylumbelliferone, were determined in wine samples using mispe-hplc method in ng.ml-1 concentration levels (sample i: 5.40.3 ng.ml-1 of scopoletin, sample ii: 9.20.1 ng.ml-1 of scopoletin, sample iii: 10.10.5 ng.ml-1 of scopoletin and 12.00.3 ng.ml-1 of 4-methylumbelliferone, sample iv: 17.20.3 ng.ml-1 of scopoletin). chromatograms of wine extracts (fig. 3) documented that mispe was suitable for extraction and pre-concentration of coumarins from wine samples. the results showed that content of coumarins in wine samples depended on the age and wine puttony. conclusions in this study, the solid-phase extraction with different types of sorbents, c18-silica, styrenedivinylbenzene copolymeric sorbents, and selective mip based sorbents were compared for isolation and pre-concentration of six coumarins. mips displayed higher selectivity and reusability. the obtained recoveries for target analytes ranged from 68.7 to 96.0 %. the results obtained demonstrated the appropriateness of solid-phase extraction with mip-umbelliferone sorbent in wine analysis. developed mispe coupled with hplc method was applicable for determination of simple coumarins in real samples and exhibited good specificity, accuracy, precision and recovery. the results showed that content of coumarins in wine samples are in ng.ml-1 concentration levels and depend on the age and wine puttony. for this purpose, further research on identification and quantification of coumarins is needed. alternatively, this study could provide a promising method for the selective separation and determination of coumarins in other samples of beverages and sample extracts too. acknowledgements this work was supported by the slovak research and development agency (grant no. apvv-15-0355). conflict of interest the authors declare that they have no conflict of interest. references barciela-alonso mc, otero-lavandeira n, bermejo-barrera p (2017) solid phase extraction using molecular imprinted polymers for phthalate determination in water and wine samples by hplc-esi-ms. microchem. j. 132: 233-237. büyüktuncel e, porgali e, özkara s (2018) catechinmolecularly imprinted cryogel for determination of catechin in red wines by hplc-dad–fluorescence detector. acta chromatogr. 30: 54-61. canas s. (2017) phenolic compounds and related properties of aged wine spirits: influence of barrel characteristics. a review. beverages 3: article number: 55. de rosso m, panighel a, dalla vedova a, stella l, flamini r (2009) changes in chemical composition of a red wine aged in acacia, cherry, chestnut, mulberry, and oak wood barrels. j. agric. food chem. 57: 1915-1920. giovannoli c, passini c, di nardo f, anfossi l, baggiani c (2014) determination of ochratoxin a in italian red wines by molecularly imprinted solid-phase extraction and hplc analysis. j. agric. food chem. 62: 52205225. kubrak t, podgórski r, stompor m (2017) natural and synthetic coumarins and their pharmacological activity. eur. j. clin. exp. med. 15: 169-175. liberatore mt, pati s, del nobile ma, la notte e (2010) aroma quality improvement of chardonnay white wine by fermentation and ageing in barrique on lees. food res. int. 43: 996-1002. lorrain b, ky k, pechamat l, teissedre pl (2013) evolution of analysis of polyhenols from grapes, wines, and extracts. molecules 18: 1076-1100. machyňáková a, lhotská i, hroboňová k, šatínský d (2017a) on-line coupling of molecularly imprinted solid-phase extraction with liquid chromatography for the fast determination of coumarins from complex samples. j. pharm. biomed. anal. 145: 144-150. machyňáková a, hroboňová k (2017b) synthesis and evaluation of molecularly imprinted polymers as sorbents for selective extraction of coumarins. chromatographia 80: 1015-1024. molinelli a, weiss r, mizaikoff b (2002) advanced solidphase extraction using molecularly imprinted polymers for the determination of quercetin in red wine. j. agric. food chem. 50: 1804-1808. ribéreau-gayon p, glories y, maujean a, dubourdieu d (2006) handbook of enology, volume 2: the chemistry bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:01 utc nova biotechnol chim (2019) 18(1): 37-43 43 of wine – stabilization and treatment, 2nd ed., john wiley & sons, ltd, chichester, p. 143-144. salagoity-auguste mh, tricard c, sudral p (1987) simultaneous determination of aromatic aldehydes and coumarins by high-preform liquid chromatography. application to wines and brandies stored in oak barrels. j. chromatogr. 392: 379-387. sanagi mm, salleh s, ibrahim waw, naim aa (2011) determination of organophosporus pesticides using molecular imprinted polymer solid-phase extraction. malaysian j. anal. sci. 15: 175-183. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:01 utc nova biotechnol chim (2020) 19(1): 61-69 doi 10.36547/nbc.v19i1.578  corresponding author: akmal84a@gmail.com nova biotechnologica et chimica comparative chemical composition of seeds of amaranth varieties introduced in uzbekistan shuhratjon s. olimjonov1, jamolitdin f. ziyavitdinov1, soyibjon s. bozorov1,2, uchqun j. ishimov1, nodir sh. berdiev1, nadjiya n. abrekova1, muydinjon m. muminov3 and akmal m. asrorov1,4, 1laboratory of proteins and peptides chemistry, institute of bioorganic chemistry, academy of sciences of uzbekistan, 83, m. ulughbek str., tashkent 100125, uzbekistan 2laboratory of physical and chemical methods of research, center for advanced technologies of ministry of innovative development of uzbekistan, 3a, talabalar shaharchasi, tashkent 100125, uzbekistan 3andijan experimental exhibition area functioning at the disposal of the scientific and practical centre for implementation of innovative projects, ministry of innovative development of uzbekistan, bobur massif, andijan region, 170800, uzbekistan 4state key laboratory of drug research, shanghai institute of materia medica, chinese academy of sciences, 501 haike road, shanghai 201203, china article info article history: received: 4th march 2020 accepted: 21st may 2020 keywords: amaranth varieties free amino acids oiliness saccharides total amino acids vitamins abstract amaranth is one of ancient cultured plants possessing valuable food quality that is still important in modern agriculture. in this work, we studied the seed chemical compositions of four plant varieties of amaranthus cruentus and amaranthus hypochondriacus species, acclimatized in uzbekistan. quantity of free amino acids, amino acids compositions of proteins, vitamins, oligoand polysaccharides were established using chemical and physical methods. our results on protein content suggest that introduction of these varieties into eco-climate of andijan region, uzbekistan, was not very favorable, though proteins resembled up to 9.4 – 13.4 % (w/w) of seed biomass. of the water-soluble vitamins tested, the vitamin b1 was most abundant (0.81 – 1.14 µg.g-1 of seed) with significant differences among the acclimatized varieties. impact of feeding rats with seeds flour of studied helios variety was established. blood levels of triglycerides, high-, lowand very lowdensity lipoproteins, catalase activity under hyperlipidemia were analyzed. we found helios variety seed flour, possibly because of high saccharides content, significantly lowered the levels of total cholesterol (-26 %) and low-density lipoproteins (-21 %). the acclimatized varieties were identified as potentially valuable food source possessing antihyperlipidemic property.  university of ss. cyril and methodius in trnava introduction the increasing number of world population necessitates to use plant resources, having nutritive quality, more and efficiently. in this regard, amaranth plant seed is a suitable raw material providing high nutrition value. the plant genus belonging to amaranthacea family contains around 70 species (berghofer and schoenlechner 2002). the species amaranthus hypochondriacus, amaranthus cruentus and amaranthus caudatus are cultivated for their seeds in latin american countries (bressani 2003). it is grown in india for both leaves and seeds (prakash and pal 1991). mailto:akmal84a@gmail.com nova biotechnol chim (2020) 19(1): 61-69 62 slovakia, hungary and italy are the european producers of amaranth. approximately 100 000 ha in russia is expected as amaranth growing area (moudry et al. 1999). four new varieties belonging to a. hypochondriacus (kharkov and lera) and a. cruentus (andijan and helios) species have been recently acclimatized in uzbekistan (bozorov et al. 2018). amaranth seed has been widely investigated. updated reviews on its chemical compositions have shown its high nutritive quality: oils, fatty acids, proteins and peptides, free amino acids, squalene, tocopherols and tocotrienols, sterols and other lipophilic compounds, carbohydrates, dietary fiber and other constituents (singh and singh 2011; venskutonis and kraujalis 2013). these papers report highly differing amounts of compounds in various amaranth varieties. therefore, in terms of the same growing conditions, studying comparative chemical compositions of different amaranth varieties was considered significant in terms of application in bakery processes. acclimatization of amaranth started in uzbekistan a decade ago. in previous work, we demonstrated oil composition of four amaranth varieties, introduced in the country (bozorov et al. 2018). in this work, we compared the quantity of water-soluble, acidic and basic polysaccharides, free and bound amino acids, some major monosaccharides as well as watersoluble vitamins. besides, we studied the effects of amaranth seed flour on lipid profile of rats. experimental chemicals and reagents all the used chemicals and reagents were of analytical grade. for hplc analysis, chemical from sigma were used. total nitrogen total nitrogen in 1 g defatted seed flour was determined by modified kjeldahl method. ammonium salts formed in boiling sulfuric acid and further ammonium was quantified titrimetrically. extraction and quantification of polysaccharides the flour samples were ground in a laboratory grinder to a size of 2 to 6 mm. to remove low molecular weight impurities and proteins, 100 grams of raw material was extracted in a soxhlet apparatus with 95 % ethyl chloroform-alcohol mixture. to isolate water-soluble polysaccharides, fat-freed flour samples were extracted with boiling water under reflux condenser, for two hours, twice. obtained extracts were filtered and concentrated. we, further, extracted pectin fractions with 1.1 % hydrochloric acid (1:10, w/v) using reflux condenser in water bath at 95 °с, for 2 hours. the extracts were filtered, concentrated and further was dialyzed against distilled water overnight, and centrifuged (10 min; 6,000 rpm). the supernatant was used for quantification. we carried alkaline extraction using 5 % naoh solution (1:10, w/v) at 60 °c in oil bath, for 1 hour. the extracts were filtered, centrifuged (10 min; 6,000 rpm) and neutralized with acetic acid. crude extracts were concentrated at 50 °c and dialyzed against distilled water overnight. freeze-dried extracts were quantified. the concentrations of polysaccharides in obtained fractions were determined by phenol-vitriolic method (saha and brewer 1994). protein quantification quantity of soluble proteins was determined by lowry method (lowry et al. 1951). extraction of amino acids and their modification one gram seed samples were extracted in 3 ml water for 4 h stirring. the supernatant was isolated after centrifugation for 10 min (3,000 rpm). higher molecular compounds were precipitated adding 10 % trichloroacetic acid (1:1, v/v) for 15 min (8,000 rpm). 200 µl aliquot was taken and lyophilized. dry extract was dissolved in 200 µl water acetonitrile-triethylamine (1:7:1) then the extract was dried. the process was twice repeated. in order to obtain phenylthiocarbamoyl (ftc) derivatives purified samples and standard amino acid were modified with water-ethanol nova biotechnol chim (2020) 19(1): 61-69 63 fig. 1. hplc chromatogram for vitamins of seeds of helios variety at 290 nm. triethylamine-phenylisothiocyanate (1:7:1:1) for 30 min (cohen and strydom 1988). preparation of total amino acid samples ground seed samples (1 g each) were hydrolyzed in 1 ml hcl (5.7 m) and concentrated trifluoroacetic acid mixture (2:1). the ampules were sealed in vacuum. the reaction was carried at 166 °c for 70 min. further the hydrolysates were freeze-dried. further ftc derivatives were obtained as described above. hplc analysis of amino acids quantity analyses of ftc derivatives were conducted in agilent technologies 1200 (column: supelco discovery hs c18, cat 56925/-u 7.5 cm / 4.6 mm, 3 µm), with buffer: b – 0.14 m ch3coona + 0.05 % tetraethyl ammonium, ph 6.4, a–mecn, gradient % b/min, 0 – 6 %, 5 min, 6 – 30 %, 30 min, 30 – 60 % 5 min, 60 – 100 % 5 min. amino acid derivatives were detected with a flow rate 1.2 ml.min-1 at 269 nm. rsd values for the mean values of amino acids’ quantity, were used for statistical evaluation. a representative chromatogram from the extract of helios seeds is depicted in fig. 1, indicating the adequacy of the method. analysis of vitamins vitamins were extracted with ten-fold 40 % ethanol. the process was carried boiling, using reflux condenser for an hour. further, the process lasted for 2 h at room temperature stirring. the extract was taken out. seed samples were extracted with five-fold volume of 40 % ethanol twice more. the sum of extracts were centrifuged (7,000 rpm) and used for hplc analysis. the vitamins were identified and quantified by hplc (agilent-1200, reverse phase eclipse xdb c18 column, 5µm, 4.6 x 150mm) at 254 and 290 nm, diode-array detector. the flow rate made 1 ml.min-1, a: 0.5 % acetic acid (ph 1.7); b: acetonitrile; the gradient of acetate buffer and acetonitrile (6 – 8 min 90:10, 9 – 15 min 80: 20, 15 – 17 min 96:4) at 25 °c, 5 µl of aliquot was used for analysis. we identified and quantified vitamins by calibration curves that were created by using standards of vitamins. animal experiments animal experiments were carried in compliance with requirements of pharmaceutical agency of uzbekistan developed in accordance with standards of research involving animals. we carried animal experiments on 50 white outbred male rats with a body weight 250 – 300 g in the vivarium of the institute of bioorganic chemistry. animals were kept in the standard condition with a balanced diet. on the sixth day of the treatment the blood samples of the animals were used for the quantification of total cholesterol (tc), triglycerides, low-density lipoproteins ldl, high density lipoproteins (hdl), very low-density lipoproteins (vldl) and catalase activity. these parameters were calculated by friedewald equation according to state standards. the atherogenic coefficient was calculated using the following formula: ac = (total cholesterol – hdl) / hdl. nova biotechnol chim (2020) 19(1): 61-69 64 table 1. total and remaining oiliness of see of amaranth varieties (% in relation to total mass). species amaranth varieties seed yield [t.ha-1] total oiliness cold compressed remaining oiliness after cold compression a. hypochondriacus kharkov 3 – 5 t 11.86±0.32 7.81±0.17 4.05±0.15 lera 1.5 – 2 t 11.20±0.40 7.55±0.23 3.65±0.17 a. cruentus andijan 1 – 1.5 t 10.07±0.14 6.39±0.06 3.68±0.08 helios 2 – 2.5 t 11.87±0.34 7.68±0.16 4.19±0.18 results and discussion obtained results show that oiliness of these amaranth seed varieties range from 10.07 to 11.87 % (v/w). during cold compression almost two third of the oil was found extracted (65.9, 67.4, 63.4 and 64.7 % respectively, v/w). the highest oiliness values were determined in kharkov and helios varieties. remaining oiliness was established by extracting with petrol (table 1). total nitrogen in amaranth seeds was established between 15.7 – 17.5 % (w/w). there is a lack of significant differences in total nitrogen in kharkov, lera and helios seeds. andijan variety sample has the lowest total nitrogen, equaled 15.7 % (w/w) in relation to defatted seeds. soluble proteins’ quantity found reversely correlated with total nitrogen; the highest quantity belonged to andijan variety that was equal to 5.1 % (w/w). kharkov and lera seeds contained 47.6 – 47.8 mg soluble proteins per one g defatted seeds (table 2). we found glucose as the major monosaccharide among the studied samples with inconsiderable differences. around 5 mg glucose was calculated per 100 mg amaranth seed flour samples. with no significant differences, fructose and mannose were determined as minor monosaccharides in all four samples. water-soluble polysaccharides made 22 – 24 % (w/w) of the seed biomass. identical mass ratios of acidic and alkali-soluble polysaccharides (1:3) were observed in lera, andijan and helios varieties. unlikely, kharkov seed flour contained higher level of acidic carbohydrates. thus, we established identical content of total carbohydrates of these four seed samples (table 2). our results indicate that, more than two third of the free amino acids in the seed composition belong to nonessential ones. aspartic acid, glutamic acid and glutamine were determined to be major amino acids among all. nonessential free amino acids in amaranthus hypochondriacus varieties were found significantly higher than the varieties of amaranthus cruentus species (table 3). we found, that histidine was the most widespread essential amino acid in the seeds of these studied amaranth varieties that its quantity is at least twice higher than valine, isoleucine, leucine and tryptophan prevailing remaining essential amino acids. differing from nonessential amino acids, species-dependent distinctions were not found among these varieties. however, andijan variety of a. cruentus contained remarkably lower quantities of valine, methionine, tryptophan table 2. weight fractions (%) of saccharides and proteins in amaranth seeds, acclimatized in uzbekistan. a. hypochondriacus a. cruentus kharkov lera andijan helios glucose 5.24±0.08 5.03±0.06 4.98±0.05 5.22±0.07 fructose 0.41±0.02 0.39±0.03 0.38±0.02 0.40±0.01 sucrose 0.23±0.01 0.21±0.01 0.22±0.01 0.23±0.01 sum 5.88 5.63 5.58 5.85 soluble polysaccharides 23.90±0.98 23.70±1.04 22.10±0.99 23.50±1.01 acidic polysaccharides 19.60±0.88 8.40±0.34 8.20±0.34 8.40±0.34 alkali-soluble polysaccharides 11.70±0.55 22.10±0.99 24.80±1.16 25.30±1.11 sum 55.2 54.20 55.10 57.20 total saccharides 61.08 59.83 60.68 63.05 total nitrogen 17.30±0.5 17.50±0.3 15.70±0.70 17.10±0.60 soluble proteins 4.76±0.02 4.78±0.05 5.10±0.02 5.00±0.01 nova biotechnol chim (2020) 19(1): 61-69 65 table 3. contents of free amino acids in the seeds of amaranth varieties (mg.g-1 dry mass, rsd ≤ 3 %). no. amino acids a. hypochondriacus a. cruentus kharkov lera andijan helios nonessential amino acids 1 aspartic acid 0.332 0.487 0.326 0.312 2 glutamic acid 0.617 0.611 0.203 0.498 3 serine 0.132 0.067 0.045 0.170 4 glycine 0.061 0.083 0.060 0.073 5 asparagine 0.062 0.083 0.059 0.074 6 glutamine 0.307 0.414 0.095 0.213 7 proline 0.040 0.044 0.042 0.041 8 tyrosine 0.084 0.101 0.051 0.079 9 alanine 0.160 0.104 0.089 0.167 sum 1.795 1.994 0.970 1.627 essential amino acids 10 threonine 0.048 0.036 0.033 0.067 11 arginine 0.057 0.068 0.066 0.054 12 valine 0.078 0.088 0.056 0.081 13 methionine 0.040 0.047 0.024 0.040 14 isoleucine 0.090 0.093 0.084 0.088 15 leucine 0.088 0.090 0.089 0.090 16 histidine 0.177 0.186 0.167 0.169 17 tryptophan 0.071 0.082 0.053 0.076 18 phenylalanine 0.039 0.053 0.042 0.046 19 lysine 0.060 0.087 0.041 0.068 sum 0.748 0.830 0.655 0.779 total 2.543 2.824 1.625 2.406 amino acids, the concentration of which are significantly higher than others, are given in bold. and lysine (table 3). the sum amounts of free amino acids were determined to make 0.16 – 0.28 % (w/w) of the seed. total amino acids concentrations were calculated as around 11.4 ± 2 % (w/w) of the seed flour (table 4). these results make enable to conclude that the seeds of these introduced amaranth varieties is a rich protein source but not of free amino acids. total amino acids contents studied in our work revealed that glutamic acid/glutamine sum were major nonessential amino acids. during the hydrolysis, glutamine and asparagine convert their acidic forms. thus, the sum of glutamic acid and glutamine, aspartic acid and asparagine are given together. at least twice higher concentrations of glutamine and glutamic acid were determined compared to other nonessential amino acids. among other nonessential amino acids, no notable differences were found, except for proline (table 4). phenylalanine was found the most widespread amino acid in the amaranth seeds. we determined thrice higher its quantity than other prevailing amino acids in kharkov, lera and andijan varieties. the lowest total amino acids belonged to helios variety. notably lower quantity of alanine, proline, arginine, methionine and phenylalanine were defined in this sample compared to other varieties (table 4). these results indicate that, studied amaranth seeds are a rich source of proteins with 9.4 – 13.4 % (w/w) related to seed mass. our results demonstrate similar amino acid ratios in these four varieties. however, highly different molar ratios of bound amino acids were reviewed in amaranth seeds (venskutonis and kraujalis 2013). thus, in terms of nutritious quality of seeds, it is possible to expect various molar ratios in different species and varieties. for instance, arginine and lysine were of the highest quality in a. cruentus (escudero et al. 2004) and a. hypochondriacus (dodok et al. 1997), whereas they were detected as low-content amino acids compared to others in another a. hypochondriacus variety (nimbalkar et al. 2012). the threonine molar content was nova biotechnol chim (2020) 19(1): 61-69 66 table 4. total amino acid contents in the seeds of amaranth varieties (mg.g-1 dry mass, rsd ≤ 3 %). no. amino acids a. hypochondriacus a. cruentus kharkov lera andijan helios nonessential amino acids 1 aspartic acid/asparagine 10.24 10.09 7.96 7.21 2 glutamic acid/glutamine 27.16 26.26 18.04 16.15 3 serine 4.80 4.49 3.53 3.68 4 glycine 5.62 5.44 4.40 4.00 5 tyrosine 5.53 4.43 6.75 5.96 6 alanine 4.97 5.11 1.84 3.58 7 proline 3.98 3.98 3.43 2.97 sum 62.30 59.80 45.95 43.55 essential amino acids 8. threonine 5.53 4.43 6.75 5.96 9. arginine 6.82 7.63 4.48 4.62 10. valine 5.09 4.87 6.08 5.57 11. methionine 3.11 2.22 1.98 1.33 12. isoleucine 7.23 6.49 6.28 5.51 13. leucine 7.79 7.02 6.93 6.26 14. histidine 5.20 5.20 6.00 7.35 15. phenylalanine 23.69 19.11 18.41 8.17 16. lysine 7.15 6.26 6.03 5.70 sum 71.61 63.23 62.94 50.47 total 133.91 123.03 108.89 94.02 amino acids, the concentration of which are remarkably higher than others, are given in bold. reverse in these three amaranth varieties. our results revealed that b1 and b2 vitamins are the main vital components in the seed of all varieties with almost no statistical differences except for vitamin b1 in lera variety. vitamin b1 was determined in the range of 0.81 – 1.14 µg.g-1. 0.33 – 0.38 µg vitamin b2 was observed in 1 g of amaranth seeds. b5, b6, b9 and pp vitamins were demonstrated as minor vitamin components in the seeds with statistical differences with the exception for b9. we found that among the studied components, vitamin pp was the most minor one in kharkov and lera varieties belonging to a. hypochondriacus, and andijan and helios varieties of a. cruentus contained the smallest amount b5 (fig. 2). data on vitamin contents in amaranth do not correspond with the obtained results in this work. several times higher contents of vitamin b2, b5 and b6 were reported in amaranth seeds. similarity belonged to b1 only (pavlík 2012). vitamin b2 and vitamin b6 were determined as the main vitamins raw amaranth seeds, that equaled 1.47 and 4.54 mg.g-1, respectively (murakami et al. 2014). vitamin b1 (thiamine), b2 (riboflavin) and b6 (pyridoxine) were found in similar quantities in amaranth seeds grown in kenya, that ranged 5 – 6 mg.g-1 (mburu et al. 2012). mihhalevski et al. (2013) demonstrated contents of vitamins in rye and wheat seeds and flours. vitamin b1 content in red rye malt variety was similar to values we measured in our samples. its content in other rye flours was defined a few-fold times higher (differing from 1.85 to 3.76 µg.g-1). the contents of b5, b6 and pp vitamins were several-fold higher in rye varieties than our findings. thus, we established that amaranth flour concedes rye and wheat flour by its vitamins contents. several-fold differences of our vitamin quantity data from these results could be linked to acclimatization in hot climate that may not be favorable for the plant. further research will require to compare annual results. a. hypochondriacus is considered to be the hybrid of a wild type a. powelli and progenitor species – a. cruentus (brenner et al. 2010). possibly, due to this relationship, previous research shows no remarkable differences in protein and/or carbohydrate contents (table 5). however, in acclimatized varieties, we observed differences in protein percentage. besides, obtained results in this work revealed significantly lower percentage of proteins in the seeds compared to other varieties nova biotechnol chim (2020) 19(1): 61-69 67 fig. 2. water-soluble vitamins contents in acclimatized amaranth varieties. data indicate average value of 3 replicates ± rsd. asterisks indicate significant difference among varieties at p < 0.05. in both species (table 5). possibly, it could be resulted by drier season in uzbekistan. for instance, the lera variety created by individual selection from a. hypochondriacus was reported to contain 20.6 % in the seed (goptsiy et al. 2008). it could be concluded that drier and hotter climate would not be a favorable condition for amaranth in terms of protein. total carbohydrates of these four varieties were determined to be average compared with available data: 60 – 63 % (w/w) of the seed mass belonged to saccharides (table 5). the seed yield in kharkov and helios make 2.0 – 2.5 and 3 – 5 tons per hectare, respectively. therefore, these varieties have been grown for the seed. andijan and lera varieties seed yield make 1 – 1.5 and 1.5 – 2.0 ton per hectare. they were chosen for their aboveground biomass (table 1). we further studied the pharmacologic effects of amaranth seed flour (helios) on lipids level in experimental rats (table 6). obtained results in twin model was accompanied with significantly table 5. comparison of proteins and carbohydrates content in a. cruentus and a. hypochondriacus seeds. variety protein % (w/w) carbohydrate % (w/w) reference a. cruentus 16.21 58.00 capriles 2008 a. cruentus (82s-1011) 14.70 bressani et al. 1987 a. cruentus (823434) 15.07 bressani et al. 1987 a. cruentus (82s-1034) 16.00 bressani et al. 1987 a. cruentus 15.68 55.41 babor et al. 1994 a. cruentus 16.89 66.09 uriyapongson and rayas-duarte 1994 a. hypochondriacus hyb. 14.02 68.23 uriyapongson and rayas-duarte 1994 a. hypochondriacus 14.23 tömösközi et al 2009 a. hypochondriacus (a-718) 13.70 bressani et al. 1987 a. hypochondriacus (a-720) 14.75 bressani et al. 1987 a. hypochondriacus (82s-1023) 15.32 bressani et al. 1987 a. hypochondriacus (82s-1024) 14.75 bressani et al. 1987 a. cruentus, andijan 10.90 60.68 this work a. cruentus, helios 9.40 63.05 this work a. hypochondriacus, kharkov 13.40 61.08 this work a. hypochondriacus, lera 12.30 59.83 this work nova biotechnol chim (2020) 19(1): 61-69 68 table 6. effects of helios seed flour on lipid profile of rats carried in twin model (n = 10). groups total cholesterol [mg.l-1] triglycerides [mg.l-1] high density lipoproteins [mg.l-1] low density lipoproteins [mg.l-1] very low density lipoproteins [mg.l-1] catalase activity [mcat.l-1] intact animals 5.20±0.03 6.00±0.06 3.10±0.05 1.20±0.04 1.10±0.03 0.079±0.002 control (hyperlipidemia + н 2 о) 8.75±0.03 +68 % 10.60±0.04 +77 % 1.60±0.05 -48 % 3.15±0.03 +74 % 2.12±0.05 +91 % 0.447±0.030 >5,6-times hyperlipidemia + amaranth flour (helios), 200 mg.kg-1 6.50±0.03 -26 %* 9.20±0.06 -13 %* 2.00±0.05 +25 * 2.49±0.04 -21 * 1.84±0.03 -13 %* 0.24±0.01 <1.9-times * stands for significant differences of the indicated parameters compared to control samples. decreased levels of total cholesterol (-26 %), triglycerides (-13 %), low density (-21 %) and very low density lipoproteins (-13 %) in comparison with hyperlipidemia model. almost twice lower catalase activity was determined in animal fed with amaranth flour compared with control. these deviations in the lipoproteins quantity indicate the positive effect of the flour. a directly proportional solid link between elevated cholesterol/ldl and coronary heart disease (nelson 2013) should serve as basis to conclude that the amaranth seed flour can be used as antihyperlipidemic nutrition (table 6). hdl, increased by 25 %, can also be estimated as positive point. more recent literature review, based on genetic analysis and clinical research, conclude that higher hdl are always beneficial for coronary heart diseases, while their lower level is always detrimental (kosmas et al. 2018). conclusions in this work we compared the chemical compositions of the seed flour of four amaranth varieties belonging to a. hypochondriacus and a. cruentus series, acclimatized in uzbekistan. it has been established that, total amino acids make 9.4 – 13.4 % (w/w) of the seed flour, more than of half of which belong to essential ones. phenylalanine, glutamic acid and glutamine were determined as major bound amino acids. dry and hot climate of uzbekistan caused total protein content to be significantly lower, compared to progenitors. contents of water-soluble vitamins in the seeds of amaranth varieties have been established. we found vitamin b1 and b3 as major compositions of this class of compounds in all of these varieties. total carbohydrates quantities were determined not to significantly differ. the detailed study of the chemical composition amaranth seeds, determining nutrition value, revealed that it can be used as rich carbohydrate source. we demonstrated important biological value of amaranth flour with antihyperlipidemic property. acknowledgement the work was supported with innovative project i-2016-5-16/3 “introduction of technology for complex processing of amaranth plants to produce feed for livestock, and also oil cake and flour for the needs of the pharmaceutical and food industry” by academy of sciences of uzbekistan. the authors express sincere thanks to researchers from andijan state university for providing amaranth seeds of the investigated varieties. conflict of interest the authors declare that they have no conflict of interest. references babor k, halásová g, dodok l, géciová r, lokaj j (1994) characterization of starch from amaranthus cruentus l. chem pap. 48: 58-63. berghofer e, schoenlechner r (2002) grain amaranth. in belton ps, taylo jrn (eds.), pseudocereals and less common cereals. grain properties and utilization potential, springer-verlag, berlin, heidelberg, pp. 219253. bozorov ss, berdiev nsh, ishimov uj, olimjonov shs ziyavitdinov jf, asrorov am, salikhov shi (2018) chemical composition and biological activity of seed oil of amaranth varieties. nova biotechnol. chim. 17: 66-73. brenner dm, baltensperger dd, kulakow pa, lehmann jw, myers rl, slabbert mm (2010). genetic resources and breeding of amaranthus. in janick j (ed.), plant nova biotechnol chim (2020) 19(1): 61-69 69 breeding reviews, wiley-blackwell, new jersey, usa, pp. 227-285. bressani r, gonzhles jm, zuiiga j, breuner m, elias lg (1987) yield, selected chemical composition and nutritive value of 14 selections of amaranth grain representing four species. j. sci. food agric. 38: 347-356. bressani r (2003) amaranth. in caballero b. (ed.), encyclopedia of food sciences and nutrition, elsevier/academic press, oxford, pp. 66-173. capriles vd, coelho kd, guerra-matias ac, areas jag (2008) effects of processing methods on amaranth starch digestibility and predicted glycemic index. j. food sci. 73, h160-4. cohen sa, strydom dj (1988) amino acid analysis utilizing phenylisothiocyanate derivatives. anal. biochem. 174: 116. dodok l, modhir aa, buchtová v, halásová g, poláček i (1997) importance and utilization of amaranth in the food industry. part 2. composition of amino acids and fatty acids. nahrung 41: 108-110. escudero nl, de arellano ml, luco jm, gimenez ms, mucciarelli si (2004) comparison of the chemical composition and nutritional value of amaranthus cruentus flour and its protein concentrate. plant food hum. nutr. 59: 15-21. goptsiy t. voroncov n, popov v, zhyravel d, gromenko s (2008). grain varieties of amaranth developed by selection at kharkiv national agrarian university and the perspectives of their use. in “amaranth – plant for the future” 5th international symposium of the european amaranth association nitra, slovak republic. 2008, pp: 97-100 kosmas ce, martinez i, sourlas a, bouza kv, campos fn, torres v, montan pd, guzman e (2018) high-density lipoprotein (hdl) functionality and its relevance to atherosclerotic cardiovascular disease. drugs context. 7: 212525. lowry oh, rosenbrough nj, farr al, randall rj (1951) protein measurement with the folin phenol reagent. j. biol. chem. 193: 265-275. mihhalevski a, nisametdinov i, hälvin k, oseka a, paalme t (2013) stability of b-complex vitamins and dietary fiber during rye sourdough bread production. j. cereal sci. 57: 30-38. moudry j, pejcha j, peterka j (1999). the effect of genotype and farming technology on the yield of amaranth amaranthus sp. in collection of scientific papers, faculty of agriculture in české budějovice. series for crop sciences. 16: 93-98. murakami t, yutani a, yamano t, iyota h, konishi y (2014) effects of popping on nutrient contents of amaranth seed. plant foods hum. nutr. 69: 25-29. mburu mw, gikonyo nk, kenji gm, mwasaru am (2012) nutritional and functional properties of a complementary food based on kenyan amaranth grain (amaranthus cruentus). afr. j. food agric. nutr. dev. 12: 5958-5977. nelson rh (2013) hyperlipidemia as a risk factor for cardiovascular disease. prim. care. 40: 195-211. nimbalkar ms, pai sr, pawar nv, oulkar d, dixit gb (2012) free amino acid profiling in grain amaranth using lc-ms/ms. food chem. 134: 2565-2569. pavlík v (2012) the revival of amaranth as a thirdmillennium food. neuro. endocrinol. lett. 33: 3-7. prakash d, pal m (1991) nutritional and antinutritional composition of vegetable and grain amaranth leaves. j. sci. food agric. 57: 573-583. saha sk, brewer cf (1994) determination of the concentrations of oligosaccharides, complex type carbohydrates, and glycoproteins using the phenol-sulfuric acid method. carbohydr. res. 254: 157-167. singh n, singh p (2011). amaranth: potential source for flour enrichment. in preedy vr, watson rr, patel vb. (eds.), flours and breads and their fortification in health and disease prevention, academic press, elsevier, oxford, uk, pp: 101-111. tömösközi s, baracskai i, schoenlechner r, berghofer e, lasztity r (2009). comparative study of composition and technological quality of amaranth: i. gross chemical composition, amino acid and mineral content. acta aliment. 38: 341-347. uriyapongson j, rayas-duarte p (1994) comparison of yield and properties of amaranth starches using wet and drywet milling processes’. cereal chem. 71: 571-577. venskutonis pr, kraujalis p (2013). nutritional components of amaranth seeds and vegetables: a review on composition, properties, and uses. compr. rev. food sci. food saf. 12: 381-412. https://en.wikipedia.org/wiki/%c4%8cesk%c3%a9_bud%c4%9bjovice nova biotechnol chim (2020) 19(2): 216-222 doi: 10.36547/nbc.v19i2.776  corresponding author: goruky@ukr.net nova biotechnologica et chimica fatty acid composition of curd spread with different flax oil content anastasiia lialyk1, oleg pokotylo1, mykola kukhtyn1, ludmila beyko1, yulia horiuk2,, svetlana dobrovolska3 and oksana mazur4 1department of food biotechnology and chemistry, faculty of engineering of machines, structures and technologies, ternopil ivan puluj national technical university, ukraine 2department of infectious and parasitic diseases, faculty of veterinary medicine and technologies in livestock, state agrarian and engineering university in podilya, ukraine 3halytskyi college named after viacheslav chornovil, ukraine 4department of foreign languages, faculty of foreign languages, ternopil volodymyr hnatiuk national pedagogical university, ukraine article info article history: received: 24th may 2020 accepted: 23rd august 2020 keywords: cottage cheese curd spread flax oil polyunsaturated fatty acids abstract the creation of new types of dairy products for functional purposes with the addition of unconventional oils as sources of polyunsaturated fatty acids (pufa) is a promising and relevant research sphere. the study aimed to investigate the fatty acid composition of experimental samples of curd spread with different content of flax oil. the fatty acid composition was determined on a hewlett packard hp-6890 chromatograph with a flame ionization detector equipped with a 100 m long sp-2560 capillary column. it is established, that the total content of saturated fatty acids in curd spread containing 8 %, 10 %, and 12 % of flax oil was reduced, and the general total content of unsaturated fatty acids increased accordingly by 5.73 %, 6.94 % and 7.31 %, compared to the control sample without flax oil. the gas-chromatographic analysis showed that flax oil is rich in omega-3 pufa due to its high content of α-linoleic acid, so adding flax oil to the spread led to an increase in its content of α-linolenic acid and, accordingly, increased the content of pufa of the omega-3 family. thus, adding 8 %, 10 % and 12 % of flax oil to the curd spread, the content of α-linolenic acid in it increased accordingly by 3.91 %, 4.52 %, and 4.69 %, compared to the control sample. curd spread with 10 % content of flaxseed oil is characterized by the most optimal fatty acid composition. the ratio of saturated fatty acids to unsaturated in this curd spread is 1.9 : 1, and the ratio between pufas of the omega-3,-6, and -9 families are 1.3 : 1: 5.3.  university of ss. cyril and methodius in trnava introduction preserving health and expanding the duration of human life is one of the modern issues. one of the key ways of tackling this issue is the creation and active introduction of those foods of mass consumption, which are not only reached in energy and plastic material but also an important source of physiologically functional ingredients into the diet. they are polyunsaturated fatty acids (pufa), fat-soluble vitamins, phospholipids, and other biologically active components (goyal et al. 2014; kukhtyn et al. 2018a). the creation of new types of dairy products for functional purposes with the addition of unconventional oils as sources of pufa is mailto:goruky@ukr.net nova biotechnol chim (2020) 19(2): 216-222 217 a promising and relevant research sphere (bermúdez-aguirre and barbosa-cánovas 2011; ganesan et al. 2014; dal bello et al. 2015). on the one hand, it is because of the presence of lactic acid microorganisms, which provide a useful antagonistic effect on the conditionally pathogenic gut flora (prado et al. 2015; kukhtyn et al. 2018b), and on the other hand, due to the increased content of pufa, especially the omega-3 family, namely linoleic acid, which cover the deficit of these fatty acids in the diet (lane et al. 2014; dal bello et al. 2017). this approach in the technological introduction of flax oil gives the prospect of making enriched cheese paste a biologically valuable functional food product. it is known that pufas of the omega-3 family enter our body either with fat from the fish of cold seas (which are eicosapentaenoic and docosahexaenoic fatty acids) or with vegetable oils such as flax, sesame, rapeseed, pumpkin, soybeans, and others, which contain different amounts of linoleic acid (bermúdez-aguirre and barbosa-cánovas 2012; jandacek 2017). reference data (asuming-bediako et al. 2014; suksombat et al. 2014; ayyildiz et al. 2015; lewinska et al. 2015) indicates that sunflower and corn oils contain a high amount of ω-6 acids and very small amount of ω-3 acids and, accordingly, do not have the appropriate fatty acid composition. soybean oil has a recommended for consumption ratio of ω-3/ω-6 pufa (1 : 10). rapeseed and mustard oils are characterized by a relatively low level of saturated fatty acids (4 – 7 %), a high level of oleic acid (33 – 59 %) and an average level of linolenic acid (9 – 11 %) and, accordingly, a favorable ratio of ω-3/ω-6 as 1: 1 – 2. olive oil is characterized by a high content of oleic acid and a low level of pufa. the content of essential α-linolenic acid in flax oil significantly exceeds the recommended levels, which indicates their high physiological value and feasibility of use as a lipid supplement to enrich food with ω-3 acid. essential γ-linolenic acid (18 %) was detected only in cedar oil. walnut and hemp oils have a fairly high content of α-linolenic acid, but their use is limited by high cost and low prevalence. therefore, scientists (goyal et al. 2014; lewinska et al. 2015; kanakri et al. 2017) believe that flaxseed oil in its biological value is in the first place to provide the body with omega-3 pufa and recommend it enter into the daily diet. the adequate content of pufas of the omega-3 family in the diet provides a whole range of functions in a healthy body due to the high, genetically determined content of these pufas in the membranes of all cells and the main pools of the organism (el abed et al. 2008; albrachtschulte et al. 2018; dinicolantonio and o’keefe 2018). also important is their role as derivatives for the formation of cellular hormones, such as prostaglandins, leukotrienes, and thromboxanes, which regulate the course of physiological and biochemical processes in cells and tissues both in normal and in various pathologies (gogus and smith 2010; browning et al. 2012; calder 2015). therefore, pufas, which are rich in omega-3s, are now regarded as "healthy fats" with several physiological benefits. in particular, consumption of products with such content helps to maintain the physiological level of triglycerides in the blood and blood pressure prevents the development of cardiovascular diseases (chowdhury et al. 2014; farvid et al. 2014). also, it has been reported that regular consumption of omega-3 fatty acids has a positive effect on the brain and nervous system activity (gogus and smith 2010; drapkina and shepel 2015). based on the above mentioned, we have developed a curd spread with additional content of flax oil as a source of omega-3 pufas (lialyk et al. 2019). the study aimed to investigate the fatty acid composition of cottage cheese, flax oil, and developed curd spread with different content of flax oil. experimental the work was carried out at the ternopil ivan puliuj national technical university in the laboratories of the department of food biotechnology and chemistry. in the process of developing the technology of a particular food product, especially with highfat content, it is advisable and important to determine the fatty acid composition of such a product. to create the curd spread according nova biotechnol chim (2020) 19(2): 216-222 218 to the recipe low-fat cottage cheese was chosen, which meets the requirements according to the requirements of ssu (state standards of ukraine) (ssu 2007). in the first stage of the experimental part of the study, the fatty acid content of raw milk and the cottage cheese of the trademark "molokiia" (which was used as a basis for the production of curd spread rich in omega-3 fatty acid) were investigated. in the second stage, the fatty acid content of flax oil was investigated. on the third stage, the fatty acid composition of curd spread with a flax oil content of 8 %, 10 %, and 12 % was investigated. gaschromatographic analysis of raw milk, low-fat cottage cheese of trademark "molokiia", flax oil and curd spread with different content of flax oil was investigated using gas-liquid chromatography on hewlett packard hp-6890 gas chromatograph with flame-ionization detector equipped with open tubular column sp-2560 with a length of 100 m (holubets and vudmaska 2015). the process of making curd spread with linseed oil included the following technological operations: 1) selection of a mixture for melting (low-fat cottage cheese and linseed oil were subjected to organoleptic evaluation and laboratory methods of quality research); 2) grinding of raw materials (raw materials were grounded into particles with a diameter of 5 – 8 mm). 3) compounding of the mixture. 4) introduction of melting salts (melting salts-sodium citrate was added to the cheese mass to increase the ph, partial transition of proteins to the soluble state and improve the melting process of the cheese mass); 5) maturation of curd mass (duration of exposure 2 h); 6) melting of the curd mass (melting time 15 min, at a temperature of 82 °c (ps-10, “ukrtechprom” ukraine)); 7) homogenization of the melted mass (duration of homogenization at p = 9.81 mpa on microtron mb 800, germany); 8) packaging (packaging of the curd product was carried out in hot way at a temperature of +60 – +75 °c in foil. the weight of the package is 50 g). 9) cooling and storage of the curd product (up to a temperature of +2 – +4 °c). statistical processing of the results was carried out using methods of variation statistics using the program statistica 9.0 (statsoft inc., usa). non-parametric methods of research were used (wilcoxon-mann-whitney test). the arithmetic mean (x) and the standard error of the mean (se) were determined. the difference between the comparable values was considered to be significant for p < 0.05. results and discussion as a result of gas chromatographic analysis of milk fat, 39 peaks were found, which corresponded table 1. the fatty acid composition of low-fat cottage cheese of trademark "molokiia" and raw milk materials (x ± se, n = 5). name of fatty acids code of fatty acids mass content of fatty acids [%] cottage cheese control sample, raw milk saturated maslinic с 4 : 0 3.16±0.11 3.55±0.12 capronic с 6 : 0 1.82±0.13 2.10±0.08 caprilic с 8 : 0 1.03±0.07 1.14±0.06 caprinic с 10 : 0 2.60±0.11 2.41±0.11 lauric с 12 : 0 2.67±0.07 2.66±0.08 myristic с 14 : 0 11.45±0.12 11.45±0.12 palmitic с 16 : 0 33.62±0.14 33.44±0.15 stearic с 18 : 0 9.75±0.11 9.68±0.16 other acids 6.76±0.10 6.56±0.11 in total saturated 72.86±0.14 72.98±0.16 unsaturated oleic с 18 : 1 22.07±0.02 21.67±0.01 linolic с 18 : 2 3.751±0.012 3.69±0.01 linolenoic с 18 : 3 0.845±0.100 0.83±0.00 arachidonic с 20 : 4 0.07±0.03 0.06±0.00 other acids 0.42±0.09 0.77±0.07 in total unsaturated 27.14±0.16 27.12±0.12 nova biotechnol chim (2020) 19(2): 216-222 219 to saturated and unsaturated fatty acids and their isomers. table 1 shows the main fatty acids. from the presented data it can be seen that in the low-fat cottage cheese of trademark "molokiia". palmitic, myristic, and stearic acids were dominant among saturated fatty acids, whereas oleic and linolenic were dominant among unsaturated fatty acids. the ratio between saturated and unsaturated fatty acids in low-fat cottage cheese of the trademark “molokiia” was 2.7 : 1. it should be noted that among unsaturated fatty acids in cottage cheese, polyunsaturated fatty acids of the omega-6 family are dominated, accounting for 26 % of the total of all fatty acids or 96 % of polyunsaturated fatty acids. the analysis of the obtained results shows that this cottage lacks the polyunsaturated fatty acids of the omega-3 family. the content of the latter in this study product is only 0.8 % of all fatty acids. thus, it can be argued that this cottage cheese is deficient in the content of pufas of the omega-3 family. therefore, when creating a curd spread based on this cottage cheese, our task was to enrich its pufa of the omega-3 family. for the enrichment of cottage cheese with omega-3 pufa in the process of making curd spread, we have chosen flax oil, which is known to be one of the richest in omega-3 family pufa because of its high content of α-linolenoic acid. since the fatty acid composition of oils, including flax oil, depends on many characteristics, we have chosen flax one, which is made from seeds of the “debiut” breed and its fatty acid composition was investigated. the results obtained are presented in table 2. as can be seen, flax oil obtained from the seeds of flax breed "debiut" is characterized by a high content of the omega-3 family pufa due to linolenoic acid, which is 53.5 % of the total table 2. the fatty acid composition of linseed oil by the method of cold-pressed flax seeds "debiut" (x ± se, n = 5). name of fatty acids code of fatty acids content of fatty acid [%] myristic с 14 :0 trace amount palmitic с 16 : 0 7.40 stearic с 18 : 0 4.20 arachidic с 20 : 4 0.50 palmitelaidic c 16 : 1 0.20 oleic с 18 : 1с 21.40 linolic c 18:2c,12c 12.80 linolenoic с 18:3c,12c, 15с 53.50 content of all fatty acids. the ratio of pufas of the omega-3, -6, and -9 families in the studied flax oil is 4 : 1 : 1.5. in general, the results of the study of the fatty acid composition of flax oil confirm the high content of α-linolenoic acid, which allows it to be reasonably used for the enrichment of curd spread. the next stage of our research was the development of technology for the production of curd spread with different contents of flax oil. the samples of cottage cheese products contained 8 %, 10 %, and 12 % of flax oil. as a result, curd products had a creamy-white color that was smooth all over and with specific for curd and slightly mustard-like taste of flax oil, without excess acidity taste. the results of the study of the fatty acid composition of curd spread with different flax oil content are presented in table 3. table 3 shows that the fatty acid composition of the studied curd spreads varied significantly depending on the content of flax oil. thus, in general, in all tested samples of curd spread with the addition of 8 %, 10 % and 12 % of flax oil the content of such saturated fatty acids as palmitic and stearic increased and the content of other, lowmolecular acids, such as oil, caprylic, capronic, caprinic, lauric and myristic, decreased. in general, the total content of saturated fatty acids in the curd spread with the addition of 8 %, 10 % and 12 % of flax oil decreased by 5.73 %; 6.94 % and 7.31 %, respectively compared to the control sample (without flax oil). the content of unsaturated fatty acids according to table 3, respectively, increases due to oleic, linolic, and linolenoic acids, and this increase is directly correlated with the increase in the content of flax oil in curd spread. these fatty acids are a key factor to a particular class of omega-3, -6, or -9 polyunsaturated fatty acids, so the ratio between these classes of omega pufas has changed accordingly. the general total content of unsaturated fatty acids in curd paste with the addition of 8 %, 10 % and 12 % of flax oil increased by 5.73 %; 6.94 % and 7.31 %, respectively compared to the control sample of curd spread without the addition of linseed oil. considering that flax oil is rich in omega-3 pufa due to the high content of α-linolenic acid, the addition of flax oil in the spread, in the first place, will lead to an increase in the content nova biotechnol chim (2020) 19(2): 216-222 220 table 3. the fatty acid composition of curd spread (cs) with different flax oil content (x ± se, n = 4). name of fatty acid mass content of fatty acids [%] control sample of cs without flax oil cs with 8 % of flax oil cs with 10 % of flax oil cs with 12 % of flax oil saturated maslinic, с 4 : 0 3.21±0.42 2.52±0.24* 2.16±0.19* 2.04±0.21* capronic, с 6 : 0 1.82±0.11 1.25±0.15* 0.78±0.13* 0.63±0.14* caprilic, с 8 : 0 1.03±0.01 0.72±0.07* 0.61±0.05* 0.54±0.04* caprinic, с 10: 0 2.60±0.19 2.16±0.13* 1.77±0.21* 1.52±0.18* lauric, с 12: 0 2.67±0.02 2.03±0.25* 1.63±0.17* 1.54±0.16* myristic с 14: 0 11.45±0.09 10.12±0.12 9.86±0.24 9.72±0.18 palmitic с 16: 0 33.62±0.21 34.04±0.18 34.19±0.28 35.04±0.35 stearic с 18: 0 9.75±0.08 10.06±0.11 10.15±0.22 10.03±0.18 other acids 6.76±0.13 5.04±0.27 4.82±0.36 4.54±0.10 in total satureted 72.91±0.54 67.18±0.52 65.97±0.34 65.60±0.37 unsaturated oleic с 18 : 1 (ω -9) 22.01±0.25 23.05±0.02 23.52±0.13 23.57±0.14 linolic с 18 : 2 (ω-6) 3.75±0.12 3.97±0.15 4.40±0.26 4.51±0.33 linolenoic с 18 : 3 (ω-3) 0.85±0.06 4.76±0.12 5.77±0.02 6.04±0.31 arachidonic с20 : 4 (ω-6) 0.07±0.01 0.07±0.01 0.06±0.02 0.06±0.01 other acids 0.41±0.02 0.21±0.03 0.28±0.05 0.22±0.03 in total unsatureted 27.09±0.25 32.06±0.36 34.03±0.26 34.40±0.27 total omega-3 0.85±0.06 4.76±0.12 5.77±0.02 6.04±0.31 total omega -6 3.82±0.12 4.04±0.15 4.46±0.26 4.57±0.33 total omega -9 22.01±0.25 23.05±0.02 23.52±0.13 23.57±0.14 ω-3/ ω-6 1 : 4.5 1.17 : 1 1.29 : 1 1.32 : 1 the ratio of saturated to unsaturated fas 2.7 : 1 2.1 : 1 1.9 : 1 1.9 : 1 * р < 0.05 – compared to control. of α-linolenic acid and, accordingly, an increase of the content of omega-3 family pufa. thus, when 8 %, 10 %, and 12 % of flax oil is added to the curd spread, the content of α-linolenic acid in the curd spread increases by 3.91 %; 4.52 % and 4.69 %, respectively compared to the control sample. we support the view of researchers (sioen et al. 2009; vella et al. 2013) that the consumption of products containing pufa of the omega-3 in the world is insufficient to provide at least 10 % of the daily rate of the recommended consumption of 2 g/day of α-linolenic acid according to eu regulation no. 1924/2006 and no. 432/2012. therefore, the development of curd spread with flax oil content is intended to cover the deficiency in the pufa of the omega-3 family and may be a good alternative to enrich the diet with biologically active ingredients. an important indicator characterizing the biological and functional value of a food product in terms of its fatty acid composition is the ratio of saturated fatty acids to unsaturated. thus, in the curd spread containing 8 %, 10 % and 12 % of flax oil the ratio between saturated and unsaturated fatty acids was 2.1 : 1, 1.9 : 1 and 1.9 : 1, compared to the control group with the ratio of 2.7 : 1. another important indicator of a food product is the ratio between omega-3 and omega-6 pufa. thus, in a curd spread containing 8 %, 10 % and 12 % flax oil, the ratio between omega-3 and omega-6 pufa was 1.17 : 1, 1.18 : 1 and 1.21 : 1. studies by other scientists (dal bello et al. 2017) also indicate a probable increase (p < 0.05) in the content of omega-3 and omega-6 pufas in dairy products with the addition of different content of flax oil. however, studies (lerch et al. 2012; puppel et al. 2013; suksombat et al. 2014) report that the fatty acid composition of milk and dairy products can be improved (increase omega-3 fatty acids) in a natural way, by the adjustment of cows' diets and feeding them on raw materials rich in linolenic acid (rapeseed, flax). we believe that this method is promising, but it requires sufficient scientific justification and the possibility of large-scale production of such milk. therefore, we believe that our studies are consistent with the data by the authors nova biotechnol chim (2020) 19(2): 216-222 221 (bermúdez-aguirre et al. 2012; dal bello et al. 2015, 2017) about the need for enrichment of yogurt, cheese, etc., with vegetable raw materials rich in pufa. also, our previous studies found (lialyk et al. 2019) that the curd spread with the content of flax oil after 14 d of storage contained at least 107 cfu.g-1 of viable lactic acid bacteria, making it a functional product. all in all, the developed curd spread with the 10 % content of flaxseed oil is characterized by the most optimal fatty acid composition, based on the biological and functional value of the food product and cost-effectiveness. the ratio of saturated fatty acids to unsaturated in this cheese paste is 1.9 : 1, and the ratio between pufas of the omega-3, -6, and -9 families is 1.3 : 1 : 5.3. considering the significant deficiency in omega-3 pufas in the diet in ukraine and a little less visible omega-9 pufa deficiency, a functional food product, which is flax curd spread, will be one of the alternatives to address the deficiencies in these pufas. the food product developed by us is patented, the standard documentation for production according to the legislation of ukraine is issued and it received the name "curd spread enriched with omega-3 fatty acids" longevity ". conclusion it was found that the studied flax oil was characterized by a high content of such polyunsaturated fatty acids as α-linolenic acid, oleic and linoleic acids, the relative content of which was 53.5 %, 21.4 % and 12.8 %, respectively. the ratio of pufas of the omega-3, 6, and -9 families in the studies flax oil is 4 : 1 : 1.5. it was found that the total content of saturated fatty acids in curd spread with 8 %, 10 %, and 12 % of flax oil decreased, and the general total content of unsaturated fatty acids increased by 5.73 %, 6.94 % and 7.31 % respectively compared to the control sample. adding 8 %, 10 %, and 12 % of flax oil to the curd spread, the content of α-linolenic acid in the curd spread changed the most and increased accordingly by 3.91 %, 4.52 % and 4.69 %, compared to the control sample. curd spread with 10 % content of flaxseed oil is characterized by the most optimal fatty acid composition, based on the biological and functional value of the food product and cost-effectiveness. the ratio of saturated fatty acids to unsaturated in this curd spread is 1.9 : 1, and the ratio between pufas of the omega-3, -6, and -9 families is 1.3 : 1 : 5.3. acknowledgement the authors would like to thank the ternopil ivan puluj national technical university for their help, support, and facilities for the conduction of this experiment. conflict of interest the authors declare that they have no conflict of interest. references albracht-schulte k, kalupahana ns, ramalingam l, wang s, rahman sm, robert-mccomb j, moustaid-moussa n (2018) omega-3 fatty acids in obesity and metabolic syndrome: a mechanistic update. j. nutr. biochem. 58: 1-16. asuming-bediako n, jaspal mh, hallett k, bayntun j, baker a, sheard pr (2014) effects of replacing pork backfat with emulsified vegetable oil on fatty acid composition and quality of uk-style sausages. meat sci. 96: 187-194. ayyildiz hf, topkafa m, kara h, sherazi sth (2015) evaluation of fatty acid composition, tocols profile, and oxidative stability of some fully refined edible 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consumption in older adults in relation to food matrices, bioactive ingredients, and health. j nutr. gerontol. geriatr. 32: 122-144. nova biotechnol chim (2019) 18(2): 84-93 doi: 10.2478/nbec-2019-0011  corresponding author: sabina.lip@gmail.com nova biotechnologica et chimica optimization of medium composition for propagation of recombinant escherichia coli sabina lipničanová1,, daniela chmelová1, andrej godány2 and miroslav ondrejovič1 1 department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava 91701, slovak republic 2 department of biology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava 91701, slovak republic article info article history: received: 18th september 2019 accepted: 14th november 2019 keywords: bacteria biomass neuraminidase plasmid dna propagation response surface methodology abbreviations: dcw – dry cell weight dns – 3,5-dinitrosalicylic acid ha – hemagglutinin lb – luria-bertani na – neuraminidase od – optical density pdna – plasmid dna pbs – phosphate-buffered saline rsm – response surface methodology tb – terrific broth abstract recombinant protein production in heterologous hosts often seems a simpler and more effective way than its production by natural producer. the secretion of recombinant protein in escherichia coli has many advantages comparing to than in insect or mammalian cells. the important factor for high-level recombinant protein production is the sufficient amount of e. coli biomass. therefore, the aim of this study was to optimize the composition of propagation medium resulting in the maximum biomass yield of recombinant e. coli as the part of fermentation strategy for neuraminidase (na) production. three independent variables including glucose, asparagine and phosphate concentrations, and four dependent variables, such as biomass yield, residual concentrations of glucose or asparagine and ph of the propagation medium after fermentation, were chosen to the optimization by response surface methodology (rsm). the optimal conditions for the maximum biomass yield expressed as dry cell weight (dcw) (16.57±0.55 g dcw.l-1) were as follows: glucose concentration of 39.37 mm, asparagine concentration of 62.68 mm and phosphate concentration of 14.80 mm. for this model, the predicted values for the responses are close to the experimental values. the yield of desired pet15b-neu plasmid from e. coli cells cultivated in optimized propagation medium was almost 23 % higher than in commonly used luria-bertani (lb) medium suggesting that asparagine may be involved in the induction of plasmid amplification.  university of ss. cyril and methodius in trnava introduction the recombinant production of desired protein can facilitate product isolation and impact its quality and activity (horga et al. 2018). the choice of suitable host affects the whole process. among microorganisms, bacteria have been widely used as the hosts for high-level expression of protein (nikerel et al. 2006). the use of gram-negative bacterial host escherichia coli has several advantages, namely the simplicity of system, fast growth linked to high cell density and cost-effective cultivation to produce recombinant protein at a relative high level (fakruddin et al. 2012; rosano and ceccarelli 2014). usually, the production of recombinant protein by e. coli host cells is two-step process. in the first step, the cells are cultivated in a simple, defined medium with a carbon source that suppresses gene expression and results in the accumulation of biomass (potvin et al. 2012; celik et al. 2012). the induction of recombinant protein synthesis is bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:20 utc mailto:sabina.lip@gmail.com nova biotechnol chim (2019) 18(2): 84-93 85 initiated by the addition of an inducer of promoter after the depletion of carbon source in the second step of production strategy. the biomass yield can be affected by the composition of production medium leading to high-level production of heterologous protein (sekar et al. 2018). thus, the yield of recombinant protein may be proportional to the final biomass production. the optimization of the propagation medium composition by the response surface methodology (rsm) is the suitable tool to increase the amount of host biomass (nikerel et al. 2006; ghosalkar et al. 2008). the sufficient biomass yield is the part of fermentation strategy for the production of neuraminidase (na) in e. coli host cells harbouring plasmid pet15b with gene for influenza virus na (pet15b-neu). na as a surface glycoprotein of influenza virus has an essential role in the infectious cycle of the virus (doyle et al. 2013). screening of new na inhibitors requires enzymatically active na which can be obtained by synthesis using recombinantly modified organisms such as e. coli (nallaiyan et al. 2010). inhibition of this enzyme through drug additives is a perspective way to cure of diseases caused by influenza virus (doyle et al. 2013; udommaneethanakit et al. 2014; hadzázi et al. 2018). the aim of this study was to optimize the composition of propagation medium by rsm to improve e. coli biomass yield leading to highlevel production of recombinant na as the perspective tool for design of novel antiviral compounds. experimental bacterial strain, media and culture conditions bacteria of escherichia coli strain bl21 (de3)plys (f– ompt hsdsb (rb –, mb –) gal dcm (de3) plyss(camr)) were obtained from agilent technologies (usa). the gene coding for na was developed through the design using the sequence deposited in genbank (accession no. km244086.1) and synthesized (atg biosynthetic, germany). propagation medium was inoculated with e. coli cell suspension at 2.0 mcfarland turbidity in the ratio of 50:1 (v/v) and incubated for 24 hours at 120 rpm. e. coli cell suspension was prepared from cells growing in lb (luria-bertani) medium (10 g.l-1 tryptone, 5 g.l-1 yeast extract, 10 g.l-1 nacl and 50 mg.l-1 ampicillin) at 140 rpm and 37 °c overnight. bacteria were transformed with the pet15b plasmid harbouring gene for influenza virus na by the standard protocol. basal propagation medium for the cultivation of recombinant e. coli consisted of 4 mm glucose, 9 mm (nh4)2so4, 10 mm nah2po4, 10 mm na2hpo4, 2.5 mm mgso4.7h2o, 34 mm nacl and the trace element solution (5 ml.l-1). the latter was composed of feso4·7h2o, coso4·7h2o, zncl2, cuso4, na2moo4·2h2o, h3bo3, nicl2, mnso4 and al2(so4)3 at the concentration of 1 g.l-1 of each compound, as recommended by sartorius (sartorius ag, germany) the ph of propagation media was adjusted to ph 7.1 with 1 m naoh and carbon source was sterilized separately. in preliminary experiments, the type of carbon (glucose, fructose or lactose) and nitrogen sources (ammonium sulfate or asparagine) in the concentration of 100 mm were tested. the effect of different concentrations of carbon and nitrogen sources (glucose and asparagine, respectively) in the range of 0 – 100 mm and phosphate concentration in the range of 0 – 64 mm on biomass yields were determined. table 1. levels of the factors tested in rsm. factor and its concentration [mm] level code -1.682 -1 0 1 1.682 glucose 23.18 30 40 50 56.82 asparagine 26.36 40 60 80 93.64 phosphate 7.27 10 14 18 20.73 experimental design the rsm was used to investigate the effect of glucose, asparagine and phosphate concentrations on the biomass yield expressed as dry cell weight (dcw), residual glucose and asparagine concentrations and final ph of propagation medium (dependent variables). the variables were tested on five level codes (-1.682, -1, 0, 1, 1.682) and are listed in table 1. the analysis of measured data was statistically evaluated by regression method according to eq. 1: 𝑌 = 𝑏0 + ∑ 𝑏𝑖 𝑋𝑖 + ∑ 𝑏𝑖𝑖 𝑋𝑖 2 + ∑ ∑ 𝑏𝑖𝑗𝑗−2 𝑋𝑖 𝑋𝑗 𝑘−1 𝑖−1 𝑖<𝑗 𝑘 𝑖−1 𝑘 𝑖−1 (1) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:20 utc nova biotechnol chim (2019) 18(2): 84-93 86 where xi are independent variables causing the y response and bi are regression coefficients describing dependences between measured properties and coded values of observed factors. after 8 h of cultivation, samples were centrifuged at 10,000 rpm for 5 min. in the supernatant, the final ph was determined, and residual carbon and nitrogen concentrations were analysed by dns (3,5-dinitrosalicylic acid) method (miller 1959) and ninhydrin reaction (kendall 1963), respectively. the sediment was washed, centrifuged and dried at 60 °c to the constant weight. biomass yield was expressed as dcw per liter of medium. extraction of plasmid dna plasmid dna (pdna) was extracted using the genejet plasmid miniprep kit (thermofisher scientific, usa) from e. coli biomass produced at 37 °c in optimized propagation and lb media. after extraction, the concentration of pdna were measured by uv-vis spectrophotometer (v-1600pc, vwr, germany) and the result was calculated according to the eq. 2. 𝑐 [µ𝑔. 𝑔−1] = ((𝐴260𝑛𝑚− 𝐴230𝑛𝑚)∗𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ∗50 µ𝑔.𝑚𝐿 −1 ) 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 [𝑔] (2) statistical analysis all experiments carried out in three parallels. data are present as the average with the standard deviation. for the evaluation of regression equations in the optimization by rsm, statgraphics plus 5.1 (statpoint technologies, inc. usa) was used. results and discussion selection of independent variables for optimization in the literature, statistical experimental design has been used to optimize the production of various types of recombinant proteins (farliahati et al. 2010; larentis et al. 2012; sekar et al. 2018). in this work, rsm was used to increase biomass yields of recombinant e. coli harbouring plasmid pet15b with gene for influenza virus na. key factors found to affect e. coli biomass yields were carbon and nitrogen sources (farliahati et al. 2010; sekar et al. 2018) and based to our preliminary studies (data not shown) also the phosphate source. therefore, the effect of these three factors on final biomass yield were evaluated (fig. 1). fig. 1. the kinetics of e. coli growth in propagation media with glucose (a), fructose (b) and lactose (c) as carbon sources (respectively). effect of carbon source on ph of medium (d). data represent mean values of three replicates. a b c d bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:20 utc nova biotechnol chim (2019) 18(2): 84-93 87 the best carbon source for e. coli biomass production was glucose (11.81±0.03 g dcw.l-1). glucose has been widely described as the appropriate substrate for the cultivation of e. coli (wang et al. 2015; jing et al. 2018; lee et al. 2018) however, as is shown fig. 1, recombinant e. coli was able to utilize effectively also pentose (fructose) and disaccharide (lactose); both of these carbon sources resulted in similar production of biomass (11.16±0.01 and 10.97±0.00 g dcw.l-1, respectively). e. coli cells entered the stationary phase of growth after 12 hours of cultivation in propagation media with glucose and fructose (fig. 1 a, b), presumably due to decreased ph in these media (fig. 1d). the consumption of carbon sources can lead to the production of organic acids, predominantly acetic acid, up to a concentration in the range of 50 – 100 mm. these concentrations are usually considered to cause the inhibition of growth (pinhal et al. 2019) and low level of protein expression (wang et al. 2016). the final ph values of media with different carbon sources were comparable (~4.8). therefore, glucose was selected as the main carbon source for followup experiments. the concentration of selected carbon source is another parameter affecting biomass production. therefore, the effect of glucose concentration in the range of 0 – 100 mm to biomass production was evaluated. glucose depletion was monitored after 8 hours (fig. 2), as increased organic acid production inhibits bacterial growth (fig. 1). it was observed that 20 – 40 mm of glucose stimulated bacterial growth (fig. 2a). farliahati et al. (2010) found that the combination of both glucose (80 mm) and inorganic nitrogen source is the best choice for e. coli biomass production. although, larentis et al. (2012) suggested that the fig. 2. the effect of glucose concentration on biomass production of e. coli (a) and its residual concentration (b) after 8 h of cultivation. data represent mean values of three replicates. fig. 3. kinetics of e. coli growth in media with ammonium sulphate (a) and asparagine (b). data represent mean values of three replicates. a b a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:20 utc nova biotechnol chim (2019) 18(2): 84-93 88 lower concentration of glucose (40 mm) in medium is beneficial to e. coli biomass production and glucose was almost depleted in the medium. maximum glucose depletion in the propagation medium plays a crucial role in the subsequent production of recombinant na, since high glucose concentrations cause the suppression of corresponding gene expression (potvin et al. 2012; celik et al. 2012). glucose concentration range of 20 to 40 mm, representing the most suitable conditions for biomass production, was associated with significant reduction of glucose levels in the propagation medium (fig. 2b). the choice of nitrogen source and its concentration are among the other factors influencing biomass yield. the appropriateness of nitrogen source, namely ammonium sulphate and asparagine, was tested (fig. 3). our results are in the contrasts to previous studies (chubukov et al. 2014; wang et al. 2016) in which ammonium belongs to the preferred nitrogen source for e. coli. the higher e. coli biomass yield was observed in propagation medium with asparagine as nitrogen source (13.43±0.02 g dcw.l-1; fig. 3b) than in medium with ammonium ions in the form of ammonium sulphate (11.81±0.01 g dcw.l-1; fig. 3a). bren et al. (2016) found that amino acid as the nitrogen source in a medium supported the growth of e. coli. therefore, the amino acid asparagine was selected as nitrogen source for further experiments and the effect of its different concentration in propagation media was tested (fig. 4). the asparagine concentration range of 40 – 60 mm stimulated bacterial growth, but this effect was absent at higher concentrations (fig. 4a). almost complete depletion of the nitrogen source in propagation media containing asparagine fig. 4. the effect of asparagine concentration on biomass production of e. coli (a) and its residual concentration (b) after 8 h of cultivation. data represent mean values of three replicates. fig. 5. the effect of phosphate concentration on biomass production of e. coli (a) and final ph of propagation media (b) after 8 h of cultivation. data represent mean values of three replicates. a b a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:20 utc nova biotechnol chim (2019) 18(2): 84-93 89 table 2. the combination of tested factors with their level codes from rsm and their effect on biomass production. run factor concentration [mm]a biomass no. glucose asparagine phosphates [g dcw.l-1] 1 50 (1) 40 (-1) 18 (1) 9.97±1.30 2 30 (-1) 80 (1) 18 (1) 12.19±0.44 3 50 (1) 80 (1) 10 (-1) 11.65±1.24 4 30 (-1) 40 (-1) 10 (-1) 9.49±0.12 5 40 (0) 60 (0) 14 (0) 16.21±0.91 6 30 (-1) 80 (1) 10 (-1) 11.12±0.64 7 50 (1) 80 (1) 18 (1) 12.36±1.12 8 30 (-1) 40 (-1) 18 (1) 11.48±0.44 9 40 (0) 60 (0) 14 (0) 16.61±0.88 10 50 (1) 40 (-1) 10 (-1) 8.65±0.59 11 23.27 (-1.682) 60 (0) 14 (0) 11.09±0.12 12 40 (0) 60 (0) 20.69 (1.682) 11.28±0.13 13 40 (0) 60 (0) 7.31 (-1.682) 8.69±0.17 14 40 (0) 93.46 (1.682) 14 (0) 9.59±0.16 15 40 (0) 26.53 (-1.682) 14 (0) 8.93±0.43 16 56.73 (1.682) 60 (0) 14 (0) 10.11±0.32 17 40 (0) 60 (0) 14 (0) 16.57±0.55 a in brackets the level codes are given. in the concentration range of 10 – 40 mm was observed (fig. 4b). one way to eliminate the problem of undesirable organic acid production is to modify the ionic strength of the buffer. in this study, pbs (phosphatebuffered saline) was used as the source of phosphate and the buffer (fig. 5). our results indicate that ph values of propagation media were maintained at phosphate concentrations of 32 and 64 mm (fig. 5b). with the decrease in salt concentration in the solution, the ph value decreases. however, it should be noted that the phosphate concentration positively influenced the biomass yield in the range of 4 to 16 mm (fig. 5a). the low biomass yields were observed in propagation media with increasing the phosphate concentrations. scheidle et al. (2011) obtained similar results. the high concentration of phosphates increases the osmolality values and significantly reduces the growth rate of e. coli cells. therefore, the suitable phosphate concentration is in the range of 4 – 16 mm for both evaluated variables (fig. 5). optimization of propagation medium based on preliminary experiments, we have chosen the factors affecting biomass yield and optimization using rsm to find the optimal conditions for maximum e. coli biomass yields (table 2). the calculated coefficient of determination (r2) for biomass yields was 0.97 thus the model could explain 97 % of the total variations in the experimental data. biomass yields varied in the range of 8.65 – 16.61 g dcw.l-1 (table 2). the residual glucose and asparagine concentrations and the final ph of the propagation medium were selected as additional dependent variables. recombinant protein production in e. coli host is a two-step process; in the second step the minimization of glucose concentration is needed prior to the induction of t7 promoter in the recombinant (pet15b) plasmid. in the experiments at center point of independent variables, glucose was almost completely consumed, with residual glucose concentrations of 6.65 – 7.25 mm. the coefficient of determination for residual glucose concentration as dependent variable was 0.99. the residual concentration of asparagine varied in the range of 5.93 – 44.59 mm with r2 value of 0.98. the final ph of propagation media varied in the range of 4.60 – 5.47 with r2 of 0.88. the final ph proportionally decreased by e. coli growth and was associated with glucose and phosphate concentrations (data not shown). for better understanding of interactions between selected variables, the 3d surface plots are shown in fig. 6. the third variable, phosphate concentration, was set as the constant (14 mm; fig. 6). table 3 summarizes the regression coefficients and analysis of variances for biomass bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:20 utc nova biotechnol chim (2019) 18(2): 84-93 90 table 3. results of variance analysis and regression coefficients of the predicted second order polynomial models of biomass and residual concentration of glucose and asparagine. effect factora biomass residual glucose concentration residual asparagine concentration ph constant -59.509 276.306 140.318 8.073 linear a 1.3307 -5.6027 -2.8562 -0.0647 b 0.6673 -2.4879 -1.0138 -0.0134 c 3.9452 -14.0071 -8.1134 -0.2812 quadratic aa -0.0178 0.0873 0.0303 0.0009 bb 0.0057 0.0208 0.0069 0.0001 cc -0.1250 0.4888 0.2792 0.0109 interaction ab 0.0019 -0.0044 0.0123 -0.0001 ac -0.0032 -0.0077 -0.0131 -7.8.10-5 bc -0.0024 0.0060 0.0156 0.0002 a concentration of glucose – a; asparagine – b; phosphates – c statistically significant differences (at p < 0.05) are shown bold. yields, glucose and asparagine concentrations and final ph of propagation medium. for biomass yields, asparagine and phosphate concentrations had the significant positive linear influence (p < 0.05) (table 3). the increase of asparagine concentration from 60 to 80 mm caused the decrease of biomass yield (fig. 6a). all tested independent variables had negative quadratic effect to biomass yields (p < 0.05) and positive quadratic effect to residual glucose and asparagine concentrations (p < 0.05). glucose concentration had negative linear effect to residual glucose concentration in propagation media after the e. coli cultivation (p < 0.05) (table 3). the increase of glucose concentration resulted in higher residual glucose concentration (fig. 6 b). the higher residual glucose concentration can be a problem to produce recombinant protein due to the suppression of t7 promoter. not surprisingly, asparagine concentration had a negative linear effect (p < 0.05) on the residual concentration of asparagine (table 3), fig. 6. 3d-surface plots showing the effect of glucose and asparagine concentrations on the biomass yield (a), effect of residual concentrations of glucose (b), asparagine (c) and effect of ph of propagation medium (d). a b c d a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:20 utc nova biotechnol chim (2019) 18(2): 84-93 91 table 4. predicted and experimental values of biomass yield, residual glucose and residual asparagine at conditions optimal for maximum biomass yield. biomass [g dcw.l-1] residual glucose concentration [mm] residual asparagine concentration [mm] predicted value 16.55 6.63 17.01 experimental value 15.91±0.43 6.27±0.72 17.20±2.55 rsm error [%] 4.02 5.74 1.10 similarly as observed for glucose (fig. 6c). for final ph of propagation medium, phosphate concentration had negative linear (p < 0.05) and positive quadratic effects (p < 0.05) (table 3). higher phosphate concentration allows to maintain the stable ph value in propagation medium (fig. 6d). moreover, as shown fig. 6d, the final ph f propagation medium affects glucose concentration, and that is in good agreement with results of kleman and strohl (1994). the final ph of propagation medium was higher at lower glucose concentration (fig. 6d). verification of model according to rsm model, the highest yield of e. coli biomass can be reached in the propagation medium with glucose concentration of 39.37 mm, asparagine concentration of 62.68 mm and phosphate concentration of 14.80 mm. optimal conditions for biomass yield were confirmed experimentally (table 4). there was no significant difference between predicted and experimental values of all tested dependent variables (p < 0.05), suggesting this mathematical model can be exploited for maximum e. coli biomass yield in the first step of fermentation strategy. moreover, biomass yield obtained in our modified optimized propagation medium was nearly 16 and 40 % higher than in complex media used as a standard culture media for e. coli cells, namely terrific broth (tb) and lb, respectively. compared to defined media such as m9 and riesenberg minimal medium (kangwa et al. 2015), we achieved more than 50 % higher biomass yield using our optimized medium. it is commonly observed that the higher yield of biomass in batch cultivation is associated with lower amplification rate of pdna compared to lb medium (o´kennedy et al. 2003). therefore, the yield of pet15b-neu obtained from cells cultivated in optimized medium was compared to pet15b-neu obtained from cells cultivated in lb medium. the yield of pet15b-neu from cells cultivated in optimized propagation medium (1.40±0.16 µg.g-1) was 22.86 % higher as from cells obtained from the cultivation in lb medium (1.08±0.14 µg.g-1). wood et al. (2017) suggested that high concentration of yeast extract in lb medium has impact on higher pdna yield. in the case of optimized propagation medium used in this study, the asparagine amino acid may be involved in the induction of plasmid amplification as it was observed for amino acids such as leucine, glycine and histidine (dorward et al. 2019). conclusions the results of the present study confirm that the key factors for the propagation of escherichia coli bl21 (de3)plys are the concentration of glucose as carbon source, concentration of asparagine as nitrogen source and concentration of phosphates. the rms model was able effectively to describe the experimental data. the optimal concentration of selected factors such as glucose, asparagine and phosphate concentrations are 39.37, 62.68 and 14.80 mm, respectively. the predicted and experimental values further showed the adequacy of this model. at the optimal conditions, the yield of e. coli biomass was 16 and 40 % higher than in tb and lb, respectively. these results suggested that optimized propagation medium for e. coli biomass production can be the suitable tool for effective recombinant protein production at relative high level. moreover, arginine in propagation medium was probably involved in the induction of plasmid amplification. acknowledgement this work was supported by research grants of the slovak bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:20 utc nova biotechnol chim (2019) 18(2): 84-93 92 research and development agency apvv-17-0239 and fund for research support of the university of ss. cyril and methodius in trnava fppv-06-2019. conflict of interest the authors declare that they have no conflict of interest. references bren a, park, jo, towbin bd, dekel e, rabinowitz jd, alon u (2016) glucose becomes one of the worst carbon sources for e. coli on poor nitrogen sources due to suboptimal levels of camp. sci. rep. 6: e24834. celik e, calik p (2012) production of recombinant proteins by yeast cells. biotechnol. adv. 5: 1108-1118. chubukov v, sauer u (2014) environmental dependence of stationary-phase metabolism in bacillus subtilis and escherichia coli. appl. environ. microbiol. 9: 29012909. dorward a, o'kennedy rd, folarin o, ward jm, keshavarzmoore e (2019) the role of amino acids in the amplification and quality of dna vectors for industrial applications. biotechnol. prog. 12: e2883. doyle m, hashem am, li c, van domselaar g, larocque l, wang j, smith d, cyr t, farnsworts a, he r, hurt ac, brown eg, li x (2013) universal anti-neuraminidase antibody inhibiting all influenza a subtypes. antiviral. res. 100: 567-574. fakruddin m, mohammad mr, bin mannan ks, chowdhury a, hossain mn (2012) critical factors affecting the success of cloning, expression, and mass production of enzymes by recombinant e. coli. isrn biotechnol. 13: 590587. farliahati mr, ramanan rn, mohamad r, puspaningsih nnt, ariff ab (2010) enhanced production of xylanase by recombinant escherichia coli dh5α through optimization of medium composition using response surface methodology. ann. microbiol. 60: 279-285. ghosalkar a, sahai v, srivastava a (2008) optimization of chemically defined medium for recombinant pichia pastoris for biomass production. bioresour. technol. 99: 7906-7910. hadházi á, li l, bailly b, maggioni a, martin g, dirr l, dyason jc, thomson rj, gao gf, borbás a, ve t, pascolutti m, von itzstein m (2018) a sulfonozanamivir analogue has potent anti-influenza virus activity. chemmedchem 13: 785-789. horga lg, halliwell s, castiñeiras ts, wyre c, matos cfro, yovcheva ds, kent r, morra r, williams sg, smith dc, dixon n (2018) tuning recombinant protein expression to match secretion capacity. microb. cell fact. 17:199. jing k, tang y, yao c, del rio-chanona ea, ling x, zhang d (2018) overproduction of l-tryptophan via simultaneous feed of glucose and anthranilic acid from recombinant escherichia coli w3110: kinetic modeling and process scale-up. biotechnol. bioeng. 115: 371-381. kangwa m, yelemane v, polat an, gorrepati kdd, grasselli m, fernández-lahore m (2015) high-level fed-batch fermentative expression of an engineered staphylococcal protein a based ligand in e. coli: purification and characterization. amb expr. 5: 1-10. kendall pa (1963) use of the ninhydrin reaction for quantitative estimation of amino groups in insoluble specimens. nature 197: 1305-1306. kleman g, strohl w (1994) acetate metabolism by escherichia coli in high-cell-density fermentation. appl. environ. microbiol. 60: 3952-3958. larentis al, nicolau jfmq, argondizzo apc, galler r, rodrigues mi, medeiros ma (2012) optimization of medium formulation and seed conditions for expression of mature psaa (pneumococcal surface adhesin a) in escherichia coli using a sequential experimental design strategy and response surface methodology. j. ind. microbiol. biotechnol. 39: 897-908. lee hw, lee hs, kim cs, lee jg, kim wk, lee eg, lee hw (2018) enhancement of l-threonine production by controlling sequential carbon-nitrogen ratios during fermentation. j. microbiol. biotechnol. 28: 293-297. miller gd (1959) use of dinitrosalicyl acid regent for determination of reducing sugar. anal. chem. 31: 426428. nallaiyan s, abbadorai rsaj, sundaramoorthy s, nelson j, veluvarthy s, sanyasi v (2010) production and application of recombinant haemagglutinin neuraminidase of newcastle disease virus. asian pac. j. trop. med. 3: 629-632. nikerel ie, öner e, kirdar b, yildirim r (2006) optimization of medium composition for biomass production of recombinant escherichia coli cells using response surface methodology. biochem. eng. j. 32: 1-6. o'kennedy rd, ward jm, keshavarz-moore e (2003) effects of fermentation strategy on the characteristics of plasmid dna production. biotechnol. appl. biochem. 37: 83-90. pinhal s, ropers d, geiselmann j, de jong h (2019) acetate metabolism and the inhibition of bacterial growth by acetate. j. bacteriol. 201: e00147-19. potvin g, ahmad a, zhang z (2012) bioprocess engineering aspects of heterologous protein production in pichia pastoris: a review. biochem. eng. j. 64: 91-105. rosano gl, ceccarelli ea (2014) recombinant protein expression in escherichia coli: advances and challenges. front microbiol. 5: 172. scheidle m, dittrich b, klinger j, ikeda h, klee d, büchs j (2011) controlling ph in shake flasks using polymer-based controlled-release discs with pre-determined release kinetics. bmc biotechnol. 11: 25. sekar a, shin y, jeong h, kim k (2018) statistical optimization of culture medium to produce recombinant viral protein by escherichia coli host for diagnostic kit to detect human immunodeficiency virus (hiv) infection. biochem. biophys. res. commun. 4: 666-671. udommaneethanakit t, rungrotmongkol t, frecer v, seneci p, miertuš s, bren u (2014) drugs against avian influenza a virus: design of novel sulfonate inhibitors of neuraminidase n1. curr. pharm. des. 20: 3478-3487. wang j, wen b, xu q, xie x, chen n (2015) optimization of carbon source and glucose feeding strategy bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:20 utc nova biotechnol chim (2019) 18(2): 84-93 93 for improvement of l-isoleucine production by escherichia coli. biotechnol. biotechnol. equip. 29: 374-380. wang j, yan d, dixon r, wang yp (2016) deciphering the principles of bacterial nitrogen dietary preferences: a strategy for nutrient containment. mbio. 7: e00792-16. wood wn, smith kd, ream ja, lewis lk (2017) enhancing yields of low and single copy number plasmid dnas from e. coli cells. j. microbiol. methods 133: 46-51. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:20 utc nova biotechnol chim (2018) 17(2): 181-192 doi: 10.2478/nbec-2018-0019  corresponding author: atmanea@gmail.com nova biotechnologica et chimica treatment of produced water by induced air flotation: effect of both tween 80 and ethanol concentrations on the recovery of pahs salima chebbi1, atmane allouche2,, marian schwarz3, souhila rabhi1, hayet belkacemi1 and djoudi merabet1 1materials technology and process engineering laboratory, university of bejaia, targa ouzemmour road, bejaia, 06000, algeria 2applied hydraulics and environment research laboratory, university of bejaia, targa ouzemmour road, bejaia, 06000, algeria 3department of environmental engineering, faculty of ecology and environmental sciences, technical university in zvolen, t. g.masaryka 24, zvolen, sk-960 53, slovak republic article info article history: received: 1st august 2018 accepted: 18th september 2018 keywords: environment ethanol induced air flotation polycyclic aromatic hydrocarbons produced water tween 80 abstract the present study investigates the application of induced air flotation (iaf) technique on pahs (pahs) removal performance from a real oilfield produced water of a separator cell. the quantification of total pahs (pahtot) was done using ultraviolet-visible spectrometry (uv-vis) according to the naphthalene calibration curve. the uv-vis spectra of naphthalene dissolved in a mixture of the binary solvent (water-ethanol) and the tween 80 showed stability in the molecular orbital of c10h8. the use of small concentration of tween 80 was revealed to be discrete in the quantification of pahtot. the flotation process was improved at the critical micelle concentration of tween 80 (cmc) of 2 % and the critical coalescence concentration of ethanol (ccc) of 0.5 ml/l for the pahtot recovery of 49.76 % and the pahtot content in the pulp of 50.24 %. at these concentrations, half of pahtot was removed from produced water pw. above the cmc and the ccc, the pahtot recovery decreased and the pahtot content in the pulp increased. it was found that there is a collector concentration at which the amount of water carrying from the pulp to the concentrate was increased and in parallel, the pahtot recovery increased and the pahtot content in the pulp decreased. both of the cmc and the ccc have promoted the decrease on the conditioning time from 30 to 10 min and the flotation time from 20 to 6 min. since the impeller speed and air flow rate were constant, the flotation of pahs was limited. the flotation kinetics of pahtot was described by the higuchi model.  university of ss. cyril and methodius in trnava introduction produced water (pw) represents the largest volume waste stream in oil and gas production operations (el-kayar et al. 1993; pardue et al. 2014). it is common that oil well production fluids in the old reservoirs are eventually composed of 90 % or more of water and only 10 % or less of hydrocarbons (edwan and qomarudin 2015). pw properties can even be varying throughout the lifetime of the reservoir (alanezi et al. 2013; akinwumi 2013; hagström et al. 2016). concentrations of total polycyclic aromatic hydrocarbons (pahtot), the main toxics and persistent in the marine environment (fisner et al. 2013), typically range from about 0.040 to about bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:39 utc nova biotechnol chim (2018) 17(2): 181-192 182 3 mg/l (lee and neff 2011; chebbi et al. 2016) and consist primarily of the most water-soluble congeners, the 2and 3-ring pah, such as naphthalene, phenanthrene (lee and neff 2011). marine animals near a pw discharge may bioaccumulate pahtot from the ambient water, their food, or bottom sediments (lee and neff 2011; almeda et al. 2013). therefore, it is important to treat the pw intended for sea disposal on the platform or at a shore treatment facility. induced air flotation (iaf) is an efficient process for removing various organic pollutants from pw (el-kayar et al. 1993; zheng and zhao 1993; painmanakul et al. 2010). according to its fundamental principles, very fine air bubbles have to be produced in the liquid to enable them to adhere to the hydrophobic particles and to float them to the surface (sylvester and byeseda 1980; li and tsuge 2006). the surface of the dispersed particles has to be hydrophobic by nature or be made hydrophobic by collectors (wang et al. 2010). tween series are known for their assistance in separating such pollutants such as hydrophobic organic compounds from water and soils (hoseini et al. 2013; lau et al. 2014; roodbari et al. 2016; gharibzadeh et al. 2016). their low toxicity and acceptable degree of biodegradability are responsible for their extensive utility (szymczyk and taraba 2016). however, flotation process requires formation of a froth layer that is to some extent stable. bubbles can collide and coalesce into larger bubbles but large bubbles are ineffective for flotation. they rise rapidly creating unfavorable turbulence and break-up of bubble-particle agglomerates (moosai and dawe 2003; bournival et al. 2015. literature studies indicated that the use of frothers increases air dispersion into fine bubbles, and reduces in coalescence of individual bubbles and decreases in the rate at which the bubbles rise to the surface (gupta et al. 2007). it is known that the flotation of particles is automatically accompanied by the recovery of water. furthermore, as the formed bubbles are large, the amount of recovered water is important. decreasing the amount of the entrained water is an important research issue in flotation process, since the large amounts of entrained water alter the oil quality. moyo et al. (2007) reported that alcohol frother entrained less amount of water. a key factor in effective flotation is strong attachment of the contaminant to the air bubble (wang et al. 2010). chemicals may be added to assist the attachment (wang et al. 2010; ofori et al. 2012). if there is such chemical intervention, the type of chemicals added, and their concentrations are important. if a full understanding of the physical processes can be gained by operators, the optimal conditions for a particular throughput and contaminant concentration reduction may be more successfully established. thence operating efficiency may be increased, and costs minimized (moosai and dawe 2003). in this research, ultraviolet–visible spectrometry (uv-vis) was employed to probe the interaction of naphthalene and the binary solvent (waterethanol). the effect of both tween 80 and ethanol concentrations on the flotation of pahs from a real oilfield pw were investigated. the flotation kinetics was studied through a kinetics experiment. first order kinetics and higuchi models were applied to describe and evaluate the flotation of pahs. uv-vis spectrometry was investigated to quantify pahtot. the main objectives of this study were to evaluate the iaf performance. experimental sites and sampling the real oilfield pw was obtained from sonatrach company of bejaia, algeria. samples were collected in situ, at the outlet of hydrocarbon storage tank, and stocked in amber glass bottles, in order to prevent the photodegradation of organic matters. chemicals the hydrochloric acid (hcl) 35 ‒ 37 % (v/v) and the nonionic collector (tween 80) were obtained from biochem chemopharma, montreal. ethanol (c2h6o) 96 % (v/v) was obtained from sigmaaldrich. naphthalene (c10h8) was obtained from panreac quimica sa (spain). experimental setup tween 80 was used as a nonionic collector. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:39 utc nova biotechnol chim (2018) 17(2): 181-192 183 the frother employed was ethanol (at 96 %). precise amounts of tween 80 were dissolved in ethanol to produce a collector-frother solution. by varying the concentrations of tween 80 in ethanol from 0.5 to 20 % and of the quantity of collector-frother solution from 0.2 to 1.5 ml added to the flotation cell, the effect of a wide range of tween 80 and ethanol concentrations on the pahtot recovery from pw by iaf could be tested. both of tween 80 and 96 % ethanol solutions and mixture were prepared. naphthalene in 96 % ethanol and a mixture of naphthalene, tween 80 and 96 % ethanol were prepared at known dosages. these different systems were analyzed by uv-vis spectrometry. the flotation tests were conducted on a real oilfield pw using a laboratory type denver flotation of 1 l capacity under the following conditions: room temperature, ph 2, impeller speed: 750 revolutions per minute (rpm), conditioning time: 10 min flotation time: 12 min. the pulp was introduced in the flotation cell of 1 l and was conditioned, during 10 min, with tween 80-ethanol during 5 min after 5 min of stirring at an appropriate dosage. air was introduced in the pulp by opening the air valve and the froth floated as a concentrate was collected during 12 min. the pahtot recovery and its content in the pulp were determined using uv-vis spectrometry analysis and then, optimal collector-frother concentrations were determined. the flotation kinetics of pahtot from a real oilfield pw at these optimal concentrations was studied. the flotation tests were done as following: after conditioning, air was injected; 2 ml of samples were taken from the froth each 2 min of flotation until the end of the process for two different condition times (5 and 10 min) and then, pahtot concentrations were determined by uv-vis spectrometry analysis. data analysis measurement of pahtot by uv-vis spectrometry samples were centrifuged for 5 min at 6,000 rpm. the dilutions were made on 0.5 ml of samples and transferred into glass pill dispensers to which were added 0.5 ml of 96 % ethanol in order to form water-96 % ethanol mixtures (v/v) for analysis by uv-vis spectrometry, similarly it has been established the naphthalene calibration curve in water-96 % ethanol (v/v). the calibration curve for naphthalene was established by obtaining optical densities on a series of dilutions made from the 100 mg/l solution in water-96 % ethanol mixture (v/v), at a wavelength of 220 nm (mistry 2009) (coefficient of regression, r2 = 0.999), measured on a shimadzu spectrometer. naphthalene is a hydrophobic compound with low solubility in water. samples were calculated from concentration dosages determined from optical densities measured at a wavelength of 220 nm, on the calibration curve for naphthalene. concentration analysis was carried out for the recovered pahtot. calculation of the pahtot recovery was done as following (lau et al. 2013), as in (eq. 1): (1) where ci and cp: concentrations of pahtot in the pw and the pulp, respectively. vi and vp: volumes of the pw and the pulp, respectively. regression model of the data is obtained using origin 6.0 scientific plotting software. kinetics the mathematical description of the effect of both cmc and ccc on the flotation of pahtot according to time can be represented by supposing the firstorder kinetics and the higuchi model (ramaswamy et al. 2007; siepmann and peppas 2011). where k (min-1) is the flotation rate constant. kh is the flotation rate constant for the higuchi model; ct (mg/l) is the concentration of the pahtot at any time and cf (mg/l) is the final concentration of the pahtot at the end of the flotation process above which pahtot concentrations weren’t appreciable. the criterion for selecting the most appropriate model was based on a goodness-of-fit test. regression model of the data is obtained using origin 6.0 scientific plotting software. (2) (3) higuchi model ) first-order bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:39 utc nova biotechnol chim (2018) 17(2): 181-192 184 results and discussion uv-vis analysis pah molecules are extremely stable in their neutral and ionized forms (crawford and tielens 1985; salama and allamandola 1995). uv-visible spectra of small pahs (2 or 3 rings) show strong absorptions in the ultraviolet corresponding to the * transitions and exhibit characteristic absorption bands of conjugated aromatic bonds with considerable fine structure, centered at “b” band with less intensity at 276 nm, due to the associated of the electronic transition with the vibration movement which disturb the symmetrical structure of the aromatic cycle (salama and allamandola 1995). the second band more intense at 220 nm is due to the more rigid and symmetrical structure state of naphthalene (salama and allamandola 1992; salama and allamandola 1993; salama and allamandola 1995). the uv–visible spectra were used for qualitative and quantitative analysis (monakhova et al. 2010; weide et al. 2015. therefore by measuring the uv absorbance of a sample containing pahs, one can quantify the total hydrocarbons (pahtot) which can be obtained via calibration (lee and neff 2007). a series of operations on data carried out on qualitative uv-visible analysis of 96 % ethanol and both naphthalene (c10h8) and tween 80, in the same solvent; at known concentrations were established. uv spectra were recorded over the wavelength range of 200 – 400 nm using single-beam uv-visible spectrometer (spectroscan 30). fig. 1 shows the uv spectra of 96 % ethanol, utilized as solvent, and c10h8 (1 mg/l) dissolved in 96 % ethanol. the reference solutions used during the scanning of both 96 % ethanol and c10h8 samples were water, 96 % and absolute ethanol, respectively. data from the uv spectrum of 96 % ethanol (1) show that the absorption of ethanol of uv light ranges from 200 – 287 nm, with both a considerable and moderate decrease in absorption between 200 – 210 nm and 210 – 240 nm, respectively, until reaching a zero absorbance at 287 nm. the full-scan uv detection of c10h8 sample in 96 % ethanol (2), with water as reference, shows 200 220 240 260 280 300 320 340 360 380 400 0,0 0,4 0,8 1,2 1,6 2,0 2,4 2,8 a b s o rb a n c e wavelength (nm) ethanol at 96% (1) reference: water naphthalene (1mg/l) in ethanol at 96% (2) reference: water naphthalene (1mg/l) in ethanol at 96% (3) reference: ethanol at 96% naphthalene (1mg/l) in ethanol at 96% (4) reference: ethanol absolute "e" band of c 10 h 8 220 nm "b" band of c 10 h 8 276 nm (1) (2) (3) (4) fine structure fig. 1. uv spectra of naphthalene (c10h8) (1 mg/l) dissolved in 96 % ethanol. in 96 % ethanol (2), with water as reference, shows the spectrum overlapping of ethanol absolute and c10h8 in the uv region where they absorb. furthermore, the shape of the three spectra of c10h8 samples is similar and no solvent effect is observed on the absorption wavelengths, except for the existence of a shift of the spectrum (2) towards higher values of absorbance, compared with the spectra with respect to 96 % (pardue et al. 2014) or absolute (4) ethanol. this difference is due to the absorption of ethanol with respect to water. in accordance with the full-scan uv detection of both c10h8 samples and 96 % ethanol, the intensity of the reference beams have suffered no light absorption. furthermore, the absorption spectra reveal that ethanol and water (polar solvents) did not destabilize the molecular orbital of c10h8 molecule. the obtained results show that despite the presence of the c10h8 molecules in a binary solvent (water-ethanol), the use of two or of one of them as reference did not influenced the shape of the obtained spectrum of the c10h8 molecules. fig. 2. molecular structure of tween 80. indexes x, y, z, and w at chromophore groups were selected as 5 (karjiban et al. 2012). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:39 utc nova biotechnol chim (2018) 17(2): 181-192 185 200 220 240 260 280 300 320 340 360 380 400 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 a b s o rb a n c e wavelength (nm) 1) tween 80 in 96% ethanol (1ml/l) reference: 96% ethanol 2) tween 80 in 96% ethanol (1ml/l) reference: water 3) tween 80 in 96% ethanol (1ml/l) reference: ethanol absolute 1 2 3 238 nm 229,5 nm 204 nm fig. 3. uv spectra of tween 80 (1 ml/l) dissolved in 96 % ethanol. the molecular structure of tween 80 molecule (fig. 2) is characterized by a chromophore group in their structure. it is an ester with system “” (c=o) conjugated with the two electron “n” in the auxochrom group (oxygen of carboxylate in  position of carbonyle) corresponding to the n* transition; it absorbs in the uv range (wuelfing et al. 2006). the uv absorption spectra (fig. 3) of tween 80 (1 ml/l) dissolved in 96 % ethanol were similar with both 96 % ethanol (1) and absolute ethanol (2) as reference solutions. the shapes of curves (obtained at 238 nm) suggest no effect of solvent. for the absorption spectrum of tween 80 (1 ml/l), with water as reference (2), we noticed two absorption bands. the first ranged from 200 – 210 nm and peaked at 204 nm, and the second ranged from 210 – 287 nm and peaked at 329.5 nm. the absorption band in the range of 200 – 210 nm is relative to ethanol, as shown in fig. 1. therefore, the choice of reference solution played an important role in both qualitative and quantitative analysis by molecular spectrometry. the absorption spectra of c10h8 (1 mg/l), tween 80 (1 ml/l) and c10h8-tween 80 in 96 % ethanol, with 96 % ethanol as a reference, are shown in fig. 4. for c10h8 (1 mg/l): in the wavelength range covered in our experiments (200 – 400 nm), c10h8 shows two distinct band systems with very different intensities. system i, peaking at 220 nm, is very strong. system ii is very weak and peaks at 276 nm. we also note that c10h8 may contribute 200 220 240 260 280 300 320 340 360 380 400 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 276 nm 220 nm 238 nm a b a b s o rb a n c e wavelength (nm) a) naphthalene (1 mg/l) in 96% ethanol b) naphthalene (1 mg/l)-tween 80 (1 ml/l)in 96% ethanol reference: 96% ethanol fig. 4. uv spectra of: a) c10h8 (1 mg/l) and b) c10h8 (1 mg/l)-tween 80 (1 ml/l) in 96 % ethanol. to the 220 nm bump (salama and allamandola 1992; salama and allamandola 1995). studies carried out by salama and allamandola (1992) strongly attest that the continuum observed in the wavelength range of 200 – 232 nm is due to the neutral naphthalene (c10h8), and in the wavelength range of 232 – 296 nm is due to fine structure of naphthalene. furthermore, there is one way in which one can examine the possible quantification of the c10h8. one extreme is to only require that the strongest c10h8 band be present (salama and allamandola 1992; lee and neff 2007). for c10h8 (1 mg/l) – tween 80 (1 ml/l): the spectrum of c10h8-tween 80 mixture in 96 % ethanol shows two distinct band systems with very different intensities. system i, peaking at 238 nm, is very strong. system ii is very weak and peaks at 276 nm. from the obtained results by the fullscan uv detection, we show that the absorption band of c10h8 (1 mg/l) is masked by that of tween 80 (1 ml/l). likewise, no effect is observed on the absorption wavelength of tween 80 (1 ml/l), 238 nm. fig. 5 shows that with the decrease in the concentration of tween 80 from 1 ml/l to 10-2 ml/l, no absorption band is observed in the uv region. however, the c10h8 (1 mg/l) – tween 80 (10-2 ml/l) full-scan uv detection showed clearly the absorption band of c10h8 at 220 nm, as was shown in fig. 1. these results have showed that the use of small concentrations of tween 80 for the flotation of pahs from pw will not affect the quantification of these organic compounds. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:39 utc nova biotechnol chim (2018) 17(2): 181-192 186 200 220 240 260 280 300 320 340 360 380 400 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 d c b 238 nm 220 nm b) naphthalene 1mg/l-tween 80 1ml/l in 96% ethanol c) tween 80 in 96% ethanol (10 -2 ml/l) d) naphthalene (1mg/l)-tween 80 (10 -2 ml/l) in 96% ethanol reference: 96% ethanol a b s o rb a n c e wavelength (nm) fig. 5. uv spectra of: b) c10h8 (1 mg/l)-tween 80 (1 ml/l), c) tween 80 (10-2 ml/l) and d) c10h8 (1 mg/l)-tween 80 (10-2 ml/l) in 96 % ethanol. effect of tween 80 concentration on the flotation of pahtot the adhesion between pah molecules and bubbles can be stimulated by adding collectors. tween 80, used as a non ionic collector, may adsorb onto the surface of the pahs. in aqueous medium, the hydrophobic effect of the carbon chains of tween 80 molecules leads to their orientation from water toward the center, forming the core of the micelle with the surface being dominated by the polar hydrophilic groups (karjiban et al. 2012). thereby, the hydrophobic groups of the free tween 80 molecules would tend to adsorb on the pahs surface by reducing the surface tension between pah-water interfaces. some pahs molecules would be trapped within the micelles formed by the tween 80 molecules and some are attached to the rising bubbles (mouton et al. 2008). the effect of tween 80 on the recovery of pahtot from pw was shown in fig. 6 as a function of concentrations. flotation tests were carried out at tween 80 concentrations ranging from 0.5 to 20 %, to recover pahtot from pw. ethanol, used as frother, was held constant in all of these tests at test dosage of 0.5 ml/1,000 ml of pw, since it was related that the flotation efficiency is obtained at lower alcohol concentrations (kowalczuk 2013; merkus 2016). it was found that frothers not only prevent bubble coalescence, but also, affect the bubble break-up process (gupta et al. 2007). edzwald (2010) reported that collectorbubble collision efficiency mechanism depends 0 2 4 6 8 10 12 14 16 18 20 22 20 30 40 50 60 70 80 y ie ld o f p a h to t ( % ) c tw80 (%) recovery efficiency (%) content in the pulp (%) fig. 6. effect of tween 80 concentration on the flotation of pahtot. strongly on bubble size, and increases with decreasing bubble size. data show that the tween 80 concentration exhibited a significant effect on the recovery of pahtot by iaf. the first micelle formed in the flotation cell is introduced as a critical micelle concentration (cmc) (painmanakul et al. 2010). at this concentration, the tween 80 molecules will start to aggregate and form micelles (moosai and dawe 2003; xu et al. 2009). the tween 80 concentration of 2 % was chosen as the cmc as a result of these experiments for both optimal pahtot recovery of 49.76 % and pahtot content in the pulp of 50.24 %. half of pahs were removed from pw. xu et al. (2009) reported that the properties of the surfactant vary markedly below and above the cmc. indeed, at tween 80 concentration below the cmc, we have observed a decrease in the pahtot recovery and an increase in the pahtot content in the pulp. it was reported that a collector at very small quantities is able to change the surface properties of particles, often with little to no influence on any surface properties (moosai and dawe 2003). practically no micelles form below the cmc, the tween 80 appear as soluble macromolecules in the aqueous medium and cannot interact with pahs (baziar et al. 2013). adsorption at the air-water interface is a function of the tween 80 concentration. the main interactions between tween 80 and pahs take place around the cmc. above the cmc, addition of tween 80 has no effect on surface tension (mouton et al. 2008). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:39 utc nova biotechnol chim (2018) 17(2): 181-192 187 0 2 4 6 8 10 12 14 16 18 20 22 0 100 200 300 400 500 600 700 800 900 1000 w a te r re c o v e ry ( m l ) c tween 80 (%) pulp concentrate fig. 7. effect of tween 80 concentration on water recovery. as the tween 80 concentration was increased above the cmc a trend of decreasing on the pahtot recovery was observed and at the same time the pahtot content in the pulp was increased up to the tween 80 concentration of 12 %. it is well reported that increasing collector concentration leads to an increase in surface adsorption and a corresponding decrease in surface tension (mouton et al. 2008). indeed, above the cmc, additional tween 80 molecules added to the pulp will simply form micelles, as this pathway is now energetically favored (shah et al. 2016). therefore, the surface tension of the liquid phase is essentially constant after the cmc and generally no further pah molecules will be driven to the surface of the flotation cell up to the tween 80 concentration above 12 % where we have observed a sudden increase in the pahtot recovery and decrease in the pahtot content in the pulp. the pahs recovery by iaf is related to the recovery of water. while the primary task is to collect particles, bubbles also transport water. moyo et al. (2007) have reported that the amount of water entrained depends on bubble size. it is known that large bubbles have a high rising velocity, so that the residence time in the flotation cell is shorter (sawyerr et al. 1998; cho and laskowski 2002). data from fig. 8 show that the amount of water carried by the bubbles into and through the froth was practically constant from the cmc to 12 % of tween 80 concentration, and was 0,0 0,5 1,0 1,5 0 10 20 30 40 50 60 70 80 90 100 y ie ld o f p a h to t (% ) ethanol concentration (ml) recovery efficiency (%) content in the pulp (%) fig. 8. effect of ethanol concentration on the flotation of pahtot. increased as the tween 80 concentration was increased above 12 %. it was also reported that as bubble rise velocity increased, the probability of collision between pah molecules and bubbles decreased because of the shorter residence time in the flotation cell (edzwald 2010; shean and cilliers 2011). but in this study, we have observed (fig. 6) that as the tween 80 concentration was increased above 12 %; the recovery of pahtot from pw was increased while the pahtot content in the pulp was decreased. it is believed that the high bubbles rise velocity, from the mixing zone to the froth zone, occurred after a rapid attachment of pah-tween 80 aggregates to bubbles in the pulp; these bubbles ascend and form stable froth height at the surface of the flotation cell. with the continuous rising of ascending bubbles, water surrounding the bubbles and in the wake of ascending bubbles continuous to transfer across the pulp/froth interface to the froth and then to the concentrate. wang et al. (2016) have established that there was an increase in the amount of water recovery from the pulp to the froth when increasing impeller speed, especially in the context of highpresence of solid particles. the obtained results (fig. 7) reveal that there was an increase in the amount of water recovery from the pulp to the concentrate when reaching a certain collector concentration (12 %) above the cmc. this phenomenon leads to the increase in the pahtot flotation and in the same time affects the concentrate quality. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:39 utc nova biotechnol chim (2018) 17(2): 181-192 188 0,0 0,3 0,5 0,8 1,0 1,3 1,5 1,8 0 100 200 300 400 500 600 700 800 900 1000 w a te r re c o v e ry ( m l ) ethanol concentration (ml/l) pulp concentrate fig. 9. effect of ethanol concentration on water recovery. pulp (circles) and concentrate (squares) are shown. effect of ethanol concentration on the flotation of pahtot in flotation systems, bubble size is one of the key factors controlling the performance of the process. there is a primary process that generates fine bubbles, and a secondary process that is coalescence. coalescence requires the out flow of liquid between adjacent bubbles, and as bubble size increases the strength of the water streamline around the bubble increases, making collision between particles and bubbles more difficult. the adsorption of alcohols on the bubble surface retards coalescence and also affects the bubble rise velocity from the mixing zone to the froth zone (cassel et al. 1975; azgomi et al. 2007). the fig. 8 gives the flotation of pahtot at ethanol concentrations varying from 0.2 through 1.5 ml/l. the cmc of tween 80 was held constant in all of these runs. ethanol molecules accumulate preferentially at the air-water interface and interact with pah-tween 80 aggregates in the pah-tobubble collision and attachment. data showed that at 0.5 ml/l of ethanol, the optimum pahtot recovery was about 48.65 % for the pahtot content in the pulp of 51.35 %. at this concentration, we arrived to recover half of the content of pahtot in the produced water. this seems to imply that a considerable part of ethanol penetrated effectively into the interfacial film, which stabilizes bubble size in the mixing zone and both increases collision rate with pahtot molecules 2 4 6 8 10 12 14 10 15 20 25 30 p a h to t c o n c e n tr a ti o n ( m g /l ) flotation time (min) conditioning time 10 min 5 min fig. 10. effect of both cmc and ccc on the flotation of pahtot at different conditioning times: 5 and 10 min and helps enable stable froth to form, that brings the collected pahtot to pass into the froth zone and to overflow from the cell. kowalczuk (2013) reported that bubble size decreases with increasing ethanol amount to a certain concentration, critical coalescence concentration (ccc), above which bubble size remains approximatively constant, and at the ccc bubble coalescence is completely prevented. according to these results, the ccc of ethanol was of 0.5 ml/l. above the ccc, we noticed the decrease in the pahtot recovery from pw and simultaneously an increase in the pahtot content in the pulp. cheng et al. (2016) related that when bubbles are too small, particles may have insufficient contact time to attach to the bubbles, or if attachment does occur, the bubbles buoyancy may be low for considerable recovery. effectively, above the ccc, the recovery of pahtot is lower and approximatively constant, 23.69 and 23.55 % for 1 ml/l and 1.5 ml/l, respectively. furthermore, the pahtot content in the pulp is higher and approximatively constant. this means that the pah-bubble attachment was occurred, but because of the constant bubble size, the very low bubble rise velocity from the mixing zone to the froth zone the pahtot recovery was low. below the ccc, the pahtot recovery was not important (10.95 %), and the pahtot content in the pulp was higher (89.05 %). this can be explained by the fact that smallest concentration of ethanol didn't prevented coalescence and therefore large bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:39 utc nova biotechnol chim (2018) 17(2): 181-192 189 bubbles were formed and bubble rise velocity was higher, because of these, the possibility of collision between bubbles and pah molecules is decreased. data from fig. 9 show that the amount of water carried by the bubbles from the pulp to the concentrate was decreased as the ethanol concentration was increased. moyo et al. (2007) have established that the adsorption of alcohol on the bubble surface leads to the transport of less water into the froth. in this study, little amounts of water were carried by bubbles into the concentrate at all ethanol concentrations. gupta et al. (2007) believed that the continued addition of ethanol above the ccc must leads to a very little effect on bubble size which leads automatically, in the same conditions, to a probable fixed amounts of water recovery. however, in this study, we have observed that, despite the increase on ethanol concentration above the ccc, the amount of water recovery into was decreased. this is probably related to the impact of the high-presence of pah molecules (fig. 8) on the water flow in the froth. effect of both cmc and ccc on the flotation kinetics of pahtot the obtained results were presented to study the effect of both cmc and ccc on the flotation of pahtot at different conditioning times: 5 and 10 min (fig. 10). the results showed for the two different conditioning times a high flotation of pahtot from pw during the initial 6 min, and then a decrease followed by stabilization. the concentration of pahtot increases with flotation time and approached an asymptote. it can be noted that the increase in pahtot concentration of was marginal if the flotation time was extended beyond 6 min. hence, 6 min may be considered as the optimal flotation time of pahtot. addition of both tween 80 and ethanol at the cmc and the ccc, respectively, significantly increased the kinetics of flotation of pahtot. it can be seen that the presence of tween 80 and ethanol at these concentrations has lead to the decrease in both flotation time of pahtot from 20 to 6 min and conditioning time from 30 to 10 min (chebbi et al. 2016) for the two conditioning times. fig. 11. effect of both cmc and ccc on the flotation kinetics of pahtot at different conditioning times as a function of concentration. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:39 utc nova biotechnol chim (2018) 17(2): 181-192 190 the formation of the froth and its subsequent removal are the final two steps in flotation. phenomena that occur in the froth phase are known to affect significantly the results of the whole process. the concentration of pahs in the uppermost pulp layers is of interest. above 6 min of flotation, pahtot molecules fall dawn in the bottom of the flotation cell and then stabilize until the end of the process. this behavior might be because of a change in froth structure caused by variation in the flotation of pahs from the pulp to the concentrate. furthermore, for a fixed flotation reagents concentrations (cmc and ccc), micelle concentration and bubble size were fixed, and since the impeller speed and the air flow rate were also kept fixed in this study, the flotation of pahtot was limited even though the flotation time has considerably decreased. fig. 11 shows a linearization of the flotation of pahtot according to the concentration for each supposed kinetics model. the following concentrations were evaluated for each conditioning time: 5 and 10 min. the data was plotted for comparison. it can be seen that the higuchi model showed a higher flotation rates of 0.07621 and 0.12036 min-1 over time for the conditioning times of 5 and 10 min, respectively. since, the first-order kinetics showed a slower flotation rates over time of 0.02049 and 0.03375 min-1 for the conditioning times of 5 and 10 min, respectively. the two supposed kinetics models assumes that the pah-bubble collision rate and bubble concentration remain constant over time. higuchi model for pah/water separation by iaf has also been found by the previous work (chebbi et al. 2016), and the optimal conditioning time in this study and under these operational conditions was found to be around 10 min as has been illustrated by the flotation rate of 0.12036 min-1. conclusions according to the results, the binary solvent (waterethanol) and tween 80 failed to destabilize the molecular orbital of naphthalene. the use of two or one of the solvents as reference to quantify pahtot had no effect the analysis. the use of tween 80 at small concentrations has been proven discrete in the quantification of pahtot. it’s presence with lower concentrations promoted the flotation of pahs. the maximum flotation of pahtot was observed at the cmc of 2 % with a recovery of 49.76 % and a content in the pulp of 50.24 %. at this concentration, half of pahtot were removed. since the impeller speed and air flow rate were constant, the flotation of pahs was limited. the water carrying begins to become an undesirable product in the recovery of oil when it reaches a certain amount. above the cmc, large amounts of water were carried. therefore, pahtot recovery decreased and pahtot content in the pw increased. it was found that at a certain concentration of tween 80 (12 %), far above the cmc, the pahtot recovery start to increase and pahtot content in the pw to decrease. this phenomenon can mislead researcher about the cmc value. addition of ethanol greatly promote the flotation of pahs, the ccc was found to be around 0.5 ml/l. contrary to the cmc, the increase in the ethanol concentration at the cmc, above the ccc, has decreased the water recovery. the kinetics experiment on the effect of both cmc and ccc on the flotation process has showed that at these operational conditions the conditioning time was decreased from 30 to 10 min, while the flotation time was decreased from 20 to 6 min as found in the previous work. it was found that the flotation kinetics of pahtot was governed by the higuchi model. acknowledgements this work was supported by university abderrahmane mira of bejaia (algeria), sonatrach company (algeria) and technical university in zvolen (slovakia). the authors are grateful for their assistance. references akinwumi a (2013) rheological effect of produced water on crude oil flow in production tubing string: a case study of agbami oil field, nigeria. int. j. sci. res. 2: 229-233. alanezi k, belkharchouch m, alali s, abuhaimed w (2013) produced water characterization in kuwait and its impact on the environment. desalin. water treat. 51: 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10.36547/nbc.877 1 nova biotechnologica et chimica sequence analysis of chloroplast psba-trnh region in citrus l. (rutaceae) species from the aegean region of turkey emre sevindik1, , gaye zeynep canbolat1, i̇layda i̇rem moral1, monika sujka2 1department of agricultural biotechnology, faculty of agriculture, adnan menderes university, south campus, cakmar, aydin, turkey 2deptartment of analysis and food quality assessment, university of life sciences in lublin, 8 skromna st., lublin 20-704, poland  corresponding author: ph.d-emre@hotmail.com article info article history: received: 22nd february 2021 accepted: 6th october 2021 keywords: citrus cpdna psba-trnh sequence analysis abstract the aim of the study was to analyze the sequence of some citrus species found in the aegean sea region of turkey based on the psba-trnh cpdna region. genomic dna was isolated from healthy and green leaves. total genomic dna was extracted using the genemark dna isolation plant kit. the psba-trnh region of chloroplast dna was amplified using primers psba and trnh. dna sequences were edited using the sequencher 5.4.6 and sequencing data were analyzed using the mega 6.0 software. the maximum likelihood (ml) tree was created to determine the relationships between citrus taxa. amplified cpdna psba-trnh sequences ranged between 426 and 470 nucleotides. the maximum likelihood phylogenetic tree was composed of two clades. the divergence values differed between 0.000 and 0.012. in addition, the sequences of some species belonging to the rutaceae family were obtained from ncbi and a maximum likelihood tree was constructed. the phylogenetic relationship between the sequence data of some species belonging to the rutaceae family taken from ncbi and citrus species was revealed. © university of ss. cyril and methodius in trnava introduction rutaceae juss. is a large tree, bush and grass family comprising around 150 – 170 genera and nearly 2.040 species (morton and telmer 2014). the genus citrus l. belongs to the aurantioideae subfamily of the rutaceae family. citrus includes economically and commercially important fruit crops, major fruit plants of the world such as limons, mandarins, sour oranges, sweet oranges, squash, grapefruits, kumquats and others. (groppo et al. 2008; baig et al. 2009; kumar et al. 2013). it is believed that especially the region extending from the north eastern india to eastward, from primary main origins of the genus citrus are in south and southeast asia from malaya archipelago to china and japan, and from south to australia (jena et al. 2009). the fruits and leaves of citrus species contain a variety of essential oils containing biologically active compounds such as vitamin c, folic acid, potassium, flavonoid glycosides, coumarins, pectin, which are important for various nutrients and human diets (othman et al. 2016; bozkurt et al. 2017; elkhatim et al. 2018; liu et al. 2018). molecular analysis to be conducted on genetic variation among the individuals of a population mailto:ph.d-emre@hotmail.com nova biotechnol chim (2021) 20(2): e877 2 may offer a tool to monitor the genetic variability of a diminishing population and evaluate the genetic consequences of fragmentation on the remaining populations (al-qurainy et al. 2014). chloroplast dna (cpdna) particularly includes non-coding regions which are maternally hereditary in angiosperms and have rich variability. these regions are significantly applied in various studies in molecular phylogenetic, population genetics and protection with a more comprehensive perspective (chen et al. 2013; gutiérrez-lópez et al. 2016). the psba-trnh intergenic region is among the most variable regions of the chloroplast genome. this region is suitable for dna barcoding studies, consists of two parts in evolutionary conservation (štorchová and olson 2007; filiz and koç 2012; yılmaz 2020). the aim of the study was to determine the sequence analysis of selected citrus l. taxa using the psba-trnh cp (dna) sequence in order to explain the phylogenetic relationships between them. experimental collection of plant materials and isolation of genomic dna the fresh green leaves from 11 populations of five species of the genus citrus: c. aurantium l., c. limon (l.) burm. f., c. paradisi macfad., c. reticulata blanco, c. sinensis (l.) osbeck, were collected from aegean region (aydın, muğla, i̇zmir, manisa-salihli) in turkey (fig.1) and brought to the plant biotechnology laboratory at aydın adnan menderes university. genomic dna was isolated using the dnaeasy plant kit (genemark) based on the manufactures protocol. then the samples were stored at -20 °c. fig. 1. location of collecting places in the aegean region, turkey (https://www.google.com/maps). pcr amplification and sequencing the entire psba-trnh region was amplified using the biometra personal thermal cycler. the pcr reactions were performed using psba and trnh primers designed by sang et al. (1997) and tate and simpson (2003) (table 1) for amplification of the psba-trnh region of cpdna. the https://www.google.com/maps nova biotechnol chim (2021) 20(2): e877 7 amplification process was carried out in 25 µl of pcr reaction volume, which contained 5.0 µl master mix (0.75 u of taq dna polymerase, reaction buffer, 2 mm mgcl2, 250 µm dntp), 1 µl for psba and 1 µl for trnh primers, approximately 2.0 µl of total genomic dna, and 17 µl of ddh2o. gel electrophoresis in 0.8 % agarose gel run in 1.0x tris-borate-edta buffer was used to separate amplicons that were stained with ethidium bromide and visualized u s i n g a uv transilluminator (fig. 2). purified pcr products were sequenced at labbiotek (i̇zmir, turkey) using an abi 3130xl genetic analyser (applied biosystems, foster city, ca, usa) with a bigdye cycle sequencing kit (applied biosystems). dna sequences were edited both manually and by the sequencher 5.4.6 programs. sequences for individual samples were obtained through at least 3 independent sequencing runs whereas sequences for each taxon were based on at least 3 independent specimens. when all the sequences of a taxon were identical, only one sequence (based on one specimen) was included in the phylogenetic analysis. table 1. cpdna psba-trnh primers and pcr amplification. primers sequences (5’-3’) pcr amplification (35 cycles) psba 5’-gttatgcatgaacgtaatgctc-3’ (sang et al. 1997) 94 oc/5 min 94 oc/45 s 50 oc/45 s 72 oc/1 min 72 oc/10 min trnh 5’-cgcgcatggtggattcacaatcc-3’ (tate and simpson 2003) fig. 2. amplified cpdna psba-trnh region. aligment and phylogenetic analysis cpdna psba-trnh sequences were aligned using the mega 6.0 alignment software (tamura et al. 2013). sequences distance values and maximum likelihood (ml) trees were created using mega 6.0 (tamura et al. 2013). to evaluate the degree of support for given clades, a bootstrap analysis (1000 replicates) was applied (felsenstein 1985). additionally, tetradium austrosinense (kr533457.1), tetradium ruticarpum (mk419238.1), melicope degeneri (eu493198.1), melicope polybotrya (eu493202.1), clausena anisate (mk260868.1), clausena lenis (kr533453.1), murraya exotica (gq435449.1), murraya paniculata (gu135341.2), glycosmis lucida (kr533450.1), glycosmis pentaphylla (gq435452.1), zanthoxylum molle (mf070225.1), and zanthoxylum bungeanum (mf070227.1) psbatrnh sequences were obtained from ncbi and were used to reveal phylogenetic relationships among citrus species. results and discussion phylogenetics aims to reveal the evolutionary relationship between all groups of organisms in the form of ancestor-lineage relationships and has become a powerful tool and starting point in many areas of biology such as taxonomy, biogeography, and developmental genetics (wei et al. 2014; sarıçam and müştak 2015; sevindik and okan 2020). recently, phylogenetic has get into a rapidly expanding area thanks to major improvements in nucleic acid and protein sequencing techniques and analyses (patwardhan et al. 2014). the sequence analyses are very useful for phylogenetic analysis in cases when the morphological characters are insufficient for information. sequence analysis methods are used in a variety of 3 nova biotechnol chim (2021) 20(2): e877 2 fields ranging from detecting the geographic origins of the living organisms to finding molecular evidence of the phylogenies (inal et al. 2017). there are many studies in citrus using various molecular markers, e.g. rapd (coletta filho et al. 1998; nicolosi et al. 2000), issr (fang and roose 1997), aflp (al-nadabi et al. 2018), ssr (jannati et al. 2009), as well as chloroplast dna (nicolosi et al. 2000; penjor et al. 2010; wali et al. 2013; oueslati et al. 2016; uchoi et al. 2016; sevindik and yalçın 2018) and nuclear ribosomal dna (nrdna) markers (amar et al. 2014; sun et al. 2015). in this study, we obtained psba-trnh sequences ranging from 426 to 470 nucleotides for 11 specimens. the highest number of nucleotides was determined for citrus sinensis (aydın) (470 bp) w h i l e the lowest for citrus sinensis (bergama) (426 bp). average nucleotide composition of psba-trnh was 42.3 % t, 11.7 % c, 28.8 % a, and 17.2 % g. (table 2). table 2. length and a, t, g and c contents of cpdna psba-trnh sequences of citrus taxa. taxa psba-trnh a [%] t [%] g [%] c [%] c. paradisi (aydın) 455.0 28.8 42.0 16.9 12.3 c. limon (aydın) 452.0 29.0 42.7 17.5 10.8 c. sinensis (salihli) 450.0 28.7 42.4 17.1 11.8 c. sinensis (muğla) 464.0 28.4 41.8 17.9 11.9 c. sinensis (aydın) 470.0 28.5 41.5 17.9 12.1 c. reticulata (bergama) 466.0 29.2 41.0 17.6 12.2 c. reticulata (aydın) 448.0 29.5 41.5 17.6 11.4 c. aurantium (aydın) 453.0 28.9 41.5 17.2 12.4 c. paradisi (i̇zmir) 434.0 28.3 43.1 16.4 12.2 c. limon (muğla) 430.0 28.6 44.2 16.7 10.5 c. sinensis (bergama) 426.0 28.6 43.7 16.7 11.0 avg. 449.8 28.8 42.3 17.2 11.7 the total length of the aligned psba-trnh sequence matrix was 483 nucleotides. the genetic distance method based on psba-trnh set was performed with mega 6.0 software. the lowest sequence divergence among the ingroup taxa was 0.000 while the highest was 0.012 (table 3). table 3. pairwise sequence distances among some citrus taxa for cpdna psba-trnh sequences using mega 6.0 software distance matrix. species 1 2 3 4 5 6 7 8 9 10 11 c. paradisi (aydın) c. limon (aydın) 0.007 c. sinensis (salihli) 0.005 0.002 c. sinensis (muğla) 0.005 0.002 0.000 c. sinensis (aydın) 0.005 0.002 0.000 0.000 c. reticulata (bergama) 0.012 0.010 0.007 0.007 0.007 c. reticulata (aydın) 0.012 0.010 0.007 0.007 0.007 0.000 c. aurantium (aydın) 0.012 0.010 0.007 0.007 0.007 0.000 0.000 c. paradisi (i̇zmir) 0.002 0.005 0.002 0.002 0.002 0.010 0.010 0.010 c. limon (muğla) 0.010 0.002 0.005 0.005 0.005 0.012 0.012 0.012 0.007 c. sinensis (bergama) 0.005 0.002 0.000 0.000 0.000 0.007 0.007 0.007 0.002 0.005 using the mega 6 program, tajma's neutrality test (tajima 1989) was calculated based on psbatrnh sequences of citrus species. numbers of sequences (m) gave one segregation site (s) revealing very low nucleotide diversity (π) of 0.005690 (table 4). maximum likelihood (ml) tree was generated using psba-trnh sequences of certain citrus species distributed in turkey. it consists of two large clades. clade 1 is divided into two subclades, a and b. subclade a consists of c. paradisi (aydın), c. paradisi (i̇zmir), c. limon (muğla), c. limon (aydın) species, whereas all c. sinensis populations are placed in subclade b. clade 2 consists of c. reticulata (aydın), c. aurantium (aydın) and c. reticulata (bergama) species (fig. 3). 4 nova biotechnol chim (2021) 20(2): e877 7 table 4. tajima’s neutrality test values based on cpdna psba-trnh of date citrus species. no. of sequences “m” no. of segregating sites “s” ps=s/n θ = ps/a1 nucleotide diversity ‘’ π ‘’ tajima test statistic ‘’d’’ 11 7 0.017115 0.005843 0.005690 -0.106371 so far, many studies have been done on citrus using a wide range of gene regions and markers. wali et al. (2013) carried out the phylogenetic analysis of citrus species using cpdna rps14 genes. they situated c. reticulata and c. aurantium c. sinensis var. malta, and c. sinensis var. fruiter species in one branch, and c. limon and c. sinensis var. mousami species in another branch. in our study, c. reticulata and c. aurantium were detected in clade 2, whereas populations of c. sinensis and c. limon in clade 1. wali et al. (2013) concluded that rps14 gene sequence was highly protected in citrus species, and it did not provide much information to form the phylogeny of citrus species. sevindik and yalçın (2018) performed phylogenetic analysis of some citrus species widespread in the aegean region based on cp(dna) trnl intron and trnl-f sequences and they located c. aurantium (aydın), c. reticulata (bergama) and c. reticulata (aydın) species in the same branch of the phylogenetic tree (fig. 3). these three species were also situated in the same branch of our phylogenetic tree formed on the basis of psba-trnh sequences. analysis of trnl intron and trnl-f sequences revealed that c. limon (aydın) and c. paradisi (aydın) belong to a fig. 4. the maximum likelihood tree generated using cpdna psba-trnh sequences and other species sequences retrieved from ncbi. 5 fig. 3. the maximum likelihood tree generated using cpdna psba-trnh sequences. nova biotechnol chim (2021) 20(2): e877 2 subsidiary group, whereas c. limon (muğla) is situated in a separate branch. generally, according to our results, populations of the same species are found to be together. the study by sevindik and yalçın (2018) showed that, based on trnl intron analysis, c. paradisi (aydın) and c. paradisi (i̇zmir) populations are located in different branches, however the results of trnl-f analysis indicated that these populations are in the same branch. our analysis of psba-trnh sequence showed that both populations are in clade 1. in case of populations of c. sinensis species, the above mentioned authors reported that they belong to different branches, both in the trnl intron tree and in the trnl-f tree. in psba-trnh tree, all populations of all c. sinensis species were collected in one group. according to psba-trnh results, populations belonging to the same species form a group, while this did not happen with trnl intron and trnl-f sequences (sevindik and yalçın, 2018). uchoi et al. (2016) analysed phylogenetically citrus species distributed in india using rbcl and matk sequences. they applied the maximum parsimony (mp) and neighbor-joining (nj) methods. in the nj bootstrap consensus tree formed based on rbcl sequence, c. reticulata and c. sinensis were grouped together, while c. limon and c. aurantium species were in different groups. in turn, in the nj phylogenetic tree based on the matk sequences, c. aurantium and c. reticulate were in one group and the species c. sinensis and c. limon were in different groups. our research has shown that c. reticulata and c. aurantium species are located in clade 2, which is partially consistent with the nj tree formed with the matk sequence. sun et al. (2015) investigated the taxonomy and phylogeny of 26 citrus materials of 22 citrus species using sequence analysis of the its region of nrdna. in their research, c. aurantium and c. sinensis were found in one group, while c. limon belonged to a separate group. according to our results, c. aurantium populations belong to clade 2 and c. sinensis and c. limon populations to clade 1. however, the its sequences results obtained by sun et al. (2015) are not consistent with ours. penjor et al. (2010) determined the phylogenetic relations of citrus and its relatives using maximum parsimony and neighbour-joining methods and cpdna rbcl gene sequence. they placed c. aurantium, c. limon, c. sinensis and c. paradisi in one group. we found that c. limon, c. sinensis and c. paradise populations belong to clade 1, while c. aurantium to clade 2. the phylogenetic relationship of some species of rutacee family (taken from ncbi) with five citrus species investigated in this study is given in fig. 4. as it is seen from the tree, citrus species are separated from tetradium ruticarpum, melicope degeneri, melicope polybotrya, clausena anisate, clausena lenis, murraya exotica, murraya paniculata, glycosmis lucida, glycosmis pentaphylla, zanthoxylum molle and zanthoxylum bungeanum species. penjor et al. (2010) grouped citrus species with fortunella, poncirus, microcitrus, eremocitrus, and clymenia. groppo et al. (2008) determined cpdna in rps16 intron analysis and situated citrus, poncirus and microcitrus in one group, whereas trnl-f analysis revealed that balsamocitrus, afraegle, citrus and clausena form one group. conclusion in this study, phylogenetic analysis of turkish citrus l. taxa using cpdna psba-trnh sequences was performed to elucidate phylogenetic relationships. according to the results of the study, the separation of citrus species in the phylogenetic tree obtained with psba-trnh sequence data was realized. however, it has been found that cpdna psba-trnh sequence populations of species belong together. the data obtained from this study will guide phylogenetic studies on both citrus and different plant species. acknowledgements this research was supported by the tubitak 2018/2209a. 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(1990), metal ions can easily interact with hydrofoil groups (oxygen, nitrogen, sulphur). these metal ions can bind four or more ligands (berg 1987). copper is one of the most critical essential microelements in living organisms, bound mainly to proteins, act as metalloenzyme (graham and welch 2000; nicolic et al. 2016). copper typically plays an essential role in redox processes in the cell (körös 1980). the organic matter part of cu is found in the chloroplasts of plants involved in the electron transport of photosynthesis. coppercontaining enzymes (polyphenol oxidase, ascorbic acid oxidase) in chloroplasts act as electron suppliers of copper (cu 2+ cu + ). copper is also crucial in the metabolic processes of proteins and carbohydrates, and it promotes the synthesis of proteins and carbohydrates. acting on the carbohydrate metabolism, copper accelerates the oxidation of glucose. its effect can be explained by the change in valence (judel 1962; pais 1999; giczi et al. 2018). copper is an essential microelement not only for plants, but it is inevitable for animal and human mailto:thomas.szakal@gmail.com nova biotechnol chim (2021) 20(1): e886 2 organisms. therefore, it is imperative to supply living organisms with copper in the form of metalloenzyme through the food chain. the most important food sources for human nutrition have plant origin. replenishing the worldwide copperdeficiency became the main task of research. oats, barley, and wheat are the most sensitive to copper deficiency. copper deficiency in cereals begins with the whitening of leaf tips and narrow, twisted leaves are formed. in poorly supplied plants, incomplete tuber or ear formation, and reduced eyes formation can be observed. in some cases the ears are empty. soil copper loss is mainly caused by extraction by plants and leaching (tölgyesi 1969). mining of the world’s copper-bearing minerals is declining due to depleting reserves. rising prices are placing an increasing burden on farmers. the salt of the metal complex is used to replace micronutrients in soils in order to save costs. copper replacement can take place in the soil or through the foliage of the plant. the possibility of compensating for the copper deficiency in soils is reduced by the fact that a larger amount of copper is required. so, the higher price of copper compounds limits such replacement. therefore, the replacement of copper through foliage has become more and more critical. the copper demand of winter wheat has been replaced for a long time by a copper-tetraamine compound produced from microelectronic waste (szakál et al. 2014). in this work, cu in the form of copper-complexes was presented on the foliage to improve winter wheat yield and quality. the selected form of copper-complex was tetraamminecopper(ii) sulphate. the suitable copper replacement criteria through foliage are that it needs to have high stability. the ligand has to be a valuable substance for the plant and needs to have a retarded effect. this compound meets the first two criteria, but tetraamminecopper(ii) sulphate cannot provide the retarded effect for the plant. thus, ion exchange of copper-tetraamine cations with synthesised zeolite anions was applied. application of such cu-complexes during the time of the experiment resulted in the improvement of the quality parameters of winter wheat. the achievements are discussed in light of critical physico-chemical parameters of applied forms of copper complexes. research has shown that zeolites are excellent adsorbents, catalysts, ion exchangers, and molecular sieves. natural zeolites are widely used in agriculture, but the synthesised zeolites are not widespread; their use as a foliar fertiliser is unknown. the production of synthetic zeolite is inexpensive and has the advantage that, unlike natural, it manufactures in high purity. as a new possibility, the ion-exchanged-synthesised zeolite was used as a foliage fertiliser in this research. zeolites are porous materials with a high specific surface area, the pores of which can vary between 0.3 and 2.0 nm. zeolites are three-dimensional, microporous, crystalline solids with a well-defined structure, containing aluminium, silicon, and oxygen as builders. zeolites are found in natural or artificially produced (synthesis) form. there are more than 60 types of zeolites in nature in volcanic or sedimentary media. the most common and most mined zeolites are chabazite, clinoptilolite, and mordenite. the number of synthesised zeolites artificially produced for different purposes is approximately 150. zeolites are the primary components of crystalline aluminium silicates to4 (t = si 4+ , al 3+ ). they are built from sio4 and alo4 tetrahedrons (flanigen et al. 2010). tetrahedrons bind on edges sharing oxygen atoms, and as a result, they can create 16 larger units assigned as secondary components. the crystal structure of zeolite is made up of secondary building blocks, while in one, two or all three directions of space, caves extend in uni-, bior tri dimensions of space (nagy et al. 1998). zeolites are formed from structural units, components, and cages (fig. 1), e.g. sodalite, zeolite a, faujasite and emt zeolite are formed from sodalite cage (schwanke et al. 2018). when building their tri-dimensional framework, sio4 tetrahedrons can be exchanged with alo4 tetrahedrons by isomorphic substitution. trivalent aluminium in alo4 tetrahedrons has a negative charge neutralised by cations, mainly alkali metals and alkali earth metals (fig. 2). the negative charge is often neutralised by na + cation (ward 1976; armbruster and gunter 2001). zeolites have a specific surface area of 800 – 1,000 m 2 .g -1 . in caves and channels of zeolites, different nova biotechnol chim (2021) 20(1): e886 3 molecules can be stored (ramsay and kallus 2000). in the channels, water, and cations can be found mostly. most of the water in the channels of zeolite is stored in the grid structure channels through absorption. a smaller amount of water forms a hydration shell that coordinates with cations. if subjected to heat, water can be removed continuously and irreversibly. with the desorption of water, the molecular-sized pores become free, thus allowing the uptake of foreign molecules and the formation of selective adsorption. most of the water can be removed at 100 – 200 o c. total dehydration occurs at 400 o c. the widespread use of zeolites is enhanced by its ion exchange capacity, variable size channels, high stability, high melting point (above 1,000 o c) insoluble in water and inorganic solvents (breck 1974; moshoeshoe et al. 2017). fig. 1. construction of a type of zeolite from a sodalite unit (schwanke et al. 2018). fig. 2. negative charge is neutralised by na + ion, si/al = 1, na-a zeolite (zhao et al. 2012). the international zeolite association (iza-sc) has created a database of zeolite structures (atlas, threeletter code); accordingly, naa-type zeolite is a member of the lta group (baerlocher et al. 2007). in this work, synthesised naa (lta) zeolite was studied and applied. the basic unit of the structure of lta is the sodalite framework. cubic octahedron is the component of a-type zeolite with a pore size of 0.4 nm that enables ion exchange and binding gases (ramsay and kallus 2000). the naa type zeolite is widely used and can be described with the molecular formula na12al12si12o48·27h2o. lta type zeolites exert high ion exchange capacity due to low ratio of si/al (~ 1), while ion exchange can be described by isotherm most clearly (nagy et al. 1998; bujnová and lesný 2004; remenárová et al. 2014). such ion exchange features appear promising for application in cu delivery to plants. copper-amine-complex copper (ii) forms complexes with nitrogencontaining ligands such as cux2n nh3, where n in general 2, 4, 5 or 6, x anion forms a bridge across the metal ions. addition of alcohol to the ammonia solution of copper sulphate (ii) results in slow crystallisation of [cu(nh3)4]so2 . h2o complex, where the four nitrogen atoms form a quadratic plane's structure around copper (ii), and there is water in the fifth position. x-ray diffraction analyses have shown that copper (ii)-tetraamine complex in solutions containing copper-amine complexes possess a distorted octahedron structure. fig. 3. species distribution of copper-amine complex as a function of ph (triki et al. 2017). the two water molecules also coordinate with the central copper ion under and above the plane determined by the four n atoms. the cu-o bond nova biotechnol chim (2021) 20(1): e886 2 distance is 2.33 å, while cu-n (equatorial) is 2.03 å. this latter bond is much longer than the cu-o(eq) (1.94 å) in copper (ii)-hexa-aqua-complex (morosin 1969; szakál 1993). in copper-tetramine complex products have been measured β1=4.14, β2=7.66, β3=10.53, β4=12.68 (bjerrum 1941). the species distribution curve of the copper-tetramine complex is in fig. 3. the solution contained cu(nh3)4 2+ ions in the highest quantity at ph 9.3. experimental all the analysis of this research was carried out at the university of pannonia, institute of materials science (veszprém, hungary). copper-containing substances tetraamminecopper(ii) sulphate was used for ion exchange. naa type synthesised zeolite (trade name: zeolon p4a) was used to modify zeolite ionexchanged with copper-tetraamine for analyses with a derivatograph and x-ray diffraction. tetraamminecopper(ii) sulphate the reaction of copper-sulphate 18 wt % solution and undiluted ammonium hydroxide 26 wt % was used to produce tetraamminecopper(ii) sulphate copper content of 12 wt % and ph 9.3 (referring to fig. 3). tetraamminecopper(ii) sulphate was crystallised by adding 100 % ethyl alcohol with cooling. crystalline tetraamminecopper(ii) sulphate was examined by x-ray diffraction and analysed with a q-1500 d mom derivatograph. ion exchange on zeolon p4a (naa) type synthesised zeolite zeolon p4a synthesised zeolite was used for the ion exchange process in the water-diluted solution of tetraamminecopper(ii) sulphate complex (ph 9.3). the structure of ion-exchanged zeolite was analysed using the x-ray diffractometer (philips pw3710). the morphology and composition of the materials' elements were studied with a scanning electron microscope (philips xl 30-esem). producing synthesised zeolite ion-exchanged by copper-tetraamine for ion exchange in zeolon p4 zeolite with tetraamminecopper(ii) sulphate, a 10 wt % suspension of synthesised zeolite in distilled water was added to the 12 wt % suspension of tetraamminecopper(ii) sulphate while stirring. based on the preliminary analyses, it was used 100 % surplus of tetraamminecopper(ii) sulphate to achieve the ion exchange. after stirring for two hours, the suspension was let to settle, and the surplus copper complex with distilled water was removed by decantation. decantation was continued until the amount of detectable copper in the aqueous phase became constant. the formed suspension was dried at 30 o c (in open space). content of cu was determined by inductively coupled plasma mass spectrometry (icp-ms), and organic nitrogen content was determined by kjeldahlmethod (o’sullivan 2011). results and discussion analysis of tetraamminecopper(ii) sulphate with derivatograph analysis of the crystalline tetraamminecopper(ii) sulphate was carried out with a derivatograph in the institution of material science university of pannonia. fig. 4 shows the derivatogram of tetraamminecopper(ii) sulphate. the figure clearly shows that during the heating process at temperature peaks of 176.0 o c, 210.5 o c, 319.5 o c and 389.5 o c mass reduction and endotherm processes occurred (dta – differential thermal analysis). mass reduction at these peaks (tga curve) (tga thermogravimetric analysis) presumably can be related to the removal of 4 ammonia content of copper-tetraamine complex through chemical decomposition. up to 632.1 o c, the four ammonia molecules could be removed entirely during the decomposition of the complex. the calculated ammonia content of the weighed 400 mg tetraamminecopper(ii) sulphate was 119.53 mg (29.88 %). the derivatograph showed data of higher values by 151.79 mg (37.95 %). besides ammonia, the water preserved in the zeolite is likely responsible for higher values. at 4 nova biotechnol chim (2021) 20(1): e886 3 higher temperatures, at tg (799.9 o c and 859.9 o c, respectively), the measured weight reduction could result from the decomposition of sulphate (decomposition products: so2, so3, and o2). fig. 4. thermal stability of tetraamminecopper(ii) sulphate (tamás korim, 2020). fig. 5. derivatogram of zeolone p4a type synthesised zeolite (tamás korim, 2020). . 5 nova biotechnol chim (2021) 20(1): e886 6 analysing zeolon p4a type synthesised zeolite and ion-exchanged zeolite with a derivatograph it was considered necessary to study whether the ion exchange does induce any changes in the zeolite. therefore, the corresponding derivatogram was compared with that of the initial zeolite. na + cations bind the negative positions of the studied zeolon p4a type zeolite. the fig. 5 shows tg, dtg and dta curves of the changes in zeolon p4a zeolite due to rising temperatures. the tg curve shows that water binds in the channels and surface of zeolite with high adsorption capacity. water desorption occurs until reaching the temperature ~ 400 o c, with a measured volume of 20.3 wt %. the maximum of the endothermic prolonged water release was 197.2 o c based on the dtg curve. due to rising temperatures, the dta curve showed an exothermic reaction at 858.8 o c, resulting in a reduction in mass of 2.3 wt %. derivatograph analysis of zeolite ion-exchanged with copper-amine the fig. 6 shows the derivatograph of zeolon p4a type zeolite treated with tetraamminecopper(ii) sulphate. following the ion exchange, the zeolite's cu content was 4.98 wt %, determined with icpms. the nitrogen content was 3.76 % (equivalent to 4.56 wt % nh3), corresponding to coppertetraamine's ammonia content in the zeolite of 85.5 %. based on these data, the presence of diand triamine forms was also hypothesized. fig. 6. derivatogram of zeolite ion-exchanged with copper-tetramine-sulphate (tamás korim, 2020) the dta curve of the tetraamminecopper(ii) sulphate shows the gradual transmission of ammonia molecules at four definite endotherm peaks. in the case of ion exchange with the coppertetraamine complex compound, the gradual release of ammonium molecules with the water remaining in the zeolite can be monitored through a single peak. a new peak providing exothermic reaction formed at the dta curve of the ion-exchanged zeolite at 772.1 o c and resulted in a slight increase in mass compared to the dta curve of zeolite. the mass of ion-exchanged zeolite was 270 mg weighed into the derivatograph. the mass of ammonia put through ion exchange was 12.35 mg. during the analysis, the temperature was increased to 510.1 o c and measured a mass loss of 64.53 mg nova biotechnol chim (2021) 20(1): e886 7 (corresponds to 23.9 % of the ion-exchanged zeolite mass). subtracting the weight of ammonia (12.35 mg) from 64.53 mg, 52.18 mg remains, what most likely gives the weight of residual water (this water mass form 19.32 % of the measured mass of ion-exchanged zeolite). energy dispersive x-ray edx analysis of zeolon p4a zeolite the fig. 7 shows the edx spectrum of zeolon p4a type of synthesized zeolite. edx records clearly show that the si : al ratio in the zeolite is close to 1 : 1 (10.16 % of si, 11.81 % of al). sodium (replaceable) content represents 16.52 %, and that present in anion sites makes out 14.05 % of atoms participating in the zeolite. fig. 7. edx spectrum of synthesised zeolite of zeolone p4a (tamás korim, 2020) edx analysis of copper ion-exchanged synthesised zeolite edx records provide reliable evidence of the progress and size of ion exchange (fig. 8). based on the recording, the edx spectrum of the ionexchanged zeolite shows that the ratio of si : al in the zeolite was approximately 1. no change in the lattice structure likely occurred due to ion exchange. this was supported by the x-ray diffraction phase analysis. x-ray diffraction analyses quality phase analyses tested cu-tetraamine, zeolite and cu-zeolite samples. the fig. 9 shows the xrd records of the three samples. the figure clearly shows that the diffractogram of zeolite and cu-ion-exchanged zeolite are identical, referring to either the diffraction peaks or their intensity ratio. this is important because if a solid solution formed due to the cu-infiltration, grid destruction would develop, followed by a change in the distance values of d-plane-network, which again would change the peak positions according to the braggequation. therefore, it can definitely be said that cu did not incorporate build into the zeolite's crystal grid structure. nova biotechnol chim (2021) 20(1): e886 8 fig. 8. edx spectrum of zeolite ion-exchanged with copper-tetramine-sulphate (tamás korim, 2020) fig. 9. xrd records of cu-tetraamine, zeolite and cu-zeolite samples (tamás korim, 2020). note: during the test, different materials were marked with colours (20729 cu-tetraamine: blue; 20723 zeolit: yellow; 20721 cu zeolit: red circle). nova biotechnol chim (2021) 20(1): e886 9 conclusion safe and quality crop production assumes to provide the crop with nutrients. copper has a primary role among the essential microelements. the efficiency of foliar application of complex compounds can be increased if their retard-effect is ensured. the natrium ion was changed with coppertetraamine ion in naa type synthesised zeolite with the high ion-exchange capacity to ensure retardation. it was possible to immobilise the cucomplex on the zeolite. derivatograph analysis confirmed heat stability of the complex ion introduced through ion exchange, the decomposition of the complex did not start on the plant surface. energy dispersive x-ray analyses (edx) revealed that si:al ratio approached 1 : 1 (si atom % = 10.16 and al atom % = 11.81) in the synthesised zeolite. this ratio did not change even after ion exchange, which ensures high ion exchange capacity. x-ray diffraction (xrd) analyses show clearly that the diffractograms of zeolite and cuion-exchanged zeolite were identical, referring to either the diffraction peaks or their intensity ratio. as a result, it can be stated that cu did not build into the zeolite's crystal grid structure. the usefulness of copper ion-exchanged zeolite as a foliar fertiliser was proved by the fact, that according to its thermoderivatogram, the release of ammonia (decomposition of copper tetramine ion) takes place above 100 º c, so there is no intraday decomposition in the foliage. after ion exchange, the measured copper content of the zeolite is 4.98 wt % (icp measurement) and the nitrogen content is 3.76 % (corresponding to 4.56 wt % nh3). based on the calculations that provided 85.5 % of the ammonia content of copper tetramine in the synthesised zeolite, it can be assumed that the triamine form is also present in the ion-exchanged zeolite. acknowledgement this work has been supported by the interreg v-a, skhu/1802/3.1/023 co-innovation program. conflict of interest authors declare that there is no conflict of interest. references armbruster t, gunter me (2001) crystal structures of natural zeolites. natural zeolites occurrence, properties, applications. in bish dl, ming dw (eds.), mineralogy and geochemistry, de gruyter, pp. 45. baerlocher c, mccusker l, olson dh (2007) atlas of zeolite framework types, 6 th revised ed., elsevier, amsterdam, 397 p. berg jm (1987) metal ion sin proteins: structural and functional roles. cold spring harb. symp. quant. biol. 52: 579-85. bjerrum (1941) metal amine formation in aqueous solutions: theory of the reversible step reaction. p. haase and son, copenhagen, denmark, 296 p. breck dw (1974) zeolite molecular sieves: structure, chemistry and use. 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zeolite membranes. membrane science and technology. 6: 373-395. remenárová l, pipíška m, florková e, augustín j, rozložník m, hostin s, horník m (2014) radiocesium adsorption by zeolitic materials synthesized from coal fly ash. nova biotechnol. chim. 13: 57-72. schwanke aj, balzer r, pergher s (2018) degradation of volatile organic compounds with catalysts-containing zeolite and ordered mesoporous silica. in martínez l, kharissova o, kharisov b (eds.), handbook of ecomaterials. springer, cham. szakál p (1993) környezetre ártalmas rézés cink tartalmú hulladékokból előállított rézés cink-komplexek mezőgazdasági hasznosítása. phd. thesis, hungary. szakál p, péntek a, barkóczi m, szakál t (2014) hulladék réz-szulfát oldatból előállított réz-komplex hatása, az őszi káposzta repcére (brassica napus l.). a magyar hidrológiai társaság kiadványa. xxi. ifjúsági napok, pp. 1-7. tölgyessy gy (1969) a növények mikroelem-tartalma és ennek mezőgazdasági vonatkozásai. mezőgazdasági kiadó, budapest. triki m, tanazefti h, kochkar h (2017) design of βcyclodextrin modified tio2 nanotubes for the adsorption of cu(ii): isotherms and kinetics study. j. colloid interface sci. 493: 77-84. ward jw (1976) zeolite chemistry and catalysis, in rabo ja (eds.), acs monographs 171, american catalysis society, washington, dc, 118 p. yamashita mm, wesson l, eisenman g, eisenberg d (1990) where metal ions bind in proteins. biophysics. 87: 56485652. zhao w, basnet b, kim ik (2012) carbon nanotube formation using zeolite template and applications, j. adv. ceram. 1: 179-193. http://jac.tsinghuajournals.com/ http://jac.tsinghuajournals.com/ http://jac.tsinghuajournals.com/en/article/showtenyearvolumndetail.do?nian=2012 nova biotechnol chim (2021) 21(1): e819 doi: 10.36547/nbc.819 1 nova biotechnologica et chimica impact of inorganic powder additives on the size and morphology of pellets of the microscopic filamentous fungus aspergillus niger sanja nosalj1,, karol jesenák2, alexandra šimonovičová1, pavol hudec3 1faculty of natural sciences, department of soil science, comenius university in bratislava, ilkovičova 6, bratislava 84215, slovak republic, 2faculty of natural sciences, department of anorganic chemistry, comenius university in bratislava, ilkovičova 6, bratislava 84215, slovak republic, 3faculty of chemical and food technology, institute of organic chemistry, catalysis and petrochemistry, slovak university of technology radlinského 9, bratislava 31237, slovak republic  corresponding author: nosalova15@uniba.sk article info article history: received: 14th january 2021 accepted: 21st april 2021 keywords: aspergillus niger fe2o microscopic filamentous fungi montmorillonite size of pellets sio2 tio2 zeolites abstract this paper has been focused on the impact of the set of microcrystal water-insoluble inorganic additives to dimension characteristics of pellets of the microscopic filamentous fungus aspergillus niger. the selection of powder substances included montmorillonite clay mineral, silicon dioxide, iron oxide, one zeolitic tuff with mineral clinoptilolite as its main component, three synthetic zeolites, na-mordenite, nay and na-zsm-5, and titanium dioxide. this group is comprised of substances that are often components of various natural waters (including contaminated waters) and also frequent components of soil. exceptions are synthetic zeolites and titanium dioxide. the impact of aforementioned substances was monitored on four aspergillus niger strains isolated from soils at four slovak locations, markedly differing in soil reaction values. three of these soil samples have been affected by considerable contamination due to previous mining activities. clay mineral montmorillonite reduced the size of the pellets regardless of the origin of strains, while the impact of other inorganic substances was either weak or variable depending on the type of inorganic substance and source location of the strains. the impact of natural zeolitic tuff was also similar, however, less significant.  university of ss. cyril and methodius in trnava introduction microscopic filamentous fungi are widely used, particularly in the pharmaceutical and food industries. the ability of microscopic filamentous fungi to absorb harmful components from the surrounding environment, including toxic metals in various forms, is also used for the decontamination of various liquid substrates (kapoor et al. 1999; žemberyová et al. 2010; ouyang et al. 2015). the most industrial applications of microscopic filamentous fungi use their biomass in the form of small pellets (krull et al. 2013; prajapati et al. 2014). the growth and final size of the pellets as well as their morphology is affected by various factors. some of the factors include the composition of the culture medium, its temperature and ph as well as the turbulence intensity of the liquid content of culture vessels or reactors. in the majority of cases, it is not only liquid solutions that are processed by biotechnological purifying methods, but also suspensions containing various small insoluble mailto:nosalova15@uniba.sk nova biotechnol chim (2021) 20(1): e819 2 particles of different shapes and sizes. such substances are not necessarily the original component of contaminated solutions; they may be added to the contaminated waters for the purpose of enhancing their purifying effect which is caused by sorption on the internal and external surfaces of these particles. the intentional addition of various inorganic powders in culture media used for the biotechnological production of various substances is different. it has been established that the effectiveness of these technologies is influenced by the morphology of the microscopic filamentous fungi pellets (znidarsič and pavko 2001; veiter et al. 2018; shmedider et al. 2019). this knowledge is used in the production of substances such as itaconic acid, citric acid (metz 1976) and certain enzymes and antibiotics (papagianni et al. 2006; teng et al. 2009). the first example of the utilization of inorganic powders in biotechnological productions was aluminum oxide and talc powder (hydrous magnesium silicate), which were used to increase the production of chloroperoxidase (znidarsič and pavko 2001). these substances facilitate the separation of individual fungi filaments, which improves their mutual contact with the surrounding solution. driouch et al. (2012) studied in greater detail the optimization of the morphology of pellets of microscopic filamentous fungi aspergillus niger for the production of enzymes. despite the existence of several works dealing with the effect of small inorganic particles on the morphology of the aforementioned fungi in complex systems, the question still remains how such particles affect the growth of the pellets in simple systems containing only a culture medium. except for the works focusing on the effect of certain clay minerals such as kaolinite, bentonite and palygorskite, relatively little attention has been paid to this topic (fomina and gadd 2002). the general conclusion is that these minerals decrease the size of these pellets. the relation between the dimensional and morphological parameters of pellets and their ability to accumulate various soluble components is not quite clear. in connection with the decontamination of waters, it is almost certainly possible to reject the claim that there is no close relationship between them. this paper strives to clarify the impact of certain inorganic microcrystal substances have on the dimensions and morphological qualities of pellets of the microscopic filamentous fungi aspergillus niger. the selection of powders included montmorillonite clay mineral, silicon dioxide, iron oxide, one natural zeolite, three synthetic zeolites, and titanium dioxide. this group is comprised of substances which are often components of various natural waters (including contaminated waters); they are also soil components. synthetic zeolites and titanium dioxide, that are due to their wide use (especially as a white pigment) among one of the industrial contaminants and despite this fact are safe to use, make an exception in this set. the common characteristics of all of the aforementioned substances is their considerable interactive surface which arises from the small size of their particles (fe2o3, sio2, montmorillonite) or (also) their internal porous structure (zeolites and montmorillonite). the impact of these substances was monitored on four aspergillus niger strains that were isolated from soil at four slovak locations markedly differing in soil reaction values. three of these soils have been affected by considerable contamination due to previous mining activities. experimental fungal strains the aspergillus niger strains were isolated from four locations whose soil reactions ranged from ph 3 (ultra acid) up to ph 9 (strongly alkaline soil environments) (čurlík et al. 2003). the first strain, marked as an – š, was isolated from the mine region of šobov near banská štiavnica. the type of soil was dystric cambisol (contaminated and eroded) without vegetation and with high aluminum and iron content. soil reaction was ultra acidic (ph 3.0). the second strain, marked as an – p, was isolated from the stream sediment of the blatina river in the environmentally burdened region of pezinok with arsenic content (363 mg.kg1) and antimony content (93 g.kg-1). soil reaction of these sediments was strong acidic (ph 5.3). the third strain came from the region slovinky and was marked as an – sl. the type of soil was alkaline https://www.google.com/search?client=firefox-b-ab&q=hydrous+magnesium+silicate&spell=1&sa=x&ved=2ahukewjy3kwoslxnahwnerqkhtxobsgqbsgaegqidbap nova biotechnol chim (2021) 20(1): e819 3 technosol with medium alkali soil reaction (ph 8.3). the fourth strain, marked as an – g, was as a control strain and was isolated from the region of gabčíkovo. the soil type was eutric fluvisol without any contamination. the soil reaction was slightly alkaline (ph 7.7) (šimonovičová et al. 2013; 2019). pellet cultivation the pellets were prepared by adding 5 ml of conidia water suspension from the stock solution in 45 ml of a sdb medium culture (sabouraud dextrose broth liquid medium, himedia, mumbai, india). one gram of powder substance was then added to the medium culture. cultivation took place over 5 days at 25 °c in 250 ml erlenmayer flasks placed with stirring at 135 rpm. (unimax 2010 shaker, heidolph, germany). after the completion of the experiment, the microscopic filamentous fungi aspergillus niger pellets were removed by filtration, washed with distilled water and placed in petri dishes. establishing pellet size the size of the pellets was established in a medium culture in transparent petri dishes. depending on their size, the number of pellets in the dish varied from 3 to 1,000 in the case of the smallest pellets. in all cases, the entire weight share of biomass represented by the microscopic filamentous fungi was cumulated in pellets. the size of the pellets was determined using a photographic recording complemented by a right-angle line raster with a known distance between the lines without using image analysis software (fig. 1). characteristics of powder forms fine iron oxide and silicone dioxide powder with particles smaller than one micrometer was used in the experiments. both chemicals were supplied by sigmaaldrich. the purity of both chemicals was in the p. a. category. fine iron (iii) oxide (sigmaaldrich, purity >97.0 %) and silicone dioxide powder (sigma-aldrich, purity ~99.0 %) with particles smaller than one micrometer was used in the experiments. zeolites three types of synthetic zeolites were used: namordenite (sio2/al2o3 = 17.5), nay (sio2/al2o3 = 4.5) and na-zsm-5 (sio2/al2o3 = 28.5) and one natural clinoptilolite. zeolites was used in the form of fine powder with a particle size of less than 5 micrometers. synthetic zeolites nay, nam (mordenite) and nazsm-5 were prepared in the research institute for petroleum and hydrocarbon gases in bratislava. clinoptilolite was a natural zeolite from east-slovakia deposits near nižný hrabovec. clinoptilolite has chemical composition (ca, k2, na2, mg)4 al8si40o96·24h2o and contains about 84 % of clinoptilolite, 8 % of crystobalite, 4 % of illite, and 3 – 4 % of plagioclase. more detailed characteristics of this rock are presented by holub et al. (2016). textural properties of zeolites were determined by physical adsorption of nitrogen at the temperature of liquid nitrogen (-196 °c) using equipment asap 2400 (micromeritics). before measuring, samples were degassed in measuring burettes at 350 °c and vacuum of 2 pa overnight. textural properties of clinoptilolite as well as all synthetic zeolites are in table 1. table 1. textural properties of zeolites. sbet vmicro st va dp [m2.g-1] [cm3.g-1] [m2.g-1] [cm3.g-1] [nm] nam 360 0.147 42.9 0.182 nay 668 0.332 38.1 0.384 nazsm-5 285 0.128 40.7 0.217 clinoptilolite 30.6 0.003 24.0 0.153 15, 38 sbet – specific surface area; vmicro – specific volume of micropores; st – external surface size; va – total pore volume; dp – size distribution of mesopores. nova biotechnol chim (2021) 20(1): e819 4 adsorption isotherms of all synthetic zeolites are typical for microporous materials – wide horizontal plateau on adsorption isotherms representing adsorption on external surface of zeolite crystals without any hystheresis loop (nay) or with very narrow horizontal hystheresis loops (nam, nazsm-5) representing voids among tiny zeolite microcrystals (fig. 1). adsorption isotherm of clinoptilolite shows wide hystheresis loop corresponding to relatively great volume of mesopores and even partially macropores. fig. 1. adsorption isotherms of zeolite samples. specific surface area sbet was calculated by treatment of adsorption data by bet-isotherm in the standard range of p/p0 = 0.05 – 0.30. specific volume of micropores vmicro and specific surface area of mesopores + external surface st was calculated by t-plot method using harkins-jura standard isotherm. total pore volume va was estimated from data of adsorption isotherm at relative pressure of about p/p0 = 0.99. pore size distribution of mesopores dp was determined from desorption part of isotherm by standard bjh (barett-joyner-halenda) method. the highest specific surface area sbet was determined for nay zeolite (almost 670 m2/g), then for mordenite nam (360 m2.g-1) and for nazsm-5 zeolite 285 m2.g-1. as it is seen from table 1, from these values only about 38 – 43 m2.g-1 was a real surface determined by t-plot (sbet values have not physical meanings for microporous materials), and nitrogen was adsorbed predominantly in micropores (0.332 cm3.g-1 for nay, 0.147 for nam and 0.128 for nazsm-5). on the other hand, clinoptilolit had quite low specific surface area sbe – only 30.6 m2.g-1 and only negligible volume of micropores vmicro = 0.003 cm 3.g-1. synthetic zeolite samples are represented by crystals of pure zeolites, and contain only micropores and external surface of crystals. clinoptilolite contained a wide range of mesopores between 5 nm and 50 nm (over 50 nm are already mesopores) with two observable maximums at 15 and 38 nm (fig. 1). peak at about dp= 3.8 nm was an artifact that does not correspond to real pores. the characteristics of the zeolite channels are shown in table 2. table 2. channel characteristics of zeolites used. zeolite channel system [nm] nam 0.65 × 0.70; 0.34 × 0.48; 0.26 × 0.57 nay 0.74 × 0.74 nazsm-5 0.51 × 0.55; 0.53 × 0.56 clinoptilolite 0.31 × 0.75; 0.36 × 0.46; 0.28 × 0.47 clay mineral the montmorillonite used in this study was acquired by the sedimentation method from a 4 % water bentonite suspension from the most famous slovak location stará kremnička – jelšový potok (slovak republic). the bentonite from this location contains calcium-magnesium montmorillonite. a detailed description of the separation method provided jesenák (1994) and jesenák and hlavatý (2000). the greater portion of montmorillonite particles was smaller than 2 μm. specific surface area sbet was 112 m 2.g-1and ethylene glycol specific surface area was 653 m2.g-1. results and discussion fungal pellets, which have the advantages of harvest ease, low fermentation broth viscosity and high yield of some proteins, have been used for a long time (zhang and zhang 2015). a wide range of microorganisms in the form of pellets have also significant application in environmental biotechnology for removal of heavy metals and nova biotechnol chim (2021) 20(1): e819 5 toxic elements from various substrates (fawzy et al. 2017). for example, bioaccumulation of some toxic metals by association of a. niger fungal pellets and green algae chlorella vulgaris has been confirmed in many experiments (šimonovičová et al. 2018; remenárová et al. 2020). aspergillus niger strains used in this study differ in the source of origin and also in characteristics such as growth rate and sporulation, production of organic acids and enzymes (šimonovičová et al. 2017; nosalj and šimonovičová 2019; šimonovičová et al. 2020). against this background we also assumed the variability in the formation of pellets. it was also confirmed during the cultivation in the sdb solution without added inorganic powders (fig. 2; fig. 3). fig. 2. shots of pellets of microscopic filamentous fungi a. niger cultivated in an sdb solution without added inorganic powders as a control (k). the shots illustrate a marked variation in the size of pellets acquired from conidia of microscopic filamentous fungi isolated from soils from various environments. a – an š (strain isolated from the locality šobov), b – an p (strain isolated from the locality pezinok), c – an sl (strain isolated from the locality slovinky) and d – an g (strain isolated from the locality gabčíkovo). fig. 3 also indicates the sizes of pellets acquired through the cultivation of the aforementioned strains without any additional inorganic powders. the variance of average values of a. niger pellets that were prepared from conidia of microscopic filamentous fungi originated from the soils from various environments and cultivated without the presence of powder microparticles was relatively large. the ratio of the average sizes of the largest and smallest pellets was 3.34. fig. 3. average sizes of pellets [mm] of microscopic filamentous fungi a. niger cultivated with the presence of particles of inorganic substances. nova biotechnol chim (2021) 20(1): e819 6 in the case of pellets acquired by cultivation with the presence of powder substances, this ratio varies from 1.30 to 4.00. the smallest (the first value) was for pellets acquired with the presence of montmorillonite, the largest (the second value) was for pellets acquired with the presence of iron oxide. with the exception of pellets acquired with the presence of natural zeolite and synthetic iron oxide, these ratios vary in a relatively narrow interval of 1.3 up to 1.76, of which it seems that this type of substance subdues the original differences observed within the referential group of pellets. a comparison was made between control samples, without inorganic particles, and the samples with these particles present. pellets acquired from conidia of microscopic filamentous fungi from various locations both experienced enlargements as well as reduction of their diameters (fig. 4). fig. 4. shots of pellets of microscopic filamentous fungi a. niger cultivated in an sdb solution with addition of inorganic powders. a – with addition of tio2; b – and d – with addition of zeolite; c – with addition of montmorillonite. the shots illustrate the significant variance in pellet size due to these substances, e.g. reduction of diameters due to montmorillonite (an sl – m) or increase of diameters due to zeolite (an g – z). the last figure also documents the differences in the size of the pellets on one petri dish, i. j. within one strain. for the comparison of the sizes of microscopic filamentous fungi pellets, the variation of these sizes within one sample of pellets must be taken into consideration. in general, this variation does not exceed one order of magnitude. however, the sizes of the largest and smallest particles do not exceed the ratio 4 : 1. in the case of control samples, this variation was within the interval 2.5 – 4.5 : 1. in the case of an – š samples cultivated with inorganic particles, the variation of this ratio was 1.3 to 3.0 : 1; in the case of an – p samples, 0.86 to 10.0 : 1; in the case of an-g samples, 2.0 to 6.0 : 1 and in the case of an – sl samples, 1.75 to 4.0 : 1. the only an-š/fe2o3 sample whose ratio was 10 : 1, was the exception compared to the relatively narrow intervals (tab. 3). statistical characteristics of the size of aspergillus niger strains pellets after cultivation with various inorganic substances was presented in table 3. if the modus and median values of sizes of analyzed pellets are taken into consideration, the following can be said regarding to effect of the aforementioned particles. while clay mineral montmorillonite definitely reduces the size of pellets (jesenák et al. 2015), regardless of the type of source material, the effect of other inorganic substances was either weak or very differentiated depending on the type of inorganic substance and source location of the strain of microscopic filamentous fungi. natural zeolite has a similar yet less distinctive effect (fig. 5). a great deal of differentiation can be observed in the an – š samples, in the case of which most of powders markedly enlarged the size of the pellets. this growth represents two to five times higher modus and median values. the exception was natural zeolite, in addition to the already mentioned montmorillonite, which lowers this value. samples of an – p and an – g strains do not markedly increase these values and in the case of an – sl samples, the size of pellets was mostly reduced. according to many authors (espinozsa-ortiz et al. 2017; garcía-reyes et al. 2017; veiter et al. 2018) fungal pellets are spherical, ellipsoidal or oval nova biotechnol chim (2021) 20(1): e819 7 masses of intertwined hyphae with a size usually in the range of several hundred micrometers to several millimeters. fungal pellets usually appear as a core of densely packed hyphae, surrounded to a large extent by a more annual dispersed or „hairy“ region that contains the radially growing portion of the hyphae (fig. 5). the inorganic substances used in the table 3. statistical characteristics of the size of aspergillus niger strains pellets [mm] after cultivation with various inorganic substances. diameter mode median variance of sizes of pellets number of pellets fungal strain an – š control sample 2.44 2 2 2 – 6 235 tio2 5.12 5 5 4 – 8 147 fe2o3 4.11 2 2 2 – 6 78 sio2 7.80 4 10 4 – 14 26 natural zeolite 1.68 1 2 1 – 3 742 na-mordenite 8.27 10 9 6 – 10 11 nay zeolite 9.62 8 8 8 – 14 16 na-zsm-5 8.06 8 8 4 – 11 15 montmorillonite 1.01 1 1 1 – 2 1,000 fungal strain an – p control sample 5.82 6 6 2 – 9 52 tio2 6.11 7 6 3 – 8 42 fe2o3 1.21 1 1 1 – 10 244 sio2 13.75 6 6 6 – 7 4 natural zeolite 2.36 2 2 1 – 4 404 na-mordenite 7.33 8 8 4 – 10 34 nay zeolite 6.85 5 5 5 – 13 40 na-zsm-5 6.12 6 6 4 – 8 49 montmorillonite 1.04 1 1 1 – 2 1,000 fungal strain an – sl control sample 8.15 9 85 4 – 10 26 tio2 5.16 5 5 4 – 7 24 fe2o3 1.93 2 2 2 – 8 178 sio2 11.21 8 10 8 – 14 12 natural zeolite 1.27 1 1 1 – 3 574 na-mordenite 12.50 10 10 6 – 18 12 nay zeolite 6.86 4 6 4 – 14 42 na-zsm-5 8.20 8 8 6 – 12 20 montmorillonite 1.04 1 1 1 – 2 1,000 fungal strain an – g control sample 5.11 5 5 3 – 8 51 tio2 3.81 3 4 2 – 12 134 fe2o3 2.87 2 2 2 – 10 101 sio2 8.88 2 7 2 – 10 9 natural zeolite 4.96 5 5 2 – 12 90 na-mordenite 12.00 8 – 28 3 nay zeolite 7.10 6 6 5 – 11 31 na-zsm-5 5.40 5 6 1 – 7 5 montmorillonite 1.02 1 1 1 – 2 1,000 nova biotechnol chim (2021) 20(1): e819 8 fig. 5. pellets of strain an – š (a) and an – g (b) cultivated in addition of montmorillonite, strain an – sl (c) and an – p (d) in addition of zeolite and strain an – p (e) and an – g (f) in addition of tio2. aforementioned experiments differ markedly in many characteristics. however, it seems that none of these characteristics plays such a significant role in the creation of these pellets that it would unidirectionally affect their size towards their enlargement or reduction within all four samples of a. niger microscopic filamentous fungi. the only explanation for the exceptional position of clay mineral montmorillonite, which always reduces the size of pellets of microscopic filamentous fungi in all cases to a greater or lesser extent, relates to the size of its mineral particles. as opposed to other inorganic powder substances, montmorillonite particles break down in water solutions into markedly smaller particles (in comparison to the primary size of the particles of other studied substances). therefore, the reduction of the size of pellets may be a result of spherical limitations in the growth of larger pellets. however, we cannot rule out the possibility that some interphase reactions between the surface of filaments of these fungi and the surface of the clay material particles also play a certain role. this may be related to the ion exchange qualities of montmorillonite. due to the markedly smaller size of its particles in comparison with zeolites, this effect could be much more distinctive in this case. conclusion the impact of the presence of water-insoluble inorganic powder additives in cultivation media and suspensions on the final size and morphology of pellets of microscopic filamentous fungus of aspergillus is complex. studies seemingly show that the primary role was played by the great differences in the size of the particles of these substances. the degree of spherical obstacles in a cultivation media affected the growth of the fungi. reduction of the size of these particles enhances various physical and chemical interactions that can contribute to changes in pellet growth. one possibility to determine more precisely the influence of montmorillonite particles size, while preserving its other characteristics, was to study the impact of its various ion exchange forms that may vary markedly. filamentous fungi play an important role not only in the bio-manufacturing of nova biotechnol chim (2021) 20(1): e819 9 value-added products, but also in bioenergy and environmental research. production and morphology of fungal pellets depends on complex factors that vary among species, and, therefore, it is not possible to establish a general methodology for the formation of fungal pellets. the use of fungal pellets is very important due to their wide range of applications in bioremediation, biosorption, bioleaching processes or metabolite production. acknowledgement the research was financially supported by the slovak national grant agency projects vega 1/0194/21 and vega 1/0658/19. conflict of interest the authors declare that they have no conflict of interest. references čurlík j, bedrna z, hanes j. holobradý k, hrtánek b, kotvas f, masaryk š, paulen j (2003) pôdna reakcia a jej úprava, 1st ed. j. suchoň, suma print bratislava, bratislava, 249 p. driouch h, hansch r, wucherpfenning t, rainer k, witmann ch (2012) improved enzyme production by biopellets of 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(2018) zníženie obsahu niklu z modelového roztoku synergickým účinkom mikroskopickej vláknitej huby a zelenej riasy za statických podmienok. phytopedon. 18: 1-7. teng y, xu y, wang d (2009) changes in morphology of rhizopus chinensis in submerged fermentation and their effect on production of mycelium-bound lipase. bioprocess. biosyst. eng. 32: 397-405. veiter l, rajamanickam v, herwig ch (2018) the filamentous fungal pellet-relationship between morphology and productivity. appl. microbiol. biotechnol. 102: 2997-3006. zahng j, zhang j (2015) the filamentous fungal pellet and forces driving its formation. crtical rev. biotechnol. 36: 1066-1077. znidarsič p, pavko a (2001) the morphology of filamentous fungi in submerged cultivations as a bioprocess parameter. morphology of filamentous fungi. food technol. biotechnol. 39: 237-252. žemberyová m, sherman, a, šimonovičová a, hagarová i (2010) bio-accumulation of as (iii) and as (v) species from water samples by two strains of aspergillus niger using hydride generation atomic absorption spectrometry. int. j. environ. anal. chem. 89: 569-581. nova biotechnol chim (2019) 18(2): 144-153 doi: 10.2478/nbec-2019-0017  corresponding author: deleibraheem2007@yahoo.com nova biotechnologica et chimica in silico toxicological analyzes of selected toxic compounds from dumpsite or contaminated soils on human health omodele ibraheem1, , toluwase hezekiah fatoki1, jesupemi mercy enibukun2, bolanle christianah faleye3 and daniel uwaremhevho momodu4 1 department of biochemistry, federal university oye-ekiti, pmb 373, oye-ekiti, ekiti state, nigeria 2 department of microbiology, federal university of technology, pmb 704 akure, nigeria 3 department of chemical science, joseph ayo babalola university, ikeji-arakeji, pmb 5006, ilesa osun state, nigeria 4 department of industrial chemistry, federal university oye-ekiti, pmb 373, oye-ekiti, ekiti state, nigeria article info article history: received: 17th july 2019 accepted: 30th september 2019 keywords: gene expression network molecular docking toxicants toxicokinetic transcription factors abstract the soil is a key component of natural ecosystems because environmental sustainability depends largely on a sustainable soil ecosystem. the objective of this study was to predict the impact of selected toxic compounds from dumpsite or contaminated soils on human health at the molecular level of biological processes. the in silico methods that were used include toxicokinetics and target gene prediction, molecular docking, and gene expressing network analysis. the result showed bisphenol a (bpa), 2,20-bis(p-chlorophenyl)-1,1-dichloroethane (ddd), 2,20-bis(p-chlorophenyl)-1,1-trichloroethane (ddt), diethylhexyl phthalate (dehp), nonylphenol (np) and tetrachlorodibenzodioxin (tcdd) as the active toxic compounds that can modulate biological system and are considered as potential cause of several diseases including cancer. the principal target genes include substance-p receptor (also known as neurokinin 1 receptor), 5-hydroxytryptamine receptor, human serotonin transporter; estrogen receptor alpha; and aryl hydrocarbon receptor. these genes implicated suz12, stat3, and trim28 as the major transcription factors while mitogen-activated protein kinases and cyclin-dependent kinases were the major kinases from the proteinprotein interaction. all the six toxicants investigated showed good free binding energies (δg) which were below 5.0 kcal.mol-1. these toxic compounds showed ligand efficiency greater than 0.25 kcal.mol-1. ha and would possibly cause fatal damage on human health. the order of in silico predicted toxicity of these compounds were bpa > ddd = ddt > tcdd > np > dehp. our results identified potential threats, which the selected toxicants can pose to public health. more importantly, it provides basis for investigation of super bugs (microorganisms) that can remediate these toxicants in our environment. environmental monitoring and modern wastes management system should be implemented and enforced in the affected countries in order to safeguard the health of the citizenry.  university of ss. cyril and methodius in trnava introduction the soil is a key component of natural ecosystems because environmental sustainability depends largely on a sustainable soil ecosystem (lombi et al. 1998). the biogeochemical cycles of contaminants have been greatly accelerated by human activities. typical contaminated sites bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:35 utc mailto:deleibraheem2007@yahoo.com nova biotechnol chim (2019) 18(2): 144-153 145 may be classified as either potential or practical, which could be quantitatively described as suspected to cause or constitute harm to human health and the environment – biotic and abiotic processes respectively. the qualitative and quantitative description of potentially contaminated and contaminated sites have been reported (who 2013). the study on soil samples of an animal burial site showed to be characterized by acute toxicity of decayed matter while the area of open waste dumping was found the most dangerous based on the amount of the contaminants (pasko and mochalova 2014). typical study has shown that more industrialized countries had higher bisphenol a (bpa) concentrations in landfill leachate than less industrialized countries (teuten et al. 2009). cases of environmental pollution and contamination in developing and developed countries have been reported (egboka et al. 1989; scu 2013). dumpsite has been huge challenge to the global health, because it serves as habitat for carcinogenic chemicals and pathogenic microbes (odeyemi et al. 2011; eze and amaeze 2016). dumpsites serve as lead for marine and coastal pollution and will account for 8 – 10 % of anthropogenic greenhouse gas emission in 2025 (iswa 2016). soil microorganisms often serve as the catalysts or promoters of reactions in the subsurface. however, the degradation of any contaminant depends on geochemical conditions and on the presence of microorganisms that are capable of adaptation (boulding and barcelona 1991). uptake of contaminants by organisms occurs by a variety of pathways, most commonly inhalation, dermal sorption and ingestion. contaminant transfer to organisms may occur by any of these routes, and the major transport route will vary according to the organism and the physicochemical properties of the contaminant (teuten et al. 2009). likelihood that health effects will occur from any exposure to a contaminant depends on the toxicity of the contaminant, which can be determined through understanding of: how harmful, how much, how long and how often the exposure occurs. however, differences in the health status, age, diet, gender, family traits and lifestyle will also affect the outcome of the level of exposure to a particular contaminant (shayler et al. 2009). the protection of human health has utilized biomonitoring as a vital tool to track and assess the level of exposure of the community to environmental pollutants as well provides measures for local and global health policies (scu 2013). characterization of a hazardous waste site provides the understanding to predict future site behavior based on past site behavior (mercer and spalding 1991). a basic assumption in performing remediation is that one cannot remediate what is not observed. consequently, an understanding of what to observe and how to go about making the observations is of utmost importance (boulding and barcelona 1991). almost all typical research of soil quality often carried out solely on the basis of chemical analyses at the oversight of the resulting effect of the level of toxicity on the organism within the ecosystem. toxicokinetics is the kinetics of toxicant absorption, distribution, metabolism and excretion (adme). the investigation of adme profiling and toxicological (adme/tox) screenings during biomonitoring and remediation processes are therefore very important. in silico prediction of toxicokinetics and impact on biomolecules will serve as an additional assessment tool for screening toxic level of soil and foster understanding of impact of pollutants in the environment considering the huge cost and time involve in bioanalytical testing as well as increasing number of toxicants nowadays. in silico prediction of key biomolecular targets of the potential toxic compound will help to ascertain the limit and possible outcome of high exposure. the understanding will help in creating public awareness that is grounded of scientific propositions. the objective of this study was to predict the potential impact of selected toxic compounds from dumpsite or contaminated soils on human health at the molecular level of biological processes. experimental in silico preparation of toxic ligands among the several known toxicants found in typical dumpsites or contaminated soils from bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:35 utc nova biotechnol chim (2019) 18(2): 144-153 146 table 1. list of 13 selected toxicants among severally found in dumpsites or contaminated soils, which have been implicated in human chronic ailments, among which 6 toxicants (in bold) showed significant predicted targets. serial no. type of contaminants smiles format 1 bisphenol a (bpa) cc(c)(c1=cc=c(c=c1)o)c2=cc=c(c=c2)o 2 2,20-bis(p-chlorophenyl)-1,1dichloroethane (ddd) c1=cc(=cc=c1c(c2=cc=c(c=c2)cl)c(cl)cl)cl 3 2,20-bis(p-chlorophenyl)-1,1dichloroethylene (dde) c1=cc(=cc=c1c(=c(cl)cl)c2=cc=c(c=c2)cl)cl 4 2,20-bis(p-chlorophenyl)-1,1trichloroethane (ddt) c1=cc(=cc=c1c(c2=cc=c(c=c2)cl)c(cl)(cl)cl)cl 5 diethylhexyl phthalate (dehp) ccccc(cc)coc(=o)c1=cc=cc=c1c(=o)occ(cc)cccc 6 hexachlorocyclohexane (hch) c1(c(c(c(c(c1cl)cl)cl)cl)cl)cl 7 octylphenol (op) cccccccccc1=cc=c(c=c1)o 8 perfluorooctanesulfonic acid (pfos) c(c(c(c(c(f)(f)s(=o)(=o)o)(f)f)(f)f)(f)f)(c(c(c(f)(f)f)(f)f)(f)f)(f)f 9 nonylphenol (np) cccccccccc1=cc=c(c=c1)o 10 perfluorooctanoic acid (pfoa) c(=o)(c(c(c(c(c(c(c(f)(f)f)(f)f)(f)f)(f)f)(f)f)(f)f)(f)f)o 11 trinonylphenylphosphine (tnpp) ccccccccc[p+](ccccccccc)(ccccccccc)c1=cc=cc=c1 12 tetrachlorodibenzodioxin (tcdd) c1=c2c(=cc(=c1cl)cl)oc3=cc(=c(c=c3o2)cl)cl 13 toxaphene (sonatox) c1c2c(c(c(c1(cl)cl)(c2(ccl)ccl)ccl)cl)cl literatures (teuten et al. 2009; valentin et al. 2013; yao et al. 2015), 13 toxicants (table 1), which were known to be critically implicated in implicated in human chronic ailments such as cancer, chronic cough, and neurological disorder, were selected and used for this study. available structures of these compounds were obtained from the pubchem compound database in structure data file (sdf) and canonical simplified molecular input line entry specification format (smiles). all file conversion to protein data bank (pdb) format were performed using pymol v2.0.7. in silico targets prediction and toxicokinetics in silico targets prediction for the toxicants were done on swisstargetprediction server, where homo sapiens was selected as target organism (diana et al. 2019). among the 13 toxicants, 6 toxicants showed significant predicted targets (i.e. active ligands; table 1), and these were then subjected to in silico absorption-distributionmetabolism-excretion (adme) screening on swissadme server (diana et al. 2017). adme screening was performed at default parameters. molecular docking the molecular docking studies for the 6 toxicants against the 6 targets were carried out according to the method described by fatoki et al. (2018a). the three-dimension (3d) structures of selected 6 targets were then obtained from rcsb protein data bank (pdb). briefly, all water molecules, hetero atoms, and multichains were removed from the crystal structure of the prepared targets using pymol v2.0.7. the gasteiger partial charges were added to the ligand atoms prior to docking. the docking parameter of each prepared ligand and each prepared target, were setup using autodock tools (adt) v1.5.6 (morris et al. 2009) and saves the output file in pdbqt format. molecular docking program autodock vina v1.1.2 (trott and olson 2010) was employed to perform the docking experiment from the command line. after docking, the ligands were analyzed and visualized using adt and pymol v2.0.7. the ligand efficiency (le) was evaluated from the equation, le = -δg/ha, where δg is the free energy of binding and ha is the number heavy atoms (non-hydrogen atoms) of the ligand (padmanabhan et al. 2016). target gene expression analyses the upstream regulatory networks from signatures of differentially expressed genes obtained from toxicants target prediction, were determined by bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:35 utc nova biotechnol chim (2019) 18(2): 144-153 147 table 2. predicted targets of the dumpsites/contaminated soils active toxicants. serial targets gene name uniprot id dumpsite toxicants no. a b c d e f 1 estrogen receptor esr1 p03372 **** **** 2 estrogen receptor beta (by homology) esr2 q92731 **** 3 5-hydroxytryptamine receptor 6 htr6 p50406 **** *** *** 4 fad-linked sulfhydryl oxidase alr gfer p55789 *** 5 carbonic anhydrase 1, 2, 3, 5a, 5b, 7, 13 ca1, ca2, ca3, ca5a, ca5b, ca7, ca13 p00915, p00918, p07451, p35218, q9y2d0, p43166, q8n1q1 *** 6 androgen receptor (by homology) ar p10275 *** ** 7 arachidonate lipoxygenase alox5, alox12, alox15 p09917, p18054, p16050 *** 8 alpha-2a adrenergic receptor adra2a p08913 *** 9 microtubule-associated protein tau mapt p10636 *** ** ** 10 sodium-dependent noradrenaline transporter slc6a2 p23975 *** *** **** 11 sodium-dependent dopamine transporter slc6a3 q01959 *** *** 12 sodium-dependent serotonin transporter slc6a4 p31645 *** *** **** 13 sodium-dependent proline transporter (by homology) slc6a7 q99884 *** *** 14 sodiumand chloride-dependent glycine transporter 1 (by homology) slc6a9 p48067 *** *** 15 sodiumand chloride-dependent neutral and basic amino acid transporter b(0+) (by homology) slc6a14 q9un76 *** *** 16 5-hydroxytryptamine receptor 2a, 2b, 2c (by homology) htr2a, htr2b htr2c, htr6 p28223, p41595, p28335, p50406 *** *** **** 17 adrenergic receptor alpha-2b, 2c (by homology) adra2b, adra2c p18089, p18825 *** *** **** 18 adrenergic receptor beta-1, beta-2, beta-3 adrb1, adrb2, adrb3 p08588, p07550, p13945 **** 19 protein kinase c alpha type, beta type, gamma type, theta type, delta type regulatory subunit prkca, prkcb, prkcg, prkcq, prkcd p17252, p05771, p05129, q04759, q05655 ** 20 tyrosine-protein phosphatase nonreceptor type 1, type 2 ptpn1, ptpn2 p18031, p17706 ** 21 opioid receptor mu-type, delta-type, kappa-type oprm1, oprd1, oprk1 p35372, p41143, p41145 ** 22 adenosine receptor a3 adora3 p33765 **** 23 substance-p receptor, k receptor tacr1, tacr2 p25103, p21452 **** 24 testis-specific androgen-binding protein shbg p04278 **** 25 vascular endothelial growth factor receptor 1, 2, 3 flt1, kdr, flt4 p17948, p35968, p35916 **** 26 aryl hydrocarbon receptor ahr p35869 **** * (20 – 40%), ** (40 – 60%), *** (60 – 80%), **** (80 – 100%) probability of binding on target. probabilities have been computed based on a cross-validation. they may therefore not represent the actual probability of success for any new molecule. (a) = bisphenol a (bpa) cc(c)(c1=cc=c(c=c1)o)c2=cc=c(c=c2)o (b) = 2,20-bis(p-chlorophenyl)-1,1-dichloroethane (ddd) c1=cc(=cc=c1c(c2=cc=c(c=c2)cl)c(cl)cl)cl (c) = 2,20-bis(p-chlorophenyl)-1,1-trichloroethane (ddt) c1=cc(=cc=c1c(c2=cc=c(c=c2)cl)c(cl)(cl)cl)cl (d) = diethylhexyl phthalate (dehp) ccccc(cc)coc(=o)c1=cc=cc=c1c(=o)occ(cc)cccc (e) = nonylphenol (np) cccccccccc1=cc=c(c=c1)o (f) = tetrachlorodibenzodioxin (tcdd) c1=c2c(=cc(=c1cl)cl)oc3=cc(=c(c=c3o2)cl)cl bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:35 utc nova biotechnol chim (2019) 18(2): 144-153 148 transcription factor enrichment analysis, proteinprotein interaction network expansion and kinase enrichment analysis, using the 50 predicted target genes on expression2kinases (x2k) webserver (clarke et al. 2018). results and discussion dumpsite/contaminated soil active toxicants and their predicted targets in this study, six toxicants from typical dumpsite or contaminated soil were found active in biological system at percentage probability greater than 20 % (table 2), although octylphenol (data not shown) was also found to be about 20 % less active than nonylphenol. fifty predicted targets (using gene name or uniprot id) were obtained (table 2) of which six were selected for further investigation based on their crucial implication on human health (table 3). these were classified mainly into: (1) membrane proteins, such as substance-p receptor (also known as neurokinin 1 receptor), 5-hydroxytryptamine receptor and human serotonin transporter; (2) nuclear proteins which include estrogen receptor alpha; and (3) transcription proteins which include aryl hydrocarbon receptor. the serotonin 2a receptor (5-ht2ar) is associated with diseases, such as bipolar disorder, depression, schizophrenia, alzheimer’s disease and parkinson’s disease (aznar and hervig 2016). structural elucidation has shown that 5-ht2ar possesses a unique sideextended cavity near the orthosteric binding site and possibly contributes to the high selectivity of ligands such pimavanserin (kimura et al. 2019). estrogen receptor (er), an allosteric signaling protein and example of nuclear receptors, is a well validated therapeutic target for the treatment of estrogen receptor positive breast cancer (burks et al. 2017; fatoki et al. 2018a). study on model compounds that targets er has provided insight on the close structural similarity with tamoxifen, the fda-approved drug which is a potent selective estrogen receptor modulator (fatoki et al. 2018a). tcdd (2,3,7,8-tetrachlorodibenzo-p-dioxin) is the best known high-affinity ligand for aryl hydrocarbon receptor (ahr), and mediates its toxicity via activation of ahr. increased levels of ahr and its target genes have been implicated in several belligerent tumors such as skin tumors, glioblastoma, or non-small cell lung cancer (schulte et al. 2017). predicted toxicokinetic properties of the toxicants the adme parameter of the selected six toxicants (table 3) showed that bisphenol a (bpa) and nonylphenol (np) were soluble and moderately soluble (respectively), and both likely have high table 3. predicted toxicokinetic parameters of the dumpsites/contaminated soils active toxicants. serial parameters selected dumpsite toxicants no. bpa ddd ddt dehp np tcdd 1 molecular weight 228.29 320.04 354.49 390.56 220.35 321.97 2 heavy atoms (ha) 17 18 19 28 16 18 3 molar refractivity 69.44 80.31 85.15 116.3 71.89 73.07 4 total polar surface area (å2) 40.46 0 0 52.60 20.23 18.46 5 consensus logp 3.6 5.44 5.89 6.17 4.51 5.29 6 esol class soluble moderately soluble poorly soluble poorly soluble moderately soluble poorly soluble 7 gastrointestinal absorption high low low high high low 8 blood brain barrier (bbb) permeant yes no no no yes no 9 p-glycoprotein substrate no no no yes no yes 10 cytochrome p450 inhibitor cyp1a2, cyp2d6 cyp1a2, ctp2c9, cyp2c19, cyp2d6 ctp2c9, cyp2c19 ctp2c9, cyp3a4 cyp1a2, cyp2c19, cyp2d6 cyp2c9 11 skin permeation log kp (cm.s -1) -5.34 -3.98 -3.56 -3.39 -3.55 -3.44 12 lipinski violation 0 1 1 1 0 1 13 bioavailability score 0.55 0.55 0.55 0.55 0.55 0.55 14 synthetic accessibility 1.43 2.37 2.37 4.12 1.63 2.71 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:35 utc nova biotechnol chim (2019) 18(2): 144-153 149 table 4. docking parameters implemented in autodocktool for selected 6 targets. serial no. selected target center grid box [points] size [points] spacing [å] 1 adrenergic receptor alpha-2a 171.339 x 21.415 x 21.578 126 x 108 x 112 0.675 (pdb id: 5uig, chain a) 2 aryl hydrocarbon receptor -8.185 x 37.203 x 215.837 126 x 126 x 126 0.375 (pdb id: 5nj8, chain a) 3 estrogen receptor -33.601 x 18.875 x -21.737 120 x 126 x 122 0.375 (pdb id: 5t97, chain a) 4 sodium-dependent serotonin transporter 36.842 x 183.210 x 142.913 126 x 100 x 126 0.475 (pdb id: 5i6z, chain a) 5 substance-p receptor 2.367 x 28.422 x -26.136 70 x 80 x 126 0.875 (pdb id: 6hll, chain a) 6 5-hydroxytryptamine receptor 2a 43.501 x -0.464 x 60.742 126 x 66 x 76 0.775 (pdb id: 6a93, chain a) gastrointestinal absorption (ga), may permeate blood-brain barrier (bbb) and could inhibit cytochrome p450 (cyp1a2 and cyp2d6). on the other hand, diethylhexyl phthalate (dehp) and tetrachlorodibenzodioxin (tcdd) were identified to possibly have high and low ga respectively, and both are poorly soluble, may not permeate blood-brain barrier (bbb), and serve as p-glycoprotein substrates as well as inhibitors of cytochrome p450 (cyp2c9). the best bioavailability score and synthetic accessibility (sa) score is 1.0 which is an indication of the amount of the compound that could reach the active site and extent of ease of synthesis of the compound, respectively (diana et al. 2017; fatoki et al. 2018b). the sa of the toxicants in this study ranged between 1.43 and 4.12, and showed that they were from or could make up synthetic materials. usually, not all contaminant found in soil are biologically available. bioavailability has been described as the physicochemical access that a toxicant has to the biological processes of an organism (allen 2002). the bioavailability of a contaminant depends on the characteristics of the soil and of the site (shayler et al. 2009). binding energy and efficiency of the toxicants the parameter for docking analysis and predicted binding site amino acid residues are shown in table 4. all the six toxicants investigated in this study show good free binding energies below –5.0 kcal.mol-1 as shown in table 5. ddt and bpa were found to have highest predicted free binding energy in four and two of the targets respectively. the interaction of some of the ligands with the protein targets are shown in fig. 1. the ligand efficiency (le) is a useful metric to assess binding affinity of compounds with respect to the number of non-hydrogen atoms (schultes et al. 2010). the toxic compounds showing le greater than 0.25 kcal.mol-1. ha will possibly cause fatal damage on human health. based on the ligand efficiency result (table 6), the order of toxicity of the compounds investigated in this study is bpa > ddd = ddt > tcdd > np > dehp. table 5. docking score for the binding free energy between the selected 6 targets and 6 active toxicants. serial selected target binding energy [kcal.mol-1] of the selected toxicants no. bpa ddd ddt dehp np tcdd 1 estrogen receptor (pdb id: 5t97) 9.1 8.5 7.3 7.5 6.7 7.4 2 sodium-dependent serotonin transporter (pdb id: 5i6z) -9.5 8.2 9.8 8.2 7.5 8.2 3 adrenergic receptor alpha-2a (pdb id: 5uig) 11.9 11.1 9.2 7.6 6.8 10.3 4 aryl hydrocarbon receptor (pdb id: 5nj8) 8.7 8.3 9.5 7.2 7.0 6.2 5 substance-p receptor (pdb id: 6hll) 9.0 8.2 9.9 7.5 7.1 9.2 6 5-hydroxytryptamine receptor 2a (pdb id: 6a93) 9.2 8.6 10.5 8.3 7.8 8.8 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:35 utc nova biotechnol chim (2019) 18(2): 144-153 150 fig. 1. binding pose and score of the 6 active toxicants, where (a) – bpa interaction with 5uig (11.9 kcal.mol-1); (b) – ddd interaction with 5t97 (8.5 kcal.mol-1); (c) – ddt interaction with 6a93 (10.5 kcal.mol-1); (d) – dehp interaction with 5i6z (-8.2 kcal.mol-1); (e) – np interaction with 5nj8 (-7.0 kcal.mol-1); (f) – tcdd interaction with 6hll (9.2 kcal.mol-1) visualized on pymol and adt, respectively. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:35 utc nova biotechnol chim (2019) 18(2): 144-153 151 table 6. ligand efficiencies (le) of docked scores of selected 6 targets and 6 active toxicants. serial no. selected target ligand efficiency of the selected toxicants [kcal.mol-1 ha] bpa ddd ddt dehp np tcdd 1 estrogen receptor (pdb id: 5t97) 0.54 0.47 0.38 0.27 0.42 0.41 2 sodium-dependent serotonin transporter (pdb id: 5i6z) 0.56 0.46 0.52 0.29 0.47 0.46 3 adrenergic receptor alpha-2a (pdb id: 5uig) 0.70 0.62 0.48 0.27 0.43 0.57 4 aryl hydrocarbon receptor (pdb id: 5nj8) 0.51 0.46 0.50 0.26 0.44 0.34 5 substance-p receptor (pdb id: 6hll) 0.53 0.46 0.52 0.27 0.44 0.51 6 5-hydroxytryptamine receptor 2a (pdb id: 6a93) 0.54 0.48 0.55 0.30 0.49 0.49 gene expression network modulated by the toxicants the overall expression network of the fifty predicted target genes obtained in this study as shown in fig. 2, highly implicated suz12 (polycomb protein) with hypergeometric p-value of 5.44 × 10-10, as the most enriched transcription factors among others, which are associated with the active toxicants from the typical dumpsite while the mitogen-activated protein kinases (mapks) and cyclin-dependent kinases (cdks) with maximum hypergeometric p-value of 2.70 × 10-15 were the major kinases from the protein-protein interaction (ppi) as shown in fig. 2. differential gene expression study has become one of the imperative methods to discover genes which are essential in diagnosis and prediction of diseases such cancer (fatoki et al. 2018b). the transcription factors such as trim28, suz12 and stat3 are associated with the proliferation of cancer cells. the result of this study was found similar with that of enrichment analysis of urethane-targeted genes which showed trim28 and suz12 as most fig. 2. overall network of target genes of active toxicants from typical dumpsite or contaminated soil generated by expression2kinases server. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:35 utc nova biotechnol chim (2019) 18(2): 144-153 152 expressed transcription factors (fatoki et al. 2018b). however, endometrial stromal tumors may be a result of a chromosomal aberration involving suz12. conclusions this study has unravelled some of the chemical compounds from typical dumpsite or grossly contaminated soil that might actively modulate biological system and are potential cause of several ailments which include cancer, bipolar disorder, schizophrenia, neurodegenerative disease and others. this study has showed the threat which toxicants pose to public health. more importantly, it provides avenue for further investigation of super bugs (microorganisms) that can remediate these toxicants in our environment through bioaugmentation, which improves the biodegradative capacities of contaminated sites. environmental monitoring and modern wastes management system should be implemented and enforced in order to safeguard the public health and prevent indiscriminate dumping of wastes in the communities and regularize human habitation far away from existing dumpsites. conflict of interest the authors declare that they have no conflict of interest. references allen he (2002) bioavailability of metals in terrestrial ecosystems: importance of partitioning for bioavailability to invertebrates, microbes, and plants. setac press. pensacola, florida, usa, 158 p. aznar s, hervig me (2016) the 5-ht2a serotonin receptor in executive function: implications for neuropsychiatric and neurodegenerative diseases. neurosci. biobehav. rev. 64: 63-82. boulding jr, barcelona mj (1991) geochemical variability of the natural and contaminated subsurface environment. in site characterization for subsurface remediation. center for environmental research information office of research and development u.s. environmental protection agency, cincinnati, usa, p. 103-122. burks he, abrams t, kirby ca, baird j, fekete a, hamann lg, kim s, lombardo f, loo a, lubicka d, macchi k, mcdonnell dp, mishina y, norris jd, nunez j, saran c, sun y, thomsen nm, wang c, wang j, peukert s (2017) discovery of an acrylic acid based tetrahydroisoquinoline as an orally bioavailable selective estrogen receptor degrader for er alpha + breast cancer. j. med. chem. 60: 2790-2818. clarke djb, kuleshov mv, schilder bm, torre d, duffy me, keenan ab, lachmann a, feldmann as, gundersen gw, silverstein mc, wang z, ma’ayan a (2018) expression2kinases (x2k) web: linking expression signatures to upstream cell signaling networks. nucleic acids res. 46, w1: 171-179. diana a, michielin o, zoete v (2017) swissadme: a free web tool to evaluate pharmacokinetics, druglikeness and medicinal chemistry friendliness of small molecules. sci. rep. 7: 42717. diana a, michielin o, zoete v (2019) swisstargetprediction: updated data and new features for efficient prediction of protein targets of small molecules. nucleic acids res. 47: w357-w364. egboka bc, enwankwor gi, orajaka ip, ejiofor ao (1989) principles and problems of environmental pollution of groundwater resources with case examples from developing countries. environ. health perspect. 83: 39-68. eze ct, amaeze nh (2016) microbiological and heavy metal characterization of soil from an open hospital waste dumpsite in enugu, nigeria. asian j. microbiol. biotechnol. environ. sci. 18: 587-595. fatoki th, awofisayo oa, ogunyewo oa, ugboko hu, sanni dm (2018a) impacts of analogy and dimerization of bioactive compounds on molecular biological functions. j. adv. med. pharma. sci. 19: 1-14. fatoki th, dutta s, oyedele as (2018b) uncovering the selective drug targets for urethane mediated cancer by network approach. .j appl. life sci. int. 19: 1-12. iswa – international solid waste association (2016) a roadmap for closing waste dumpsites the world’s most polluted places. iswa’s scientific and technical committee work-program 2015-2016, 124 p. kimura tk, asada h, inoue a, kadji fmn, im, d, mori c, arakawa t, hirata k, nomura y, nomura n, aoki j, iwata s, shimamura t (2019) structures of the 5-ht2a receptor in complex with the antipsychotics risperidone and zotepine. nat. struct. mol. biol. 26: 121-128. lombi e, wenzel ww, adriano dc (1998) soil contamination, risk reduction and remediation. land contamin. reclamat. 6: 183-187. mercer jw, spalding cp (1991) site characterization overview. in site characterization for subsurface remediation. center for environmental research information office of research and development u.s. environmental protection agency, cincinnati, usa, p. 12-21 morris gm, huey r, lindstrom w, sanner mf, belew rk, goodsell ds, olson aj (2009) autodock4 and autodocktools4: automated docking with selective receptor flexibility. j. comput. chem. 30(16): 2785-2791. odeyemi, at, faweya eb, agunbiade or, ayeni sk (2011) bacteriological, mineral and radioactive contents of leachate samples from dumpsite of ekiti state government destitute centre in ado-ekiti. arch. appl. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:35 utc nova biotechnol chim (2019) 18(2): 144-153 153 sci. res. 3(4): 92-108. padmanabhan b, ramu m, mathur s, unni s, thiyagarajan s (2016) identification of new inhibitors for human sirt1: an in-silico approach. med. chem. 12(4): 347-361. pasko oa, mochalova tn (2014) toxicity assessment of contaminated soils of solid domestic waste landfill. iop conf. ser. earth environ. sci, 21: 012044 schulte kw, green e, wilz a, platten m, daumke o (2017) structural basis for aryl hydrocarbon receptor-mediated 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bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:35 utc nova biotechnol chim (2019) 18(2): 115-123 doi: 10.2478/nbec-2019-0014 corresponding author: baquralhilly_79@yahoo.com; mohammed79@agre.uoqasim.edu.iq nova biotechnologica et chimica deleterious amino acid substitutions with a series of putative damaging effects on egg components are revealed in the ovalbumin gene family; an in silico approach mohammed baqur s. al-shuhaib department of animal production, college of agriculture, al-qasim green university, 8-al qasim, babil 51001, iraq article info article history: received: 18th january 2019 accepted: 14th april 2019 keywords: chickens egg production oviduct prediction single nucleotide polymorphism abstract this study was conducted to identify the most deleterious nonsynonymous single nucleotide polymorphisms (nssnps) in the ovalbumin gene family, including ovalx, ovaly, and oval genes, which are involved in the synthesis of the most important components in the chickens’ eggs using a comprehensive in silico approach. ten different computational servers were utilized to prioritize the possible deleterious effects of the retrieved nssnps in terms of structure, function, and stability. results indicated entirely damaging effects of h365p in ovalx, i167t in ovaly, and v209g, l231p, f307c, and s317p in oval proteins. further prediction tools showed that all of these deleterious nssnps were positioned in variable locations within several α-helix motifs in all studied ovalbumin proteins. furthermore, all witnessed nssnps were predicted to be resided in the receptors binding sites, signifying remarkable involvement of such nssnps in damaging of the altered proteins. in conclusion, the present study provides the first inclusive data with regard to the most deleterious nssnps in ovalx, ovaly and oval genes in chickens. the present bioinformatics data may be useful for breeders who intend to raise chickens for egg production, in such a way the presence of any of these deleterious nssnps in any selected breed may possess several damaging effects on the egg components, which may impair egg production. therefore, it can be stated that breeders have to confirm the absence of any of these deleterious nssnps before being proceeded further for large-scale egg-production purposes. university of ss. cyril and methodius in trnava introduction the ovalbumin gene family in gallus gallus is composed of three homologous genes located on chromosome 2, namely ovalbumin (oval), ovalbumin-related protein x (ovalx), and ovalbumin-related protein y (ovaly). oval, ovalx, and ovaly proteins belong to the serine protease inhibitor (serpin) family whose members share the same overall tertiary structure of eight to nine α-helices and three beta-sheets. all these three genes are mainly expressed in the oviduct (sugimoto et al. 2001), and more specifically by tubular gland cells of the chicken’s magnum (rehault-godbert et al. 2013). sequences alignment of these three genes have shown a sequence identity of 73 % between oval and ovalx, of 72 % between oval and ovaly, and 82 % between ovalx and ovaly genes (da silva et al. 2015). although oval, ovaly, and ovalx proteins have exerted high homology in their primary sequences, distinct and subtle physicochemical and structural differences have exhibited among them that might be associated bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:28 utc mailto:baquralhilly_79@yahoo.com nova biotechnol chim (2019) 18(2): 115-123 116 with specific function on each one. in addition to their major localization in the egg white (hashim et al. 2019), oval, ovalx, and ovaly proteins have also been detected in all other egg compartments, including eggshell, egg yolk, and vitelline membrane (mann et al. 2008). oval is the major egg white protein and might be a source of amino acids for the developing embryo (liu et al. 2015). oval is synthesized in the tubular gland cells in the hen’s oviduct. it is well known that such a synthesis mechanism is under hormonal control with a consequent effect on egg white formation. oval accounts for about 54 % of the total proteins of egg white and more than 80 % of the total proteins of egg yolk (rehaultgodbert et al. 2018). however, the current hypothesis is that oval serves as a source of amino acids for the developing embryo. the synthesis of oval is upregulated in the magnum in the early onset of the postovulation period (zhao et al. 2016). ovalx gene and its closest neighbour ovaly gene, are highly similar genes and have a close relation with oval. both have likely arisen by duplication events from a common ancestral oval gene because the two gene-coding sequences share highly significant similarity with that of the oval (da silva et al. 2015). though ovalx and ovaly properties have been described recently, their biological functions remain to be undefined. with regard to ovalx, it has been shown to exhibit antimicrobial activities against pathogens via its positively charged heparinbinding domain exposed at its surface (rehaultgodbert et al. 2013). these activities suggest considerable participation for ovalx in egg innate defense mechanisms (dombre et al. 2017; dasilva et al. 2019). meanwhile, the role of ovaly has not been explored yet but because its physiological function is not known. however, increasing data in the literature have shown that its abundance was affected during incubation, suggesting a potential involvement of ovaly in some aspects of fertilization or embryonic development (qiu et al. 2012). thus, any deleterious amino acid substitution in these proteins may have a series of damaging effects on their biological activity leading to a series of negative consequences on eggs formation. irrespective of ovalbumin genes, the recent computations of amino acid substitutions have been applied on several genetic markers to predict a variety of physiological effects in birds, such as chickens (yakubu et al. 2017), ostriches (alshuhaib et al. 2018), and quails (al-shuhaib et al. 2019). up to date, no comprehensive analyses have presented to highlight this aspect in oval, ovalx, and ovaly genes and their altered proteins. therefore, this study aims to exploit the advent of computational biology to present the most deleterious nonsynonymous single nucleotide polymorphisms (nssnps) that may be involved in preventing of oval, ovalx, and ovaly proteins in undergoing their roles in the proper egg white formation. experimental molecular modelling the uniprotkb codes for oval, ovalx, and ovaly proteins were a0a2h4y7u9, a0a1d5pi58, and e1btf4, respectively. no tertiary structures were found in the deposited protein databank to cover the full studied proteins. thus, the 3d structure of the involved oval, ovalx, and ovaly proteins were generated using the phyre2 (protein homology/analogy recognition engine) software (kelley et al. 2015). the generated models were validated using ramachandran plot (laskowski et al. 2001) and qmean (qualitative model energy analysis) z-score generated by swiss model structure assessment validation tools (waterhouse et al. 2018). protein data bank formats of the generated models were used as a template for predictions. snps retrieval all the amino acid substitutions of the three ovalbumin genes of chickens were comprehensively investigated in this study, namely oval (gene id: 396058), ovalx (gene id: 420898), and ovaly (gene id: 420897) genes. a total of 30 nssnps were retrieved were retrieved from ensemble genome browser 97 (https://asia.ensembl.org/index.html), including 6 nssnps for oval, 15 nssnps for ovalx, and 9 nssnps for ovaly genes. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:28 utc https://asia.ensembl.org/index.html nova biotechnol chim (2019) 18(2): 115-123 117 prediction of nssnps effects on proteins structure, function, and stability all nssnps were prioritized in terms of their effects on protein structure, function, and stability using ten different in silico tools. sift (sorting intolerant from tolerant snps) (pauline et al. 2003), provean (protein variation effect analyzer), predict snp (bendl et al. 2014), mapp (multivariate analysis of protein polymorphism) (chao et al. 2008), polyphen (polymorphism phenotyping) (adzhubei et al. 2013), and snap2 (screening for non-acceptable polymorphisms 2) (smigielski et al. 2000) were utilized to assess the effect of each nssnp on protein structure and biological activity. meanwhile, mcsm (pires et al. 2012), sdm (worth et al. 2012), i-mutant2 (capriotti et al. 2005), and cupsat (parthiban et al. 2006) tools were used to evaluate the effects of the same nssnps on proteins stability upon mutations. prediction of ligand binding sites of the most deleterious nssnps the potential effect of each nssnp on altering protein binding capability was predicted using set of protein-receptors or protein-ligand binding tools, including coach (wu et al. 2018), tm-site (zhang et al. 2005), s-site (yang et al. 2013), cofactor (zhang et al. 2017), finsidte (brylinski et al. 2008), and concavity (capra et al. 2009) prediction tools. energy analyses of proteins after being mutated with deleterious nssnps two sets of parameters were utilized to assess energy changes upon mutation. the total energy after minimization parameters and rmsd (root mean square deviation) values were calculated by deepview/swiss-pdbviewer (johansson et al. 2012) and yasara (krieger et al. 2014) tools, respectively. results the generation of optimal tertiary models of oval family genes was performed via phyre2 server. the validation of the generated models that carried out by procheck and qmean tools indicated that the generated models were being within the accepted limits (table 1). considering the ramachandran plot, models were considered qualified when they showed 90 % or more within the favoured regions. results showed 91.3 % of the amino acid residues of ovalx and ovaly were suited in the favoured regions, while the amino acid residues of oval exhibited 90.0 % in the same region. likewise, both ovalx and ovaly did not show any amino acid residues in the disallowed region, while oval showed only 2 amino acid residues (0.6 %) in the same region. qmean z-score outputs were further proven the current models by giving -2.60, -2.20, and -1.92 for ovalx, ovaly, and oval, which, however, they were under the accepted standards. table 1. three-dimensional structures validations for the phyre2 generated models of ovaly, ovalx, and oval proteins in chickens using procheck tool and swiss model assessments server. characteristics ovalx ovaly oval residues in most favoured regions 324 (91.3 %) 324 (91.3 %) 320 (90.9 %) residues in additional allowed regions 27 (7.6 %) 27 (7.6 %) 29 (8.2 %) residues in generously allowed regions 4 (1.1 %) 4 (1.1 %) 1 (0.3 %) residues in disallowed regions 0 (0.0 %) 0 (0.0 %) 2 (0.6 %) number of non-glycine and non-proline residues 355 (100 %) 355 (100 %) 352 (100 %) number of end-residues 1 1 1 number of glycine residues 20 20 19 number of proline residues 12 12 14 total number of residues 388 388 386 qmean z-score -2.60 -2.20 -1.92 cβ -1.49 -1.51 -0.77 all atoms -2.32 -1.92 -1.60 solvation 0.23 0.48 0.53 torsion -2.47 -2.15 -2.02 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:28 utc nova biotechnol chim (2019) 18(2): 115-123 118 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:28 utc nova biotechnol chim (2019) 18(2): 115-123 119 table 3. predictions of ligand binding sites for the most deleterious missense snps in the ovalx, ovaly, and oval proteins in chickens. protein name snp id aa change coach tm-site s-site co factor findsite concavity ovalx rs738624565 h365p peptide (pdb id: 4au2d) – multiple ligands – – – ovaly rs1057687043 i167t – – – – ligand (pdb id: 1br8b00) – oval rs733410745 v209g peptide (pdb id: 3dy0a) ligand (1jrra_bs01) – – – – rs734546333 l231p peptide (pdb id: 3dy0a) ligand (1lq8e_bs01) – ligand (pdb id: 1iz2a) – – rs731320904 f307c – – – – – binding residue rs733774347 s317p – – multiple ligands – ligand (pdb id: 1azxa00) – in order to evaluate the effect of each nssnp on protein structure, biological activity, and stability, a variety of computational tools were utilized, including sift, provean, predict snp, mapp, polyphen, snap2, mcsm, sdm, i-mutant2, and cupsat. cumulative results showed an entirely deleterious effect for a total six nssnps for the ovalbumin gene family, including h365p (rs738624565) for the ovalx gene, i167t (rs1057687043) for the ovaly gene, and v209g (rs733410745), l231p (rs734546333), f307c (rs731320904), and s317p (rs733774347) for the oval gene (table 2). more analyses were conducted on these most deleterious nssnps to explore their role to cause such extreme alteration in the mutant proteins. the ability of these risky nssnps to change the binding activity with other proteins was investigated by utilizing six prediction tools, including coach, tm-site, s-site, cofactor, findsite, and concavity. all the utilized prediction tools revealed obvious participation of all the included risky nssnps in several anticipated intensities with respect to their binding with ligands or receptors (table 3). these findings may be due to the critical positioning taken by most of these nssnps in their proteins. considering h365p and i167t, their positioning in the α-helix of the ovalx and ovaly, respectively may be involved in their deleterious effects (fig. 1 a and b). the 3d visualizations of the normal and mutant oval proteins were added another layer of confirmation of such deleterious effects caused by v209g, l231p, f307c, and s317p. this was due to their positioning within variable locations in the α-helix (fig. 2). the total energy values for the native ovalx, ovaly, and oval structures and their mutant structures were performed. both h365p and i167t table 4. the total energy after minimization parameters and rmsd values for wild type and the most deleterious mutant forms of ovaly, ovalx, and oval proteins in chickens. the total energy after minimization and rmsd values were calculated by deepview/swiss-pdbviewer and yasara tools, respectively. protein name nssnp id amino acid change total energy after minimization rmsd ovalx wild type -15699.480 rs738624565 h365p -15431.578 0.0219 a ovaly wild type -13697.646 rs1057687043 i167t -13707.831 0.0068 a oval wild type -17478.184 rs733410745 v209g -17381.457 0.0218 a rs734546333 l231p -17443.225 0.0085 a rs731320904 f307c -15665.894 0.0309 a rs733774347 s317p -15635.917 0.0095 a bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:28 utc https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000043252;r=2:67714111-67721182;t=ensgalt00000067317;vf=15238280 https://zhanglab.ccmb.med.umich.edu/biolip/qsearch_pdb.cgi?pdbid=4au2 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000019551;r=2:67700354-67711715;vf=15237987 https://zhanglab.ccmb.med.umich.edu/coach/output/ch051932/protein/findsite/complex5.pdb https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000012869;r=2:67682465-67690037;t=ensgalt00000037195;vf=15237550 https://zhanglab.ccmb.med.umich.edu/biolip/qsearch_pdb.cgi?pdbid=3dy0 https://zhanglab.ccmb.med.umich.edu/coach/output/ch051940/protein/tmsite/complex2_1jrra_bs01_iii.pdb https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000012869;r=2:67682465-67690037;t=ensgalt00000037195;vf=15237548 https://zhanglab.ccmb.med.umich.edu/biolip/qsearch_pdb.cgi?pdbid=3dy0 https://zhanglab.ccmb.med.umich.edu/coach/output/ch051940/protein/tmsite/complex3_1lq8e_bs01_iii.pdb https://zhanglab.ccmb.med.umich.edu/biolip/qsearch_pdb.cgi?pdbid=1iz2 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000012869;r=2:67682465-67690037;t=ensgalt00000037195;vf=15237524 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000012869;r=2:67682465-67690037;t=ensgalt00000037195;vf=15237522 https://zhanglab.ccmb.med.umich.edu/coach/output/ch051940/protein/findsite/complex3.pdb https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000043252;r=2:67714111-67721182;t=ensgalt00000067317;vf=15238280 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000019551;r=2:67700354-67711715;vf=15237987 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000012869;r=2:67682465-67690037;t=ensgalt00000037195;vf=15237550 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000012869;r=2:67682465-67690037;t=ensgalt00000037195;vf=15237548 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000012869;r=2:67682465-67690037;t=ensgalt00000037195;vf=15237524 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000012869;r=2:67682465-67690037;t=ensgalt00000037195;vf=15237522 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000043252;r=2:67714111-67721182;t=ensgalt00000067317;vf=15238280 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000019551;r=2:67700354-67711715;vf=15237987 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000012869;r=2:67682465-67690037;t=ensgalt00000037195;vf=15237550 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000012869;r=2:67682465-67690037;t=ensgalt00000037195;vf=15237548 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000012869;r=2:67682465-67690037;t=ensgalt00000037195;vf=15237524 https://asia.ensembl.org/gallus_gallus/variation/summary?db=core;g=ensgalg00000012869;r=2:67682465-67690037;t=ensgalt00000037195;vf=15237522 nova biotechnol chim (2019) 18(2): 115-123 120 fig. 1. three-dimensional structure of the most deleterious nonsynonymous snps in the ovalx and ovaly proteins, namely i90m, f109s, and y127f. a. positioning of the amino acid substitution h365p in the ovalx protein. b. positioning of the amino acid substitution i167t in the ovaly protein. the generated structures were visualized by pymol software. did not show a noticeable deviation in the total energy minimization values compared with their wild type counterparts. considering oval deleterious mutations, both f307c and s317p mutant models showed an increase in energy minimization than the native structure (table 4). these less favourable changes indicated more deleterious nature of both f307c and s317p models than the v209g and l231p counterpart. discussion ovalbumin family are the most essential proteins in hens’ reproductive systems and are an ideal source for embryo development. though several data have increasingly been provided with regard to oval, ovalx, and ovaly dna sequences and its translation (ren et al. 2017; dasilva et al. 2019), no comprehensive study has been conducted to analyze the consequences of their nssnps on ovalbumin family. in this study, the retrieved nssnps of the ovalbumin gene family were comprehensively highlighted in terms of their effects on structure, biological activity, and stability. the present study witnessed six entirely deleterious nssnps in the ovalbumin gene family by recruiting up to 10 different in silico tools. however, all of these deleterious amino acid substitutions were found to be localized within several positions in the α-helix motif of ovalx, ovaly, and oval proteins. this localization may reflect a similar pattern of these substituted amino acid residues to induce these deleterious effects (abrusán and marsh 2016). in order to explore the possible roles of the observed nssnps in such deleterious effects, several in silico algorithms were used to perform this task. moreover, the possible mechanisms of these nssnps in the interruption of these mutant proteins with regard to its binding with receptors were analyzed. noticeable positioning of all these risky nssnps in binding sites with their corresponding receptors was notified. these findings indicated that the observed amino acid substitutions were involved in the binding network of the ovalx, ovaly, and oval protein, which may provide a clear modification in the affinity of interaction with corresponding receptors. whatever the pattern each nssnp takes in the interruption of the ovalbumin family, the current finding bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:28 utc nova biotechnol chim (2019) 18(2): 115-123 121 fig. 2. three-dimensional structure of the most deleterious nonsynonymous snps in the oval protein, namely v209g, l231p, f307c, and s317p. the generated structures were visualized by pymol software. indicated that these nssnps seriously affect the structure, function, and stability of ovalbumin family, which have a series of deleterious consequences on the biological pathways in which these proteins involved, such as egg defense activity, embryonic fertilization and development (liu et al. 2015; akazawa et al. 2019). accordingly, its noteworthy to distinguish that both h365p and i167t, being the most deleterious nssnps in ovalx and ovaly, respectively, should take priority in terms of reducing antimicrobial activity in case of ovalx (guyot et al. 2016), or damaging embryonic development in case of ovaly (da silva et al. 2015). this observation is particularly interesting, knowing that once these amino acid substitutions detected in a particular breed, serious precautions should be taken for breeders with respect to the handling of this breed. this is due to the presence of these snps in ovalx and its ovaly homolog, which may hamper the eggs production in this mutant population. the same precautions could also be applied to the v209g, l231p, f307c, and s317p mutant oval proteins. however, these amino acid substitutions deserve more attention as embryo development and the synthesis of many egg components are sensitive to alterations in oval architecture (lin et al. 2016). keeping in mind the wide availability of these proteins in all the eggs compartments (dombre et al. 2017), the altered structure, biological activity, stability, or binding characteristics of ovalbumin proteins family seem to disrupt several biological pathways undertaken by chicks’ embryos to take their schedules tasks of development and proper formation. it can be stated that the following nssnps h365p, i167t, v209g, l231p, f307c, and s317p should be absent in ovalbumin family in any chicken populations desired to be raised for higher egg production purposes. conclusions the present cumulative in silico approach indicated the presence of entirely deleterious six nssnps in ovalbumin family, including h365p in ovalx, i167t in ovaly, and v209g, l231p, f307c, and s317p in oval. as long as more deleterious nssnps were witnessed in a particular breed, more serious indication for the reduced eligibility of this bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:28 utc nova biotechnol chim (2019) 18(2): 115-123 122 breed for egg-production purposes should be concluded. this study provides the first comprehensive in silico guide for breeders to investigate the competency of raising chicken breeds for the large-scale egg production purposes. acknowledgement this study has not received any fund from any agency or institution. conflict of interest the authors declare that they have no conflict of interest. references abrusán g, marsh ja (2016) alpha helices are more robust to mutations than beta strands. plos comput. biol. 12: e1005242. adzhubei i, jordan 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mj, luo h, lin h (2016) ovalbumin expression in the oviduct magnum of hens is related to the rate of egg laying and shows distinct stress-type specific responses. j. anim. physiol. anim. nutr. 100: 876-883. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:28 utc nova biotechnol chim (2019) 18(1): 66-71 doi: 10.2478/nbec-2019-0009  corresponding author: stefan.demcak@tuke.sk nova biotechnologica et chimica using of wooden sawdust for copper removal from waters stefan demcak1,, magdalena balintova1, maria demcakova2, inga zinicovscaia3,4, nikita yushin3 and marina v. frontasyeva3 1 institute of environmental engineering, faculty of civil engineering, technical university of kosice, vysokoskolska 4, sk-042 00 kosice, slovakia 2 institute of experimental physics, slovak academy of sciences, watsonova 47, sk-040 01 kosice, slovakia 3 frank laboratory of neutron physics, joint institute for nuclear research, joliot-curie str. 6, 1419890 dubna, russia 4 horia hulubei national institute for r&d in physics and nuclear engineering, 30 reactorului str. mg-6, bucharest – magurele, romania article info article history: received: 18th january 2019 accepted: 14th april 2019 keywords: heavy metals water treatment adsorption wooden sawdust abstract the heavy metal removal from wastewater is very important due to their persistent character in aquatic environment. the use of wooden sawdust is emerging as a potential alternative to the existing conventional technologies for the removal of metal ions from aqueous solutions. the aim of this work is to study the cu(ii) removal of from water by unconventional waste products including the wooden sawdust of poplar, cherry, spruce and hornbeam. the ft-ir spectra of the studied wooden sawdust confirmed the presence of functional groups that have potential for heavy metal binding. the highest efficiency of cu(ii) removal was observed for poplar wooden sawdust at static (86 %) and dynamic (88 %) adsorption experiments. data obtained by neutron activation analysis revealed that ion exchange is also a mechanism of metal removal by the selected wooden sawdust.  university of ss. cyril and methodius in trnava introduction the heavy metal ions are discharged to the aquatic environment by various branches of industries as mining, tanneries, batteries, paper industries, agrochemicals, etc. the heavy metals are toxic and accumulate in living bodies causing serious diseases and disorders (celik and demirbaş 2005; singovszka et al. 2016). the toxic heavy metals such as cr, se, v, cu, co, ni, cd, hg, as, pb, and zn can be considered as the most hazardous metals toxic for human even at low concentrations (gardea-torresdey et al. 1998). the most commonly used methods for removal of metal ions from wastewaters are chemical precipitation, ion exchange, chemical oxidation/reduction, reverse osmosis, electro dialysis, ultra-filtration, etc. (gardea-torresdey et al. 1998; zhang et al. 1998). however, these techniques have limitations such as less efficiency at low concentrations or production of secondary sludge, which furthers the disposal, is costly (ahluwalia and goyal 2005). another technology suitable for treatment of wastewater is adsorption of heavy metals by activated carbon. however, the high cost of activated carbon and its loss during the regeneration restricts its wide application (imamoglu and tekir 2008). in last two decades is booming for the use of low cost organic materials for heavy metal ions removal. it started with the application of biomass and agricultural waste materials as peat, rice husk, leaves or seeds (bailey et al. 1999; vieira and bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:12 utc mailto:stefan.demcak@tuke.sk nova biotechnol chim (2019) 18(1): 66-71 67 table 1. examples of adsorption capacity of selected natural adsorbents. adsorbent adsorption capacity [mg.g-1] references cu(ii) zn(ii) ni(ii) pb(ii) cd(ii) chitosan 222.0 75.0 164.0 16.4 8.5 (bailey et al. 1999; babel and kurniawan 2003) zeolites 5.1 5.5 4.5 175.0 137.0 (bailey et al. 1999; babel and kurniawan 2003) clays 1.2 52.9 – 4.3 11.4 (bailey et al. 1999; babel and kurniawan 2003) peats 19.6 13.1 11.7 – 22.5 (bailey et al. 1999; babel and kurniawan 2003) bark 0.7 0.8 – 400.0 32.0 (bailey et al. 1999; demcak et al. 2017b) sawdust 3.9 3.7 – 7.3 – (bailey et al. 1999; holub et al. 2016) volesky 2000; demcak et al. 2017a). the major advantages of adsorption on natural sorbents in comparison to conventional methods include low cost, high efficiency, minimization of chemical or biological sludge, no additional nutrient requirement, regeneration of biosorbents, and possibility of metal recovery (leng et al. 2015). the table 1 represents the adsorption capacities of selected natural adsorbents for the heavy metals removal. wooden materials or wastes are cheap sorbent materials (ngah and hanafiah 2008; ahmaruzzaman 2011). the benefits of application of wooden adsorbent materials (by-products or wastes) for heavy metals removal from wastewaters are determined by their high removal selectivity, good adsorption capacity and possibility of regeneration. in several studies (shukla et al. 2002; šćiban et al. 2007; asadi et al. 2008) it has been reported that the wooden adsorbent materials such as straw, tree bark, peanut skins, wood sawdust, moss and peat are a suitable replacement of industrial produced sorbents with comparable efficiencies of pollutants removal. the wooden sawdust is a perspective low-costs adsorbent material for the removal of heavy metal ions, some types of acid, basic dyes, and other unwanted compounds from wastewaters (demcak et al. 2017b). the efficiency of the wooden sawdust adsorption processes is also affected by the composition of the wastewater where the formation of complex compounds of metal cations with wood sawdust functional groups can take place resulting in the decrease or a failure of the heavy metals adsorption (ahmad et al. 2009). the adsorption properties of wooden sawdusts are influenced by their composition. the wooden sawdust are mainly composed from lignin, cellulose and hemicellulose, carbohydrates, and phenolic compounds, which contain carboxyl, hydroxyl, sulphate, phosphate, and amino groups, the main metal binding (gardea-torresdey et al. 1990; crini 2006). the application of wooden materials such as wooden sawdust for heavy metals removal brings many benefits for the protection of environment, where the contaminated water could be treated. it brings for the timber industry a new opportunity for the utilization of wooden sawdust as promised cheap adsorbents (shukla et al. 2002). the paper deals with a cu(ii) ions removal from model solutions by poplar, spruce, cherry and hornbeam wooden sawdust under static and dynamic conditions. the selected wooden sawdust were analysed by infrared spectrometry for characterization of functional groups, which can be responsible for metal binding. efficiency of cu(ii) ions removal was analysed by colorimetric method and changes of ph values were also measured. neutron activation analysis was used to determine the elemental composition of wooden sawdust (raw and culoaded). experimental sorbent-sorbate characterisation the wooden sawdust of poplar, spruce, cherry, and hornbeam species of locally available wood was sieved, and only the fractions with a particle size under 2.0 mm were used for the experiments. the model solution with initial concentrations of cu(ii) ions 10 mg.l-1 was prepared by dissolving of cuso4·5h2o in deionised water. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:12 utc nova biotechnol chim (2019) 18(1): 66-71 68 sorption experiment the batch adsorption experiments were carried out in two modes (static and dynamic conditions). in both adsorption experiments, 1 g of dry wooden sawdust were mixed with 100 ml of model solutions (initial concentration of dissolved cu(ii) ions was 10 mg.l-1). in static conditions, the sorbent-sorbate interaction time was 1 day. to determine the contact time required for equilibrium sorption in dynamic condition the samples were analysed in different time intervals (5, 10, 15, 30, 45, 60 and 120 min). in all causes, at the end of the adsorption experiments, wooden sawdust was removed by filtration through a laboratory filter paper. residual concentrations of cu(ii) ions present in solutions were determined by colorimetric method and ph changes was also measured. in both cases, the percentage efficiency of ion removal η removal was calculated using the following equation (eq. 1): , (1) where c0 is the initial concentration of appropriate ions [mg.l-1] and ce equilibrium concentration of ions [mg.l-1]. all adsorption experiments were carried out in triplicate under the batch conditions and results are given as arithmetic mean values. apparatus and instrumentation the ir measurements of the wooden sawdust were performed on a bruker alpha platinum-atr spectrometer (bruker optics, ettingen, germany). a total of 24 scans were performed on each sample in the range of 4,000 to 400 cm-1. concentrations of the cu(ii) in solution were determined using the colorimetric method with a colorimeter dr890, (hach lange, germany) and the appropriate reagent. the input ph value of model solution was measured by ph meter inolab ph 730 (wtw, germany). the elemental composition of raw and cu-loaded wooden sawdust was determined by neutron activation analysis (naa) at the pulsed fast reactor ibr-2 of the frank laboratory of neutron physics, jinr, dubna, russia. for naa analysis samples of approximately 0.3 g, dried at 40 °c to constant weight, were packed in polyethylene foil bags for short-term irradiation or in aluminium cups for long-term irradiation. a total of 5 elements: na, mg, k, ca, and ba were determined. more details concerning the irradiation time and gamma spectra processing can be found in (frontasyeva 2011). the difference between the certified and measured content of elements of the certified material varied between 1 and 10 %. results and discussion infrared spectra of wooden sawdust the functional groups present in natural materials as alcoholic, carbonyl, phenolic, amide and amino, sulfhydryl groups etc. have affinity for heavy metal ions to form metal complexes or chelates. the mechanism of biosorption process includes chemisorption, complexation, adsorption on surface, diffusion through pores and ion exchange (sud et al. 2008). the functional groups of poplar, spruce, cherry, and hornbeam were determined using ftir spectroscopy (fig. 1). the metal adsorption capacity of wooden sawdust is influenced with the presence of surface structures of -oh (3,650 – 3,000 cm-1 and 1,700 – 1,600 cm-1), -cooh (1,750 – 1350 cm-1 and 1,250 – 1,000 cm-1), and -nhx (3,337 cm -1) functional groups that are present in organic materials (ricordel et al. 2001). according to the literature (kidalova et al. 2015), the structure of wooden sawdust is primarily formed by cellulose, hemicellulose, and lignin. the detailed ft-ir spectra of wooden sawdust fig. 1. infrared spectra of selected wooden sawdust. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:12 utc nova biotechnol chim (2019) 18(1): 66-71 69 characterisation and the band assignments are available in the literature (ricordel et al. 2001; schwanninger et al. 2004; stevulova et al. 2014; ghosh et al. 2015; kidalova et al. 2015; zhang et al. 2015). static adsorption study the results of cu(ii) removal from the model solution with initial concentration 10 mg.l-1 by the selected wooden sawdust are shown in table 2. the poplar, spruce, cherry, and hornbeam wooden sawdust exhibit a very good efficiency for cu(ii) removal. the best absorption property was revealed for the poplar wooden sawdust with efficiency about 86 %. it is comparable to the results of research šćiban et al. (2007). they found that 2 g of poplar wooden sawdust in 100 ml of contaminated water is efficient for removal of the most of the dissolved cu(ii). table 2. results of static adsorption experiments with the selected wooden sawdust (c0(cu(ii)) = 10 mg.l -1). sorbent cu(ii) initial ph = 5.8 ce η ph [mg.l-1] [%] poplar 1.42 85.8 5.2 spruce 1.90 81.0 4.9 cherry 2.30 77.0 4.9 hornbeam 2.72 72.8 5.2 the adsorption experiments were accompanied by decreasing of ph values in solutions. the monitoring of the ph changes in the solutions during adsorption is an important controlling parameter that could be used to describe the heavy metals removal mechanisms. since ph affects the surface charge of the adsorbent, the degree of ionization and speciation of sorbate during adsorption (rahman and islam 2009). shukla et al. (2002) observed that at decreasing of ph values, the positively charged metal ion species can compete with h+ and be absorbed at the surface of the sawdust through the ion exchange mechanism. on the other hand, some heavy metals may cause the releasing of ohinstead of h+ when they are adsorbed by sawdust materials, and therefore result in an increase in ph. decrease of ph in studied systems indicate on ion exchange of cu(ii) and h+ ions. kinetic adsorption study the poplar wooden sawdust exhibited under static conditions the best adsorption properties. from this reason these sawdust was used for detailed study under dynamic adsorption mode. the efficiency of cu(ii) removal and ph changes over the experimental time are shown in table 3. obtained data show high rate of cu(ii) removal from model solution after 5 min of sorbent-sorbate interaction (approximately 88 %). the rest of the adsorption experiment can be considered as a relative settled with slower changes of removal efficiency. from this result we could suppose that the removal of cu(ii) ions might occur as a two-stage process. at the beginning of the sorbent-sorbate interaction, the increase in ph is accompanied with ionexchange. in the second stage, the adsorption of metals ions takes place at stabilised ph values. the maximum efficiency of cu(ii) removal from model solution (98 %) was achieved after 30 min. changes of ph values in solutions were also observed. a significant change of ph value was revealed at the begging of dynamic experiment. holub et al. (2013) observed that an intensive ph change is caused by the high initial concentration of heavy metals in solution that are involved in the intensive ion exchange with the adsorbent. the change of ph was recorded after 5 min of the adsorption where ph values increase from 5.8 to 6.1. it could be caused by the mechanism of ion exchange between cu(ii) and chemical elements in the poplar wooden sawdust. after the completion of the ion exchange, the ph beganto decrease gradually to the approximately input values. neutron activation analysis of wooden sawdust the naa data obtained for raw and cu-loaded wooden sawdust are presented in table 4. obtained results reveal the decrease of content of all determined elements in cu-loaded wooden sawdust showing that ion exchange is one of the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:12 utc nova biotechnol chim (2019) 18(1): 66-71 70 table 3. results of metal sorption efficiencies η and changes of ph values over the experimental time at dynamic. time [min] 0 5 10 15 30 45 60 120 ce [mg.l-1] 10.0 1.22 0.88 0.72 0.24 0.68 0.50 1.12 η [%] 0.0 87.8 91.2 92.8 97.6 93.2 95.0 88.8 ph 5.8 6.1 5.9 5.8 5.7 5.7 5.7 5.7 mechanisms of copper biosorption. thus, the amount of potassium (k) in the cu-loaded wooden sawdust after the 24-hour adsorption experiments decreased under the detection limit. the most pronounced decrease of calcium (ca) content, approximately 10 times, was observed for spruce cu-loaded sawdust. significant decrease of sodium (na) content was observed for spruce and hornbeam wooden sawdust, while of magnesium (mg) for spruce and cherry wooden sawdust. the content of barium almost in the same degree for all studied sorbents was decreased. table 4. chemical composition of natural wooden sawdust and cu-loaded wooden sawdust (selected elements). element average content of elements in adsorbents [mg.kg-1] na mg k ca ba poplar 12.5±0.9 264±18.5 1,790±286 1,810±181 32.1±1.6 poplar_cu(ii) 12.3±0.9 174±12.2 < 151.0* 1,520±152 24.5±1.23 spruce 5.8±0.4 497±34.8 287±45.9 5,210±521 5.4±0.54 spruce_cu(ii) 1.8±0.1 37.4±2.62 < 63.0* 495±49.5 4.0±0.40 cherry 21.2±1.5 175±12.3 606±97 1,040±104 6.5±0.52 cherry_cu(ii) 15±1.1 8.7±0.61 < 232.0* 697±69.7 3.7±0.29 hornbeam 48.6±3.4 376±26.3 1,570±267 4,350±435 30.2±1.51 hornbeam_cu(ii) 11.3±0.8 220±15.4 < 214.0* 2,660±266 26.4±1.32 * values under detection limits conclusions the wooden materials as sawdust, in their natural form or after some physical or chemical modification, are promising by-products that can be used in the removal of metal ions. the ft-ir analysis of the wooden sawdust confirmed the presence of the functional groups that are able to bind heavy metal ions as was also confirmed by static adsorption experiments with efficiencies of cu(ii) ions removal approximately 85 %. at dynamic conditions approximately 88 % of cu(ii) ions were removed by the poplar wooden sawdust after 5 min of sorbent-sorbate interaction. the maximum removal efficiency approximately 98 % of cu(ii) was observed after 30 min. the changes of ph were recorded after 5 min of adsorption that was caused by the ion exchange between cu(ii) in model solutions and chemical elements in poplar wood sawdust. the neutron activation analysis revealed a decrease of concentration of the certain elements (k, na, mg, ba, and ca) after the adsorption, indicating that ion exchange is one of the mechanisms of heavy metals removal by wooden natural sorbents. acknowledgement this work has been supported by the slovak grant agency for science (grant no. 1/0419/19). conflict of interest the authors declare that they have no conflict of interest. references ahluwalia ss, goyal d (2005) removal of heavy metals by waste tea leaves from aqueous solution. eng. life sci. 5: 158-162. ahmad a, rafatullah m, sulaima o, ibrahim mh, chii yy, siddique bm (2009) removal of cu(ii) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 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sawdust. bioresour. technol. 98: 402-409. shukla a, zhang yh, dubey p, margrave jl, shukla ss (2002) the role of sawdust in the removal of unwanted materials from water. j. hazard. mater. 95: 137-152. singovszka e, balintova m, holub m (2016) heavy metal contamination and its indexing approach for sediment in smolnik creek (slovakia). clean technol. environ. policy 18: 305-313. stevulova n, cigasova j, estokova a, terpakova e, geffert a, kacik f, singovszka e, holub m (2014) properties characterization of chemically modified hemp hurds. materials 7: 8131-8150. sud d, mahajan g, kaur mp (2008) agricultural waste material as potential adsorbent for sequestering heavy metal ions from aqueous solutions – a review. bioresour. technol. 99: 6017-6027. vieira rh, volesky b (2000) biosorption: a solution to pollution? internat. microbiol. 3: 17-24. zhang li, zhao li, yu y, chen c (1998) removal of lead from aqueous solution by non-living rhizopus nigricans. water res. 32: 1437-1444. zhang p, dong sj, ma hh, zhang bx, wang yf, hu xm (2015) fractionation of corn stover into cellulose, hemicellulose and lignin using a series of ionic liquids. ind. crops prod.76: 688-696. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:12 utc nova biotechnol chim (2021) 20(1): e809 doi: 10.36547/nbc.809 1 nova biotechnologica et chimica potential of the indirect and direct beneficial effects of the use of trichoderma koningii, aspergillus niger and mucor sp. on eggplants plants: plant growth and systemic resistance induction abdulnabi abbdul ameer matrood 1 , abdelhak rhouma 2,  1 department of plant protection, college of agriculture, university of basra, iraq 2 higher agronomic institute of chott mariem sousse, university of sousse, tunisia  corresponding author: abdelhak.rhouma@gmail.com article info article history: received: 30 th december 2020 accepted: 7 th may 2021 keywords: chlorophyll content peroxidase activity plant growth solanum melongena l. trichoderma koningii abstract several pathogens fungi responsible for total yield losses are worldwide spread notably in iraq. the alternatives strategies to decrease disease development are those able to destruct a total or partial population density using eco-friendly approach treatments. in this investigation, we demonstrate the symbiotic interaction with trichoderma koningii, aspergillus niger and mucor sp. on the eggplant plants growth and development, and on the defence response induction. the results revealed that the highest fungal frequency from eggplant rhizosphere was registered for a. niger, followed by mucor sp. and t. koningii. seeds treatment with t. koningii showed a higher value of length of shoots (2.83 cm), roots (3.00 cm), and leaves (3.50 cm). obtained results revealed that t. koningii ameliorates the seedling fresh (3.91 g), dry weight (0.24 g), and accelerates plant length (48.67 cm). obtained results revealed increasing of peroxidase activity (12.53, 12.68, and 11.28 10-1 units.g.ml.min -1 , respectively) and chlorophyll content (2.11, 1.70, and 1.90 mg.g -1 fresh weight, respectively) eggplants treated with combination mucor sp. + a. niger + t. koningii, t. koningii + mucor sp., and t. koningii alone. to control pathogens fungi within integrated management strategies, the biological control should be taken into consideration.  university of ss. cyril and methodius in trnava introduction several phytopathogenic fungi cause eggplants plant (solanum melongena l.) damage, resulting in total crop loss. on the other hand, eggplants contribute quite a bit in the agricultural economy in iraq. recently, it can be produced throughout the year in greenhouses (offseason and early crops) as well as in the field (seasonal culture). the eggplant production in iraq was reported to be around 102,452 thousand tons with an average 12.26 t.ha -1 (saeed omar and mohmmed 2020). iraqi annual economic yield losses due to several diseases have been estimated more than 70 % and about 50 – 100 % decrease in plant yield have been reportedly (hussein 2016; 2018; el-debaiky 2018). unfortunately, strategies to manage plant pathology employed by iraqi farmers were fungicides (matrood et al. 2020). the massive application of synthetic chemicals against fungal pathogens poses human health hazards and increases environmental pollution (rhouma et al. 2016; 2020). therefore, alternatives strategies are required for mailto:abdelhak.rhouma@gmail.com nova biotechnol chim (2021) 20(1): e809 4 phytopathogens control. biological control is the best alternative and eco-friendly approach for such treatments, defined as a total or partial destruction of phytopathogens fungi by other organisms, which occur routinely in nature (rhouma et al. 2018; matrood et al. 2020). the plants and seeds treatment with beneficial microorganisms including mucor sp., aspergillus sp., trichoderma sp. could eliminate impacts of abiotic, biotic, and physiological stresses (mondal et al. 2000; yedidia et al. 2001; mastouri et al. 2010). trichoderma species are widely used as biocontrol agents (bca) against phytopathogens since the 1920s (heydari and pessarakli 2010). trichoderma sp. are plant symbionts and can stimulate plant growth and development (fresh and dry weight, yields, plant length, foliar area, root volume, etc.) by increasing the macronutrients and micronutrients solubility (altomare et al. 1999; azarmi et al. 2011; abd-el-kareem et al. 2019). latha et al. (2009), rhouma et al. (2018) and inayati et al. (2020) pointed out that the good colonization of trichoderma spp. on the roots can lower the damages of various phytopathogens and the harmful stresses caused by environmental. it has been documented that some trichoderma species can induct systemic resistance; a mechanism triggered after the colonization of trichoderma spp. and regulated the plants by a cascade of specific signal transduction (kavitha and umesha 2008; lorito et al. 2010; kangasjärvi et al. 2012). this filamentous fungus rarely inducts systemic acquired resistance (hermosa et al. 2012; martínez-medina et al. 2014). plants naturally have dormant defense genes in healthy plants which are activated by biotic or abiotic inducers. trichoderma spp. induced systemic resistance by stimulating these genes. induced resistance is persistent and generally non specific to biotic and abiotic constraints (safin et al. 2020; matrood et al. 2021). phoka et al. (2020) and safin et al. (2020) revealed that the treatment with trichoderma spp. increased catalase and peroxidase activities and that this increase could be related to lignification. al-askar et al. (2016) and ghoniem abeer et al. (2021) noted that the total chlorophyll content in plant leaves was significantly increased in response to foliar spraying and root inoculation of trichoderma spp. plant peroxidases, which play an important role in the growth and differentiation of plants, are widely distributed in higher plants. in addition, they are also enzymes responsible for the lignification of the cell wall and destructive process such as aging and senescence (velazhahan and vidhyasekaran 1994; deborah et al. 2001; nath et al. 2015). in addition to systemic resistance induction, trichoderma species are known to have beneficial physiological/growth-promoting effects on plants, including delaying of leaf senescence, greater stress tolerance, exudation of plant growth regulators, solubilization of phosphates, micronutrient and minerals (fe, mn and mg), and secretion of exogenous enzymes, siderophores and vitamins. these physiological effects may contribute to greater yield and plant vigor to overcome biotic and/or abiotic stresses (azarmi et al. 2011; abd-el kareem et al. 2019; matrood and rhouma 2021). the aim of this investigation was to i) evaluate the substantial differences in the response to the symbiotic interaction with t. koningii, a. niger and mucor sp. on the eggplant plants growth and development, and ii) examine the defence response induction from treated eggplant plants separately or simultaneously with t. koningii, a. niger and mucor sp. experimental fungal community eggplant rhizosphere was collected in 2020 from four greenhouses (9 m x 60 m) located in basra iraq (chatt-el-arab, abu al-khaseeb, hartha and az zubayr) at 10 – 20 cm depth. for each greenhouse, soil samples were mixed into a single one. nine soil samples (100 g) per replicate (3 replicates) were collected in sterile polythene bags from each greenhouse (rhouma et al. 2019; 2020). the soil-borne fungi number was determined by the method of dilution-plate according to boughalleb-m’hamdi et al. (2017). the fungal species identification was carried by observing the macroscopic (growth, colour, aspect of the colony) and microscopic characterization (mycelium, conidiophore, conidia, resistance structures, sexual 2 nova biotechnol chim (2021) 20(1): e809 3 form), after a series of sub-culturing until purification. the fungal species were identified by using blue cotton as a mounting liquid and with reference to different identification keys. promoting growth and development of eggplant seeds and seedlings by using trichoderma koningii, aspergillus niger, and mucor sp. three fungal species namely; a. niger, mucor sp., and t. koningii (highest fungal frequency) were used for this assay. fungal cultures were developed on pda medium at 25 °c for 4 days. four discs (1 mm) of each species were transferred to erlenmeyer flasks containing 50 ml of pdb (potato dextrose broth). flasks were incubated at 25 °c for 7 days (for a. niger and mucor sp.) and 4 days (for t. koningii) in an orbital shaker. the conidial suspensions were filtered through whatman’s filter paper and were adjusted 10 8 cfu.ml -1 using a haemocytometer. eggplant (cv. barcelona) seeds were sterilized by soaking in 3 % solution of naocl for 3 min and washed with sterilized distilled water three times. the seed eggplants were treated by dipping into the flask containing a conidial suspension of the different antagonists for 30 min. one control was performed by inoculating the seeds with sterilized distilled water (negative control). the treated eggplant seeds were transferred on the surface of petri dishes containing cotton balls soaked in sterilized distilled water. in each petri dish, 10 seeds were placed (with a total of 10 petri dishes for each replicate (three replicate)). the plates were incubated in the dark at 25 ± 2 °c for 10 days, and then examined the length of the shoot (ls) (cm), root (lr) (cm) and leaf (ll) (cm). ls, lr and ll were assessed on 100 eggplant seedlings per treatment and per replicate (rhouma et al. 2018; matrood et al. 2020). germinated eggplant seeds were placed in a pot containing a mixture of peat (50 %) and vermiculite (50 %) with 3 seedlings per pot. the pots were placed in a greenhouse for 21 days. for each treatment, eggplant plants were randomly distributed with 60 plants per replicate (3 replicates), and the entire experiment was repeated twice. the evaluation parameters were measured within 21 days following inoculation. after determination of the fresh weight (fw) (g), eggplant plants were placed in an oven at 60 °c for 48 h to determine the dry weight (g) (dw). the plant length (pl) (cm) was measured using a flat rule. fw, dw and pl were assessed on 45 eggplant plants per treatment and per replicate (boughalleb-m’hamdi et al. 2018; rhouma et al. 2018). eggplant leaves were collected at different sampling moments (5, 10, 15 and 21 days after inoculation) to isolate airborne pathogens. the fragments leave of the eggplant (0.5 – 1 cm) were sterilized by soaking in 3 % solution of naocl for 2 min and washed with sterilized distilled water 3 times. the samples were dried and inserted on the surface of petri dishes (9 cm) containing pda medium amended with streptomycin (60 µg.ml -1 ). in each petri dish, seven fragments were placed (with total of 30 petri dishes of each treatment). the plates were incubated in the dark at 25 ± 2 °c for 5 – 7 days, and then examined for fungal analysis. the fungal species identification was carried by observing the macroscopic and microscopic characterization after a series of subculturing until purification. the airborne pathogens isolation was assessed on 15 eggplant plants per treatment and per replicate (rhouma et al. 2016; matrood et al. 2020). peroxidase activity and chlorophyll content in eggplant plant treated separately or simultaneously with a. niger, mucor sp., and t. koningii sterilized (soaking in 3 % solution of naocl for 2 min and washing with sterilized distilled water 3 times) eggplants seeds (cv. barcelona) were placed in a pot (50 cm diameter) containing a mixture of peat and vermiculite (1 : 1) at the rate of one seedling in each pot. the experimental design was a randomized complete block and arranged in three blocks each of 10 pots per treatment, and the entire experiment was repeated twice. the treatment applications were occurred after 30 days of the eggplant plant growing by inoculated the roots with conidial suspension (10 ml) of the different antagonists (10 8 cfu.ml -1 ). treatments were applied separately or simultaneously as follows: a. niger alone, mucor sp. alone, t. koningii alone, a. niger and mucor sp. inoculated simultaneously, a. niger and t. koningii inoculated simultaneously, t. koningii and mucor sp. inoculated simultaneously, nova biotechnol chim (2021) 20(1): e809 4 mucor sp., a. niger, and t. koningii inoculated simultaneously and control (inoculated with distilled water). the treated plants were placed in a greenhouse for 21 days (rhouma et al. 2018; matrood et al. 2020). peroxidase activity and chlorophyll content were conducted 10 days after inoculation and were evaluated on five eggplant leaves per treatment and per replicate (three replicates). plant tissue extraction for enzyme activities were prepared by freezing a 0.1 g of leaf samples in liquid nitrogen to stop the activity of proteolytic, followed by homogenizing with extraction buffer (1 : 5) (0.1 m phosphate buffer + 0.5 mm edta, ph = 7.5), and by centrifugation at 15,000 × g for 20 min at 4 °c. peroxidase (pox) activity was assayed according to castillo et al. (1984). 3 ml of reaction mixture composed of 0.5 ml guaiacol, 1 ml phosphate buffer, 0.5 ml h2o2, 0.1 ml enzyme extract, and 0.9 ml water. the absorbance was examined at 470 nm. chlorophyll content (cchl) was estimated by rinsed the eggplants leaves in 85 % acetone solution according to mackinney’s method and assessing its absorbance by spectrophotometer at λ = 663 nm and λ = 645 nm (mackinney 1941). arnon (1949) formulated the work done by mackinney’s to get chlorophyll concentration shown in equation (eq. 1): cchl = 20.21 a645 + 8.02 a663 (1) statistical analysis the data were analyzed by anova using the spss version 20.0 statistical software (spss, sas institute, usa), to evaluate parameter values differences. differences between treatments were determined by least significant difference (lsd) test at 5 % of significance level. results and discussion fungal community the results of fungal species isolated from eggplant rhizosphere of four experimental greenhouses are presented in table 1. the list includes 15 species belonging to 13 genera. obtained data showed that trichoderma koningii, aspergillus niger, and mucor sp. were recovered from all sampling greenhouses at 10 – 20 cm depth. the highest fungal frequency was registered for a. niger, followed by mucor sp., and t. koningii. another antagonistic fungus may be used in biological control was recovered from the four sites (a. flavus, penicillium sp., purpureocillium sp., chaetomium sp., trichoderma sp., and paecilomyces sp.) as well as pathogenic fungus may be caused plant disease (sclerotinia sp., fusarium sp., macrophomina sp., rhizoctonia solani, cladosporium sp., and alternaria sp.). table 1. fungal community isolated from eggplant rhizosphere. locations fungal isolates chatt-el-arab trichoderma koningii aspergillus niger a. flavus mucor sp. fusarium sp. penicillium sp. purpureocillium sp. abu al-khaseeb trichoderma koningii a. niger a. flavus mucor sp. chaetomium sp. trichoderma sp. hartha trichoderma koningii penicillium sp. a. niger mucor sp. macrophomina sp. sclerotinia sp. az zubayr trichoderma koningii penicillium sp. a. niger alternaria sp. mucor sp. rhizoctonia solani cladosporium sp. paecilomyces sp. fusarium sp. this prevalence in plants rhizosphere is supported by previous investigations undertaken by cwalinaambroziak and wierzbowska (2011), gaddeyya et al. (2012), onyimba et al. (2014), and boughallebm’hamdi et al. (2017). sangeetha et al. (2020) studied fungal diversity in cultivable fields. these authors noted that 15 species belonging to more than 6 genera were recorded at 10 – 20 cm with the nova biotechnol chim (2021) 20(1): e809 3 highest species density for aspergillus spp. and penicillium spp. these findings are in concordance with ratna kumar et al. (2015) showing that aspergillus and penicillium species were dominant in all agricultural fields due to high sporulation capacity. rosas-medina et al. (2020) and sangeetha et al. (2020) pointed out that the species fungal diversity and conidia dispersion has been varied according to the specific conditions, the ecological factors of each soil, the geographical area, the climatic conditions, the host physiology and the specificity of the colonized plant tissue. promoting growth and development of eggplant seeds and seedlings by using trichoderma koningii, aspergillus niger, and mucor sp. table 2. shoot, root, and leaf length of eggplant seedlings treated separately with trichoderma koningii, mucor sp., and aspergillus niger. treatments length of shoot [cm] length of root [cm] length of leaf [cm] t. koningii 2.83 3.00 3.50 a. niger 2.56 2.83 3.14 mucor sp. 2.83 2.67 3.32 control 2.10 2.50 2.00 lsd a <0.05 <0.05 <0.05 a probabilities associated with individual f tests. data are the average of 100 eggplant seedlings per treatment and per replicate (3 blocks). data presented in table 2 indicated clearly that the three treatments exerted a significant increasing (< 0.05) on shoot (ls), root (lr), and leaf (ll) length of eggplant seedlings after 10 days of incubation. seeds treatment with t. koningii showed a higher value of ls (2.83 cm), lr (3.00 cm) and ll (3.50 cm) compared to the negative control (2.10, 2.50, and 2.00 cm, respectively). the effect of three treatments on the fresh (fw) and dry (dw) weight and plant length (pl) is shown in table 3. all treatments increased significantly (<0.01) the fw, dw, and pl as compared with the negative control (2.22 g, 0.13 g, and 35.23 cm, respectively). results showed that t. koningii were found effective to increase the fw (3.91 g), dw (0.24 g), and pl (48.67 cm). several trichoderma species had beneficial effects on plant growth (crop yield increasing, seedling fresh weight and foliar area increasing, root volume development, secondary roots proliferation, etc.) and inducted resistance to both biotic and abiotic stresses. lindsey and baker (1967) reported that the treated tomato plants by trichoderma spp. revealed a significant augmentation of fresh weight (8 %) and height (28 %) plants in sterile conditions. table 3. fresh and dry weight and plant length of eggplant treated separately with trichoderma koningii, mucor sp. and aspergillus niger. treatments fresh weight [g] dry weight [g] plant length [cm] t. koningii 3.91 0.24 48.67 a. niger 2.28 0.16 43.00 mucor sp. 2.83 0.21 41.67 control 2.22 0.13 35.23 lsd a <0.05 <0.01 <0.05 a probabilities associated with individual f tests. data are the average of 45 eggplant plants per treatment and per replicate (3 replicates). several trichoderma species had beneficial effects on plant growth (crop yield increasing, seedling fresh weight and foliar area increasing, root volume development, secondary roots proliferation, etc.) and inducted resistance to both biotic and abiotic stresses. lindsey and baker (1967) reported that the treated tomato plants by trichoderma spp. revealed a significant augmentation of fresh weight (8 %) and height (28 %) plants in sterile conditions. windham et al. (1986) revealed that the amended cucumber by the propagules of trichoderma spp. ameliorated the seedling emergence (30 %) comparing to controls. lynch et al. (1991) explained the beneficial effect of t. harzianum on lettuce plants, which increased the emergence rate and enhanced the dry weights. rabeendran et al. (2000) showed that the treated cabbage seedlings by trichoderma spp. are enhanced shoot (91 – 102 %) and root (100 – 158 %) dry weight, and leaf area (58 – 71 %) under glasshouse assays. yedidia et al. (2001) depicted that trichoderma spp. increased shoot length (45 %), cumulative root length (75 %), root (95 %) and leaf (80 %) area, and dry weight (80 %). mastouri et al. (2010) demonstrated that the tomato seeds treatment with t. harzianum increased the vigour of seedling and accelerated the germination of seeds by triggering the plant physiological protection. lorito et al. (2010) noted that trichoderma spp. ameliorated the plant growth through the phytohormones and several secondary metabolites production. 5 nova biotechnol chim (2021) 20(1): e809 4 similarly, yedidia et al. (2001) pointed out that the roots treatment by trichoderma spp. enhancd the phosphorus and iron availability to plants. these authors observed a significant augmentation in shoot length, dry weight and leaf area. altomare et al. (1999) showed that trichoderma spp. can solubilize many plant nutrients. the uptake of nutrient may stimulate by trichoderma spp. in two ways by changing the anchorage of root system or by the substances exudation increasing the availability of nutrient (nitrogen, phosphorus, iron, potassium, etc.) to plants (mastouri et al. 2010; azarmi et al. 2011). in the same sense, altomare et al. (1999), benítez et al. (2004), rudresh et al. (2005), molla et al. (2012) confirmed that trichoderma spp. augments the macronutrients and micronutrients solubility. different airborne pathogens at different sampling moments (5, 10, 15, and 21 days after inoculation) are presented in table 4. a total of 8 species belonging to 4 genera were identified from eggplant leaves. the genera with the highest species number were alternaria (4) and cladosporium (2). present results are in analogy with boughalleb-m’hamdi et al. (2017), sangeetha et al. (2020). table 4. airborne pathogens contribution at different sampling moments (5, 10, 15, and 21 days after inoculation). treatments sampling moments 5 dai 10 dai 15 dai 21 dai t. koningii alternaria alternata a. niger cladosporium sp. cladosporium sp. alternaria sp. a. alternata cladosporium sp. a. solani mucor sp. a. alternata a. alternata a. alternata alternaria sp. control cercospora sp. cladosporium sp. cercospora sp. cladosporium sp. a. alternata alternaria sp. cercospora sp. botrytis cinerea a. alternata a. solani alternaria sp. data are the average of 15 eggplant plants per treatment and per replicate (3 replicates), dai: days after inoculation, absence of airborne pathogens. peroxidase activity and chlorophyll content in eggplant plant treated separately or simultaneously with a. niger, mucor sp., and t. koningii the change of peroxidase activity and chlorophyll content from eggplants treated separately or simultaneously with t. koningii, mucor sp. and a. niger were increased significantly comparing to the negative control (5.81 10 -1 units.g -1 .ml -1 .min -1 and 0.70 mg.g -1 fw) (table 5). there was increasing in peroxidase activity in treated plants with mucor sp. + a. niger + t. koningii (12.53 10 -1 units.g -1 .ml 1 .min -1 ), t. koningii + mucor sp. (12.68 10 -1 units.g -1 .ml -1 .min -1 ), and t. koningii (11.28 10 -1 units.g -1 .ml -1 .min -1 ). however, the combination of a. niger and mucor sp. (8.06 10 -1 units.g -1 .ml 1 .min -1 ) cause peroxidase activity decrease indicating that there was t. koningii-specific response in peroxidase activity changes (table 5). chlorophyll content was higher in eggplants leaves when combined with microorganisms nonpathogenic (mucor sp. + a. niger + t. koningii) with 2.11 mg.g -1 fw. t. koningii (1.90 mg.g -1 fw) inoculation increases the chlorophyll content as well as a. niger + t. koningii (1.73 mg.g -1 fw) treatment (table 5). bcas induce the activity of peroxidase to protect the plants from biotic and/or abiotic damage (gusain et al. 2014; ahmad et al. 2015). the peroxidase activity was implicated in the self and growth regulation (respiration, photosynthesis, etc.) (kavitha and umesha 2008). this activity was increased in plants treated separately or simultaneously with by trichoderma spp. comparing to plants treated only with water as reported by latha et al. (2009), saksirirat et al. (2009), and heydari and pessarakli (2010). yedidia et al. (2000) reported a higher level of peroxidase, ß-1,3-glucanases, and chitinase when cucumber plants were treated with trichoderma spp. 6 nova biotechnol chim (2021) 20(1): e809 5 compared to controls. abd-el-kareem et al. (2019) revealed that all tested trichoderma species increased significantly the peroxidase activity. these authors’ also pointed out that the mixture of t. harzianum, t. viride, and t. koningii attained the highest increase of activity of peroxidase with 150 %. mastouri et al. (2012), zehra et al. (2017), herrera-téllez et al. (2019) and phoka et al. (2020) documented that the pretreatment of tomato plants with trichoderma suppressed reactive oxygen species (ros) by enhancing mechanisms antioxidant defense and increased catalase and peroxidase activities. peroxidase helps in conversion of h2o2 to water and oxygen (gusain et al. 2014; ahmad et al. 2015). table 5. comparison of peroxidase activity and chlorophyll content of eggplant leaves recorded by eggplant plants treated separately or simultaneously with trichoderma koningii, mucor sp., and aspergillus niger. treatment peroxidase activity [10 -1 units.g -1 . ml -1 .min -1 ] chlorophyll content [mg.g -1 fw] t. koningii 11.28 1.90 a. niger 10.78 1.43 mucor sp. 9.14 1.66 a. niger + mucor sp. 8.06 1.67 a. niger + t. koningii 9.74 1.73 t. koningii + mucor sp. 12.68 1.70 mucor sp. + a. niger + t. koningii 12.53 2.11 control 5.81 0.70 lsd a < 0.05 < 0.01 a probabilities associated with individual f tests. data are the average of 5 eggplant leaves per treatment and per replicate (3 replicates). the chlorophyll content related to biophysical conditions indicating the plants health and plays critical role in photosynthesis (moharan and dutta 2016; inayati et al. 2020). photosynthesis plays an important role in the physiology of plant and a critical process in the regulation of plant defense (kangasjärvi et al. 2012; pérez-bueno et al. 2019). durairaj et al. (2018) pointed out that the increase chlorophyll content in plant influence photosynthesis and enchanted the yield. harman (1992), rawat et al. (2012), vitti et al. (2016), doni et al. (2017), fu et al. (2018) and zhao‐ying et al. (2018) demonstrated an increase in chlorophyll content in many plant species treated with trichoderma sp.. azarmi et al. (2011) showed that the chlorophyll content augmented when plants were inoculated with trichoderma spp. chlorophyll content was increased in trichodermatreated plants as was observed in melon and cacao plants inoculated. this result suggests an optimal physiological status of plants (martínez-medina et al. 2009; 2013; tchameni et al. 2017). inayati et al. (2020) demonstrated that the changes of chlorophyll content from treated vigna radiata leaves by trichoderma spp. could be defense mechanisms part to limit the availability of nutrients to the r. solani. conclusions based on the current results, it was deduced that t. koningii (alone or with other antagonistic fungi) could be employed in leaves treatments as bca’s to induce s. melongena of systemic resistance, through a specific signal transduction cascade. this antagonistic fungus allowed not only the induction systemic resistance but also the good ability to stimulate plant growth and development by increasing macronutrients and micronutrients solubility. the systemic resistance induction of s. melongena by t. koningii against soilborne and airborne pathogens represents a subject of future research. acknowledgment the authors are grateful to the review editor and the anonymous reviewers for their helpful comments and suggestions to improve the clarity of the research paper. dr. 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rs (2017) synergistic effects of plant defense elicitors and trichoderma harzianum on enhanced induction of antioxidant defense system in tomato against fusarium wilt disease. bot. stud. 58: 44. zhao‐ying l, chan‐ying j, tong‐tong z, yuan c, zhi‐hong y, zhi‐hua l, rong‐shu z (2018) effects of trichoderma asperellum combined application of growth and photosynthesis charcteristics of populus davidiana x p. alba var pyramidlis. bull. bot. res. 38: 64-74. 10 nova biotechnol chim (2018) 17(1): 48-57 doi: 10.2478/nbec-2018-0005  corresponding author: zahir1979@hotmail.com nova biotechnologica et chimica modeling of the soybean oil bleaching and optimization of its conditions in the refining process for environmental interest zahir henache, abdelhamid boukerroui and imad kashi laboratory of materials technology and process engineering (ltmgp), faculty of exact sciences, university of bejaia, targa ouzemour road, 06000 bejaia, algeria article info article history: received: 20th april 2018 accepted: 19th june 2018 keywords: bleaching bleaching clay rejects modeling pigments pollution soybean oil abstract in this work, we propose mathematical models describing the soybean oil bleaching process as a function of its parameters (temperature: 80 – 120 °c, clay dosage: 0.25 – 2 %, contact time: 10 – 30 min). the crude soybean oil visible spectrum shows three values of maximum wavelength (max). a value at 426 nm corresponding to the chlorophyll-a, the values at 451 and at 479 nm were assigned to β-carotene pigment. the models were developed using multiple linear regression analysis (mlra) and were performed with matlab programming language. the input variables are the temperature (x1), the clay dosage (x2) and the contact time (x3). the output parameter is the bleaching capacity (y in % uptake). statistical analysis methods were used to analyze and to confirm the reliability of the selected models. the optimal bleaching conditions for the soybean oil were: temperature 100 °c; clay dosage 2 % w/w and contact time 30 min. the highest bleaching capacity was found to be 81.04 % at 426 nm, 90.60 % at 451 nm and 93.66 % at 479 nm. the developed models allowed predicting the bleaching capacity representing the removal of the β-carotene and chlorophyll-a pigments present in the crude soybean oil at each max. also they allowed a better control of the most influencing parameters on the bleaching step and contribute to the optimization of the spent bleaching clay rejects by optimizing the amount of bleaching clay used in the refining process; consequently, to reduce risks of pollution.  university of ss. cyril and methodius in trnava introduction it is well known that the crude edible oil contains undesirable substances such as soap residues, free fatty acids, phosphatides, trace metals and coloring pigments (dijkstra 2013; gil et al. 2014; pal et al. 2015). those substances degrade the quality of the oil by altering the taste and color properties, thus limits the conservation and the use of this product (boukerroui and ouali 2002), for that reason, it is imperative to refine it to produce a stable product with desired color and a pleasant taste (mohdaly et al. 2017). this color is due to the presence of pigments in the crude edible oil, such as chlorophyll-a and β-carotene (junmao et al. 2008; pohndorf et al. 2016). the bleaching operation is an important step in the refining process of crude edible oil, it is carried out by various types of porous materials with a strong bleaching power which are known as bleaching earth or bleaching clay activated by hot acid solutions (foletto et al. 2011; komadel 2016) or microwave irradiation (boukerroui and ouali 2002; foletto et al. 2013). other types of materials are commonly used in the edible oil bleaching process such as diatomaceous earth (larouci et al. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:36 utc nova biotechnol chim (2018) 17(1): 48-57 49 2015), activated kaolin (aung et al. 2015), activated sepiolite (tian et al. 2014) and rice hull silicate (gil et al. 2014). these materials remove the unwanted compounds such as the coloring pigments (chlorophyll-a and β-carotene) and other impurities (soap residues, traces of heavy metals etc.) contained in the crude edible oil (sabah et al. 2007; pohndorf et al. 2016). normally, these undesirable elements are removed by adsorption process (nwabanne et al. 2013). the region of bejaïa (algeria) has two edible oil refineries (cogb-labelle and cevital). they produce huge quantities of waste called spent bleaching clays, which are discharged directly in the rubbish dump without any treatment that would prevent contamination of the environment. this presents a serious problem regarding the solid wastes management, storage and the harmful effects they would cause (boukerroui and ouali 2000; meziti and boukerroui 2011). in order to reduce the waste products, some vegetal oil refining factories have opted to reduce the amount of the bleaching clay used in the bleaching step. moreover, the reduction of the bleaching earth added to the bad controls of temperature and contact time causes an incomplete decolorization (boukerroui et al. 2018). in the other hand, they have proceeded by increasing the temperature processing (240 – 270 °c) in the deodorization step to remove all the thermally degradable pigments, undesirable elements and volatile compounds, such as alcohols, ketones and aldehydes (silva et al. 2014), as a result, these substances volatilized during the deodorization increase air pollution (boukerroui et al. 2018). the aim of this study was to propose mathematical models governing the bleaching of soybean oil in order to optimize the bleaching conditions which are temperature (°c), clay dosage (% w/w) and contact time (min). furthermore, this study contributes to a better control of the most influencing physicochemical parameters (temperature, contact time and clay dosage) on the decolorization step while keeping a better quality of oil – in order to reduce risks of pollution that presents a potential threat to our region by optimizing the quantities of the bleaching clay used in the refining process. in this study we suppose the variation in bleaching efficiency with the three mentioned parameters is linear with respect to the parameters of the selected models, i.e. . the most general linear models (glm) used are derived from the following equation (boumehrat and gourdin 1991; neuilly 1993; cornillon and matzner-lober 2007; moussaceb 2012) (eq. 1): y= (1) where y is the bleaching efficiency or capacity, is a regular function, is the bleaching parameter (i.e. temperature, contact time and/or clay dosage), are the model coefficients to be calculated and p is the number of coefficients. the modeling procedure was performed using the matlab language and statistical analysis methods. experimental materials the bleaching clay (bc) used in this study is produced and commercialized by taiko clay group (malaysia). the major chemical components of bc are as follows: sio2 (76.2 %), al2o3 (11.2 %), fe2o3 (2.7 %), mgo (0.8 %), cao (2.3 %), na2o (0.6 %), k2o (0.6 %) and loss on ignition (5.7 %). the bc and all edible oils used in this study were kindly donated by edible oil refining factory (cevital, bejaia, algeria). all reagents used in this study are analytical grade. bleaching operation the oil treatment experiments were achieved under a vacuum of 25 – 30 mm hg to avoid undesired side-reactions (kheok and lim 1982). the dried clay was weighed corresponding to the various clay dosages (0.25 – 2 % w/w) and was added at the degummed and neutralized crude edible soybean oil (neutral oil) preheated at the predetermined temperature in an oil bath. the bleaching experiment was carried out over a temperatures range of 80 – 120 °c. the mixture was continuously agitated under those conditions for the desired contact time in the range 10 – 30 min (see table 1). the oil was then filtered through whatman no.5 filter paper to separate the clear oil and clay (aung et al. 2015; boukerroui at al. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:36 utc nova biotechnol chim (2018) 17(1): 48-57 50 (2) (3) 2018). the absorbance measurements of the neutral (unbleached) and treated (bleached) oils were conducted on uv-visible spectrophotometer (model: uv-1800 shimadzu uv spectrophotometer) to evaluate the amount of pigment removed. the oil sample was diluted with petroleum ether (1:4 v/v) and the bleaching capacity of the clay was determined by measuring absorbance at 426 nm, at 451 nm and at 479 nm, which were the maximum absorptions observed in the wavelength range between 400 and 600 nm. the bleaching capacity, or % uptake, was calculated by applying the following equation (nguetngam et al. 2008; aung et al. 2015; boukerroui et al. 2018) (eq. 2): where 0 a and a are the absorbance of unbleached (neutral oil) and bleached (treated) oil, respectively, at the maximum absorbance wavelength of the unbleached oil. all bleaching parameters used in this study are given in the table 1. the details of the bleaching procedure used have been better described in previous studies (boukerroui and ouali 2000, 2002). table 1. bleaching parameters of soybean oil samples. bleaching parameters temperature [°c] clay dosage [%] contact time [min] x1 x2 x3 80 0.25 10 90 0.50 15 100 0.75 20 110 1.00 25 120 1.25 30 1.50 1.75 2.00 quality analysis of neutral and bleached soybean oils the quality parameters of neutral and bleached oil samples including color, humidity, free fatty acids, iodine, peroxide and saponification values, phosphorus content were determined. they were performed according to the aocs (1998) official methods: lovibond colour (cc 13e-92), using a lovibond tintometer color scale at 70 °c, phosphorus (ca 12-55), free fatty acids (ca 5a-40), peroxide value (cd 8-53) and saponification value (cd 3-25). numerical methods mathematical and statistical aspects the general linear models (glm) are largely used to modeling the physicochemical phenomena (boumehrat and gourdin 1991; neuilly 1993; moussaceb 2012); the majority of the statistical models used are special cases of the glm. in this study, the modeling of the soybean oil bleaching is carried out by using the multiple linear regression analysis (mlra) which is based on the variation of the following parameters: x1 (°c), x2 (%) and x3 (min). the multiple linear regression model is a generalization of the simple linear regression model when the explanatory variables are in finite numbers (cornillon and matzner-lober 2007). the multiple linear regression model may be expressed as follows (eq. 3): where βj (j =1...p) are the coefficients of the model and fj (j =1...p), the regular functions which may be polynomial, exponential, trigonometric or logarithmic function. to predict the bleaching capacity (y) as a function of temperature (x1), clay dosage (x2) and contact time (x3), we used the mlra to develop these models. the linear systems resulting from the mlra were resolved using the gauss method. the modeling process consists of changing the parameter p in the range [1, pmax] to obtain different linear models: . the best model ( ) with reasonable accuracy should satisfy different statistical criteria which are residual variance, coefficient of determination (r2), student test and fisher-snedecor test (moussaceb 2012). in this study, the confidence limit of the student and fisher-snedecor tests is 95 % (α = 0.05), as supported by the following bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:36 utc nova biotechnol chim (2018) 17(1): 48-57 51 hypotheses (h) for student test (eq. 4): against , j =1…p. (4) and for fisher-snedecor test (eq. 5): (5) we also assume that our experimental results can be well-fitted by the gaussian function (neuilly 1993). modeling approach to find out a correlation model that would best describe the relationship between bleaching capacity and the bleaching parameters (temperature, clay dosage and contact time), we tested several models, mainly polynomial, exponential, trigonometric and logarithmic ones. the coefficient of determination (r2) is the main statistical criteria, which determines the choice of any eligible model (neuilly 1993). a best model has r2 approaching unity (neuilly 1993). moreover, the selected model must satisfy other statistical criteria, which are the residual variance and the student as well as fisher–snedecor tests. in the light of the results obtained in this study, the experimental data of soybean oil bleaching has shown that the polynomial model has the maximum value of r2 and thus the highest reliability. results and discussion spectra of neutral and bleached soybean oils the absorbance spectra between 400 and 600 nm of the neutral and bleached soybean oils at different conditions of refining are represented in fig. 1. the visible spectra are characterized by three absorption maxima occurred at 426, 451 and 479 nm (fig. 1). similar result has been reported by many authors (sarier and guler 1988; kondal et al. 2001; boukerroui et al. 2018). the maximum absorption at 426 nm is attributed to the band of chlorophyll-a, while those observed at 451 and 479 nm are assigned to the bands of β-carotene (gonzales-pradas et al. 1994; boukerroui et al. 2018). the neutral oil spectrum shows that the carotenoids are the predominant pigment in soybean oil (kondal et al. 2001). fig. 1. visible absorption spectra of neutral (no), refined (ro) and bleached (bo) soybean oils at different conditions of refining. the absorbance peaks intensity at each max decrease in the bleached soybean oil samples spectra due to the reduction of carotenoids and chlorophyll compounds present in the crude oil, they are lower than those observed in neutral and refined soybean oil spectra, these peaks disappear in the bleached oil at the optimum conditions, this can be explained by the fact that the coloring pigments were adsorbed and removed by the clay during the bleaching operation. the color decrease was maximal at the optimum decolorization conditions (100 °c, 2 %, 30 min). the effect of activated bleaching clay on edible oil and our findings on physicochemical parameters influence are consistent with many other studies (silva et al. 2013; pal et al. 2015; boukerroui et al. 2018). the absorbance measurements at 426, at 451 and at 479 nm and the bleaching capacity calculated using eq. (2) were used to develop our models of the removal of chlorophyll-a and β-carotene pigments. the results obtained from eq. (2) were used in the eq. (3) in order to develop these models. modeling of the bleaching capacity at different wavelength oil samples treated with bleaching earth at the conditions listed in table 1 were analyzed in the visible light by measuring their absorbance at 426, 451 and 479 nm. the values of the bleaching capacity calculated for the cited parameters bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:36 utc nova biotechnol chim (2018) 17(1): 48-57 52 table 2. statistical criteria of the model governing the bleaching of the soybean oil at λmax = 426 nm. residual variance coefficient of determination [%] fisher-snedecor test calculated value tabulated value ƒ(n-p, p-1, α) 15.89 10-4 99.99 7.89 108 2.1 student tests: t(n-p, α/2) = 1.98 t(β1) t(β2) t(β3) t(β4) t(β5) t(β6) t(β7) t(β8) t(β9) t(β10) t(β11) 9.6 103 9.9 103 9.7 103 1.8 103 8.5 102 2.8 102 2.6 102 3.6 102 9.5 103 2.2 104 2.3 103 (temperature, clay dosage and contact time) are introduced into eq. 3. the results of the numerical computation of y using matlab generated a mathematical relationship, which was used to develop the models. the mathematical model at λmax = 426 nm is expressed as follows (eq. 6): this model (eq. 6) was selected on the basis of the statistical criteria presented in table 2. the mathematical model (eq. 7) at λmax = 451 nm is as follows (table 3): this model (eq. 7) was selected on the basis of the statistical criteria presented in table 3. the mathematical model at λmax= 479 nm is (eq. 8): this model (eq. 8) is selected on the basis of the statistical criteria presented in table 4. table 3. statistical criteria of the model governing the bleaching of the soybean oil at λmax = 451 nm. residual variance coefficient of determination [%] fisher-snedecor test calculated value tabulated value ƒ(n-p, p-1, α) 8.23 10-6 99.99 9.42 108 2.1 student tests: t(n-p, α/2) = 1.98 t(β1) t(β2) t(β3) t(β4) t(β5) t(β6) t(β7) t(β8) t(β9) t(β10) t(β11) 1.1 104 1.1 104 1.1 104 2 103 1.4 103 1.1 102 4 102 5.2 102 1.1 104 2.5 104 3 103 the variation of the soybean oil bleaching capacity as a function of clay dosage and contact time at fixed temperature and at different maximum wavelengths (426, 451 and 479 nm) is presented in fig. 2, fig. 3 and fig. 4 (respectively). all the curves show that the bleaching capacity varies significantly the function of the experiment parameters, it increases with increase in the temperature, the clay dosage and the contact time reaching values of 81.04 % (6) (7) (8) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:36 utc nova biotechnol chim (2018) 17(1): 48-57 53 table 4. statistical criteria of the model governing the bleaching of the soybean oil at λmax = 479 nm. residual variance coefficient of determination [%] fisher-snedecor test calculated value tabulated value ƒ(n-p, p-1, α) 9.73 10-6 99.99 7.99 108 2.1 student tests: t(n-p, α/2) = 1.98 t(β1) t(β2) t(β3) t(β4) t(β5) t(β6) t(β7) t(β8) t(β9) t(β10) t(β11) 1.1 104 1.1 104 1.1 104 2.1 103 1.9 103 3.7 102 1.8 102 3.2 102 104 2.4 104 2.8 103 fig. 2. variation of the bleaching capacity as a function of clay dosge and contact time at fixed temperature and at different temperatures: 80 °c (a), 90 °c (b), 100 °c (c), 110 °c (d), 120 °c (e) at λmax = 426 nm. at max = 426 nm, 90.60 % at max = 451 nm and 93.66 % at max = 479 nm under the optimum operating conditions (100 °c, 2 %, 30 min). the obtained results indicate that the simulated and experimental bleaching capacity have comparable values. this confirms that the proposed models best describe the soybean oil bleaching phenomena. in addition, for the temperature of 120 °c, the values of the bleaching capacity decrease as a result of destruction of some of the active sites. similar results have been reported by the following authors (kheok 1982; srasra et al. 1989; christidis et al. 1997; james et al. 2008(. moreover, these results reveal that the removal of the a b c d e bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:36 utc nova biotechnol chim (2018) 17(1): 48-57 54 fig. 3. variation of the bleaching capacity as a function of clay dosge and contact time at fixed temperature and at different temperatures: 80 °c (a), 90 °c (b), 100 °c (c), 110 °c (d), 120 °c (e) at λmax = 451 nm. carotenoids from the crude edible oil (451 nm, 479 nm) by the bleaching clay used in this study is easier than the removal of the chlorophyll-a pigments (426 nm) present in the soybean oil (kondal et al. 2001). validity of the selected models the validity of the selected models was analyzed using the following statistical criteria: residual variance, coefficient of determination (r2), the student and fisher-snedecor tests (tables 2, 3 and 4). the results indicate a low value of residual variance (15.89.10-4 for λmax = 426 nm; 8.23 10 -6 for λmax= 451 nm and 9.73 10 -6 for λmax = 479 nm), this suggests that it does not remain other useful information to be brought to the phenomenon of bleaching. in addition, they reveal that the maximum value of r2 (99.99 %) is close to unity for the different wavelengths (426, 451 and 479 nm), this confirms the goodness of simulated fit to observed data. moreover, the calculation of the model coefficients using both the student and fisher-snedecor tests gives higher values than the tabulated ones, which implies the accuracy of the models and all the coefficients are retained. furthermore, the experimental and the predicted bleaching capacity have comparable values. that confirms the reliability of the adjustment. the three developed mathematical models show the values corresponding to each coefficient (βj = β1, β2,…, β11), which are supposed to describe the individual effect of each parameter as well as the effect of the parameters interactions on the bleaching capacity. our findings reveal that interactions between the analyzed parameters substantially influence the bleaching capacity. similar results have been reported by many authors (christidis et al. 1997; boukerroui and ouali 2000, 2002; nwabanne et al. 2013). the models coefficients sign interpretation it is seen that the three elaborated models present a similarity between the coefficients signs of the different terms (linear, quadratic and multiple), a b c d e bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:36 utc nova biotechnol chim (2018) 17(1): 48-57 55 fig. 4. variation of the bleaching capacity as a function of clay dosage and contact time at fixed temperature and at different temperatures: 80 °c (a), 90 °c (b), 100 °c (c), 110 °c (d), 120 °c (e) at λmax = 479 nm. which implies that the bleaching capacity of the bleaching clay at each maximum wavelength varies in the same way (increase or decrease). the examination of these models reveals that the positive values of the coefficient of temperature (x1), clay dosage (x2) and contact time (x3) indicate that an increase in these factors involves an increase in the bleaching capacity, similar results have been found by the following authors (ajemba et al. 2013; nwabanne et al. 2013). however, the clay dosage and the temperature respectively have a more significant effect on bleaching capacity since their coefficients are higher, while the negative values of the other coefficients mainly those of quadratic and multiple terms cause a decrease in the bleaching capacity. determination of quality criteria for neutral and bleached soybean oils the quality of any edible oil is evaluated by the knowledge of its quality criteria such as free fatty acids content, phosphorus content, peroxide, iodine and saponification values. the results of the neutral and bleached soybean oils analysis at the optimum bleaching conditions (2 %, 100 °c, 30 min) are summarized in table 5. according to the results mentioned in this table, we noted that free fatty acids content, the iodine and the saponification values remain unchanged. furthermore, humidity, phosphorus content and the peroxide value decreased after the soybean oil bleaching process, this implies that the bleaching clay eliminates these substances contained in the neutral oil, as reported by many authors (pohndorf et al. 2016; boukerroui et al. 2018). in addition it is noted that the bleached soybean oil is fitted into the quality standards recommended for bleached oils and adopted by edible oil refining factory (cevital, bejaia, algeria), except for the peroxide value which decrease from 24.06 (meq o2/kg) to 12.06 (meq o2/kg) after bleaching but remain slightly higher than the standard value and that is due to the oil storage conditions (oxygen and light) which romote the formation of peroxides. a b c d e bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:36 utc nova biotechnol chim (2018) 17(1): 48-57 56 table 5. quality criteria of neutral and bleached soybean oils. oil neutral oil bleached oil standard values humidity [%] 1.3 0.28 ≤ 2 phosphorus [ppm] 4.75 0 ≤ 2 free fatty acids [%] 0.06 0.06 ≤ 0.08 iodine value 128 128 124-139 peroxide value [meq o2/kg] 24.06 12.06 ≤ 10 saponification value [mg koh/g] 190 190 189-195 color tests red 9.8 3.8 ˂ 5 yellow 1.8 41 ˂ 50 conclusions analysis of the visible spectrum of crude soybean oil shows maximum wavelengths at 426 nm (chlorophyll-a) and at 451 and 479 nm (β-carotene). the bleaching clay application on soybean oil shows a decrease of maximum absorbance value due to a progressive elimination of pigments present in the oil, which depends on the physicochemical parameters (temperature, % dosage and contact time). our findings suggest that the removal of chlorophyll-a and β-carotene pigments present in the soybean oil, represented by the bleaching capacity (% uptake at max), can be expressed under its modeled form. mathematical models governing the decolorization process at each maximum wavelength (written in matlab) depend of three parameters which are; temperature, contact time and clay dosage. the model equations, calculated at the maximum wavelength (426, 451 and 479 nm), are linear and second order polynomial. the results reveal that the decolorization process is highly influenced by the analyzed parameters, which is combined to the influence that results from interactions between them. the color decrease was maximal at the optimum bleaching conditions (2 %, 100 °c, 30 min) and the soybean oil bleached at these conditions is fitted into the quality standards recommended for bleached oils. the established models present an environmental interest since they allowed optimizing the amount of the bleaching clay used in the soybean oil bleaching process and contribute to the optimization of the bleaching clay wastes by optimizing the quantities of spent bleaching clay used in the refining process; consequently, to reduce risks of pollution. references aocs (1998) official methods sand recommended practices of 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cheriet7, noureddine bouras4,8 1department of microbiology and biochemistry, faculty of science, university mohamed bouadiaf of m'sila, bp 166 m’sila 28 000, algeria. 2department of biology, faculty of sciences, university 1 ahmed benbella of oran, algeria. 3agro-pastoralism research center (aprc), djelfa, algeria. 4laboratoire de biologie des systèmes microbiens (lbsm), ecole normale supérieure de kouba (ens-kouba), algiers, algeria. 5laboratoire de valorisation et bio-ingénierie des ressources naturelles (lvbrn), university of algiers 1, algiers, algeria 6laboratory of applied microbiology, department of biology, faculty of sciences, university 1 ahmed benbella of oran, algeria. 7laboratory of bacteriology research, department of biology, faculty of sciences, tunisian institute of veterinary research, university el manar-tunis, tunis-tunisia. 8department of biology, faculty of natural and life sciences and earth sciences, university of ghardaïa, ghardaïa, algeria  corresponding author: mourad.guetouache@univ-msila.dz article info article history: received: 13th march 2021 accepted: 31st march 2022 keywords: biological inhibition lactic acid bacteria traditional dairy products abstract a total of 33 lactic acid bacteria (lab) were isolated from 5 samples of traditional dairy product (jben) collected from various livestock farms in oued el malha, djelfa, the ouled naïl range of north-central algeria. the taxonomic study of the strain b04 using a multiphase approach based on morphology, physiology, molecular (16s rrna) and phylogenetic analyses allowed correlating this strain to enterococcaceae. the difference of the 16s rrna gene sequence of the isolate toward the most closely related genus enterococcus was more than 6 % suggesting that the strain b04 represents a new genus. the results of the evaluation of some physiological tests indicated that b04 exhibited good biological activities including acidifying, proteolytic and bacterial inhibition. the strain b04 was antagonistic toward staphylococcus aureus atcc 6538, escherichia coli atcc 25922, and bacillus cereus atcc 10876. introduction dairy products and milk, as commonly consumed products, are rigorously investigated for their biochemical and physiological functions (moussaid et al. 2021). traditional dairy products are made by means of ancient artisanal processes, using milk or mixtures of milk from different animal species (guetouache and guessas 2018). several types of fermented milk products have been reported throughout the world. these products differ in their taste and their consistency. in north of african countries, the most popular dairy products are jben, klila, zebda, rayeb, lben, bouhezza, medghissa, and takammarit. traditional dairy products are cultural goods that deserve to be studied, characterized, and protected (guetouache and guessas 2019; leksir et al. 2019). “jben” cheese is mailto:mourad.guetouache@univ-msila.dz nova biotechnol chim (2022) 21(2): e904 2 manufactured with the raw goat or sheep milk, voluntarily acidified and coagulated by coagulating enzymes of plant origin from wild thorny plant, cardoon flowers, pumpkin seeds, or artichoke (ouadghiri et al. 2005). after a moderately heating (55 – 75 °c) of “lben” to avoid degradation during the storage phase, the coagulum obtained is called “klila”, which is consumed as a fresh cheese (benlahcen et al. 2017; adewumi 2019). the fresh butter obtained after churning “rayeb” (curdled milk) is called “zebda”. by shaking “rayeb” added with a quantity of warm water (40 – 50 °c) the fat globules agglomerates and lipid globules appear on the surface and are recovered at the end of the churn. “zebda” is furthermore produced in middle eastern countries and is known as “zobdeh” (fao 1990). in traditional fermented dairy foods, the lactic acid bacteria (lab) microflora plays an essential role giving out the dairy products’ particular features and native flavors (zuo et al. 2014). in addition to their role in food fermentation, labs can improve nutritional value, texture, shelf life, flavor, and safety through the production of bioactive substances (viana de souza at al. 2017). labcontrolled fermentation is also considered a nextgeneration strategy for functional foods (moslehishad et al. 2013). many labs can be used as a starter culture for cheese fermentation due to its ability to release lipases, proteases, or β-galactosidases to produce a unique flavor, texture, and aroma (juan et al. 2016). in cheese fermentation, the most important characteristics and functions of labs are: (1) the transformation of lactose in milk toward small molecular monosaccharides, glucose and galactose, which boost the cheese flavor genesis (blaya et al. 2018); (2) the destruction of proteins into free amino acids and peptides in cheese, and (3) the dissolution of lipids into fatty acids (konkit and kim 2016). lab belongs to gram-positive bacteria which also include aerobic and facultative anaerobic, acidtolerant, non-sporulating, either rod-shaped or spherical, bacteria. as a commercial starter cultures labs must have acidification, proteolytic, autolytic, and antimicrobial activities, and be resistant to bacteriophages. in this group are included the various major genera namely: aerococcus, alloiococcus, enterococcus, lactobacillus, lactococcus, leuconostoc, oenococcus, pediococcus, streptococcus, carnobacterium, symbiobacterium, vagococcus, tetragenococcus, weissella, atopobium, and bifidobacterium (gutiérrez-cortés et al. 2017). it is well known that traditional dairy foods production is based on the metabolic activity of labs to ferment milk sugars, especially glucose and galactose, so to produce lactic acid and aroma substances that give typical flavors and tastes to fermented products. moreover, labs end products provide an effective form of natural preservation of many widely used dairy products such as, antimicrobial metabolites so called bacteriocins, which are considered safe and natural preservatives, with great potential to be used on their own, or synergistically with other methods in food preservation (marshall 2005). in recent years, many of labs, isolated from different fermented food systems in distinct parts of the world have been experimented for their probiotic potentiality and capacity to produce industrially relevant substances (ranadheera et al. 2012). then, the aim of the present work was to isolate and characterize lactic acid bacteria from different traditional dairy products (jben, klila, and zebda) and to characterize the different groups of microflora, acidifying power and antimicrobial producing bacteria using classical and molecular methods. experimental samples collection five samples of traditional dairy product (jben) produced traditionally from sheep milk and without adding starter culture were collected from different area of the rural region (oued el malha) of djelfa province (fig. 1). samples were brought to the laboratory at 4 °c in an icebox under sterile conditions, and stored in laboratory under refrigeration at 4 °c. all samples were analyzed immediately after delivery. the ph value of the different samples was measured using a hanna instruments hi 8424 digital ph meter (leighton buzzard, uk). the ph probe was calibrated using standards of ph 4.0 and 5.6 (vwr international ltd., poole, uk) at 25 ºc. nova biotechnol chim (2022) 21(2): e904 3 fig. 1. the study area. isolation and phenotypic identification twenty-five grams of jben sample were transferred to 225 ml of sterile 2 % sodium citrate solution, in an erlenmeyer flask. using a stomacher laboratory blender (colwarth stomacher 400c seward laboratory, uk) all the dairy samples were homogenized and decimal dilutions (10-1 to 10-5) were prepared by mixing 10 ml with 90 ml of 0.1 % (w/v) sterile peptone water (merck kgaa, darmsadt, germany), according to the international dairy federation (idf) standard 122b (1992). lactic acid bacteria were isolated and identified on three culture media: mrs agar (merck kgaa, darmstadt, germany) a selective medium for counting and isolating lactobacilli, after incubation at 37 °c for 48 – 72 h, m17 agar (merck kgaa, darmstadt, germany) a selective medium for counting and isolating lactococci after incubation at 30 °c for 48 – 72 h, and eliker agar (merck kgaa, darmstadt, germany) a selective medium for counting and isolating streptococci and lactobacilli after incubation at 37 °c for 48 h. for each batch of the fifteen dairy products samples, three colonies were taken randomly from mrs, m17, and eliker agar plates. consequently, a total of 135 colonies were gathered from each medium. all isolates were purified on mrs agar slant at 4 °c and subcultured every 4 weeks. bacterial concentrations were calculated with the formula (eq. 1): n = ʃc/[(n1 + 0.1 n2)] d (1) where c is the sum of counted colonies; n1 is the number of the plates for the first dilution ratio; n2 is the number of the plates for the second dilution ratio; d is the first dilution ratio and expressed as colony forming unit per gram of sample (cfu.g-1) (abraha et al. 2017). the selected isolates were purified by repeated seeding on the agar media, lab strains were kept on mrs agar slant at 4 °c and sub-cultured every 4 weeks. prior to use, lab strains were activated in mrs broth at 30 °c for 24 h and sub-cultured in mrs agar at 30 °c for 24 h. the isolates were identified according to their morphological, physiological, and biochemical characteristics. all isolates were tested for morphology and catalase production and gram stain (norris et al. 1982). catalase-negative and gram-positive isolates were checked for gas production from glucose in mrs and m17 broth containing inverted durham tubes (song et al. 2018). growth at different temperatures (4, 10, 37, and 45 °c), growth in different ph values (4.0, 6.5, and 9.6) and growth in 10 % nacl broth were tested for coccusand bacillus-shaped isolates. detection of arginine dihydrolase was tested in bromocresol purple medium (merck kgaa, darmstadt, germany) (thomas 1973). search for citratase was done in kempler and mckay medium (merck kgaa, darmstadt, germany) (lee et al. 1986). the isolate was inoculated into methyl red voges proskauer medium (merck kgaa, darmstadt, germany), and then was incubated for 24 h at 30 °c. a total of three drops of koh and two drops of the alphanova biotechnol chim (2022) 21(2): e904 2 naphthol reagent were added to the medium. a positive result was interpreted as color change into the pink. all the strains were tested for fermentation of sugars. this is done by choosing the following sugars: glucose, arabinose, cellobiose, mannitol, melibiose, raffinose, ribose, lactose, rhamnose, sorbitol, xylose, trehalose, maltose, sucrose, galactose, and fructose by adding the specific sugar to the man rogosa and sharp broth medium (mrs-bcp) (merck kgaa, darmstadt, germany). after incubation, acid production was indicated by a change in color, and gas production was detected by observation of gas collection in the inverted durham tubes (thakur et al. 2017). dna extraction from pure cultures lab isolate sharing some of the screened properties was further identified by molecular biology. dna extraction was performed according to liu et al. 2000 by adding 500 μl of a lysis solution composed of 400 mm tris-hcl (ph 8.0), 60 mm edta (ph 8.0), 150 mm nacl, and 1 % sodium dodecyl sulfate (sds). the colony fractions are well crushed with sterile cones and incubated at room temperature for 15 min. a volume of 150 μl of a ph 4.8 solution composed of 5 m potassium acetate and 11.5 % glacial acetic acid was added and vortex briefly and then centrifuged for one minute at 12,000 × g. a volume of 400 μl of supernatant are recovered and transferred to another sterile eppendorf tube, then added with the same volume of isopropanol and mixed briefly by inversion, before being centrifuged for 2 min at 12,000 × g. the pellet is washed with 300 μl of 70 % ethanol by centrifugation for one minute at 12,000 × g. the dna obtained (the pellet) was dried overnight at ambient temperature and resuspended in 40 μl of sterile double distilled water and stored at -20 °c before using. amplification and sequencing of 16s ribosomal dna the purified dna was used as a template for 16s rrna gene amplification on the automatic thermal cycler (biorad mycycler™ thermal cycler, biorad laboratories, inc., hercules, usa). the primers of 16s rrna gene were 27f (5’-agagtttgatcctggctcag-3’) and 1492r (5’-ggttaccttgttacgactt-3’). the amplification carried out in a volume of 50 μl of reaction mixture composed of 25 – 50 ng of genomic dna, 0.5 μm of each primer 27f and 1492r, 1x pcr buffer containing mgcl2 (10 mm tris-hcl, ph 9.0, 50 mm kcl, 1.5 mm mgcl2, 0.1 % triton x-100, 0.2 mg.ml-1 bsa), 200 μm of the mixture dntps and 1.5 u of taq dna polymerase. for amplification, the following conditions must be observed: initial denaturation of the dna template at 98 °c for 4 min, 30 cycles of denaturation at 94 °c for 1 min, hybridization at 52 °c for 1 min, and extension at 72 °c for 2 min. at the end the reaction mixture was maintained at 72 °c for 10 min for final elongation and then cooled to 4 °c. the size of the pcr product was determined by agarose gel electrophoresis in 0.8 % agarose gel containing ethidium bromide and visualized by uv light and documented by the gel doc xr+ gel documentation system (bio-rad laboratories, inc., hercules, usa). pcr products were submitted to beckman coulter, inc. (high wycombe, uk) for purification and sequencing. the same primers, sense: 27f and antisense: 1492r, were used for amplification. phylogenetic study once the sequences have been determined, the raw sequences obtained (800 to 1,100 bp) were processed using the mega 7.0 software (molecular evolutionary genetics analysis program, version 7.0) (kumar et al. 2016) to build the sequence of 16s rrna. in this treatment, the antisense sequence is inverted and then aligned with the sense sequence; the two sequences are then fused at the 3' end for the first and 5' for the second to have a single 16s rrna sequence about 1,500 bp (depending on the quality and length of the initial sequences). the 16s rrna sequence thus determined is compared, to blast, to the homologous sequences of reference species listed in the ncbi genomic library. this comparison was made through the ez biocloud server (yoon et al. 2017) with the sequences of all validated standard 4 nova biotechnol chim (2022) 21(2): e904 3 species of bacteria. the sequence of the bacterial strain is aligned with the reference sequences by the clustal w tool (larkin et al. 2007), and then the set of aligned data is used for phylogenetic analysis with the mega 7.0 software. the evolutionary distances were computed as described jukes and cantor (1969) and are in the units of the number of base substitutions per site. the algorithms for constructing topologies of phylogenetic trees are those of the neighbor-joining method (saitou and nei 1987). statistical validation of established phylogenetic links is performed by the bootstrap test, the values of which are based on the result of 1000 analyses (felsenstein 1985). acidifying activity of isolates potentiometric (ph measurement) and titrimetric methods were used to determine acid production capacity (villani et al. 2001). to do this, isolates from frozen stocks were activated in mrs and m17 broth for 24 h at 30 °c for lactococci and 37 °c for isolates of enterococci and lactobacilli and night cultures of 100 μl were inoculated in 10 ml of sterile uht skimmed milk broths. after 3, 6, 9, and 24 h, 2 ml aliquots were removed aseptically and ph changes were recorded using a ph meter (orion model 250 a, orion research inc., boston, ma) and acidity titratable during incubations at 30 °c and determined 37 °c. proteolytic activity the proteolytic activity of the strain b04 was tested using mrs agar containing 2 % skimmed milk. after incubation, the proteolytic strains would form the clearing zone around colonies. the zones were measured in mm (jini et al. 2011). as for the proteolytic activity residual proteins concentrations in the culture were estimated by a coomassie g250 binding procedure (bradford 1976). antibacterial activity the agar well diffusion method was used to assess the antimicrobial activity of the isolate b04. their antimicrobial properties were tested against four major pathogenic bacteria: bacillus cereus atcc 10876, staphylococcus aureus atcc 6538, pseudomonas aeruginosa atcc 27853, and escherichia coli atcc 25922. the pathogenic bacteria were seeded in mh agar medium and allowing them to solidify at 45 °c. wells were prepared at the agar with a sterile cylinder 6 mm diameter. fifty micro-liters of the filtered cell-free supernatant of test strain were separately placed into the wells. the plates, prepared in duplicate, were kept at 4 °c for 24 h into the agar and then incubated at 37 °c for 24 h. after incubation, the antimicrobial activity was determined by measuring the diameter of the inhibition zones around the well using caliper in mm clearing zone diameter around the well more than 1mm was measured as a positive result (akabanda et al. 2014). statistical analysis statistical data analysis was conducted using the microsoft excel 2007 (microsoft corporation, redmond, wa, usa). all determinations were represented as mean values ± standard deviation of triplicate experiments. results and discussions physicochemical analysis the ph of samples was from 4.20 ± 0.39 to 5.72 ± 0.57. these values are similar to those found by rhiat et al. (2013). isolation and identification of lab this study was carried out for main purpose to isolate and characterizes of large number of lactic acid bacteria from traditional dairy product (jben) and characterization of different groups of microbiota. the results obtained from the present study demonstrated that there is diversity of lactic acid bacteria; with 65 lactic acid bacteria belonging to the genus lactobacillus (33.85 %), lactococcus (32.31 %), leuconostoc lactis (23.07 %), enterococcus (4.62 %). these bacteria have good biochemical and biotechnological properties, which puts it at the forefront of industrial microbiology. after enumeration on the appropriate culture 5 nova biotechnol chim (2022) 21(2): e904 2 media, mrs, m17, mse, and elliker, the results are as follows: 2.2 x 104 to 2.1 x 106 cfu.ml-1, 1.1 x 102 to 1.7 x 103 cfu.ml-1, 2.0 x 10 to 1.8 x 102 cfu.ml-1, and 2.2 x 10 to 2.8 x 104 cfu.ml-1, respectively. all isolates were gram-positive and catalase negative bacteria; among the isolated lactic acid strains, one strain named b04 was purified and identified (fig. 2, 3); its characteristics are shown in table 1. table 1. morphological, physiological, and biochemical characteristics of the isolated lab strain b04. characteristics strain b04 gram staining catalase test motility gas production from glucose hydrolysis of: adh citrate growth at different temperature (°c): 04 10 37 45 growth at different ph: 4.0 6.5 9.6 growth in the presence of 10% nacl sugars fermentation: arabinose cellobiose mannitol melibiose raffinose ribose lactose rhamnose sorbitol xylose trehalose maltose sucrose galactose fructose + v + + dr dr + + + + + + + + + + dr + + + + + (+) – positive reaction, (-) – negative reaction, v – variable, dr – delayed reaction, adh – alcohol dehydrogenase. after extraction and amplification of the 16s rrna of the isolate b04, the corresponding sequence is determined, and then aligned and compared with the sequences of the bacteria contained in the genomic databases deposited at the genbank. among the isolates selected in this work, the almost complete 16s rrna gene sequence (1,500 bp) of the isolate b04 was determined and deposited in the genbank data library under the accession numbers ky746350.1. the blast performed for the isolate b04 shows its proximity to the species enterococcus lactis (bt159t), which appeared to be the closest, with a similarity of 91.22 %. neighbor-joining phylogenetic analysis shows the relationship between the isolate b04 and the typical strains of the closest species of the genus enterococcus (fig. 4). the similarity rate between 16s rrna sequence is less than the critical threshold for qualifying an isolate as a new species that is 98.65 % (kim et al. 2014) and a new genus that is 94 % similar to 16s rrna sequences (yarza et al. 2008). these results indicate that the strain b04 belongs to a new genus. the confirmation of these results can be possible with whole genome sequencing then digital dna-dna hybridization and further chemotaxonomic analyses. fig. 2. macroscopic appearance of colonies of a lactic acid bacterium b04 after 48 h of incubation at 30 °c, seeded by streaks on mrs agar medium. fig. 3. microscopic image showing the appearance of grampositive stain of the strain b04. 6 nova biotechnol chim (2022) 21(2): e904 3 fig. 4. phylogenic tree based on 16s rrna gene sequences showing the position of the strain b04 and its related species of the genus enterococcus. the optimal tree is shown. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. numbers at nodes indicate percentages of bootstrap support based on neighbor-joining analysis of 1,000 resampled datasets. bar, 0.010 substitutions per nucleotide position. this analysis involved 7 nucleotide sequences. all ambiguous positions were removed for each sequence pair (pairwise deletion option). numbers in the parentheses indicate the genbank accession numbers. physiological and antibacterial properties of lab isolated from traditional dairy products the diminution of ph of the milk is due to the production of lactic acid from lactose fermentation (marroki et al. 2011). the lactic acid values of analyzed samples showed that the isolate b04 was highly acidifying isolate that coagulate milk before 18 h of incubation and the remaining isolates coagulate milk after 18 to 24 h of incubation. the amount of lactic acid was measured (15 – 25 °c). the acidity increases with the time in a variable way to arrive until 75.00 ± 02.11 °d (°d: dornic degree; this result represents the mean ± standard deviation of three replicates) after 24 h with isolate b04. it has been found that lab b04 isolate presented antimicrobial activity against bacillus cereus atcc 10876, staphylococcus aureus atcc 6538 and escherichia coli atcc 25922. the strain lab b04 was completely inactivated by α-chymotrypsin and pepsin. these results suggest that the biochemical nature of the molecule produced is peptidic. the inhibitory compounds produced by the isolate showed great resilience to thermal treatments. the bacteriocin has proved stable over a wide ph range with all peptides, now some antimicrobial activity in the ph ranges from ph 4.0 – 7.0. bacteriocin is very sensitive to ph its stability was detected at a ph range of 3.5 to 6.5. in this study, bacteriocin produced by the strain b04 had the same profile and was active at ph values 4.0 to 6.0. based on the results, it can be concluded that the bacteriocin produced develops a positive activity against staphylococcus aureus atcc 6538 and escherichia coli atcc 25922 (table 2, fig. 5). table 2. antibacterial activity of lactic acid bacteria strains against some pathogenic bacteria. strain diameter of inhibition zone [mm] on pathogenic strains s. aureus e. coli b. cereus p. aeruginosa b04 17.33 ± 3.06 9.33 ± 2.52 9.67 ± 1.15 0 7 nova biotechnol chim (2022) 21(2): e904 3 fig. 5. antibacterial activity of strain b04 compared to four species of labs against (a) escherichia coli atcc 25922 and (b) staphylococcus aureus atcc 6538 by the diffusion method on mueller-hinton agar after 24 h of incubation at 30 °c in the darkness. 1 – lactobacillus brevis, 2 – lactobacillus intestinalis, 3 – leuconostoc lactis, 4 – strain b04, 5 – lactobacillus plantarum. conclusions the selected strain of lactic acid bacterium b04 has high acidifying power and proteolytic activity and a strong bactericidal effect against pathogenic strain staphylococcus aureus atcc 6538 and a medium effect against bacillus cereus atcc 10876 gramnegative pathogenic strain escherichia coli atcc 25922. based on these results, it can be concluded that traditional dairy products are cultural goods that deserve to be studied, characterized, and protected. furthermore, it would be interesting to enhance the physicochemical and biological investigations for the transformation of milk into different dairy products through the intervention of controlled industrial fermentations. acknowledgements the authors would like to acknowledge mohamed bouadiaf university of m’sila for their willingness and providing laboratory facility. corresponding author: particularly thank the private laboratory ensemble doumi quality control and in particular doumi boubaker director of laboratory, which allowed me to finalize my doctorate within the laboratory. m. guetouache gratefully acknowledge the receipt of funding from ahmed benbella university of oran algeria. conflict of interest the authors declare that they have no conflict of interest. references abraha a, bikila t, alemu s, muktar y (2017) bacillus cereus isolation and load from raw cow milk sold in markets of haramaya district, eastern ethiopia. int. j. food contam. 4: 15. adewumi ga (2019) health-promoting fermented foods. in melton l, shahidi f, varelis p (eds.), encyclopedia of food chemistry, vol. 3. elsevier, amsterdam, 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cow’s milk. j. appl. microbiol. 90: 430-439. yarza p, richter m, peplies j, euzeby j, amann r, schleifer kh, ludwig w, glockner fo, rosselló-móra r (2008) the all-species living tree project: a 16s rrna-based phylogenetic tree of all sequenced type strains. syst. appl. microbiol. 31: 241-250. yoon sh, ha sm, kwon s, lim j, kim y, seo h, chun j (2017) introducing ezbiocloud: a taxonomically united database of 16s rrna gene sequences and whole-genome assemblies. int. j. syst. evol. microb. 67: 1613-1617. zuo fl, feng xj, chen ll, chen sw (2014) identification and partial characterization of lactic acid bacteria isolated from traditional dairy products produced by herders in the western tianshan mountains of china. lett. appl. microbiol. 59: 549-556. 9 nova biotechnol chim (2018) 17(2): 132-139 doi: 10.2478/nbec-2018-0014  corresponding author: katarinakulich@gmail.com nova biotechnologica et chimica determination of selected phenolic acid and majoritarian avenanthramides in different varieties of naked oats (avena sativa l.) grown in slovakia katarína kulichová1,, mária maliarová1, jozef sokol1, katarína lašáková1 and michaela havrlentová2,3 1department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, 917 01, slovak republic 2department of biotechnologies, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, 917 01, slovak republic 3research institute of plant production, national agricultural and food centre, bratislavská cesta 122, piešťany, 921 68, slovak republic article info article history: received: 11th november 2018 accepted: 17th december 2018 keywords: hplc –dad avenanthramides phenolic acids oats abstract oats are important cereals. oats are a good source of protein and lipids, polyphenolics, phenolic acids, flavonoids and avenanthramides. avenanthramides is phenolic group, which is unique in oats and have antioxidant activity, anti-inflammatory, anti-atherogenic and anti-proliferative effect. the aim of study is determination of the majoritarian avenanthramides (2c, 2p and 2f) and phenolic acids (p-coumaric and ferulic) in selected varieties of oat (avena sativa l.) grown in two consecutive years using the hplc method. the oats were exposed to ultrasound supported extraction (two 15 min cycles).the simultaneous separation was performed using c18 type of stationary phase. the method showed a good linearity in the concentration range 0.04 – 5.24 µg/ml for p-coumaric acid, 0.04 – 5.13 µg/ml for ferulic acid, 0.19 – 24.5 µg/ml for avenanthramide 2c, 0.53 – 17.1 µg/ml for avenanthramide 2p, 0.8 – 25.6 µg/ml for avenanthramide 2f. correlation coefficients were higher than 0.9997. detector operated at a wavelength 320 nm. the repeatability of the method was evaluated in three concentration levels with satisfactory results for each analyte. the content of both phenolic acids is significantly lower (50– 100-times) compared to the total content of avenanthramides in both years’ harvests for all analyzed varieties. content of total avenanthramides was the highest in varieties racoon (723.28 mg/kg) followed by oliver (578.59 mg/kg) and kamil (384.17 mg/kg).  university of ss. cyril and methodius in trnava introduction oats are cereals containing β-glucan, and are a good source of protein and lipids, several antioxidants (including e.g. vitamin e), phytic acid, sterols, polyphenolics, phenolic acids (pas), flavonoids and avenanthramides (avns). these are concentrated in the outer gates layers (menon et al. 2016). pas and phenolic alkaloids, notably the avns, are present either in the ‘free form’ as soluble conjugates, or as insoluble bound forms (shewry et al. 2008). avns are a group of unique soluble bioactive compounds that are absent in other food crops. oats contain a unique group of approximately 40 different types of avns that consist bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:24 utc nova biotechnol chim (2018) 17(2): 132-139 133 of an anthranilic acid derivatives and hydroxycinnamic acid derivatives (boz 2015). avns from oats exhibit (potent) antioxidant activity in vitro and in vivo (thomas et al. 2018). amides of anthranilic acid are a group of naturally occurring phenolic amides in oats. three avenantramides are most represented in the oat grains: n-[3', 4'-dihydroxy-(e)-cinnamoyl]-5hydroxyanthranilic acid (2c), n-[4'-hydroxy-(e)cinnamoyl]-5-hydroxyanthranilic acid (2p) and n-[4'-hydroxy-3'-methoxy-(e)-cinnamoyl]-5hydroxyanthranilic acid (2f) (maliarová et al. 2015). both animal studies and human clinical trials confirmed that oat antioxidants have the potential of reducing cardiovascular risks by lowering serum cholesterol, inhibiting ldl oxidation, they are anti-carcinogenic and attenuating platelet aggregation and peroxidation (chen et al. 2004). dietary avns supplementation increases antioxidant capacity in the biological tissues, thereby reduces steady-state formation of reactive oxygen species and oxidative tissue damage (li et al. 2017). increasing the daily intake of whole grain cereals by 90 g has been associated with reduction in mortality from cardiovascular disease by 27 %, total cancer by 15 %, respiratory disease by 22 %, diabetes by 51 % and infectious diseases by 26 % (whitehead et al. 2014). it has been reported that gallic acid, vanillic acid, caffeic acid, ferulic acid, and p-coumaric acid are the main pas in oats (xu et al. 2009). experiments in vitro have shown that avns have significant antioxidant capabilities, with 10– 30-times higher radical scavenging activities than caffeic acid, ferulic acid, and vanillin (emmons et al. 1999). for this reason, avns have become the subject of our study. there are several studies that were focused on the identification and quantification of avns in oats, oat products, but also directly in clinical biological materials. in the study by chu et al. (2013) the contents of avns in 7 oat varieties from canada were reposted. oats and products present data for oats from china were studied and published in several other studies (ren et al. 2011; li et al. 2017; chen et al. 2018), husked oat from finland (multari et al. 2018). several studies assessed avns content in human materials such as plasma (chen et al. 2007) and urine (schär et al. 2018). however, data on content of avns in oats and oats products are still rather limited. the aim of our study was the identification and quantification of avns as well as selected phenolic compounds: p-coumaric acid (pca), ferulic acid (fa), avenanthramide 2c (avn 2c), avenanthramide 2p (avn 2p) and avenanthramide 2f (avn 2f) in oat varieties growing in slovakia, in commercially available products from local markets and oatmeal and extruded oatbread. experimental material analyzed samples included 17 different oat varieties. in addition, 6 commercially available products – 4x oat flakes (ravita, coop jednota, vince, and the gluten free kroner, all purchased from local market), 1x oat flour from new oat breading line (lo) (obtain from celpo spol. s.r.o., slovakia) and1x extruded oatbread. varieties of oat differ in their morphological, agronomical, phytopathological, and other characteristics. oat was harvested in the summer of 2014, 2015 from the research field in the research and breeding station at vígľaš-pstruša, slovakia (48º32'n, 19º10'e). the following varieties were included in the study – variety name (country of origin, year of harvest): 100 260 cn (united kingdom, 2014), avenuda (czech, 2014), bayan 2 (china, 2014), dunajec (ps – 191) (slovakia, 2014), hronec (ps – 166, 2014) (slovakia, 2014), kamil (slovakia, 2014), oliver (slovakia, 2014), racoon (united kingdom, 2014), tatran (slovakia, 2014), važec (ps – 176) (slovakia, 2014), ac percy (canada, 2015), avenuda (czech, 2015), avenuda x atego (slovakia, 2015), expression (united kingdom, 2015), fussion (united kingdom, 2015), izák (czech, 2015), racoon (united kingdom, 2015). all varieties of oat (avena sativa l.) were provided by the gene bank of the slovak republic (research institute of plant production, piešťany, slovakia). all varieties came from the same period of oat breeding in our region, originating from different countries as indicated below. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:24 utc nova biotechnol chim (2018) 17(2): 132-139 134 preparation of extracts samples of oat were milled by common laboratory mill (120 w, 230 v, 50 hz the size of particles 5 – 10 µm). samples of flour for each variety (0.30.01 g) were extracted by 2 x 3 ml 70 % (v/v) water solution of methanol at 55 °c. the samples were extracted using ultrasound 2 x 15 min. the samples were then centrifuged at 6,500 g for 15 min. the supernatants were kept at –20 ºc until hplc analysis was carried out. the supernatants were immediately centrifuged repeatedly before analysis and then directly injected without dilution into the hplc system. extraction solutions (supernatants) were injected three times. hplc analysis hplc separation, identification and quantification was performed using waters hplc system (waters, usa) equipped by waters 1525 binary pump, waters 2998 photodiode array (pda) detector, waters 2707 autosampler, thermostat waters model column heater 1500 series, waters symmetry c18 column (75 × 4.6 mm i.d., 3.5 μm) with adequate security guard column and software empower 2. for gradient elution were prepared two phases, phase a was 0.1 % water solution of formic acid and phase b was 0.1 % methanol solution of formic acid. the gradient was non-linear: 0 – 8 min 26 % solvent b, 8 – 18 min of 42 to 45 % solvent b, 18 – 21 min 100 % solvent b. similarly, the nonlinear gradient for phenolic compounds was used by authors zeng et al. (2016). a flow rate was used of 1.0 ml/min. the column temperature was set at 35 °c and the injection volume was 20 μl. detector operated at a wavelength 320 nm. validation of method for the validation of the hplc-dad method, parameters such as linearity, limits of detection (lod), limits of quantification (loq), precision, and accuracy were evaluated. the calibration curves of analytes were constructed after injection of standard solutions (three concentration levels, three replicate injections of each solution). the lod of the method were calculated as a signal to noise ratio of 3 using the injection of series of more diluted standard solutions of analytes. the loq were evaluated using signal to noise ratio of 10. intra-day and inter-day precisions of the method were evaluated for three preparations of sample within one day and five days, making triplicate injections under the working conditions and (expressed as rsd %). results and discussion extraction in the study maliarová et al. (2015) were established the optimal conditions for the extraction of avenanthramides 2c, 2p and 2f from oat grain using response surface methodology (rsm). sample extracts were prepared in 70 % methanol. comparison of retention times from hplc and uv spectra of sample peaks and standards was used for identification of selected phenolic compounds in extracts of oats. ultrasound supported extraction was proved most advantageous compared to eppendorf thermo-mixer and to environmental shaker-incubaton for pca, fa and avns (fig. 1). at the same time, optimal time of ultrasound supported extraction was investigated. the optimum extraction time was 2 times 15 min (fig. 2). this extraction was showed maximum yield and short time of extraction. the advantages of using extraction of ultrasound in food and natural products were been published too by chemat et al. (2017). results from extraction demonstrated, that for the following analysis of phenolic compounds: fa, pca, avn 2c, avn 2p, avn 2f it is best to use ultrasound-supported extraction. highest yield of phenolic compounds have been achieved in two time cycles for 15 min of sonification. hplc analysis reversed phase separation mode is most commonly used for hplc separation of both pas (pca, fa) and avns. the work was focused on development of hplc method for separation and determination of two pas and three avns (2c, 2p, 2f). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:24 utc nova biotechnol chim (2018) 17(2): 132-139 135 fig. 1. used extraction techniques for the determination of phenolic compounds (mg/kg): ferulic acid (fa), p-coumaric acid (pca), avenanthramide 2c (2c), avenanthramide 2p (2p), avenanthramide 2f (2f) (from three replicates). for separation of phenolic compounds was used analytical column symmetry c18 (75 x 4.6 mm; 3.5 µm). the mobile phase consisted of methanol with formic acid and ultra pure water with formic acid. the gradient was non-linear. similarly, the non-linear gradient for phenolic compounds was used by authors zeng et al. (2016). gradient elution profiles were investigated, which resulted in effective separation of target compounds in analysis time less than 20 min. phenolic compounds allow uv absorbance. spectrophotometric detection of pas and avns was realised by dad detector operated in the interval from 220 to 400 nm, where the absorption maxima of target compounds are presented (310 nm for pca, 320 nm for fa, 340 nm for avn 2c, 320 for avn 2p and 330 nm avn 2f). as a result, the 320 nm (excitation) wavelength was chosen for the purpose of this study (fig. 3). validation of method the analytical data were determined under optimized separation and detection conditions. fig. 2. yield of phenolic compounds (mg/kg): p-coumaric acid (pca), ferulic acid (fa), avenanthramide 2c (2c), avenanthramide 2p (2p), avenanthramide 2f (2f) by ultrasound supported extraction at various sonication time cycles (from four replicates). parameters including lod, loq, and linear range were calculated to demonstrate the validation of the developed hplc-dad method. the calibration curves of the five analytes were created after the injection of a mixed standard solution. results showed good linear relationship for each analyte (coefficient of determination in the range 0.9997 – 0.9999). the lod of the method were calculated as a signal to noise ratio of 3 using the injection of series of more diluted standard solutions of analytes. the loq were evaluated using signal to noise ratio of 10. lod/loq of pca was 18/68 ng/ml; lod/loq of fa was 28/101 ng/ml; lod/loq of avn 2c was 20.3/88.8 ng/ml; lod/loq of avn 2p was 22.8/85.6 ng/ml and lod/loq of avn 2f was 28/108.8 ng/ml. analytical parameters of the method used are listed in table 1. intra-day precisions were evaluated for retention time in range 0.37 – 1.04 % rsd and for area 0.47 – 2.18 % rsd. inter-day precisions (5 days) were evaluated for retention time in range 0.72 – 1.35 % rsd and for area 1.19 – 3.09 % rsd. eppendorf thermo-mixer environ. shaker-incubator ultrasons selecta eppendorf thermo-mixer environ. shaker-incubator ultrasons selecta bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:24 utc nova biotechnol chim (2018) 17(2): 132-139 136 fig. 3. overlay of two chromatograms: separation of standard solution (red line) and real sample of oat variety važec (blue line). identified peaks are labelled by name and chemical structures of separated analytes. table 1. analytical parameters of the hplc method. analyte concentration range [µg/ml] r [min] regression equation r2 wavelength [nm] pca 0.04 – 5.24 6.19 y = 137780x – 2282.3 0.9998 320 fa 0.04 – 5.13 7.41 y = 112910x – 3019.3 0.9999 320 avn 2c 0.19 – 24.5 13.49 y = 75748x – 17047.0 0.9998 320 avn 2p 0.53 – 17.1 16.17 y = 110824x – 2944.8 0.9999 320 avn 2f 0.80 – 25.6 17.22 y = 82557x – 4287.9 0.9997 320 in table 2 are listed values of determination of five studied polyphenols in oats. the highest content of pca was determined in the varieties oliver 2014 (2.60 mg/kg), kamil 2014 (2.05 mg/kg), racoon 2014 (1.78 mg/kg). the highest content of fa in oat was determined in the varieties kroner 6.27 mg/kg, oliver 2014 (5.72 mg/kg) and 100 260 cn 2014 (5.66 mg/kg). the highest content of avn 2c in oat was determined in varieties the racoon 2014 (288.22 mg/kg), oliver 2014 (133.13 mg/kg) and 100 260 cn 2014 (109.91 mg/kg). samples of racoon 2014 (230.09 mg/kg), oliver 2014 (153.31 mg/kg) and kamil 2014 (118.38 mg/kg) contain the largest amount of avn 2p. the highest content of avn 2f was detected in the varieties oliver 2014 (292.15 mg/kg), racoon 2014 (204.98 mg/kg) and kamil 2014 (180.94 mg/kg). in general, the highest content of all five studied polyphenols was found in the varieties: oliver 2014, racoon 2014 and kamil 2014. content of total avenanthramides in this study was the highest in the varieties racoon (723.28 mg/kg) followed by oliver (578.59 mg/kg) and kamil (384.17 mg/kg). the racoon, oliver, kamil and 100 260 cn from the year 2014 are the most exciting varieties for purpose of cultivation and breeding. the racoon, oliver, kamil and 100 260 cn can improve the nutritional and health benefits properties of oats. values of pas (pca, fa) and avns (2c, 2p and 2f) of oat flour (lo) and of extruded oatbread were similar. this finding suggests that the biotechnological process for lo preparations retains the nutrition quality of processed oats. the biotechnological production process used does not reduce the content of studied phenolic compounds. thus, the content of health beneficial substances remains preserved for the consumer. content of pca and fa was higher in oat breads as much as in the flour. it is possible to explain by partial bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:24 utc nova biotechnol chim (2018) 17(2): 132-139 137 table 2. summary of content (mg/kg) of five analytes in 18 samples of oats (avena sativa l.) expressed as averages and standard deviations calculated from three replicates. pca fa avn 2c avn 2p avn 2f avns year variety m±std rsd [%] m±std rsd [%] m±std rsd [%] m±std rsd [%] m±std rsd [%] s 2014 oliver 2.60±0.02 a 0.76 5.72±0.09 a 1.52 133.13±3.42 b 2.57 153.31±3.31 b 2.16 292.15±32.12 b 10.99 578.59 100 260 cn 1.39±0.04 b 2.67 5.66±0.15 a 2.66 109.91±7.55 d 6.87 106.45±6.77 d 6.36 138.75±7.43 d 5.36 355.12 kamil 2.05±0.21 c 10.3 4.72±0.53 b 11.26 84.84±2.80 c 3.30 118.38±4.42 c 3.74 180.94±5.98 c 3.31 384.17 racoon 1.78±0.18 d 10.29 4.98±0.31 c 6.20 288.22±11.38 a 3.95 230.09±9.47 a 4.11 204.98±6.29 a 3.07 723.28 bayan 2 0.96±0.07 e 7.63 5.37±0.25 d 4.73 73.52±1.74 e 2.37 66.60±2.11 e 3.17 120.40±2.37 e 1.97 260.52 hronec 1.08±0.01 f 1.7 5.08±0.16 c 3.16 52.93±2.19 g 4.15 65.71±3.62 ef 5.51 94.56±4.01 g 4.25 213.19 dunajec 0.88±0.02 eg 2.26 3.05±0.08 e 2.77 62.58±1.36 f 2.17 71.42±1.77 e 2.48 103.62±1.16 fg 1.12 237.62 avenuda 1.04±0.10 ef 10.9 2.81±0.26 f 9.11 74.25±4.12 e 5.54 64.12±2.95 ef 4.61 110.61±2.90 ef 2.62 248.99 važec 0.78±0.05 gh 6.74 2.51±0.13 g 4.99 34.84±0.52 h 1.50 25.45±1.37 h 5.39 42.18±2.14 h 5.07 102.46 tatran 0.88±0.01 egh 0.36 2.40±0.03 g 1.33 33.58±2.22 h 6.61 31.37±0.44 g 1.40 52.62±0.43 h 0.81 117.57 2015 avenuda 0.68±0.02 a 2.25 4.55±0.04 a 0.94 9.57±0.20 e 2.13 3.92±0.10 e 2.54 6.84±0.46 c 6.79 20.33 ac percy 0.65±0.07 b 11.10 3.13±0.19 b 5.,95 10.30±0.58 e 5.66 3.72±0.09 e 2.34 4.39±0.38 d 8.73 18.40 avenuda-atego 0.57±0.02 c 2.91 3.11±0.37 b 11.90 15.27±1.56 d 10.25 9.17±0.84 d 9.11 9.74±1.23 b 12.65 34.18 fussion 0.53±0.03 d 5.55 2.54±0.08 c 3.26 36.13±2.08 a 5.75 24.29±0.50 a 2.05 16.71±1.91 a 11.46 77.12 racoon trace – 2.43±0.04 c 1.68 27.48±1.16 b 4.21 21.50±0.41 b 1.88 9.38±0.27 b 2.84 58.35 expression trace – 2.37±0.04 c 1.84 20.52±2.47 c 12.03 11.97±1.26 c 10.48 6.37±0.58 c 9.14 38.87 izák 0.79±0.03 e 3.68 1.40±0.01 d 1.05 9.15±0.39 e 4.24 5.06±0.18 f 3.50 5.21±0.51 d 9.70 19.42 cap kroner 1,03±0,11 10.99 6.27±0.54 8.63 6.97±0.05 0.76 4.39±0.33 7.52 6.00±0.35 5.80 17.36 ravita 0.88±0.10 10.84 5.66±0.53 9.32 32.31±2.77 8.58 15.33±1.12 7.28 34.64±2.09 6.05 82.28 jednota 0.74±0.06 8.29 4.65±0.50 10.68 22.04±1.82 8.27 10.18±0.81 7.94 21.39±1.96 9.17 53.61 vince 0.80±0.03 3.34 4.43±0.07 1.57 27.07±1.00 3.70 12.98±0.20 1.56 21.55±0.80 3.73 61.61 oatbred 1.32±0.11 8.63 3.72±0.25 6.70 48.61±2.04 4.20 64.73±2.60 4.01 75.28±2.39 3.18 188.62 flour 0.84±0.03 3.32 3.48±0.20 5.79 52.79±2.37 4.50 67.16±2.87 4.28 78.49±3.67 4.67 198.44 cap – commercially available products; different letters indicate significant differences at p < 0.05. hydrolysis of bound pas during extrusion (kováčová et al. 2007). it is stated that the quantity of free ferulic acidin grains is 0.1 – 0.5%, most of it bound with polysaccharides and sterols (zhao et al. 2008). in the study by xu et al. (2009) was reported that gallic acid, vanillic acid, caffeic acid, ferulic acid, and p-coumaric acid are the main pas in oats. the content of fa in oat bran was 33 mg/100g (boz 2015). in the study by chen et al. (2018) was reported that fa was in range 1.32 – l8.98 μg/g. the concentration of p-coumaric acid in oats is not very much published. there are studies of the presence of p-coumaric acid in other cereals. the p-coumaric acid (97.87 – 211.03 mg/kg) and o-coumaric acid (126.53 – 575.87 mg/kg) were determined in the corn (hung 2014). the intervention diet (60 g oat bran) contained 28.6 mg of total phenolics (24 % in the soluble fraction), with fa being the predominant pas (16.8 mg) followed by pca (3.3 mg) and the three avns totally amounting to 2.5 mg (schär et al. 2018). fa, pca and avn 2p accounted only for small percentages of the total excreted phenolics and avns 2f and 2c were not detected, suggesting that these dietary forms are subject to extensive metabolism (schär et al. 2018). avns content in whole oat extracts was 3.7 to 48.41 mg/kg for avn 2c, 1.05 to 43.77 mg/kg for 2p and 3.66 to 58.63 mg/kg for 2f in the varieties of oats from canada (chu et al. 2013). in oat, values of avns were as follows: 1.16 to 70.06 µg/g for avn 2c, 2.3 to 74.7 µg/g for avn 2p and 1.11 to 69.74 µg/g for avn 2f (chen et al. 2018). the latter results indicate that low amount of hydroxycinnamic acids and high amount of hydroxybenzoic acids may be characteristics of the oats (avena nuda l.) from china (chen et al. 2018). avns content of oat milling fractions ranged from 323.7 to 775.5 μg/g, while the free phenolic acids content was much lower (103.5 – 194.6 μg/g). the study by li et al. (2017) reported that the concentrations of avn 2f were higher than those of the other two avns. concentrations of total avns in this study ranged from 22.1 to 471.2 mg/kg. for comparison, concentrations of total avns in our study ranged from 17.36 to 723.28 mg/kg, which presents high concentration values of avns. in the varieties of oat were observed year-on-year fluctuations of selected phenolic compounds. in the oat varieties from the year 2014, the analytes were bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:24 utc nova biotechnol chim (2018) 17(2): 132-139 138 determination in significantly higher concentration than in the oat varieties from the year 2015. this observation was caused by the meteorology. in march-july 2014, higher average temperatures and lower precipitations (%) were measured, reflecting significantly different concentrations of all studied polyphenols. statistical analysis the data were expressed as the means with standard deviation and were analyzed by the analysis of variance (one way anova) and pearson product-moment correlation coefficient. statistical significance was defined as p < 0.05. the analysis was carried out with statistical software ibm spss 22 using the post hoc test lsd. conclusions oat is a cereal with a rich source of health beneficial phytochemicals. we evaluated the contents of phenolic compounds present in oat grown in slovakia and selected the most interesting varieties with a high content of nutritional values. a simple reversed-phase hplc-dad method was developed for simultaneous determination of pca, fa, avns 2c, 2p and 2f. the separation of selected phenolic compounds by hplc was rapid, efficient and reproducible, and the method presented acceptable results for sensitivity, linearity, precision, and accuracy. the study has shown that the oat varieties grown in 2014 have a significantly higher content of selected polyphenols than the oat varieties of 2015. the most exciting varieties for purpose of cultivation and breeding are oliver, racoon, kamil and 100 260 cn. the hplc method has potential to be used in analysis of oats and product of oats since currently there is an interest on characterization and determination of biologically active compounds in cereal. acknowledgement this work was financially supported by the grants kega 006ucm-4/2018, vega 1/0919/17, vega 1/0534/16 and apvv-17-0113. references boz h (2015) phenolic amides (avenanthramides) in oats – a review. czech j. food sci. 33: 399-404. emmons chl, peterson dm, paul gl (1999) antioxidan capacity of oat (avena sativa l.) extracts. 2. in vitro antioxidant activity and contents of phenolic and tocol antioxidants. j. agric. food. chem. 47: 4894-4898. hung pv (2014) phenolic compounds of cereals and their antioxidant capacity. crit. rev. food sci. nutr. 56: 25-35. chemat f, rombaut n, meullemiestre a, turk m, perino s, fabiano-tixier as, abert-vian m (2017) review of green food processing 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https://www.ncbi.nlm.nih.gov/pubmed/?term=gong%20e%5bauthor%5d&cauthor=true&cauthor_uid=27513581 https://www.ncbi.nlm.nih.gov/pubmed/27513581 nova biotechnol chim (2020) 19(2): 240-247 doi: 10.36547/nbc.v19i2.592  corresponding author: l.cheban@chnu.edu.ua nova biotechnologica et chimica obtaining phycobiliprotein-containing nostoc linckia (roth.) born. et flah biomass via bioconversation of waste water from reticulating aquaculture systems (ras) larysa cheban, yevdokiia turianska and mykhailo marchenko department of biochemistry and biotechnology, chernivtsi national university named after y. fedkovych, kotsiubynsky 2 str., chernivtsi 58012, ukraine article info article history: received: 2nd july 2020 accepted: 23rd november 2020 keywords: bioconversion nostoc linckia phycobiliprotein pigments recirculating aquaculture systems waste water abstract the paper studies the possibility of bioconversion of waste water from recirculating aquaculture systems (ras). for this purpose, cyanobacteria nostoc linckia (roth.) born. et flah. was grown on waste water from ras. in the process of cultivation, indicators of total mineralization, ph, and culture density were monitored. in the waste water, the content of various forms of nitrogen (nh4 +, no3 -, no2 -), the content of p and s before and after the growth of cyanobacteria were determined it was noted that as a result of growing n. linckia in ras waste water, a decrease in the content of various forms of nitrogen, sulfur and phosphorus is observed. also, the content of phosphates decreased from 35×10-3 to 5×10-3 mg.l-1 and the content of sulfates from 95×10-3 to 25×10-3 mg.l-1. moreover, the resulting biomass of cyanobacteria contained 57 % of proteins, 20 % of lipids and 11% of carbohydrates. it was found that n. linckia biomass is a promising source of phycobiliprotein pigments: phycocyanin, allophycocyanin and phycoerythrin.  university of ss. cyril and methodius in trnava introduction in recent years, due to a significant increase in demand for aquatic food, land-based aquaculture is developing rapidly and intensively around the world. however, the continuous development of intensive aquaculture is tempered by certain problems, and the issue of wastewater disposal is especially acute (mook et al. 2012; chen et al. 2015). wastewater discharged from fisheries is usually concentrated with nutrients (nitrogen, phosphorus) and mainly consists of fish waste products and feed residues. bioconversion makes it possible to dispose of various wastes in wastewater. this is the conversion of substances and energy under the influence of living organisms or in their cells. this process is inherent in any biological system, since the conversion of substances and energy occurs in metabolic reactions and in the processes of energy and substance exchange between living organisms and the environment. under the influence of cells, metabolites turn into structurally related compounds. various microorganisms were used to disinfect the contaminated environment, including bacteria, fungi, algae and plants (vidali 2001; leung 2004). in controlled conditions, organic waste degrades to harmless levels under the influence of microorganisms (nayyef and amal 2012; lu et al. 2019). growing cyanobacteria in the aquaculture wastewater ensures the removal of fish waste products and simultaneously, production of biomass that can be used to produce valuable mailto:l.cheban@chnu.edu.ua nova biotechnol chim (2020) 19(2): 240-247 241 products such as aquaculture feed, medicines, natural dyes, and biofuels (michels et al. 2014). the advantages of cultivating members of cyanobacteria genus are higher yields, use of non-agricultural land, nutrients restoration from wastewater, and the efficient capture of carbon. moreover, adaptation to a wide range of environmental conditions makes them a rich source of important and useful metabolites. these organisms have the potential to meet a wide range of global needs, but realization of this potential calls for lower production costs. nostoc linckia roth. born. et flah is a thallogen heterocyst cyanobacterium capable of nitrogen fixation (fokina et al. 2011). due to the sufficiently large size of thallome and a significant amount of mucus, it does not require complex approaches in the post-cultivation separation of biomass from the nutrient medium. like most tallogens, n. linckia is responsive to changes in the nutrient medium and builds up biomass rapidly. these cyanobacteria are unpretentious to the composition of nutrient media (gupta and rastogi 2008) and are therefore considered an excellent choice for use in various biotechnological processes. biomass of cyanobacteria is characterized by a high content of amino acids and proteins (rosales-loaiza et al. 2017). the high content of basic nutrients allows using n. linckia as animal feed, specifically, in aquaculture. a variety of photo-synthetic pigments, which are used in the food, cosmetic and pharmaceutical industries, permits the use of this species as their producer. recently, photosynthetic pigments such as phycobiliproteins, which are usually present in cyano-bacteria, have attracted attention. c-phycocyanin is an active antioxidant able to suppress the proliferation of tumor cells, reduce the concentration of tumor necrosis factor in tissues, inhibit oxidative stress, prevent lipid peroxidation, dna damage, destruction of cell membranes and cell death (soundarapandian and muruganandham 2008; sinha et al. 2011). phycocyanin allows to get the so-called anatomical portrait of a person (it selectively accumulates in atherosclerotic plaques and neoplasms, the localization of which is determined by ultrasound scanning) (kardash et al. 2012). most synthetic dyes are toxic, so interest in unconventional sources of raw materials for natural dyes has increased significantly. one of such promising food colors is protein phycoerythrin, an absolutely non-toxic red pigment with pronounced orange fluorescence, which is now widely used in the food, cosmetic and medical industries. in the united states, coloring materials based on this pigment are patented as fluorescent markers for immunodiagnostics. there is also evidence of the use of phycoerythrin and phycocyanin in oncology as radioprotectors. as a nutritional supplement, these pigments can improve the color of salmon flesh and also positively influence health and fertility in cattle (valuta et al. 2015). the aim of our work was to use recirculating aquaculture systems (ras) waste water for growing cyanobacteria and obtain phycobiliprotein-containing biomass of nostoc linckia (roth.) born. et flah. experimental biological material and cultivation conditions studies were conducted using the monoculture of n. linckia (roth.) born et flah, which is maintained in the collection of the department of biochemistry and biotechnology, yurii fedkovych chernivtsi national university. museum n. linckia (roth.) born. et flah. (hpdp453) culture was obtained from the collection of the institute of hydrobiology of the nas of ukraine. n. linckia (roth.) is a thallogen heterocyst cyanobacterium (guiry and guiry 2020), characterized by the ability to proliferate rapidly. this, as well as the ease of separation of biomass from the culture fluid makes this species a convenient object for biotechnological research. cyanobacteria were cultivated in ras waste water and on the standard fitzgerald’s no. 11 medium in the modification of zehnder and gorhem as a comparison medium (zolotareva et al. 2008). water was taken from the mechanical filter of the ras, aliquoted, sterilized in an autoclave at a temperature of 121 °c for 30 min and standardized at ph (7.5 – 8.0) and total salinity (495±5 ppm) (khydyi et al. 2016). cultivation was carried out in 500-ml size erlenmeyer flask under climate room conditions: nova biotechnol chim (2020) 19(2): 240-247 242 21±2 °с, illumination with fluorescent lamps of about 2,500 lux and a photoperiod light/darkness 16 : 8 h. the inoculation was carried out in the ratio of inoculum : nutrient medium – 1 : 10 in the sterile laminar box (cheban et al. 2015). cyanobacteria under these conditions were cultured for 50 days. under these conditions, cyanobacteria were grown for 50 days. in the process of cultivation, indicators of total mineralization, ph, and culture density were monitored. the content of various forms of nitrogen (nh4 +, no3 -, no2 -), the content of p and s before and after the growth of cyanobacteria were determined in the waste water. the quantitative content of mineral elements in ras waste water was determined according to generally accepted photocolorimetrically methods; nitrates were determined with phenol disulfonic acid, nitrites using griss reagent, ammonia with nessler's reagent, sulfates by the method of ozerov and phosphates using a molybdenum mixture (arsan et al. 2006). the density of the biomass was determined from the culture density indicators using an optical indicator at 750 nm with carywin uv 60 software (agilent, usa). the transition from optical density units (d750) to the absolute dry biomass (asb) value was carried out through the empirical coefficient k: asb = k × d750 (hevorhyz et al. 2008). the coefficient k (k = g.unit opt. density.l-1) for the culture was determined experimentally in three independent reruns. using the obtained values, a growth curve was drawn for the culture of n. linckia. after the cultivation, the isolation of algae cells from the culture medium was carried out by centrifugation at 6,000 rpm within 15 min on "herauses" biofuga stratos. in the irrigated biomass, the content of proteins, lipids and carbohydrates, chlorophyll а, carotenoids and phycobiliproteins was determined. hydrophilic components of the biomass were extracted with 0.1 m phosphate buffer solution with a ph of 7.4 (musienko et al. 2001). extraction of hydrophobic pigments was performed with 100 % acetone. optical density of the pigments was measured spectrophotometrically in the wavelength range of 400 – 800 nm with on a carywin uv 60 (agilent, usa), and to obtain their concentration, a formulaic calculation has been put forward (geffrey and humphrey 1975). determination of total protein content was carried out according to the method of lowry (lowry et al. 1951). lipids were extracted using the folch method and determined by the presence of sulfuric acid and phosphorus-vanillin reagent (knight et al. 1972). the amount of total carbons was determined by the color reaction with the anthrone reagent (olennikov and tanhaeva 2006). the obtained indicators were calculated as a mass fraction (%) based on dry biomass. analyses of phycobiliprotein content phycobiliprotein complex was obtained with 0.2 m phosphate buffer, ph 7.0, at a biomass : extractant ration of 1 : 5. the concentration of phycobilin pigments was determined spectrophotometrically at analytical wavelengths of 450 – 700 nm using carywin uv 60 (agilent, usa). the purity of the obtained fractions was calculated using standard formulas as the optical density ratio at d620 / d280 (patil et al. 2006). data analysis all data are presented as mean ± se. the significance of differences in the results was evaluated with one-way anova. statistical analysis was computed using ms excel software and statistica 6.0. mean values were considered significantly different at p ≤ 0.05 according to student’s criterion. results and discussion to ensure normal functioning of the cell culture of cyanobacteria, a sufficient content of essential nutrients in the culture medium is necessary. this has significant implications on the growth of microalgae and synthetic processes in them. enriched with salts of nitrogen, phosphorus, and sulfur, ras waste water has a qualitative composition of the main mineral elements similar to that of the fitzgerald’s medium (cheban et al. 2015; khydyi et al. 2016). microalgae best absorb nitrogen in nitrate or ammonium form, however, when using the nitrate form of nitrogen, an intensive increase in biomass is observed, while nova biotechnol chim (2020) 19(2): 240-247 243 table 1. physical and chemical parameters of waste water from ras (mean ± se, n = 4). characteristics of culture media waste water from ras fitzgerald’s medium no3 [mg.l-1] 43.2±0.7 83.2±2.1 no2 [mg.l-1] 0.64±0.04 – nh4 + [mg.l-1] 0.51±0.02 – po4 3[mg.l-1] 0.031±0.030 0.040±0.030 so4 2[mg.l-1] 0.094±0.020 0.031±0.010 ph 7.0 – 8.0 7.0 – 8.0 total mineralization [ppm] 371.0 – 477.0 332.0 – 547.0 conductivity [µs.ml-1] 555.0 – 693.0 452.0 – 690.0 redox potential [mv] 211.4 – 213.9 176.9 – 245.0 ammonium ions are able to suppress the development of the culture (parshikova et al. 2010; tejido-nunez et al. 2019). with nitrogen deficiency, inhibition of algoculture growth is observed, which affects the content of basic nutrients in biomass. ras waste water is a suitable medium for cyanobacteria cultivation since it has a high content of nitrogen compounds (ammonium, nitrite and nitrate), phosphates and dissolved organic carbon (lananan et al. 2014) coming from fish waste products and food residues (crab et al. 2007). this composition of discharged water promotes the growth of various microorganisms, including microalgae and cyanobacteria. the quality composition of ras waste water and the fitzgerald’s medium was found to be similar in terms of basic mineral elements. in both cases, there is a sufficient amount of nitrogen, phosphorus, carbon. studies shown that the physical and chemical parameters of ras waste water are fully comparable with fitzgerald’s medium (table 1). therefore, for our research, ras waste water was used as a nutrient medium. it was noted that n. linckia grows exponentially during the first 30 days with subsequent stabilization (fig. 1). from the day 40 of cultivation, there was a decrease in the amount of the biomass was observed, which is explained by a decrease in the available nutrients in the medium. this is confirmed by the decrease in the total mineralization index. the growth pattern of the culture was the same both in waste water and in the control medium. the amount of biomass obtained, taking into account the simultaneous use of media, did not significantly differ. the main advantage of using cyanobacteria for waste water treatment is their availability, autotrophy, high reproduction rate and high ratio of body surface area to cell number. these bacteria are photosynthetic organisms that assimilate n and p during growth. microalgae-based technologies are a promising solution for aquaculture waste water treatment (gao et al. 2016; guldhe et al. 2017; egloff et al. 2018). fig. 1. n. linckia biomass (a) and total mineralization (b) cultured at waste water from ras: fitzgerald medium (diamonds), waste water from ras (squares). note: * – significant difference relative to control values (p ≤ 0.05). nova biotechnol chim (2020) 19(2): 240-247 244 table 2. biochemical composition of n. linckia biomass under various cultivation conditions (mean ± se, n = 4). investigated indicators fitzgerald’s medium waste water from ras total proteins [%] 56.6±2.6 55.1±2.3 total lipids [%] 19.8±0.8 20.1±0.8 carbohydrates [%] 10.7±0.4 11.3±0.5 chlorophyll а [mg.g-1] 17.4±0.8 16.2±0.8 carotenoids [mg.g-1] 11.3±0.5 9.5±0.4 note: * – significant difference relative to control values (p ≤ 0.05). one of the most important properties of the species n. linckia is its high protein content, which makes this genus a candidate for the continuous production of fertilizers and protein supplements in animal feed (habib et al. 2008). during the cultivation of the cyanobacteria n. linckia, a biomass was obtained with the following content of basic nutrients and pigments (table 2). when cultivating a culture of n. linckia on ras waste water, a significant decrease in the content of various forms of nitrogen, sulfur, and phosphorus is observed (fig. 2). as it turns out, in the process of growing biomass there is a complete absorption of the ammonium form, and the amount of nitrate form decreases by 8-times. also, as a result of nitrification, ammonium could be classified as nitrite. it should be noted that ammonium and nitrate nitrogen under certain conditions are equivalent sources of nitrogen for cyanobacteria. the predominant absorption of ammonium nitrogen occurs when nh4 + is the only source of nitrogen (shilova et al. 2020). in cyanobacteria, ammonium, either extracted from the external environment or formed intracellularly, is incorporated into carbon skeletons mainly through the glutamine synthetase/glutamate synthase cycle (herrero et al. 2001). the content of phosphates and sulfates decrease by 7and 4-times, respectively. therefore, growing cyanobacteria on ras waste water not only allows obtaining protein-rich biomass, but also significantly purifies waste water during bioconversion. cyanobacteria, unlike higher plants and bacteria, contain, in addition to chlorophyll and carotenoids, water-soluble pigments – phycobiliproteins – chromoproteins, the non-protein part of which includes phycobilin chromophores. they are the main light-absorbing pigments in cyanobacteria (valuta et al. 2015). since n. linckia is a promising source of phycobilins, we developed a scheme for the preparation of phycobilin protein complex and ways to improve it. this scheme includes stages of the steps of isolating a pigment preparation, spectroscopy and purity testing, and determining the amount of pigment. it is known that the extraction efficiency of any cell components depends on the extractant used. the phycobiliprotein complex of cyanobacteria dissolves well in phosphate buffer or alcohol solutions is well soluble in phosphate buffer or in alcohol solution, the latter also allow increasing the shelf life of the obtained preparation. typically, these compounds are extracted at room fig. 2. the content of various forms of nitrogen, phosphorus and sulfur in waste water from ras before (gray columns) and after cultivation of n. linckia (black columns). note: * – significant difference relative to control values in waste water from ras before cultivation (p ≤ 0.05). nova biotechnol chim (2020) 19(2): 240-247 245 temperature, but with chilled extractants. so as an extractant we used 0.2 m phosphate buffer with a ph of 7.0. to ensure complete extraction of phycobiliproteins, this operation was performed three times. phycobiliproteins are the main components of cyanobacteria cells: it is known that in some species they can make up to 40 % of the cellular protein. three compounds were found in our phycobiliprotein preparation, the absorption maxima of which correspond to phycoerythrin (560 nm), (620 nm) and alophycocyanin (650 nm) (fig. 3). it should be noted that phycoerythrin is not always present in the cells of cyanobacteria, it is characteristic mainly for filamentous forms, n. linckia, in particular. fig. 4. the content of phycobiliproteins in n. linckia cells when cultured on waste water from ras: fitzgerald’s medium (gray columns), waste water from ras (black columns). note: * – significant difference relative to control fizgerald medium values (p ≤ 0.05). there is a link between the amount of pigments and the conditions of nitrogen supply, which is possibly due to the chemical structure and functions of the main components of the photosynthetic system. so, when cultivating n. linckia in ras waste water, its biomass revealed c-phycocyanin – 12.2 mg.g-1 (1.2 %), phycoerythrin – 23 mg.g-1 (2.3 %), alophycocyanin – 25.7 mg.g-1 (≈ 2.6 %) (fig. 4). the amount of analyzed compounds in n. linckia biomass did not differ significantly with the nutrient medium. it is obvious that the same growing conditions, ph of the medium and the amount of basic nutrients in both applied nutrient media make it possible to obtain the amount of phycobiliproteins in the biomass, almost identical in both cases. table 3. purity index by optical density of the phycobylprotein preparation derived from biomass n. linckia (mean ± se, n = 4). investigated indicators fitzgerald’s medium waste water from ras phycoerytryn [odu] 1.42 1.35 c-phycocianin [odu] 0.78 0.64 alophycocianin [odu] 1.62 1.54 note: * – significant difference relative to control values (p ≤ 0.05). we also calculated the purity of individual fractions of phycobiliprotein pigments according to standard formulas (table 3). degree of purification is determined by an indicator measured by the ratio of optical densities at two wavelengths: d620/d280. in the aqueous extract, this value should be at least 0.3 – 0.4 (kardash et al. 2012). fig. 3. absorption spectra of phycobiliproteins from the biomass of n. linckia, where: fitzgerald’s medium (filled line), waste water from ras (dashed line); 1 – phycoerythrin, 2 – c-phycocyanin, 3 – alophykocyanin. nova biotechnol chim (2020) 19(2): 240-247 246 thus, the use of ras waste water as a nutrient medium can reduce the costs of biomass cyanobacteria production, as well as cleanse ras waste water from excess nutrients and use it as a reverse.the use of the obtained biomass as a source of the phycobiliprotein complex allows receiving a cheaper product with the components of high purity. so, the waste water from ras can be used as an alternative medium for the cultivation of n. linckia. the resulting biomass in terms of total protein, lipids, carbohydrates, and basic photosynthetic pigments does not significantly differ from that grown on fitzgerald’s control medium. the performed operations have shown that cleaning ras waste water by growing cyanobacteria n. linckia on it is possible. such bioremediation measures allow pursuing several goals simultaneously: to purify water from chemical agents and obtain valuable cyanobacterial biomass. these approaches are now widely used both for microalgae and cyanobacteria (ansari et al. 2017; tejido-nunez et al. 2019). understandably, to complete the task, additional examinations for the presence of bacterial contamination and the safety of the obtained biomass are required (halfhide et al. 2014; fulbright et al. 2018). however, even at this stage, it can be concluded that the cultivation of cyanobacteria in ras waste water is a promising trend in the field of aquaculture. conclusion the chemical composition of ras waste water is comparable to the fitzgerald’s medium in terms of the amount of various forms of nitrogen, phosphorus and sulfur. this makes it possible to use ras waste water as an alternative medium for growing the cyanobacteria n. linckia. this technology eliminates chemical pollution of water, significantly reduces the content of nitrites and nitrates as well as the level of sulfur and phosphorus in water. the biomass grown in waste water has a similar content of proteins, lipids and carbohydrates as in the control medium. from this biomass, a highly purified preparation of phycobilin protein pigments can be obtained, containing c-phycocyanin (12.2 mg.g-1), phycoerythrin (23 mg.g-1) and allophycocyanin (25.7 mg.g-1). conflict of interest the authors declare that they have no conflict of interest. references ansari fa, singh p, guldhe a, bux f (2017) microalgal cultivation using aquaculture wastewater: integrated biomass generation and nutrient remediation. algal res. 21: 169-177. arsan om, davydov oa, dyachenko tm (2006) methods of hydroecological studies of surface waters. national academy of science of ukraine, kiev. cheban l, malischuk i, marchenko m (2015) peculiarities of cultivation desmodedesmus armatus (chocl.) hegew. in the wash water from ras. arch. pol. fish. 23: 155162. chen s, yu j, wang h, yu h, quan x (2015) a pilot-scale coupling catalytic ozonation–membrane filtration system for recirculating aquaculture wastewater treatment. desalination 363: 37-43. crab r, avnimelech y, defoirdt t, bossier p, verstraete w (2007) nitrogen removal techniques in aquaculture for a 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biotechnologica et chimica variable accumulation of cadmium in flax (linum usitatissimum l.) mária pavlovičová1, zuzana gerši2, monika bardáčová3, petra ranušová3, miroslav horník3 and ildikó matušíková3, 1 department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, sk-917 01 trnava, slovak republic 2 department of biology, faculty of natural science, university of ss. cyril and methodius in trnava, nám. j. herdu 2, sk-917 01 trnava, slovak republic 3 department of ecochemistry and radioecology, faculty of natural science, university of ss. cyril and methodius in trnava, nám. j. herdu 2, sk-917 01 trnava, slovak republic article info article history: received: 12th may 2020 accepted: 7th june 2020 keywords: hyperaccumulators metal accumulation metal stress pr proteins abstract given the potential use of flax in metal-contaminated soil remediation programs, the uptake and accumulation of cadmium was studied in five varieties of flax (linum usitatissimum l.) including var. belinka, escalina, jitka, marina and krasnoder. early stage plants in hydroponics were exposed to cd to assess their tolerance by means of growth parameters and content of photosynthetic pigments. all varieties exerted tolerance indexes in the range of 63 – 89 %, while oxidative stress was not detected in either variety. chitinase enzymes were analyzed in leaf protein extracts since activities of these enzymes have previously been correlated with plant tolerance to cd. however, total enzyme activities remained unchanged in presence of cd in all flax varieties. a more detailed analysis of these enzymes identified up to 3 chitinase isoforms upon separation of leaf protein extracts in polyacrylamide gels, and their quantification confirmed responsiveness to cd for each of them. the obtained data were interpreted in light of metal uptake rate, which we measured using gammaspectrometry in growth media spiked with 109cd and in the plant tissue. the variety jitka showed the most sensitive to cd and accumulating fast and the highest amounts of metal. in contrast, the variety belinka appeared most tolerant, accumulating the least cd in a slow rate. activation of chitinase isoforms correlated with more sensitive varieties and suggests activation of general defense mechanisms. the obtained data suggest the variety jitka as most promising for phytoremediation programs and the var. belinka as the most suitable when avoidance of potential health risk is of interest.  university of ss. cyril and methodius in trnava introduction crop plants grown in heavy metal (hm) contaminated areas represent a considerable health risk after entering the food chain (chen et al. 2016a). even in areas with low contamination, metals can cause phytotoxicity and consequently yield losses, affecting livestock or people by consuming large quantities of the produced crops (guo et al. 2020). though data from the lucas topsoil survey assign the immense majority of european agricultural land as adequately safe for food production, a continentwide survey highlighted large areas where precautionary measures are needed, and estimated that 6.24 % of the agricultural land needs local mailto:ildiko.matusikova@ucm.sk nova biotechnol chim (2020) 19(1): 70-79 71 assessment and eventual remedial action (tóth et al. 2016). remediation of soils is, however, extremely costly therefore phytoremediation is currently emphasized as a new, noninvasive and publicly acceptable technology that removes pollutants from the environment or to render them harmless by stabilization. the plants usable for phytoremediation should have very high metal removal rate that depends on the plant biomass harvested and metal concentration in harvested biomass (sumiahadi and acar 2018). attempts to reduce the cd pollution evoked deeper research on plant species that can well tolerate and accumulate metals in their tissues. hyperaccumulating plants have the capacity to concentrate more than 100 mg.kg-1 of dry matter (dm) in harvested organs compared to usual species that accumulate 0.05 – 0.2 mg of cd kg-1 of their dry leaves. according to the global hyperaccumulator database (http://hyperaccumulators.smi.uq.edu.au/), few species within up to 7 plant families are known to hyperaccumulate cd. most of these slow growing plants produce a low biomass in contrast to some other metal-accumulating crop species such as sunflower, maize, tobacco or fast growing trees (poplar, willow). the phytoextraction potential and high biomass production renders the latter interesting in the phytoremediation concept to overcome the mass limitation of hyperaccumulators (vassilev et al. 2002). flax (linum usitatissimum l.) has been suggested in phytoremediation strategy by several authors (bjelková et al. 2011; guo et al. 2020) as this species exerted better cd accumulation and tolerance capacity than some other crops such as cotton and hemp (angelova et al. 2004; shi and cai 2009). furthermore, flax as an annual crop species can remove considerable quantities of heavy metals from the soil every year, has a well elaborated large-scale agrotechnology and plethora of industrial uses (griga and bjelková 2013). although a protein map of mature flax seeds from the chernobyl remediation area in ukraine was prepared (klubicová et al. 2011) and changes in flax protein profiles during growth in the presence of cadmium ions are known (szalata et al. 2009, 2014), the profile and activity of specific groups proteins and enzymes in flax remains unclear. tolerance of flax to heavy metals (cadmium) is attributed to phytochelatins (najmanova et al. 2012), cadmium-binding proteins (oomah et al. 2007), but also sorption processes of cellulose fibers (rezić 2013). furthermore, chitinase enzymes have been shown to respond flexibly to the presence of different types of metals and contribute to tolerance (metwally et al. 2005; mészáros et al. 2013). identifying biomarkers which could help to identify varieties that are tolerant at the same time accumulate high amounts of metal (cadmium) would be useful for bioremediation programs. the present work was therefore devoted to studying a set of flax varieties bred in europe and compare their tolerance/cd uptake rate with previously published results. experimental plant material and experimental setup different varieties of flax (linum usitatissimum l.) – jitka, marina, belinka, krasnoder and escalina were provided by the gene bank of slovakia (nafc, research institute of plant production, piešťany, slovakia). seeds were surface-sterilized for 5 min. in 0.5 % (v/v) sodium hypochlorite and washed twice in distilled sterile water. after germinating on wet filter paper for 5 days the plants were cultivated in hydroponic conditions in plastic containers with 500 ml of 1/4 hoagland medium (ph 5.6) (hoagland 1920) in a growth chamber (kbwf 720, binder) under 16h/8h photoperiod, photosynthetic photon flux (ppf) level 120 μmol.m-2.s-1, at 28 ºc (day)/ 18 ºc (night) temperature and 60 % relative humidity. after 10 days a set of plants was exposed to 50 mg.l-1 cd2+ (in a form of cdcl2) spiked with 109cd for the period of 72 h. another set of plants was further grown under the same conditions in media without cd. the experiment was repeated 3-times and for each flax variety. physiological measurements and oxidative stress shoots were removed from roots with scalpel and their length and weight were determined. tolerance indexes were calculated as average value http://hyperaccumulators.smi.uq.edu.au/ nova biotechnol chim (2020) 19(1): 70-79 72 of the stressed plant character to average value of the non-stressed plant character and expressed in %. the content of photosynthetic pigments was determined according to lichtenthaler and wellburn (1983). the level of occurring oxidative stress was measured through the level of malondialdehyde (mad) according to karabal et al. (2003). analyses of chitinase enzymes crude protein extracts were isolated from 0.2 g flax shoots. the material was homogenized in presence of liquid nitrogen using a laboratory tissue lyzer (retsch mm 400). to the powderized tissue was added 320 μl of cold extraction buffer consisting of 0.1 mol.l-1 sodium acetate (ph 5.2) with 100 mm fenylmethylsulfonylfluorid (sigma). the samples were vortexed 3-times for 15 s, while during brakes (min. 15 s) keeping on ice. the samples were gradually centrifuged twice at 12,000 rpm at 4 °c for 15 min. the crude extracts were measured for protein content using standard bradford method, aliquoted and stored at -80 °c. total chitinase activity in samples was measured fluorimetrically (fluoroskan ii microtiterplate reader, titertek, finland) in 20 μl protein extracts mixed with 30 μl of 300 μm 4-methylumbelliferyl-β-d-n,n´,n´´-triacetylchitotrioside in 0.1 m sodium citrate buffer (ph 3.0). after incubation at 37 °c for 1 h the reactions were stopped with 150 μl of 0.2 m na2co3. the measurement was performed using excitation and emission filters 355 nm/450 nm and the enzyme activity was expressed as picomoles of methylumbelliferone generated per µg of soluble protein per hour. chitinase isoforms were detected in protein aliquots (20 μg). samples were separated in 12.5 % (w/v) sds-containing polyacrylamide slab gels with 0.01 % glycol chitin as enzyme substrate (gálusová et al. 2015) at a constant voltage of 120 v at 8 °c for 2 h. the samples were not heat-treated prior to loading. the separated proteins in gels were re-naturated in 1 % (v/v) triton x-100 in 50 mm sodium acetate buffer (ph 5.0) overnight. the gels were washed in sodium acetate buffer without detergent for 1 h and stained with 0.01 % (w/v) fluorescent brightener 28 (pan et al. 1991) and uv-illumination. enzyme profiles were digitally photographed (uvp biodoc-it system) and quantified using scion image software (http://www.scioncorp.com) (gálusová et al. 2015). gammaspectrometry cadmium uptake and content in plant tissue was measured by gammaspectrometry using a radioisotope 109cd. the isotope was used to spike the cd solution in liquid samples of culture medium (bq.ml-1) and to measure the assimilated metal in washed and dried plant biomass (bq.g-1 tissue). the measurements were implemented using a scintillation gammaspectrometer type 76bp76/3 (scionix, the netherland) with well type nai(tl) crystal. calibration of the instrument and calculation of radioactivity were realized using a library of analyzed radionuclides and the program scintivision-32 (ortec, usa). results and discussion several fibre flax and linseed varieties have been shown to tolerate cd in soil containing up to 1 g of metal per kg of soil, while exerting no visible symptoms of toxicity or with minimal growth reduction or developmental aberrations in the stems (bjelková et al. 2011). the plantlets of assayed flax varieties were grown in hydroponic conditions and exposed to short-term cd stress. we observed no visible symptoms of metal toxicity such as necrosis or chlorosis. the tolerance indexes expressed based of fw data ranged between 63 – 85 % (fig. 1a), similarly to previous studies with adult plants of some other flax varieties (shi and cai 2009; bjelková et al. 2011; douchiche et al. 2012). in contrast, doses of 10, 20 and 40 mg cdcl2 per kg of soil were toxic but sublethal and inhibited flax growth (hosman et al. 2017). the reduction in plant growth in the presence of cadmium is attributed to reduce water absorption due to its osmotic effect, lack of nutrition as a result of ion imbalance, and to decline in many metabolic activities due to its toxicity (singh et al. 2016). most tolerant plants to cd appeared here the variety krasnoder, the growth of which was http://www.scioncorp.com/ nova biotechnol chim (2020) 19(1): 70-79 73 fig. 1. effect of cadmium on growth parameters of the tested flax varieties by means of fresh weight (a) and length of shoots (b). values above columns indicate tolerance indexes. data are given for control (white columns) and cd-treated plants (black columns) and represent means ± se (n = 3). asterisks indicate significant effect of the metal at p ≤ 0.05 (*), p ≤ 0.01 (**) or p ≤ 0.001 (***). unaffected also in regard of shoot length (fig. 1b). on the contrary, the variety escalina appeared the least tolerant to cd (fig. 1a), though the shoot length dropped most obviously for the variety jitka (fig. 1b). genotype-dependent sensitivity to cd has been described for flax (bjelková et al. 2011; saastamoinen et al. 2013; praczyk et al. 2015) but also other species (socha et al. 2015). fig. 2. effect of cadmium on content of malondialdehyde as indicator of oxidative stress in the tested flax varieties. data are given for control (white columns) and cd-treated plants (black columns) and represent means ± se (n = 3). the shoots were analyzed for content of mad as measure of ongoing oxidative stress. basic levels significantly differed among the varieties (fig. 2). it has been speculated that higher basic levels of peroxidation presume an a priori more efficient detoxification mechanism against metal stress and/or might lead to a ‘‘standby’’ state of defense for a faster and/or stronger response in the more tolerant cultivars (mészáros et al. 2013). the data, however, revealed minor alterations in mad levels in either of the flax cultivars, indicating to efficient antioxidative system and/or tolerance to cd. noteworthy, mad content and rate of membrane peroxidation have been reported to depend on cd concentration in the flax var. vicking (belkadhi et al. 2010). photosynthetic pigment contents the contents of pigments in the (control) leaves of flax varieties varied (fig. 3). according to mitchell and bakker (2014), the emergence of such variability is driven by plasticity and plant ontogenesis rather than adaptation to the environment. the mentioned authors confirmed the intraspecific variability of the content of photosynthetic pigments in hypochaeris radicata, as well as its impact on the resistance to abiotic stress such as drought or light. the importance of intraspecific variability in plant functional properties in structuring communities and managing ecosystem processes is becoming increasingly important, but the mechanisms governing the ongoing changes are still unknown. the most significant negative impact of cd on chlorophyll a content was in the variety krasnoder (fig. 3a); it points to damage to photosystems i and ii. in contrast, the stressed variants in the belinka and escalina varieties contained more chlorophyll a than the corresponding controls (p < 0.05). increased chlorophyll levels have previously been observed as a consequence of mild drought (avramova et al. 2012), and by some others as a consequence nova biotechnol chim (2020) 19(1): 70-79 74 of short drought (parida et al. 2007; nikolaeva et al. 2010). in most cases, however, chlorophyll loss occurs in the leaves when exposed to heavy metals (dobroviczká et al. 2013; maglovski et al. 2017). the effect of cd on the chlorophyll b content was recorded only in the jitka variety (fig. 3b). this type of pigment is part of the psii light collection antenna system. unchanged content of chl b under metal stress was also observed in other plant species like soybean (dobroviczká et al. 2013) and wheat (maglovski et al. 2017). the ratio of chlorophylls a/b, which is an indicator of the functionality and light adaptation of the photosynthetic apparatus, was influenced by cadmium in the variety krasnoder (fig. 3c). such a decrease results from faster degradation of chl a than chl b (kummerová et al. 2010) due to disorganization of chloroplast membranes (feng et al. 2010) and changes in their sizes and densities (sun et al. 2012). unchanged values of chl a/b in the other flax varieties (fig. 3c) indicate good plant condition and relative tolerance to cadmium. the content of carotenoids in individual varieties of flax was also variable, while the highest content was detected in the variety jitka (fig. 3d). this parameter is genetically determined and may indicate a better defense against stress (chen et al. 2016b); this assumption, however, is not supported here by growth parameters. under cd stress, we recorded an increased content of car in the varieties belinka (p < 0.01), marina (p < 0.05) and escalina (p < 0.05), in contrast to fig. 3. effect of cadmium on photosynthetic parameters of the tested flax varieties. indicated are data for content of chlorophyll a (a), chlorophyll b (b), their ratio chla/chlb (c), content of carotenoids (d) and ratio of chla/chlb to carotenids (e). data are given for control (white columns) and cd-treated plants (black columns) and represent means ± se (n = 3). asterisks indicate significant effect of the metal at p ≤ .05 (*), p ≤ 0.01 (**) or p ≤ 0.001 (***). nova biotechnol chim (2020) 19(1): 70-79 75 fig. 4. effect of cadmium on chitinase enzymes in the tested flax varieties. total chitinase activity (cht) was measured fluorimetrically (a). the activity of isoforms separated in polyacrylamide gels (b) was quantified. integrated densities of bands were obtained for the isoform of 35 kda (c) and 26 kda (d). data are given for control (white columns) and cd-treated plants (black columns) and represent means ± se (n = 3). asterisks indicate significant effect of the metal at p ≤ 0.05 (*), p ≤ 0.01 (**) or p ≤ 0.001 (***). the variety jitka in which the content of car decreased (fig. 4d). increased car levels have also been reported by chaneva et al. (2010) and may reflect the involvement of compensation mechanisms with a likely short effect in order to obtain resources. nevertheless, a decrease in car content in presence of heavy metals has been reported more frequently (gálusová et al. 2015). the carotenoid content was not affected in the individual varieties to the same extent as the chlorophyll contents, in agreement with the observations of stoeva et al. (2005). the weight ratio of chlorophylls a and b to total carotenoids (a + b)/(x + c), as an indicator of the "greenness" of the plant, was deceased by cd in the krasnoder and marina varieties (both at p < 0.05). conversely, increasing the ratio in the jitka variety (p < 0.01) is likely a form of a compensatory mechanism for obtaining resources under stress (fig. 3e). though changes in pigment concentrations and their ratios are a good indicator of stress effects on plants (maglovski et al. 2019), their contents are affected by a number of internal factors as well, including genotype and leaf type (dobroviczká et al. 2013), age and type of leaf tissue (hédiji et al. 2010). the differences caused by cadmium are attributed to variability in the accumulation of glutathione and phytochelatins, which chelate and/or detoxify the metal (gaudet et al. 2011). activities of defense enzymes – chitinases the total activity of chitinase enzymes was different for individual varieties of flax (fig. 4a), as already described for peas (metwally et al. 2005) or soybeans (socha et al. 2015). however, we did not notice any significant changes in the presence of cd in any of the varieties. since the total activity of chitinases is the result of the action of several enzyme isoforms, we separated the flax protein extracts in polyacrylamide gels. the chitinase enzyme profile in flax has not been studied in more detail in relation to the response to heavy metals. we detected the presence of two protein fractions nova biotechnol chim (2020) 19(1): 70-79 76 fig. 5. content of cadmium in the roots of the tested flax varieties measured by gammaspectrometry using spiked 109cd. data represent means ± se (n = 3). with chitinolytic activity of ~ 26 and 35 kda, in all tested flax varieties (fig. 4). in addition to them, we also detected an isoform of chitinases of ~ 65 kda in the belinka and krasnoder varieties (fig. 4b). by quantifying the intensity of the activities of the individual fractions, we found that all isoforms were significantly responsive to cadmium ions, but not in the same way or to the same extent. the ~ 35 kda chitinase activity was inhibited in the jitka variety by cd, while in all other varieties it was induced (fig. 4c). this induction was most pronounced (almost threefold) in the krasnoder variety. the trend of the activities of the isoform ~ 26 kda was the same (fig. 4d), but the most significant induction was observed in the variety belinka (almost double). the ~ 65 kda isoform was detectable only in control plants of the belinka and krasnoder varieties, but due to cadmium its activity fell below the level of detectability (fig. 4d). the obtained results confirmed the high responsiveness of flax chitinases to the given concentration of cadmium, but we did not notice any change in their profiles (békésiová et al. 2008), nor differences in profiles between varieties (mészáros et al. 2013). dynamics of uptake and accumulation of cd we observed differences in the accumulation of cd in the dry matter of young plants of different varieties of flax. the highest amounts were determined after 10 days of cultivation in medium with 109cd in the variety jitka and escalina, on the contrary the least in the variety belinka (fig. 5). previously, variable cd accumulation in four soybean cultivars has been reported (bardáčová et al. 2017) and shown to coincide with tolerance to metal stress, nevertheless, metal accumulation and tolerance should be considered as independent traits (goolsby and mason 2015). the high cd intake of these varieties is relatively fast during the first three days. on the contrary, the low metal accumulation of the belinka variety is associated with the relatively slowest intake fig. 6. uptake of cadmium (%) by roots of the tested flax varieties during first three days of experiment. data were measured by gammaspectrometry using spiked 109cd and are presented as average values (n = 3). nova biotechnol chim (2020) 19(1): 70-79 77 (fig. 6). linear cd depletion rate from media has been described previously especially at low concentrations of metals and likely refers to combination of both apoplastic cd flow towards the xylem through channels or transporters and symplastic uptake by roots (hu et al. 2013). however, metal uptake by roots may show specific kinetics and rates depending on the concentration of metal in the environment, soil characteristics (e.g. presence of various adsorbents or chelating agents), but also on plant defense and sequestration strategies (pipíška and remenárová 2014; bardáčová et al. 2017). plant strategies in metal uptake and allocation are key to their survival. high metal intake in the jitka variety correlated with suppressed growth (fig. 1) and reduced content of individual pigments (fig. 3). these results are consistent with a good transfer of cd to the aboveground parts of this variety (bjelková et al. 2011). at the same time, both detected isoforms of chitinases were inhibited in the jitka variety (fig. 4). this may indicate that metabolic processes (including enzyme synthesis) have been adversely affected by metal (békésiová et al. 2008). therefore, we evaluate the jitka variety as the most sensitive to cd among the tested flax varieties. in contrast, the belinka and krasnoder varieties accumulated relatively low amounts of cd, had less inhibited growth or pigment content (fig. 1 and 3, respectively) and were characterized by a strong induction of both chitinase enzymes (fig. 4). interestingly, complete inhibition of the 65 kda isoform was also detected in these varieties. this suggests the specificity of regulation, as well as the different roles of individual chitinases in the defense of flax against cadmium toxicity. the belinka and krasnoder varieties are the most tolerant within the selected set of flax varieties. in this study, they accumulated even less cd than the escalina variety, which in a previous study (bjelková et al. 2011) accumulated up to 4 to 5-fold lower amount cd (per ha per season as related to particular soil cd concentration) than the other 10 varieties, including the var. jitka. the differences detected among varieties were relatively stronger compared to studies performed on field trials in naturally contaminated soils which rarely exceeded 0.5 – 1 mg cd kg-1 soil (grant et al. 2000). for example guo et al. (2020) examined 18 different flax and linseed cultivars in a field trial for cd concentrations in plant tissues but they recognized very small differences. though it is assumed that genotype effects turn more obvious with high concentration of cd in soil, they still were lower than expected also in soil containing more than 10 mg cd kg-1 (bjelková et al., 2011). this suggests higher sensitivity of young plants. at the same time, bioavailability of the contaminating metal depends on many soil factors which likely restrict the uptake kinetics, and provides time to activate defense mechanisms to cope stress despite of long exposure and higher total uptake. in addition, seasonal changes have to be considered as well since all these factors are believed to over the effect of genotype itself (marquard et al. 1990; grant et al. 2000; guo et al. 2020). conclusions of the flax varieties examined we recognized the variety jitka as best cd accumulator, confirming the results of previous studies. in addition to impact of cd on pigment content, activity of chitinase isoforms appears to coincide with plant tolerance/sensitivity to cd. though genotype differences tend to dismiss under field trial, laboratory experiments still can provide a good mean for pre-selection of varieties promising for phytoremediation programs. on the other hand, phloem transport is the basis for cd redistribution in shoots and phloem is the productive part of flax processed into linen textiles, therefore the safety of raw material reutilization has to be considered. from this point of view the variety belinka appeared as the most appropriate. conflict of interest the authors declare that they have no conflict of interest. acknowledgements this work was supported by the slovak research and development agency under the contract no. apvv-150051 and by the operational programme research and development for the project implementation nova biotechnol chim (2020) 19(1): 70-79 78 of the research of plant genetic 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2, , abu tholib aman3, lutfan lazuardi4, tri wibawa3 1doctoral program, faculty of medicine, public health and nursing, universitas gadjah mada, jl. farmako sekip utara, yogyakarta 55281, indonesia 2department microbiology, school of medicine and health sciences, atma jaya catholic university of indonesia, jl. pluit raya no. 2, jakarta 14440, indonesia 3department microbiology, faculty of medicine, public health and nursing, universitas gadjah mada, jl. farmako sekip utara, yogyakarta 55281, indonesia 4department of health policy and management, faculty of medicine, public health and nursing, universitas gadjah mada, jl. farmako sekip utara, yogyakarta 55281, indonesia  corresponding author: yulia.tnarwati@gmail.com article info article history: received: 1st april 2021 accepted: 8th november 2021 keywords: blactx-m blashv blatem esbl escherichia coli abstract the use of antibiotics in veterinary and human treatment can cause the development of antibiotic resistance in bacteria for β-lactam antibiotics. worldwide, this resistance has become a growing concern in public health. limited data are currently available regarding extended spectrum β-lactamase (esbl) escherichia coli in indonesia. the current study determined the prevalence and characteristics of esbl genes of e. coli in retail chicken meat and humans in indonesia. two hundred eighty retail chicken meat were randomly collected from various modern and traditional markets in jakarta (70 retail sourced chicken meat from modern markets and 210 from traditional markets). the prevalence of e. coli from the chicken meat sold at traditional markets was 97.14%, which was significantly higher than those of the modern markets with 78.57 % (p < 0.05). the prevalence of esbl-producing e. coli isolated from chicken meat sold at traditional market was 40.47% and the modern market was 35.71 % and the prevalence of esbl-producing e. coli isolated from chicken meat was 38.09 %, which is significantly higher than those of the clinical sample (with average 5.57 %). the most predominant gene is blatem in 54.54 % as a single gene or mixed with other genes followed by blactx-m in 44.31 % and blashv gene was only found in three isolates in 1.13 %. this study found that isolates from both the broiler chicken meat and clinical samples were having the same molecular characteristics. it is speculated that there is a relationship between them. however, this needs to be substantiated further. introduction one of the health problems occurring all over the world is the bacterial antibiotic resistance phenomenon, which causes a serious decline in the quality of health service. according to a recent study, thousands of patients die each year from bacterial infections resistant to more than one type of antibiotic (li and webster 2018). veterinary and human treatments of infections often use antibiotics mailto:yulia.tnatwati@gmail.com nova biotechnol chim (2022) 21(1): e935 2 not only for control but also prevention of infectious diseases. the major cause of gramnegative bacteria resistance to β-lactam antibiotics is the production of β-lactamases (eiamphungporn et al. 2018). gram-negative bacteria demonstrate increased resistance against newly developed β-lactam antibiotics, which are the most commonly used treatment for bacterial infections. these variant enzymes are known as extended-spectrum β-lactamases (esbls) (shaik et al. 2015). bacteria can resist penicillin, cephalosporins (first. second, and third generations) as well as aztreonam due to esbl enzymes through hydrolysis of these antibiotics (chishimba et al. 2015). in the past, infections with esbl-producing bacteria tended to be exclusively hospitalassociated, also known as nosocomial, but today such infections are commonly occurring among community-dwelling patients without a history of hospitalization or antimicrobial use (eiamphungporn et al. 2018). e. coli is a commensal microbiota present in human and animal intestines and is also known as one of the most important pathogens (conway and cohen 2015). some e. coli pathotypes cause intestinal and extraintestinal diseases such as diarrhea, urinary tract infections, septicemia, and neonatal meningitis (safitri et al. 2017; rojas-lopez et al. 2018). recent research concerning esblproducing e. coli in human subjects and animals has demonstrated a significant threat to public health. those studies show that many foods can be sources of contamination from esbl-producing e. coli and represent a growing global health problem (le et al. 2015a, 2015b; bhoomika et al. 2016; hoang et al. 2017; falgenhauer et al. 2019). public health is directly affected by the presence of esbl-producing bacteria in poultry-based food because e. coli can contribute to the spreading of resistance genes in animals and humans. such bacteria are known to be able to exchange resistant genetic material among strains (da costa et al. 2013). resistant gene transfer generally can occur through three mechanisms: conjugation, transduction, and transformation. horizontal gene transfer among bacterial species is the most common cause of antibiotic resistance. bacteria are becoming more efficient in adapting to environmental changes using a variety of mechanisms, compared to random mutations. the most commonly emerging transfer routes for gene transfer between bacterial cells are through transformation and conjugation. the antibioticresistant gene transfer from commensal bacteria to pathogenic bacteria relies on the transfer mechanisms of availability, selective pressure, and the density of the donor, nutrients, and the recipient bacteria (marshall et al. 2009). indonesia is currently undergoing significant development and has become the world's fourth most populous country. unfortunately, limited data are currently available on the prevalence and molecular characteristics of esbl-producing e. coli in indonesia. in the present study, we investigated the prevalence and molecular characteristics of esbl-producing e. coli isolated from chicken meat and clinical isolates from three hospitals in jakarta, indonesia. experimental identification and bacterial isolation between october 2018 and march 2019, a total of 280 chicken carcasses were randomly collected from various modern and traditional markets in jakarta (70 retail-sourced chicken carcasses samples from modern markets and 210 from traditional markets). a total of 280 chicken meat samples were collected. among these, 70 samples were collected from 70 modern markets sales points (supermarkets) while another 210 samples were collected from 53 traditional markets (at each traditional market, we collected 4 samples each from a different seller). because in traditional markets in indonesia there are many sellers of chicken meat in one market, so we chose 4 samples of chicken meat each from different sellers in one market. so, 52 × 4 = 208, and only 2 samples were taken from the last market. all samples were collected and placed into sterile sampling bags, then immediately taken to the laboratory in a thermobox container maintained at 4 °c. 25 g chicken meat samples were homogenized in buffered peptone water (bpw) (product code: 100908, merck, germany) from each sample then 0.1 ml sample aliquots were swabbed on nova biotechnol chim (2022) 21(1): e935 2 brilliancetm e. coli/coliform selective agar (product code: cm1046, oxoidtm, england) then incubated 24 h at 37 ºc. colonies with a typical red color were collected from each plate. identification of e. coli was done using microbacttm gramnegative identification system 12a (product code: mb1073, oxoidtm, england). human clinical e coli isolates were obtained from a hospital clinical microbiology laboratory in three hospitals (royal taruma hospital, atmajaya hospital, and husada hospital) in the jakarta area. the clinical specimens comprised sputum, blood, pus, urine, and feces. antimicrobial susceptibility testing and phenotypic detection of esbl-producing e. coli the disc diffusion test by kirby-bauer’s method was performed using muller-hinton agar plates (product code: 103872, merck, germany) according to the clinical and laboratory standards institute standards (clsi) (clinical laboratory standard institute 2017). antibiotics used for this study were: tobramycin (tob), gentamicin (gen), cefazolin (kz), cefuroxime (cxm), aztreonam (atm), amoxicillin (aml), levofloxacin (lev), ciprofloxacin (cip), trimethoprimsulfamethoxazole (sxt), amoxicillin-clavulanate (amc), cefoxitin (fox), fosfomycin (fos), meropenem (mem), and amikacin (amk) (oxoidtm, england). suspected esbl-producing isolates were tested for esbl-production by double-disc synergy test (ddst), 5 antibiotics were used for ddst namely ceftriaxone (30 µg), ceftazidime (caz) (30 µg), and cefotaxime (cxm) (30 µg), amoxicillin-clavulanic acid (20/10 µg), aztreonam (atm) (30 µg). the amoxicillin clavulanic acid disc was placed at the center and these discs were placed at a distance of 30 mm apart (center to center). the enhancement of the zone of inhibition of any one of the three discs toward the disc containing clavulanic acid at 37 °c after 24 h incubation was indicative of a potential esbl-positive organism (drieux et al. 2008). genetic characterization of esbls producing isolates dna extraction was performed using the boiling method as follows: isolated bacteria were cultured in luria bertani broth (product code: m1245, himedia®, india) overnight, then 2 ml of the culture were centrifuged for 10 min at 8,000 rpm. the pellet was used after the supernatant was discarded and mixed with 200 µl of sterile distilled water. samples were then boiled in a dry bath for ten minutes and immediately chilled on ice for 5 min. the samples were then centrifuged at 6,000 rpm, and the supernatant which contained the nucleic acid material was pipetted off and stored for genetic testing (kaur and aggarwal 2013). multiplex pcr analysis was performed for the simultaneous detection of 𝑏𝑙𝑎ctx-m, 𝑏𝑙𝑎tem, and 𝑏𝑙𝑎shv genes that are responsible for esbl activity, using primers as described in table 1 (lim et al. 2009; le et al. 2015b). multiplex pcr plus kit (product code: 206152, qiagen®, usa) was used to detect esbl genes, using primers as described in table 1. pcr amplification products were visualized by gel electrophoresis in a 2 % agarose gel for 30 min. table 1. multiplex pcr (le et al. 2015b; lim et al. 2009). primers sequences 5’ 3’ pcr conditions product [bp] tem-410f ggtcgccgcatacactattctc initial denaturation at 95 ºc for 5 min, followed by 35 cycles of denaturation at 95 ºc for 30 s, annealing at 90 °c for 90 s, extension at 72 ºc for 90 s, and a final cycle of amplification at 68 °c for 10 min 372 tem-781r tttatccgcctccatccagtc shvf-287f ccagcaggatctggtggactac 231 shvr-517r ccgggaagcgcctcat ctx-m f atgtgcagyaccagtaargt 593 ctx-m r tgggtraartargtsaccaga 3 nova biotechnol chim (2022) 21(1): e935 2 statistical analysis the statistical analysis was performed using the chi-square test with the level of significance, p = 0.05. results a total of 259 e. coli isolates were collected from 280 chicken meat sold in traditional and modern markets in jakarta. the prevalence of e. coli from the chicken meats sold at traditional markets was 97.14 % (table 2) which was significantly higher than those of the modern markets with 78.57 % (p < 0.05). phenotypic detection showed that the average prevalence of esbl-producing e. coli isolated from chicken meat was 35.71 % from the modern markets and 40.47 % from the traditional markets. table 2. isolated bacteria e. coli and esbl-producing e. coli from chicken meat and clinical samples. % (numbers) e. coli esbl-producing e. coli broiler chicken meat traditional market 97.14 % (204/210) 40.47 % (85/210) modern market 78.57 % (55/70) 35.71 % (25/70) clinical isolates rsa rsrt rsh 17.90 % (43/239) 13 % (39/300) 17.4 % (101/579) 3.35 % (8/239) 7.33 % (22/300) 6.05 % (35/579) rsa – rumah sakit atmajaya/atmajaya hospital; rsrt – rumah sakit royal taruma/royal taruma hospital; rsh – rumah sakit husada/husada hospital. table 3. antibiotic susceptibility profiles of esbl-producing e. coli isolates. % (numbers) traditional (n = 85) modern (n = 25) clinical (n = 65) aml 1.18 % (1) 8.00 % (2) 3.08 % (2) amc 65.88 % (56) 68.0 % (17) 58.46 % (38) atm 17.65 % (15) 44.00 % (11) 6.16 % (4) caz 45.88 % (39) 28.00 % (28) 1.54 % (1) cxm 4.71 % (4) 12.00 % (3) 1.54 % (1) kz 2.35 % (2) 8.00 % (2) 0 % (0) fox 88.24 % (75) 72.00 % (18) 95.38 % (62) lev 48.24 % (41) 48.00 % (12) 3.08 % (2) cip 3.53 % (3) 0 % (0) 0 % (0) sxt 16.47 % (14) 40.00 % (10) 33.85 % (22) amk 100 % (85) 100 % (25) 100 % (65) tob 54.94 % (45) 48.00 % (12) 27.69 % (18) gen 24.71 % (21) 28.00 % (7) 50.77 % (33) fos 9412 % (80) 92.00 % (23) 96.92 % (63) mem 100 % (85) 100 % (25) 100 % (65) aml – amoxicillin, amc – amoxicillin-clavulanate, atm – aztreonam, caz – ceftazidime, cxm – cefuroxime, kz – cefazolin, fox – cefoxitin, lev – levofloxacin, cip – ciprofloxacin, sxt – trimethoprim-sulfamethoxazole, amk – amikacin, tob – tobramycin, gen – gentamicin, fos – fosfomycin, mem – meropenem. 4 nova biotechnol chim (2022) 21(1): e935 5 the antibiotic resistance profiles of all 175 esblproducing e. coli isolates are shown in table 3. low percentages of susceptibility to aml (1.18 – 8 %), kz (0 – 8 %), cip (0 – 3.53 %) and high rate of susceptibility to fos (92 – 96 %). most of the clinical isolates showed a lower susceptibility percentage to almost all the tested antibiotics than those isolates from chicken meat, except for gen, which was significantly higher than those isolates from the chicken meat (p < 0.05). most notably, all the isolates exhibited susceptibility to mem and amk. all of the 175 e. coli isolates (110 isolates from the chicken meat and 65 isolates from the clinical samples) which were phenotypically confirmed as esbl were genetically characterized using multiplex polymerase chain reaction (pcr) (fig. 1). the results showed that the most predominant gene was blatem (54.54 %) as a single gene or mixed with other genes, followed by blactx-m (44.31 %) while the blashv gene which was only found in three isolates (1.13 %) (table 4). in five of the isolates, the process of multiplex pcr did not detect any genes responsible for the esbl phenotype. since in this study only three genes were investigated, the results suggested that those five isolates harbored other genes responsible for esbl production. fig. 1. the electrophoresis result of pcr products indicates genes blactx-m with a band of 593 bp, blatem 372 bp, and blashv 231 bp. table 4. distribution of esbl types among e. coli isolates. number [%] traditional modern clinical ctx-m 13 (16 %) 3 (12 %) 8 (13 %) tem 25 (31%) 12 (48 %) 14 (22 %) shv 0 (0 %) 1 (4 %) 1 (2 %) ctx-m/tem 44 (54 %) 8 (32 %) 40 (63 %) ctx-m/tem/shv 0 (0 %) 1 (4 %) 0 (0 %) discussion the worldwide prevalence of esbl-producing e. coli is rapidly increasing both in hospitals and in the communities. a possible connection between esbl-producing bacteria in retail meat products and humans has been suggested (leverstein-van hall et al. 2011; odwar et al. 2014). this study successfully isolated e. coli in almost all chicken meat bought from traditional markets (97.1 %) and in a significant proportion from modern markets (78.6 %). in another study, the level of e. coli contamination in chicken meat had a comparable result with 78 % (odwar et al. 2014). other result from traditional markets was also high as reported in greece is 91 % (dakic et al. 2014). in indonesia, e coli found in chicken meat varied. e. coli was reported in 62 % of samples of chicken meat from traditional markets in surabaya (safitri et al. 2017), while 90.03 % of e. coli were found in chicken meat also from traditional markets in nova biotechnol chim (2022) 21(1): e935 5 surabaya (wardhana et al. 2020). e. coli was found in the clinical samples in this study in 13 – 17 % of samples, while 12 % of clinical samples from a hospital in manado were reported positive for e. coli (palit et al. 2018). the significant difference in the results of e. coli percentage isolated from chicken meat bought from the traditional versus the modern markets can be caused by several conditions. generally, modern markets tend to be more hygienic, and the broiler chicken meat sold in modern markets is usually wrapped in plastics. meanwhile, in traditional markets, the chicken meat is left exposed to the air where it is easier to become contaminated through contact with the buyers or the sellers themselves. personal hygiene of the sellers in the modern market tends to differ from traditional markets, where the modern market seller usually uses a uniform, gloves, and apron. stall conditions in traditional markets are generally not wellmaintained, where the sellers often use a big open table shared with other chicken meat sellers and sometimes with sellers of other products. the presence of chickens that are still alive, where the chicken meat is sold is common in traditional markets. market sanitation in traditional markets tends to be poor due to the accumulation of garbage in many places, lack of adequate wash facility, and usage of the wooden cutting board. frequently, the meat products are placed at room temperature due to not having a refrigerator to store the meat (mukti et al. 2013). when measuring the sensitivity of the isolates, it was especially notable that all the isolates exhibited susceptibility to mem and amk. the same result was found in nigeria, indicating that the sensitivity of esbl-producing e. coli to amk was 100 % (alo et al. 2012). in the study by uyanik et al. (2021), the authors reported that all esbl-producing e. coli isolates were resistant to amk and mem, which is parallel with the outcome of the current study (uyanik et al. 2021). it was further reported that the sensitivity of esbl-producing e. coli isolates to mem from urinary infection samples was 100 % (soltani et al. 2014). in this study, nearly half (40.47 %) of the e. coli isolated from broiler chicken meat sold in traditional markets were positive for esblproducing e. coli. the research conducted in belgia (smet et al. 2008) had the same result (45 %). also, more than 40 % of samples were found positive for esbl-producing e. coli isolated from chicken meat (le et al. 2015a). in this study, phenotypic detection of esbl-producing e. coli from a clinical sample of the three selected hospitals varied between 3.35 – 7.33 %. genetic characteristics analysis showed that the most predominant gene was blatem as a single gene or mixed with other genes and the blashv gene was found in only three isolates. a study in erbil, iraq found that the blatem gene was the most frequent in esbl-producing e. coli isolated from thalassemia patients followed by the blactx-m gene (pishtiwan and khadija 2019). other studies in india, iraq, and iran reported blatem as predominantly found in isolates of esblproducing e. coli from patients with urinary tract infections (yazdi et al. 2012; jena et al. 2017; michael and saadi 2019). research in korea, had similar results to our study, wherein the blatem gene was found as the most common esbl gene in esbl-producing e. coli isolated from chicken meat (lim et al. 2015). chicken as one of the common food-producing animals has the potential as a reservoir for esblproducing bacteria. the esbl-producing bacteria can be transmitted from animals to humans, potentially causing the spread of zoonotic diseases. esbl-producing bacterial infections from consuming food-producing animals can lead to limited options in the treatment of patients. such conditions can cause an increase in the occurrence of the diseases, increase the cost of treatment, and extend the treatment period and possibly cause death (khosbayar et al. 2014). research in the netherlands suggested a relationship between contamination of chicken meat with drug-resistant bacteria and appearance of esbl genes in humans, isolates from both the chicken meat and human samples showed that most e. coli harboring blactxm-1 or blatem52 and belong to clusters containing strains from both sources (overdevest et al. 2011). for these reasons, the poultry industry has been considered a potential reservoir for transmission of esbl-producing e. coli from poultry to humans are most likely through the food chain (leversteinvan hall et al. 2011). this study found that isolates from both the broiler chicken meat and clinical samples were having the same molecular 6 nova biotechnol chim (2022) 21(1): e935 5 characteristics. accordingly, the most frequent esbl gene was blatem followed by blactx-m and blashv. it is speculated that there is a relationship between them. however, this needs to be substantiated further. for instance, the genetic relations between bacteria are investigated by methods such as pulsed-field gel electrophoresis (pfge) and multi-locus sequence typing (mlst). in our study, we investigated the presence of blatem, blactx-m, and blashv genes. however, the subgroups of these genes were not investigated, which is a limitation of this study. detection of these genes alone may not indicate an association between hospital-acquired and chicken-derived isolates. therefore, further investigation is needed to strengthen the preliminary findings of this study. conclusion the result of this study affirmed the presence of esbl-producing e. coli in chicken meats sold in traditional and modern markets in jakarta. this study found that isolates from both the broiler chicken meat and clinical samples were having the same molecular characteristics, the most predominant esbl gene was blatem followed by blactx-m and blashv. it is speculated that there is a relationship between them. however, this needs to be substantiated further. these findings emphasize the need for continuous surveillance to detect esbl-producing e coli from food and clinical samples, strict guidelines for the antibiotics used in veterinary and human medicines for treatment, control, and prevention of infectious diseases. further research is needed to determine the relationship between contamination of chicken meat with drug-resistant bacteria and the appearance of esbl genes in human isolates. acknowledgments we sincerely thank the head of atma jaya hospital, royal taruma hospital, husada hospital, and the head of clinical laboratory, atma jaya hospital, royal taruma hospital, and husada hospital, jakarta, indonesia, and dr. enty tjoa, department of microbiology, school of medicine and health sciences, atma jaya catholic university of indonesia, jakarta, indonesia for 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wardhana dk, effendi mh, harijani n, ooi hk (2020) detection of extended-spectrum-beta-lactamase (esbl) producing escherichia coli in meat chicken from traditional market in surabaya, east java, indonesia. indian j. public. health. res. dev. 11: 1353-1357. yazdi m, nazemi a, mirinargasi m, jafapour m, sharifi (2012) genotypic versus phenotypic methods to detect extended-spectrumβ-lactamase (esbls) in uropathogenic escherichia coli. ann. biol. res. 3: 24542458. 8 nova biotechnol chim (2020) 19(2): 208-215 doi: 10.36547/nbc.v19i2.738  corresponding author: b.bakchiche@lagh-univ.dz nova biotechnologica et chimica fatty acid, mineral content and antioxidant activities of algerian fat bee pollen boulanouar bakchiche1,, hadjira guenane1, bihter sahin2, mehmet öztürk2, mosad a. ghareeb3 and maria da graça miguel 4 1laboratory of process engineering, laghouat university, laghouat 03000, algeria 2department of chemistry, faculty of science mugla sitki kocman university, mugla, turkey 3medicinal chemistry department, theodor bilharz research institute, kornaish el nile, warrak el-hadar, imbaba (p.o. 30), giza 12411, egypt 4universidade do algarve, faculdade de ciências e tecnologia, departamento de química e farmácia, mediterranean institute for agriculture, environment and development, campus de gambelas, faro 8005139, portugal article info article history: received: 27th may 2020 accepted: 4th november 2020 keywords: antioxidants bee pollen dpph gc/ms mineral content abstract bee pollen is known for its nutritional value, and therefore it is used in the treatment of some health disorders as food supplements. herein, the chemical fatty acid profile and mineral contents of two algerian bee pollen collected from different regions were investigated, along with their physicochemical properties such as specific gravity, refractive index, and acid, saponification, and iodine values. in addition, their total phenolic contents (tpc) were also investigated. gas chromatography/mass spectrometry (gc/ms) analysis revealed that the bee pollens mainly contained palmitic (17.0 and 26.5 %), oleic (8.5 and 10.1 %), linoleic (15.7 and 12.6 %) and linolenic acids (27.2 and 26.3 %), respectively. the saponification values were 178.54 and 175.73 mg koh.g-1, while the specific gravity 0.915 and 0.924, the refractive index 1.465 and 1.464, and acid values 22.04, and 10.01 mg koh.g-1 oil. the iodine values were, however, 44.42 and 32.90 mg i2.g -1 fat, respectively. potassium and sodium were the main detected elements in both pollen samples with variable percentages. in the dpph assay, the ic50 was 4.88 and 1.73 mg.ml -1 for samples 1 and 2, respectively, abts and phosphomolybdenum assays supported dpph assay results. it was concluded that the fat part of bee pollen by honeybees (apis mellifera l.) is a promising source of naturally occurring antioxidants and nutrients.  university of ss. cyril and methodius in trnava introduction honeybees (apis mellifera l.) produce bee pollen to feeding their larvae at the early stage. bee pollen, constituting a mixture of pollens from flowers belonging to diverse species, and are agglutinated by nectar and honeybee mouth secretions that contain diverse enzymes (e.g., amylase, catalase) (campos et al. 2008; denisow and denisow-pietryk 2016). although the great chemical variability of bee pollen owing to the heterogeneity of plant species in diverse geographic zones and the inherent climatic diversity where hives are located, it is mainly constituted by proteins, essential amino acids, reducing sugars, lipids, nucleic acids (particularly rna), and crude mailto:b.bakchiche@lagh-univ.dz nova biotechnol chim (2020) 19(2): 208-215 209 fig. 1. map of the sampling area (sample 1: na and sample 2: sa). fibre. this means that the variability found is mostly quantitative (denisow and denisow-pietryk 2016). this composition makes bee pollen high value in human nutrition (denisow and denisowpietryk 2016; abdelnour et al. 2019). besides major compounds, bee pollens include minerals, vitamins, fatty acids, phytosterols, terpenes, phenols and flavonoids, and carotenoid pigments. the bioactivities of bee pollen such as antimicrobial, antioxidant, anti-inflammatory, anticarcinogenic, anti-allergenic, antiatherosclerotic, and hepatoprotective, can be attributed to the presence of those bioactive metabolites (denisow and denisow-pietryk 2016). such properties have led to the utilization of bee pollen as a valuable dietary supplement and with a great potential for being used as medicine (denisow and denisow-pietryk 2016; abdelnour et al. 2019). despite the well-known importance of bee pollen, the algerian bee pollen has a lack of information (rebiai and lanez 2012; 2013 and 2014). the present short communication aimed at contributing to unravel on the mineral and fatty acid composition of the bee pollen collected from two different locations of algeria. in addition, the total phenolic content and antioxidant activity were also reported. experimental bee pollen and reagents dried bee pollen was obtained in april 2019 from two different regions of algeria. na is from north algeria (blida) in which olea europeae, eucalyptus sp. were present along with pimpinella anisum, trifolium repens, daucus carota, rubus sp., acacia sp. and eryngium sp.; and sa from south algeria (laghouat) (fig. 1), in which pergamum hermala, trifolium sp., echium, acacia sp., and ziziphus sp. could be found, according to the beekeepers' information. all solvents, standards and reagents used in this study were of high analytical grade and purchased from sigma-aldrich (st. louis, mo, usa). fat extraction using the soxhlet method one hundred gram of pollen was ground in a mortar and placed in a cartridge, which was introduced into the soxhlet extractor using hexane (350 ml) as an extraction solvent. the operation consisted of several cycles until the complete extraction of the fat (2 h). then, the solvent of the extract was evaporated using a rotary vacuum evaporator for recovering the fat extract (converti et al. 2009). preparation of extracts each bee pollen sample (10 g) was dissolved in 50 ml of hexane and extracted three times with 100 ml of 60 % aqueous methanol at room temperature (2 h). the mixture was centrifuged at 3,000 rpm for five min. the lower phase was collected and used to analyze total phenolic content. determination of physicochemical properties specific gravity and refractive index were determined at room temperature (25 °c) using a densitometer and a refractometer, respectively. acid, saponification, and iodine values were determined according to standard procedure (aocs 2003). preparation of methyl esters of fatty acids the fat extract (25.0±0.1 mg) was weighed in a 25 ml volumetric flask and 1.5 ml of 0.5 m methanolic sodium hydroxide was added and heated in a water bath, at 50 °c, for five min. nova biotechnol chim (2020) 19(2): 208-215 210 then, 1.5 ml of bf3 (boron trifluoride)-methanol was added, and the mixture was heated for 5 min, at 80 °c. after cooled, the flask was completed with a saturated solution of sodium chloride. then the mixture was transferred to a separator funnel, 5 ml of hexane was added, the funnel was shaken and then left to stand for layer separation. the upper hexane layer was submitted to evaporation using a rotary evaporator. before analysis with gc/ms, the mixture was diluted with hexane (1 : 25; v/v) (öztürk et al. 2014). analysis of methyl esters of fatty acids by gas chromatography/mass spectrometry (gc/ms) gc/ms analyses of fatty acids were performed using a varian saturn 2100t equipped with an electron ionizer, and an ion trap analyzer and db-1 ms fused silica nonpolar capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm). the carrier gas was helium. the injector and ion source temperatures were 250 °c and 120 °c, respectively. the initial oven temperature was held at 100 °c for 5 min, then increased up to 238 °c with 3 °c.min-1 increments and held at this temperature for 9 min. the injection volume was 0.2 μl with a split ratio of 1 : 20, 70 ev ionization energy was used for ei-ms (electron ionizationmass spectrometry) spectra. the mass range was from m/z 28-450 amu. the fatty acid methyl ester standard mixture (fame supelco™ 37 catalog no: 47885-u) was used for the comparison and quantification of the gc chromatograms. the library search was also carried out using wiley-2008 gc/ms libraries to identify fatty acids. mineral analysis of bee pollen for minerals determination, 0.5 g of each sample was taken and heated in a muffle furnace at 400 ºc. the ash obtained was cooled, dissolved in 5 ml of 6 n hcl and allowed to stand for 30 min. it was later filtered, and its volume made up to 50 ml with deionized water (ullah et al. 2012). the elements fe, cu, pb, and ni, in previously mineralized samples were analyzed with an atomic absorption-emission spectrometer (solaar 969, unicam ltd., cambridge, uk). the k and na contents were determined by using a pfp7 industrial flame photometer (united kingdom). total phenolic content determination the total phenolics content of extracts was determined by the folin & ciocalteu′s method (gao et al. 2000). briefly, 0.1 ml diluted extract solution was shaken for 1 min with 200 µl of folin-ciocalteu's reagent and 2 ml of distilled water. the mixture was shaken, and 2 ml of 20 % na2co3 were added. after 30 min, the absorbance at 750 nm was evaluated using a spectrophotometer (beckman coulter du-640, ca, usa). the results were expressed as gallic acid equivalents. determination of antioxidant potential dpph free radical scavenging activity free radical scavenging capacity was measured using the dpph assay, according to brandwilliams et al. (1995). briefly, 0.2 ml of extracts diluted in methanol was added to a 60 µm dpph in methanol (1.8 ml). the dpph solution in the absence of the extract was used as the control, and methanol was used as blank. the samples were incubated for 1 h in the dark at room temperature, and the decrease in absorbance was spectrophotometrically measured at 515 nm (shimadzu uv-b382). measurements were performed in triplicate. the scavenging effect percentage was calculated from the formula: percentage of inhibition (eq. 1): [%] = [(abscontrol – abssample)/abscontrol] × 100 (1) the percentage was plotted against the samples, and ic50 values were estimated (concentration of samples able to scavenger 50 % of the dpph free radicals). the sample concentration providing 50 % inhibition (ic50) was calculated from that graph. determination of antioxidant activity using abts cation radical scavenging activity the abts radical cation decolorization method is based on the reduction of abts•+ radicals by antioxidants. a volume of 15 μl of aqueous methanolic extract was added to 1.485 ml of abts•+ solution, and the mixture was kept nova biotechnol chim (2020) 19(2): 208-215 211 at 30 °c. the absorbance was measured at 734 nm. measurements were performed in triplicate. the values were presented as ic50 values according to that reported for dpph scavenging activity (dorman and hiltunen 2004). total antioxidant capacity measured through the phosphomolybdenum assay the total antioxidant capacity was determined by the method of prieto et al. (1999). the methanolic extract (0.3 ml) was mixed with 3 ml of reagent solution (0.6 m sulphuric acid, 28 mm sodium phosphate, and 4 mm ammonium molybdate) and to left to incubate in a water bath at 95 ºc, for 90 min. after cooling, the absorbance of the green phosphomolybdenum complex was measured at 695 nm. in the case of the blank, 0.4 ml of methanol was used replacing the sample. the antioxidant activity was determined using a standard curve of ascorbic acid solution at different concentrations as the standard. results and discussion the fat obtained from bee pollen samples were characterized by the determination of saponification value, specific gravity, refractive indices, acid, and iodine values. the fatty acid and mineral composition, and the antioxidant activity of bee pollens were evaluated. fat characteristics the saponification value is inversely proportional to the molecular weight of the fat (mello and pinheiro 2012). the values found in the present work (table 1), lower than 190 – 200 mg koh.g-1 are characteristic of oils predominantly constituted by triglycerides in which fatty acids have 18 carbon table 1. some physical and chemical properties of pollen fat. characteristics na sa specific gravity [at 25 °c] 0.92±0.00 0.92±0.00 refractive index [at 25 °c] 1.47±0.00 1.46±0.00 acid value 22.04±0.15 10.01±0.12 saponification value 178.54±2.40 175.73±2.20 iodine value (wijs method) 44.42±1.60 32.90 ±1.20 mean ± standard deviation (n = 3). atoms, can be partly explained by the presence of arachidic and tricosanoic acid in concentration higher than 1 % (table 2). the specific gravity lower than 1.0 reveals that both samples are less dense than water, falling within the narrow range reported for vegetable oils (0.900 – 0.925) (yahaya et al. 2012; tesfaye et al. 2016; yildiz and dasgupta 2016; ogunlade and aremu 2019). the refractive index is within the recommended acceptable levels for edible oils (1.445 and 1.47) (yahaya et al. 2012; ogunlade and aremu 2019). the acid value represents the quantity of free fatty acids arising from hydrolytic processes. according to the results obtained, na had higher amounts of free fatty acids than sa. for example, and according to tesfaye et al. (2016), the acid value of bee wax is highly influenced by agroecology, whereby the differences found in these two samples, although not being waxes, may partially be explained by these factors. in addition, the acid value is also a relative measure of rancidity, since free acidity generally occurs during decomposition of triglycerides (miguel et al. 2005). moreover, unsaturated fatty acids are more exposed to oxidation processes than saturated ones. the iodine value is a measure of the degree of unsaturation of fat, and at the same time, it is a measure of vulnerability to oxidation. higher values of iodine value mean higher oxidation of vulnerability (ngassapa et al. 2012). the iodine values for samples na and sa were 44.42 and 32.90 mg i2.g -1 fat, respectively (table 1), which are in accordance with the values found for total unsaturated fatty acids in both samples. na was richer in unsaturated fatty acids than sa (table 1). fatty acid composition of pollen fat the fatty acid compositions of both bee pollens are shown in table 2. the palmitic, oleic, linoleic, and linolenic acids were the major fatty acids in samples na and sa. the remaining fatty acids were predominantly saturated ones, namely, myristic acid, stearic acid, arachidic acid, and tricosanoic acid, respectively. except for tricosanoic and sebacic acids, the remaining fatty acids are common in bee pollen in different percentages (campos et al. 2008; özcan et al. 2019). tricosanoic acid was previously reported nova biotechnol chim (2020) 19(2): 208-215 212 table 2. the fatty acid compositions (%) of algerian bee pollens. fatty acid/compound (iupac nomenclature) fatty acid/compound na [%] sa [%] lauric acid dodecanoic acid c12:0 1.6±0.1 4.4±0.2 sebacic acid decanedioic acid 0.5±0.0 4.4±0.2 myristic acid tetradecanoic acid c14:0 1.6±0.1 3.1±0.2 palmitic acid hexadecanoic acid c16:0 17.0±0.9 26.5±1.3 unidentified unidentified 7.0±0.4 2.5±0.1 linolenic acid (9z,12z,15z)-octadeca-9,12,15-trienoic acid c18:3 27.2±1.4 26.3±1.3 linoleic acid (9z,12z)-octadeca-9,12-dienoic acid c18:2 15.7±0.8 12.6±0.6 oleic acid (9z)-octadecenoic acid c18:1 8.5±0.4 10.1±0.5 stearic acid octadecanoic acid c18:0 2.2±0.1 2.5±0.1 arachidic acid eicosanoic acid c20:0 4.4±0.2 2.8±0.1 tricosanoic acid tricosanoic acid c23:0 1.5±0.1 1.0±0.1 17-pentatriacontene 17-pentatriacontene 12.9±0.7 3.8±0.2 as being present in citrus and clover bee bread (bee pollen that underwent lactic acid fermentation) collected in turkey (kaplan et al. 2016). the presence of decanedioic acid or sebacic acid, a dicarboxylic fatty acid, has been reported in acacia and palm honey (hegazy and el-hady 2009). the presence of these fatty acids may be explained by the presence of citrus and acacia species near to the hives where bee pollen was collected. the unsaturated fatty acids constituted 51 and 49 % of the oils of samples 1 and 2, respectively, while the saturated fatty acids were 29 and 45 %, respectively; nevertheless, na had a higher percentage of the alkane 17-pentatriacontene (12.9 %) than sa (3.8 %) (table 2). this compound has been found as a dominant hydrocarbon in propolis (bayram and gerçek 2017). mineral content the mineral content of fat bee pollen is depicted in table 3. potassium was the main element present in both fat bee pollen samples, although with significant differences (1497.51 and 513.16 mg.kg-1 in na and sa, respectively). the second most important element was sodium, at the same level of potassium. sodium was at higher concentration in sa, where potassium was in lower amount, and na with higher concentration of potassium had lower concentration of sodium. the third most important element was iron, but significantly lower than potassium and sodium. in contrast to k that was in higher amounts in na, the highest amount of fe was verified in sa (table 3) such as for sodium. copper was present in similar amounts in both samples ( 3 mg.kg-1). sodium, lead and nickel elements were not detected in both samples (table 3). pollen grains are a significant source of potassium (k) that remains in lipid fraction, as observed in the present work. other elements constitute bee pollen grains, but they were not detected in the present work (e.g., phosphorus, calcium, sulfur, magnesium), which contents change depending on the location that involves plant varieties and climatic conditions (liolios et al. 2019; özcan et al. 2019). the values of k found in our samples differed significantly from those previously reported (liolios et al. 2019; özcan et al. 2019) for bee pollen grains from different regions of turkey, russia, and greece, which can be partially explained by the different matrix used for the determination of mineral elements. in our case, fixed oils were the matrices used in contrast to the general studies that use bee pollen grains (liolios et al. 2019; özcan et al. 2019). the present work showed that the fat samples from bee pollen also contain minerals, although much less when compared to the bee pollen matrix. table 3. mineral composition of bee pollen fat. mineral content [mg.kg-1]; dw na sa na 235.21±47.52 1142.47±95.04 k 1497.51±38.67 513.16 ±116.01 fe 6.27±0.80 22.52±2.62 cu 3.20 ±0.33 3.12±0.14 pb nd nd ni nd nd mean ± sd (n = 3); dw: dry weight; nd: not detected. nova biotechnol chim (2020) 19(2): 208-215 213 table 4. phenolic content and antioxidant activities of bee pollen. na sa ascorbic acid phenolic content [mg gae/g]; dw 7.68±2.64 29.85±5.10 – ic50 [mg.ml -1] dpph assay 4.88±1.02 1.73±0.08 0.001±0.000 ic50 [mg.ml -1] abts assay 2.57±0.07 3.37±0.36 0.003±0.000 tac 0.04±0.00 0.06±0.00 – gae: gallic acid equivalent; dw: dry weight; tac: total antioxidant capacity; –: not determined. phenolic content and antioxidant activity several biological properties have been attributed to bee pollen (denisow and denisow-pietrzyk 2016). for this reason, the antioxidant activity was determined in both samples from different regions of algeria. first of all, the total phenol content was evaluated and significant difference was found, being sa much richer in phenols than na. the total phenol content observed in na is within the range reported by özcan et al. (2019) for turkish and russian bee pollen grains, but lower when compared to sa. such difference may explain the best activity of sa in what concerns the ability for scavenging dpph free radicals. the concentration needed for scavenging 50 % of dpph free radicals (ic50 = 1.73 mg.ml -1) of sa was much lower than ic50 = 4.88 mg.ml -1 for na (table 4). if a correlation seems existing between phenol content and dpph scavenging activity, the same was not observed when the assay for determining the antioxidant activity was that for scavenging the abts free radicals (table 4). this finding can be attributed to the different capacity of diverse structures of phenols for scavenging dpph or abts (shalabi and shanab 2013; badanai et al. 2015). several antioxidant natural products (compounds and/or extracts) have been reported for their ability to scavenge free radicals and to attenuate their harmful effects (boulanouar et al. 2013, 2019; ghareeb et al. 2018a, b, c; sobeh et al. 2018; ghareeb et al. 2019; cheraif et al. 2020). the activities found were significantly inferior to those verified for the reference (ascorbic acid) with ic50 values of 0.001 and 0.003 mg.ml -1, in dpph and abts scavenging activities, respectively. furthermore, the two samples showed total antioxidant capacity values of 0.04 and 0.06, for samples na and sa, respectively. the phosphomolybdenum assay is based on the reduction of mo (vi) to mo (v) by the tested bee pollen sample and subsequent creation of a green-colored [phosphate = mo (v)] complex at acidic ph (ghareeb et al. 2013; abdelaziz et al. 2018; hamed et al. 2019). conclusion the fats extracted from bee pollen samples have a saponification index lower than 190 mg.kg-1 koh, which may be due to the presence of arachidic and tricosanoic acids (c20 and c23) in percentages > 1 %. the remaining physicochemical parameters are within the range for fixed oils. the fatty acids of pollen fat samples with percentages higher than 10 % were palmitic, oleic, linoleic, and linolenic acids. among other minor fatty acids, tricosanoic and sebacic acids were detected, which indicate the presence of acacia and other species near the hives. potassium and sodium were the most important minerals present in bee pollen fat samples; nevertheless, their amounts deeply varied depending on the sample. bee pollen extracts had different amounts of total phenols, which were determinant in the antioxidant activity, although more significant when the dpph assay was used. these results show the importance of floral origin of bee pollen. the biological properties attributed to these bee products are highly dependent on the floral origin. in addition, and in our particular case, the presence of some fatty compounds generally absent in other examples of bee pollen, can be considered as biomarkers: sebacic acid and acacia. such approach must be deeply studied. conflict of interest the authors declare that they have no conflict of interest. nova biotechnol chim 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methods. am. j. anal. chem. 7: 611-616. nova biotechnol chim (2022) 21(1): e995 doi: 10.36547/nbc.995 1 nova biotechnologica et chimica response surface methodology designation for optimization of lactic acid production by streptococcus thermophilus isolated from industrial yogurt mina adibi1, nafiseh sadat naghavi2,, dina sadat zohrabi1 1department of biology, nourdanesh institute of higher education, meymeh, isfahan, iran 2department of microbiology, falavarjan branch, islamic azad university, isfahan, iran  corresponding author: nafiseh_naghavy@yahoo.com article info article history: received: 31st may 2021 accepted: 16th august 2021 keywords: industrial yogurt lactic acid streptococcus thermophiles response surface methodology abstract using inexpensive sources is a crucial point in the production of valuable compounds such as lactic acid. in the present study, the production of lactic acid by an efficient isolate of s. thermophilus was optimized using whey as a carbon source and yeast extract as a nitrogen source. mrs culture medium with 6.5 % nacl was used for the isolation of streptococci from industrial yogurt samples and the selected isolate was identified using the 16s rrna gene sequencing. evaluation of lactic acid production was done by the randox method. lactic acid production by the selected isolate was optimized by response surface methodology (rsm) in mrs broth regarding to different concentrations of whey and yeast extract. the isolate that produced the highest amount (8.9 g.l-1) of lactic acid within 52 h growth in mrs broth was identified as a strain of s. thermophilus by molecular identification. optimization of concentrations of carbon and nitrogen sources resulted in the induction of lactic acid production by streptococcus thermophilus up to 24.18 g.l-1 in the presence of 3.5 % of whey and 5 % of yeast extract as external carbon and nitrogen sources in the mrs medium, which was similar to the value predicted by rsm. yeast extract was found to be more effective on lactic acid production than whey. optimization of lactic acid production by low-cost substrates whey and yeast extract resulted in high induction of lactic acid production by s. thermophilus compared to some other studies that used other cost-effective substrates and/or bacteria isolated from traditional dairy products. introduction lactic acid with the formula c3h6o3 is a chiral alpha-hydroxy acid with a molar mass of 190.08 g.mol-1 (hujanen et al. 2001). the use of lactic acid in industries is widespread due to its low price and bioactivities that make it suitable for use as a preservative, antioxidant, flavoring, prebiotic, cryoprotectant, viscosifier, and acidifier. also, polylactic acid produced by its polymerization has more applications in the industry than its monomer, especially in the plastics production industry. lactic acid is a chemical used in the textile industry as a stabilizer and helps with the better fabric absorption of the dye. this organic acid is also involved in the production of varnish and ink and leather tanning. it is also used as a moisturizer and lubricant in cosmetics and added to food to create a sour taste. lactic acid is an important compound in the production of dairy products including yogurt and cheese and as a starting material in the pharmaceutical industries (reddy et al. 2008). since the production of pure optical isomers of lactic acid is not possible by chemical methods, the mailto:nafiseh_naghavy@yahoo.com nova biotechnol chim (2022) 21(1): e995 2 use of fermentation methods for its production has been more interested nowadays. the biological production of lactic acid accounts for more than half of the world's total production (kotzamanidis et al. 2002). biological production of lactic acid via cost-effective carbon and nitrogen sources such as whey, milk powder, yogurt powder, and curd powder is expected to provide a suitable market for lactic acid (reddy et al. 2008; abedi and hashemi 2020). lactic acid bacteria (labs) that are found in plants, meat, and dairy products are commonly used to produce lactic acid with high production and yield (ayana et al. 2011). streptococcus is one of the most important genera in this group. most streptococci are catalase and oxidase negative facultative anaerobes. the only species in this genus used in the production of dairy products is streptococcus thermophilus. lactobacilli are important bacteria in the large group of labs, are known as starter cultures and key players in the microbial flora of fermented dairy and meat products such as yogurt and sausages, respectively. the synergistic effects between the two bacterial species are the basis of lactose metabolism and high lactic acid production in milk (brashears et al. 2005; ayana et al. 2011). due to the fact that the fermentation methods for the production of lactic acid using inexpensive sources, such as whey obtained from dairy factories, is an important phenomenon (abedi and hashemi 2020), in this study, the production of lactic acid by a potent strain of s. thermophilus isolated from industrial yogurt was optimized using whey as carbon source and yeast extract as nitrogen source. experimental isolation of bacteria as in previous study the addition of nacl salt has been used for stimulation of the growth of lactic acid producing isolates specially lactococcus and streptococcus species (medveďová et al. 2019), in the present study steps for the purification of streptococci was done in 1 – 7 % nacl in mrs agar (de man et al. 1960) and it was observed that the streptococcus sp. was able to tolerate up to 6.5 % nacl. therefore, in order to purify this isolate, industrial yogurt samples were cultured in mrs agar medium containing 6.5 % nacl followed by incubation at 37 °c for 48 h. also, as lactose has been proposed for induction of the growth of lactobacilli (rezvani et al. 2017), in the present study the purification steps for lactobacilli was done in 1 – 5 % lactose in mrs agar and it was observed that the lactobacillus sp. grows best in the presence of at least 2 % lactose. therefore, the purification steps for this isolate from industrial yogurt samples were done in mrs agar medium containing 2 % lactose followed by incubation at 39 °c for 48 h in a candle jar. the microscopic features of the isolated bacteria were analyzed after gram staining (taligene pars, iran) using the manufacturer instructions. the culture media and chemicals were obtained from merck corporation (germany). llactate assay the 48 h cultured bacteria were centrifuged at 4,000  g for 20 min. then, the amount of lactic acid in the supernatant was measured based on the amount of pyruvate and hydrogen peroxide catalyzed by lactate oxidase enzyme by randox lc2389 kit (randox laboratories ltd, uk) following instructions of the manufacturer. the production of lactic acid was measured every 3 h during 4 days of incubation along with the bacterial growth curve was plotted through assessment of the culture turbidity at a wavelength of 550 nm. molecular identification of the selected isolate total dna was extracted using the dn8115c kit (sinaclon, iran). the extracted dna quality was determined based on optical density ratio at 260/280 nm wavelengths using a spectrophotometer (agilent, uv-vis-nir), and its quantity was measured by thermo scientific™ nanodrop 2000 full-spectrum (desjardins and conklin 2010). then, a fragment in the 16s rdna gene was amplified by using universal primers, rw01 (5´-aactggaggaaggtggggat-3´) and dg74 (5´-aggaggtgatccaaccgca-3´) in a hot started polymerase chain reaction (pcr). the primers were constructed by bioneer nova biotechnol chim (2022) 21(1): e995 3 corporation (daejeon, south korea). the amplification protocol was performed in 4 steps included 1: 95 ºc for 60 s; 2: 94 ºc for 60 s, 58 ºc for 50 s, and 72 ºc for 60 s (repeated 6 times); 3: 94 ºc for 60 s, 56.5 ºc for 60 s, and 72 ºc for 60 s (repeated 37 times); and 4: 72 ºc for 5 min. the pcr mixture involved 1x pcr buffer, 1.5 mm mgcl2, 0.5 mm dntp (cinnagen, tehran, iran), 0.5 µm of each primer, 1 unit taq dna polymerase (cinnagen, tehran, iran), and 1 µg of the extracted dna in a total volume of 50 µl. the size of pcr products were detected by agarose gel electrophoresis. the amplified fragment was then sequenced and analyzed in the blast server (http://www.ncbi.nlm.nih.gov/blast; greisen et al. 1994; fantin et al. 2013). optimization of lactic acid production by the selected isolate by response surface methodology (rsm) based on the results from previous studies conducted by one factor at a time experiments for lactic acid production using low-cost carbon and nitrogen sources (nancib et al. 2001; schepers et al. 2002; altaf et al. 2005; bernardo et al. 2016), in the present study, whey powder (11.5 % protein, 65 % lactose, and 2 % lipid content with 8.5 % ash and 0.15 % acidity) and yeast extract (merck, germany) were used in the experiments which designed by rsm methodology, to optimize lactic acid production by the most potent lab that was isolated from industrial yogurt. for this purpose, the first pre-trials were conducted by supplementing different concentrations of whey powder and yeast extract to mrs medium. then based on the obtained results for lactic acid production in each concentration, the minimum and maximum levels for each source were entered into the design expert software and the software designed the experiments to be done. statistical analysis the data were collected as mean ± standard deviation according to the amount of the produced lactic acid and entered the software for analysis by one-way analysis of variance anova using qualitek 4 software. f test was used for comparing the results of experiments. significance levels were considered as p ≤ 0.01. results molecular identification of the selected isolates among the isolated bacteria, 2 isolates produced high amounts of lactic acid in the mrs medium. these isolates were a gram-positive rod and a gram-positive coccus. fig. 1 shows the 370 bp band resulting from amplification of a fragment in the 16s rrna coding gene by using universal primers in the genome of 2 initially selected isolates (a and b; fig. 1). fig. 1. amplification of the fragment in the 16s rrna coding gene resulted in a 370 bp band (lines 1 and 2: the isolates a and b, respectively) according to the 50 bp dna marker (line 3). the alignment of the amplified 16s fragment in the gene bank for 2 initially selected isolates indicated that the bacteria were a strain of streptococcus thermophilus and strains of lactobacillus delbrueckii subsp. bulgaricus. the phylogenetic trees in fig. 2 show the relationships of the isolates with the close strains. nova biotechnol chim (2022) 21(1): e995 3 fig. 2. phylogenetic trees of the isolated strains: a* is related to the strains of s. thermophilus with 99.5 % identity and b* is related to the strains of lactobacillus delbrueckii subsp. bulgaricus with 99.6 % identity. growth curve and lactic acid productivity of the isolated bacteria the growth and lactic acid production curves by the 2 selected isolates during 60 h growth in mrs broth at 37 °c for s. thermophilus and 39 °c for l. delbrueckii subsp. bulgaricus are shown in fig. 3 and 4. the highest amount of lactic acid (8.9 g.l-1) was obtained at 52nd h in mrs broth by s. thermophilus. at this point, the bacterium was at the end of the logarithmic growth stage. this productivity was significantly higher than the productivity of l. delbrueckii subsp. bulgaricus which produced 3.913 g.l-1 lactic acid at 57th h in mrs broth (p < 0.05). therefore, the s. thermophilus isolate was selected for the production optimization process. fig. 3. the growth curve and lactic acid production by s. thermophiles. 4 nova biotechnol chim (2022) 21(1): e995 2 fig. 4. the growth curve and lactic acid production by l. delbrueckii subsp. bulgaricus. determination of optimal conditions for lactic acid production by streptococcus thermophiles the results from one factor at time experiments for initial optimization of lactic acid production by s. thermophilus in different concentrations of whey and yeast extract sources are shown in fig. 5 and 6. the highest amounts of lactic acid were produced at 6 % concentrations of both whey and yeast extract. therefore, the minimum (3.5 %) and maximum (6.5 %) levels for each source were entered the design expert software, and the software designed 13 experiments by rsm to be done (table 1). fig. 5. lactic acid production by s. thermophilus in different concentrations of yeast extract. the isolate represented the most productivity in the concentration of 6 %. different letters indicate a significant difference between the results (p < 0.05). fig. 6. the lactic acid production by s. thermophilus in different concentrations of whey. the isolate represented the most productivity in the concentration of 6 %. different letters indicate a significant difference between the results (p < 0.05). table 1. experiments designed by the design expert software. experiment no. whey [%] yeast extract [%] 1 4 4 2 4 6 3 6 4 4 6 6 5 5 3.5 6 5 6.5 7 3.5 5 8 6.5 5 9 5 5 10 5 5 11 5 5 12 5 5 13 5 5 the results obtained from designed experiments are shown in fig. 7. as seen, the highest lactic acid 5 nova biotechnol chim (2022) 21(1): e995 2 production by s. thermophilus (24.18 g.l-1) was obtained in the presence of 3.5 % whey and 5 % yeast extract as supplementary carbon and nitrogen sources in the mrs medium (experiment no. 7). this result had no significant difference with the result obtained from experiment no. 6 (21.01 g.l-1 lactic acid productivity) in which 5 % whey and 6.5 % yeast extract were added as supplementary carbon and nitrogen sources to the mrs medium. fig. 7. comparison of lactic acid production by s. thermophilus between 13 experiments designed by design expert software. different letters indicate a significant difference between the results (p < 0.05). statistical analysis of data table 2 shows the results from anova analysis. a quadratic correlation was seen between the effect of examined factors and lactic acid production. also, a significant correlation was seen between the designed model and the studied factors, and the lack of fit between the results was not significant. the obtained values had normal distribution with no significant difference from the expected values. according to f-test and p-values, the effect of yeast extract supplementation on lactic acid production was significantly greater than the effect of whey supplementation (p = 0.0341). table 2. results from anova analysis to investigate the quadratic model of lactic acid production. source sum of squares degree of freedom mean squares f-value p-value significance model: quadratic 401.92 5 60.38 5.23 0.0256 significant yeast extract (a) 105.94 1 105.94 6.90 0.0341 whey (b) 14.67 1 14.67 0.96 0.3610 a2 120.33 1 120.33 7.84 0.0266 b2 71.80 1 71.80 4.67 0.0674 residual 107.50 7 15.36 lack of fit 88.76 3 29.59 6.31 0.0536 non-significant pure error 18.85 4 4.69 total 509.42 12 normal distribution that observed around the center point by the 5 best results from 13 experiments is shown in fig. 8. the central red dot is the result of repeated experiments with central amounts of 5% cheese and 5% yeast extract, and the distribution of the best results is shown on the blue lines. other parts of the blue lines are the values predicted by the software for the results. based on statistical analysis, the obtained lactic acid production was 24.18 g.l-1 under optimal conditions. it was not significantly different from the predicted value that was expected by rsm. 6 nova biotechnol chim (2022) 21(1): e995 2 fig. 8. two-dimensional distribution of the best test results obtained from design expert software. the red point in the center is the central test, a and b indicate yeast extract and whey factors, respectively. discussion whey is a by-product of the dairy industry, accounting for 85 – 95 % of milk volume and retaining 55 % of milk nutrients. lactose is low in whey. all 9 essential amino acids are found in whey protein. the most abundant of these nutrients are lactose, soluble proteins, lipids, and mineral salts. the presence of lactose and other nutrients for the growth of microorganisms makes it one of the potential substrates for the production of various biological products through biotechnological processes (panesar et al. 2007). in the present study, the lactic acid bacteria s. thermophilus and l. delbrueckii subsp. bulgaricus were purified from industrial yogurt samples with some modifications in traditional mrs medium enriched by 6.5 % nacl for streptococcus spp. and by 2 % lactose for lactobacillus spp. the isolate was identified by a molecular method. s. thermophilus was reported to be more sufficient for lactic acid production than l. delbrueckii subsp. bulgaricus. rezvani et al. (2017) compared five lactobacillus strains for biomass yield and lactic acid production. the results showed that l. bulgaricus was the most efficient strain which produced the highest biomass and lactic acid yield using whey supplemented with lactose and yeast extract as carbon and nitrogen sources. other species including l. fermentum, l. casei, and l. lactis had less productivity than l. bulgaricus. the strain of l. delbrueckii had the least biomass and lactic acid production. this result is in coincidence with the present study. in this study, the lactic acid production by the particular strain of s. thermophilus with high lactic acid production was optimized by response surface methodology (rsm) by changing the concentrations of whey powder and yeast extract as low-cost carbon and nitrogen sources. the highest lactic acid production by streptococcus thermophilus (24 g.l-1) was obtained in the mrs medium containing 5 % yeast extract and 3.5 % whey powder during 52 hours of incubation. this rate of production was consistent with, or in some cases greater than, the amount of lactic acid produced by other researchers (nacib et al. 2001; benkun and yao 2007; panesar et al. 2007; prachamon and boonmee 2008; zhang and vadlani 2013). hujanen et al. (2001) optimized the process variables and carbon concentrations for lactic acid production by lactobacillus casei nrrl b-441. the lactic acid yield was proportional to the initial glucose concentration (80 – 160 g.l-1) in the medium. the highest concentration of lactic acid that was obtained during fermentation was 118.6 g.l-1. rsm was used to optimize the process variables. the optimum temperature and ph for lactic acid production were 35 °c and 6.3, respectively. malt extract along with yeast extract (4 g.l-1) seemed to be an economical alternative to yeast extract alone (22 g.l-1), although fermentation time was slightly longer and the usage of glucose as the initial carbon source was not cost-effective. in a similar study, lu et al. (2016) achieved a lactic acid concentration of 83 g.l-1 using a combination of high-cost sources, including glucose and xylose, with a 36 h fermentation by lactococcus lactis. zhang and vadlani (2013) used a recombinant strain of l. delbrueckii to produce lactic acid with a purity of 99 % at the rate of 36.3 g.l-1 using a combination of glucose and xylose as the carbon source. inexpensive substrates have also been used for the production of lactic acid similar the present study. for instance, prachamon and boonmee (2008) studied the effect of sugarcane extract as a substrate for lactic acid production. they cultured five strains of lactic acid bacteria with different carbon sources such as glucose, sucrose, and sugarcane . 7 nova biotechnol chim (2022) 21(1): e995 3 extract. the results showed that when glucose or sucrose was used as the substrate, there was no significant difference in the concentration of lactic acid produced. the production rates were 18.19 – 20 g.l-1 and 17.78 – 20.77 g.l-1, respectively. when using sugarcane extract as the substrate, the concentration of produced lactic acid was decreased to 5.56 – 10.3 g.l-1. panesar et al. (2007) conducted a study on the fermentation of whey to produce lactic acid using l. casei. the effect of various process parameters such as ph, temperature, inoculation size, inoculation age, stirring, and incubation time was investigated to increase the conversion of whey lactose to lactic acid. process conditions optimization led to the production of 33.73 g.l-1 lactic acid after the 36 h of incubation period. inexpensive sources other than whey also have been used for the production of lactic acid by bacteria. benkun and yao (2007) examined the low-cost production of lactic acid using rice husk as the only carbon source in solid-state fermentation and produced 18 g.l-1 of lactic acid. nancib et al. (2001) studied the production of lactic acid by fermentation using l. casei. different nitrogen sources were compared for their efficiency in lactic acid production. none of the used nitrogen sources produced as much lactic acid as the yeast extract which resulted in 20 g.l-1 lactic acid production. in the present study, the results obtained by the experiments designed by rsm showed that increasing the concentration to more than 6 % for yeast extract and 3.5 % for whey had an inhibitory effect on lactic acid production in rsm designed experiments. data analysis also showed that lactic acid production is affected more by the concentration of yeast extract compared to that of whey. similar results have been obtained in the study of benaissa et al. (2017) that used sweet whey as a base medium supplemented with yeast extract, and/or tomato juice for the growth of lactobacilli and lactic acid production. obtained results showed that the bacteria produced more biomass on whey supplemented with yeast extract than those produced with tomato juice. they concluded that whey is too poor to provide the needs of lactobacilli and optimization of growth medium through the addition of yeast extract (1 %) and/or tomato juice (30 %) as inexpensive substrates can promote lactic acid production by these bacteria similar to the case took place with mrs medium in the current study. conclusion in the present study, high lactic acid production (24.18 g.l-1) was obtained by the isolated s. thermophilus through supplementation of 3.5 % whey powder and 5 % yeast extract as respective low-cost carbon and nitrogen sources in the production medium. the optimization process by response surface methodology (rsm) resulted in elevated lactic acid production at more levels than some other studies that used other expensive substrates or optimized the production condition by designations in which a single factor has been optimized in each experiment at a time. acknowledgments the authors of the paper thank the research management of nourdanesh institute of higher education, meymeh, isfahan, iran for their technical support. conflict of interest the authors declare that they have no conflict of interest. references abedi e, hashemi smb (2020) lactic acid productionproducing microorganisms and substrates sources-state of art. heliyon. 6: e04974. altaf m, naveena bj, reddy g (2005) screening of inexpensive nitrogen sources for the production of l (+) lactic acid from starch by amylolytic lactobacillus amylophilus gv6 in single step fermentation. food tech. biotech. 43: 235-239. ayana iaaa, el-deen ag, el-metwally ma (2011) behavior of certain lactic acid bacteria in the presence of pesticides residues. int. j. dairy sci. 6: 44-57. benaissa m, halima zk, karam ne (2017) development of a sweet whey-based medium for culture of lactobacillus. afr. j. biotechnol. 16: 1630-1637. benkun q, yao r (2007) l-lactic acid production from lactobacillus casei by solid state fermentation using rice straw. bioresources 2: 419-429. bernardo mp, coelho lf, sass dc, contiero j (2016) l-(+)lactic acid production by lactobacillus rhamnosus b103 from dairy industry waste. braz. j. microbiol. 47: 640646. 8 nova biotechnol chim (2022) 21(1): e995 2 brashears mm, amezquita a, jaroni d (2005) lactic acid bacteria and their uses in animal feeding to improve food safety. adv. food nut. res. 50: 1-31. de man jc, rogosa d, sharpe me. (1960) a medium for the cultivation of lactobacilli. j. appl. bacteriol. 23: 130-135. desjardins p, conklin d (2010) nanodrop microvolume quantitation of nucleic acids. j. vis. exp. 45: 25-65. fantin ys, neverov ad, favorov av, alvarez-figueroa mv, braslavskaya si, gordukova ma, karandashova iv, kuleshov kv, myznikova ai, polishchuk ms (2013) base-calling algorithm with vocabulary (bcv) method for analyzing population sequencing chromatograms. plos one 8: e54835. greisen k, loeffelholz m, purohit a, leong d (1994) pcr primers and probes for the 16s rrna gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. j. clin. microbiol. 32: 335-351. hujanen m, linko s, linko yy, leisola m (2001) optimisation of media and cultivation conditions for l (+) (s)-lactic acid production by lactobacillus casei nrrl b-441. appl. microbiol. biotechnol. 56: 126-130. kotzamanidis ch, roukas t, skaracis g (2002) optimization of lactic acid production from beet molasses by lactobacillus delbrueckii ncimb 8130. world j. microbiol. biotechnol. 18: 441-448. lu h, zhao x, wang y, ding x, wang j, garza e, manow r, iverson a, zhou s (2016) enhancement of d-lactic acid production from a mixed glucose and xylose substrate by the escherichia coli strain jh15 devoid of the glucose effect. bmc biotechnol. 16: 1-10. medveďová a, šipošová p, mančušková t, valík ľ (2019) the effect of salt and temperature on the growth of fresco culture. fermentation 5: 2-10. nancib n, nancib a, boudjelal a, benslimane c, blanchard f, boudrant j (2001) the effect of supplementation by different nitrogen sources on the production of lactic acid from date juice by lactobacillus casei subsp. rhamnosus. bioresour. technol. 78: 149-153. panesar ps, kennedy jf, gandhi dn, bunko k (2007). bioutilisation of whey for lactic acid production. food chem. 105: 1-14. prachamon t, boonmee m, hamsupo k (2008). lactic acid production using sugar cane juice as a substrate. kku res. j. (gs) 8: 1-8. reddy g, altaf md, naveena bj, venkateshwar m, kumar ev (2008) amylolytic bacterial lactic acid fermentation-a review. biotechnol. adv. 26: 22-34. rezvani f, ardestani f, najafpour g (2017) growth kinetic models of five species of lactobacilli and lactose consumption in batch submerged culture. braz. j. microbiol. 48: 251-8. schepers aw, thibault j, lacroix c (2002) lactobacillus helveticus growth and lactic acid production during phcontrolled batch cultures in whey permeate/yeast extract medium. part i. multiple factor kinetic analysis. enzyme microb. technol. 30: 176-186. zhang y, vadlani pv (2013) d-lactic acid biosynthesis from biomass-derived sugars via lactobacillus delbrueckii fermentation. bioprocess biosyst. eng. 36: 1897-1904. 9 nova biotechnol chim (2020) 19(1): 98-108 doi: 10.36547/nbc.v19i1.582  corresponding author: hornik@ucm.sk nova biotechnologica et chimica factors affecting the analysis of 2-deoxy-2-fluoro[18f]-d-glucose in plant tissues by a commercial pet system vanda adamcová1, klára kuglerová1, katarína ondreičková2, marcela gubišová2, jozef gubiš2, juraj lesný1, peter kováč1,3 and miroslav horník1, 1department of ecochemistry and radioecology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic 2national agricultural and food centre – research institute of plant production, bratislavská cesta 122, piešťany, sk-921 68, slovak republic 3biont inc., karloveská 63, bratislava, sk-842 29, slovak republic article info article history: received: 15th may 2020 accepted: 10th june 2020 keywords: 2-[18f]fdg pet plant tissues uptake quantification 3d imaging abstract some works showed that commercial pet systems and 2-deoxy-2-fluoro[18f]-dglucose (2-[18f]fdg) can be used in plant studies to analyze the transport and allocation of photoassimilates. the aim of this work was to evaluate the effect of characteristics of plant tissues, applied solution and of the phenomenon of “escaping positrons” on the visualisation and quantification of the distribution of 2-[18f]fdg, as a model of photoassimilates, in the tissues of selected plants by a commercial pet system. experiments included pepper plants immersed with the root system or stem into a 2-[18f]fdg solution showed that the accumulation of 2-[18f]fdg was several times higher in the aboveground parts that were not directly exposed in a 2-[18f]fdg solution than in the parts directly immersed in the solution. individual experiments carried out by exposing the excised leaves of radish plants in the solutions of 2-[18f]fdg containing 100-fold higher concentrations d-glucose (cglu) compared to the original 2-[ 18f]fdg solution showed that with increasing cglu there were significant changes in the distribution of 2-[ 18f]fdg in the leaf blade. the last factor studied was the phenomenon known as "escaping positrons" and defined by annihilation of released positrons during radioactive decay of a given emitter 18f outside the studied object, whose thickness exceeds the lower limits of resolution of pet systems it was found that the covering the leaf of tobacco, after exposure in a 2-[18f]fdg solution, by a parafilm, aluminium foil or copper thin plate caused significant changes in the quantitative data obtained in the pet analysis.  university of ss. cyril and methodius in trnava introduction rapidly growing human population increases requirements on food, energy and other natural resources. advances in molecular biology techniques developed crops that are more resistant to pathogen microorganisms, viruses, insects and abiotic stress, such as temperature, nutrient limitation, etc. and they are widely available to improve food and energy production (wang et al. 2014). soil provides most of the essential elements required for the growth of plants. these elements are absorbed by the root system and then are transported into the leaves via the xylem and photoassimilates and other nutrients are translocated from the leaves to the maturing organs via the phloem. for centuries, scientists have been fascinated by water and sugar nova biotechnol chim (2020) 19(1): 98-108 99 transport in plants because these processes are important for plant growth and cycle. if we aspire to explain plant growth or to increase the productivity of agriculture or forestry, it is crucial to understand the dynamics and the controls of water and sugar flow, as well as their interactions. therefore, an accurate understanding of the movement of metabolites and elements across the plant body is of paramount importance in plant science research (suzui et al. 2019). it is also essential to unravel how transport in plants affects their functioning and their ability to withstand stress, and also which conditions determine whether a plant thrives or dies (hubeau and steppe 2015). research focused on the distribution and dynamics of photoassimilates in plants is important for increasing food production. imaging methods based on radioisotopes application represent unique technics to understand the kinetics of elements and bioorganic compounds in the plant body (suzui et al. 2019). furthermore, non-invasive radioisotopes imaging techniques, which can image the temporal changes in radioisotopes distributions, are effective for investigating the dynamics and distributions of elements and molecules under in vivo conditions. in plant studies, they are powerful tools for the elucidation of physiological functions (kurita et al. 2020). the field of radiotracer imaging involves the application of radioisotope-labelled compound (radiotracer) to analyze the uptake and distribution of corresponding non-labelled analogue, thus helping to shed light on the underlying physiology or diagnostics (fatangare and svatoš 2016). one of these radiotracer imaging systems and approaches is positron emission tomography (pet). pet is a non-destructive imaging method based on the injection of radiotracer labelled with a positronemitting radionuclide. this method represents a highly sensitive nuclear medicine technique that enable to visualize the disposition and kinetics of radioisotopes in vivo. the advantage of the technique lies in the wide range of available radiotracers that produce image contrast directly related to underlying physiology, metabolic pathways, or molecular targets (berg and cherry 2018; moein et al. 2019). pet scanners consist of a cylinder of scintillation detectors coupled to fast electronics, feeding into software. the detectors collect coincident pairs of 511 kev γ-photons emitted at 180o, which originate from the site where a positron produced by radioactive decay annihilates with an electron in the surrounding material. the generated three-dimensional (3d) images map the distribution of a positron-emitting radionuclide. pet is an ultra-sensitive technique allowing the detection of very low amounts of radiotracer (< 10-16 moles, 10 kbq) and a good spatial resolution between 1 and 6 mm, depending on the construction of the detector and the image reconstruction algorithm (thorpe et al. 2019). pet has been primarily used for clinical (human) and preclinical (animal) applications. a common use of pet in clinical practice is for the diagnosis of various cancers and in preclinical applications. pet is also often used in drug development through animal-based imaging studies. additionally, pet has proven to be very useful particularly in plant biology research applications. medical imaging techniques are rapidly expanding in the field of plant science (weisenberger et al. 2013; hubeau and steppe 2015). pet can be used to study whole-plant transport and nutrient allocation by quantifying the distribution of a positronemitting radioisotope non-invasively, over a timecourse, and with a spatial resolution of a few millimetres (karve et al. 2015). the advantages of the pet systems mainly include the capability of providing dynamic images of positron-emitting isotopes under real time and in vivo conditions without damaging the plants. the commonly used positron-emitting radioisotopes include 18f, 15o, 11c and 13n, which are usually bound to bioorganic molecules to form a radiopharmaceutical targeting a particular uptake pathway. the most widely used radiopharmaceutical for imaging human is 2-deoxy2-[18f]fluoro-d-glucose (2-[18f]fdg), which detect malignant tumours with faster glucose uptake than a benign tissue. the 2-[18f]fdg can accumulate in any cell that has the ability to metabolise glucose, including those in subsurface anaerobic environments (thorpe et al. 2019). in this glucose analogue the hydroxyl group at the c-2 position has been replaced by a 18f radioisotope with a half-life t1/2 =109.8 min (fatangare and svatoš 2016). there are several works that evaluate the possibility of using commercial pet systems in visualization and quantification of uptake and transport of 2-[18f]fdg in plant tissues. tsuji et al. (2002) nova biotechnol chim (2020) 19(1): 98-108 100 first reported on 2-[18f]fdg uptake and distribution in tomato plants. hattori et al. (2008) described 2-[18f]fdg translocation in intact sorghum plants and suggested its use in tracing photoassimilates translocation in plants. 2-[18f]fdg has been used to study glycoside biosynthesis in plants as a measure of induction of plant response (defense) (ferrieri et al. 2012). moreover, tran et al. (2017) synthetized 1´-[18f]fluoro-1´-deoxysucrose and 6-[18f]fluoro-6-deoxysucrose ([18f]fds) analogues and studied the role of sucrose transporter (sut) proteins in wild-type and mutant (sut1 mutant plants of maize (zea mays l.) by phosphor imaging plate system. in previous papers, the application potential of a commercial pet system in the characterization of the uptake and distribution of 2-deoxy-2fluoro[18f]-d-glucose labelled with the positronemitting isotope 18f (2-[18f]fdg) in plant tissues of tobacco (nicotiana tabacum l.) or giant reed (arundo donax l.) plants was evaluated (partelova et al. 2017, 2016, 2014). the aim of this work was to evaluate the effect of plant tissues characteristics, concentration of d-glucose in a 2-[18f]fdg solution and the phenomenon of “escaping positrons” on the visualisation and quantification of the uptake and distribution of 2-[18f]fdg in the tissues of selected plants by a commercial pet systems. experimental plant material plants of pepper (capsicum annuum l.) and radish (raphanus sativus l.) as a model of vegetables and plants of tobacco (nicotiana tabacum l.) were chosen as studied objects. seeds of pepper and radish plants were purchased as commercially available seeds (seva seed, ltd. valtice, czech republic and semo, inc. smržice, czech republic) and seeds of tobacco plants were obtained from the gene bank of the slovak republic. seedlings of the mentioned plant species were obtained from the seeds germinated and cultivated in an inorganic carrier perlite watered with hydroponic medium according to hoagland (hoagland 1920). for these purposes, hoagland media (hm) diluted with deionized water (0.054 μs.cm-1) at a volume ratio 1 : 3 (25 % concentration strength) were used, whereby the full strength medium (100 % hm) contained macroelements (in mg.l-1) mgso4.7h2o – 370; kno3 – 404; cacl2 – 444; nah2po4.2h2o – 292; na2hpo4.12h2o – 46.5; feso4.7h2o – 17.9; nano3 – 340; nh4cl – 214; nh4no3 – 160; and microelements (in mg.l-1) h3bo3 – 8.5; na2moo4.2h2o – 0.06; mnso4.5h2o – 5.0; znso4.7h2o – 0.66; cuso4.5h2o – 0.8. the cultivation conditions – photoperiod 16 h light/8 h dark (max. photosynthetic photon flux density ppfd = 200 μmol.m-2.s-1), temperature max. 28 ºc (light)/min. 18 ºc (dark) and relative humidity 60 – 80 % were provided by the growth chamber for plant cultivation (kbwf 720, binder, d). after 6 – 8 weeks of the cultivation, the uniform plants, in terms of height (approx. 15 cm), weight (approx. 1 g; w.w.), root size, number of leaves and developmental stage, were removed from the perlite and the root system was carefully washed with tap water to remove adherent particles. production of 2-[18f]fdg a radioisotope 18f as a positron emitter (t1/2 = 109.5 min) in the form of fanions was prepared from the molecules of h2 18o via the reaction 18o (p, n) 18f using the cyclotron cyclone 18/9 (iba, belgium). the key intermediate 1,3,4,6-tetrao-acetyl-2-[18f]fluoro-d-glucopyranose for the production of 2-[18f]fdg was obtained from the precursor 1,3,4,6-tetra-o-acetyl-2-otrifluoromethanesulfonyl-β-d-manno-pyranose and using the phase-transfer catalyst aminopolyetherpotassium complex (kryptofix® 222) and produced [18f]fas well. finally, 2-[18f]fdg was prepared by the acid hydrolysis of mentioned intermediate. the procedures of 18f production and 2-[18f]fdg synthesis were realized by the company biont inc. (bratislava, slovak republic). the final solution of 2-[18f]fdg, as a radiopharmaceutical product under the trade name biontfdg, was supplemented with nacl (9.0 g.l-1) and showed the following physico-chemical parameters (determined/evaluated by): opalescence (organoleptically) – clear, colourless solution; ph (colorimetrically) – 6.8 – 7.2; specific volume radioactivity (ionization chamber) – 1.6 – 2.8 gbq.ml-1; half-life decay (ionization chamber) – 109.5 min; chemical nova biotechnol chim (2020) 19(1): 98-108 99 concentration of 2-[18f]fdg (liquid chromatography) – < 0.0016 mg.ml-1; concentration of kryptofix® 222 (thin-layer chromatography) – < 0.22 mg.ml-1; radiochemical purity of 18f in the form of 2-[18f]fdg and 2-deoxy2-[18f]fluoro-d-mannose (thin-layer chromatography) – 97 % – 99 %; concentration of glucose (liquid chromatography) – 0.0762 mg.ml-1; concentration of acetonitrile (gas chromatography) – 0.03 mg.ml-1; concentration of ethanol (gas chromatography) – 0.037 mg.ml-1. before the application in the experiments, a 2-[18f]fdg solution was diluted with deionised water at a volume ratio of 1 : 9. 2-[18f]fdg uptake and pet analysis the uptake of 2-[18f]fdg was carried out by immersing the root system of the intact plant, the stem after the removing the root system with a scalpel, the petiole of the excised leaf or by immersing the excised apex of the leaf blade into small plastic vials containing of 2-[18f]fdg solution with known volume activity of 18f and d-glucose concentration. thus, the visualization and quantification of 2-[18f]fdg uptake and distribution in the tissues of selected plant parts were realized in two transport ways, in acropetal and basipetal (opposite) direction of the solute transport within the conductive tissues as well. to avoid spillage and evaporation of the water, the plastic vials were covered with a parafilm over the neck of the vial. in the case of leaf samples, the third developmentally youngest leaf branch was excised with a scalpel. the exposure was realized under laboratory conditions (temperature 23 °c – 25 °c; relative humidity 40 % – 45 %) during 30 min. after this time, the immersed plant parts were washed in deionized water by submersion, dried between cellulose towels and weighed (w.w.). subsequently, the studied objects were carefully placed on the scanning bed of the micropet system explore vista pre-clinical pet scanner (ge healthcare, spain) and fixed with parafilm strips so that the individual leaves did not overlap or overlap across the boundary of the tomograph bed. the acquisition of primary data was initiated using the program explore vista ver. 3.1 under conditions of a static mode during 2 min for every length of the scanning bed position (2 cm). the total number of bed positions was chosen depending on the length of the analyzed plant part. a commercial micropet system consisted of 18 detection modules arranged in a circle with a diameter of 11.8 cm. one detection module was represented by 13 × 13 arrays of 1.45 mm × 1.45 mm × 7.0 mm lutetium oxyorthosilicate (lso) crystals and 1.45 mm × 1.45 mm × 8.0 mm gadolinium orthosilicate single (gso) crystals and was located in time coincidence with the seven opposite modules. this arrangement gives an effective transverse field-of-view of 6.7 cm and an axial fieldof-view of 2.0 cm. the whole system provides a spatial resolution of 1.5 mm and an absolute sensitivity of the central point source of 1.9 % at the 250 – 700 kev energy window used. the control of mentioned micropet system, acquisition and reconstruction of the obtained data in the form of pet images (3d; voxel size 0.3875 mm × 0.3875 mm × 0.775 mm) were performed using the program explore vista ver. 3.1 analysis & visualization software that includes the mathematical algorithms 3d fore (fourier rebinning algorithm) / 2d osem (2d ordered subset expectation maximization). within the reconstruction process, the obtained 2d sinograms were also corrected for accidental coincidences, radioactive decay and dead-time. amide.exe ver. 1.0.4. software was used to visualize 3d pet images after their reconstruction, which allows the reduction of the maximum threshold to improve the visual quality of the regions with a lower accumulated activity. determination of 18f activity gamma-spectrometric analysis was carried out to determine the residual radioactivity of 2-[18f]fdg solution, as well as in the measurement of accumulated 18f activity in individual parts of studied plants. for these purposes, a scintillation gamma-spectrometer with a nai(tl) well type crystal 54bp54/2 (scionix, netherlands) and scintivision-32 software (ortec, usa) were used. standard solutions of 109cdcl2, 2-[18f]fdg and 137cscl with defined activities 101 nova biotechnol chim (2020) 19(1): 98-108 102 at a given time were applied to calibrate (γ-photon energies and measurement efficiency) of the scintillation gamma-spectrometer and a library of 109cd (eγ = 88.04 kev), 18f (eγ = 511 kev) and 137cs (eγ = 661.66 kev) was built. in the case of samples showing high 18f activity (> 1.105 bq) outside the detection limits of the scintillation gamma-spectrometric system used, these samples were analyzed by ionisation chamber curiementor (3ptw, germany). results and discussion root system as a barrier for 2-[18f]fdg transport in the first part of the work, the effect of the characteristics of plant tissues on the uptake and distribution of 2-[18f]fdg, as a model of photoassimilates, and on the visual and quantitative side of the pet analysis was studied. in this way, the experiments with intact pepper (c. annuum l.) plants immersed with the root system or cut stem into a 2-[18f]fdg solution including the pet analysis using a commercial micropet primarily developed for animal objects were carried out. it is generally known that the root system of plants acts as a natural selective barrier determining the solute transport. in this context, we focused on the comparison of the uptake and distribution of 2-[18f]fdg in tissues of pepper plants after 30 min of their exposure by immersing the root system of the intact plant and the stem after the removing the root system into a 2-[18f]fdg solution with known 18f activity and concentration of d-glucose 7.62 mg.ml-1. as can be seen from fig. 1, for both objects analyzed, we obtained visually evaluable pet images. as was found in our previous work partelová et al. (2014), the visual interpretability of pet records regarding the distribution of 2-[18f]fdg in plant tissues as well as the translocation of 2-[18f]fdg from the application part (root system or leaf petiole) to other parts of the plant are significantly affected by the concentration of d-glucose in the applied solution 2-[18f]fdg. in the present experiments, we based these findings and therefore increased the initial concentration of d-glucose in dilute solution of 2-[18f]fdg, which is applied in pet diagnostic tests, 1,000-fold to the concentration of 7.62 mg.ml-1. from the obtained pet quantitative data (table 1), it can be claimed that the accumulation of 2-[18f]fdg expressed in terms of tfc values was several times higher in the aboveground parts that were not directly exposed in 2-[18f]fdg solution than in the parts directly immersed in the solution of 2-[18f]fdg. tfc represents coincidence transfer factor defined as the ratio of the numc (number of analyzed coincidences within the pet analysis) in the non-immersed parts of the plant to the numc in the parts of the plant immersed into a 2-[18f]fdg a b fig. 1. imaging of 2-[18f]fdg distribution in the tissues of pepper (c. annuum l.) plants immersed with the root system (a) or cut stem (b) into a 2-[18f]fdg solution: a. 100 mbq and 7.62 mg.ml-1 of d-glucose; b. 20 mbq and 7.62 mg.ml-1 of d-glucose. after 30 min of exposure plants were analyzed by a commercial micropet system with the acquisition time 2 min for bed position. the maximum threshold th (thresholding-based methods) was reduced to 10 %. nova biotechnol chim (2020) 19(1): 98-108 103 solution. thus, this parameter quantitatively describes the translocation of 2-[18f]fdg within the evaluated plant parts. in the comparison of the analyzed pepper plants, it was found that in the case of plant immersed in a 2-[18f]fdg solution with the stem after removal of the root, the tfc value was 2-times higher than in the case of intact pepper plant immersed in a solution of 2-[18f]fdg by the root system (tfc = 9.4). also, it can be concluded that there was a significantly higher translocation of 2-[18f]fdg and tfc values compared to the values found in previous work (partelová et al. 2014) in the case of tobacco plants (n. tabacum l.), as well as in the case of giant reed plants (a. donax l.) (partelová et al. 2017). in the work partelová et al. (2014), there were reached the values of tfc = 0.04 at d-glucose concentration 0.00762 mg.ml-1 and tfc = 0.21 at 0.762 mg.ml-1, which indicated a significant accumulation of 2-[18f]fdg in the root system and its minimal transfer to the aboveground parts of plants. thus, it is clear that the concentration of d-glucose will play an important role in the distribution of 2-[18f]fdg as a model molecule of photoassimilates within plant tissues in terms of its translocation. at the same time, it must be stated that these processes and characteristics will also depend on the study object – on the given plant species, even in the case of plants originating from the same family (family solanaceae, e.g. tobacco, pepper, and tomato). these facts must be taken into account in terms of pet analysis and its relevant applicability as a non-invasive analytical method. after the pet analysis, the pepper plants were divided into individual parts (root, stem, developmentally older and younger leaves) and analyzed for the accumulated activity of 18f by direct gamma-spectrometry (fig. 2a). based on the obtained results, it can be concluded that in the case of a pepper plant immersed by the root system in a 2-[18f]fdg solution, the accumulated 18f activity decreased proportionally from the submerged root to the aboveground parts, with the lowest 18f activity measured in the developmentally younger leaves. it was found that the removal of the root system resulted in increased translocation and distribution of 2-[18f]fdg within the stem and accumulation of 2-[18f]fdg in developmentally younger leaves in comparison with cotyledon (primary leaves), taking into account the fact of 5-fold lower applied activity of 18f in the case of a pepper plant immersed with the stem into a 2-[18f]fdg solution. this result was also confirmed from the point of view of percentage evaluation of the distribution of 18f activity in individual parts of pepper plants of the total amount of 18f activity accumulated by the plant (fig. 2b). comparing the distribution of 18f activity in the aboveground parts of pepper plants (fig. 2b), the most significant difference can be observed between a plant exposed through the root system (intact plant) and a plant exposed through the stem (plant without root system), especially in the case table 1. initial parameters defining the experiments and obtained quantitative data (variables) from the pet analysis of pepper (c. annuum l.) plants immersed with the root system or cut stem into a 2-[18f]fdg solution containing 7.62 mg.ml-1 of d-glucose. for details, see fig. 1. parameter/ variable intact plant stem mp [g] 0.789 0.671 a0 [mbq] 100 20 numc 4,966 3,457 tfc 9.4 18.0 ev [cm3] 16.73 4.35 pv 143,778 37,351 tgv 11 804,100 63 672,100 max pv 4,557 10,228 mp – fresh weight of the plant biomass; a0 – initial applied activity of 2-[ 18f]fdg solution; numc – number of analyzed coincidences within the pet analysis; tfc – coincidence transfer factor defined as the ratio of the numc in the non-immersed parts of the plant to the numc in the parts of the plant immersed into a 2-[ 18f]fdg solution; ev – estimated volume within the pet analysis; pv – pixel volume; tgv – total gray value; maxpv – maximum pixel value. nova biotechnol chim (2020) 19(1): 98-108 104 fig. 2. a. accumulated 18f activity in the form of 2-[18f]fdg within the individual parts of pepper (c. annuum l.) plants after their 30 min exposure by immersion of the root system or cut stem (plant without root system) into a 2-[18f]fdg solution containing 7.62 mg.ml-1 of d-glucose. b. distribution expressed in percentage of the total 18f activity accumulated by the plant. of older leaves. for the plant without root system, an increased percentage of accumulated 18f activity was confirmed in developmentally older leaves (35 % of the total 18f activity accumulated by the plant), while in the case of plant immersed in a solution of the 2-[18f]fdg by the root system the ratio of 18f activity accumulated in developmentally older leaves represents only 21 %. in the case of plant immersed in the solution of 2-[18f]fdg by root system, a more significant accumulation of 18f activity can be observed, especially in the stem and primary leaves, which accounted for up to 61 % of the total 18f activity accumulated by the plant. effect of d-glucose concentration to confirm the effect of the concentration of d-glucose (cglu) in the applied solution of 2-[18f]fdg on the translocation and distribution of 2-[18f]fdg from the conductive tissues to parenchymal leaf blade cells, the experiments with excised leaves of radish (r. sativus l.) were carried out. in the first experiment, an excised radish leaf was exposed with a petiole in the solution of 2-[18f]fdg with cglu = 0.00762 mg.ml -1 and initial 18f activity a0 = 8 mbq for 30 min under laboratory conditions. subsequent pet analysis showed only a slight a b fig. 3. imaging of 2-[18f]fdg distribution in the tissues of radish (r. sativus l.) leaves immersed with the petiole into a 2-[18f]fdg solution: a. 8 mbq and 0.00762 mg.ml-1 of d-glucose; b. 8 mbq and 0.762 mg.ml-1 of d-glucose. after 30 min of exposure leaves were analyzed by a commercial micropet system with the acquisition time 2 min for bed position. the maximum threshold th (thresholding-based methods) was reduced to 10%. a b nova biotechnol chim (2020) 19(1): 98-108 105 table 2. initial parameters defining the experiments and obtained quantitative data (variables) from the pet analysis of excised leaves of radish (r. sativus l.) immersed with the petiole into a 2-[18f]fdg solution containing 0.00762 mg.ml-1 (leaf a) or 0.762 mg.ml-1 (leaf b) of d-glucose. for details, see fig. 3. parameter/ variable leaf a leaf b ml [g] 0.439 0.435 a0 [mbq] 8.0 8.0 cglu [mg.ml -1] 0.00762 0.762 numc 1 235,000 2 693,000 tfc 4.92 9.03 ev [cm3] 3.156 4.898 pv 27,121 42,090 tgv 3 746,360 7 968,240 max pv 5,589 9,404 ml – fresh weight of the leaf; a0 – initial applied activity of 2-[ 18f]fdg solution; cglu – initial concentration of d-glucose in a 2-[18f]fdg solution; numc – number of analyzed coincidences within the pet analysis; tfc – coincidence transfer factor defined as the ratio of the numc in the non-immersed parts of the leaf to the numc in the parts of the leaf immersed into a 2-[18f]fdg solution; ev – estimated volume within the pet analysis; pv – pixel volume; tgv – total gray value; maxpv – maximum pixel value. transfer of 2-[18f]fdg from the major venous to the secondary veins of the radish leaf blade (fig. 3). in contrast, in the case of pet analysis of radish leaf exposed under the same conditions as in the previous experiment, but in the solution of 2-[18f]fdg with a 100-fold higher cglu, obtained 3d pet record clearly indicates the increase of the translocation of 2-[18f]fdg into the secondary veins of the leaf blade. the observed increase in translocation of 2-[18f]fdg was also confirmed on the basis of quantitative data obtained from the pet analysis (table 2), such as numc, tgv (total gray value) and especially tfc, the values of which determine and clearly correlate with the increased distribution of 2-[18f]fdg within the radish leaf blade. we suggest two hypotheses which may explain these findings. both are associated with phloem transport of photoassimilates. the first hypothesis is related to the mechanism of sugar transport in plant conductive tissues. in plant physiology, the mechanisms responsible for sugar transport within plants are well known that sugars produced by photosynthesis are transported through the phloem to the sink tissues. the second hypothesis is based on the effect of environmental conditions on the composition and viscosity of phloem sap, which determines the rate of phloem flow and translocation of nutrients or photoassimilates (van bel and hess 2008; liu et al. 2012). in this context, it is known that if the sugar concentration is low, only small amounts of energy are transferred from the source to the sink tissues. on the contrary, if it is too high the viscosity of the phloem sap impedes the flow. effect of “escaping positrons” in the last type of experiments, we focused on the phenomenon of "escaping positrons", which is related to the annihilation of positrons outside plant tissues as very thin structures. this negative effect in the case of plant structures and organs (especially leaves) significantly affects the qualitative and quantitative aspects of pet analysis of the uptake, transport and distribution of radioindicators in living plants. the mentioned phenomenon was previously studied and described by some authors through the covering the analyzed plant object with different types of plastics or metal foils. alexoff et al. (2011) measured the magnitude and distribution of escaping positrons from the leaf of tobacco (n. tabacum l.) for the radionuclides 18f, 11c and 13n using a commercial small-animal pet scanner. they were used a thin plate of plexiglass to prevent the escape positrons and the annihilation of positrons outside the studied object. in the fig. 4 can be seen the distribution of 2-[18f]fdg after 30 min of exposure of tobacco (n. tabacum l.) leaf immersed with excised leaf tip into a 2-[18f]fdg solution containing 0.00762 mg.ml-1 d-glucose and initial 18f nova biotechnol chim (2020) 19(1): 98-108 106 fig. 4. imaging of 2-[18f]fdg distribution in the tissues of tobacco (n. tabacum l.) leaf immersed with the excised apex of the leaf blade into a 2-[18f]fdg solution (a0 = 40 mbq; cglu = 0.00762 mg.ml -1). after 30 min of exposure leaf was analyzed by a commercial micropet system with the acquisition time 2 min for bed position and under conditions: (a) uncovered leaf; (b) leaf covered by a parafilm; (c) leaf covered by an aluminium foil of thickness 0.1 mm; or (d) leaf covered by a copper plate of thickness 0.5 mm. the maximum threshold th (thresholding-based methods) was reduced to 10%. table 3. obtained quantitative data (variables) from the pet analysis of tobacco (n. tabacum l.) leaf under conditions: uncovered leaf (leaf a); leaf covered by a parafilm (leaf b); leaf covered by an aluminium foil of thickness 0.1 mm (leaf c); or leaf covered by a copper plate of thickness 0.5 mm (leaf d). for details, see fig. 4. parameter/variable leaf a leaf b leaf c leaf d ev [cm3] 2.027 3.314 2.478 3.061 pv 17,415 28,480 21,291 26,307 tgv 5 659,020 6 931,595 6 167,039 7 321,765 mean tgv/ev 2 792,370 1 831,720 2 020,860 1 800,080 mean tgv/pv 325.0 213.2 235.2 209.5 max pv 15,855 9,656 11,510 9,665 ea [mbq] 1.043 1.269 1.134 1.339 ev – estimated volume within the pet analysis; pv – pixel volume; tgv – total gray value; mean tgv/ev – mean total gray value divided by estimated volume of the leaf; mean tgv/pv – mean total gray value divided by pixel volume of the leaf; maxpv – maximum pixel value; ea – estimated 18f activity accumulated in the leaf within the pet analysis. activity a0 = 40 mbq. thus, the transport of photoassimilates in the basipetal way in this experiment was studied. after the primary pet analysis of tobacco leaf without covering (a), this leaf was subsequently covered with a parafilm (b), aluminium foil (c; thickness 0.1 mm) or copper thin plate (d; thickness 0.5 mm). based on the obtained 3d pet images (fig. 4), it can be claimed that visually there were no significant changes in imaging the distribution of 2-[18f]fdg in tobacco leaf tissues. however, quantitative data obtained from the pet analysis indicate significant changes in the values of the defined variables. in the table 3, it can be seen that in the case of covering the leaf with a parafilm or copper plate, higher values were obtained for the parameters or variables, such as estimated volume of the visualized leaf (ev), pixel volume (pv), as well as the total gray value (tgv) and estimated activity of 2-[18f]fdg according to the linear regression equation tgv vs. va (volume activity of 18f) compared to uncovered leaf. on the other hand, a significantly lower value of maximum pixel value (max pv) was found, which defines the maximum value of pixel intensities in the entire 3d image of the object. these changes in the values of the analyzed parameters or variables can be explained nova biotechnol chim (2020) 19(1): 98-108 107 by the principle that the covering materials – parafilm or copper thin plate used to cover the tobacco leaf showed the ability to absorb part of the "escaping positrons" as well as γ-photons released during the annihilation process, which could result in reduced radioactive hot spots in the leaf blade. this fact is also confirmed by the lower values of max pv estimated for these cases. in this context, it must be added that lower values of max pv can lead in the reconstruction of 3d pet images to obtain richer quantitative information in the form of higher tgv values (also in relation to ea) due to the fact that sites with lower pixel intensities within the studied object were not excluded to such an extent as in the case of wider ranges of pixel intensities (higher values of max pv). these facts can also result in higher values of ev and pv as well. conclusions in the last two decades, pet has undergone significant development, aimed primarily to increase the spatial resolution of scanned objects, thus not only improving the existing application capabilities, but enhancing its advantages in terms of obtaining quantitative results. an interesting fact is that the development of this modern imaging technique was to a significant extent also related to the studied objects belonging to research in the field of plant biology. thus, while following specific methodological approaches, pet is included in the group of quantitative nuclear analytical methods. due to the size and shape of plant objects compared to conventional pet objects, obtaining quantitative analytical results in the study of plants encounters difficulties resulting from thin structures of plant tissues. our results showed that commercial pet systems, developed primarily for small laboratory animals, can also be used for the non-invasive 3d imaging and quantitative evaluation of in vivo radiotracers dynamics in plants with sizable organs or structures (in micrometres; e.g. leaves) which are below the limits of the spatial resolution of these imaging systems. we also found that in order to obtain relevant results in plant research focused on plant production or on the study of the accumulation of toxic organic substances (e.g. pesticides) or toxic metals, it will be necessary to take into account various factors influencing pet analysis. as our results showed, these factors include the characteristics of the studied plant tissues and the processes taking place in them, the chemical characteristics of the applied radioindicator solution – the chemical concentration of the carrier as well as the phenomenon of "escaping positrons" occurring in plants as thin objects. acknowledgement this work was supported by the slovak research and development agency under the contract no. apvv-15-0098. conflict of interest the authors declare that they have no conflict of interest. references alexoff, dl, sdewey sl, vaska p, krishnamoorthy s, ferrieri r schueller m schlyer dj, fowler js (2011) pet imaging of thin objects: measuring the effects of positron range and partial-volume averaging in the leaf of nicotiana tabacum. nucl. med. biol. 38: 191-200. berg e, cherry sr (2018) innovations in instrumentation for positron emission tomography. semin. nucl. med. 48: 311-331. fatangare a, svatoš a (2016) applications of 2-deoxy-2fluoro-d-glucose (fdg) in plant imaging: past, present, and future. front. plant sci. 7: 483. ferrieri ap, appel h, ferrieri ra, schultz jc (2012) novel application of 2-[18f]fluoro-2-deoxy-d-glucose to study plant defenses. nucl. med. biol. 39: 1152-1160. hattori e, uchida h, harada n, ohta m, tsukada h, hara y, suzuki t (2008) incorporation and translocation of 2-deoxy-2-[18f]fluoro-d-glucose in sorghum bicolour (l.) moench monitored using a planar positron imaging system. planta 227: 1181-1186. hoagland dr (1920) optimum nutrient solution for plants. science 52: 562-564. hubeau m, steppe k (2015) plant-pet scans: in vivo mapping of xylem and phloem functioning. trends plant sci. 20: 676-685. karve aa, alexoff d, kim d, schueller mj, ferrieri ra, babst ba (2015) in vivo quantitative imaging of photoassimilate transport dynamics and allocation in large plants using a commercial positron emission tomography (pet) scanner. plant biol. 15: 273. kurita k, miyoshi y, nagao y, yamaguchi m, suzui n, yin y-g, ishii s, kawachi n, hidaka k, yoshida e, takyu s, tashima h, yamaya t (2020) fruit pet: 3-d imaging of carbon distribution in fruit using openpet. nucl. instrum. methods phys. res. 954: 161843. nova biotechnol chim (2019) 18(2): 98-108 108 liu dd, chao wm, turgeon r (2012) transport of sucrose, not hexose, in the phloem. j. exp. bot. 63: 4315-4320. moein mm, nakao r, amini n, abdel-rehim m, schou m, halldin c (2019) sample preparation techniques for radiometabolite analysis of positron emission tomography radioligands; trends, progress, limitations and future prospects. trends anal. chem. 110: 1-7. partelová d, uhrovčík j, lesný j, horník m, rajec p, kováč p, hostin s (2014) application of positron emission tomography and 2-[18f]fluoro-2-deoxy-d-glucose for visualization and quantification of solute transport in plant tissues. chem. pap. 68: 1463-1473. partelová d, horník m, lesný j, rajec p, kováč p, hostin s (2016) imaging and analysis of thin structures using positron emission tomography: thin phantoms and in vivo tobacco leaves study. appl. radiat. isot. 115: 87-96. partelová d, kuglerová k, konotop y, horník m, lesný j, gubišová m, gubiš j, kováč p, matušíková i (2017) imaging of photoassimilates transport in plant tissues by positron emission tomography. nova biotechnol. chim. 16: 32-41. suzui n, tanoi k, furukawa j, kawachi n (2019) recent advances in radioisotope imaging technology for plant science research in japan. quantum beam sci. 3: 18. thorpe cl, williams ha, boothman ch, lloyd jr, morri k (2019) positron emission tomography to visualise in-situ microbial metabolism in natural sediments. appl. radiat. isot. 144: 104-110. tran tm, hampton cs, brossard tw, harmata m, robertson jd, jurisson ss, braun dm (2017) in vivo transport of three radioactive [18f]-fluorinated deoxysucrose analogs by the maize sucrose transporter zmsut1. plant physiol. biochem. 115: 1-11. tsuji a, uchida h, yamashita t, matsuhashi s, ito t, mizuniwa c, ishioka ns, watanabe s, sekine t (2002) uptake of 18fdg and 13noin tomato plants. in: saidoh m, toraishi a, namba h, itoh h, tanaka s, naramoto h, et al. (eds.), tiara annual report 35: 103-104. van bel aje, hess ph (2008) hexoses as phloem transport sugars: the end of a dogma? j. exp. bot. 59: 261-272. wang q, mathews aj, li k, wen j, komarov s, o`sullivan ja, tai, y-c (2014) a dedicated high-resolution pet imager for plant sciences. phys. med. biol. 59: 5613-5630. weisenberger ag, kross b, lee sj, mckisson j, mckisson je, xi w, zorn c, howell cr, crowell, as, reid cd, smith m (2013) nuclear physics detector technology applied to plant biology research. nucl. instrum. methods phys. res. 718: 157-159. nova biotechnol chim (2018) 17(2): 103-114 doi: 10.2478/nbec-2018-0011  corresponding author: salampradeep@gmail.com nova biotechnologica et chimica characterization and assessment of biosurfactant producing indigenous hydrocarbonoclastic bacteria: potential application in bioremediation pranjal bharali1, salam pradeep singh2,3,, yasir bashir3, nipu dutta4, bolin kumar konwar3 and chingakham brajakishor singh2 1department of environmental science, nagaland university, hq: lumami, zunheboto, 798627, nagaland, india 2institute of bio-resources and sustainable development, takyelpat, imphal, 795001, manipur, india 3department of molecular biology and biotechnology, tezpur university, tezpur, 784028, assam, india 4department of chemical sciences, tezpur university, napaam, tezpur, 784028, assam, india article info article history: received: 13th march 2018 accepted: 20th august 2018 keywords: bacillus circulans biodegradation bioremediation biosurfactant hydrocarbonoclastic pseudomonas aeruginosa abstract petroleum and hydrocarbons contamination can be remediated by physical, chemical or biological methods. among these, in situ bioremediation is considered to be environmentally friendly because it restores the soil structure, requires less energy input and involves the notable removal after degradation of biosurfactant. the present study involves the characterization and assessment of biosurfactant producing indigenous hydrocarbonoclastic bacteria and their potential application in bioremediation processes. three bacterial strains were isolated from various crude oil contaminated environments and characterized using standard identification techniques. the results clearly demonstrate the capability of utilizing hydrocarbon and biosurfactant produced by the bacterial strains. 16s rdna sequencing followed by blast analysis revealed their similarity to pseudomonas aeruginosa. the physico-chemical characterization of the biosurfactants revealed significant surface properties with stability at extreme temperature conditions (up to 121 ˚c), ph (5 – 8) and salinity (up to 4 %). further, the mass spectrometry confirmed predominance of di-rhamnolipids in biosurfactant mixtures. the biosurfactants were found to be efficient in the removal of crude oil from the contaminated sand suggesting its applicability in bioremediation technology. further, improved discharge of crude oil at elevated temperatures also confirms their thermo-stability which, could be exploited in microbial enhanced oil recovery processes. thus, the applications of biosurfactants produced by the indigenous hydrocarbonoclastic strains appeared to be advantageous for bioremediation of petroleum-contaminated environments.  university of ss. cyril and methodius in trnava introduction soil which is accidentally contaminated with petroleum hydrocarbons can be remediated by physical, chemical or biological methods. among all, in situ bioremediation is considered to be environmentally friendly because it restores the soil structure, requires less energy input, and involves the notable removal after degradation of biosurfactant. although most of the hydrocarbons are biodegradable, the rate of biodegradation in the environment is limited due to their hydrophobicity or less accessibility to microbes and low aqueous solubility (radzuan et al. 2017). one of the approaches to enhance biodegradation of crude oil contamination is the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:14 utc mailto:salampradeep@gmail.com nova biotechnol chim (2018) 17(2): 103-114 104 use of biosurfactants which increase the solubility of hydrophobic substrates/oils in an aqueous medium to enhance their bioavailability leading to higher oil degradation (zhang et al. 2017). the treatment of crude oil contaminated soil and environments through the indigenous biosurfactant producing bacteria having the capacity to degrade the petroleum hydrocarbons seems to be advantageous (kuppusamy et al. 2017). most of the bacteria such as pseudomonas putida, p. aeruginosa, p. saccharophila, flavobacterium sp., burkholderia cepacia, rhodococcus sp., and mycobacterium sp. etc. dwelling in the crude oil contaminated environment have the capacity to utilize the components of crude oil as the source of carbon and energy for their growth. they tend to secrete biosurfactants which in turn help in the degradation of crude oil components (radzuan et al. 2017). moreover, the production of biosurfactant at the site of treatment with the producer bacteria doesn’t require rigorous testing like that of the chemical surfactants because of their environment-compatible nature (pristas et al. 2015; rahman et al. 2016). therefore, the application of biosurfactants in bioremediation might be more acceptable from the social point of view. thus there is a need for increased production of biosurfactants and their characterization. the present study focuses on the application of indigenous bacterial strains for bioremediation processes as there is no need for them to get established in a new environment and they can coexist with the already inhabiting microbiota in the soil. their population density can be enhanced by supporting their growth factors. moreover, these bacteria don’t need to add biosurfactant manually being able to produce biosurfactants by utilizing the contaminating petroleum hydrocarbons. the study involves the isolation, characterization, and application of the biosurfactants along with their producer strains in bioremediation processes. experimental isolation of biosurfactant producing hydrocarbonoclastic bacteria the environmental samples includes crude oilcontaminated soils of oil fields, petroleum sludge and waste residual crude oil from the dump sites of oil and natural gas corporation (ongc), jorhat, assam; petroleum sludge from the oil fields of ongc, sibsagar, assam; contaminated soil samples from the different oil depots of tezpur, assam. the bacterial strains were isolated from the above samples using enrichment culture technique on mineral salt medium (msm) (bharali and konwar 2011). morphologically different bacterial colonies thus obtained were further purified on msm agar plates with or without 0.1 % (v/v) n-hexadecane to eliminate autotrophs and agarutilizing bacteria. the procedure was repeated and isolates showing pronounced growth on n-hexadecane were selected and preserved for further characterization (parthipan et al. 2017). screening for biosurfactant production by the selected strains was done using de novo ring method, qualitative drop-collapse test and oil displacement test (bodour and maier 1998). detection and quantification of biosurfactant produced by the selected strains were determined by three independent tests viz. cetyl triammonium bromide (ctab) agar test (siegmund and wagner 1991), blood agar test (johnson and boesemarrazzo 1980) and orcinol assay (chandrasekaran and bemiller 1980). molecular and morphological characterization of selected isolates the 16s rrna gene sequencing of the selected bacterial isolates was performed and the similarity search for the sequences thus obtained was done using ncbi blast. the sequences showing maximum similarity from the blast search were taken for phylogenetic analysis using mega 5.1 (kumar et al. 1994). on the basis of partial 16s rrna gene sequencing as well as with the use of ncbi blast, these three bacterial isolates obp2, obp3 and obp4 were found to be closely related to pseudomonas aeruginosa with 99 % similarity in all the strains. the partial 16s rrna gene sequences of the bacterial strains obp2, obp3 and obp4 were deposited in the genbank database under the accession numbers 1568199, 1568206 and 156820, respectively. phylogenetic tree was constructed using the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:14 utc nova biotechnol chim (2018) 17(2): 103-114 105 neighbour-joining method and all the three bacterial isolates were identified as pseudomonas aeruginosa using mega 5.1 (kumar et al. 1994). on the basis of colony morphology, physiology and biochemical characterization details three selected bacterial isolates were found to be under the genus pseudomonas. isolation of biosurfactants each culture broth was centrifuged at 12,000 rpm for 20 min at 4 ˚c. 6 n hcl was used to adjust the ph of the culture supernatant at 2.0 and kept overnight at 4 °c. the precipitate thus obtained was extracted thrice with an equal volume of ethyl acetate. the upper organic phase was collected and dried. the brownish oily residue left behind was then dissolved in 3.0 ml of 0.1 m sodium phosphate buffer (ph 7.2) (mulligan and gibbs 2004). chemical characterization of biosurfactants the thin layer chromatography technique (tlc) was used to detect the carbohydrate and lipid moieties using a mobile phase system consisting of chloroform: methanol: water (65 : 15 : 2). anthrone, ninhydrin and iodine fumes were used as developing agents for carbohydrate, amino acids and lipid, respectively (silva et al. 2017). the ir spectrum of the isolated biosurfactant was recorded in potassium bromide pellet using an ftir spectrophotometer (nicolos impact 410). the liquid chromatographic and mass spectroscopic (lc-ms) study of the isolated biosurfactant samples was carried out using the standard protocol (haba et al. 2003) on a perkin elmer system. the carrier gas was helium, maintained at the flow rate of 1.5 l.min-1. physical characterization estimation of surface activity the surface tension of the cell-free culture supernatants was measured using a digitalized tensiometer (krűss tensiometer k9 et/25) at 25±1 °c (bodour and maier 1998). the interfacial tension was measured against diesel. each experiment was repeated thrice. critical micelle concentration of the isolated biosurfactant samples was determined by measuring the surface tension of the aqueous biosurfactant at different concentrations up to the constant value of surface tension. further, the surface tension of aqueous biosurfactant samples was determined at their critical micelle dilutions (cmd) of 10 and 100-times, i.e., cmd−1 and cmd−2, respectively. each measurement was repeated in triplicates. emulsification activity (e24) and foaming index (f24) the emulsification index (e24) (in %) was measured using the standard method described by kumar et al. (2015). different hydrocarbons were used for testing the emulsification efficiency. 20 ml of each biosurfactant solution (1 g.l−1) was transferred to a glass measuring cylinder and compressed n2 gas was passed through the solution at a flow rate of 0.5 l.min−1 for 2 min (zaman 2010; garcía-reyes et al. 2017). the foaming index (f24) (in %) of the biosurfactant samples was calculated after 24 h. stability in surface properties to determine the stability in relation to the temperature, 10 ml of each cell-free culture supernatant was exposed to various temperature viz. 4 °c, 25 °c, 37° c, 50 °c, 75 °c, and 100 °c for 60 min and 121 °c for 30 min (i.e. autoclaving) separately. after the treatment, the cell-free culture supernatants were kept at laboratory temperature (lt) (20±1 °c) for 20 min after which the surface tension of each culture supernatant was measured at normal concentration (i.e. without dilution), at cmd−1 and cmd−2 and the emulsification index against diesel was measured. to study the ph, the stability of the biosurfactant and the ph of the culture supernatant were adjusted to different ph values (2 – 11). the surface tension of each treated culture supernatant was also measured at normal concentration, and emulsification index against diesel was measured. similarly, the effect of nacl concentrations (1 – 5 %) on the surface tension bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:14 utc nova biotechnol chim (2018) 17(2): 103-114 106 table 1. influence of ph on the surface activity of biosurfactant produced by p. aeruginosa strains in 2 % n-hexadecane supplemented medium at normal and critical micelle dilutions (cmd−1 and cmd−2). bacterial strains surface tension [mn/m] ph cell-free culture supernatant cmd-1 cmd-2 p. aeruginosa obp2 2 41.5±0.58 48.9±0.78 63.7±0.31 5 37.8±0.35 41.0±0.23 58.3±0.46 7 37.6±0.11 40.7±0.83 57.6±0.29 8 38.1±0.34 41.5±0.69 58.8±0.36 11 39.7±0.53 43.2±0.45 61.0±0.53 2 40.4±0.36 46.3±0.93 63.5±0.29 p. aeruginosa obp3 5 35.7±0.31 38.7±0.67 56.8±0.49 7 35.4±0.19 38.2±0.30 55.6±1.00 8 36.5±0.83 39.5±0.47 57.4±0.19 11 38.7±0.62 42.5±0.61 60.8±0.38 2 38.5±0.43 43.3±0.60 57.1±0.51 p. aeruginosa obp4 5 33.7±0.76 39.8±0.71 50.3±0.94 7 33.1±0.93 39.5±0.35 48.7±0.54 8 33.6±0.51 39.7±0.29 51.4±0.66 11 35.8±0.23 41.4±0.72 54.9±0.39 values represent mean of three replicates. of the culture supernatant at normal concentration was studied and emulsification activity was determined. synthetic surfactant sodium dodecyl sulphate (sds) was used as a standard and the assays were carried out in triplicates. possible industrial application of biosurfactant ‒ sand pack column experiment the sand pack column experiment was carried out as described by suthar et al. (2008). a vertical glass column with a dimension of 25 cm × 3cm (internal diameter) was packed with 150 g of acidwashed sand. the column was initially saturated with brine (5 % nacl solution), followed by flooding with crude oil supplied by oil and natural gas corporation limited (ongcl), assam, india. the column was further flooded with brine until no further oil appeared in the effluent. finally, the cell-free culture supernatant of stationary phase of growth was pumped into the column. the oil released from the column after exposure to various temperatures (20, 50, 70 and 90 °c) was measured. each experiment was repeated in triplicates. soil washing experiment crude oil contaminated sand samples, each weighing 20g, were transferred to 250 ml erlenmeyer flasks containing 100 ml aqueous biosurfactant solution of various concentrations (0.001, 0.005, 0.007, 0.01 and 0.1 %; w/v) and kept at 150 rpm for 24 h at laboratory temperature (lt) (20±1 °c). the contaminated sand samples were separated, dried and washed twice with dichloromethane. the solvent part was removed and the residual oil was determined gravimetrically (chen et al. 2017). results and discussion isolation and characterization of potential biosurfactant producing hydrocarbonoclastic bacteria a total of 52 cultivable isolates were obtained based on colony morphology analysis. on further experimentation, 3 isolates were found to be the potential users of hydrocarbons and producers of biosurfactants as determined by their biomass yield and surface properties. thus, causing a reduction in the surface tension of the culture medium from 37.6±0.51 mn.m−1 to a minimum of 33.2±0.79 mn.m−1, while growing on n-hexadecane. the bacterial isolates were designated as obp2, obp3 and obp4. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:14 utc nova biotechnol chim (2018) 17(2): 103-114 107 among the screening techniques, all the selected bacterial strains showed positive results in hemolysis assay, ctab agar assay, and glycolipid quantification test. all three bacterial strains exhibited a distinct zone of hemolysis in blood-agar plates containing 2 % (v/v) goat blood. hemolysis assay is widely used for the screening of biosurfactant producing bacteria and could be used for the quantification of glycolipid-type biosurfactant produced by bacteria (banat 1993; yonebayashi et al. 2000). all the three bacterial strains could grow on ctab agar plates forming blue halos around the colonies. the ctab agar test is another technique used for the detection of glycolipid-type biosurfactant production by the bacterial colonies in the culture plate directly (irorere et al. 2017). aparna et al. (2012) reported screening of rhamnolipid producing thermophilic hydrocarbon-degrading pseudomonas aeruginosa strains by ctab agar method from the soil contaminated with petroleum products. the concentration of biosurfactant present in the culture medium of the bacterial strains was determined using the orcinol assay and the production was 8.8±0.6, 9.2±1.1 and 9.8±0.5 for obp2, obp3 and obp4, respectively. the genus pseudomonas is reported to be the most versatile group due to its inherent ability to utilize a diverse range of substrates as the carbon source, particularly those found in petroleum (saikia et al. 2012). bordoloi and konwar (2007) reported the predominance of the bacterial genus pseudomonas in the north eastern region of india especially in the petroleum contaminated environments of assam and assam-arkan basin, ongc. physical characterization of biosurfactants the surface tension of the culture medium of bacterial strains obp2, obp3 and obp4 were drastically reduced by the bacterial strains from 68.5 to 37.6±0.51, 35.5±0.38, 33.2 ± 0.79 mn.m−1, respectively. reductions in the ift of diesel containing culture supernatant of bacterial strains obp2, obp3 and obp4 as compared to the control culture medium without any bacteria are 3.4±0.48, 2.8±0.93, 2.2±0.28 mn.m−1 respectively. the biosurfactant produced by the bacterial strains was able to reduce the surface tension of the culture medium significantly. further, they were effectively reducing the ift between diesel and fig. 1. effect of (a) ph, (b) temperature, and (c) salinity (nacl) on the emulsifying properties (e24) of culture supernatants of p. aeruginosa strains against diesel. results represent the mean of three independent experiments ± standard deviation. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:14 utc nova biotechnol chim (2018) 17(2): 103-114 108 table 2. influence of temperature on the surface activity of biosurfactant produced by p. aeruginosa strains in 2 % n-hexadecane supplemented medium at normal and critical micelle dilutions (cmd−1 and cmd−2). bacterial strains exposure to 60 min at the temperature [°c] surface tension [mn.m-1] cell-free culture supernatant cmd-1 cmd-2 p. aeruginosa obp2 4 38.3±0.41 43.0±0.25 58.1±0.29 25 37.5±0.38 40.3±0.48 57.6±0.38 37 37.6±0.28 40.8±0.39 57.5±0.65 50 37.8±0.37 41.1±0.52 57.8±0.75 75 38.0±0.51 41.6±0.41 58.2±0.28 100 39.4±0.20 43.9±0.40 59.3±0.47 121 (for 30 min) 39.2±0.51 44.2±0.35 59.1±0.76 p. aeruginosa obp3 4 36.8±0.39 39.6±0.77 56.8±0.87 25 35.5±0.38 38.4±0.93 55.3±0.54 37 35.3±0.40 38.3±0.38 55.5±0.39 50 35.3±0.19 38.7±0.84 55.9±0.82 75 35.9±0.36 39.4±0.62 56.4±0.39 100 38.3±0.72 41.5±0.65 59.2±0.35 121 (for 30 min) 38.2±0.10 41.8±0.94 59.6±0.62 p. aeruginosa obp4 4 34.7±0.39 40.6±0.54 49.5±0.82 25 33.2±0.91 39.8±0.77 48.7±0.22 37 33.2±0.32 39.7±0.83 48.8±0.91 50 33.5±0.17 39.7±0.39 49.2±0.30 75 33.7±0.63 40.2±0.98 49.7±0.45 100 34.0±0.72 43.8±0.74 51.4±0.76 121 (for 30 min) 34.6±0.83 44.1±0.59 51.2±0.98 data represent mean values, n = 3. water. it is considered as an important factor in oil recovery because capillary number increases with the decrease in the interfacial tension (hajibagheri et al. 2017). the cmc values were found to be in the range of 105±0.34, 90±0.58, 65±0.94 mg.l−1 respectively. the cmc values obtained in this investigation differed within the strains as well as the other reported strains of p. aeruginosa. previously, a range of cmc values between 10 ‒ 230 mg.l−1 was reported for the rhamnolipids isolated from the different microbial sources. such variation in the cmc values might be due to the intrinsic variability of the rhamnolipids accumulated and the complexity of its composition, number and proportions of homologues. the presence of unsaturated bonds, branching and length of the aliphatic chain of the rhamnolipid collectively affect the cmc and surface tension values between the rhamnolipids produced. in most of the instances, reduction in the surface tension at cmd-1 was almost similar to that of normal, whereas the cmd-2 caused a slight increase in the surface tension of the system due to the higher dilution. however, the biosurfactant of the bacteria strains maintains significant extent of their surface property which indicates their possible application in meor technology. addition to the surface and interfacial tensions, stabilization of an oil-water emulsion is usually used as an indicator of surface activity. the bacterial strains showed a wide difference in the emulsification activity against the test hydrocarbons. the cell-free culture supernatant of the bacterial strains exhibited appreciable emulsification indices against tested hydrophobic substrates especially against diesel, n-hexadecane followed by n-octadecane and crude oil. further, the preference for hydrophobic substrates and the behavior of emulsification activity (e24) were quite different among these p. aeruginosa strains. this may be because most of the microbial surfactants are substrate specific causing bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:14 utc nova biotechnol chim (2018) 17(2): 103-114 109 solubilization or emulsification of hydrocarbons at different rates. fig. 2. thin-layer chromatogram of biosurfactants produced by p. aeruginosa strains when cultivated in mineral salt medium supplemented with n-hexadecane. (a) p. aeruginosa obp2, (b) p. aeruginosa obp3, and (c) p. aeruginosa obp4. f1-f3 represents fractions that exhibit positive results of surface activity. the water-oil emulsions were found to be compact and remained stable at laboratory temperature (lt) (20±1 °c) for more than one month suggesting a possible application of the biosurfactants in the bioremediation process for enhancing the availability of the recalcitrant hydrocarbons. moreover, the ability of the biosurfactants to emulsify specifically the crude oil products might facilitate their microbial assimilation, which could be useful for the bioremediation of petroleum contaminated environments. additionally, the ability of the biosurfactants to emulsify the vegetable oil also suggests their potential application in the pharmaceutical and cosmetic industries. the cell-free culture supernatant of each bacterial strain produced stable foam with the foaming index (f24) in the range of 50.4 ‒ 59.8 %. foams produced by the cell-free culture supernatants of all three bacterial strains remained relatively stable up to 24 h. such characteristics of biosurfactants indicate their possible application in coal and mineral froth-flotation as a frothing and co-frothing agent (garcía-reyes et al. 2017). biosurfactants produced by the bacterial strains at normal cmd-1 and cmd-2 concentrations were found to stable and showed optimum surface activity at ph 5 ‒ 8 (table 1). the emulsification activity (e24) of the cell free culture supernatant of the bacterial strains against diesel was quite stable at ph 5-8, though the optimum emulsifying activity was observed between ph 7 ‒ 8 (fig. 1a). considerable reduction in the stability as well as in the surface activity of biosurfactants was observed beyond ph 8. such decrease in the surface activity might be due to the alteration of surfactant structures at the extreme ph conditions. increase in ph from 5 ‒ 8 caused an increase in the negative charge on the polar head of the rhamnolipid molecule (pka 5.6) enhancing its solubility in water. however, below ph 5, table 3. influence of salinity on the surface activity of biosurfactant produced by p. aeruginosa strains in 2 % n-hexadecane supplemented medium at normal and critical micelle dilutions (cmd−1 and cmd−2). bacterial strains nacl concentration [%] surface tension [mn.m-1] cell-free culture supernatant cmd-1 cmd-2 p. aeruginosa obp2 0 37.4±0.43 40.6±0.48 57.7±0.26 2 37.6±0.26 40.9±0.71 57.5±0.40 3 37.9±0.39 41.0±0.39 57.8±0.40 4 40.5±0.48 43.7±0.51 59.6±1.00 5 46.6±0.65 50.3±0.30 61.9±0.49 p. aeruginosa obp3 0 35.4±0.28 38.3±0.83 55.5±0.72 2 35.4±0.76 38.5±0.62 55.6±0.49 3 35.7±0.39 39.9±0.96 55.6±0.49 4 37.3±0.49 41.0±0.70 58.3±0.02 5 43.1±0.65 48.8±0.52 59.8±0.39 p. aeruginosa obp4 0 33.1±0.38 39.7±0.30 48.6±0.56 2 33.3±0.29 39.6±0.73 48.6±0.75 3 33.6±0.48 39.7±0.40 48.9±0.95 4 36.0±0.20 42.5±0.67 50.4±0.34 5 39.6±0.29 48.2±0.93 53.7±0.94 a b c bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:14 utc nova biotechnol chim (2018) 17(2): 103-114 110 table 4. chemical composition of rhamnolipid mixture produced by p. aeruginosa strains as determined by mass spectroscopic analysis. bacterial strains rhamnolipid congeners pseudomolecular ion [m/z] p. aeruginosa obp2 rha-c8:2 302 rha-c12:2 357 rha-c10-c10 501 rha-c10-c12:1 527 rha-rha-c10-c12 531 rha-rha-c12-c10 531 rha-rha-c10-c10 648 rha-rha-c10-c12:1 675 p. aeruginosa obp3 rha-c8:2 302 rha-c10 333 rha-c8-c10 477 rha-rha-c10 480 rha-c10-c10 502 rha-c10-c12:1 529 rha-rha-c10-c12 531 rha-rha-c12-c10 531 rha-rha-c8-c10 622 p. aeruginosa obp4 rha-c8:2 302 rha-c10 332 rha-c12:2 358 rha-c8-c10 477 rha-c10-c10 502 rha-c10-c12 532 rha-rha-c8-c10 622 rha-rha-c10-c10 648 rha-rha-c10-c12:1 673 rha-rha-c10-c12 679 surface activity decreased due to the protonation of the rhamnolipid molecules affecting their precipitation (xia et al. 2011). biosurfactants maintained their surface activity after exposure to temperatures from 4 ‒ 100 ˚c. the cell-free culture supernatants also exhibited stable surface activity even after autoclaving at 121 ˚c for 30 min (table 2). such thermal stability of biosurfactants indicates its utility in those industries where wet sterilization is of principal importance. the emulsification activity (e24) of the culture supernatant of each bacterial strain against diesel was quite stable at all the tested temperatures (fig. 1b). emulsions were found to be stable up to one month at lt. emulsions were found to be stable up to one month at laboratory temperature (20±1 °c). it is interesting to note that biosurfactants retained about 53 % of their original emulsifying properties even after autoclaving, indicating their thermal stability which broadens the scope of their application in microbial enhance oil recovery (meor) process. the biosurfactants retained their surface activity up to the addition of 5 % nacl at both normal and cmd-1 conditions (table 3). the culture supernatants retained significant surface activity at cmd-2. emulsification activity (e24) of culture supernatants against diesel remained almost unchanged upto the addition of 3 % nacl (fig. 1c). increase in the salt concentration beyond 4 %, caused a significant reduction in the emulsification activity (e24) of culture supernatants. such reduction in e24 might be due to the formation of na+‒rhamnolipid complexes that reduce surface tension values. the stability of culture supernatants against higher ph and salinity suggests their applicability in bioremediation of marine environments and in industries where high salinities and ph prevail (bharali et al. 2014). chemical characterization of biosurfactants the tlc derived fractions showed a positive reaction to sugars with orcinol reagents and lipids with iodine vapours but possessed negative reaction to amino groups with ninhydrin. the lower bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:14 utc nova biotechnol chim (2018) 17(2): 103-114 111 fractions of the average rf value of 0.32 while the other with an average rf value of 0.71 (fig. 2). the separated fractions in tlc plates that show positive results to sugars with orcinol reagents and lipids with iodine vapors clearly indicates the presence of both glycosyl units and lipid moieties on the same spots. this confirmed the glycolipid nature of the biosurfactant produced by the bacteria. the lower fraction with the average rf value of 0.32 indicated the presence of di-rhamnolipids while other with an average rf value of 0.71 for mono-rhamnolipid molecules. these observations were found to be consistent with the reports of garcía-reyeset et al. (2017). ftir spectrum of obp2 biosurfactant exhibited the intense characteristic peak at 3434.00 cm-1 representing –oh groups. the intense stretching bands at 2927.92, 2845.91 and 1731.95 cm-1 corresponds to cm-1 –ch2, –ch3 and –c=o groups, respectively. in case of obp3 biosurfactant the peak at 3403.76 cm-1 indicates –oh groups. other characteristic peaks at 2924.99, 2858.66 and 1725.97 cm-1 represents –ch2, –ch3 and –c=o stretching bands, respectively. the ftir spectrum of obp4 surfactant had an intense peak at a frequency of 3416.40 cm-1 referring to the presence of –oh groups. peaks at 2929.43 and 2863.50 cm-1 represent stretching bands of –ch2 and –ch3 groups. another characteristic peak at the frequency of 1732.60 cm-1 shows the presence of carbonyl stretching. all three spectra showed the same essential adsorption bands for the rhamnolipid type biosufactant; only the relative areas under the various absorption bands are slightly different. the appearance of additional bands in the spectra might be the result of contamination of polypeptides and polysaccharides from cell debris during the extraction of biosurfactant from the culture supernatant. the mass spectrometric analysis of the biosurfactant samples produced by p. aeruginosa strains shows a characteristic peak at 163, 171, 193, 205, 339 m/z. the homologs structures present in the tested biosurfactant samples were identified through m/z values (table 4). the ms investigation of the biosurfactant samples established the results of the tlc with peak values appearing at 171, 193, 205, 339 m/z indicated the presence of lipids and peak value at 163 for carbohydrate moiety (aparna et al. 2012). a total of twelve rhamnolipid homologs were identified from the biosurfactants produced by the bacterial strains which included both mono and di-rhamnolipids. the results clearly showed the predominance of di-rhamnolipids over the monorhamnolipids and typically the main rhamnolipids were found to be rha-c10-c10, rha-rha-c8-c12 and rha-rha-c10-c12. in the literature, the number of rhamnolipid homologues reported varies from 4 to 28 (wittgens et al. 2017). the difference between the rhamnolipid composition and predominance of a particular type of congener in the present investigation was probably due to the factors like type of carbon substrate (benincasa and accorsini 2008), culture conditions, the age of the culture and the strains of p. aeruginosa used. possible industrial application of biosurfactant the aqueous biosurfactant solutions of the selected bacterial strains could efficiently separate the crude oil from the contaminated sand (fig. 3). fig. 3. removal of crude oil from the contaminated sand after washing with biosurfactant solution (bs) produced by p. aeruginosa strains. cfcscell free culture supernatant, sds-sodium dodecyl sulphate. values are the mean of three independent experiments ± standard deviation. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:14 utc nova biotechnol chim (2018) 17(2): 103-114 112 the maximum removal of crude oil by the biosurfactants was achieved up to a range of 44.2 – 52.0 % within their cmc, but by increasing the concentration beyond the cmc the removal of crude oil from the contaminated sand could not be enhanced. such behaviour might be due to the reduction in the interfacial tension between crude oil and the sand particles, as a result the capillary force that holds them together in the sand-oil mixture gets reduced. such reductions in the tension further increase the contact angle between the oil and soil particles and change the wet ability of the system. this results in the mobilization of the crude oil from the sand-oil mixture to the aqueous solution. differences in the separation behavior of the biosurfactants samples in the washing experiment suggests that the separation process also dependent on the physico-chemical properties of the biosurfactant and combined behaviour of surfactant/crude oil/sand systems. efficient washing off of crude oil from the contaminated sand could be achieved even with synthetic surfactant sds. it is now well established that synthetic surfactants are more recalcitrant than the petroleum hydrocarbons and potentially toxic to the environment (wyrwas et al. 2012; de et al. 2015). hence, use of biosurfactant seems to be more advantageous. the investigation of saturated sand pack column with the biosurfactant samples demonstrated their efficiency in the recovery of residual crude oil. the available residual oil in the sand pack column was mobilized during the passage of the cell-free culture broth containing biosurfactant which began to exude with the effluent. nearly, 6.4 – 11.4 % of the residual crude oil was recovered from the saturated sand pack column using cell-free culture supernatant of the bacterial strains as compared to the control. this is due to the fact that biosurfactants start to decrease the interfacial tension at oil/brine interface which in turn increases the capillary number of the system. further, increase in capillary number lowers the residual oil saturation in the column and increases the recovery process (hajibagheri et al. 2017). the recovery process from the saturated sand pack column was unaffected between 70 − 90˚c, which indicates the stability of biosurfactants. such property is very crucial for their application of biosurfactants in meor where they have to withstand the prevailing extreme temperatures inside the oil reservoirs. conclusions the selected three bacterial strains were found to be effective in producing biosurfactant in the presence of n-hexadecane and efficient in utilizing petroleum hydrocarbons. on the basis of biochemical and genotyping analysis the bacteria were identified as three different strains of pseudomonas aeruginosa. biosurfactants produced by these strains exhibited excellent surface properties and remained stable while exposed to extreme conditions of high temperature, ph and salinity suggesting their potential industrial applications. chemical characterization using standard techniques confirmed the production of rhamnolipids with the predominance of di-rhamnolipids. the efficiency of isolated biosurfactants in removal and washing experiments suggests its possible applications in bioremediation. the enhanced release of crude oil at higher temperatures also confirms their thermostability that finds potential applications of crude oil recovery processes such as meor. the application of such hydrocarbon utilizing and biosurfactant producing indigenous bacterial strains seems advantageous for bioremediation processes in local instead of using foreign strains. contributions of authors pb, sps designed the experiments, analyzed the data. pb, yb, nd, bkk, conducted most of the experiments. sps, cbs provided the experimental materials and performed few experiments. pb, sps, ym, nd, bkk, cbs, ds wrote the manuscript. all authors read and approved the manuscript. the authors declare that they have no competing interests. acknowledgement pb acknowledges ongc, india for the fellowship and funding the research. the help from chemical sciences, tezpur university, tezpur, assam, india, for various chemical analyses is gratefully acknowledged. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:14 utc nova biotechnol chim (2018) 17(2): 103-114 113 references aparna a, srinikethan g, smitha h (2012) production and characterization of biosurfactant produced by a novel pseudomonas sp. 2b. colloids surf. b biointerfaces 95: 2-29. banat im (1993) the isolation of a thermophilic biosurfactant producing bacillus sp. biotechnol. lett. 15: 591-594. benincasa m, accorsini fr (2008) pseudomonas aeruginosa lbi production as an integrated process using the wastes from sunflower-oil refining as a substrate. bioresour. technol. 99: 3843-3849. bharali p, konwar bk (2011) production and physicochemical 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zhang y, zhao q, jiang j, wang k, wei l, ding j, yu h (2017) acceleration of organic removal and electricity generation from dewatered oily sludge in a bioelectrochemical system by rhamnolipid addition. bioresour. technol. 243: 820-827. zaman au (2010) comparative study of municipal solid waste treatment technologies using life cycle assessment method. int. j. environ. sci. 7: 225-235. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 10:14 utc nova biotechnol chim (2018) 17(1): 16-28 doi: 10.2478/nbec-2018-0002  corresponding author: r.tandlich@ru.ac.za nova biotechnologica et chimica adsorptive removal of ciprofloxacin and isoniazid from aqueous solution cyril dube1, roman tandlich1, and brendan wilhelmi2 1environmental health and biotechnology research group, division of pharmaceutical chemistry, faculty of pharmacy, rhodes university, grahamstown, 6140, south africa 2department of biochemistry and microbiology, rhodes university, 6140, south africa article info article history: received: 1st april 2018 accepted: 20th june 2018 keywords: adsorption ciprofloxacin isoniazid wastewater antimicrobial resistance abstract this paper describes study of ciprofloxacin and isoniazid removal from aqueous solutions using coal fly ash (fa), kaolinite, perlite, talc and vermiculite. the adsorptive features of the adsorbents were evaluated for ciprofloxacin and isoniazid with regards to the effects of contact time, ph, the solid/liquid ratio and antibiotic concentration. all adsorbents were sterilised by dry heat before use to avoid the proliferation of antimicrobial resistance by the bacteria present on the adsorbents during experiments. the regression correlation coefficients indicate that the langmuir model gives the best fit for the sorption of both antibiotics onto fa and talc, ciprofloxacin onto kaolinite, and isoniazid onto perlite and vermiculite with r2 values ranging from 0.908 – 0.999. the freundlich isotherm best describes the sorption of ciprofloxacin onto vermiculite and isoniazid onto kaolinite with r2 values of 0.999 for both. the tempkin model best describes the sorption of ciprofloxacin onto perlite with an r2 = 0.997. the values of the freundlich exponent, 1/n, range from 0.221 – 0.998, indicating a favourable adsorption of ciprofloxacin and isoniazid onto the adsorbents. the heat of sorption, b, calculated from the temkin plots has values ranging from 0.018 – 10.460 j/mol, indicating a physical adsorption process (physisorption). adsorption equilibrium was achieved after 30 min for both antibiotics and the kinetic data obtained conforms best to the pseudo-second order equation with r2 values ranging from 0.998 – 0.999. the removal of ciprofloxacin and isoniazid by all adsorbents except fa was strongly influenced by the ph suggesting that electrostatic interactions play a major role in the adsorption processes.  university of ss. cyril and methodius in trnava introduction the prevailing emergence of antimicrobial resistance amongst bacteria remains a serious health and economic problem. it is estimated that about 700 000 deaths occur worldwide every year as a result of antibiotic resistance (bengtssonpalme and larsson 2016). antibiotics are extensively used worldwide in human and veterinary medicine for therapeutic, prophylactic and growth enhancing purposes (chen et al. 2016). consequently, they are relentlessly discharged into the environment and are therefore considered to be “pseudo-persistent” environmental pollutants (yao et al. 2015). antibiotics in wastewater from domestic, hospital, farm and industrial effluent (wu et al. 2016) are released into wastewater treatment plants (wwtp) which act as the major port of entry of antibiotics into different facets of the environment (luo et al. 2014). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:27 utc nova biotechnol chim (2018) 17(1): 16-28 17 the application of adsorption technology to remove wastewater pollutants has proven to be one of the most versatile water treatment methods which is efficient, environmentally friendly and easy to operate (yu et al. 2016). naturally occurring rock minerals such as kaolinite, perlite, talc and vermiculite and waste-derived materials such as coal fly ash (fa), a mineral residue, are potential alternatives owing to their porous nature, chemical inertness, relatively low costs and availability in commercially bulk quantities (ahmaruzzaman 2010; kolvari et al. 2015; tian et al. 2016). in the present study, the removal of ciprofloxacin and isoniazid from aqueous media by sorption onto kaolinite, perlite, talc, vermiculite and fa in a batch system was investigated. the adsorption of antibiotics at the solid–liquid interface is strongly influenced by the nature of the structural groups present on the solid surface, the molecular structure of the antibiotic being adsorbed and the environment of the aqueous phase. therefore, the effect of varying parameters such as the solid/liquid ratio, ph, contact time, initial antibiotic concentration was also investigated. langmuir, freundlich and temkin isotherms were used to analyse the data. experimental reagents and chemical ciprofloxacin (purity ≥ 98%, cas # 85721-33-1) and isoniazid (purity ≥ 99%, cas # 54-85-3) were purchased from sigma-aldrich (pty.) ltd. (johannesburg, rsa). all other chemicals were of analytical grade and were used as received without any further purification. milliq water was obtained from a milli-q® rios™ and academic a10 water purification system (merck millipore, bedford, usa). fa was obtained from ash resources (pty.) ltd. (randburg, rsa). ground talc was purchased from micronized products (pty.) ltd. (johannesburg, rsa). raw kaolinite was obtained from the local strowan mine (grahamstown, rsa). perlite and vermiculite, both in the expanded form, were obtained from sunshine nursery (grahamstown, rsa). preparation and characterization of adsorbents the adsorbents were ground using a mortar and pestle and passed through a 0.315 µm mesh sieve to ensure uniform particle size. afterwards, the adsorbents were washed with milliq water, in the ratio 1:10 (g/ml), by stirring on a model str-mh magnetic stirrer (fmh laboratory products, rsa) at speed 5 for 24 hours. the suspensions were then separated by vacuum filtration and dried for 12 hours in a memmert ulm600 oven (memmert, western germany) at 105 oc. all adsorbents and glassware were sterilised by dry heat at 160 oc and left to air dry for 24 hours in an la-1200 bii laminar-flow hood (vivid air, rsa). characterization of the adsorbents was done by determining the ph, cation exchange capacity (cec) using the ammonium acetate saturation method (sumner and miller, 1996), specific surface area (ssa) using the ethylene glycol monomethyl ether (egme) method (tandlich and balaz 2011), total organic carbon content using the loss on ignition (loi) method (salehi et al. 2011), surface morphology was observed on a tescan vega lmu scanning electron microscope (sem) (tescan, czechoslovakia), mineralogical composition using (xrd) patterns were recorded using a bruker d8 discover machine (bruker, germany) equipped with the psd lynx eye detector, using cu-k radiation (λ = 1.5405 å, nickel filter). the fourier transform infrared (ftir) spectra were obtained at room temperature from an average of 10 individual scans using perkin elmer version 10.4 spectrophotometer (perkin elmer, port elizabeth, south africa) in the spectral range of 4,000 to 650 cm−1 having a resolution of 4 cm−1 by the kbr disc method. furthermore, xrd patterns and ftir spectra were used to investigate the adsorption mechanisms. adsorption studies all adsorption experiments were carried out by the batch equilibration method at room temperature (25±1 oc) in duplicates. aqueous stock solutions of concentration 1.0 g/l were prepared by dissolving the antibiotic powders in milliq water. in the adsorption kinetics studies, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:27 utc nova biotechnol chim (2018) 17(1): 16-28 18 0.1 ± 0.001 g and 1.0 ± 0.01 g were added to sterile 30 ml centrifuge tubes containing 10 ml ciprofloxacin and isoniazid solution, respectively. the concentration of the antibiotic which was obtained from the dilution of the stock solutions was 25 mg/l. the mixtures were shaken on a junior orbit shaker (lab-line instruments inc., melrose park, ill) at a constant speed of 220 rpm for a fixed time interval (5, 10, 15, 30, 60, 120, 240 and 480 min) in the dark. after shaking, the mixtures were separated by centrifugation on a mixtasel centrifuge (selecta®, barcelona, spain) at 700 rcf for 10 min and passing the supernatants through a 0.45 µm syringe filter. antibiotic solutions which were not mixed with adsorbents were used as controls to assess the influence of phase separation on the initial antibiotic concentrations. the filtrates were then diluted accordingly with milliq water. the concentrations of the antibiotic solutions were determined spectrophotometrically using a cintra 10 gbc 916 uv/vi̇s spectrophotometer (gbc scientific equipment (pty) ltd., melbourne, australia) wavelengths of maximum absorbance, 277 nm and 262.5 nm for ciprofloxacin and isoniazid, respectively. the wavelengths of maximum absorbance of the antibiotics were predetermined spectrophotometrically after eliminating the background influence of the aqueous phase by performing an automatic baseline correction with a blank solution. calibration curves were established by using ten standards in the range 0 to 7.5 mg/l and 0 to 27 mg/l for ciprofloxacin and isoniazid, respectively. to investigate the effects of the solid/liquid ratio on antibiotic sorption, dosages of 0.100, 0.175, 0.250 g were used for ciprofloxacin solutions and 0.5, 1.0 and 1.5 g were used for isoniazid solutions. influence of ph was evaluated by adjusting the initial ph of the antibiotic solutions ranging from 3 to 12 using 0.1 m aqueous solutions of hcl or naoh. to obtain adsorption isotherms, solutions of different antibiotic concentrations (5 – 250 mg/l) were made by diluting the stock solutions with milliq water. the ciprofloxacin and isoniazid solutions were mixed with 0.1 ± 0.001 g and 1.0 ± 0.01 g adsorbent, respectively. the mixtures were shaken at 220 rpm for 40 min to allow adsorbents to reach equilibrium adsorption. the initial ph of antibiotic solutions was at the optimum for maximum adsorption for each adsorbent. the amounts of antibiotic adsorbed and % removal were calculated by mass balance using the formula (eq. 1 and 2): (1) (2) where qe (mg/g) is the amount of antibiotic adsorbed at equilibrium, w (g) is the weight of the adsorbent, v (l) is the volume of antibiotic solution, co (mg/l) is the initial antibiotic concentration in solution and ce (mg/l) is the concentration of antibiotic remaining in solution at equilibrium. results and discussion characterization physicochemical properties of sorbents the physical-chemical properties of adsorbents are important parameters in adsorption processes and are often manipulated to enhance the adsorption process. the physicochemical properties of the adsorbents are shown in table 1. most of the values are in agreement to those reported in literature; specific surface area (saa) of talc and kaolinite (ouchiar et al. 2015), cation exchange capacity (cec) of vermiculite (sutcu 2015), ph of vermiculite (rashad 2016a), ph of perlite (rashad 2016b), cec and saa of perlite (tekin et al. 2010), cec of fa (visa 2016), saa of fa and cec of talc (dellisanti et al. 2009). table 1. physicochemical properties of adsorbents. adsorbent ph cec [cmol/kg] saa [m2/g] average loi [%] fa 12.2 9.46 5.5 0.60 talc 8.6 12.02 6.3 0.16 perlite 5.3 41.21 3.1 0.86 kaolinite 7.6 15.15 20.2 0.47 vermiculite 7.5 86.52 5.9 0.20 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:27 utc nova biotechnol chim (2018) 17(1): 16-28 17 effect of contact time on antibiotic sorption contact time is a very important parameter that influences the effectiveness of the adsorption process. the adsorption is almost instantaneous and increased sharply initially, after which it slows as it approaches a state of adsorption equilibrium after 30 min. this is because initially, the sorbent sites are vacant resulting in a higher frequency of interactions between the sites and antibiotic molecules. as time lapses the sorption sites continue getting occupied and gradually most sites become saturated. the few if any remaining vacant sites become more difficult to occupy due to repulsive forces between the antibiotic molecules on the surface of the adsorbent and those in the bulk solution (singh and choden 2014). the highest rate and extent of adsorption of both ciprofloxacin and isoniazid are exhibited by kaolinite. whereas, vermiculite and fa show both the lowest rate and extent of adsorption of isoniazid and ciprofloxacin, respectively. adsorption equilibrium for isoniazid and ciprofloxacin was achieved in 30 min by all adsorbents and was kept at 40 min for the rest of the experiments. table 2. kinetic parameters for adsorption of ciprofloxacin onto fa, kaolinite, perlite, talc and vermiculite. derived from pseudo-first-order, pseudo-second order and intraparticle diffusion models. pseudo-first order pseudo-second order intraparticle diffusion adsorbent k1 [min−1] qe.c [mg/g] r2 k2 [mg/g.min] qe.c [mg/g] r2 kid [mg/g.min0.5] c [mg/g] r2 fa 0.1870 0.3309 0.9945 0.9343 0.9319 0.9999 0.0650 0.6190 0.9889 talc 0.1803 0.3351 0.9719 0.9649 1.8875 0.9999 0.0776 1.5300 0.9222 perlite 0.0829 0.2037 0.9943 1.4248 1.1375 0.9997 0.0454 0.9194 0.9989 kaolinite 0.1819 0.2735 0.9776 1.3500 2.2341 0.9999 0.0521 1.9875 0.9996 vermiculite 0.2006 0.2979 0.9842 1.2110 1.9662 0.9999 0.0635 1.6744 0.9270 adsorption kinetics the non-equilibrium stages of adsorption were used to make a kinetic evaluation of the adsorption behaviours of ciprofloxacin and isoniazid by the adsorbents. the adsorption rate constants were estimated using the linearised forms of the following kinetic models based on the r2 values and amount of antibiotic adsorbed per unit weight of adsorbent. the pseudo first-order rate model expressed as (eq. 3, 4 and 5): (3) the second-order rate model expressed as: (4) the intra particle diffusion model is expressed as: (5) where qe and qt (both in mg/g) are the amounts of antibiotic adsorbed per unit mass of adsorbent at equilibrium and time t (min), respectively, k1 (min−1) is the rate constant of the pseudo-first-order model, k2 (mg/g.min) is the rate constant of the pseudo-second-order model, kid (mg/g.min 0.5) is the rate constant of the intra-particle diffusion model. values of k1 and qe can be calculated from the slope and intercept of the linear plot of log (qe qt) versus t, respectively. values of k2 and qe can be calculated from the intercept and slope of the linear plot of t/qt versus t, respectively. the value of kid can be calculated from the slope the linear plot qt versus t0.5 and c (mg/g) is the intercept (nithya et al. 2016). parameters and regression coefficients for the treatment of ciprofloxacin and isoniazid data according to each kinetic equation are presented in table 2 and table 3, respectively. based on the r2 values, the pseudo-second-order model gives the better fit for the adsorption of both ciprofloxacin and isoniazid onto all the adsorbents, with r2 values ranging from 0.9987-0.9999, compared to the pseudo-first and intra-particle diffusion model. the data of equilibrium adsorbed amount calculated from the pseudo-second-order kinetics (qe.c) are also close to the amounts adsorbed at equilibrium in experiments. this suggests that the sorption of ciprofloxacin 19 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:27 utc nova biotechnol chim (2018) 17(1): 16-28 20 and isoniazid obey pseudo-second order kinetics. it is interesting to note that when employing the pseudo-first order model, close to equilibrium the value of qt approaches qe resulting in qe-qt becoming smaller and smaller. ln qe-qt takes very large and uncertain values compromising the fit table 3. kinetic parameters for adsorption of isoniazid onto fa, kaolinite, perlite, talc and vermiculite. derived from pseudofirst-order, pseudo-second order and intraparticle diffusion models. pseudo-first order pseudo-second order intraparticle diffusion adsorbent k1 [min−1] qe.c [mg/g] r2 k2 [mg/g.min] qe.c [mg/g] r2 kid [mg/g.min0.5] c [mg/g] r2 fa 0.1630 0.0527 0.9677 5.2455 0.1374 0.9987 0.0105 0.0857 0.9984 talc 0.1370 0.0201 0.9830 12.052 0.0503 0.9986 0.0044 0.0287 0.9997 perlite 0.1745 0.0387 0.9859 6.0785 0.0693 0.9990 0.0077 0.0287 0.9980 kaolinite 0.1361 0.0594 0.9960 4.0844 0.1555 0.9995 0.0134 0.0899 0.9973 vermiculite 0.1671 0.0161 0.9974 12.782 0.0273 0.9998 0.0034 0.0100 0.9894 of the model. on the other hand, the pseudosecond-order gives a more accurate fit of the experimental data with giving almost perfect r2 values because in the plots when the majority of the data is at equilibrium the data points are well aligned because the value of t/qt ≃ t/qe when qt ≃ qe (simonin 2016). interaction between the antibiotics and the sorbent surfaces the sem images of the adsorbents indicated that characteristics of all sorbents were similar to what is reported in literature (hongo et al. 2012; sprynskyy et al. 2011; zhang et al. 2017). results of the xrd analyses indicated that the crystalline structure of fa and kaolinite was affected by the sorption of ciprofloxacin and isoniazid. crystaline structure of perlite and talc was not affected by the sorption of either of the two antibiotics. both antibiotics could have been intercalated between the inner layers of vermiculite. ftir spectral analysis can be used to identify specific functional groups present within the si-containing or crystalline phases of the adsorbents. this assists in understanding the adsorption mechanisms and interactions between the antibiotics and the adsorbents. the ir spectra of the adsorbents are presented in fig. 1. fa has a heterogeneous structure consisting of a glassy surface layer (sio2) and crystalline components such as quartz, mullite and magnetite. hence, its ftir (fig. 1a) exhibits a broad band in the range 1,200 – 900 cm-1 with a centre around 1,090 cm-1 which is attributed to the asymmetric stretching vibration peaks of sio-si or si-o-al bonds of sio2 or alo4 tetrahedrons which overlap in this region (böke et al. 2015; onutai et al. 2016). the shoulder visible between 1,074 and 1,069 cm-1 corresponds to the si-o-si stretching vibration. after shaking with the ciprofloxacin and isoniazid solutions, the peak of the centre of the broad band corresponding crystalline structure of fa shifted to lower wavenumbers around 1,080 and 1,060 cm-1, respectively. this indicates a possible interaction of the oxygen with the antibiotics resulting in a greater number of non-bridging oxygen atoms (shearer et al. 2016). the bands at 3,340 and 1,635 cm-1 are respectively assigned to the o-h stretching and bending of absorbed water indicating the presence of the antibiotic molecules on the surface of the adsorbent (timakul et al. 2016; ul haq et al. 2014). the ir spectra of kaolinite (fig. 1b) reveal that raw kaolinite exhibits bands in the range 3,694 – 3,620 cm-1 corresponding to stretching vibrations of oh groups coordinated to al3+ ions. the peaks at 3,694, 3,672 and 3,620 cm-1 exhibited on ftir spectra of kaolin are assigned to the in-phase stretching vibrations of inner surface oh groups. the peak at 3,654 cm-1 is attributed to the out of phase stretching of inner o-h groups (zhu et al. 2016). all three peaks are also present in the spectra of kaolin after shaking with both antibiotic solutions indicating that no intercalation took place, instead, hydrogen bonding or ligand exchange might have occurred on the external surface of kaolinite (spence and kelleher 2012). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:27 utc nova biotechnol chim (2018) 17(1): 16-28 21 intercalation would have resulted in the reduction of electrostatic attractive forces between the antibiotics and the inner surface oh groups of the layers thus separating them (castrillo et al. 2015). the bands around 3,400 and 1,650 cm-1 correspond to stretching and bending vibrations of water molecules, respectively. the peaks at 933 and 908 cm-1, which are attributed to al-oh deforming vibration and al-oh bending vibration move to higher wave number and an increase in intensity (zhu et al. 2016). the presence of quartz in the kaolinite used is revealed by the low-intensity doublet with bands at 798 and 780 cm-1 (król et al. 2016). 4500 4000 3500 3000 2500 2000 1500 1000 500 50 60 70 80 90 100 50 60 70 80 90 100 50 60 70 80 90 100 4500 4000 3500 3000 2500 2000 1500 1000 500 t ra n s m it ta n c e ( % ) wavnumber (cm -1 ) f t ra n s m it ta n c e ( % ) f + i a t ra n s m it ta n c e ( % ) f + c 4500 4000 3500 3000 2500 2000 1500 1000 500 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 4500 4000 3500 3000 2500 2000 1500 1000 500 t ra n s m it ta n c e ( % ) wavnumber (cm -1 ) t t ra n s m it ta n c e ( % ) t + i b t ra n s m it ta n c e ( % ) t + c 4500 4000 3500 3000 2500 2000 1500 1000 500 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100 4500 4000 3500 3000 2500 2000 1500 1000 500 t ra n s m it ta n c e (% ) wavnumber (cm -1 ) p t ra n s m it ta n c e (% ) p + i c t ra n s m it ta n c e (% ) p + c 4500 4000 3500 3000 2500 2000 1500 1000 500 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 4500 4000 3500 3000 2500 2000 1500 1000 500 t ra n s m it ta n c e ( % ) wavnumber (cm -1 ) k t ra n s m it ta n c e ( % ) k + i d t ra n s m it ta n c e ( % ) k + c 4500 4000 3500 3000 2500 2000 1500 1000 500 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 4500 4000 3500 3000 2500 2000 1500 1000 500 t ra n s m it ta n c e ( % ) wavnumber (cm -1 ) v t ra n s m it ta n c e ( % ) v + i e t ra n s m it ta n c e ( % ) v + c for perlite (fig. 1c), the band at 1,014 cm-1 of the perlite ftir spectra represents si-o-si asymmetric stretching vibration (sun and wang 2015) which shifted to a lower wavenumber after shaking with the antibiotic solutions. this suggests that interaction occurred, and based on the abundance of hydroxyl groups that exist on the inner surface of perlite, possibly hydrogen bonding and electrostatic attraction occurred with the functional groups on the antibiotics. it is also noteworthy that the bands at 778 and 3,342 cm-1 are attributed to stretching of o-h and si-oh respectively (sun and wang 2015), shifted to higher wavenumbers suggesting a reduction in bond strength possibly resulting from weaker electrostatic attractions between the perlite and both antibiotics. the peak fig. 1. ftir spectra of adsorbents. (a) f is the spectrum of fa, f + c is a spectrum of fa after mixing with ciprofloxacin solution, f + i is the spectrum of fa after mixing with isoniazid solution. (b) t is the spectrum of talc, t + c is the spectrum of talc after mixing with ciprofloxacin solution, t + i is the spectrum of talc after mixing with isoniazid solution. (c) p is the spectrum of perlite, p + c is the spectrum of perlite after mixing with ciprofloxacin solution, p + i is the spectrum of perlite after mixing with isoniazid solution (d) k is the spectrum of kaolinite, k + c is the spectrum of kaolinite after mixing with ciprofloxacin solution, k + i is the spectrum of kaolinite after mixing with isoniazid solution (e) v is the spectrum of vermiculite, v + c is the spectrum of vermiculite after mixing with ciprofloxacin solution, v + i is the spectrum of vermiculite after mixing with isoniazid solution solution. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:27 utc nova biotechnol chim (2018) 17(1): 16-28 22 at 1,045 cm−1 is the characteristic absorption peak of the telescopic vibration of si-o-si, and the shift to lower wave number after shaking with the antibiotic solutions is attributed to hydrogen bonding (wei et al. 2014). for talc (fig. 1d), the bands ranging between 3,700 – 3,400 cm-1 are attributed to the stretching vibrations of o-h associated with si (si-oh) and mg (mg-oh) (sprynskyy et al. 2011). characteristically, the band at 3,678 cm-1 is associated with o-h in the 3 mg region (mg3-oh) of talc (ngally sabouang et al. 2015). this band showed a significant change decrease in intensity after shaking with isoniazid solutions and increase in intensity after shaking ciprofloxacin solutions, respectively. this strongly suggests a possible interaction of the mg-oh with the antibiotics. the same explanation can be given for shifts of wavenumbers of the band at 664 cm-1 which is assigned to the o-h stretching vibrations associated with mg-oh. 2 4 6 8 10 12 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 q e ( m g /g ) ph fa talc perlite kaolinite vermiculite a 2 4 6 8 10 12 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 0.28 q e ( m g /g ) ph fa talc perlite kaolinite vermiculite b fig. 2. effect of ph on the adsorption of (a) ciprofloxacin and (b) isoniazid onto fa, kaolinite, perlite, talc and vermiculite. amount adsorbed at equilibrium (qe) presented as the mean ± standard deviation (n = 3). a significant shift to lower wavenumbers and a marked increase in the intensity of the band at 3,400 cm-1 corresponding to si-oh groups was observed after shaking with isoniazid solution. this is an expected binding site for isoniazid as the antibiotic is most likely to bind to surface hydroxyls by hydrogen interaction through ring nitrogen lone pairs directly, or indirectly through water bridges (akyuz et al. 2010). the ir spectra of vermiculite (fig. 1e) show bands at 3,414 and 999 cm−1 which represent the o-h stretching vibrations of si-oh groups and si-o-si groups of the silicate framework of vermiculite, respectively (tran et al. 2015). the peak 1,637 cm-1 is attributed to the o-h bending vibration (oliveira et al. 2015). band at 999 cm-1 shifted to higher wavenumbers and there was an increase in the signal intensity after shaking with the antibiotic solutions which indicating interaction, between the sorbent and the antibiotic molecules. the peaks at 667 and 729 cm-1 which are attributed to the stretching vibrations of x-o-si, where x = mg, al or fe and si-o & al-o, respectively (hongo et al. 2012), they also shifted to higher wavenumbers after shaking with the antibiotics solutions. the adsorption at 1,645 cm−1 is primarily due to water directly coordinated to the exchangeable cations of the clay (tran et al. 2015). the significant shift of this band to lower wavenumber after shaking with ciprofloxacin suggests that cation exchange was one of the mechanisms of adsorption in the removal of ciprofloxacin by vermiculite. influence of ph the adsorption mechanism is generally dictated by the physical and chemical interactions which occur between the adsorbate and adsorbent (zhao et al. 2016). the presence of highly charged groups on the surfaces of all the adsorbents make them very sensitive to ph changes and this results in a significant influence on the degree of interaction between the antibiotics and adsorbents. the influence of ph on the removal of ciprofloxacin and isoniazid from aqueous solutions by fa, kaolinite, perlite, talc and vermiculite is depicted in fig. 2. a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:27 utc nova biotechnol chim (2018) 17(1): 16-28 23 there is a strong dependence on ph exhibited by the adsorption of ciprofloxacin and isoniazid by all adsorbents except fa. in general, the net surface charge of mineral oxides such as silica and alumina, tend to become more positive under acidic conditions i.e. when ph is lowered resulting in the adsorption of anionic species and decrease in the adsorption of cationic species (alkan et al. 2005). however, looking at fig. 2, most ciprofloxacin was adsorbed within the ph range 3-5 by all adsorbents except fa. in this ph range, ph < 5.90, ciprofloxacin predominantly exists as a cationic species as a result of the basic nitrogen in the piperazine ring being protonated to form a positively charged ciprofloxacin ion (pka 1). under basic conditions, ph > 8.89, the carboxylic acid functional group is deprotonated to form an anionic species (pka 2). at neutral ph, 5.90 ≥ ph ≥ 8.89, ciprofloxacin occurs as a zwitterion (chang et al. 2016; nageswara rao et al. 2008). this, therefore, suggests that cation exchange is the dominant mechanism of adsorption of ciprofloxacin by kaolinite, talc, perlite and vermiculite at lower ph values. there is a significant drop in the adsorption of ciprofloxacin as ph approaches pka 1 indicating that the cation is no longer the dominant species. the general minimum variation in adsorption of ciprofloxacin that is observed at ph ≥ pka 1 suggests that the positive charge on the ciprofloxacin cation still contributes to adsorption via cation exchange mechanism and the rapid decline when ph was approaching pka 2 indicates that cation exchange is no longer the dominant mechanism of adsorption (wu et al. 2010). despite the decrease, the continued adsorption of ciprofloxacin at ph ≥ pka 2 indicates that another mechanism is responsible for adsorption, possibly hydrogen bonding and/or hydrophobic interactions due to π–π stacking. it is also highly likely that ciprofloxacin and isoniazid can interact with the si-oand al-o functional groups on the surfaces of the adsorbents via complex formation and/or ion exchange. it is, therefore, important to note that although one mechanism dominates at certain ph, other mechanisms may also be involved adsorption process. the nitrogen and oxygen groups present in the structure of isoniazid enable it to form complexes with adsorbents as a ligand. the amino group hydrogens and carbonyl group oxygen of isoniazid are capable of forming hydrogen bonds with the adsorbents (akyuz and akyuz, 2008). isoniazid is also amphoteric and has multiple ionizable groups. it speciates as a function of ph and has two pka values, pka 1 = 3.53 and is a result of the protonation of the heterocyclic nitrogen atom under acidic conditions. pka 2 = 11.40 and is ascribed to the proton loss from the amide nitrogen atom aggravated by basic conditions. at рн < 5, isoniazid exists as three species in equilibrium: one neutral and two positively charged. in the ph range 5 – 9, isoniazid has an uncharged molecular form. deprotonation occurs gradually with increasing ph above 9 resulting in a negatively charged species (blokhina et al. 2015). as seen in figs. 2 and 3 the high adsorption of isoniazid by kaolinite and perlite at ph 3 suggests that cation exchange is the main mechanism of adsorption and this is further confirmed by the drastic decline in adsorption when ph ≥ pka 1 of isoniazid since the cationic species is no longer dominating. the invariance in adsorption of isoniazid exhibited by all the adsorbents in the ph range 5 – 10 suggests that mechanism of adsorption is consistent throughout the range and since isoniazid exists predominantly as a neutral species in this range adsorption is most likely attributed to hydrogen bonding and hydrophobic interactions between the isoniazid and adsorbents. the decrease in adsorption observed at ph ≥ 10 is most likely due to electrostatic repulsion between the negatively charged isoniazid species and the highly negative surface charge of the adsorbent. interestingly, the adsorption capacities were relatively stable throughout the entire ph range when fa was used as an adsorbent for both ciprofloxacin and isoniazid, indicating that ph had no significant influence on adsorption. this shows that fa has excellent environmental adaptability. therefore, in contrast to the other adsorbents, hydrophobic interactions are probably more important than electrostatic forces in the adsorption of ciprofloxacin and isoniazid by fa. the pyridine ring of isoniazid probably makes it capable of forming hydrophobic bonds especially when bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:27 utc nova biotechnol chim (2018) 17(1): 16-28 24 the cyclic nitrogen forms a hydrogen bond the rest of the ring becomes more electronegative. the aromatic ring found in ciprofloxacin is also rich in delocalised electrons. effect of initial antibiotic concentration the effect of initial antibiotic concentration on the removal of ciprofloxacin and isoniazid by the adsorbents is presented in fig. 3. the amount of both ciprofloxacin and isoniazid adsorbed increases with increase in initial antibiotic concentration, which is most probably due to increasing concentration gradient of the antibiotic between the solution and adsorbent particles, resulting in a greater driving force of the mass transfer of the antibiotic molecules from the bulk solution to the solid phase (singh et al. 2016). however, it is evident that the percentage of antibiotic removed decreases with increase in initial antibiotic concentration. this is because at lower antibiotic concentrations, the ratio of the initial number of antibiotic molecules/ions to the available surface area of adsorbent is larger, and vice versa (nithya et al. 2016). kaolinite removed all ciprofloxacin to concentrations below the detection limit for ciprofloxacin solutions which had an initial concentration less than 50 mg/l. the removal percentages of the ciprofloxacin after shaking for 30 min increased in the order fa < perlite < talc < vermiculite < kaolinite while those of isoniazid increased in the order vermiculite < talc < perlite < fa < kaolinite. the order remained the same despite the change in initial antibiotic concentrations. effect the solid/liquid ratio on antibiotic sorption the adsorption of ciprofloxacin and isoniazid was studied by varying the solid/liquid ratio by adjusting the adsorbent dosage from 0.10 to 0.25 g and 0.5 to 1.5 g, respectively while keeping other parameters constant. it is clear that an increase in the solid/liquid ratio results in a greater percentage removal due to increased contact between the the solute molecules and the adsorbent however, it is more interesting to note that the amount of antibiotic loaded per unit adsorbent at equilibrium tends to decrease with increase in the solid/liquid ratio. this is because at higher doses aggregation of the adsorbent particles may occur resulting in a reduction of freely accessible sorption sites for the solute (nithya et al. 2016). hence, the adsorption capacity of adsorbent is not fully utilised. adsorption isotherms the reason for employing adsorption isotherms was to analyse the relationship between the equilibrium concentration of antibiotic in solution and the amount adsorbed at the adsorbents surface. furthermore, adsorption isotherms provide constant values which enable us to compare the adsorption capacities for different adsorbents for ciprofloxacin and isoniazid. the linearised plots of the langmuir (eq. 6), freundlich (eq. 7) and temkin (eq. 8) adsorption isotherms were used to analyse the fa talc perlite vermiculite 10 20 30 40 50 60 70 80 90 100 % o f r e m o v a l 5 mg/l 50 mg/l 100 mg/l a fa talc perlite vermiculite 0 1 2 3 4 5 6 7 8 9 q e ( m g /g ) fa talc perlite kaolinite vermiculite 0 10 20 30 40 50 60 70 % o f r e m o v a l 25 mg/l 50 mg/l 100 mg/l b fa talc perlite kaolinite vermiculite 0.0 0.1 0.2 0.3 0.4 0.5 0.6 q e ( m g /g ) fig. 3. effect of initial antibiotic concentration on the adsorption of (a) ciprofloxacin and (b) isoniazid onto fa, perlite, talc, vermiculite and (b) isoniazid onto fa, kaolinite, perlite, talc and vermiculite. % removal and amount adsorbed at equilibrium (qe) presented as the mean ± standard deviation (n = 3). a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:27 utc nova biotechnol chim (2018) 17(1): 16-28 25 table 4. adsorption isotherm constants and correlation coefficients for adsorption of ciprofloxacin onto fa, kaolinite, perlite, talc and vermiculite. langmuir freundlich temkin adsorbent kl [l/mg] qm [mg/g] r2 1/n kf [mg/g] r2 kt [l/mg] b [j/mol] r2 fa 0.017 3.123 0.989 0.942 0.051 0.960 e-1.494 0.927 0.694 talc 0.064 6.083 0.998 0.667 0.436 0.993 e-0.035 1.215 0.928 perlite 1.505 0.819 0.978 0.221 0.392 0.953 e3.478 0.122 0.997 kaolinite 0.002 500.0 0.995 0.993 0.778 0.992 e-1.427 10.460 0.930 vermiculite 0.039 11.93 0.998 0.901 0.115 0.999 e-0.245 2.056 0.826 experimental data. the regression line correlation coefficients (r2) of the plots were used to establish the model which best described the data. (6) (7) (8) in eq. 6, 7 and 8, qe is the equilibrium adsorption capacity of the adsorbent, i.e. the equilibrium solid phase/sorbed concentration of the antibiotic in question (mg/g), ce is the concentration of antibiotic in the aqueous phase at equilibrium. the term of qm (mg/g) is the equilibrium solid phase/sorbed concentration of the antibiotic in question required to achieve monolayer coverage of the adsorbent surface (mg/g). at the same time, qm and kl (sorption coefficient for the antibioti in question; l/mg) are adjustable parameters of langmuir isotherm. kf (mg/g) and n are freundlich parameters representing the adsorption constant and the adsorption intensity, respectively. kt (l/g) is the temkin isotherm equilibrium binding constant and b (j/mol) is the temkin’s heat of adsorption (can et al. 2016). the adsorption isotherm parameters and r2 values obtained from fitting the experimental data obtained at 25±1 oc to linear plots of the isotherms values are presented in table 4 and table 5. in order to quantify the applicability of the isotherm models, the r2 values were used. these revealed that the langmuir model was the best fit for the sorption of ciprofloxacin onto fa, talc and kaolinite with r2 ≥ 0.99, which implies that adsorption was predominantly a monolayer physical process. the freundlich and temkin isotherms gave the best for the sorption of ciprofloxacin onto vermiculite and perlite, respectively both with r2 ≥ 0.99. the langmuir isotherm also gave the best fit for the adsorption of isoniazid onto fa, talc, perlite and vermiculite exhibiting r2 values in the range 0.90 – 0.99, the freundlich isotherm was the better fit for the sorption of isoniazid onto kaolinite with r2 ≥ 0.99. the freundlich model mainly describes adsorption onto surfaces with no uniform energy distribution which means the adsorption is heterogeneous (guan et al. 2017). however, r2 values only give us an indication of the degree to which the experimental data agree with each other. it is, therefore, important to also take into account the isotherm parameters when assessing the applicability of an isotherm. the freundlich constant, 1/n is a function of the strength of adsorption in the adsorption process and ideally, it should have a value less than one during a normal adsorption process which is an indication that the adsorption sites are homogenous in energy and there is no interaction between the adsorbed species. when 1/n assumes a value greater than 1, the interactions become weak and adsorption becomes unfavourable (can et al. 2016). the values of the freundlich exponent, 1/n, range from 0.221 – 0.998, indicating a favourable adsorption of ciprofloxacin and isoniazid onto the adsorbents. the heat of sorption, b, calculated from the temkin plots has values ranging between 0.018 – 10.460 j/mol, indicating a physical adsorption process (physisorption). unlike the langmuir and freundlich isotherm models, the temkin isotherm takes into account the interactions between adsorbents and antibiotics in solution which is based on the assumption that the free energy of adsorption is simply a function of surface coverage (abdelnaeim et al. 2016). again, looking, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:27 utc nova biotechnol chim (2018) 17(1): 16-28 26 table 5. adsorption isotherm constants and correlation coefficients for adsorption of isoniazid onto fa, kaolinite, perlite, talc and vermiculite. langmuir freundlich temkin adsorbent kl x 10 -3 [l/mg] qm [mg/g] r2 1/n kf x 10 -3 [mg/g] r2 kt [l/mg] b [j/mol] r2 fa 1.134 7.092 0.999 0.983 9.727 0.998 e-2.245 0.2752 0.953 talc 0.879 2.481 0.990 0.998 1.973 0.987 e-2.776 0.1098 0.917 perlite 45.910 0.130 0.986 0.319 2.488 0.923 e-0.502 0.0259 0.948 kaolinite 18.002 0.824 0.994 0.697 25.991 0.999 e-1.804 0.1925 0.968 vermiculite 15.365 0.099 0.908 0.486 0.619 0.775 e-1.557 0.0183 0.790 at the regression r2 values obtained (0.694 – 0.997), this isotherm is applicable to the description of majority data equilibrium data and gave the best fit for the adsorption of ciprofloxacin by perlite. comparing the isotherms applied, the langmuir gave the best fit, followed by the freundlich isotherm and the least fit was obtained from the temkin isotherm model. kaolinite exhibited the greatest removals of both ciprofloxacin and isoniazid from aqueous solutions, achieving removals of up to 99 % and 55 %, respectively. the results indicate that the adsorption of ciprofloxacin and isoniazid mainly by surface coverage of the adsorbents and this corresponds with the findings from the kinetic studies which revealed that adsorption was mainly controlled external mass transfer. conclusions this work presented the feasibility of adsorption of ciprofloxacin and isoniazid using fa, kaolinite, perlite, talc and vermiculite. the adsorbents were used without any chemical modification and can therefore significantly cut costs for wastewater treatment. 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1466-1471. zhao h, liu x, cao z, zhan y, shi x, yang y, zhou j, xu j (2016) adsorption behavior and mechanism of chloramphenicols, sulfonamides, and non-antibiotic pharmaceuticals on multi-walled carbon nanotubes. j. hazard. mater. 310: 235-245. zhu x, zhu z, lei x, yan c (2016) defects in structure as the sources of the surface charges of kaolinite. appl. clay sci. 124-125: 127-136. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 08:27 utc nova biotechnol chim (2019) 18(1): 18-24 doi: 10.2478/nbec-2019-0003  corresponding author: jana.sedlakova@upjs.sk nova biotechnologica et chimica the first evidence of acidithiobacillus albertensis in weathered ore samples from active gold mine hodruša-hámre (slovakia) jana sedláková-kaduková1,, jana kisková1, lenka maliničová1, ivana timková1, stanislav jeleň2 and peter pristaš1 1 department of microbiology, institute of biology and ecology, faculty of science, pavol jozef šafarik university in košice, šrobárova 2, sk-041 54 košice, slovak republic 2 earth science institute, slovak academy of sciences, ďumbierska 1, sk-974 01, banská bystrica, slovak republic article info article history: received: 28th february 2019 accepted: 5th may 2019 keywords: autotrophic bacteria acidithiobacillus albertensis gold mine abstract sulphur-oxidising autotrophic bacterial communities in deep biosphere from weathered ore samples from active gold mine hodruša-hámre, slovakia were analysed using cultivation approach followed by dna extraction, pcr amplification and 16s rrna gene analyses. indirect measurement of ph changes in cultivation media evidenced the presence of acidophilic bacteria with active production of acids. the decrease of ph was observed at the beginning of isolation and later ph in range of 1.5 – 2 was maintained in both, sulphuric acid and thiosulphate, media. the presence of homogenous population of gram-negative rods was proved by gram staining. molecular analyses have revealed that the population of sulphur-oxidising bacteria in gold mine is dominated by a single species of aciditiobacillus genus, identified as a. albertensis, suggesting the low level of autotrophic bacteria diversity in deep deposits. for the first time this species was isolated from weathered rocks of a gold mine subsurface environment.  university of ss. cyril and methodius in trnava introduction in the past years, our perspective on the earth’s biosphere has expanded from just the terrestrial and oceanic realms to include deep subterranean and subseafloor environments (inagaki et al. 2003). the deep terrestrial subsurface environments such as those exemplified by deep mines represent an emerging area for exploring microbial populations with bewildering arrays of metabolic capabilities (rastogi et al. 2010). majority of these environments are characterised as extreme because of their hostile life conditions such as extreme temperature, ph, pressure, low oxygen content, no light and toxic metals presence. the existence of microorganisms in such environment is of increasing scientific and practical interest because subsurface microorganisms with novel metabolic properties may be of potential value to industry for applications in bioremediation and biotechnology (takai et al. 2001). microbes in the deep terrestrial subsurface environment are considered to play a key role in deposition and weathering the minerals being a part of geochemical processes and are the only life forms that have been encountered in the deeper regions of the earth´s crust (li et al. 2017). microbial communities existing in deep subsurface soil especially in gold mines have been studied very rarely and therefore they remain largely uncharacterized. the banská hodruša au + ag, pb, zn, cu deposit at the rozália mine (48°27´n, bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:54 utc nova biotechnol chim (2019) 18(1): 18-24 19 18°51´e) is the last operating gold mine in slovakia and it offers a unique opportunity for direct exploration of the mining-impacted deep subsurface environment. it occurs in the central zone of the large middle miocene banská štiavnica stratovolcano (15.0 – 10.7 mil. years) within the banská štiavnica – hodruša ore district, which belongs to the largest ore districts in the carpathian arc, famous for ag-au mining since the middle ages. the subhorizontal vein system occurs in 400 – 650 m depth, it is about 1.2 km long, hosted by andesite, near to the flat roof of the premineralization subvolcanic granodiorite pluton. the deposit consists of two parts, separated by a thick sill of quartz-diorite porphyry. the eastern part is currently mined, and the western part has already been depleted (kubač et al. 2018; koděra et al. 2018). gold-sulphide ore from epithermal vein deposit banská hodruša consists of gold, electrum, galena, sphalerite, chalcopyrite, and pyrite. minor amounts of tellurides are present in the form of hessite, petzite and other rare au sulphides and tellurides. gangue minerals are represented by abundant quartz and adularia, clay minerals and carbonates. the ore has excellent metallurgical properties, content of gold is in range of 5 – 600 g.t-1, with average 20 – 30 g.t-1 and maximum achievable gold recovery by flotation is 96 – 98 % (chovan et al. 2016). microbial diversity present in hodruša-hámre mine have not been reported yet. in the present study our objective was to elucidate the existence and composition of sulphur-oxidising autotrophic bacterial population in the weathered ore samples of hodruša-hámre gold mine, slovakia, by culturebased molecular methods. sulphur oxidising ts strain of acidithiobacillus albertensis species was isolated for the first time in europe and basic microbiological characteristics and phylogenetic relatedness of the strain were determined. experimental sample collection the samples (500 g each) were collected along the junction of the drift wall and the floor in vein blanka. yellow mats were covering the rock surface around the sample colleting site. the area was not disturbed by any type of activities including human trafficking from the end of 2000. the weathered ore was collected 10 cm below the surface with sterile spatula. the temperature at the time of sampling was 19 °c, it was measured using vwr eu 620-1259 thermometer. the samples were transported to the laboratory in sterile zip lock sacks on ice and in refrigerator. isolation of autotrophic bacteria six grams of homogenized soil sample was resuspended into 100 ml of sulphuric acid with ph 1.5 and 100 ml of thiosulphate medium. the composition of thiosulphate medium was 90 ml of mineral medium and 10 ml of 10 % (w/v) na2s2o3, mineral medium was composed of 1.2 g kh2po4, 0.2 g k2hpo4, 0.75 g mgcl2·6h2o, 0.15 g cacl2·2h2o, 0.5 g nh4cl, 0.5 g na2co3 into 1,000 ml of deionised water. few drops of concentrated hcl were added into sterilised mineral media to dissolve precipitates. both cultures were cultivated aerobically at 25 °c and ph values of media were measured once a week for 237 d using vwr ph 110 ph meter fresh medium (20 ml) was added into culture flask after 29 d of cultivation. dna isolation, 16s rrna gene amplification, rflp analysis and sequence analysis genomic dna was extracted from bacterial cultures as described by chen et al. (2009). amplification of 16s rdna gene was performed using fd1 (5’-agagtttgatcctggctcag-3’) and rp2 (5’-acggctaccttgttacgactt-3’) universal bacterial primers according to vandžurová et al. (2013). the amplification products (0.5 µg of dna) were analysed by restriction fragment length polymorphism (rflp) method using alui, hpaii, haeiii, hhai, ecori, and psti (thermo scientific, usa) restriction endonucleases according to the manufacturer’s instruction. the restriction fragments were separated by electrophoresis in 1 % (w/v) agarose gel. the 16s rdna amplicon of the culture from thiosulphate medium was cloned into the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:54 utc nova biotechnol chim (2019) 18(1): 18-24 20 ptz57r/t vector using an instaclone pcr cloning kit (thermo fisher scientific, usa) following manufacturer’s instructions. recombinant plasmids were isolated with genelutetm plasmid miniprep kit (sigma-aldrich, usa), checked on 1 % (w/v) agarose gel and purified using wizard® sv gel and pcr clean-up kit (promega, usa). sequencing was performed using sanger dideoxy sequencing method using plasmid specific primers by gatc biotech sequencing facility (gatc biotech ag, germany). sequences from both primers were assembled in cap3 sequence assembly program (http://doua.prabi.fr/software/cap3) (huang and madan 1999) and submitted to the genbank database under accession number mh796351. the sequences were taxonomically classified using blastn analysis against a database of 16s rdna sequences of the type strains of bacteria and archaea (http://www.ncbi.nlm.nih.gov/blast) (altschul et al. 1990). to elucidate phylogenetic relatedness of ts isolate the 16s rrna sequences of acidithiobacillus spp. were downloaded from genbank database and aligned using clustalw algorithm. the phylogenetic tree was constructed using neighbor joining method with 1000 bootstrap replicates. for all phylogenetic analyses mega software ver. 7 (kumar et al. 2016) was used. results and discussion the presence and activity of sulphur-oxidizing bacteria in both media was indirectly observed by changes of the medium ph values (fig. 1). in medium containing sulphuric acid only a slight increase of ph was observed, the ph value increased from 1.5 up to 2.6 at the 35th day followed by the decrease to 1.6 later on. after that time no significant changes in ph were observed till the end of monitoring period at day 237. in thiosulphate medium with initial ph 4 the significant decrease of ph was observed to 2.1 after 17th days of cultivation followed by the decrease to 1.54 after 94 d. this ph value remained unchanged until the end of the observation period. values of ph from 5.8 to 6.4 were measured in control experiments without bacteria added so it is evident that bacterial activity was responsible for the ph decrease. fig. 1. changes of ph during cultivation of bacteria from weathered ore samples from hodruša-hámre gold mine in two cultivation media (the arrow indicates the addition of 20 ml of fresh media into cultivation flasks at 29 d of cultivation). changes of ph in both cultivation media suggested acidifying activity of bacteria indirectly confirming presence of the bacteria. as no carbon source was added to the cultivation media bacterial growth and ph changes observed have to be accounted for the presence of autotrophic bacteria. under direct microscopic observation a morphologically homogenous population of small (approximately 1x0.5 µm) gram-negative rods were observed only in thiosulphate medium (fig. 2). bacterial dna was, however, isolated from both media. rflp analysis of the amplified 16s rrna gene revealed that the identical rflp banding pattern were generated for both, h2so4 and thiosulphate media (data not shown). lack of diversity observed in this experiment indicated the presence of highly genetic related autotrophic sulphur-oxidising bacteria in both media. the culture growing in thiosulphate medium designated as ts strain was used for further analyses. no heterotrophic growth of ts strain on lb medium with added glucose nor iron – oxidation activity on 9k medium (silverman et al. 1959) were observed. the amplification and sequence analysis of 16s rrna gene was used for molecular identification of ts isolate. analysis of obtained 16s rrna sequence against the prokaryotic_16s_ribosomal_rna database bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:54 utc http://doua.prabi.fr/software/cap3 http://www.ncbi.nlm.nih.gov/blast nova biotechnol chim (2019) 18(1): 18-24 21 fig. 2. bright field photomicrograph of gram-stained a. albertensis ts isolate growing in thiosulphate medium (magnification 640x). the image was taken using motic ba310 microscope with digital camera attached. scale bar 10 µm. available at ncbi site (http://www.ncbi.nlm.nih.gov/blast) showed that 16s rrna sequence of ts isolate is highly similar to the 16s rrna sequences of multiple acidithiobacillus spp. with highest similarity to the a. albertensis dsm 14366/atcc 35403 strain with similarity as high as 99.7 %. similarity to other well established species of acidithiobacillus genus were lower e.g. a. thiooxidans (98.7 %), a. ferrooxidans (97.7 %) or a. ferridurans (97.6 %), respectively. multiple sequence alignment placed 16s rrna sequence of ts isolate to the large well supported clade (bootstrap support 100, see fig. 3) of a. albertensis sequences. based on the morphological, metabolic and phylogenetic analyses the autotrophic sulphuroxidising ts isolate cultivated from weathered ore samples from active gold mine hodruša-hámre could be classified as a. albertensis. the bacteria of acidithiobacillus genus are gramnegative obligatory acidophilic chemolithotrophic autotrophs capable of growth utilising inorganic sulphur compounds as sole energy source (karavaiko et al. 2003). due to increasing industrial and environmental impact of acidithiobacilli there is profound interest in biology, genetics, and genomics of these bacteria (pristas et al. 2018). the genus comprises 7 species which occur world-wide in a range of natural fig. 3. unrooted phylogenetic tree documenting relatedness of a. albertensis ts isolate (underlined) to other acidithiobacillus spp. 16s rrna gene sequences. genbank accession numbers are shown in parentheses. the tree was constructed using neighbor joining algorithm implemented in mega software ver. 7 (kumar et al. 2016). number at nodes are bootstrap values after 1000 repetition, only values over 80 are shown. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:54 utc http://www.ncbi.nlm.nih.gov/blast nova biotechnol chim (2019) 18(1): 18-24 22 table 1. list of acidithiobacillus sp. isolated from european mines (modified from nuñez et al. 2017). data on a. albertensis characterised in our study are shown bold. and industrial settings (kelly and wood 2000), six members of the genus, except a. albertensis, were already isolated in european industrial areas (table 1). although a. albertensis species was for the first time described by bryant et al. already in 1983 (bryant et al. 1983), it is not frequently studied species and there are very limited number of strains available, mainly from china territory (liu et al. 2008). in previous studies this species was isolated from acidic soil adjacent to a sulphur stockpile in canada (bryant et al. 1983), from acid-mine drainage of copper ore rich of sulphur in china (xia et al. 2007), from copper ore sludge in china mine (xingyu et al. 2010), from acidic river rio agrio in argentina (urbieta et al. 2012) and from operating zinc sulphide heap processing the ore from red dog mine in alaska (lizama et al. 2012). for the first time a. albertensis bacterium was isolated from gold mine subsurface environment and it is for the first time when this bacterium was isolated from europe territory. preliminary report on ts isolate characterization appeared in pristas et al. (2018). ts isolate of a. albertensis, characterised in our study, shows typical features of the species. it is acidophilic, mesophilic, obligatory chemoautotrophic, aerobic, gram-negative rods, not producing spores. the strain is unable neither to grow heterotrophically nor to catalyse the dissimilatory oxidation of ferrous iron. the sequence analysis placed 16s rrna sequence of ts isolate to the large cluster of a. albertensis sequences with similarity values as high as 99.7 %. to the cluster some environmental sequences fall as well (uncultured bacterium clone amd-a49, genbank accession no. kc620631) from acid mine drainage sample in china. the sequence comparison confirmed close relationship of a. albertensis and a. thiooxidans species (fig. 3) forming a cluster well separated from ferrous ions oxidizers. the by-05 strain of a. albertensis was employed for bioleaching of metal sulphides ores (xia et al. 2007) so probably biotechnological utilisation of the species might be oriented more to sulphur dissolution or as a part of bioleaching consortium with iron-oxidising bacteria to metal dissolution. conclusions the subsurface biosphere in gold deposits was very rarely studied, however, according to the newest findings become evident that bacteria actively contribute and regulate biogeochemical cycles in this environment. the diversity of culturable microorganisms involved in sulphur oxidation was investigated in the weathered rocks of gold mine hodruša-hámre, slovakia. just the single species of acidithiobacillus albertensis was isolated from the samples suggesting the low level of biodiversity of autotrophic bacteria in deep subsurface deposits. in this study we for the first time reported autotrophic bacterial diversity and isolation of a. albertensis from solid sample of weathered rocks in deep gold mine. although a. albertensis is not an unknown species, there is still very little known about these bacteria and their potential country species sample type ore type austria a. ferrooxidans water uranium finland a. ferriphilus na zinc a. ferrooxidans na copper france a. ferridurans water zinc, lead a. ferrooxidans water zinc, lead germany a. ferrooxidans na uranium a. ferrooxidans soil na a. ferrooxidans water copper a. ferrooxidans slurry copper a. ferrooxidans soil coal a. ferrooxidans soil uranium norway a. ferrivorans water copper romania a. ferrooxidans soil sulphide a. ferrooxidans soil zinc slovakia a. ferrooxidans water copper a. albertensis soil gold spain a. ferrooxidans water nickel a. ferrivorans soil copper united kingdom a. caldus soil coal a. ferrooxidans soil coal a. ferrooxidans soil copper a. ferrooxidans water tin a. ferrooxidans na pyrite a. thiooxidans soil coal bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:54 utc nova biotechnol chim (2019) 18(1): 18-24 23 in bioleaching of metals from ores or metal-bearing waste. further studies of functional bacteria and potential new functional species need to be carried out to explore their specific contributions to gold biogeochemical cycling. acknowledgement the work was financially supported by research agency of the ministry of education, science, research and sport of slovak republic under the vega projects 1/0229/17 and sk-pl-18-0012. sj acknowledges support from vega project 1/0560/15 and apvv-15-0083 project from slovak research and development agency. conflict of interest the authors declare that they have no conflict of interest. references altschul sf, gish w, miller w, myers ew, lipman dj (1990) basic local alignment search tool. j. mol. biol. 215: 403-410. bryant rd, mcgroarty km, costerton jw, laishley ej (1983) isolation and characterization of a new acidophilic thiobacillus species (t. albertis). can. j. microbiol. 29: 1159-1170. chen h, yang b, chen x (2009) identification and characterization of four strains of acidithiobacillus ferooxidans isolated from different sites in china. microbiol. res. 164: 613-623. chovan m, jágerský i, delaney v, žitňan p, kubač a, bačík p, troppová d, mikuš t (2016) mineralogy of ore dressing products from banská hodruša au (ag, pb, cu) epithermal deposit. acta geol. slov. 8: 203-216. huang x, madan a (1999) cap3: a dna sequence assembly program. genome res. 9: 868-877. inagaki f, takai k, hirayame h, yamato y, nealson kh, horikoshi k (2003) distribution and phylogenetic diversity of the subsurface microbial community in a japanese epithermal gold mine. extremophiles 7: 307-317. karavaiko gi, turova tp, kondraťeva tf, lysenko am, kolganova tv, ageeva sn, muntyan ln, pivovarova ta (2003) phylogenetic heterogenity of the species acidithiobacillus ferrooxidans. int. j. syst. evol. micr. 53: 113-119. kelly dp, wood ap (2000) reclassification of some species of thiobacillus to the newly designated genera aciditihobacillus gen. nov., halothiobacillus gen. nov. and thermithiobacillus gen. nov. int. j. syst. evol. micr. 50: 511-516. koděra p, kubač a, vojtko r, uhlík p, lexa j, chovan m (2018) field stop 1: hodruša hámre, rozália gold mine. in joint 5th central-european mineralogical conference and 7th mineral sciences in the carpathians conference, excursion guide, bačík p, fridrichová j (eds.), banská štiavnica, june 26-30, 2018, p.16-21. kubač a, chovan m, koděra p, kyle rj, žitňan p, lexa j, vojtko r (2018) mineralogy of the epithermal precious and base metal deposit banská hodruša at the rozália mine (slovakia). miner. petrol. 112: 705-731. kumar s, stecher g, tamura k (2016) mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. mol. biol. evol. 33: 1870-1874. li m, tian h, wang l, duan j (2017) bacterial diversity in linglong gold mine, china. geomicrobiol. j. 34: 267-273. liu xy, chen bw, wen jk (2008) dominance of acidithiobacillus at ore surface of zijinshan commercial low-grade copper bioleaching heap. trans. nonferrous met. soc. china. 18: 1506-1512. lizama hm, jensen se, stradling aw (2012) dynamic microbial populations in heap leaching of zinc sulphide ore. min. eng. 25: 54-58. nuñez h, moya-beltrán a, covarrubias pc, issotta f, cárdenas jp, gonzález m, atavales j, acuña lg, johnson db, quatrini r (2017) molecular systematics of the genus acidithiobacillus: insights into the phylogenetic structure and diversification of the taxon. front. microbiol. 8: 30. pristas p, kiskova j, timkova i, malinicova l, luptakova a, kusnierova m, sedlakova-kadukova j (2018) genetic variability in acidithiobacillus spp. – a working horse of environmental biotechnologies. nova biotechnol. chim. 17: 125-131. rastogi g, osman s, kukkadapu r, engelhard m, vaishampayan pa, andersen gl, sani rk (2010) microbial and mineralogical characterizations of soils collected from the deep biosphere of the former homestake gold mine, south dakota. microb. ecol. 60: 539-550. silverman mp, lundgren dg (1959) studies on the chemoautotrophic iron bacterium ferrobacillus ferrooxidans. i. an improved medium and a harvesting procedure for securing high cell yields. j. bacteriol. 77: 642-647. takai k, moser dp, onstott tc, spoelstra n, pfiffner sm, dohnalkova a, fredrickson jk (2001) alkaliphilus transvaalensis gen. nov., sp. nov., an extremely alkaliphilic bacterium isolated from a deep south african gold mine. int. j. syst. evol. micr. 51: 12451256. urbieta ms, toril eg, aguilera a, giaveno ma, donati e (2012) first prokaryotic biodiversity assessment using molecular techniques of an acidic river in neuquén, argentina. microb. ecol. 64: 91-104. vandžurová a, bačkor p, javorský p, pristaš p (2013) staphylococcus nepalensis in the guano of bats (mammalia). vet. microbiol. 164: 116-121. xia jj, peng aa, he h, yang y, liu xd, qiu gz (2007) a new strain acidithiobacillus albertensis by-05 for bioleaching of metal sulfides ores. trans. nonferrous met. soc. china. 17: 168-175. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:54 utc nova biotechnol chim (2019) 18(1): 18-24 24 xingyu l, bowei ch, jiankang w, renman r (2010) leptospirillum forms a minor portion of the population in zijinshan commercial non-aeration copper bioleaching heap identified by 16s rrna clone libraries and real-time pcr. hydrometallurgy 104: 399-403. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:54 utc nova biotechnol chim (2018) 17(1): 74-85 doi: 10.2478/nbec-2018-0008  corresponding author: jalcam@unsa.edu.pe nova biotechnologica et chimica nutritional composition, total phenolic compounds and antioxidant activity of quinoa (chenopodium quinoa willd.) of different colours yemina karen diaz-valencia1, juan josé alca1,, maria antonia calori-domingues2, sonia jackeline zanabria-galvez1 and sandra helena da cruz2 1department of food industry engineering, universidad nacional de san agustín de arequipa, independencia avenue s/n, arequipa 04000, peru 2department of agri-food industry, food and nutrition, college of agriculture “luiz de queiroz”, university of sao paulo, pádua dias avenue 11, piracicaba, brazil article info article history: received: 2nd january 2018 accepted: 30th april 2018 keywords: antioxidant minerals nutrition phenolics starch quinoa abstract quinoa (chenopodium quinoa willd.) has been nutritionally highlighted when compared to other grains. in recent years the research on this pseudocereal has increased. in this work, six quinoa samples were studied: three from peru, one from brazil and two commercial samples. the samples were physically and physicochemically characterized, including macroand micronutrient analysis, phenolic compounds content and antioxidant activity. black, red and white samples showed as main difference the size, weight, ashes and dietary fibre content. black samples were the smallest and lightest and had the lowest starch content but presented the highest levels of ashes and dietary fibre. the protein content (16.9 %) in the white brazilian variety was higher than the others. red and black samples had the highest levels of most minerals analysed. the antioxidant capacity measured by the dpph method was higher for black and red samples in comparison with the white ones. however, the white brazilian variety showed a significantly higher antioxidant capacity measured by the abts assay. with regard to the phenolic content, a difference was found between the samples which ranged from 55.5 to 95.5 g gae 100 g-1. the colour of the grain was found as not related to a higher content of phenolic compounds. because their compositions are generally similar to light-coloured grains, and in some parameters such as dietary fibre and content of some micronutrients are superior, the grains of dark-coloured quinoa varieties (rpp, bcp) would have to be explored to develop foods that take advantage of this colour diversity.  university of ss. cyril and methodius in trnava introduction quinoa (chenopodium quinoa willd.) has been cultivated by the inhabitants of the andes for human consumption for centuries and in recent years it has increased its sowing and market value in part to its excellent nutritional characteristics (navruz-varli and sanlier 2016). the lightcoloured varieties had been the most preferred by the consumers and also the most studied. several studies had already reported the nutritional profile of these varieties (kozioł 1992; ando et al. 2002; aluwi et al. 2017) indicating also the presence of some anti-nutrients like phytates and saponins. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:30 utc nova biotechnol chim (2018) 17(1): 74-85 75 the relationship between the content of saponins and the colour of the quinoa grains has been studied showing that the whites are sweeter (lower saponins content) than the yellow ones (souza et al. 2004). with regard to the phenolic acid content of quinoa of different colours, the published results were inconclusive. some have reported that the content of phenolic acids and flavonoids did not differ much between samples of different colours, highlighting only some variety while others of similar colour presented similar values to the rest (repo-carrasco-valencia et al. 2010). on the other hand, comparing one cream-coloured quinoa sample with other white ones, the first has been reported to have a higher content of total polyphenols and scavenging activity (repocarrasco-valencia et al. 2011). other authors have pointed out that for red quinoa varieties, the ones that were redder would have greater antioxidant activity and higher content of phenolic compounds (abderrahim et al. 2015). it is reported that the content and characteristics of starch and protein from different quinoa lines vary due to the environment and genetic and agronomic differences (lindeboom 2005). in the same way, the variation in the mineral content of quinoa of different varieties is due to the interactions of genotype and environmental parameters (prado et al. 2014). the content of certain minerals was higher in varieties that were of a determined colour. physically, some coloured varieties were reported to be heavier and larger than the white ones (apaza et al. 2013). thus, our aim was to determine some characteristics of different quinoa varieties in order to confirm possible relationships between their values and the colour. experimental quinoa samples three samples of quinoa (chenopodium quinoa willd.) grains were obtained from the national agricultural research institute (inia) of peru: inia 415 “pasankalla”, inia 420 “negra collana” and inia “salcedo”, coded as rpp, bnp and wsap for this study. rpp, bnp and wsap were released in 2006, 2008 and 1995 respectively (apaza et al. 2013). two commercial samples from arequipa, peru were purchased for the study (coded as rcp and bcp for this study) and a brazilian quinoa sample (brs “syetetuba”, coded as wsyb, released in 2006 according to spehar et al. 2011) was obtained from the farm of the federal university of brasilia. rpp and bnp samples were sown in 2011 and harvested in 2012 (puno, peru), wsap was sown in 2013 and harvested in 2014 (puno, peru), wsyb was harvested in 2014 (brasilia, brazil), the commercial varieties (rcp and bcp) were both acquired in 2014. the samples of quinoa grains are shown in fig. 1. fig. 1. quinoa grains of different colors. rpp: inia 415 “pasankalla”, rcp: red peruvian commercial sample, bnp: inia 420 “negra collana”, bcp: black peruvian commercial sample, wsyb: brs “syetetuba”, wsap: inia “salcedo”. saponin removal the saponin content in the samples was evaluated by a semiquantitative method described by kozioł bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:30 utc nova biotechnol chim (2018) 17(1): 74-85 76 (1990). this method is based on the tensioactive properties of the saponins, which form a stable foam whose height is related to the saponin content in the grains after being dissolved in water and shaken. the percentage of saponins was obtained with eq. 1. (1) preparation of quinoa samples the quinoa grains were washed with distilled water (at 37 °c) in the ratio 1:2 (quinoa: distilled water). then the grains were shaken by hand for 20 min with distilled water and rinsed. this procedure was performed twice and then the grains were dried at 37 °c for 12 hours in a hot air oven. the dried grains were stored in polypropylene bags in a cold storage chamber (10 °c) for two months before use (august 2014). for the colour evaluation, four sub-samples were taken from these stored grains. for the determination of size distribution and weight of thousand grains eight sub-samples were taken. three, two and six subsamples were taken to the physical-chemical, elemental and antioxidant activity analysis respectively. for all cases, each of these subsamples were processed according to the corresponding methodology described in this work. colour evaluation the colour of the quinoa grains was evaluated using the colourimeter chroma meter 200b (minolta co., osaka, japan) by the cie 1976 l*a*b* system obtaining the values of l* (lightness), a∗ and b∗ (opponent colour axes). the hue angle (h°) and chroma (c∗) were calculated according the eq. 2 and 3. size distribution and weight of thousand grains the dried quinoa grains after saponin removal were analyzed by size. for this purpose, tyler/mesh sieves number 12, 14 and 20, corresponding to openings of 1.41 mm, 1.19 mm, and 0.841 mm respectively, were used. the amounts retained in each sieve were weighed and expressed in percentages. in addition, 100 whole grains without impurities were counted manually, in 8 replicates, for the determination of the weight of one thousand grains. after weighing using a sartorius bl210s analytical balance (sartorius, germany), the mean, standard deviation and coefficient of variation of the measurements were calculated. the result of a thousand grains was calculated by multiplying by 10 the average weight of the repetitions if the coefficient of variation was less than 4 %. as the weight of a thousand grains of a sample varies according to the moisture content the results obtained were standardized to 10 % moisture, which was the average moisture obtained from the grains at the time of evaluation. physical-chemical analysis the grains previously washed were ground in a marconi ma-090 hammer mill (marconi ltd., piracicaba, brazil) with a 20 mesh screen (0.841 mm). the milled grains were evaluated for moisture using an a&d mx-50 moisture analyzer (a&d co. ltd., tokyo, japan). the ash content (calcination in muffle at 550 °c for two hours), lipids (extraction with ethyl ether using soxhlet extractor) and crude protein (kjeldhal digestion with the factor 6.25 for conversion of the total nitrogen content) were determined according to the methods 923.03, 920.39c and 979.09a described by aoac (2005). the starch content of the milled grains of each sample was determined by the megazyme total starch kit (aa / amg) ktsta 09 (megazyme, ireland). macroand microminerals content macrominerals phosphorus, potassium, calcium, magnesium, sulfur and microminerals iron, zinc, copper, manganese, aluminum, sodium, and lithium were determined using a methodology based on the method 953.01 by aoac (2005). for all elements, nitroperchloric digestion was used, except for boron, which was extracted by dry digestion in muffle according to malavolta et al. (1997). the quantification of the elements was performed in an atomic absorption spectrophotometer, (2) (3) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:30 utc nova biotechnol chim (2018) 17(1): 74-85 77 the lithium was quantified in a coupled plasma mass spectrometer (icap 7400 icp-oes analyzer, thermo fisher scientific, usa). the results were expressed as mg 100 g−1 of dry basis product. determination of phenolic compounds the content of total phenolic compounds (tpc) was determined according to the folin-ciocalteu spectrophotometric method (singleton et al. 1999) using gallic acid as standard. the hydroalcoholic extracts were obtained according to the methodology of repo-carrasco-valencia et al. (2010), with modifications. a sample containing 5 g of milled grains was dissolved in 20 ml of 95 % ethanol. after homogenization for 1 min the samples were storaged at -4 °c for 18 hours. then the extracts were centrifuged at 29,000 g for 20 min in an eppendorf 5810 r refrigerated centrifuge at 4 °c (eppendorf ag, hamburg, germany) and filtered using whatman filter paper no. 2 and stored in amber bottles under refrigeration at 7 °c until the time of analysis. aliquots of 0.5 ml of the hydroalcoholic extract were transferred to test tubes, where 4 ml of distilled water and 0.5 ml of the folin-ciocalteu solution were added. then the tubes were shaken in a vortex mixer and put aside for 3 min. after the addition of 0.5 ml of a 4 % (w/v) sodium carbonate solution, the tubes were vortexed again and held for two hours at room temperature and protected from light. immediately after, the absorbance reading was performed in a femto 700s spectrophotometer at 740 nm (sao paulo, brazil). the results were expressed in milligrams of gallic acid equivalent (gae) per gram (dry basis). evaluation of antioxidant capacity the evaluation of the antioxidant capacity of the hydroalcoholic extracts obtained for the determination of phenolic compounds was performed using the 2,2-diphenyl-1-picrylhydrazyl radical (dpph) and 2,2'-azino-bis(3ethylbenzthiazoline-6-sulphonic acid) (abts) assays. for the dpph assay the methodology proposed by brand-williams et al. (1995) was used. an aliquot (500 μl) of solution was transferred to a test tube containing 3 ml of 95 % ethanol and 300 μl of the dpph solution. the solution was held out of the light for 45 min previously to the absorbance reading at 515 nm using a spectrophotometer and compared with a blank. a standard curve was obtained using trolox solutions from 5 to 50 μm. the trolox equivalent antioxidant capacity (teac) was obtained with eq. 4 and expressed as μmol teac g−1 db (dry basis). the methodology proposed by re et al. (1999) was used to evaluate the reduction of the abts radical. an aliquot (30 μl) was transferred to a test tube containing 3.0 ml abts (7 mm). the solutions were kept in the dark for 6 min and the absorbance was read at 734 nm. the standard curve was obtained from 500 to 2000 μm using trolox solutions. the results were expressed in μmol teac g−1 db using a blank as a reference. statistical analysis the results were subjected to analysis of variance (anova) and the tukey test for comparison of means (p < 0.05) using the sas statistical software version 9.3 (sas institute inc., cary, nc, usa). results and discussion saponin content the quinoa grains of the different samples were evaluated for saponin content before and after saponin removal. the grains presented low initial saponin content, from nearly zero (≤ 0.01 % db for rpp and bcp) to ≤ 0.04 % db (rcp, bnp and wsap), except for wsyb which presented a high content (0.34% db). bnp and rpp were considered as free from saponins by apaza et al. (2013) like wsyb according to spehar et al. (2011), which differed from our results. kozioł (1992) suggested, as possible explanation, the different environmental conditions of grain production or the different methodologies used to determine the saponin (4) bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:30 utc nova biotechnol chim (2018) 17(1): 74-85 78 table 1. lightness (l*), opponent colour axes (a* and b*), chroma (c*) and hue angle (h°) of quinoa grains. four replicates. values followed by the same letter in the column do not differ (tukey test, p < 0.05). sample l* a* b* chroma (c*) h° rpp 33.4±1.4c 16.2±1.2 25.4±2.8 30.2±2.9a 57.4±1.5b rcp 30.8±1.5d 18.9±0.9 22.1±2.1 29.1±2.1a 49.4±1.7 c bnp 14.1±1.8e 7.3±1.5 4.9±0.9 8.8±1.7c 34.2±4.4e bcp 13.2±2.6e 6.9±1.6 5.7±1.5 9.1±1.6c 39.4±9.3d wsyb 66.0±0.9b 2.9±2.7 23.1±0.7 23.2±0.7b 82.8±0.5a wsap 76.1±1.2a 2.1±0.3 24.8±1.0 24.9±1.0b 85.3±0.7a content. our results showed that after saponin removal procedure, the saponin content was 0.01 % (dry basis) for bsyb and 0 % for the other cultivars. according to this author the saponins are the main antinutritional factors of the quinoa grain but they can be removed by wet or abrasive methods, reaching levels not exceeding 0.01 %, as observed in our samples because such substances are concentrated in the outer layers of the grains. it has been reported that some plantbased saponins can form complexes with iron and zinc, which would reduce absorption in rats (southon et al. 1988). however, saponins have very low toxicity to other mammals (malinow et al. 1982) and do not affect protein availability in the case of quinoa (ruales and nair 1992). color evaluation the studied quinoa samples presented different colour parameters as shown in table 1. in the literature consulted the saponin content was related to the colour; reporting that the yellow cultivars had more saponin than the white ones (souza et al. 2004). the values observed for h° (above 80) suggest that the samples considered white could be classified with a tendency to yellow. however, even though the white brazilian sample (wsyb) had the highest content of saponins, the peruvian white sample (wsap) had a similar content to the samples of different colour, so it is not possible to confirm with our data the relationship between colour and content of saponins in quinoa grains of different colours. the differences observed for h°, among the grains of the same colour, rpp and rcp and bnp and bcp samples, may be due to the commercial samples (rcp and bcp) are not standardized and a mixture of different colours may have occurred. medina et al. (2010) tried to use image analysis to determine the geographic origin of different quinoa grains but proved that it was impossible to use the colour of the grains for this purpose. however, it has been stated that the colour of grains of dark quinoa varieties is an indicator of the presence of betacyanins (low l* values), while the colour of lighter varieties is related to a higher betaxanthin content (escribano et al. 2017). thus, the main use of measuring the colour of quinoa grains has been as a characteristic associated with their composition. as regards manufactured products (based on quinoa flour), table 2. distribution of quinoa grains by size and weight of one thousand grains. for size distribution n = 4 whereas for the weight of one thousand grains n = 8. values followed by the same letter in the row do not differ (tukey test, p < 0.05). tyler mesh no. (aperture in mm) percentage with respect to the weight of one thousand grains [%] rpp rcp bnp bcp wsyb wsap 12 (1.41) 86.2 83.9 12.6 19.6 78.2 85.7 14 (1.19) 13.4 13.9 81.0 74.7 21.2 13.7 20 (0,841) 0.4 2.2 6.4 5.4 0.4 0.4 bottom (< 0.841) 0.0 0.0 0.0 0.3 0.2 0.2 one thousand grains weight [g] 3.71±0.06c 4.11±0.09a 2.30±0.07e 2.06±0.05f 3.88±0.06b 3.12±0.09d coefficient of variation [%] 1.6 2.1 3.2 2.3 1.6 2.9 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:30 utc nova biotechnol chim (2018) 17(1): 74-85 79 table 3. physical-chemical composition of quinoa grains. three replicates. values followed by the same letter in the column do not differ (tukey test, p < 0.05). quinoa samples ash [%] protein [%] lipid [%] total dietary fibre [%] starch [%] rpp 2.51±0.02b 14.0±0.3b 7.33±0.11a 14.30±0.01b 56.02±0.03 b rcp 2.58±0.02b 12.5±0.6b 6.70±0.14b 13.90±0.06b 54.56±0.01 d bnp 2.96±0.10a 14.1±0.1b 6.67±0.10b 18.20±0.00a 49.25±0.07 e bcp 3.01±0.09a 14.0±0.2b 7.34±0.11a 19.70±0.22a 47.22±0.09 f wsyb 2.32±0.01c 16.9±0.2a 6.84±0.08b 8.70±1.67c 55.39±0.05 c wsap 2.36±0.02c 13.7±0.1b 7.29±0.03a 10.50±0.45c 59.72±0.05 a it has been observed that there is an effect on the colour of the final product in relation to the percentage of flour used, but it was not indicated whether the pigments of quinoa grains played a role in this characteristic (demir and kilinç 2017). size distribution and weight of thousand grains the red and white samples showed larger grains than the black ones (table 2). this is consistent with the data from apaza et al. (2013) who reported the average diameter of bnp, rpp and wsap as 1.60, 2.10 and 2.00 mm, respectively. the same authors also reported the weight of thousand grains for bnp, rpp and wsap as 2.03, 3.51 to 3.72 and 3.10 to 3.70 g, respectively. this trend is followed by our data, which showed that black samples are lighter than the others. values above 3.0 g per thousand grains are considered as large grains (spehar et al. 2011) and considered more desirable in the growing quinoa market. physical-chemical analysis all the samples had moisture levels below 12 % (from 8.3 to 11.2 %), being thus suitable for storing according to spehar (2006). the black samples presented the highest values for the ash content (table 3), around 3 % db, followed by the red and white samples. these values were similar to those found by repo-carrasco-valencia et al. (2010) for the cultivars rpp (2.5 %) and wsap (2.4 %). all the samples analysed had a similar protein content, with only one of the white samples (wsyb) standing out. with respect to lipid content, a sample of each colour (rpp, bcp, wsap) was particularly noteworthy. the protein content of the samples was also similar to those observed by miranda et al. (2012), who reported values from 11.32 to 16.10 % db. repo-carrasco-valencia et al. (2010) reported values for lipids ranging from 5.2 to 6.8% for white, black and red cultivars. meanwhile, ando et al. (2002) found 6.5 % of lipids in the white cultivar real, which was slightly lower than our results for samples of the same colour. the content of lipids of all samples was higher than other cereals such as rice, corn, wheat and barley, which present 2.2 %, 4.7 %, 2.3 % and 1.9 %, respectively (kozioł 1992). the two white samples had the lowest values of dietary fibre, followed by the two red samples. the dietary fibre content of black samples is comparable to that of hulled barley and superior to that of raw white rice (17.3 % and 1.3 % respectively, according to usda (2017). the variation in the ash, protein, lipid, and fibre contents among quinoa samples can be attributed to the production site (climate, soil, temperature, management) and to the genetic characteristics of the cultivars (lindeboom 2005; repo-carrasco-valencia et al. 2011). the main component found in the samples was starch (table 3). the observed results for starch are close to the ranges already reported in previous studies. the composition of quinoa grains of different colours (red, yellow and white) was evaluated by bruin (1964), reporting the starch content ranged from 58.1 to 64.2 % and the white samples showed the highest levels. on the other hand, lindeboom (2005) evaluated 8 lines of quinoa grains founding that the starch content varied from 48.3 to 62.5 % db, but the colour of the grains was not specified. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:30 utc nova biotechnol chim (2018) 17(1): 74-85 80 table 4. contents of macrominerals and microminerals in mg 100 g⁻¹ db, except for li. two replicates. values followed by the same letter in the row do not differ (tukey test, p < 0.05). element quinoa samples rpp rcp bnp bcp wsyb wsap p 578±13a 588±13a 512±14b 490±7b 510±12b 492±4b k 699±1b 593±5c 815±33a 693±1b 559±10c 663±1b ca 73±3b 109±0a 71±7b 118±1a 69±5b 77±2b mg 243±7bc 264±5a 226±6cd 246±0ab 239±5bcd 220±1d s 185±5a 174±5ab 158±14ab 144±1b 177±16ab 164±2ab fe 5.63±0.15cd 5.25±0.03d 7.28±0.18bc 17.2±1.10a 8.59±0.09b 4.74±0.03d zn 3.70±0.05b 3.79±0.04b 2.98±0.06d 3.36±0.04c 5.12±0.08a 3.17±0.03cd b 1.34±0.05a 1.16±0.03b 1.01±0.01cd 0.92±0.01d 1.11±0.02bc 0.80±0.02e cu 0.47±0.01b 0.50±0.01b 0.47±0.04b 0.57±0.00a 0.48±0.01b 0.51±0.01ab mn 6.90±0.15c 3.41±0.02e 9.48±0.11a 7.99±0.06b 2.53±0.07f 5.53±0.10d al 2.21±0.05dc 2.82±0.25c 1.84±0.01d 5.87±0.20a 4.54±0.19b 1.79±0.07d na 17.5±0.29c 36.9±0.56b 11.8±0.32d 49.3±0.17a 17.1±0.06c 16.6±0.38c li (µg kg-1) 0.14±0.01b 0.58±0.02a 0.14±0.02b 0.65±0.08a 0.17±0.02b 0.19±0.00b macroand micromineral content a high content of p and k was observed, confirming that these two elements represent the greatest amount of minerals in quinoa grains (bruin 1964; mota et al. 2016). red samples showed the highest phosphorus content with respect to the others samples. rpp and rcp had more p content that varieties from chile (285.6 to 526.36 mg 100 g−1 db according to miranda et al. 2012) or bolivia (123.72 to 330.96 mg 100 g−1 db according to prado et al. 2014). the content of p in quinoa is higher than that found in maize (210 mg 100 g−1), wheat (323 mg 100 g−1), rice (95 mg 100 g−1) and barley (221 mg 100 g−1) (usda 2017). it was reported that the potassium content of quinoa milled grains was 639.3 mg 100 g−1 (ando et al. 2002), whereas (prado et al. 2014) showed a wider range (from 498.47 to 995.24 mg 100 g−1). those results were consistent with ours although no colour effect has been observed and just bnp stood out from the rest. quinoa has higher potassium contents than other cereals and pseudocereals of interest as corn, wheat, rice and barley (usda 2017) and amaranth (nascimento et al. 2014). it has been reported that even after quinoa grains were cooked, potassium and phosphorus remained the main elements (mota et al. 2016). the commercial samples rcp and bcp had the higher calcium content, with no grain colour effect observed. other studies had reported broader ranges in the concentration of this mineral. varieties analyzed by miranda et al. (2012) ranged from 25.15 to 116.60 mg 100 g−1, whereas prado et al. (2014) reported a range from 77.10 to 211.29 mg 100 g−1. the saponin removal process could decreased the percentage of calcium because this mineral is more concentrated in the pericarp followed by the embryo and perisperm (konishi et al. 2004). magnesium was present in more concentration than reported for other quinoa varieties (miranda et al. 2012; prado et al. 2014) but close to 250 mg 100 g−1, informed by kozioł (1992). however, it has been recorded values as high as 502.0 mg 100 g−1 (konishi et al. 2004). in comparison, the magnesium content of hard red winter wheat and hulled barley was reported as 126 and 35 mg 100 g−1 respectively (usda 2017). although there is no recommended intake intake for sulphur, and that deficiencies of this mineral are not known (sizer and whitney 2003), our results were consistent with those of bruin (1964), who reported a range from 150 to 220 mg 100 g−1. the genetic characteristics influence the mineral composition of the quinoa grains, as well as the type of soil where they are cultivated due to the chemical or mineral composition of the soils and fertilizers used (vega-gálvez et al. 2010; nascimento et al. 2014). in relation to the bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:30 utc nova biotechnol chim (2018) 17(1): 74-85 81 microminerals evaluated (table 4), it was observed that the iron content was in the range previously reported by miranda et al. (2012), who reported a range from 4.82 to 7.19 mg 100 g−1, except for bcp which stood out from the rest with 17.2 mg 100 g−1. however, prado et al. (2014) reported a wider range from 4.76 to 24.04 mg 100 g−1, evidencing the great variation that can exist between the different samples of quinoa grains, as well as in this study. it was reported that the hard red winter wheat and hulled barley had just 3.19 and 3.60 mg 100 g−1 of iron respectively, whereas the raw medium-grain white rice had 0.80 mg 100 g−1 (usda 2017). the zinc content in the samples was similar to the range reported by miranda et al. (2012) (from 2.73 to 5.01 mg 100 g−1) and prado et al. (2014) (from 1.65 to 4.22 mg 100 g−1). one white sample (wsyb) had more zinc content than the red and black samples although the other white sample (wsap) show a similar value to the black ones, so it was not possible to establish a colour-based relationship with our data. red samples (rpp and rcp) had the highest boron content. these results were similar to those of bruin (1964), who reported a range from 0.92 to 1.29 mg 100 g−1, but lower than those of karyotis et al. (2003) who reported a completely different range (from 3.4 to 4.7 mg 100 g−1). boron is considered to be essential for animals, but the requirement for humans is still under study (sizer and whitney 2003). as far as the authors know, the presence of aluminium in quinoa was analysed previously only by bruin (1964), who reported a range from 6 to 10 mg 100 g−1. our results are lower than those but since aluminium is not known to have a biological function in animal or human organisms (stahl et al. 2017), it is not considered an essential nutrient but an anti-nutrient. a black sample (bcp) had the higher copper content although the other black showed a similar value than the ones of different colour. overall, our results were in the range reported by prado et al. (2014) (from 0.35 to 1.12 mg 100 g−1) for varieties harvested in bolivia. however, copper content of quinoa harvested in chile showed a different and higher range: from 0.75 to 1.52 mg 100 g−1 (miranda et al. 2012). both black samples (bnp and bcp) presented higher content of manganese than the red ones and lastly by the white ones. our results for black and red samples were higher than those reported by prado et al. (2014) (from 1.55 to 3.85 mg 100 g−1) and the black samples even surpassed the levels reported by miranda et al. (2012) (from 2.36 to 6.47 mg 100 g−1). with regard to sodium and lithium content, black and red samples showed the highest levels. in the case of sodium, our results were higher than those by prado et al. (2014) (from 1.74 to 7.33 mg 100 g−1) and karyotis et al. (2003) (0.34 to 2.13 mg 100 g−1) but lower than those reported by bruin (1964) (from 11 to 22 mg 100 g−1). regarding the lithium, both commercial samples (bcp and rcp) had the highest content. nascimento et al. (2014) observed lithium contents of 7.95 g 100 g−1, being higher than those found in the present study. however, grains of bolivia quinoa showed levels as high as 7.5 mg 100 g−1 (figueroa et al. 2013). these authors have attributed the high lithium content to the salar de uyuni (bolivia), which is considered to be among the areas with the highest lithium content in the world, so the dissimilar data found in the literature on the micromineral contents in quinoa grains could probably be explained by the type of soil and climate from which the cultivars come from. table 5. content of total phenolic compounds present in quinoa grains. six replicates. values followed by the same letter in the column do not differ (tukey test, p < 0.05). samples tpc [mg gae 100 g-1] rpp 61.1±3.9bc rcp 65.4±4.0b bnp 95.9±3.6a bcp 55.5±3.7c wsyb 66.4±5.6b wsap 59.6±4.4bc content of total phenolic compounds one of the black samples (bnp) showed the higher content of phenolic compounds (table 5). the rpp cultivar was analysed reporting a content of 53.8 mg gae 100 g−1 (repo de carrasco and zelada 2008), lower than that found in the present study. fifteen quinoa cultivars evaluated by the same authors presented values ranging from 35.3 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:30 utc nova biotechnol chim (2018) 17(1): 74-85 82 to 139.9 mg gae 100 g−1, a range that encompasses our results. other studies presented values of phenolic compounds above 3.75 mg gae g−1, using a different methodology for the extraction process (using methanol and then acetone as extraction solvents, as reported by paśko et al. (2009). repo-carrasco-valencia et al. (2011) evaluated four different quinoa (blanca de juli, kcancolla, la molina and sajama) cultivars from peru and observed phenolic compounds values higher than ours ranging from 142 to 197 mg gae 100 g−1. it has been noted that the content of secondary plant metabolites, such as phenolic compounds, varies from generation to generation, depending on environmental factors (verma and shukla 2015). these factors include soil nutrients and rainfall (borges et al. 2013). because our samples were obtained from different locations in a different year, the effect of these factors on phenolic compounds should be considered, as some plant species have been reported to produce more phenolic compounds and antioxidant activity in highland and semi-arid climates (kumar et al. 2017). it was expected that the black and red samples had a higher tpc value than the white ones since a previous study reported that an improvement in tpc was accompanied by an increase in the pigments content in quinoa grains (abderrahim et al. 2015). the same authors reported a negative correlation between tpc and lightness (l) value (r = -0.619, p = 0.024). however, our results did not allow us to confirm this relationship because the other black sample (bcp) had the lowest value of tpc, so other factors besides pigmentation must have influence on the content of phenolic compounds in quinoa grains. in this regard, escribano et al. (2017) quantified some pigments (amaranthin, iso-amaranthin, betanin, iso-betanin, dopaxanthin, dopamine-bx, proline-bx and one unknown-bx) of rpp, bnp, wsap and 26 other quinoa varieties of different colors, detecting a concentration of amaranthin and iso-amaranthin of 0.8 mg kg-1 (for each pigment) for rpp only, while the pigment content of the wsap and bnp varieties was undetectable, although the colour varieties showed higher antioxidant activity. however, tang et al. (2015b) presented data showing that the tpc content in canadian commercial samples of dark-coloured quinoa is greater than that of white samples, with ferulic acid being the main contributor to the tpc value of black samples. therefore, it would be necessary to further quantify the content of ferulic acid and other phenolic compounds to elucidate whether the storage conditions or genotype of the present bcp sample are responsible for its low tcp value. evaluation of antioxidant capacity the antioxidant capacity of the studied quinoa samples, evaluated by the dpph method are shown in table 6. it was previously reported that the antioxidant activity of the black quinoa grain samples was approximately 5.6 μm trolox equivalent g−1, while for the white and red samples it was between 4.4 and 4.8 μm trolox equivalent g−1 (tang et al. 2015a), respectively. our results shown a similar trend, being the coloured grains those with the highest antioxidant activities. however, an unidentified bolivian quinoa sample presented 38.84 μm trolox equivalent g−1 (paśko et al. 2009), a value much higher than ours. a wide range of activity results was also reported by repo de carrasco and zelada (2008), who evaluated the antioxidant capacity of 15 quinoa cultivars by the dpph method, obtained values ranging from 117.5 to 2,400.5 μg trolox g−1, although the grain colour was not specified. the same authors presented values for amaranth in the range of 556.49 to 660.90 μg trolox g−1. for six varieties of barley, ondrejovič et al. (2014) determined antioxidant activity of 200 to 1,400 µg trolox g−1. regarding the percentage of inhibition, the black and red samples had higher values than the white ones which also presented the lowest contents of phenolic compounds and antioxidant capacity. analyzing the results of antioxidant capacity determined by the abts assay (table 6), it can be observed that one white sample (wsyb) presented the highest antioxidant capacity, followed by one of the black samples (bcp). repo-carrascovalencia et al. (2011) studied four light-coloured quinoa cultivars (blanca juli, kcancolla, la molina 89 and sajama) which presented higher values from 2,351.9 to 3,689.5 μg trolox g−1, being the yellow cultivar (la molina 89) the one with the highest value. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:30 utc nova biotechnol chim (2018) 17(1): 74-85 83 overall the results of antioxidant capacity are lower than those presented in the literature for quinoa. these large differences with respect to our results may be partly explained by the different extraction table 6. antioxidant activity of quinoa grains using dpph and abts and percentage of inhibition. six replicates. values followed by the same letter in the row do not differ (tukey test, p < 0.05). quinoa samples rpp rcp bnp bcp wsyb wsap dpph [µm teac g-1] 5.67±0.29b 4.35±0.20c 5.99±0.33ab 6.18±0.31a 3.33±0.39d 1.95±0.16e dpph [µg teac g-1] 1,419.1 1,088.8 1,499.2 1,546.8 833.5 488.1 inhibition [%] 45.0±2.4a 34.4±1.6b 47.6±2.7a 47.4±2.6a 23.5±3.3c 12.0±1.3d abts [µm teac g-1] 2.02±0.19c 1.79±0.07d 1.98±0.10c 2.25±0.08b 2.78±0.14a 1.95±0.12cd abts [µg teac g-1] 505.6 448.0 495.6 563.2 695.8 488.1 inhibition [%] 12.92±1.85bc 10.59±0.71d 12.45±0.98c 14.59±0.39b 18.67±1.42a 10.32±1.25d methodologies, so we could not accurately estimate the effect of colour. conclusions this study has determined several nutritional characteristics of quinoa varieties of different colours (black, red and white) obtained from different sites (arequipa and puno regions, in peru) in different harvest years (2012 and 2014). physically, the red samples were larger and heavier than the white ones, and these in turn were larger and heavier than the black ones, confirming previous reports. regarding the main anti-nutrient, one of the white samples (wsyp) had the highest content of saponins while bcp and rpp had a content of less than 0.01 % db. the lipid and protein content were similar for all the samples but black samples presented the lowest starch content. black and red samples had more content of ashes and dietary fibre than the white ones indicating a possible correlation just for this two components. samples of red and black colour stood out for their macrominerals content, especially rcp for mg and bnp for k. the microminerals analysis revealed the same trend, with bnp standing out in most results although it was matched in aluminium content by wsyb. this white sample had the highest zinc content. the black samples showed the highest and lowest levels of phenolic compounds, while the red and white samples had similar results. regarding the antioxidant activity, black and red samples showed the highest activities, highlighting bcp using the dpph method. using the abts method there was no clear differentiation, although wsyb showed increased antioxidant activity and inhibition percentage. thus, the relationship between the colour of the samples and the antioxidant activity could not be 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for fluctuation in plant secondary metabolites. j. appl. res. med. arom. plants. 2: 105113. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 09:30 utc nova biotechnol chim (2020) 19(2): 192-199 doi: 10.36547/nbc.v19i2.774  corresponding author: ioyeyemi@unimed.edu.ng; lovetayo2000@yahoo.com nova biotechnologica et chimica the protective effect of the aqueous extract of sida acuta burm.f on lead nitrate-induced genotoxicity ifeoluwa temitayo oyeyemi department of biological sciences, university of medical sciences, ondo, nigeria article info article history: received: 28th april 2020 accepted: 10th august 2020 keywords: anti-genotoxicity chromosome aberration mitotic index sida acuta abstract this study investigated the protective effective of sida acuta leaf extracts against the genotoxic effect of lead nitrate, a toxic heavy metal that easily permeate the ecosystem. the genotoxic and anti-genotoxic effects of the aqueous extract of s. acuta on onion cells (allium cepa l.) was evaluated using the allium cepa l. assay. onion bulbs were exposed to 0.25 – 2.5 mg.ml-1 concentrations of the plant extract for analyses of induction of cytogenetic damage. there was observed a concentration-dependent decrease in mitotic index of the a. cepa roots cells compared to the negative control. lead nitrate significantly induced chromosomal aberration in a. cepa root cells. this effect, however, was significantly ameliorated by the s. acuta leaf extract. this effect was demonstrated by the lower frequency of chromosome aberrations in lead nitrate treated root cells after exposure to the extract. furthermore, the extract restricted the extent of lead-induced cytological aberrations in a. cepa. the findings in this study suggested the mitodepressive, antiproliferative and anti-genotoxic potentials of the extract.  university of ss. cyril and methodius in trnava introduction plants are source of bioactive compounds with numerous benefits to mankind. some of them act as antioxidants and anti-mutagens while others act through various mechanisms to prevent or cure diseases and or modulate the harmful effects of xenobiotic effects. this has led to increase in consumption of herbs and herbal supplements worldwide for various reasons. some people believed it can cure diseases that orthodox medicine are unable to cure (welz et al. 2018) while others believe it is affordable, natural, and thus non-toxic. however there are reports of some medicinal plants being toxic in man or animals (oyeyemi et al. 2015; ruan et al. 2019). hence the need to establish the safety of medicinal plants. sida acuta, is a common wireweed, a species of the flowering plants of the mallow family (malvaceae). it is an herbaceous weed widely distributed in the tropical and subtropical countries (jindal et al. 2012). renowned for its phytoremediation properties, it acts as a phytoextractor and phytostabilizer of heavy metals (gupta and sinha 2007; putshaka et al. 2015; ameh et al. 2019) suitable for decontamination of metals in waste contaminated sites. the different parts of the plants are also used traditionally for medicinal purposes. it is used to treat wound (adetutu et al. 2011), liver disorders (ekpo and etim 2009), malaria (iyamah and idu 2015), nervous and urinary diseases, blood and bile disorders (sreedevi et al. 2009), headaches, infectious diseases, rheumatism (enemor et al. 2013), asthma, renal inflammation, colds, ulcers and worm infections (caceres et al. 1987; fokou mailto:ioyeyemi@unimed.edu.ng mailto:lovetayo2000@yahoo.com nova biotechnol chim (2020) 19(2): 192-199 193 et al. 2015). it has been reported to have antioxidant (adetutu et al. 2011; nwankpa et al. 2015), antineoplastic and antiproliferative (jang et al. 2003; pieme et al. 2010; mallikarjuna et al. 2013), hepatoprotective (sreedevi et al. 2009), neuroprotective (benjumea et al. 2016; owoeye and salami 2017; owoeye et al. 2017), antimicrobial (oboh et al. 2007; adetutu et al. 2011; akinnibosun and pela 2015), antiplasmodial (karou et al. 2003; nguyen-pouplin et al. 2007), analgesic (konaté et al. 2012), anti-hyperglycemic and anti-inflammatory activities (arciniegas et al. 2017). phytochemicals present in the leaves include alkaloids, saponins, tannins, steroids, glycosides and phenolic compounds (akinnibosun and pela 2015). despite its wide usage and potential health benefit, it has been reported to be neurotoxic (eluwa et al. 2013; enemor et al. 2013), although non-toxic to the liver and kidney (konaté et al. 2012). however, little is known about its genotoxic and anti-genotoxicity effects. studies of genotoxicity and anti-genotoxicity are valuable to establish the safety and efficacy of herbs or natural products (bast et al. 2002). allium cepa assay is a widely used plant assay for evaluation of genotoxic potential of different compounds/mixtures including medicinal plants (oyeyemi and bakare 2013; sharma et al. 2018). it detects genomic mutation (bonciu et al. 2018) with results comparable to those obtained in animal cell lines and is therefore an excellent model to determine the potential genotoxicity of compounds or mixtures. in view of the widespread use of s. acuta in traditional medicine and dearth of information on its anti-genotoxic potentials, this study seeks to investigate the genotoxic and anti-genotoxic effect of the aqueous extract of the leaves of s. acuta. genotoxicity tests can confirm the efficacy of a phytoremediation agent and also give an insight into the safety of medicinal plants for consumption. experimental collection of plants leaves of s. acuta were collected within ondo city and authenticated by a taxonomist at the department of forestry, federal university of technology, akure, nigeria. voucher specimen was deposited in the herbarium of the same department (futa herbarium: 149). the leaves were air dried and pulverised using electronic blender. the pulverised leaves were extracted by boiling in water at 100 °c as practised by the local people. the extract was filtered and concentrated using rotatory evaporator at 60 °c. the concentrated extract was stored at 4 °c until use. allium cepa assay allium cepa (onion) bulbs of same size were procured from a local market. these were sun dried for two weeks. the dried a. cepa bulbs were used for the modified a. cepa assay (fiskesjö 1997; oyeyemi and bakare 2013) to evaluate the genotoxic and anti-genotoxic effect of the extract. for the genotoxicity test, onions bulbs were placed on 100 ml beakers filled with tap water for 24 h. after 24 h, three onions per concentration variant were transferred into beakers with four different concentrations (0.25 – 2.5 mg.ml-1) of the aqueous extract of s. acuta (esa) for 48 h to permit two complete cell cycles. these concentrations were chosen based on the result of our preliminary range finding test where 0.25 – 20 mg.ml-1, were tested. the test sample was changed after the first 24 h. tap water served as negative control and lead nitrate (10 ppm) served as positive control. after 48 h exposure to esa, the meristematic cell region of the roots were excised and prepared for microscopic analysis. onion bulbs were rooted in tap water for 24 h, transferred into lead nitrate (pbno3) solution for 24 h and then into the extract for another 24 h. negative and positive controls are tap water and pbno3 respectively. as in the genotoxicity study, the meristematic cell regions of the roots were excised after 24 h exposure to the extract and prepared for microscopic analysis. esa extracts at the above mentioned concentration range (0.25 – 2.5 mg.ml-1) were assessed for protective effect against lead, under the same pb conditions applied. slide preparation for both the genotoxicity and anti-genotoxicity studies was carried out as previously described (oyeyemi and bakare 2013). briefly, the root tips (cut at 48 h) were fixed in nova biotechnol chim (2020) 19(2): 192-199 194 table 1. summary of the cytological effects of aqueous extracts of sida acuta on allium cepa cells. extract concentration [mg.ml-1] no. dividing cells mitotic index [mean ± sd] sticky chr. disturbed spindle f lag chr. a scattered / disoriented ch. total aberration frequency of aberrant cells [mean ± sd] 0 221 5.53±2.34 2 7 – – 6 13 28 0.73±0.36 0.25 237 5.93±2.02 2 – – 9 5 30 46 1.15±0.17* 0.5 164 4.10±0.64 1 – 1 3 4 25 34 0.85±0.37 1.25 110 2.75±0.96* – – – 4 3 16 23 0.68±0.45 2.5 94 2.35±1.44* – – – – 6 16 22 0.55±0.33 pbno3 184 4.60±0.69 14 7 2 1 1 32 57 1.43 ±0.22* extract c = 0 mg.ml-1 = negative control (tap water), pbno3 ̶ c = 0.01 mg.ml -1, chr. ̶ chromosome, f ̶ fragment, a ̶ anaphase bridge, * significantly different from negative control, ● significantly different from positive control. ethanol:glacial acetic acid (3 : 1 v/v), hydrolyzed in 1 n hcl at 60 °c for 5 min and then washed in distilled water. root tips were squashed on each glass slide and stained with acetocarmine for 10 min. six slides were prepared per concentration, out of which four were scored. a total of 1,000 cells per slide and 4,000 cells per concentration were scored for both the genotoxicity and anti-genotoxicity studies. the mitotic index (mi) and frequency of aberrant cells were calculated as follows: statistical analysis statistical analysis was performed using ibm-spss 23.0 software. data were presented as mean ± standard deviation. analysis involving comparison of means was carried out using oneway analysis of variance (anova) followed by duncan posthoc test where necessary. p-value of < 0.05 was considered significant. results genotoxicity study this study investigated the potential genotoxic effect of the aqueous extract of the leaves of s. acuta using the a. cepa assay. the esa extract caused a reduction in mi of onion cells after 48 h exposure compared with the negative control. the reduction in mi was significant (p > 0.05) at 1.25 and 2.5 mg.ml-1 (table 1). the tested doses of s. acuta did not significantly induced chromosomal aberrations except at the lowest concentration (0.25 mg.ml-1). the frequency of aberration decreased with increasing concentration (table 1). the frequency of aberration at 1.25 and 2.5 mg.ml-1 is lesser than the background frequency observed in the control. there were normal chromosomes (fig. 1) while various types of aberrations which include spindle aberration (fig. 2a), vagrant chromosome (fig. 2bd), c-mitosis (fig. 2e), disturbed spindle (fig. 2f), anaphase bridge (fig. 2f-2g), sticky chromosome (fig. 2h-2i) polar deviation at telophase (fig. 2j). anti-genotoxicity study in the anti-genotoxicity study, the ability of the esa extract to mitigate lead nitrate induced genotoxicity in a. cepa cells was investigated. the extract significantly (p < 0.05) reduced mi across the tested esa concentrations compared to both the negative and positive controls. lead nitrate significantly (p < 0.05) increased the frequency of chromosome aberrations compared to that of the negative control. this frequency was restored to level comparable to that of the negative control by the extract (table 2). discussion humans are constantly exposed to genotoxins in the environment. some of these only cause alteration in the somatic cells, which although may not be passed onto the next generation, may have serious health implications. herbs have been consumed over the ages for the prevention nova biotechnol chim (2020) 19(2): 192-199 195 table 2. summary of the cytological effects of the aqueous extracts of sida acuta on a. cepa cells pre-treated with lead nitrate. extract concentration [mg.ml-1] no. dividing cells mitotic index [mean ± sd] sticky chr. disturbed spindle f lag chr. a scattered / disoriented ch. total aberration frequency of aberrant cells [mean ± sd] 0 221 5.53± 2.34 2 7 – – 6 13 28 0.70±0.36● 0.25 65 1.63±1.48*● 1 6 – – – 7 14 0.35±0.37● 0.5 61 1.53±0.32*● 1 – – – 1 6 8 0.20±0.28● 1.25 51 1.28±0.30*● – – – – – – 0 0.00±0.00● 2.5 32 1.05±0.13*● – – – – – 2 2 0.05±0.10● pbno3 195 4.89±0.62* 36 20 3 8 6 23 96 2.40±0.61* extract c = 0 mg.ml-1 = negative control (tap water), pbno3 ̶ c = 0.01 mg.ml -1, chr. ̶ chromosome, f ̶ fragment, a ̶ anaphase bridge, * significantly different from negative control, ● significantly different from positive control. and treatment of several ailments with the presumption that they are safe (elgorashi et al. 2002). however, concern has been raised over the safety of these herbs as some herbs with therapeutic effects have also been reported to be toxic (oyeyemi et al. 2015; amadi et al. 2018; ruan et al. 2019). this study investigated the genotoxicity and anti-genotoxicity of the aqueous extract of sida acuta, a widely used medicinal plants. our data showed a decline in the mi in both the genotoxicity and anti-genotoxicity studies. this is in line with previous reports on several medicinal plants and/or natural products (shetty et al. 2017; sharma et al. 2018). suppression of mi correlates with cytotoxicity in plant cells (shetty et al. 2017) and is thus an indication that s. acuta extracts at given concentrations are cytotoxic in this study. this suggested that the extract interfered with the cell cycle (oyeyemi and bakare 2013), inhibited dna synthesis and cell cycle progression, which resulted into decreased cellular proliferation (qin et al. 2015). this could probably be the basis of the antiproliferative effect of s. acuta reported in various cancer cell lines (pieme et al. 2010; thondawada et al. 2016). the chromosome aberration induced by the extract in this study was, however, very low and comparable with that observed in the negative control. the extract at the high concentrations reduced the background frequency of chromosome aberration. this implies it is not genotoxic and can even prevent occurrence of spontaneous damage to the genetic material. the chromosome aberrations observed were physiological aberrations due to spindle inhibition (spindle aberration at anaphase (fig. 2a), vagrant chromosomes (fig. 2b-2d), c-mitosis (fig. 2e) disturbed spindles (fig. 2f) and disoriented chromosomes (fig. 2j) or chromatin dysfunction such as stickiness (fig. 2h). this shows that the extract probably contains spindle inhibitor(s). inhibition of mitotic spindle is an important strategy in the development of anticancer drugs, as it inhibits the cell cycle progression (zhu et al. 2016). the observed genotoxicity could due to the presence of alkaloids and tannins in this plant fig. 1. normal chromosomes observed in allium cepa cells exposed to aqueous extract of sida acuta: (a) interphase; (b) prophase; (c) metaphase; (d) anaphase; (e) telophase. nova biotechnol chim (2020) 19(2): 192-199 196 fig. 2. chromosomal aberrations induced in allium cepa root cells by the aqueous extract of sida acuta.: (a) spindle aberration at anaphase; (b, c) vagrant chromosome at anaphase; (d) vagrant chromosome at metaphase; (e) c-mitosis; (f) disturbed spindle with anaphase bridge; (g) anaphase bridge; (h, i) sticky chromosome; (j) polar deviation at telophase. (oboh and onwukaeme 2007). these phytochemicals have been associated with chromosomal damage (oyeyemi and bakare 2013). heavy metals are big menace in the environment. they threaten human health as they easily enter human body through the food chain. lead (pb2+) is one of the most toxic heavy metals (abdullah et al. 2014). lead moves into and throughout ecosystems and contaminate the vegetation, air, water and soil (samal et al. 2017). the toxicity of lead nitrate in several systems has been reported. its genotoxicity in a. cepa has been established (oyeyemi and bakare 2013; atoyebi et al. 2015). esa significantly mitigated the genotoxicity induced by pbno3 in this study as the frequency of chromosome aberration was reduced below that observed in the negative control. this buttressed the phytoremediation ability of sida acuta, as phytoremediation agents are known to deplete the genotoxicity of effluents or any other environmental contaminants (di gregorio et al. 2015; basílico et al. 2017). the observed chromosomal aberration are also structural and not numerical. structural chromosomal alterations occur as a result of dna breaks, inhibition of dna synthesis or replication of altered dna. chromosomal aberrations such as chromosome bridges and breaks, are indicators of a clastogenic action while abnormal segregation of chromosomes (lag chromosome, scattered/disoriented chromosome and c-metaphases) occur either spontaneously or by the action of aneugenic agents (nefic et al. 2013). pbno3 induced disoriented chromosomes predominantly, showing that it is an aneugenic agent. however esa, mitigated this aneugenic effect demonstrating its antigenotoxic effect. one of the mechanisms of lead induced genotoxicity is oxidative stress (dai et al. 2012; taie et al. 2019). esa having antioxidant property (subramanya et al. 2015) possibly inhibited bioaccumulation of pb2+ in a. cepa cells. s. acuta is phytostabilizer of several heavy metals (ameh et al. 2019) with the ability to inhibit the mobility/bioavailibity of pb2+, thus preventing pbno3 induced oxidative stress and ultimately dna damage and or genotoxicity. noteworthy, the observed anti-genotoxic effect was accompanied by severe cytotoxicity as indicated by very low mi. in oocytes, when there is dna damage, the spindle assembly checkpoint induces mitotic index arrest to inhibit the progression of cells harbouring the dna damage (marangos nova biotechnol chim (2020) 19(2): 192-199 197 et al. 2015). this pathway is not naturally activated in somatic cells (collins et al. 2015). s. acuta being a natural spindle inhibitor probably activates this pathway in cells harbouring dna/chromosomal damage resulting in the arrest of the cell cycle progression in those cells. prolonged mitotic inhibition leads to inhibition of further cell proliferation or induction of apoptosis (hain et al. 2016). hence the complete inhibition of proliferation observed at the high concentration. alkaloid isolated from s. acuta triggered apoptosis in human gastric adenocarcinoma cells (ahmed et al. 2011). the low mi observed in the antigenotoxic study may corroborate the apoptosis inducing effect of the plant. in this study, the aqueous extract of s. acuta showed antiproliferative, mitodepressive and antigenotoxic effects. its ability to modulate the mitotic spindle is probably one of its key mechanisms of action. however, caution should be taken against indiscriminate consumption of the extract as it has the potential to be cytotoxic. conclusion this study shows the potential of s. acuta to inhibit lead induced genotoxicity. s. acuta is a spindle inhibitor with the potential to interfere with cell cycle and inhibit mitosis. this buttresses its potential as an anticancer agent. there is a need to further explore the effect of s. acuta on cell cycle and proliferation so as to gain further insight into its molecular mechanism of action. acknowledgement i acknowledge miss oluwatomisin ojo for her assistance in 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(malvaceae) for its anti-oxidant and anti-cancer activity. der pharma chem. 8: 96-402. welz an, emberger-klein a, menrad k (2018) why people use herbal medicine: insights from a focus-group study in germany. bmc complement. altern. med. 18: 92. zhu l, xiao f, yu y, wang h, fang m, yang y, sun h wang l, sheng y (2016) ksp inhibitor sb743921 inhibits growth and induces apoptosis of breast cancer cells by regulating p53, bcl-2, and dtl. anticancer drugs 27: 863-872. nova biotechnol chim (2020) 19(2): 183-191 doi: 10.36547/nbc.v19i2.654  corresponding author: sestinova@saske.sk nova biotechnologica et chimica environmental risk assessment of metal-contaminated areas using different bioassays oľga šestinová, jozef hančuľák and lenka findoráková department of environment and hygiene in mining, institute of geotechnics, slovak academy of sciences, watsonova 45, košice 04001, slovak republic article info article history: received: 2nd september 2020 accepted: 25th october 2020 keywords: avoidance bioassays heavy metals phytotoxkit soils toxicity abstract mining activities in the areas krompachy and rudňany-markušovce were focused on mining and processing of copper and mercury ore and left harmful effects on the region of eastern slovakia. the aim of this study is using different screening methods (xrf, phytotoxkit and earthworm bioassays) for environmental risk assessment of metal-contaminated areas. elemental analysis by x-ray fluorescence spectrometry indicated severe pollution of studied soils by cu, ni, as and hg, which exceeded limit values. significant positive correlation is found between pb and zn occurrence in the agricultural soil from krompachy: kluknava, and for the contents of particular metals in soil from permanent grass vegetation in kolinovce locality, namely between pb and ni, pb and zn, and between hg and zn contents. a 7-day bioassay and avoidance test with the dendrobaena veneta was used to assess the environmental risk of heavy metals in soils. the earthworms mortality was very little influenced by metals in krompachy soils, but rather affected by rudňany soils tailing. phytotoxkit results for soils from krompachy showed inhibition in germination by 32 % and 29 % for sinapis alba and lepidium sativum, respectively. results of the average percentage of growth inhibition by lepidium sativum was 28 % and 24 % for sinapis alba. on the other hand, soil samples from rudňany tailing showed 56 % of germination inhibition by sinapis alba, and 49 % for lepidium sativum, respectively. results of the average percentage of grow the inhibition by lepidium sativum was 48 %, and 52 % for sinapis alba rudňany tailing soils. the significant results (p < 0.05) of the avoidance percentages of dendrobaena veneta for tested soils were within the range 80 – 100 % in soils rudňany-markušovce tailing after 48 h. the variable toxicity of contaminated soils demonstrated the efficiency and usefulness of the phytotoxkit and earthworm bioassays as a useful tool for evaluation of soil ecotoxicity. the results supported the expected negative impact of the soil samples on the region eastern slovakia.  university of ss. cyril and methodius in trnava introduction soil is a dynamic and complex system that provides a habitat for different microorganisms, flora, animals, and humans. these days’ soils are exposed to anthropogenic contamination with heavy metals that create a risk in many ecosystems as well as human health. slovakia is note due to metal ore mines and their processing. the soil chemical reduction by heavy metal contamination, especially in eastern slovakia is an actual issue of societal matter. from the occurrence point of view of ores and industrial mining, it is one of the most significant geological formations mailto:sestinova@saske.sk nova biotechnol chim (2020) 19(2): 183-191 184 in slovakia (demková et al. 2017; maity et al. 2018; šestinová et al. 2019a). mining activities in the region eastern slovakia, krompachy and rudňany-markušovce are focusing on copper and mercury ore mining and processing. hence, the determination of the total heavy metal matter is not adequate to score the environmental problem, which is inseparable to a contaminated soil. for assessment of soil quality, bioassays could be effective tools to scale the potential contaminants toxicity aimed at bioavailability fraction (hundrinke et al. 2003; loureiro et al. 2005; šestinová et al. 2019c). phytotoxkit (phytotoxkit 2004) is an alternative test procedure which is giving estimation of the chemical compounds biological effects on plants. phytotoxicity bioassays with a higher plant collated on seed germination and root elongation growth measurements were conducted with different terrestrial plant species (wang 1991; garcialorenzo et al. 2009). data on phytotoxicity studies were thought over the toxicity evaluation of trade chemicals (günter and pestemer 1990), industrial effluents, dangerous wastes, and leachates (vasseur et al. 1998; czerniawska-kusza and kusza 2011). recently, phytotoxicity tests with terrestrial plants are accepting increasing attention also in assessing soil toxicity. given to its shortexposure periods and its high sensitivity, macrobiotics was successfully used as appropriate help for screening; hence they contribute to a responsible risk assessment for soil environments (persoone et al. 2003). earthworms are commonly used as model organisms to evaluate the ecological risk of various pollutants on soil ecosystems since are considered as fundamental organisms for proper soil functionality (aldaya et al. 2006; latha and basha 2019).furthermore, as major agents of the soil fauna biomass they contribute to soil aeration and drainage (davies et al. 2003) as well as decomposition of organic matter that can be reduced as a consequence of pollution. ability of organisms to evade contaminated soils serves as an indicator of soil ecotoxicity. nannoni et al. (2014) has reported on variable bioconcentration of metal trace elements in earthworms at urban, peri-urban and garden levels depending on extent of pollution. avoidance tests performed on soils from contaminated sites usually score and the response of organisms to a combination of different soil properties (e.g. structure, content of organic matter) with the effects of the contaminants. soil properties outside the range of physiological requirements of the test species strongly influence the avoidance response to pollutants. avoidance tests with most abundant soil organisms can be used as an early screening tool assessment. this study is devoted to evaluate the environmental risk in study soils using tests of ecotoxicity, phytotoxkit microbiotest, earthworm acute toxicity and avoidance tests. the presented approach contributes to the future resolution of a local pollution problem in metalcontaminated environments. experimental samples and analysis soils were collected from two areas krompachy (kr) and rudňany-markušovce (rm). mining activities by the metallurgical treatment of complex metals and copper ores left negative effects on the region eastern slovakia, krompachy and rudňanymarkušovce tailing (angelovičová et al. 2015; šestinová et al. 2019a). the markušovce tailing is situated between the markušovce and rudňany villages (spišskogemerské rudohorie mountains). its construction and operation are connected with former and current mining activities on the rudňany and poráš-zlatník polymetallic ore deposits. the thickness of deposited materials varies in the dependence on initial ground levels and it may achieve maximally about 38 m3. recently, the settling pit is in operation by the sabar, ltd. markušovce. altogether 9 800,000 tons of the tailings of so-called flotation sands at the density of 1.59 t.m-3 and a total volume of 6 200,000 m3 are deposited in the settling pit (jakabský et al. 2010; hredzák et al. 2019). in the year 2015, the two sampling sites were localised in the surrounding krompachy town sampled were agricultural soil in kluknava: (kras) and permanent grass vegetation soil in kolinovce: (krpgvs). the soils were collected from a depth of 20 ‒ 40 cm into bags. soils were homogenized, dried at room temperature, and sieved through 2-mm sieve nova biotechnol chim (2020) 19(2): 183-191 185 (weight 5 kg). trough x-ray fluorescence spectrometer model xepos 3 the concentrations of cu, zn, ni, pb, as and hg in soils and control reference soil (crm-tm52) were measured. all the analyzed samples were conducted in triplicate. soil phytotoxicity evaluation of soil phytotoxicity was based on germination and seedling growth of the two terrestrial plants (mustard sinapis alba and garden cress lepidium sativum), by use the phytotoxkit microbiotest (oecd 208). the soil was covered by the filter plate and after that seeds of the plant were placed on top of the filter in a single row. the test plates with the cover, they were placed vertically and incubated for 72 h at 25 °c. five replicates were performed for the sample set. root growth inhibition was measured after 72 h. the test was considered to be valid if the number of germinated seeds in the control which was at least 90 %. pictures of the test plates were analyzed through the program image tools. the percent inhibition of seed germination (isg) and inhibition of root growth (irg) for the plant were calculated using (eq. 1):    %100   irgisg (1) where a is the mean seed germination or root length in the control (mm); and b is the mean seed germination or root length in the test soil (mm). the system of toxicity classification generated by persoone et al. 2003 was used to forecast the soil toxicity: pe (percent toxic effect) < 20 %, no significant toxic effect, class i, no acute hazard; 20 % < pe < 50 % significant toxic effect, low toxic sample, class ii, low acute hazard; 50 % < pe < 100 % significant toxic effect, toxic sample, class iii, acute hazard; pe-100 % (single test), class iv, high acute hazard; pe-100 % (all tests), class v, very high acute hazard. acute bioassays with dendrobaena veneta the acute bioassays were based on earthworm acute toxicity tests, (oecd 207). from a local supplier adult earthworms were purchased. ten earthworms (and c.w. – control worms) were placed to a plastic box (9 cm × 9 cm × 3 cm) control worms were added to 100 g of crm soil. distilled water was added and the boxes were maintained at laboratory temperature for 7 d. earthworms’ mortality for each soil was estimated and compared with corresponding control. finally, the earthworms were lyophilized; the samples were mineralized and analyzed by atomic absorption spectrometry (aas variant, australia) to determine the metals concentrations in warm tissues (šestinová et al. 2019b). avoidance bioassays with dendrobaena veneta the avoidance tests with earthworms were based on iso guideline 17512-1/2008 – avoidance test for determining the quality of soils and effects of chemicals on behaviour: test with the earthworms. every replicate was composed of a plastic box: 23 cm × 13 cm × 6 cm. the box was distributed into two parts by a divider. every part was filled with 250 g dry weight of one of the three test soils (kr-as, kr-pgv, rm) and a control soil (crm) on the opposite. later, the divider was eliminated and 10 adult earthworms were located onto the middle line. the test containers were over casted by a plastic wrap perforated with pinholes to facilitate air exchange. the test containers were incubated at 20±2 °c and a 16 : 8 h light : dark photoperiod for 48 h. subsequently, the vessels were divided again with the divider at the end of the period, and the earthworms were counted on each side of the replicates. in the midline of the test container an earthworm found was counted as 0.5 earthworms. earthworms found on the underside of the lid of the test were counted as dead. soil ph was measured at the beginning and the end of the test period. the avoidance rates of worms to different soils were calculated according to eq. 2, and expressed as a percentage:     %100(%)    n tc r (2) where r = avoidance; c = number of worms in the control (c0) condition; t = number of worms in each dose in the same soil; n = total number of worms. positive values account for avoidance of worms in test soil, while neutral or negative responses represent indifference or preference of the test substance (hund-rinke et al. 2003). nova biotechnol chim (2020) 19(2): 183-191 186 table 1. basic properties of analyzed soils samples from agricultural (kr-as) and permanent grass vegetation soils in krompachy (kr-pgvs), and from rudňany-markušovce (rm). soils kr-as kr-pgvs rm ph/ h2o 6.5 6.9 8.1 ph/ kcl 5.2 7.2 7.8 eh [mv] 582 580 686 dry weight [%] 97.9 94.3 99.6 organic matter dry weight [%] 5.6 7.3 9.8 grain size [%] sand 3.7 4.5 4.2 silt 30.2 24.7 27.2 clay 66.1 70.8 68.6 dry weight (stn en 75791), organic matter dry weight (stn en 12879). statistical analysis statistical analyses were done through spss ver. 9.0 software. by the pearson matrix correlation the statistical dependence between total metal concentrations was evaluated. correlation analysis was used as a comparison of the potential relationships among the data. the certified river sediment (lgc6187) was used to validate data. control earthworms (crm) fended in noncontaminated soil was used. results and discussion soil physical and chemical properties, including grain size of the studied soils, are presented in table 1. we recorded acidic ph values for the soil samples (kr-as), near-neutral ph values for the soils from kr-pgvs and near-alkaline ph values for the soil samples from rm. the organic matter dry weight of all soils ranged between 5.6 to 9.8 %. the soil organic matters content could reduce metal concentrations in soil solution, and then, their potential availability and toxicity to earthworms (lukkari et al. 2006; šestinová et al. 2019b). all soil samples included the sand, silt and many clay fractions (66.1 – 70.8). soil types were silty-clay texture for all soil samples. some studies have demonstrated that variations on ph, electric conductivity, salinity, redox potential, cation exchange capacity (cec), clay minerals, texture, permeability, porosity and moisture content influence the behaviour of metals in soil (das et al. 2011; angelovičová et al. 2015; šestinová et al. 2019a). due to the heavy metal limits in the slovakia soils (law no. 220/2004), we found quite extensive contamination with cu, hg, as, and ni in all studied localities (kr-as, krpgvs and rm) (table 2). it was found that after 7-days exposure, earthworms in some instances caused decrease of metal concentrations in contaminated soils; such drop was estimated between 5 – 20 % for cu, 4 – 19 % for hg, 5 – 17 % for as, 4 – 15 % for pb, 7 – 14 % for ni and 2 – 11 % for zn, for all studied soils. most processes, including direct dumping of wastes from ore extraction and manufacturing, may bring heavy metals into the surface soil. usually, soil properties determine the bioavailability of toxic heavy metals (šestinová et al. 2017; maity et al. 2018; bravo et al. 2019). acute toxicity tests results and the metals concentrations of the d. veneta tissue are demonstrated in table 2. the earthworms mortality (n) was little effected by krompachy soils, and rather influenced by rudňany tailing soils. the higher pollutant concentrations (heavy metals) may be evaluated by the acute test, which propose dose-dependent effects on mortality. less polluted soils require for reliable risk assessment more sensitive assays such as behavioral tests (avoidance bioassays). pearson correlation analysis among heavy metals in the soils is demonstrated in table 3. statistical correlations at the levels p < 0.05 and p < 0.01 were considered significant. significant positive correlation was founded between pb and zn (r = 0.68, p < 0.05) in the kr-as soil, for the kr-pgvs soil was found between pb and ni (r = 0.86, p < 0.05), pb and zn (r = 0.66, p < 0.05), and hg and zn (r = 0.79, p < 0.05) (table 3). significant positive correlation was identified among as and zn (r = 0.90, p < 0.05) and hg and pb (r = 0.65, p < 0.05) for the rm-tailing soil (table 3). significant correlation was not be identified for as in the krompachy soils (kr-as and kr-pgvs). to know the heavy metals source the correlation analysis is a suitable way. many authors dealing with the eastern slovakia environmental pollution has founded that the soil environment contamination by arsenic is related not only to anthropogenic impact but also to nova biotechnol chim (2020) 19(2): 183-191 187 table 2. heavy metal content and corresponding worm mortality (n) in soils from the region of eastern slovakia (average ± standard deviation). locality metal concentration in soils cu zn ni pb as hg mortality [mg.kg-1] [mg.kg-1] [mg.kg-1] [mg.kg-1] [mg.kg-1] [mg.kg-1] [%] no treatment kr-as 172±56 220±54 62±14 75±25 50±12 3.2±1.0 – kr-pgvs 325±74 478±82 81±14 109±9 75±19 20.4±2.5 – rm 634±98 35±15 98±21 31±5 104±28 82.5±8.1 – crm 3.2±4.0 6.5±5.0 11±2 26±8 1.4±1.0 0.1±0.0 – metal concentration in soils after 7-days bioassay kr-as 156±49 215±36 53.2±8.0 71.5±19 46±6 2.61±1.71 n = 2 kr-pgvs 309±30 452±62 75±15 104±11 71±13 18.5±1.0 n = 4 rm 511±99 31±10 91±21 26.5±3.0 86.5±19.0 79.2±0.1 n = 6 crm 2.8±1.0 6.0±2.0 12±4 24±3 0.9±1 0.10±0.01 n = 1 metal concentration in d. veneta tissues/7-days kr-as 12.1±15 102.5±17.5 2.4±1.1 7.8±9.5 6.3±2.1 0.12±0.03 – kr-pgvs 17.1±10.0 122±19 2.6±1.3 5.6±1.6 7.6±2.5 0.54±0.35 – rm 24.7±21 105±11 19.1±11.5 14.2±4.2 5.2±1.9 2.27±1.15 – c. w. 4.3±2 1.5±1.2 2.1±0.8 2.4±0.2 0.4±0.0 0.47±0.10 – limit 70 200 60 115 30 0.75 – kr-as – agricultural soil and kr-pgvs – soils from permanent grass vegetation both from krompachy, tailing in rudňanymarkušovce; control reference material – crm; law no. 220/2004 – limit; control worm – c.w. the geochemical effects of mineralized zones (hronec et al. 2008; kučerová et. al. 2014; angelovičová et al. 2015; demková et al. 2017). the individual heavy metals presence is caused by mining and smelting activities, as confirmed by significant positive correlations. table 3. correlation relationship between heavy metals in soils from studied localities. zn ni pb as hg agricultural soil: krompachy cu 0.072 -0.37 0.2 0.3 0.06 zn 0.37 0.68 -0.31 0.09 ni -0.08 0.16 0.22 pb -0.73 0.6 as -0.71 permanent grass vegetation soil: krompachy cu -0.37 0.16 0.059 -0.33 -0.39 zn 0.57 0.66 0.25 0.79 ni 0.86 0.39 -0.007 pb -0.01 0.22 as -0.16 tailing soil: rudňany-markušovce cu 0.41 -0.42 -0.71 0.43 -0.22 zn -0.86 -0.16 0.9 -0.09 ni 0.18 -0.83 0.35 pb -0.26 0.65 as -0.4 impact on plant performance was further tested. phytotoxkit results for soils from krompachy showed inhibition in germination by 32 % and 29 % for sinapis alba and lepidium sativum, respectively (fig. 1). results of the growth inhibition average percentage by lepidium sativum was 28 %, and 24 % for sinapis alba. on the other hand, soil samples from rudňany tailing showed 56 % of germination inhibition by sinapis alba, and 49 % for lepidium sativum. results of the growth inhibition average percentage by lepidium sativum revealed 48 % and 52 % for sinapis alba for rudňany tailing soils, see in fig. 2. soils of the rm-tailing have high as (104 mg.kg-1) and hg (82 mg.kg-1) contents. the growth inhibition average percentage for l. sativum was 28 %, 24 % for s. alba in the two soils studied from the krompachy area, as shown in fig. 2. the growth inhibition results showed that the potential toxicity is lower for the krompachy soils. rm-tailing showed growth inhibition percentage lower than 50 % and the values are lower than those for germination inhibition. many agents that might have an impact on this plant response are present. the elements with similar physicochemical properties might interfere in enzymatic pathways, compete during transport and affect nova biotechnol chim (2020) 19(2): 183-191 188 fig. 1. index of inhibition in seed germination (isg, in %) of sinapis alba and lepidium sativum in the six series of the soil samples, krompachy soils (kr-as and kr-pgvs), rudňany tailing soils (rm-tailing) and control reference sample (crm). data represent average of n = 5. accumulation rate (valerio et al. 2007; fu et al. 2019). fargašová (1999) reported a strong antagonistic effect between ni and elements like mo, cu, mn, and described a significant stimulatory effect on s. alba seedlings cultivated hydroponically. the potential toxicity data were described by the use of the toxicity classification system proposed by persoone et al. 2003. the control reference sample (crm) belonged to class i (no acute hazard).all soil samples (kr-as and kr-pgvs) belonged to classes ii (slight acute hazard) and soil samples (rm-tailing) belonged to classes iii (acute hazard). this sample are characterized by a very high total concentration of heavy metals (cu, ni, as and hg). the results of the avoidance percentages of dendrobaena veneta for three tested soil are shown in fig. 3. avoidance tests with earthworms were controlled by dual combinations of each test soil vs. the corresponding reference soil. the test was performed in a two chamber system. soils were not acutely lethal to earthworms. the distribution of the worms found in the double control was within the range 10 – 40 % for soils from kr-as, while from kr-pgvs locality they were 40 – 80 % after 48 h. on the contrary, significant (p < 0.05) avoidance by d. veneta was 80 – 100 % in soils from rudňany–tailing. the habitat soils function was controlled to be limited if less than 20 % of the organisms (on average) were found in the soil. this indicates an impact on properties and deputizes the ‘‘habitat function’’. soils of limited function are where 80 % of the total animals are found in the control soil and 20 % in the polluted soil (latha and basha 2019). behaviour is a final integrated result of sensory, hormonal and metabolic processes. fig. 2. index of root growth inhibition (irg, in %) of the sinapis alba and lepidium sativum in the six series of the soil samples, krompachy soils (kr-as and kr-pgvs), rudňany tailing soils (rm-tailing) and control reference sample (crm). data represent average of n = 5. nova biotechnol chim (2020) 19(2): 183-191 189 the habitat function of soil is involved in the interaction with biotic and abiotic components of the environment (maity et al. 2018). in our case, when we compared all the results of the used bioassays, we obtained agreement in the evaluations so that the highest soil ecotoxicity was demonstrated in soil from rudňany-markušovce tailling. capowiez et al. (2006) suggests that behaviour seems to be a promising (efficiency) biomarker in earthworm studies, as it may give various endpoints which can perhaps be linked to soil functioning. avoidance response tests reflect this behaviour of the earthworms, which works on the principle of preference or avoidance of substrates after a specific exposure period. the sensory cells in the epithelium of mouth region enable the earthworms to avoid adverse habitats (stephenson et al. 1998). according to manzo et al. (2014) a larger tests number with higher sensitivity together with a tailored weights attribution to endpoints and matrices would improve the final site evaluation. the results show that earthworm avoidance behaviour is an ecologically relevant parameter for assessing toxic metal spiked soils. information combining from screening methods oriented on ecological receptors at risk by using available chemical methods and ecotoxicity tests have great benefit for further research of metal-contaminated sites. advantage of combined methods is that the results can be compared and statistical analyzed together and so it is better to estimate the risk of ecosystems contamination. thus, the tools of combined screening with environmental risk assessment in a certain area should be able to indicate effects, but also rapid, easy to apply and cheap (semenzin et al. 2008). conclusions to human health and other organisms the heavy metals contaminated soils are one of the environmental issues considered to be a serious threat. the toxicity and metal bioavailability were studied in soils from krompachy and rudňanymarkušovce in eastern slovakia. average cu, ni, hg and as concentrations are well above the limit levels, defining the need to apply a risk assessment procedure in order to determine and quantify potential environmental risks both to ecosystems. all soil samples from krompachy locality belonged to slight acute hazard (classes ii) and soil samples from rudňany-markušovce (rm-tailing) belonged to acute hazard (classes iii). the abundance of the worms found in the double control was within the range of 10 % – 40 %, and for the soils from krompachy it was 40 % – 80 % after 48 h. on the contrary, significant (p < 0.05) avoidance by d. veneta of 80 % – 100 % was estimated in the soils rm-tailing. the results of the used bioassays coincided and pointed to the highest soil ecotoxicity soil from rudňany-markušovce tailing. toxicity tests allowed us to carry out a preliminary screening of the contaminated areas ecotoxicological risk. the obtained results show that the phytotoxkit test with sinapis alba and lepidium sativum, and avoidance test with earthworms (dendrobaena veneta) can be considered as suitable tools in the soil contamination screening evaluation. fig. 3. avoidance responses of the earthworm (d. veneta) in the six series of three different soil samples, krompachy soils (kr-as and kr-pgvs), and rudňany tailing soils (rmtailing). data represent average of n = 5. nova biotechnol chim (2020) 19(2): 183-191 190 acknowledgments this research was funded by the slovak grant agency for the vega (grant no. 2/0165/19). conflict of interest the authors declare that they have no conflict of interest. references aldaya mm, lors ch, salmon s, ponge j-f (2006) avoidance bio-assays may help to test the ecological significance of soil pollution. environ. pollut. 140: 173180. angelovičová l, bobuľská l, fazekašová d (2015) toxicity of heavy metals to soil biological and 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robinson et al. 2001). the removal of dyes (colour) from wastewater is, therefore, a challenge to the related industries. due to the variability of the organic dyes and the resultant waste solution, wastewater containing dyes is difficult to treat using classical methods. these methods have lot of disadvantages such as transfer of pollutants from one phase to another; some of them are nondestructive, costly methods and could generate secondary waste products such as sludge which may need further disposal (el-desoky et al. 2010; lu and liu 2010; jonstrup et al. 2011; chhabra et al. 2015). among the chemical processes, fenton’s oxidation is one of the earliest advanced oxidation processes (aop) that was successfully used in dye degradation. the simplicity and relative bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:34 utc mailto:aseoud2002@yahoo.fr nova biotechnol chim (2018) 17(2): 160-171 161 low cost of the process made it a promising and attractive treatment method for the effective decolourization and degradation of dyes (arslan et al. 2000; meric et al. 2004). in fenton system, ferrous ions react with hydrogen peroxide at acidic ph producing hydroxyl radicals (ho●) with powerful oxidizing abilities to degrade organic pollutants (neamtu et al. 2003; el-desoky et al. 2010) (eq. 1): h2o2 +fe 2+ → ho● +oh+ fe3+ (1) hydroxyl radicals may react with ferrous ions to form ferric ions or react with organics (eq. 2, 3): ho● + fe2+ → oh+ fe3+ (2) ho● + organics → products (3) however, at high ph values in the fenton process (ph > 4) ferric ions precipitate onto various iron hydroxyls, this leads to production of sludge containing high amount of fe (iii), which need to be treated by safe disposal methods. other kind of problems could occur, such as the generation of aromatic amines and high reagent costs (neamtu et al 2003; lu and liu, 2010). plant peroxidases are potential catalysts that could be used in the degradation and/or elimination of aromatic pollutants (mohan et al 2005). these enzymes can catalyze the transformation of organic pollutants present at very low concentrations (akhtar and husain 2006). peroxidases from gourd or bitter gourd are highly effective in decolourizing wide spectrum of industrially important azo dyes (akhtar et al. 2005; boucherit et al. 2013). maximum decolourization efficiency is obtained with a limited amount of enzyme under mild conditions (schmitt et al. 2012). further, in certain cases enzyme can lead to forming toxic aromatic amines (ali et al. 2013). from the above discussion it seems that use of either fenton or enzymatic process could generate some drawbacks. combined chemical-biological processes were also carried out by many researchers for treating recalcitrant coloured compounds. thus, the coloured solutions were treated in a sequential or mixed batch with a chemical oxidation to partially degrade recalcitrant organic compounds. this pretreatment leads to a discoloured solution, which is readily biodegradable by bacteria or enzymes (karimi et al. 2009; mandal et al. 2010; oller et al. 2011). karimi et al. (2009) showed that sequential coupling of enzymatic and photo-fenton processes were promising solutions for decolourization of pulping waste water. similarly, mandal et al. (2010) achieved total decoulourization by sequential fenton-biological process. nevertheless, such results were obtained under high hydrogen peroxide (111 g/l) and iron sulfate (6 g/l) concentrations. the duration of the process (3 days) was also an inconvenient. besides, some dyes with complex structures could inhibit microbial growth and their biodegradation byproducts could be more toxic. on the other hand, the advanced oxidation process by fenton’s reagents is not cost effective while the biological treatment by micro-organisms is too much time consuming (lucas et al. 2007). it is therefore quite interesting to explore the feasibility of a hybrid, fenton-enzymatic process, in the treatment of dye pollution as an alternative to limited individual treatments. in this context we studied the effectiveness of the combined sequential or mixed fenton-enzyme process under optimal conditions of individual processes. attention was focused on the effects of iron concentration and contact time on combined process efficiency comparatively with individual processes. the proposed strategy aims to obtaining the highest qualitative and quantitative efficiency by reducing the process duration and reactants concentrations. experimental dye direct azo dye investigated: c.i. direct yellow 106 (dy106, c.i. 40300), was provided by soitex (textile manufacturing unit in tlemcen (algeria), and which was purchased from ciba colours ltd. uv-visible region was selected at 396 nm. the structure of dye is showed in fig. 1. reagents for enzymatic treatment cucurbita pepo (courgette) peroxidase (c-peroxidase) was extracted from fresh courgette fruit, of local market. acetone, buffers solutions, hydrogen peroxide (30 % v/v), were obtained from sigma chemical co. (st. louis, mo, usa). others bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:34 utc nova biotechnol chim (2018) 17(2): 160-171 162 chemicals were of analytical grade and were used without further purification. fig. 1. chemical structure of dy106. reagents for fenton treatment hydrogen peroxide (30 %, v/v), hydrochloric acid (37 %), sodium hydroxide, and fe2+ ion as feso4.7h2o, all were purchased from sigma-aldrich corporation. (st. louis, mo, usa). enzymatic treatment enzymatic assay activity of c-peroxidase was assessed by colorimetric estimation using phenol and hydrogen peroxide as substrates and 4-aminoantipyrene as a chromogen (nicell and wright 1997). treatment conditions batch experiments were conducted in a glassy reactor with a working volume of 0.l l. reactor contents were kept under magnetic stirring. these experiments were carried out under the following conditions: initial dye concentration (varying from 10 to 110 mg/l), hydrogen peroxide concentration (0.1 ‒ 7.5 mm), enzymatic activity (0.16 ‒ 3 iu/ml) and ph (2.0 – 10.0). the effect of ph on decolourization was investigated by performing different type of buffers including: 20 mm potassium chloride/hcl buffer (ph 2), 20 mm potassium phthalate/hcl buffer (ph 3, 4), 20 mm potassium phthalate/naoh buffer (ph 5), 20 mm potassium phosphate/naoh buffer (ph 6, 7, 8), 17.5 mm borax/naoh buffer (ph 9, 10). fenton treatment the effects of ph, concentrations of hydrogen peroxide, ferrous ions and dye were investigated in order to determine the optimal conditions for the oxidation of organic dye. all fenton experiments were run at 24 °c in a working volume of 0.1 l containing 50 mg/l of dye solution with initial ph in range (2 to 8). in order to initiate fenton reaction, 8 mm of h2o2 and 1 mm of fe+2 were added to reaction mixture. magnetic stirrer was used to provide continuous mixing during fenton experiments. the reaction was immediately stopped after 15 min by ph adjustment to 10. the mixture was centrifuged at 4,000 rpm for 10 min. quantitative estimation of the residual dye concentration after treatment with fenton was carried out on the supernatant at the maximum wavelength 396 nm. all experiments were conducted in duplicate and their average values were reported. enzymatic-fenton treatment after optimization of enzymatic and fenton treatments separately, different configurations (for fixed dye concentration of 50 mg/l) of combined enzymatic-fenton treatment were conducted as follows: sequential batch of enzymatic-fenton, or fenton-enzymatic processes and mixed batch. the objective was to determine the best combination which gives the highest yield, the shortest time duration and minimum residual byproducts for minimum ferrous dose. all combined treatment experiments were conducted in glass reactors containing 0.1 l of reaction mixture. sequential batch fenton-enzyme sequential batch in the first stage, aqueous dye solution was treated by fenton treatment (ft). initial conditions were: 50 mg/l of dye concentration, 2.5 mm of fe2+ and 20 mm potassium chloride/hcl buffer (ph 2). the reaction was started by addition of 8 mm of h2o2. after 15min of fenton’s treatment, the mixture was centrifuged after neutralisation with naoh (1 m). the enzymatic reaction was started after ph adjustment to 2 by addition of 1.7 iu/ml bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:34 utc nova biotechnol chim (2018) 17(2): 160-171 163 of c-peroxidase. the treated samples were neutralized to a ph of 10, by ammonium liquor after 10 min. the mixture was centrifuged and the supernatant was analysed by uv-visible spectrophotometer. enzyme-fenton sequential batch the reaction mixture contained initially 50 mg/l of dye, 1.7 iu/ml of c-peroxidase in 20 mm potassium chloride/hcl buffer (ph 2). the reaction was started by addition of 1 mm of h2o2. at the end of the enzymatic treatment (2 min), the mixture was centrifuged. the supernatant was then treated with fenton process by addition of fe+2 (2.5 mm) and h2o2 (7 mm). at the end of the process (10 min), the ph was adjusted to 10 by adding ammonium liquor and centrifuged. the supernatant was analysed by uv-visible spectrophotometer. mixed batch for the mixed batch treatment, a volume of 100 ml was prepared as follows: 50 mg/l of dy106, and enzymatic activity 1.7 iu/ml of c-peroxidase, 50 mm of potassium chloride/hcl buffer (ph 2) and 2.5 mm of fe2+. the reaction was started by addition of 8 mm of h2o2. after 30 min, the reaction was stopped by adjusting ph to 10 with ammonium liquor. the mixture was centrifuged and the supernatant separated and analyzed by uvvisible spectrophotometer. in all cases of combined process, fe2+ concentration was optimized in order to reach a maximum yield of decolourization. for mixed process, kinetics was followed by withdrawal of aliquots of 5 ml of treated dye solution at different time intervals. the decolourization efficiency (de) was expressed as the percentage ratio of the decolourized dye concentration to that of initial one and was calculated as follows (eq. 4): (4) where a0 – initial dye absorbance at 396 nm; at – dye absorbance at time t at 396 nm. for enzymatic process, the initial decolourization rate (idr) was calculated from the slope of the dye concentration versus time curve for different initial dye concentration (results not shown). the optimal concentration of 50 mg/l was obtained through a one factor at a time experimental strategy, by choosing initial reaction rate and degradation yield as responses, for both enzymatic and fenton process. then, the same concentration was chosen for the combined processes. analytical methods dye decolourization was measured by monitoring the absorbance with a uv-visible spectrophotometer (model perkin-elmer 550 a) based on the maximum absorbance. the maximum absorbance was obtained by preparing a solution in a selected buffer with a known concentration and measuring the absorbance by uv-visible at different wavelengths from 190 to 700 nm. uv-visible spectral analysis (between 200 to 500 nm) was also carried out in order to evaluate changes in absorption spectrum before and after treatment and, eventually, detect the appearance or disappearance of any by-products. high performance liquid chromatography (hplc) was carried out on a shimadzu equipped with a uv detector (spd10a) fixed at 254 nm. samples were analyzed on a c18 column. an isocratic method with the 0.025 m phosphate buffer (ph 3.0): acetonitrile mobile phase was employed in the separation (40 : 60) (v/v). the injection volume was 20 µl. the flow rate was kept at 1 ml/min during the run under 25 °c. phytotoxicity studies phytotoxicity tests were conducted to assess the impact of the treated and untreated coloured water on growth of plants. this method was developed to ascertain the toxicity of polluted liquid samples on seed germination and root elongation as an indication of the possibility to use it in irrigation or recycling for industrial or domestic purposes (di salvatore et al. 2008). the ethyl acetate extracted products of dy106 degradation obtained from different treatments and the original dye were dissolved in sterile distilled water to make a final concentration of 100 ppm. the experiments were carried out on common bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:34 utc nova biotechnol chim (2018) 17(2): 160-171 164 beans (phaseolus vulgaris l.), a fast growing and sensitive plant. ten seeds of plant were sowed into a plastic sand pot. the sand pot was prepared by adding 20 g washed sand into the plastic pot. then, each pot was irrigated with 5 ml (100 ppm) of each solution per day. control set was executed using distilled water at the same time. germination rate (%), length of plumule (shoot) and radicle (root) were recorded after 7 days (parshetti et al. 2006). results and discussion enzymatic treatment, optimal ph, dye, h2o2, enzyme concentrations and time duration the variation of dye removal (for initial concentration of 50 mg/l) by free peroxidase at various ph values is depicted in fig. 2. about 73.7 % of dye was removed at ph 2 under the specified experimental conditions of dye (50 mg/l), h2o2 (1 mm) and enzymatic activity (1.4 iu/ml). the efficiency of enzymatic dye removal decreased for higher ph values between 3 and 10. similar results were established with other plant peroxidases for the treatment of different azo dyes. maximum degradation was observed within the ph range of 2 to 5 (mohan et al. 2005; kalsoom et al. 2015). the concentration of the dye has significant influence on enzymemediated reaction. enzymatic reactions are significantly affected by concentration of substrate (fig. 3). if the amount of substrate fig. 2. effect of ph on dye decolourization by c-peroxidase. (t = 24 °c, 50 mg/l of dy106, 1 mm of h2o2 and 1.4 iu/ml of c-peroxidase). increases gradually for a constant enzyme concentration, the rate of reaction will increase to reach a maximum constant value (more than 9 mg/min.l). however, the decolourization efficiency decreased for the same change of dye concentration. similar results were obtained with different type of dyes by plant peroxidases within the concentration range of 20 – 50 mg/l (mohan et al. 2005; bhatti et al. 2012; ahmedi et al., (2012). maximum decolourization of solar blue a and solar flavine 5g has been reported with dye dose of 20 mg/l for a peroxidase activity of 12 iu/ml, h2o2 dose of 0.8 and 0.7 mm, respectively, at ph 4 (bhatti et al. 2012). also ahmedi et al. (2012) showed that optimal concentration of congo red giving maximum decolourization by turnip peroxidase is 50 mg/l for a dose h2o2 of 50 mm, peroxidase activity of 0.45 iu/ml at ph 2. mohan et al. (2005) obtained maximum decolourization of acid black 10 bx with enzyme activity of hrp of 2.94 iu/ml. the optimal dye concentration of 50 mg/l was selected as the intersection point between initial rate and efficiency of decolourization curves. in order to find out optimal h2o2 concentration, experiments were carried out by varying peroxide concentration and keeping other experiment conditions unchanged. hydrogen peroxide was considered as a co-substrate. it contributes in the catalytic cycle of peroxidase, to oxidize the native enzyme into a reactive intermediate radical, which accepts the aromatic substrates and converts them fig. 3. effect of initial dye concentration on idr and de by c-peroxidase. (t = 24 °c, 1 mm of h2o2 and 1.4 iu/ml of c-peroxidase). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:34 utc nova biotechnol chim (2018) 17(2): 160-171 165 fig. 4. effect of h2o2 dose on rate and efficiency of decolourization by c-peroxidase. (ph 2, t = 24 °c, 50 mg/l of dy106, and 1.4 iu/ml of c-peroxidase). into radicals which may further polymerize or degrade into small products (kalsoom et al. 2015). so, from fig. 4 is appeared that the dy106 decolourization efficiency and initial decolourization rate by c-peroxidase is affected by the concentration of hydrogen peroxide. it is clear that a concentration of 1 mm of h2o2 could be considered as critical dose. at lower concentrations the highest efficiency was noticed, while an inhibition effect took place at peroxide concentrations above 1 mm. the optimal of h2o2 dose will be taken as 1 mm. comparable results were reported by using other plant peroxidases for decolourization of many azo dyes (jamal et al. fig. 6. effect of contact time on residual dye and de after enzymatic treatment by c-peroxidase. (t = 24 °c, 50 mg/l of dy106, 1 mm of h2o2 and 2.25 iu/ml of c-peroxidase). fig. 5. effect of enzyme activity on rate and efficiency of decolourization by c-peroxidase. (ph 2, t = 24 °c, 50 mg/l of dy106, 1 mm of h2o2). 2011). the optimal enzyme activity in the reaction medium was also determined. from fig. 5 it is obvious that the efficiency and decolourization rate increased with increasing enzyme concentration to reach maximum values (89.5 %, 8 mg/l.min) when enzyme activity was above 2.25 iu/ml. no further improvement was noticed for higher enzyme concentrations. mohan et al. (2005) obtained with horseradish peroxidase (hrp) activity of 2.2 iu/ml a maximum decolourization yield of 84 % of acid black 10 bx. after 2 min, the residual dye remained constant (de = 89.45 %). the kinetics of enzymatic treatment of dy106 solution was then followed under optimal ph, dye fig. 7. effect of ph on dye decolourization by fenton treatment. (t = 24 °c, 50 mg/l of dy106, 1 mm of fe2+, 2 mm of h2o2). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:34 utc nova biotechnol chim (2018) 17(2): 160-171 166 concentration, h2o2 dose and enzyme activity. at regular time intervals, one vial was removed and analyzed for the residual dye concentration (fig. 6). a contact time of 2 min for enzymatic treatment will be considered for the rest of the study fenton treatment, optimum ph, h2o2, fe2+, initial dye concentrations and time duration it is well known that ph is an important parameter in fenton process. it controls the production of hydroxyl free radicals and the concentration of ferrous ions (sun et al. 2007) as illustrated in eq. 1. from fig. 7 it is clear that a degradation yield of 88.7 % was obtained at ph 2 at constant concentrations of h2o2 (2 mm) and fe+2 (1 mm), respectively. the degradation of dy106 was significantly influenced by ph. decolourization decreases with increasing ph. similar results were obtained with different types of dyes and it was proved that maximum degradation was achieved at ph ranges between 2 ‒ 4 (neamtu et al. 2003; meric et al. 2004; sun et al. 2007). the other interesting aspect of this result is the concordance with optimal ph range of the enzymatic process, which facilitates the design of the combined process without the necessity of ph adjustment. data on fig. 8 illustrate the relationship between decolourization efficiency (de) and initial concentration of h2o2. the selection of an optimal fig. 8. effect of h2o2 dose on dye decolourization by fenton treatment. (ph 2, t = 24 °c, 50 mg/l of dy106, 1 mm of fe2+). h2o2 concentration for the degradation of the dyes by fenton’s reaction is important from an economical and practical point of view (neamtu et al. 2003). results indicate that the decolourization of dy106 increased with increasing h2o2 concentration from 0.5 to 8 mm. no significant increase was noticed for higher concentrations up to 16 mm. sufficient amount of hydroxyl radicals were produced leading to higher decolourization yield (panda et al. 2011). the optimal h2o2 dose giving maximum decolourization (93.7 %) was 8 mm. iron in its ferrous and ferric forms acts as a catalyst and requires a working ph below 4 (nidheesh et al. 2013). in this work, concentrations of fe2+ were varied to obtain the optimal concentration for fenton treatment. results are shown in fig. 9. the decolourization of dy106 solution increased with initial catalyst concentration till it reached 98.6 % when (fe2+) was 2.5 mm. further increase of (fe2+) dose leads to the decrease of decolourization yield to reach 78.8 % at ferrous concentration of 10 mm. dye removal is directly proportional to catalyst (fe2+) concentration. this is mainly caused by the increase of ho● radical concentration, which promotes the degradation efficiency of pollutants. nevertheless, many studies have revealed that the use of excess concentration of fe2+ could lead to the self-scavenging of ho● radical by fe2+ and thus induce the decrease in degradation rate of pollutants (joseph et al. 2000). fig. 9. effect of fe2+ dose on decolourization by fenton treatment. (ph 2, t = 24 °c, 50 mg/l of dy106, 8 mm of h2o2). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:34 utc nova biotechnol chim (2018) 17(2): 160-171 167 fig. 10. effect of initial dye concentration on decolourization by fenton treatment. (ph 2, t = 24 °c, 2.5 mm of fe2+, 8 mm of h2o2). initial concentration of dye is an important parameter in practical applications (modirshahla et al. 2007). therefore, the effect of dye concentration on the decolourization efficiency was investigated at different initial concentrations of dy106. results are presented in fig. 10. we observed that the decolourization efficiency decreased slowly with increasing the initial dye concentration. the highest efficiency (98.7 %) was obtained for dy106 concentrations below 50 mg/l. for higher concentrations, the yield decreased. this could be explained by the unbalanced concentrations of dye and hydroxyl radicals for a fixed iron concentration. therefore, the optimal dye concentration was taken as 50 mg/l. fig. 11 provides an effect of reaction time duration on dy 106 decolourization and dye removal efficiencies by fenton reaction. further, it was noticed that decolourization increases with contact time under optimal conditions. the residual dy106 concentration decreased rapidly during the first 10 min (de =98.6 %) and then slowed down as time goes on. similar results were obtained by sun et al. (2007) for decolourization of 50 mg/l of amido black 10b (de= 69.3 %) under specified conditions. combined enzymaticfenton treatment the major purpose of this integrated process was to reduce the operational concentration of reagents, fig. 11. effect of time duration on dy 106 decolourization by fenton treatment. (ph 2, t = 24 °c, 50 mg/l of dy106, 2.5 mm of fe2+, 8 mm of h2o2). particularly the ferrous ions concentration used in fenton’s reagent, to achieve the highest decolourization efficiency of dy106. data indicate that both enzymatic and fenton processes as individual treatments occur under similar conditions of ph (2), ambient temperature, optimal dye concentration (50 mg/l) in the presence of hydrogen peroxide in each enzymatic and fenton treatment. similarities in mechanisms of both processes could be also exploited for the development of a hybrid process in order to reduce the dose of iron in fenton process (less than 2.5 mm), alternatively, improve decolourization efficiency of enzymatic process (the efficiency was 89.5 %). the reduction of certain by-products e generated by either fenton or enzymatic processes could be also performed by sequential or mixed processes. for this purpose, the effect of fe2+ for all combined processes was studied under constant conditions of h2o2 dose (8 mm), enzyme activity (1.7 iu/ml), ph 2, temperature (24 °c) and dye concentration (50 mg/l). on the other hand, contact time was optimized for mixed combined process. as seen in fig. 12, the decolourization capacity was significantly affected when fe2+ concentrations were below 1 mm, for all combined process under specific conditions. it was also noticed that the sequential fenton-enzyme treatment (fe) and mixed process, were more efficient when fe2+ dose varied from 0.125 to 1.25 mm, which gives bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:34 utc nova biotechnol chim (2018) 17(2): 160-171 168 fig. 12. effect of fe2+ dose on decolourization efficiency by fenton and different combined processes. (ph 2, t = 24 °c, 50 mg/l of dy106, 8 mm of h2o2 and 1.7 iu/ml of c-peroxidase). efficiency between 88 and 98 %. for fe2+concentrations above 1 mm, efficiencies of fe, mixed and fenton processes were comparable. a similar result (91 %) was obtained for sequential biological fenton process for decolourization of remazol red with fe2+ dose of 1 mm (jonstrup et al. 2011). finally, the effect of contact time on decolourization efficiency for mixed process (fig. 13) showed that more than 98 % of colour was eliminated in 5min. according to kinetic data shown on fig. 6 and fig. 11, enzymatic process was faster than fenton treatment. this indicates that during mixed treatment, the enzymatic process starts firstly and may generate specified, readily degradable products by fenton reaction. no comparison could be shown with results that were obtained by sequential chemical-biological process which needs 39 min for fenton and 72 days for the enzymatic treatments (mandal et al. 2010). several reviews (guieysse and norvill 2014) confirmed this table 1. optimal conditions and efficiencies of different treatment processes for the dy106. fig. 13. effect of time duration on decolourization efficiency by mixed process. (ph 2, t = 24 °c, 50 mg/l of dy106, 0.8 mm of fe2+, 2 mm of h2o2 and 1.7 iu/ml of cperoxidase). observation. recapitulation of optimal treatment conditions by all processes and efficiency (de) are listed in the table 1. uv-visible spectra and hplc analysis of the dy106 treated by different chemical enzymatic process despite the technical performance results that were obtained in terms of de, the treated solutions were also analyzed by uv-visible spectroscopy and hplc chromatography in order to compare the qualitative efficiency of each combined treatment. analysis was performed on samples before and after treatment under optimal conditions: ph 2, 8 mm of h2o2, 0.8 mm of fe 2+ and 50 mg/l of dy106. before treatment of dy106 aqueous solution (50 mg/l), uv-visible spectra consisted of two main characteristic absorption bands (fig. 14). one is in uv region at 212 nm and another in visible region at 396 nm. uv band is characteristic of adjacent rings (transition →*), whereas visible band owns to long conjugated  system linked by azo groups (transition n→*) (silverstein et al. 1991). it is clear that the adsorption peak at 396 nm completely disappeared after each process treatment. this indicated that under optimized experimental conditions (ph 2, fe2+ dose of 0.8 mm, h2o2 dose of 8 mm and dy106 concentration of 50 mg/l) process enzyme fenton f-e e-f mixed ph 2 2 2 2 2 dye [mg/l] 50 50 50 50 50 h2o2 [mm] 1 8 8 8 8 ae [iu/ml] 2.25 00 1.7 1.7 1.7 fe2+ [mm] ---2.5 1 1 0.8 contact time [min] 2 10 20 20 5 de [%] 89.5 98.6 98.3 87.0 98.4 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:34 utc nova biotechnol chim (2018) 17(2): 160-171 169 fig. 14. uv-visible spectra of dy106 untreated and treated solutions by: c-peroxidase, fenton, sequential and mixed combined process. (ph 2, t = 24 °c, 50 mg/l of dy106, 0.8 mm of fe2+, 8 mm of h2o2 and 1.7 iu/ml of c-peroxidase). the chromophore azo group -n=nwas destructed. in the uv zone, the disappearance of the band at 212 nm was also observed, but other cases could be discussed such as the appearance of a new peak at 265 nm after enzymatic treatment probably due to the formation aromatic amine intermediate or other products (hailei et al. 2009), strong absorption bands at 300 nm and between 200 and 250 nm after fenton treatment or complete disappearance of dy106 characteristic bands after sequential fe, ef and mixed combined processes, with minimum absorption for the mixed combined treatment (compared to the sequential one). further, with fenton oxidation, results showed that the destruction of the aromatic rings was difficult. this could be due to the lowest energy band which is assigned to the n→* transition related to the azo group. therefore, ho● radical attacks firstly the –n=n– group of the lowest energy leading to the opening of the –n=n– double bonds. the long conjugated π systems were then destructed, and consequently a maximum of decolourization was achieved (el-desoky et al. 2010). also, the absence of bands in the uv zone after treatment by combined processes shows that the by-products which were generated after the first treatment were eliminated during the second one. the oxidation of aromatic amines by bitter gourd peroxidase and fenton's reagent was previously thoroughly studied (karim and husain 2009; casero et al. 1997). this is clearly confirmed in table 2 which compares retention time and peak areas of solutions before and after treatment by combined processes. results show that there is a certain synergy between the two processes which conducted to the elimination of compounds which were generated by individual processes after sequential or mixed processes. the synergetic aspect could be also demonstrated by further analysis with nmr spectroscopy and mass spectrometry. phytotoxicity studies coloured textile effluents pose serious health and environmental problems. they can be rejected directly and thus return to agricultural or industrial activities. thus, the importance of conducting phytotoxicity tests for enzyme, fenton and combined treated processes and untreated dye solutions, in order to evaluate the possible use of the treated aqueous solutions in the irrigation or industrial fields, is appeared. results in table 3 show that the germination of p. vulgaris l. in the case of the treated dy106 (100 ppm) by simultaneous peroxidase-fenton, did not significantly differ from their germination in distilled water (96.66 % vs. 95.03 %). at the same found to be lower in the case of germinated seeds by fenton, enzyme and untreated solutions than seeds germinated that were irrigated with distilled water and simultaneous peroxidase-fenton treated solution. moreover, germination was also influenced by the quality of the effluent that was table 2. retention time and peak areas of hplc spectra of dy106 and treated solutions. process retention time [min] peak area [mv.s] dy 106 2.242 1444000 4.025 2770254 6.817 69299 enzyme (e) 2.433 72301 3.075 884384 fenton (f) 2.997 985432 5.013 83532 f-e 2.292 427785 3.050 688916 e-f 2.283 503835 3.033 347095 mixed 2.283 474616 3.042 438942 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:34 utc nova biotechnol chim (2018) 17(2): 160-171 170 table 3. phytotoxicity tests on growth of p. vulgaris l. irrigated in the absence or presence of raw or treated dy106 solutions. growth parameters distilled water dy106 [100 ppm] enzymatic treatment fenton treatment mixed treatment germination [%] 96.66 28.46 46.92 63.85 95.03 radicle [cm] 5.300.3 1.090.1 2.280.2 1.570.1 3.890.4 plumule [cm] 11.750.2 6.660.3 6.210.3 70.5 10.420.2 generated after individual treatments (63.85 % for fenton vs. 46.92 % for peroxidase). this indicates that the azo-dye dy106 was more toxic than degraded products obtained after fenton or enzymatic treatments. alternatively, treated water from combined processes could be considered as non-toxic, which could help promoting it´s reuse. conclusions from the findings of this work it can be concluded that c-peroxidase and fenton reagent have potential in the decolourization of dy106 under specified experimental conditions. the decolourization performance by enzymatic treatment depends on ph, enzyme activity and initial dye concentration. similarly, fenton process efficiency was affected by ph, h2o2, fe 2+ and dye concentrations, h2o2 and fe 2+. optimal operating conditions regarding yield and decolourization rate were determined for both enzymatic and fenton processes. applied individual processes present some drawbacks; these include high ferrous dose in the case of fenton process or the generation of toxic or unknown compounds even in the case of enzymatic treatment. due to similarities existing between enzymatic and fenton processes (ph 2 and the presence of hydrogen peroxide and iron as key elements, and optimal dye concentration), the mixed and sequential combined enzymaticfenton processes resulted in improvements such as the reduction of ferrous dose, minimizing degradation time and increasing yield. elimination of (possible) by-products of the fenton process showed that there is a certain synergy with enzymatic process. thus, combined approaches were effective in degrading components of the colored solutions, especially at lower iron concentrations, with comparison to sequential processes. references ahmedi a, abouseoud m, couvert a, amrane a (2012) enzymatic degradation of congo red by turnip (brassica rapa) peroxidase, z. naturforsch. c, 67: 429-436. akhtar s, khan aa, husain q (2005) partially purified bitter gourd (momordica charantia) peroxidase catalysed decolourization of textile and other industrially important dyes. bioresour. technol. 96: 1804-1811. akhtar s, husain q (2006) potential of immobilized bitter gourd (momordica charantia) peroxidase in the removal of phenols from polluted water. chemosphere 65: 1228-1235. arslan i, balcioglu ia, bahnemann dw (2000) advanced chemical oxidation of reactive dyes in simulated dye house effluents by ferrioxalate fenton/uv-a and tio2/uv-a processes. dyes pigm. 47: 207-218. ali l, algaithi r, habib hm, souka u, rauf ma, ashraf ss (2013) soybean peroxidase-mediated degradation of an azo dye-a detailed mechanistic study. bmc biochem. 14: 1-13. banat im, nigam p, singh d, marchant r (1996) microbial decolourization of textile-dye-containing effluents. a review. bioresour. technol. 58: 217-227. bhatti hn, kalsoom u, habib a (2012) decolourization of direct dyes using peroxidase from raphanus sativus (f04 sl). j. chem. soc. pakistan 34: 257-262. boucherit n, abouseoud m, adour l (2013) degradation of direct azo dye by cucurbita pepo free and immobilized peroxidase. j. environ. sci. 25: 1235-1244. casero i, sicilia d, robio s, bendito dp (1997) chemical degradation of aromatic amines by fenton’s reagent. water res. 31: 1985-1995. chhabra m, mishrab s, sreekrishnan tr (2015) combination of chemical and enzymatic treatment for efficient decolourization/degradation of textile effluent: high operational stability of the continuous process. biochem. eng. j. 93: 17-24. di salvatore m, carafa a, carratu c. (2008). assessment of heavy metals phytotoxicity using seed germination and root elongation tests: a comparison of two growth substrates. chemosphere 73: 1461-1464 el-desoky hs, ghoneim mm, zidan nm (2010) decolourization and degradation of ponceau s azo-dye in aqueous solutions by the electrochemical advanced fenton oxidation. desalination 264: 143-150. guieysse b, norvill zn (2014) sequential chemicalbiological processes for the treatment of industrial wastewaters: review of recent progresses and critical assessment. j. hazard. mater. 267: 142-152. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:34 utc nova biotechnol chim (2018) 17(2): 160-171 171 hailei w, ping l, min p, zhijun z, zhijun z, guangli y., guosheng l., jianming y. (2009) rapid decolourization of azo dyes by a new isolated higher manganese peroxidase producer: phanerochaete sp. hsd. biochem. eng. j. 46: 327-333. jamal f, qidwai t, pandey pk, singh d (2011) catalytic potential of cauliflower (brassica oleracea) bud peroxidase in decolourization of synthetic recalcitrant dyes using redox mediator. catal. commun. 15: 93-98. jonstrup m, punzi m, mattiasson b (2011) comparison of anaerobic pre-treatment and aerobic post-treatment coupled to photo-fenton oxidation for degradation of azo dyes. j. photoch. photobio. a 224: 55-61. joseph jm, destaillats h, hung hm, hoffmann mr (2000) the sonochemical degradation of azobenzene and related azo dyes: rate enhancements via fenton’s reactions. j. phys. chem. a 104: 301-307. kalsoom u, bhatti hn, asgher m (2015) characterization of plant peroxidases and their potential for degradation of dyes: a review. appl. biochem. biotechnol. 176: 1529-1550. karim z, husain q (2009) redox-mediated oxidation and removal of 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cod removal from wastewater containing reactive black 5 using fenton’s oxidation process. chemosphere 54: 435-441. mohan sv, prasad kk, roa nc, sarma pn (2005) acid azo dye degradation by free and immobilized horseradish peroxidase (hrp) catalysed process. chemosphere 58: 1097-1105. modirshahla n, behnajady ma, ghanbary f (2007) decolourization and mineralization of c.i. acid yellow 23 by fenton and photo-fenton processes. dyes pigm. 73: 305-310. neamtu m, yediler a, siminiceanu i, kettrup a (2003) oxidation of commercial reactive azo dye aqueous solutions by the photo-fenton and fenton-like processes. j. photochem. photobiol. a 161: 87-93. nicell ja, wright h (1997) a model of peroxidase activity with inhibition by hydrogen peroxide, enzyme microb. technol. 21: 302-310. nidheesh pv, gandhimathi r, ramesh st (2013) degradation of dyes from aqueous solution by fenton processes: a review. env. sci. pollut. res. 20: 2099-2132. oller i, malato s, sánchez-pérez ja (2011) combination of advanced oxidation processes and biological treatments for wastewater decontamination-a review. sci. total environ. 409: 4141-4166. panda n, sahoo h, mohapatra s (2011) decolourization of methyl orange using fenton-like mesoporous fe2o3sio2 composite. j. hazard. mater. 185: 359-365. parshetti g, kalme s, saratale g, govindwar s (2006) biodegradation of malachite green by kocuria rosea mtcc 1532. acta chem. eng. 53: 492-498. robinson t, mcmullan g, marchant r, nigam p (2001) remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. bioresour. technol. 77: 247-255. schmitt s, de souza r, bettin f, pinheiro dillon aj, borges valle ja, andreaus j (2012) decolourization of aqueous solutions of disperse textile dyes by oxidoreductases. biocatal. biotransfor. 30: 48-56. silverstein rm, bassler gc, morrill tc (1991) spectrophotometric identification of organic compounds, 5th edition, wiley, new york, usa, 419 p. sun jh, sun sp, wang gl, qiao lp (2007) degradation of azo dye amido black 10b in aqueous solution by fenton oxidation process. dyes pigm. 74: 647-652. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:34 utc nova biotechnol chim (2019) 18(1): 44-51 doi: 10.2478/nbec-2019-0006  corresponding author: akmal84a@gmail.com nova biotechnologica et chimica characterization of a novel lipase from pseudomonas aeruginosa nodir sh. berdiev1, jamolitdin f. ziyavitdinov1, akmal m. asrorov1,2,, shukhratjon s. olimjonov1 and shavkat i. salikhov1 1 laboratory of proteins and peptides, institute of bioorganic chemistry, academy of sciences of uzbekistan, 83, m. ulughbek str., tashkent, uzbekistan 2 shanghai institute of materia medica, cas, 501 haike rd, shanghai 201203, china article info article history: received: 27th april 2019 accepted: 15th july 2019 keywords: amino acid sequence cotton seed-oil lipase activity pseudomonas aeruginosa abstract lipases cleaving oils into fatty acids and glycerol are of great interest for the use in increasing the efficiency of fuels. in this work, a novel lipase from pseudomonas aeruginosa, p. aeruginosa a12, was isolated by ion-exchange and hydrophobic chromatography. the purity of lipase was shown by electrophoresis and its molecular weight was estimated to be ~ 31.6 kda. the whole amino acid sequence was analyzed by an lc-ms/ms method. temperatureand ph-dependent optimum of the enzyme compiled 30 °c and 7.5, respectively. the obtained enzyme exhibited 79 % similarity of amino acid sequence to a lipase isolated from the same strain of p. aeruginosa. thus, the novel lipase was determined to belong to i.1 subfamily of bacterial true lipases. three dimensional structure of the isolated lipase isoform was modeled based on obtained sequences. amino acids forming the catalytic domain were shown in the model. lid domain is suggested to be in the open conformation. these results provide a potential alternative for enzymatic digestion of fuel oils and serve for the development of fundamental knowledge of lipase activity.  university of ss. cyril and methodius in trnava introduction lipases (ec 3.1.1.3) are triglyceride ester hydrolases that cleave ester bonds in glycerides (balcao et al. 1996). bacteria are considered as their most reliable source that can express them even in coal mines (ali et al. 2015) or geothermal springs (shahinyan et al. 2017). several isoforms of lipases have been isolated from bacterial cultures (verma and sharma 2014) and found applications in food, dairy, and detergent industries (gupta et al. 2004; sharma et al. 2013). lipases largely differ by biochemical parameters. the enzymes isolated from p. aeruginosa reveal different mw, ph and temperature optimum. a 54 kda lipase isolated from p. aeruginosa was determined to have ph optimum in the range 6 – 9 (karadzic et al. 2006). the 29 kda lipase with high activity towards olive oil was found to have optimum at moderate temperatures (gilbert et al. 1991). activity of this lipase, suggested to be a metalloprotein, was inhibited by metal ions such as hg2+, zn2+, cu2+, ag2+ and fe2+. its temperature and ph optimum were defined to be 55 ºc and 6.9, respectively (borkar et al. 2009). the active center of 70 % of lipases isolated from filamentous bacteria includes the amino acid sequence gly-his-ser-leu-gly-gly-ala, and the oxyanion gap consists of the sequence ile-val-valtyr-leu-val-ile-ala-val-ser-phe-arg-gly; these two active sites are separated by 72 amino acids (mancheño et al. 2003). the ser-16, asp-116 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:03 utc mailto:akmal84a@gmail.com nova biotechnol chim (2019) 18(1): 44-51 45 and his-291 as triad were concluded reveal catalytic activity of lipase isolated from aeromonas hydrophila mainly found in regions of warm climate (brumlik and buckley 1996). the threedimensional structures of lipases show their active site association with α-helices and a central β-sheet including active ser (arpigny and jaeger 1999). lack of disulphide bridges in lipases likely increases their conformational flexibility under cold conditions (alquati et al. 2002). in this work a new isoform of 31.6 kda lipase was isolated from p. aeruginosa strains. all biochemical parameters were determined and its full amino acid sequence was determined by mass-spectrometry analysis. three dimensional structure of the lipase was modeled using swissmodel software. lipases with specific properties are promising tools for obtaining biodiesel fuels from fats. experimental growing bacterial cultures the cultivation was performed in magnetic rocking chair in 250 ml erlenmeyer flasks with a circular rotation of 100 rpm, at a temperature of 25 – 26 °c. then, the resulting cultural liquid was filtered, centrifuged at 6,000 rpm, and lyophilized. lipolytic activity of three bacterial strains pseudomonas aeruginosa, chromobacterium violaceum and bacillus stearothermophilus were studied at the earliest stage of our studies. bacteria were grown on a nutrient medium containing 1 % (v/v) cottonseed oil. the growth dynamics and kinetic curves of lipase accumulation in bacterial cultural liquids were observed during 4 d of cultivation in 12 h intervals. purification and preparative isolation of lipase the lipase enzyme was purified from lyophilized bacterial cells as described. lyophilized culture fluid of p. aeruginosa with a specific activity of 129 u.mg-1 was used for purification of lipase as a source for enzyme preparation. at the beginning, an enzyme preparation in the amount of 1 g was dissolved in 20 mm phosphate buffer (ph 8.0, 65 ml) and dialyzed in the same buffer. further the enzyme solution was concentrated to 10 ml using a 10 kda membrane (millipore), to the protein concentration 2.2 mg.ml-1. chromatographic purification after dialysis of protein extract, purification was performed on deae-tsk 650 column (16 mm – 200 mm) using 20 mm phosphate buffer (ph 8.0). single-stage gradient of 0 – 0.2 m and 0.2 – 1.0 m nacl in the same buffer was used. the flow rate was 0.5 ml.min-1. the proteins present in the eluate were monitored by measuring the absorption at 280 nm. further purification of bacterial lipase was carried out on the column (16 mm – 200 mm) filled with phenyl-tsk 650. phosphate buffer (20 mm, ph 8.0) was used with a gradient of ethyl alcohol (0 – 60 %; v/v) in the same buffer. the flow rate of eluent was 0.5 ml.min-1. the proteins present in the eluate were monitored by measuring the absorption at 280 nm. lipase activity determination of lipase activity was carried in various buffer systems with a ph from 5 to 11, at room temperature, using the ph-stat method in time-dependent and temperature-dependent manner. tributyrin (98 %, sigma aldrich) was used as a substrate. temperature-dependent activity was determined in 0.1 mm sodium phosphate buffer (ph 7.5), and the enzyme solutions were incubated in a water bath at the range of 20 – 60 °c. the enzyme activity was measured after 1 h after incubation. aliquots were taken every 60 min for 15 h. ph-dependent lipase activity was studied in various buffer systems in the ph range of 5 – 11 at room temperature. aliquots were taken every 60 min for 15 h. the quantity of soluble proteins was determined by lowry method (lowry et al. 1951). isolation and purification of proteins from pa gel standard sds-page was performed with 15 µl aliquot of purified proteins and staining was carried out by silver-stain method (switzer et al. 1979). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:03 utc nova biotechnol chim (2019) 18(1): 44-51 46 the spot (fraction) corresponding to the purified lipase was isolated from the gel. sds residues were removed by treating with 40 % (v/v) methanol, 5 % (v/v) acetic acid, and incubation at room temperature for 15 min. the solution was discarded. the stain was removed by incubating with 50 mm ammonium bicarbonate in 50 % (w/v) acetonitrile for 15 – 20 min at 56 – 60 °с. the procedure was repeated a few times until complete destaining. then, gel with the sample was washed with deionized water 3-times and were further reduced with 50 – 65 mм dtt in 50 mm ammonium bicarbonate and incubated for 45 min at room temperature. it was subsequently alkylated with 100 µl of 20 mm iodoacetamide in 50 mm ammonium bicarbonate, keeping in dark for 50 – 60 min. gel samples were dehydrated in acetone incubating until they turned white colour. preparation of protein solution for lc-ms/ms analysis lipase was digested with trypsin at 37 °с for 15 h. the reaction was terminated by the addition of formic acid. in order to extract enzyme from the gel, gel sample was dried at 45 °с, treated with 0.1 m trifluoroacetic acid, and kept in ultrasonic bath for 10 min and further was centrifuged for 5 min (12,000 rpm). the solution was collected into 1.5 ml tube. the procedure was repeated. extract was lyophilized (virtis 2kbtes) for mass analyses. mass spectroscopy analysis the digested protein samples (2 – 5 µl) were subjected to chip (150 µm  43 mm) zorbax sb c18 column in agilent technologies 1260 cap pump with a flow rate of 4 µl/min, elution rate 0.6 µl.min-1. b solution was managed as following: 5 % (3 min), 80 % (25 – 30 min). the solution was degassed in agilent technologies 1260 µ-degasser. mass detection was performed in chip-q-tof lc-ms agilent technologies 6520в series mass spectrometer. mass data was recorded in positive ionization mode (ionization source: chip interface, gas flow rate: 4 l.min-1, 350 °c, skimmer current power: 65 v, ms/ms: 50-2400 m/z, mobile phase: a – 0.1 % formic acid, b – acetonitrile + 0.1 % formic acid, v/v in all cases), and partial sequences were identified with spectrum mill database. 3d structure homology modelling a 3d model of p. aeruginosa a12 was built by swiss-model modeling program based on crystal structure lipase pao1. lid region was also predicted comparing with the same lipase exhibiting 79 amino acid sequence similarity (nardini et al. 2000) with the p. aeruginosa a12. results and discussion bacterial strains c. violaceum, b. stearothermophilus, p. aeruginosa with high lipase production were cultivated as described. the growth dynamics of cells and also kinetic curves of lipase activity in bacterial cultures were determined during 4 d of cultivation period in 12 h intervals. the data obtained using 1 % (v/v) cottonseed oil as inductor of lipases show that the lipase activity in cultured bacteria c. violaceum reached the maximum after 48 h and 84 h were determined as the maximum point for b. stearothermophilus and p. aeruginosa (fig. 1). p. aeruginosa had the greatest ability to synthesize the enzyme, which is rapidly secreted into the culture medium after 36 h of cultivation, reaching the maximum for 84 h of growth. the lipase secreting abilities of the strains’ cultures were as follows: c. violaceum < b. stearothermophilus < p. aeruginosa. (fig. 1). fig. 1. total lipase activities of bacterial cultures of strains c. violacheum (circles), p. aeruginosa (squares) and b. stearothermophilus (triangles) (n = 4, rsd ± 5%). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:03 utc nova biotechnol chim (2019) 18(1): 44-51 47 fig. 2. electrophoretic analysis of bacterial lysate at individual steps of lipase extraction procedure. line 1 – marker proteins; 2 – concentrated bacterial extract; 3 – after deae-tsk 650 column; 4 – after phenyl-tsk 650 column. for further studies, p. aeruginosa was cultivated in an optimized nutrient medium for 72 – 96 h; again, 1 % cottonseed oil was used for the induction of the enzyme.the degree of enzyme purification after each stage was determined by electrophoretic analysis. purification was repeated until a single fraction of 31 kda lipase was obtained (fig. 2). at the first purification process culture liquid, the specific activity of which equaled 129.0 u.mg-1, was dialyzed against water and concentrated. the specific activity of the obtained extract equaled 262.7 u.mg-1. at the next stage of purification ionexchange chromatography was employed. the quantity of the obtained lipase fractions (12 – 15 fractions in fig. 3 a) was a total of 22 mg protein in 10 ml, and the specific activity made 723.0 u.mg-1. in the obtained chromatograms five protein peaks were observed. the second peak belonged to a lipase (fig. 3 a) was used for further purification. the lipase was completely separated from other proteins by hydrophobic interaction chromatography. the volume of the collected fraction compiled 5.5 ml with a specific activity 1009 u.mg-1 and the total protein made 0.825 mg (3 – 6 fractions in fig. 3 b). fig. 3. column chromatography purification of novel lipase p. aeruginosa a12. chromatograms were obtained by ionexchange chromatography (a) and hydrophobic interaction chromatography (b). the optical density of fractions (280 nm) is presented as squares, and corresponding lipase activities (u.mg-1) as filled triangles (n = 4, rsd ± 5 %). the overview data of protein quantity and enzyme activity concerning novel p. aeruginosa a12 lipase purification steps are presented in table 1. we determined the properties of the purified lipase p. aeruginosa a12. the results showed that the optimal ph value is 7.5 (fig. 4), at which the lipase retains 95 % of the initial activity for 15 h (data not shown). high activity of the enzyme was observed in the range of ph 7 – 8 (fig. 4). table 1. purification of lipase from the culture fluid of p. aeruginosa (n = 5, rsd ≤ 4 %). purification stage volume [ml] protein [mg.ml-1] total protein [mg] specific activity [u.mg-1] culture liquid 65.0 0.90 58.5 129.0 conc. liquid after dialysis 10.0 2.20 22.0 261.7 deae-tsk 650 4.0 0.60 2.40 723.0 phenyl-tsk 650 5.5 0.15 0.825 1,009 a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:03 utc nova biotechnol chim (2019) 18(1): 44-51 48 fig. 4. optimum conditions of lipase isolated p. aeruginosa (n = 4, rsd ± 5 %). the enzyme incubated for 1 h at a temperature of 30 °c showed a maximum specific activity of 1,009 u.mg-1; after 15 h the activity was reduced to 90 %, equalled to 101 u.mg-1 (data not shown). at higher temperatures, the lipase activity rapidly dropped. at 50 °c, for instance, the enzyme activity in one h reduced to 85 % (to 115.2 u.mg-1), and to 99 % in 15 h, respectively (fig. 4). in this work, we purified a total of 82.5 mg homogeneous enzyme of 31 kda with 1,009 u.mg-1 specific activity from 10 g of lyophilized bacterial preparation. the obtained parameters for enzymatic activity of this lipase (ph and temperature) correspond to other literature data (table 2). lipase isolated from p. tolaasii demonstrated highest enzyme activity at ph 7.0 and 35 °c. at 21 °c and ph 7.0, it retained activity for 48 h, and was inhibited by edta, o-phenanthroline, pmsf (phenylmethylsulfonyl fluoride, a serine protease inhibitor) but reactivated by ca+2 (baral and fox 1997). biochemical analysis and rna sequencing revealed a new p. aeruginosa strain, t1, revealing a lipase activity in the presence of mineral salts, which can degrade different types of edible oils at 30 °c. noteworthy, it was active in the broad range of 15 – 50 °c (hasanuzzaman et al. 2004). further, the ph and temperature optima of partially purified lipase from p. aeruginosa aau2 was reported as 7.5 and 40 °c, with preference for longer carbon chain fatty acid esters (bose and keharia 2013). depending on growing conditions of bacterial strains, lipases differ by their ph and temperature optimum (table 2). the novel lipase, studied in this work, was found to be strongly inhibited by methanol; efficiency of transesterification was dramatically reduced by concentrations of 1 – 4 m (data not shown). on contrary higher concentration of ethanol enhanced formation of mono-, di-, and triglycerides (data not shown). p. aeruginosa a12 revealed maximum activity at 30 °c. table 2. biochemical parameters of lipases isolated from pseudomonas strains. bacterial strain mw [kda] ph [optimum] temperature [optimum] reference p. aeruginosa a12 31.6 7.5 30 °c this work pseudomonas aeruginosa 54 11.0 70 °c karadzic et al. 2006 pseudomonas tolaasii 7.0 35 °c baral and fox 1997 pseudomonas aeruginosa km110 8.0 45 °c mobarak-qamsari et al. 2011 pseudomonas aeruginosa ef2 29 gilbert et al. 1991 pseudomonas aeruginosa srt 9 29 6.9 55 °c borkar et al. 2009 pseudomonas aeruginosa aau2 7.5 40 °c bose and keharia 2013 pseudomonas aeruginosa 7.0 30 °c zouaoui et al. 2012 pseudomonas sp.lp1 8.0 40 °c kanimozhi et al. 2010 pseudomonas gessardii 7.0 37 °c veerrapagu et al. 2013 pseudomonas fluorescens ke38 43 8.0 45 °c gokbulut et al. 2013 pseudomonas aeruginosa sp. 58 9.0 55 °c gaonkar et al. 2018 pseudomonas aeruginosa ps-1,2 50 7.0 37 °c saadatullah et al. 2018 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:03 utc nova biotechnol chim (2019) 18(1): 44-51 49 fig. 5. comparison of amino acid sequences of lipases isolated in this work p. aeruginosa a12 and pao1 (by nardini et al. 2000; protein data bank deposit number 1ex9). *amino acids determining catalytic triad such as ser82, asp229, and his251 are shown with a black triangle. lid region was indicated as shown by khan et al. 2017. for comparison, enzyme activity of the lipase from p. aeruginosa reached maximum at 45 °c and ph range 6 – 9, and was inhibited by zn+2 and cu+2 to 32 and 17 %, respectively (mobarak-qamsari 2011). the other 54 kda lipase from p. aeruginosa had highest activity at ph 11 and 70 °c, while was inhibited strongly by zn2+, hg2+, cu2+ and slightly also by ca2+ and mg2+ (karadzic et al. 2006). methyl p-nitrophenyl n-hexylphosphonate was also reported to inhibit lipase activity (rotticci et al. 2000). ph and temperature tolerance of p. aeruginosa a12 correspond to other lipases isolated from other strains of p. aeruginosa, found in ph interval 6 –10 (mobarak-qamsari et al. 2011; zouaoui et al. 2012; gokbulut et al. 2013). structure of the lipase from p. aeruginosa lc ms/ms analysis revealed 14 fragments covering the whole sequence of the peptide, proved by comparison with amino acid sequence of other lipase from p. aeruginosa. the amino acid sequence of p. aeruginosa a12, identified in this work, was compared with the 32 kda lipase from p. aeruginosa pao 1 consisted of 285 amino acids (nardini et al. 2000), the structure of which was earlier established in crystal. results showed 79 % similarity between these two proteins with 60 amino acid difference (fig. 5). in blast analysis, the isolated isoform of lipases showed similarity to i.1 subfamily of ec 3.1.1.3 bacterial true lipases (messaoudi et al. 2010). besides, it exerts high amino sequence similarity to the p. aeruginosa lipase consisted of 311 amino acids (identity ~ 77.5 %, e-value ≤ e-140). three dimensional structure of the isolated lipase was modeled using swissmodel software analysis (fig 6 a). the catalytic triad involving ser-asp-his is indicated in dot and line mode (fig 6 b). spatial proximity of these three amino acids explains its structure-activity relationship of the novel lipase. protein modeling enables to suggest the conservation of catalytic triad – ser82, asp229, and his251 (fig. 5 and fig.6). lid domain in open conformation that make the active site cavity to solvent and substrate (nardini et al. 2000) is suggested to be similar in p. aeruginosa a12. homology modeling revealed all α-helices and β-sheets, available in pao1 lipase, are conserved in p. aeruginosa a12 which is 79 % identical. similar trends were suggested in lipases from rhizopus chinensis (yu et al. 2014). lid regions in lipases have been reviewed well and by their lid domains they were divided into three groups: ones without lids, seconds lipases with one loop or one helix and lipases with two or more helixes lids (khan et al. 2017). homology modeling enables us to conclude that the novel lipase p. aeruginosa a12 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:03 utc nova biotechnol chim (2019) 18(1): 44-51 50 fig. 6. 3d structure homology modelling of the lipase. (a) crystal structure of pao 1 lipase (nardini et al. 2000). (b) 3d cartoon model of p. aeruginosa a12. (c) 3d tube model of p. aeruginosa a12 with structural positions of catalytic triads, indicated in dot and line mode. lid region among three α-helices was demonstrated in pao and p. aeruginosa a12. belongs to the third group that at least three α-helixes are located over the active site (fig. 6). based on observations lipases containing larger lipases with two or more α-helixes are expected thermostable (khan et al. 2017). p. aeruginosa partially agrees with these observations that at 40 °c still twice lower activity was detected. however, at 50 °c lipase activity of p. aeruginosa a12 strongly dropped, 5-fold lower enzyme activity than that at 30 °c was determined. conclusions in conclusion, a novel 31.6 kda lipase isoform was isolated from p. aeruginosa strain. the ph and temperature optima were determined as 7.5 and 30 °c (respectively). enzyme activity was found to be induced by ethyl alcohol and inhibited by methyl alcohol. modeling three dimensional homology structure proved the spatial proximity of ser-asp-his catalytic triad revealed open conformation of lid region. the enzyme can be potentially interesting for biotechnological means because of its ph tolerance in the interval 6 –10 and temperature tolerance with a 2-fold difference in activity from 20 till 40 °c. acknowledgement we appreciate the support of the cas president's international fellowship initiative (pifi) postdoctoral researcher (2019pb0076) and shanghai "one belt one road" young scientist cooperation fellowship (18430740800). conflict of interest the authors declare that they have no conflict of interest. references ali f, bahadar s, ur rahman kh, khan i, zeb a, khan j, daud m. hassan a, rabbani nkh, khattak aa, jadoon fkh, hayat a (2015) isolation and identification of lipase producing bacteria from coal mines of darra adam khel, khyber pakhtoonkhwa. pakistan. int. j. biosci. 7: 187-191. alquati c, gioia ld, santarossa g, alberghina l, fantucci p, lotti m (2002) the cold‐active lipase of 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purification and properties of lipase from pseudomonas aeruginosa. afr. j. biotechnol. 11: 12415-12421. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:03 utc https://mic.microbiologyresearch.org/search?value1=e.+jane+gilbert&option1=author&noredirect=true https://mic.microbiologyresearch.org/search?value1=alex+cornish&option1=author&noredirect=true https://mic.microbiologyresearch.org/search?value1=colin+w.+jones&option1=author&noredirect=true https://link.springer.com/journal/253 https://www.sciencedirect.com/science/article/pii/s1389172306706338#%21 https://www.sciencedirect.com/science/article/pii/s1389172306706338#%21 https://www.sciencedirect.com/science/article/pii/s1389172306706338#%21 https://www.sciencedirect.com/science/article/pii/s1389172306706338#%21 https://www.sciencedirect.com/science/journal/13891723 https://www.sciencedirect.com/science/journal/13891723 https://www.ncbi.nlm.nih.gov/pubmed/?term=mobarak-qamsari%20e%5bauthor%5d&cauthor=true&cauthor_uid=22347589 https://www.ncbi.nlm.nih.gov/pubmed/?term=kasra-kermanshahi%20r%5bauthor%5d&cauthor=true&cauthor_uid=22347589 https://www.ncbi.nlm.nih.gov/pubmed/?term=moosavi-nejad%20z%5bauthor%5d&cauthor=true&cauthor_uid=22347589 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3279805/ nova biotechnol chim (2020) 19(1): 19-29 doi 10.36547/nbc.v19i1.574  corresponding author: hassanienmohamed@hotmail.com, mhassanien@uqu.edu.sa nova biotechnologica et chimica biosafety of genetically modified wheat (triticum aestivum) hi-line 111 mohamed abd-elfattah mohamed hassan1, rafaat mohamed elsanhoty2, mohamed fawzy ramadan3,4, and mohyeldeen ali osman5 1food technology research institute, agriculture research center, 9 algamaa street, giza, egypt 2department of industrial biotechnology, institute of genetic engineering and biotechnology, sadat city university, egypt 3department of agricultural biochemistry, faculty of agriculture, zagazig university, zagazig 44519, egypt 4deanship of scientific research, umm al-qura university, makkah, saudi arabia 5department of biochemistry, faculty of agriculture, cairo university, egypt article info article history: received: 8th february 2020 accepted: 13th april 2020 keywords: genetic modification gmo histopathology rats safety assessment serum biochemistry transgenic wheat abstract genetically modified (gm) crops were approved for edible use in several countries but their biosafety for organisms remains to be crucial. the objectives of this work were to compare gm wheat (triticum aestivum) hi-line 111 (gmw) with native non-gmw wheat (ngmw) to find the differences, if any, in their biosafety. three groups of albino rats (rattus norvegicus) were used to study the biosafety of gmw for 30 days. group 1 was fed on a basal diet (control), and group 2 on a control diet with 30 % replacement of starch with ngmw, while group 3 was fed on the control diet with 30 % replacement of starch with gmw. there were no significant signs of adverse impacts noted in the clinical appearance of animals fed on gmw in terms of initial body weight, absolute or relative organ weights and serum profile in comparison with the control group. however, slight histopathological changes were observed in the organs of animals fed on gmw. though our results demonstrate gmw biosafety regarding its biochemical parameters, however, detailed description of submucosal edema and further studies on allergenic potential with long feeding periods should be performed to conclude its impacts on health.  university of ss. cyril and methodius in trnava introduction agricultural biotechnology and genetic engineering have opened an avenue in the development of genetically modified (gm) plants with improved abiotic stress tolerance, herbicide or insect resistance, and nutritional value (sthrestha et al. 2008; raybould 2012; elfattah et al. 2016; sun et al. 2019). with the international commercialization of gm organisms (gmos), gm crops were engineered to solve problems such as weed management, disease, improving functional properties and enhancing production efficiency (shin et al. 2013; klymiuk et al. 2018). genetic modification introduces new genetic information, new genes, and new compounds in the cells of food-producing organisms. they might alter the cellular metabolism of the foodproducing organisms in unanticipated ways. these new proteins might also be toxic or cause allergy (taylor 1997). in addition, the food-producing organism could fail to produce some nutrients or vitamins. thus, gm foodstuffs could lack some nutrients that are present in the corresponding nongm foods. genetic engineering could cause unintended adverse changes in the food chemical composition or food characteristics, thereby rendering foodstuffs to be unsafe (elsanhoty mailto:hassanienmohamed@hotmail.com mailto:mhassanien@uqu.edu.sa nova biotechnol chim (2020) 19(1): 19-29 20 et al. 2004, 2013; herman et al. 2009). based on the criteria for safety, some gm crops have been approved for food or feed uses in several countries (oguchi et al. 2010). consumer concerns regarding gm products related to unexpected health impacts that might arise from gm products consumption (dona and arvanitoyannis 2009; martinez-povida et al. 2009). as recommended by food safety agencies, biosafety and nutritional assessment of gm crops is an important aspect (chassy 2010). pressure from public demand and consumers has led some countries to label gmo in foods (ramadan and elsanhoty 2012; elsanhoty et al. 2013; castigliego et al. 2015; turkec et al. 2016). many countries have imposed biosafety laws and surveillance programs to regulate gmos uses (de jong 2010). within eu countries, regulations and laws on the traceability and labeling of gmos require mandatory labeling of food and/or feed containing gm ingredients above > 0.9 % (the commission of the european communities 2003a, b). eu has adopted a zero-tolerance policy towards the low-level presence of unauthorized gmos in foods, with a 0.1 % threshold for the permissible presence of unauthorized gmo in the feed (the commission of the european communities 2011; turkec et al. 2016). many studies were performed to investigate the safety of gm food and feed (elsanhoty et al. 2004, 2006; he et al. 2008; magaña-gómez and de la barca 2009; appenzeller et al. 2009a,b; snell et al. 2012). feeding trials in which rats were fed gm foods for prolonged periods were reported (brake and evenson 2004; brake et al. 2004). some experiments indicated histopathological abnormalities in organs or tissues and clinical impacts (appenzeller et al. 2009a,b; snell et al. 2012; kaspareit and rittinghausen 2013; abdo et al. 2014). some reports assessed the safety of gm plants and reported changes in the serum parameters of animals treated with gm foodstuffs (elsanhoty et al. 2004, 2006; hammond et al. 2006). they noted some differences between animals fed on gm potato spunta and its control as well as gm corn (mon 810) and its control. however, monitoring of biosafety and nutritional quality for gm food and/or feed require detailed knowledge of the plant composition. wheat (triticum aestivum l.) is one of the most important cereals that is globally cultivated (pigolev et al. 2018; sun et al. 2019). the gm wheat hi-line 111 (gmw) was transformed with plasmid pab1 harboring the full-length hva1 cdna to transform immature gmw hi-line 111 cv. hi-line embryos (hong et al. 1988; lanning et al. 1992; sivamani et al. 2000). the chemical composition of gmw and non-gmw (ngmw) line as well as the physical and rheological characteristics of the dough were studied (elfattah et al. 2016). the gmw under study was modified by introducing of barley hva1 gene, encoding for a member of the group 3 late embryogene sis abundant (lea) proteins to increase drought tolerance, biomass production and water use efficiency (sivamani et al. 2000; elfattah et al. 2016). our work is aimed to assess the safety of gmw in comparison with the conventional wheat line or ngmw upon animals feeding study. experimental materials gmw hi-line 111 and its conventional ngmo control line were cultivated and harvested under the same conditions in the agricultural genetic engineering research institute (ageri, giza, egypt). the hva1 expression cassette, containing the hva1 gene under control of the maize ubi promoter was introduced into t. aestivum l. cv. hi-line genome via particle bombardment of immature embryos (elfattah et al. 2016). grain samples were freeze-dried prior to mixing to feed in further investigations. the genetic elements in gmw were reported (elfattah et al. 2016). non-gmw contained 10.7 moisture (%), 15.6 total protein, 1.61 ash (%), 2.13 lipids (%), 2.63 crude fiber (%), and 69.9 total carbohydrates (%). gmw contained 10.1 moisture (%), 15.9 total protein, 1.61 ash (%), 2.73 lipids (%), 2.71 crude fiber (%), and 69.5 total carbohydrates (%). experimental conditions experimental procedures involving the animals and their care were carried out according to the guidelines for care and use of laboratory nova biotechnol chim (2020) 19(1): 19-29 21 table 1. the composition of the basal and experimental diet (g.100 g-1). ingredient cda [g.100 g-1 diet] experimental diet group 2 group 3 caseinb 20.0 20.0 20.0 corn oil 5.0 5.0 5.0 cellulose 5.0 5.0 5.0 salt mixture 3.50 3.50 3.50 vitamin mixture 1.0 1.0 1.0 starch 55.0 25.0 25.0 cholin bitartarate 0.2 0.2 0.2 dl methionine 0.3 0.3 0.3 sucrose 10.0 10.0 10.0 ngmw 30.0 gmw 30.0 a cd means control diet according to reeves et al. (1993). b casein contained ≥ 80 % protein. animals in biomedical research as promulgated by the who. fifteen weaning male albino rats (average weight was 65 g) were provided from the institute of ophthalmology research (giza, egypt). the animal experiment was ethically approved before collecting the data by the animal subjects committee of the sadat city university (egypt). the animals were housed individually in well-aerated cages with screen bottoms and fed on a basal diet as described by reeves et al. (1993). salt and vitamins mixtures were prepared according to aoac (2000). humidity and temperature were maintained at 60 % and 25 °c, respectively. the animals were randomly sorted into three groups each group contains 5 animals. group 1 was fed on control diet according to reeves et al. (1993), and group 2 was fed on the control diet with a replacement of 30 % from starch by ngmw, while group 3 was fed on the control diet with a replacement of 30 % from starch by gmw. table 1 presents the diet composition and animal groups. during the experiment (30 days), the diet consumed and the body weight were measured every day. the body weight and the rest of the feed were measured to compare some performance parameters such as food intake, body gain, daily body weight gain, final body weight, and feed efficiency. blood biochemistry and serum analysis at the end of the experiment (30 days), blood samples were collected from treated groups from the orbital venous plexuses using a capillary tube. blood was centrifuged for 15 min at 3,000 rpm. to recover the serum, which kept at -18 °c until analysis. serum was analyzed according to schermer (1967). kidney function parameters (urea, and creatinine) and liver function enzymes (alt, and ast) were analyzed using kits obtained from bio-diagnostic (cairo, egypt). blood glucose was measured according to trinder (1969). serum total cholesterol (tc) was determined according to thomas (1992). glutamate-pyruvate aminotransferase (gpt), and glutamate oxaloacetate aminotransferase (got) activates were measured at 540 nm (reitman and frankel 1957). serum urea was determined at 580 nm according to patton and crouch (1977). total lipids (tl) content was measured by the reaction of tl with the sulfophosphovanillic mixture (coudon and bouige 1973). low-density lipoprotein (ldl)cholesterol was calculated as the difference between tc and the high-density lipoprotein (hdl)-cholesterol content of the supernatant after precipitation of the ldl by the polyvinyl sulphate in the presence of polyethylene glycol monomethyl ether (demacker et al. 1984). triacylglycerols (tag) content was analyzed as described by fossati and prencipe (1982). organ weights, gross necropsy, and histopathology at the end of the experimental period (30 days), the rats were slaughtered. the organs of each animal (kidney, liver, heart, spleen and testes) were weighed. kidneys, liver, spleen, and stomach tissues from those organs as well as macroscopically evident lesions were fixed in 4 % buffered formaldehyde for the histological test. tissues were embedded in paraffin and sections, 4 – 6 lm thick, stained by the standard hematoxylin-eosin (h & e) for light microscopy. the intact small intestines were flushed with a 0.9 % nacl solution and the length was recorded (drury and willington 1980). statistical analysis results are presented as means ± confidence intervals (p < 0.05). means were considered as significantly different, if their confidence nova biotechnol chim (2020) 19(1): 19-29 22 table 2. changes in the body weights of animal groups at the end of the experimental period (30 days)a. parameter animal group control group ngmw b group gmwc group initial body weight [g] 65.2 ± 4.64a 65.6 ± 4.87a 65.8 ± 4.44a final body weight [g] 108.4 ± 4.74b 147 ± 4.64a 142.6 ± 7.16a body weight gain [g] 43.2 ± 4.69b 81.4 ± 4.75a 76.8 ± 5.8a the same letter in the same row is not significantly different (p > 0.05). amean ± standard error (n = 5) bngmw = non genetically modified wheat cgmw = genetically modified wheat intervals were not overlapping. results from the animal studies were presented as mean ± sd where appropriate according to gomez (1984). results and discussion biosafety monitoring research performed on gm wheat based on the application of the principle of substantial equivalence, which adopted by leading international food and regulatory bodies such as who (who 1995). a new food derived from gm crop was found to be equivalent to its non-modified counterpart and safe as the food or feed from the non-modified plant varieties. substantial equivalence might be classified into; substantially equivalent, substantial equivalent expects for some defined traits, and lack of equivalence (momma et al. 2000). the present experiment was performed to study the nutritional effects and safety of gmw. a rat performance study was conducted to compare gmw with ngmw. clinical observations and body weight during the experimental period, no treatmentrelated signs of adverse impacts in the clinical appearance of rats were noted. the body weights of animals were comparable at the beginning of the experiment, while throughout the study period there was an increase in body weight of ngmw and gmw groups (table 2). this increase may be due to the high nutrient value of wheat that replaced starch in the feeding formula. differences between groups were tested for statistical significance (p < 0.05). the results revealed no statistically significant differences between groups in the initial body weight, while at the end of the experiment, there were statistically significant differences between control and other groups. the results agreed with previous reports (elsanhoty et al. 2004, 2006; abdo et al. 2014), where no statistically significant differences between groups observed in the initial body weight of rats during feeding of gm potato spunta, and bt-corn (mon810: ajeeb yg) during feeding study. however, the results disagreed with some studies (zhu et al. 2004; séralini et al. 2007; zhu et al. 2015), where some gmos (roundup tolerant and mon863) increased the body weight. zhu et al. (2015) found significant differences in organ weights among dietary groups fed on gm rice (expressing cry1ab/1ac protein) and non-gm rice. séralini et al. (2007) reported significant table 3. changes in the relative organ weight (%) of animal groups at the end of the experimental period (30 days) a. parameter animal group control group ngmw b group gmwc group final body weight [g] 108.4 ± 4.740b 147 ± 4.640a 142.6 ± 7.160a liver [%] 3.916 ± 0.068a 3.597 ± 0.241a 3.438 ± 0.093a spleen [%] 0.459 ± 0.032a 0.489 ± 0.045a 0.432 ± 0.051a kidney [%] 0.718 ± 0.054a 0.737 ± 0.044a 0.759 ± 0.017a stomach [%] 0.757 ± 0.024a 0.652 ± 0.044b 0.632 ± 0.026b the same letter in the same row is not significantly different (p > 0.05). amean ± standard error (n = 5) bngmw = non genetically modified wheat cgmw = genetically modified wheat nova biotechnol chim (2020) 19(1): 19-29 23 table 4. changes in the serum biochemical values of animal groups at the end of the experimental period (30 days) a. parameter animal group control group ngmw b group gmwc group got [u.l-1] 10.6 ± 0.39a 11.4 ± 0.81a 10.8 ± 0.58a gpt [u.l-1] 11.2 ± 1.53a 10.6 ± 0.51a 10 ± 1.76a cholesterol [mg.dl-1] 71.2 ± 10.0a 63.2 ± 10.9a 69.2 ± 17.7a hdl cholesterol [mg.dl-1] 64.6 ± 22.7a 43.8 ± 8.87a 57 ± 13.0a triglyceride [mg.dl-1] 73.8 ± 5.46a 62.6 ± 18.6a 54.6 ± 7.45a total lipids [mg.dl-1] 517.4 ± 26.0a 379.6 ± 89.6ab 257 ± 72.8b creatinine [mg.dl-1] 0.396 ± 0.02a 0.396 ± 0.02a 0.506 ± 0.05a urea [mg.dl-1] 106.2 ± 10.6a 85.8 ± 10.1a 79.2 ± 10.5a the same letter in the same row is not significantly different (p > 0.05). a mean ± standard error (n = 5) b ngmw = non genetically modified wheat c gmw = genetically modified wheat variation in the weights of rats fed on mon 863 compared to the control animals. the findings of the present study showed no significant differences in the absolute body weights of animals fed on gmw in comparison with ngmw and control groups. relative organ weight the impact of feeding diets containing 30 % gmw and ngmw on the relative weights of some organs (kidney, liver, spleen, and stomach) to the animal body weight is presented in table 3. the relative ratio of liver to the body weight recorded a value of 3.91 %, 3.59 %, and 3.43 % for control, ngmw group, and gmw group, respectively. the relative ratios of spleen weight for control and ngmw group (0.43 – 0.48 %) were in lower levels as the value resulting from feeding on gmw. in addition, comparable findings were noted for the relative weights of the kidney (table 3). from these findings, it is clear that there is no significant statistical differences between the studied groups. however, ngmw group, and gmw group showed a significantly lower range of relative ratio for stomach weight (-14 %) and (-16.5 %), respectively. these findings agree with elsanhoty et al. (2004) and poulsen et al. (2007) who found an increase in the relative weight of the small intestine (+10 %) in rats fed gm rice, andan increase in the absolute and relative weight of the adrenals. on the other hand, poulsen et al. (2007) found statistically significantly reduce in the absolute weight of the adrenals (-15 %) in male rats fed on gm rice. similar results were reported in the previous study on gm potato spunta (elsanhoty et al. 2004), wherein no significant differences in the spleen, kidney and heart weights were observed after 1.5 months of feeding. kilic and akay (2008) reported that the liver weights were increased in females' rats, but there were no significant differences in males. séralini et al. (2011) reported that bt-corn or gm soybean caused significant differences in organs’ weights, especially liver and kidney because of these organs responsible for the detoxification. the obtained results disagree with abdo et al. (2014) who reported significant differences in all organs weights among different groups after 45 days of feeding study. effect of gmw on serum parameters the effects of feeding ngmw, and gmw on serum biochemical parameters that reflecting kidney and liver functions are given in table 4. the results showed no significant differences among the studied parameters in all groups. the health conditions of all groups coincided with normal values of gpt, got, cholesterol, hdlcholesterol, tag, tl, creatinine, and urea. the results indicated that the kidney and liver functions were in a good state without suffering from acute infections. the total cholesterol was also in the normal physiology level. the results revealed that the general health and metabolic process of the animals were not affected by feeding on gmw, wherein there were no significant differences (p < 0.05) between groups. the exception was in the values of total lipids, nova biotechnol chim (2020) 19(1): 19-29 24 fig. 1. liver sections of control (a), ngmw group (b) and gmw group (c). control group (a) and ngmw group (b) showing the normal histological structure of hepatic lobule, while gmw group (c) showing leucocytes in the hepatic sinusoids (arrows) (h&e x 200). wherein there were significant differences (p < 0.05) between the control group and gmw group. these results agree with elsanhoty et al. (2004), who reported no significant difference in serum biochemical values between groups fed gm potato spunta and non-gm potato. also, the results agreed with poulsen et al. (2007) who reported that male rats fed on gm rice had a lower plasma concentration of potassium as well as the levels of protein, and albumin. abdo et al. (2014) reported from subchronic feeding study on rats fed on gm bt-corn that there were many alternations in the organs weights, hematology, and serum biochemical parameters in bt-corn group after 45 days but changes were increased after 90 days. the obtained results of lipid parameters fig. 2. kidney sections of control (a), ngmw group (b) and gmw group (c). the control group (a) and ngmw group (b) showing normal histological structure, while gmw group (c) showing slight vacuolations of endothelial liming glomerular tufts (arrows) (h&e x 200). disagreed with gab-alla et al. (2012) who reported an increase in all lipid parameters (except for hdlcholesterol) of rats’ blood fed on mon810-ajeeb yg after 91 days of starting the experiment. histopathological profile microscopically examination of liver in the control group exhibited the normal histological structure of hepatic lobule (fig. 1a). the liver of the rat from the ngmw group showed no histopathological changes (fig. 1b). however, the liver of rat from group 3 showed the presence of leucocytes in the hepatic sinusoids (fig. 1c). concerning kidney, examined sections from control, and non-gmw group revealed b c a b a a c nova biotechnol chim (2020) 19(1): 19-29 25 no histopathological changes (fig. 2a and b). meanwhile, kidneys of rat from gmw group showed no changes except slight vacuolations of endothelial lining glomerular tufts (fig. 2c). regarding spleen, examined sections from the control group, non-gmw group and gmw group revealed no histopathological changes (fig. 3a, b and c). macroscopically examination of the stomach from control revealed the normal histological structure of gastric layers (fig. 4a), and examined sections from the ngmw group showed no histopathological changes (fig. 4b). moreover, the stomach of the rat from gmw group showed submucosal edema associated with few leucocytic cells infiltration (fig. 4c). these results indicated that the submucosal edema could be caused by gmw. to explain the nontoxicity of gmw, a detailed description of submucosal edema is needed (fig. 4c). these results agree with kilic and akay (2008) and gab-alla et al. (2012) who reported minimal histopathological changes in kidney and liver in rats fed on gm corn. the results of histopathological examination agree with the results obtained by ewen and pusztai (1999) and trabalza-marinucci et al. (2008) who observed no histological differences in pancreas, duodenum, liver, spleen, mesenteric lymph nodes, c b a fig. 3. spleen sections of control (a), ngmw group (b) and gmw group (c). control group (a), ngmw group (b) and gmw group (c) showing normal histological structure (h&e x 200). nova biotechnol chim (2020) 19(1): 19-29 26 cecal appendix, rumen, and abomasum sections between control group and gm maize-fed groups in sheep and lambs. the differences between the groups (individuals) can fall under individual variability or due to the particular location of the individual histological cuts. conclusions this research aimed to study the biosafety of gmw hi-line 111. no statistically differences in the biochemical parameters were noted between animals fed on gmw and rats fed on ngmw. when it comes to gmw, which contains a gene from barley, with both the plants belonging to the same genus, we perhaps cannot expect a significant impact on parameters in the serum of the experimental animals compared to commercial wheat of the same strain. based on the obtained data, it could be concluded that the gmw has no adverse health or toxic effects. however, a detailed description of submucosal edema and further studies on allergenic potential with long feeding periods might be needed. conflict of interest the authors declare that they have no conflict of interest. e e c b a fig. 4. stomach sections of control (a), ngmw group (b) and gmw group (c). the control group (a) and ngmw group (b) showing normal histological structure, while gmw group (c) showing submucosal edema (e) associated with few leucocytic cells infiltration (arrows) (h&e x 200). nova biotechnol chim (2020) 19(1): 19-29 27 statements on compliance with ethical standards the procedures followed were in accordance with the institutional and national ethical standards of the responsible committee on animal/human experimentation. references abdo me, barbary om, el-sayed shaltout eo (2014) feeding study with bt corn (mon810: ajeeb yg) on rats: biochemical analysis and liver histopathology. food nutr. sci. 5: 185-195. aoac international (2000) official methods of analysis of aoac international. 17th ed. gaithersburg, md, usa, association of analytical communities. appenzeller lm, malley l, mackenzie sa, hoban d, delaney b (2009a) subchronic feeding study with genetically modified stacked trait lepidopteran and coleopteran resistant (das-ø15ø7-1×das-591227) maize grain in sprague-dawley rats. food chem. toxicol. 47:1512-1520. appenzeller lm, munley sm, hoban d, sykes gp, malley la, delaney b (2009b) subchronic feeding study of grain from herbicide-tolerant maize dp-ø9814ø-6 in sprague-dawley rats. food chem. toxicol. 47: 22692280. brake dg, evenson dp (2004) a generational study of glyphosate-tolerant soybeans on mouse fetal, postnatal, pubertal and adult testicular development. food chem. toxicol. 42: 29-36. brake dg, thaler r, evenson dp (2004) evaluation of bt (bacillus thuringiensis) corn on mouse testicular development by dual parameter flow cytometry. j. agric. food chem. 52: 97-102. castigliego l, armani a, tinacci l, gianfaldoni d, guidi a (2015) two alternative multiplex pcrs for the identification of the seven species of anglerfish (lophius spp.) using an end-point or a melting curve analysis real-time protocol. food chem. 166: 1-9. chassy bm (2010) food safety risks and consumer health. new biotechnol. 27: 534-544. coudon b, bouige d (1973) automatic method for determination of total serum lipids, using the sulfophosphovanillic reaction. ann. biol. clin. 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infrared spectroscopy and the iron determination also and exhibited very low organic matter content – the sum of n, c, h and s content less than 20 % and fe content around 40 %. prepared samples consist of iron(ii) amino acid complex [fe(glyh)(so4)]n, known as iron food supplement (fegs) as the only sulphur containing compound. its content varies from 26 % up to 46 %. ferrous glycinate was obtained in the samples collected initially after the reaction in very low content < 7 %. iron(iii) hydroxy-oxide feo(oh) was obtained in the range 40 – 65 %. the feo(oh) content increased with the decreasing fegs content. formation of inorganic feo(oh) suggest the ongoing redox reaction under aerobic condition therefore, it is suggested as a biomineralization reaction model.  university of ss. cyril and methodius in trnava introduction the term biomineralization was introduced into chemistry three decades ago by professor r.j.p. williams group (webb et al. 1986) on ferritin isolation and the wide current usage of this term was well explained in editorial (bertazzo 2015) as "at the core of the biomineralization discipline is the inspiration for research drawn from diverse tissues presenting minerals synthesized by a living organism", e.g. magnetite production by magnetic microbes (prozorov 2015), or silica formation in diatoms (hildebrand and lerch 2015), or coral biomineralization (falini et al. 2015), or mineralization processes in the skeletons (dean et al. 2015), and/or cardiovascular calcification (hutcheson et al. 2015). possible explanation of the oxide formation by ferritin biodegradation together with the oxide observation in human spleen (boča et al. 2013a; kopáni et al. 2015) and observation of iron(iii) oxide deposits in human basal ganglia (boča et al. 2013b) are the inspiring points of our interests in this field. the understanding of chemical reactions occurring in the human brain is very important for health and treatment of some serious diseases. the iron oxide mixture formation in two steps reactions of ferrous salts with some amino acids in alkaline medium were explained by second step oxidation of iron(0) nanoparticles formed in anaerobic first step reaction (klačanová et al. 2013; kišš et al. 2017). the presented paper is dealing with analysis of the reaction products of selected amino acid mailto:katarinamitalova@gmail.com nova biotechnol chim (2021) 20(1): e808 2 glycine with mohr’s salt under different aerobic reaction conditions. experimental mohr’s salt (ammonium iron(ii) sulfate hexahydrate) (nh4)2fe(so4)2·6h2o analytical grade, was bought from aldrich and used without further purification. l-glycine (analytical grade, centralchem) was used as received. samples were prepared by reaction of glycine and mohr’s salt aqueous solutions. glycine (100 mmol, 7.507 g) dissolved in warm water was added under stirring to mohr’s salt (50 mmol, 19.607 g) dissolved in warm water and the reaction mixture was stirred for 30/60/120 min with the temperature kept at 60 °c. the reaction mixture was then cooled to room temperature and the formation of precipitate was observed. the time of aging is listed in table 1. the solid precipitate was isolated by filtration under reduced pressure. the products were dried at laboratory temperature. the filtrates were left for further product formation. elemental analyses were carried out on a chnso flasheatm 1112 automatic elemental analyzer. inductively coupled plasma atomic emission spectrometer, model 5100 icp-oes (agilent, usa) was used for iron content determination. infrared spectra (4,000 – 400 cm-1) were measured using the nicolet 5700 ft-ir spectrophotometer at room temperature using the atr technique. table 1. reaction conditions of samples prepared. sample glycine[mmol] / water [cm3] mohr’s salt [mmol] / water [cm3] reaction time [min] filtered off after [h] km 04.2.2 20 / 50 10 / 20 30 24 km 04.3.2 20 / 50 10 / 20 30 24 km 13.1.3 100 / 250 50 / 100 30 48 km 14.1.2 100 / 250 50 / 100 60 24 km 14.1.3 100 / 250 50 / 100 60 48 km 15.1.1 100 / 250 50 / 100 120 0 km 15.1.2 100 / 250 50 / 100 120 24 km 15.1.3 100 / 250 50 / 100 120 48 results and discussion the experiments were realized according to the procedure published (ghasemi et al. 2012) as the procedure for preparation of iron-amino acid chelates [fe(gly)2] as growth stimulator used in nutrient solution culture. the initial light green colour of mohr's salt solution has rapidly changed to darker brown-green colour due to iron(ii) glycine complex formation and proceeding reactions were recognized by further colour changes to brown shades and usually some browncoloured solid products formation were observed. the obtained brownish products were analyzed by elemental analysis and results are shown in table 2. it should be stressed that all the obtained samples show in addition to the rather high sulphur content very "low organic matter content" (table 2). the data show nitrogen-to-carbon stoichiometric ratio ν(n) : ν(c) ≈ 1 : 2 that corresponds to the ratio of the amino acid used in the reaction – glycine. moreover the stoichiometric ratio ν(n) : ν(s) is roughly close to 1 : 1 which means that the representation of amino acid and sulphur in the obtained samples is approximately 1 : 1 which is more similar to therapeutic "ferrous sulfateglycine complex" (rummel 1959) than to "ironamino acid chelate" (ghasemi et al. 2012). infrared spectra of all samples are similar, and the fig. 1 gives as an example two of them together with the spectrum of glycine, the amino acid used. all spectra show the broad bands presence in the region 3,300 – 3,500 cm-1 that could be assigned to o–h vibrations probably of uncoordinated water nova biotechnol chim (2021) 20(1): e808 3 molecules present in samples. absence of o–h vibration in the spectrum of glycine is logically explained by existence of "zwitter ion" structure +nh3ch2coo – in the solid state (drebushchak et l. table 2. elemental analysis and calculated stoichiometric ratios of selected samples. sample elemental analysis / stoichiometric ratio n [%] / ν(n) c [%] / ν(c) h [%] / ν(h) s [%] / ν(s) sum[%] nchs km 04.2.2 3.26 / 1.19 4.68 / 2.00 2.80 / 14.2 6.25 / 1 17.0 km 04.3.2 -* 4.54 / 2.11 2.63 / 14.5 5.74 / 1 12.9 km 13.1.3 2.94 / 1.22 5.61 / 2.72 2.66 / 15.4 5.51 / 1 16.7 km 14.1.2 2.80 / 1.11 4.14 / 1.92 2.64 / 14.6 5.76 / 1 15.3 km 14.1.3 3.08 / 1.12 4.63 / 1.96 2.74 / 13.8 6.29 / 1 16.7 km 15.1.1 -* 2.86 / 2.08 1.85 / 16.0 3.67 / 1 8.40 km 15.1.2 3.01 / 1.17 4.41 / 2.00 2.71 / 14.7 5.88 / 1 16.0 km 15.1.3 3.44 / 1.19 4.95 / 2.00 2.79 / 13.4 6.62 / 1 17.8 * low nitrogen content evaluated as zero by running computer programme. 2002). the region from 3,000 to 3,300 cm-1 can be assigned to the n–h vibrations of the amino group. this confirms the presence of amino acid in the samples. difference in the spectra of glycine and samples is a region around and below 3,000 cm1 that could be assigned to the hydrogen-bonding present in the samples which differ from that one in pure glycine spectrum. the presence of glycine in samples could be confirmed by bands in the region 1,700 – 1,300 cm-1 – the coo vibrations of the carboxyl group. very strong and rather broad bands in the region 1,100 – 1,000 cm1 could be assigned to the s–o vibration of sulphate group. 4000 3500 3000 2500 2000 1500 1000 500 20 40 60 80 1060 cm -1 kmi15.1.1 kmi15.1.2 glycine t / a .u . wavenumber / cm -1 fig. 1. comparison of the infrared spectra of the samples prepared along with the infrared spectrum of the amino acid glycine used. nova biotechnol chim (2021) 20(1): e808 4 elemental analysis, along with the infrared spectroscopy, suggested that products contained glycine and the sulphate group. a search through the cambridge crystallographic database has shown that there are only two ferrous glycine complexes with a solved structure. the first complex is [fe(glyh)2(h2o)4][fe(h2o)6](so4)2, where glyh = glycine, (fig. 2 at the top) containing two different iron(ii) cations together with two sulphate anions (ougey et al. 2013). the second complex found is polymeric catena-[{fe(h2o)4}(μglyh){fe(h2o)2(so4)2}(μ-glyh)]n (fig. 2 at the bottom) (ougey et al. 2013). both complexes show common stoichiometric formula {fe2(glyh)2(so4)2(h2o)x} where x = 10 or 6. just recently the third polymeric complex structure [fe(glyh)2(h2o)4][fe(h2o)6](so4)2 catena-[{fe(h2o)4}(μ-glyh){fe(h2o)2(so4)2}(μ-glyh)]n fig. 2. structures of the hexa-aqua-iron(ii)-bis(ammonioacetato)-tetra-aqua-iron(ii) sulfate complex (glycfe01 above), and the catena-[bis(μ2-ammonioacetato)-hexa-aqua-bis(sulfato)-di-iron(ii)] complex (udopio below) (ougey et al. 2013). nova biotechnol chim (2021) 20(1): e808 5 ferrous glycine sulfate (fegs) was published (dinnebier et al. 2016) as structure of dietary supplement [fe(glyh)(so4)]n (fig. 3). there are two conclusions drawn out of all three structures mentioned above. at first, all three complexes exhibit the same stoichiometry ν(fe) : ν(glyh) : ν(so4) = 1 : 1 : 1 and they differ only by the decreasing water to iron(ii) stoichiometry. moreover, the fegs food supplement is prepared by the reaction of glycine with iron(ii) sulphate, and it should be taken as one of the principal components in our products formation. fig. 3. structure of the complex ferrous glycine sulfate [fe(glyh)(so4)]n (anivok) (dinnebier et al. 2016). in particular conclusion of the qualitative analysis above, it could be stressed that studied reactions gave, due to their complexities, the reaction products composed of at least three iron containing components. the ferrous glycine sulfate fegs, or its "hydrated forms" are surprisingly at the top to be accepted due to the elemental analysis and sulphate spectral evidence. the ferrous glycinate fe(gly)2, listed also as fe-glycine chelate (ghasemi et al. 2012), could be partly accepted as iron and glycine containing minor component in addition to the major fegs one. the minority of the fe(gly)2 in our products (despite the suggestion (ghasemi et al. 2012) is based on the elemental analysis results only few samples gave the ν(c) : ν(s) ratio higher than 2 : 1. moreover, the formation of the fe(gly)2 in the reaction fe(so4) with glycine could be to greater extent seen after addition of more naoh (ashmead 1980). finally, the third completely inorganic component feooh should be included into the list, and that allows to analyze the composition of obtained products. two products km 13.1.3 and km 15.1.1 have been recently analyzed for iron content (table 3) and that together with sulphur and carbon allowed us to calculate all three iron compounds content. the calculation is based on assumptions that the fegs is only component with sulphur content (3rd column in table 3) and the total carbon content comprise of the glycine in the fegs and glycinate anion presence in fe(gly)2 (4 th column in table 3). the total iron content could be found only if samples contained calculated amount of inorganic iron oxide formally as feooh ≈ ½(fe2o3∙h2o). the obtained results are interesting for the possibility to explain consistently on the base of the elemental analysis the composition of prepared samples, e.g., km 13.1.3 contains 84.6 % of iron containing compounds and similarly the content of these compounds in km 15.1.1 is 91.6 %. the calculation based on these data has shown that hydrogen content in mentioned components is far less than hydrogen content determined in elemental analysis (table 2). it shows together with the infrared spectra (presence of the broad absorption nova biotechnol chim (2021) 20(1): e808 6 bands between 3,500 and 3,300 cm-1 in all samples) that water content should be included into the composition of the prepared samples. for these two samples evaluated, water could be up to 10 % or 5 % in km 13.1.3 or km 15.1.1, respectively. the results shown above allowed us to do some table 3. elemental analysis and calculated iron compound content. sample elemental analysis / iron compound content fe [%] s% / fegs [%] c% / fe(gly)2 [%] feooh [%] km 13.1.3 35.9 5.51 / 39.0 5.61 / 6.28 39.3 km 15.1.1 47.3 3.67 / 26.0 2.86 / 0.46 65.1 approximations for those samples where iron could not be determined because of the insufficient number of samples. the results (table 4) show that all samples exhibit over 40 % of fegs content and practically all samples have shown carbon content approximately equal to glycine content in the fegs. the estimated feooh content could be in the range 40 – 45 % that gives the total iron content in range 35.9 – 38.2 % and finally, the water content from 10.5 to 12.5 %. table 4. elemental analysis and calculated/estimated iron compound content. sample elemental analysis / stoichiometric ratio s% / fegs [%] c% / fe(gly)2 [%] feoohest [%] fecalc [%] km 04.2.2 6.25 / 44.3 4.68 / 40 35.9 km 04.3.2 5.74 / 40.7 4.54 / 1.00 44 37.8 km 14.1.2 5.76 / 40.8 4.14 / 45 38.2 km 14.1.3 6.29 / 44.6 4.63 / 40 36.0 km 15.1.2 5.88 / 41.6 4.41 / 0.02 42 36.5 km 15.1.3 6.62 / 46.9 4.95 / 40 36.5 the estimated data all together gives us the chance come to conclusion concerning the reaction procedure under study. first of all, data are in good agreement with the conclusion the mohr's salt in solution primarily gives "ferrous sulfate-glycine complex" containing fe : glyh ratio 1 : 1. the excess of glycine in all experiments (fe : glyh ratio 1 : 2 was used) apparently allows the fe(gly)2 formation, but probably the formed product undergoes some further reactions in solution and only a small part of this substance is included into the final solid product. this rather complicated solid phase vs solution equilibria could be to some extent demonstrated on km 15 samples that were from the reaction mixture filtered off at different times. the initially (just after cooling down the reaction mixture) separated part of product km 15.1.1 contains the lowest 26.0 % fegs content and the highest 65.1 % content of feooh. the increasing content of fegs and simultaneously decreasing content of feooh in samples km 15.1.2 (separated after 24 h) and km 15.1.3 (filtered off after 48 h) could be taken as data supporting the idea of these reactions between the forming solid phase and solution. at this state of experiments one cannot give any proof which of two glycine containing components undergo the biomineralization procedure leading to the formation of iron(iii) product – feooh, but it is clear that this study brings some suitable results. conclusions in conclusion, we can say that the formation of the products fegs and fe(gly)2 containing glycine in the form of a molecule or chelating anion together with iron(ii) was proved. it was also shown that some of the products undergo the decomposition together with oxidation iron(ii) to iron(iii) reactions that altogether could be called as biomineralization reactions and they probably lead to iron(iii) oxide formation. nova biotechnol chim (2021) 20(1): e808 7 this research was funded by agency apvv (apvv-190087) and ucm grant fppv-35-2021. furthermore, we would like to thank assoc. prof. m. horník, phd. for his kind and great support of this work by guaranteeing the iron content analysis of samples at geoanalytical laboratories of state geological institute of dionýz štúr in spišská nová ves. conflict of interest the authors declare that they have no conflict of interest. references ashmead, hh (1980) soluble iron proteinates. us. patent. 4 216 144. bertazzo s (2015) biomineralization. semin. cell dev. biol. 46: 1. boča r, dlháň ľ, kopáni m, mrázová v, miglierini m (2013a) deposits of iron oxides in the human spleen. polyhedron. 66: 65-69. boča r, kopáni m, miglierini m, čaplovičová m, mrázová v, dlháň ľ (2013b) magnetic and non-magnetic ironoxide deposits in basal ganglia. in costa a, villalba e (eds.), horizons in neuroscience research, nova science publishers, new york, usa, pp. 135-214. dean mn, ekstrom l, monsonego-ornan e, ballantyne j, witten pe, riley c, habraken w, omelon s (2015) mineral homeostasis and regulation of mineralization processes in the skeletons of sharks, rays and relatives (elasmobranchii). semin. cell dev. biol. 46: 51-67. dinnebier re, runčevski t, hinrichsen b (2016) crystal structure of the dietary supplement ferrous glycine sulfate. z. anorg. allg. chem. 642: 306-310. drebushchak tn, boldyreva ev, seryotkin yv, shutova es (2002) crystal structure study of the metastable β modification of glycine and its transformation into the αmodification. j. struct. chem. 43: 835-842. falini g, fermani s, goffredo s (2015) coral biomineralization: a focus on intra-skeletal organic matrix and calcification. semin. cell dev. biol. 46: 17-26. ghasemi s, khoshgoftarmanes ah, hadadzadeh h, jafari m (2012) synthesis of iron-amino acid chelates and evaluation of their efficacy as iron source and growth stimulator for tomato in nutrient solution culture. j. plant growth regul. 31: 498-508. hildebrand m, lerch sjl (2015) diatom silica biomineralization: parallel development of approaches and understanding. semin cell dev. biol. 46: 27-35. hutcheson jd, goettsch c, rogers ma, aikawa e (2015) revisiting cardiovascular calcification: a multifaceted disease requiring a multidisciplinary approach. semin. cell dev. biol. 46: 68-77. kišš ľ, rapta p, boča r, miglierini m, čaplovičová m, martinka m, žemlička l, fodran p (2017) nanoscale iron particles formed from the metalloprotein-like structures prepared using ferrous ions in the presence of sodium glutamate and bovine serum albumin. mon. chem. 148: 2019-2029. klačanová k, fodran p, šimon p, rapta p, boča r, jorík v, miglierini m, kolek e, čaplovič ľ (2013) formation of fe(0) nanoparticles via reduction of fe(ii) compounds by amino acids and their subsequent oxidation to iron oxides. j. chem. 2013: 961629. kopáni m, miglierini m, lančok a, dekan j, čaplovičová m, jakubovský j, boča r, mrázová, v (2015) iron oxides in human spleen. biometals. 28: 913-928. oguey s, jacquier y, neels a, stoeckli-evans h (2013) csd communication glycfe01 & udopio, private communication 2013. prozorov t (2015) magnetic microbes: bacterial magnetite biomineralization. semin. cell dev. biol. 46: 36-43. rummel w (1959) therapeutic iron complex. us. patent. 2 877 253. webb j, mann s, bannister jv, williams rjp (1986) biomineralization of iron isolation of ferritin from the hemolymph of the limpet patella-vulgata. inorg. chim. acta. 124: 37-40. acknowledgement nova biotechnol chim (2018) 17(2): 193-200 doi: 10.2478/nbec-2018-0020  corresponding author: zita.tokarova@ucm.sk nova biotechnologica et chimica synthesis and structure-physicochemical properties relationship of thiophene-substituted bis(5,4-d)thiazoles zita tokárová and anna biathová department of chemistry, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk-917 01, slovak republic article info article history: received: 28th june 2018 accepted: 25th september 2018 keywords: chemosenzors π-conjugation dithioxamide fluorescence structure-physicochemical properties thiazolothiazole thiazolo[5,4-d]thiazole thiophene-2-carbaldehydes uv-vis abstract substituted thiophene-2-carbaldehydes 1a–d were utilized in the synthesis of symmetrically substituted thiazolo[5,4-d]thiazoles 3a–d. bis(5,4-d)thiazoles with thiophene core at the termini are the most employed in the chemistry of materials but exhibit insufficient solubility in majority of organic solvents with notable impact on the low yields of products. accordingly, the synthetic approach towards 2,5-dithiophen-2-yl-thiazolo[5,4-d]thiazole (3a) and its substituted derivatives 3b–d is discussed under the various reaction conditions. appropriate structural characterisations are included with emphasis on relationship between structure and physicochemical properties highlighting the uv-vis and fluorescence.  university of ss. cyril and methodius in trnava introduction thiazolo[5,4-d]thiazoles are an important class of bicyclic aromatic systems constructed from two [3.3.0]-fused thiazole rings acting as the electrondeficient moiety due to the presence of imine backbone (c=n, tztz, fig. 1) (smirnova et al. 2006). the parent compound of this class was misstated as 2,2´-diaryl-4,4´-bisthiazethine (tze, fig. 1). later, the 2,5-diaryl-thiazolo[5,4d]thiazoles were established as thermodynamically favoured over bithiazetines (johnson et al. 1970). since the thiazoles are generally known as key structural units in a wide variety of natural products (i.e. abafungin, tiazofurin) (arshadi et al. 2017) the fused bis(5,4-d)thiazoles have been initially screened as inhibitors of central nervous system and antibacterial agents. the high toxicity (ld50 ≥ 0.5 g/kg) associated with most of pattern tztz derivatives when tested as cns depressants, and unsufficient antibacterial activity against various strains of bacteria have excluded thiazolo[5,4-d]thiazoles suitable for further biological applications (ketcham and mah 1971). re-interest in potential biological applications of these compounds was highlighted quite recently. owing the rigid planar structure in the ground state and upon coordination (millan et al. 2018) accompanied with the colorimetric change and often also with the fluorescent quenching thiazolo[5,4-d]thiazoles can effectively sense s n s n r r tztz 1 2 3 4 5 6 7 8 a b c d n sr n s r tze fig. 1. structure of thiazolo [5,4-d]thiazole (tztz) with atoms and bonds numbering and the structure of mis-stated 2,2´-diaryl-4,4´-bisthiazethine (tze). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:43 utc mailto:zita.tokarova@ucm.sk nova biotechnol chim (2018) 17(2): 193-200 194 table 1. coordination of tztz-based frameworks with variety of cations and their applications as chemosensors and other coordination compounds with miscellaneous utilizations. tztz-type compound cation/s colorimetric change and/or fluorescent quenching / type reference n s n s nn o o o oo o o o cu 2+ color change from yellow to dark green (red shift: 400 nm ligand → 700 nm complex) selective fluorescent quenching jung et al. (2012) n s n s nn cu 2+ color change from yellow to blue (red shift: 400 nm ligand → 600 nm complex) selective fluorescent quenching zhang et al. (2014) n s n s o o o o oo al 3+ cr 3+ fluorescent „on-off“ change upon coordination with cation jung et al. (2012) n s n s ag + h + uv-vis change upon coordination protonization based on pearson´s principle of soft-soft acidbase/colorimetric change olgun and güflen (2013) n s n s nn zn 2+ – /solvothermal charact./ millan et al. (2018) s n n s nn n n n n ru 2+ fluorescence as the function of the redox state of complex with ruthenium hua et al. (2017) s n n s nn zn 2+ mixed-valence state characterised by ivct# band in nir (6,576 and 8,202 cm−1) in mofs* hua et al. (2018) *mofs = metal organic frameworks with paddle-wheeled unit constructed of two monodentate fashion coordinated zn(ii). #ivct band = through-space intervalence charge transfer. variety of cations (jung et al. 2012; sivaraman et al. 2018). in some of these compounds the bivalent sulphur atom makes close contact with electrondonating nitrogen/oxygen (table 1), which is isosteric to the hydrogen-bonding interactions as important features of bioactivity in pharmaceuticals and natural drugs (beno et al. 2015) what re-opens the possibility of research on the design of bis(5,4d)thiazoles as bioactive compounds. however, these applications have lagged behind the use of tztz units as monomeric building blocks in photovoltaics (song et al. 2017) or co-polymers in semiconductors such as organic field emitting transistors (ofets) (ando et al. 2005) (fig. 2). fig. 2. number of publications on tztz in the period 1978 – 2018 (bevk et al. 2013) and our search update (june 2018). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:43 utc nova biotechnol chim (2018) 17(2): 193-200 195 s n n s s s r r ox. scheme 1. synthesis of thiophene-based thiazolo[5,4-d]thiazoles 3a–d according to ketcham´s method (johnson and ketcham 1960). the structure of intermediate formed during the reaction process is presented in brackets. the carbonyl substrate acting as the oxidizing agent of appropriate intermediate. reaction conditions used in our approach are following: method a: dmf (153 °c); method b: o-dichlorbenzene (180 °c); method c: nitrobenzene (211 °c). despite the significant interest in applications of bis(5,4-d)thiazoles the synthetic chemistry has only been explored to a minor extent (dessi et al. 2014; dessi et al. 2015; papernaya et al. 2016). the most of procedures towards (hetero)aryl substituted thiazolo[5,4-d]thiazoles are still based on the condensation of dithioxamide (2) with an excess of aldehyde (johnson and ketcham 1960). the reactions are most often performed in high boiling point solvents, such as dimethylformamide (dmf, 153 °c); n,n-dimethylacetamide (dmac, 165 °c) or under solvent free conditions (≤ 200 °c). in general, the solubility of thiophene-substituted tztz derivatives is the most supressed affecting the tedious work-up and low yields. accordingly, in our research the synthesis of a series of thiophene-substituted thiazolo[5,4-d]thiazoles 3a–d is discussed under various reaction conditions (scheme 1). compounds 3a–d are important intermediates for design of low molecular mass type chemosensors and building blocks for (opto)electronic materials. in this context, the physicochemical data (uv-vis and fluorescence) are analyzed. experimental general all commercially available chemicals were used as received without further purification. solvents were purified by standard methods and dried if necessary. reactions were monitored by thin layer chromatography (tlc) on plates percoated with silica gel (sigel 60 f254). melting points were recorded on a kofler hot plate apparatus and are uncorrected. the infrared spectra were taken on agilent cary 630 ftir spectrometer with diamond atr. elemental analyses were obtained using a flash ea 2000 chns/o-oea analyser. 1h nmr spectra (400 mhz) were measured as solutions in deuterated dimethylsulfoxide (dmsod6) on a bruker biospin type instrument, products were reported relative to tetramethylsilane (tms, 0.0 ppm). absorption spectra (uv-vis) of solutions (c = 1.10-5 mol.l-1) in dimethylformamide were recorded on a uv 1650pc spectrometer (schimadzu, jpn) and the fluorescence on a rf 5301 pc type instrument (shimadzu, jpn). bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:43 utc nova biotechnol chim (2018) 17(2): 193-200 196 synthesis method a. to the mixture of dithioxamide (2, 10.0 mmol; 1.10 g) in n,n-dimethylformamide (25 ml) solution of appropriate thiophene-2carbaldehyde 1a–d (40.0 mmol) in dmf (15 ml) was added at room temperature. the reaction mixture was stirred at the boiling point of dmf (153 °c) for 6 hours. the colour of the reaction changed to dark after the substrates became completely dissolved. after the reaction was stated as completed (tlc control), the reaction mixture was cooled to room temperature and diluted with water (15 ml). crude mixture was extracted with chloroform (3 x 25 ml). combined organic layers were dried with mgso4, filtered and the solvent was evaporated. the dark crude product was analyzed (1h nmr) and then purified by flash column chromatography with chloroform as eluent. note. in the crude residue the traces of the appropriate product were identified according to 1h nmr only the substrates were retrieved in majority. method b. to the mixture of dithioxamide (2, 10.0 mmol; 1.10 g) in o-dichlorobenzene (25 ml) the solution of appropriate thiophene-2carbaldehyde 1a–d (40.0 mmol) in o-dichlorobenzene (15 ml) was added at room temperature. the reaction mixture was stirred at the boiling point of o-dichlorobenzene (180 °c) for 12 hours. the colour of the reaction changed to dark after the substrates became completely dissolved. after the reaction was stated as completed (tlc control), the mixture was cooled to room temperature and washed with water (25 ml) afterwards the slow precipitation of crude product occurred. the immiscible mixture was left to cool down to 0 °c overnight and the precipitate was filtered. the crude product was crystalized from methanol. note. the mixture of products 3a–d and the substrate 1a–d was achieved with the ratio ¼ (aldehyde in majority). method c. to the mixture of dithioxamide (2, 10.0 mmol; 1.10 g) in nitrobenzene (25 ml) the solution of appropriate thiophene-2-carbaldehyde 1a–d (40.0 mmol) in nitrobenzene (15 ml) was added at room temperature. the reaction mixture was stirred at the boiling point of nitrobenzene (211 °c) for 5 hours. the colour of the reaction changed to dark after the substrates became completely dissolved. after the reaction was stated as completed (tlc control), the mixture was cooled to room temperature and diethylether was added to precipitate the product (15 ml). the precipitate was filtered, washed with diethylether (2x25 ml) to get rid of unreacted dithioxamide. the crude residue was crystallized from methanol with 15 – 25 % addition of n,n-dimethylformamide to increase the solubility. note. finally, pure products 3a–d (fig. 3) were isolated from the crude mixture after crystallization (methanol/dmf). fig. 3. products isolated from mixture after crystallization (methanol/dmf) with atom numbering at thiophene core (corresponding with 1h nmr signals, table 2). 2,5-di-thiophen-2-yl-thiazolo[5,4-d]thiazole (3a) dark brown solid after the crystallization from methanol/dmf (ratio: 4/1). yield 30 % (3.7 g, method c) starting from thiophene-2-carbaldehyde 1a (40.0 mmol, 4.5 g). mp 228 – 235 °c. anal. calc. for. c12h6n2s4 (306.45 g.mol -1): c 47.03, h 1.97, n 9.14, s 41.85 %. found: c 47.29, h 2.09, n 9.70, s 41.25. 2,5-bis-(3-methylthiophene-2-yl)-thiazolo[5,4d]thiazole (3b) brown-yellow solid after the crystallization from methanol/dmf (ratio: 6/1). yield 31 % (4.1 g, method c) starting from 3-methylthiophene-2carbaldehyde 1b for. c14h10n2s4 (333.97 g.mol -1): c 50.27, h 3.01, n (40.0 mmol, 5.0 g). mp 216 – 222 °c. anal. calc. 8.37, s 38.34 %. found: c 49.95, h 3.14, n 8.10, s 38.50. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:43 utc nova biotechnol chim (2018) 17(2): 193-200 197 2,5-bis-(5-ethylthiophene-2-yl)-thiazolo[5,4d]thiazole (3c) brown solid after the crystallization from methanol/dmf (ratio: 6/1). yield 33 % (4.8 g, method c) starting from 5-ethylthiophene-2carbaldehyde 1c (40.0 mmol, 5.6 g). mp 200 – 205 °c. anal. calc. for. c16h14n2s4 (362.00 g.mol -1): c 53.00, h 3.89, n 7.73, s 35.38 %. found: c 53.24, h 3.87, n 7.89, s 35.50. 2,5-bis-(4-bromo-3-methylthiophene-2-yl)thiazolo[5,4-d]thiazole (3d) black solid after the crystallization from methanol/dmf (ratio: 3/1). yield 22 % (4.3 g, method c) starting from 4-bromo-3methylthiophene-2-carbaldehyde 1d (40.0 mmol, 8.2 g). mp 296 – 300 °c. anal. calc. for. c14h8 br2n2s4 (489.79 g.mol -1): c 34.16, h 1.64, n 5.69, s 26.05 %. found: c 34.52, h 1.96, n 5.18, s 26.42. results and discussion synthesis thiophene-substituted thiazolo[5,4-d]thiazoles are among all tztz-based the most utilized in the optoelectronic materials, but in the same time are one of the most difficult for synthesis. designed compounds 3a–d were synthesized by initial onestep condensation reaction of appropriate thiophene-based substrate 1a–d with dithioxamide (2, scheme 1). the carbonyl substrate/dithioxamide ratio 4/1 was used as the optimal for successful reaction proceeding. the aldehyde primarily undergo the condensation reaction (–hc=o → –c=nh) followed by subsequent cyclization but, in the same is essential for final aromatization as oxidizing agent (scheme 1). our initial approaches using dmf (method a, 153 °c, 6 hours) and o-dichlorobenzene (method b, 180 °c, 12 hours) have failed in products formation. nitrobenzene was found to be optimal (method c, 211 °c, 5 hours) and the target thiophene-substituted thiazolo[5,4-d]thiazoles 3a–d were isolated in moderate yields (22 – 33 %) and satisfactory purity upon crystallization. in practice, nitrobenzene aside allows the better maintenance of the reaction temperature has few limitations. as the solvent is highly toxic and the products are usually not easily precipitable from the reaction mixture. this can be avoided by the use of n-butanol or performance of the reaction under microwave irradiation (mwo). still, the solubility of carbonyl substrates in n-butanol could be insufficient and not all of the substrates are liquids as is required for mwo assisted reactions. structure characterisation previously published 2,5-di-thiophen-2-yl(3a) (jung et al. 2010; dessi et al. 2013) was selected as the representative compound for the structure characterisation. full conversion of carbonyl substrates 1a–d towards corresponding thiazolo[5,4-d]thiazoles 3a–d was assigned by the disappearance of carbonyl proton (–hc=o) characteristic at δ ≈ 9.5 ppm and further corroborated by the ftir spectra where the stretching vibrations of carbonyl group (1,680 – 1,750 cm-1) vanished and the characteristic stretch vibration at ν ≈ 1,250 cm-1 was observed for c=n (table 2). the chemical shifts of heteroaromatic protons h-3´, h-4´, h-5´ revealed doublets for 3a– c and singlet for 3d in the range of 7.39 – 6.04 ppm. the most intense downfield shift (paramagnetic) because of methyl´s deshielding effect to 7.39 table 2. structure and product yields together with the specific chemical shifts (δ/ppm) in 1h nmr and vibrations (ν/cm-1) in ir spectra for target thiophene-substituted thiazolo[5,4-d]thiazoles 3a–d. compound r yield 1h nmr [δ/ppm] ir [ν/cm-1] [%] h-3´ h-4´ h-5´ c=n 3a h 30 6.99 6.97 6.40 1,250 3b 3´-ch3 31 7.01 7.39 1,260 3c 5´-ch2ch3 33 6.70 6.70 1,250 3d 4´-br-3´-ch3 22 7.35 1,250 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:43 utc nova biotechnol chim (2018) 17(2): 193-200 198 and 7.35 ppm for h-5´is associated with 2,5-bis-(3methylthiophene-2-yl)-thiazolo[5,4-d]thiazole (3b) and its c4´-bromo-substituted analogue 3d. on the other hand, the protons of tztz bearing the unsubstituted thiophene ring for compound 3a and with ethyl group in c5´ for compound 3c, respectively, appear in the range of 6.99 – 6.40 ppm in accordance with the similar thiophenesubstituted derivatives. the complete structural characterisations are part of our subsequent research and herein are presented as the complementary statement supported by the elemental analysis (with deviation max. 0.4 %). compounds 3a–d were prepared as useful tools for the design of (opto)electronic materials when taking in account the further possibilities of the substitution and functionalization arising from the electronic effects in the structure of parent compounds (fig. 4). accordingly, the uncommon nucleophilic substitutions (snar, tokárová et al. 2017) are expected to the c4´of thiophene core, but with certainty this position will stay unaffected and 3-substituted thiophene-2-carbaldehyde have to be used when c3´-substituted tztz products are required (papernaya et al. 2016). the electrophilic substitutions (arse) are predicted to occur at the adjacent β-position (c4´) (dessi et al. 2013). on the c-α (= c5´) the radical substitutions (sr) are quite common offering the possibility of ubsequent alkylation via the cross-coupling manner (jung et al. 2010). s n s n s s 3a-d r r + un af ec ct ed a rs e sr (s n ar ) fig. 4. general structure of thiophene-substituted thiazolo[5,4-d]thiazoles 3a–d presented herein, showing the electronic effects in the structure with prediction of possible substitution patterns. uv-vis and fluorescence the absorption (fig. 5a) and fluorescence spectra (fig. 6) of 3a–d were examined in dmf solutions (1.10-5 mol.dm-3). the absorption maximum of representative, the 2,5-di-thiophen-2-ylthiazolo[5,4-d]thiazole (3a), observed at 390 nm is assigned to the n→π*of bis(5,4-d)thiazole c=n group, while the less intense maximum at 275 nm to the n→σ* (c–n). the slight red-shift to 401 nm (n→π*) is observed for 2,5-bis-(3methylthiophene-2-yl)-thiazolo[5,4-d]thiazole (3b) as a result of electron-donating character of methyl group at c3´ involved in the extension of the π-conjugation length. the 2,5-bis-(5ethylthiophene-2-yl)-thiazolo[5,4-d]thiazole (3c) showed similar absorption bands as 3a at 390 nm (n→π* / c=n) and 270 nm (n→σ* / c–n) as the consequence of almost no influence of ethyl group at c5´ to π-conjugation of the whole system. the blue shift to 311 nm (n→π*) for 2,5-bis-(4-bromo-3-methylthiophene-2-yl)thiazolo[5,4-d]thiazole (3d) agrees with the fact, that the electron-donating halo-substituent at c4´ has the strong influence on decrease of the π-conjugation (andicsová-eckstein et al. 2017). the solutions of the bis(5,4-d)thiazoles 3a–c in dmf showed fluorescent (fl) emissions in the range of 450-470 nm when excited at the absorption maximum wavelengths and the colors were observed as yellow under a uv-lamp. for 3d, with br at c4´, the fluorescence is strongly quenched as typical behaviour of similar, halogensubstituted thiophene-based compounds (romiszewski et al. 2014). conclusions the synthesis and optical properties of a series of thiazolo[5,4-d]thiazoles bearing the substituted thiophene ring at the termini have been investigated. thiophene core in the target bis(5,4d)thiazoles 3a–d offers the possibility of subsequent functionalization according to suggested substitution pattern (fig. 2) as one of the key requisites in materials design. exchange of hydrogen to bromoor other halo-substituent seems to play an important role in blue-shifted uv-vis and hindered fluorescence. on the other hand, the alkyl-substitution along with position on which bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:43 utc nova biotechnol chim (2018) 17(2): 193-200 199 fig. 5. uv-vis spectra of 3a–d. position of the thiophene core is attached significantly influences the optical properties – i.e. red-shifted uv-vis with alkyl at c3´ no change in uv-vis with alkyl at c5´. accordingly, the substitution of the thiophene moiety in thiophenecontaining thiazolo[5,4-d]thiazole could be fig. 6. fl emission of 3a–c. recognized and further managed according to requirements on these compounds as building blocks for (opto)electronical materials (i.e. as monomers for copolymers of different nor ptype semiconductors, π-spacers or electron-withdrawing moieties for dye sensitised solar cells, ligands). acknowledgments the support by the ministry of education of the slovak republic – vega 1/0534/16 is appreciated. references andicsová-eckstein a, tokár k, kozma e, tokárová 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slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 11:43 utc nova biotechnol chim (2022) 21(1): e978 doi: 10.36547/nbc.978 1 nova biotechnologica et chimica lc-esi-ms/ms analysis, toxicity and anti-anaemic activity of rubia tinctorum l. aqueous extract fatima zohra houari1,, ramazan erenler2, sena bakir3,4, esra capanoglu3, ahmed hariri1 1laboratory of bioconversion, microbiology engineering and health safety, faculty of sciences of nature and life, mustapha stambouli university of mascara, sidi saïd 29000, algeria 2plant research laboratories, department of chemistry, faculty of arts and sciences, tokat gaziosmanpasa university, tokat, turkey 3department of food engineering, faculty of chemical and metallurgical engineering, istanbul technical university, maslak, istanbul 34469, turkey 4department of gastronomy and culinary arts, faculty of tourism, recep tayyip erdogan university, ardesen, rize 53400, turkey  corresponding author: fz.houari@univ-mascara.dz article info article history: received: 22nd may 2021 accepted: 8th october 2021 keywords: antianaemic phytochemical analysis rubiaceae toxic effect abstract the present study investigated the chemical profile, toxicity, and anti-anaemic activity of rubia tinctorum root aqueous extract against phenylhydrazine induced hemolytic anaemia. phenolic compounds were analyzed by lc-esi-ms/ms; acute toxicity test was evaluated by administering a single dose of 2,000 mg.kg-1 of the extract; anaemia was induced by administration of 40 mg phenylhydrazine by intraperitoneal injection for 2 days. moreover, the anti-anaemic activity was evaluated by measuring the haematological parameters of rats treated with iron and aqueous extract for 15 days. the lc-esi-ms/ms analysis results revealed the presence of 31 phytochemical compounds, among them, citric acid was found as the most abundant. no signs of toxicity or death were recorded, indicating that the ld50 of r. tinctorum root extract is higher than 2,000 mg.kg-1. furthermore, the aqueous extract increased red blood cell levels by 69.82 and 71.67 % in the groups treated with 200 and 400 mg.kg-1 of the extract, respectively. besides, a significant increase in hemoglobin from 12.05 ± 0.15 to 12.9 ± 0.52 g.dl-1 was noted in rats treated with 400 mg.kg-1 of extract. thus, the data indicate that the root extract could be considered a natural source for the treatment of anaemia. introduction nowadays, many people suffer from anaemia. it is a global public health phenomenon affecting developed and developing countries via severe consequences for human health and economic and social development (who 2008). the most exposed are infants, children in intensive growth, older adults, and pregnant women. it is thought to be responsible for 3.7 % to 12.8 % of maternal deaths during pregnancy and childbirth in africa and south asia (khan et al. 2006). several factors are responsible for the spread of anaemia: malnutrition, attacks by blood parasites such as trypanosomes, plasmodium, and helminths. it has also been reported that the high demand for the mailto:fz.houari@univ-mascara.dz nova biotechnol chim (2022) 21(2): e978 2 developing fetus during pregnancy is a factor in anaemia. there are many types of anaemia. one of them is hemolytic anaemia, the most frequent sort defined as the destruction or elimination of red blood cells (rbcs) from the circulation before their average life span of 120 days (dhaliwal et al. 2004). it is caused by the abnormality of the constituents of rbc (hemoglobin, an enzyme of energy metabolism of rbc or constitutive protein of the membrane-extrinsic factor). also by infectious agents, mechanical factors, toxins, etc. (loustau et al. 2011). since ancient times, medicinal plants have been considered essential products for the health of communities and individuals. they contain bioactive substances used in traditional medicine and as precursors in the synthesis of valuable drugs at the industrial level. interestingly, many plants including phyllanthus emblica l., moringa oleifera l., boerhavia diffusa l., hemidesmus indicus l., centella asiatica l., ageratum conyzoides l., hemidesmus indicus l., momordica charantia l., phyllunthus amarus l., punica granatum l., ocimum tenuiflorum l., solanum americanum l., ichnocarpus frutescens l. are used in the treatment of anaemia, as they are sources of iron and minerals (silja et al. 2008). rubia tinctorum belongs to the rubiaceae family, comprising 6,000 species (sharifzadeh et al. 2014). it is also known as “el foua”(ezzaki et al. 2021), growing in the northern regions of africa, the mediterranean regions of spain, and some areas of asia, and it is cultivated in the central and western regions of iran (sharifzadeh et al. 2014). rubia tinctorum has been widely used as a source of red color with its variants for textile dyeing for thousands of years (degano et al. 2009). in addition, several ethnobotanical studies have reported its use in the treatment of various diseases, such as diarrhea (el haouari and rosado 2016), rheumatism, cardiovascular diseases (jouad et al. 2001), and kidney stones (agarwal and varma 2015). moreover, it is really appreciated by moroccan women as a purgative after childbirth (ezzaki et al. 2021). besides, there are numerous biological studies on r. tinctorum that have demonstrated its therapeutic potential as antiplatelet agent, antitumor, hepatoprotective, antimicrobial, diuretic, and stones inhibitory activities (sharifzadeh et al. 2014; marhoume et al. 2019; eltamany et al. 2020; hoseinzadeh et al. 2020). to the best of our knowledge, no studies have been performed on the anti-anaemic activity of r. tinctorum extract so far. thus, this study aims to evaluate the anti-anaemic potential of r. tinctorum aqueous extract on an experimental model of phenylhydrazine-induced hemolytic anaemia and to identify its phytochemical compounds. experimental chemicals distilled water, ammonium formate, acetonitrile, formic acid, sulfuric acid (98 %), chloric acid, hydrochloric acid, ammonium thiocyanate, sodium chloride (9 %), phenylhydrazine, oroofer plus (bottle of 100 ml as a drinkable solution, iron iii poly maltose hydroxide, 20 mg. ml-1 of iron). plant material the rubia tinctorum plant was gathered in june 2018 in sidi bel abbes (north-west algeria). it was identified by dr. righi k, department of botany, university of mustapha stambouli, mascara, algeria. the plant material was shadedried in a room for 15 days and pulverized using an electrical grinder. a voucher specimen plant has been deposited at the herbarium of the sciences faculty, mustapha stambouli university (ham 02 74). preparation of aqueous root extract the aqueous extract of rubia tinctorum l. was prepared by maceration, using 10 g of powders of the root soaked in 100 ml of distilled water for 2 days with constant stirring. the mixture was filtered through whatman filter paper no. 1. thereafter the extract was freeze-dried. the obtained residue was maintained at 4 °c (bruneton 1999). nova biotechnol chim (2022) 21(1): e978 2 lc-esi-ms/ms analysis the aqueous root extract of rubia tinctorum was subjected to lc-esi-ms/ms analysis. the compounds were separated on an agilent poroshell 120 ec-c18 column in reverse phase (100 mm × 3.0 mm, 2.7 μm). the column temperature was fixed at 25 °c and the volume of injection was 5 μl. the mobile phase consisted of a linear gradient of acetonitrile -20 mm ammonium formate (ph 3.0) (15/100; v/v). the injection rate was 1 ml.min-1. the elution gradient was eluent a (water + 5mm ammonium formate) and eluent b (acetonitrile + 0.1 % formic acid), the solvent flow rate was set to 0.250 ml.min-1and the gradient was as follows: 1 min 40 % a – 60 % b; 2 min 70 % a – 30 % b; 3 min 70 % a – 30 % b; 4 min 40 % a – 60 % b; 5 min 10 % a – 90 % b. the agilent 6460 triple quadrupole mass spectrometer system model tandem mass spectrometer and the electrospray ionization source (esi) operating in negative and positive ionization modes were used. the conditions of the lc-esi-ms/ ms analysis were as follows: capillary temperature 350 °c, nebulizer n2 gas flow rate 15 l.min-1, fragmentor voltage 4,400 v. the collision energies (ce) were optimized to generate optimal phytochemical fragmentation and maximum transmission of the desired product ions. lc-ms/ms method validation analysis to quantify 37 phytochemicals (17 flavonoids, 15 phenolic acids, 3 non-phenolic organic acids, 1 phenolic aldehyde, and 1 benzopyrene) in rubia tinctorum extract, a comprehensive lc-ms/ms method was optimised and validated (yilmaz et al. 2018). spike and non-spike standard solutions were used to determine the performance characteristics of the method. the developed method has been validated in terms of linearity, inter-day and intra-day precision (repeatability), accuracy (recovery), limits of detection and quantification (lod/loq), and relative standard uncertainty (u % at 95 % confidence level (k = 2)). the parameters are listed in table 1. determination of na, ca, k in a glass flask, 3 g of plant powder (roots) was mixed with 8 ml of concentrated h2so4 (98 %) and 2 ml of hclo3 (60 %) for 24 h, all placed at a temperature of 80 °c using a sand bath until the digestion material became a white powder. after that, 8 ml of distilled water was added to the powder. finally, the mineral elements were measured by a flame atomic absorption spectrophotometer (nv202 spectrophotometer) (aboud 2010). determination of iron (fe) the iron was quantified spectrophotometrically by the thiocyanate method. for this, the sample was prepared using 5 g of the root powder, placed in a muffle furnace at 600 °c for 3 h until completely transformed into ashes. ashes were mixed with 1 m hydrochloric acid and 5 ml of distilled water and then filtered. thiocyanate solution was prepared using 38 g of ammonium thiocyanate (nh4scn), completed with distilled water to a final volume of 500 ml. for the standard curve, optical density was determined at 490 nm for five standard iron solutions (iron concentrations: 2, 4, 6, 8, 10 × 10 – 5 m) mixed by vortexing with 10 ml of thiocyanate solution. iron content in the root sample was determined using 10 ml of the sample placed in a dry test tube, then mixed with 10 ml of the prepared ammonium thiocyanate solution. the optical density of the mixture was read at 490 nm. iron content was deduced from the standard curve. the results were expressed in mg iron.100 g-1 of the sample (bhuvaneswari et al. 2015). experimental animals healthy male rats (aged 8 weeks, weighing 162 – 233 g) were used for acute and anti-anaemic studies. they were obtained from the animal house unit of the university of mustapha stambouli, mascara (algeria), and kept in the experiment room under normal environmental conditions with a temperature of 22 ± 3 °c, relative humidity of 30 – 70 %, and 12 h light/dark cycle, with free access to a regular diet and water. 3 nova biotechnol chim (2022) 21(2): e978 2 the experimental protocols were approved by the animal research ethics committee of the mascarian university, mustapha stambouli (arecm) according to the adelaide university animal ethics committee (ethics number m/76/98). acute toxicity test to assess the acute oral toxicity of rubia tinctorum aqueous extract, the protocol of the organization for economic cooperation and development (oecd 2002; kifayatullah et al. 2015) was used with slight modifications. the procedures were carried out in two stages. for each stage 3 wistar rats were used. twelve male rats weighing 170 ± 7.5 and 232 ± 7.02 g were randomly divided into two groups of 6 each. a single dose of aqueous extract was dissolved in distilled water at 2,000 mg.kg-1 body weight, and it was administered to the first group by oral gavage while the control group received only nacl 9 %. all animals were submitted to overnight fasting. the animals were under surveillance for the first 4 h after administration, and no food was given during this time. after that, they were observed for 14 days once a day for physical and behavioural changes. relative organ weight to determine the relative weight, on the 15th day of the experiment, the animals were sacrificed (anesthesia), the weights of the following organs were recorded in grams: liver, lungs, kidneys, heart, and spleen. relative organ weight was calculated as follow (eq. 1; kifayatullah et al. 2015): (organ weight/body weight of the rat (on the day of sacrifice)) × 100 % (1) the percentage change in body weight the percentage change in body weight of the experimental animals was calculated (eq. 2; desai and singh 2009): (final body weight-initial body weight / final body weight) × 100 (2) analysis of hematological parameter for toxicity tests on the 15th day, all the animals were sacrificed by anesthesia. the blood was collected from each rat into an edta tube by an abdominal puncture. blood parameters were assessed using an automatic counter (abacus 380). analysis of biochemical parameter biochemical analyses were performed on their serum after the centrifugation of the blood. the biochemical parameters, including aspartate transaminase (ast), alanine transaminase (alt), glycemia, urea, and creatinine were determined for the control groups and treated groups. all analyses were performed using a clinical chemistry analyzer (mindray ba-88a). anaemia test it was carried out according to the approach used by (pandey et al. 2016), with some modifications. anaemia was induced in rats by intraperitoneal injection of phenylhydrazine (phz) at 40 mg.kg-1 for 2 days. rats that presented anaemia with a hemoglobin concentration of less than 13 g.l-1 were used in the study (diallo et al. 2008). the anaemic rats were divided into five groups (6 animals per group) and treated daily for 15 days. all four groups of animals were treated with phenylhydrazine (phz) except group (i). the first group (control group) received (nacl (0.9); 10 ml.kg-1 body body weight). group ii (negative group) received (phz i.p. 40 mg.kg-1 body weight) once daily for 2 consecutive days + (nacl (0.9); 10 ml.kg-1 body weight). group iii (positive control) received (phz i.p. 40 mg.kg-1 body weight) once daily for 2 consecutive days + standard treatment (orofer plus). group iv received (phz i.p. 40 mg.kg-1 body weight) once daily for 2 consecutive days + extract (200 mg.kg-1). group v received (phz i.p. 40 mg.kg-1 body weight) once daily for 2 consecutive days + extract (400 mg.kg-1). all tested drugs were administered orally. 4 nova biotechnol chim (2022) 21(1): e978 2 analysis of hematological parameters for antianaemic activity tests the blood was collected from each rat into an edta tube by ocular puncture before the induction of anaemia (day 1); during the test (day 3); and at the end of the test (day 15), were evaluated for blood parameters using an automatic counter (abacus 380) (pandey et al. 2016). statistical analysis data analysis was carried out using spss ibm 23 statistical package. analysis of variance (anova) with fisher’s lsd tests and t-test were used. the data were presented as mean ± standard error, p<0.05 was considered statistically significant. results and discussion lc-esi-ms/ms analysis in the present study, aqueous extracts of rubia tinctorum roots were assessed for phytochemical composition, acute toxicity, and antianaemic activity thereof. the percentage extraction yield of the extracts was recorded as 18.8 %. the phytochemical profile of r. tinctorum root aqueous extracts investigated by lc-esi-ms/ms equipment report fig. 1 and table 1. lc-esi-ms/ms analysis of rubia tinctorum aquenos aqueous extract revealed the presence of many phytochemical compounds. the highest amount was attributed to citric acid (165.80 µg.mg1) followed by ascorbic acid (64.99 µg.mg-1), vanillic acid (52.88 µg.mg-1), epicatechin (50.78 µg.mg-1), and 4-hydroxybenzoic acid (49.93 µg.mg-1), and eleven other compounds with concentrations varying between 1.028 – 47.42 µg.mg-1. fig. 1. lc-esi-ms /ms chromatogram of rubia tinctorum aqueous extract. a – citric acid; b – 5-o-caffeoylquinic acid; c – 4hydroxyibenzoic acid; d – vanillic acid; e – trans-ferulic acid. although rubia tinctorum roots are widely used in traditional medicine (manojlovic et al. 2005), a few studies were found in the literature on the phytochemicals composition of aqueous extract thereof. according to the previous findings, a large variety of phenolic compounds were identified in the rubia tinctorum root. a study by aboud (2010) revealed the presence of anthocyanidins, chalcone, and kaempferol. similarly, a previous investigation using lc-esi-ms/ms reported the presence of further compounds that are munjistin, pseudopurpurin, rubiadin, ruberythric acid, pseudopurpurin, lucidin, primeveroside, and nordamnacanthal (lajkó et al. 2015). thus, comparing these results, the richness of r. tinctorum root in terms of phenolic compounds’ diversity can be confirmed. 5 nova biotechnol chim (2022) 21(2): e978 2 mineral composition of aqueous extract table 2. shows the mineral content of rubia tinctorum roots. the results revealed the macro mineral elements (sodium (na), potassium (k), calcium (ca), and micro mineral elements (iron (fe)) at varying concentrations. these results are consistent with those of aboud (2010), who indicated that the concentrations of sodium (na), potassium (k), and iron (fe) in the root of rubia tinctorum were higher, whereas calcium (ca) was found to be at the lowest concentration. table 2. mineral composition of rubia tinctorum root. the data present the mean value of three replicates ± sd. ppm – parts per million; na – sodium; k – potassium; ca – calcium and fe – iron. study of acute toxicity in the present study, the acute toxicity of the extract was investigated using a single dose of 2000 mg/kg/body weight of the extract on 12 rats. the mortality or morbidity was determined after 14 days. besides morbidity, behavioral changes such as respiration, temperature, convulsions, diarrhea, shaking, and itching were examined during the period of the test, as reported in table 3. the administration of a single dose of aqueous extract did not cause any sign of toxicity (morbidity or mortality) in all animals. similarly, no behavioral changes were observed in rats. on the other hand, our data showed no significant change (p>0.05) in the liver, kidney, heart, and pancreas weights in either treated or untreated rats after 15 days (table 4). besides, macroscopic observations of the organs displayed neither morphological alterations nor changes in their color texture. the effect of the aqueous extract of rubia tinctorum on haematological parameters is summarized in table 5. we observed an increase in the platelet count (plt), mean corpuscular hemoglobin concentration (mch), mean corpuscular volume (mcv) in treated rats (p>0.05). however, white blood cells (wbc), red blood cells (rbc), hemoglobin (hb), and lymphocytes (lmy) were significantly decreased. regarding the biochemical parameter levels, the results are shown in table 6. compared to the control group, no significant changes (p>0.05) in creatinine (crea), glutamate-pyruvate transaminase (tgp), triglycerides (triglyc) and cholesterol levels were observed. moreover, a significant decrease in urea and cholesterol was detected (p<0.05). according to our results, dl50 is superior to 2,000 mg.kg-1. a single dose of 2,000 mg.kg-1 did not produce any visible signs or symptoms of toxicity in all treated animals. in addition, we observed no changes in the rat’s behavior, no toxic symptoms, no changes in hematological parameters, and no deaths. therefore, based on the ocde method of acute toxicity, the rubia tinctorum aqueous extract can be considered a non-toxic substrate (oecd 2002). table 3. clinical signs in acute oral toxicity study of rubia tinctorum aqueous extract in wistar rats exposed for a dose 2,000 mg.kg-1. observation control group treated group temperature food intake rate of respiration change in skin eye color diarrhea general physique coma death drowsiness nr nr nr no nr np nr no no np nr nr nr no nr np nr no no np notes: nr – normal; no – not observed; np – not present. mineral component na k ca fe content. ppm 0.115 ± 0.01 1.68 ± 0.01 0.043 ± 0.02 3.32 ± 0.07 6 nova biotechnol chim (2022) 21(1): e978 2 table 1. rubia tinctorum l. roots aqueous extracts lc-esi-ms/ms analysis. compound rt ion transitions [m/z] ion. mode r2 dw [µg.mg-1] linearity range [μg. l-1] lod [μg.l-1] loq [μg.l-1] recovery [%] tartaric acid 1.696 149-87 negative 0.999 nd 62.5-2,000 12.309 37.3 100.7 citric acid 1.793 191.1-111 negative 0.999 165.8091 125-2,000 6.237 18.9 100.55 ascorbic acid 1.802 175.1-114.9 negative 0.999 64.9996 62.5-2,000 7.75 23.5 99.6 fumaric acid 1.821 115-71.1 negative 0.999 16.0948 31.25-2,000 6.43 19.5 100.77 maleic acid 1.821 115-71.2 negative 0.999 nd 62.5-2,000 6 18.2 99.8 chicoric acid 1.989 472.8-310.5 negative 0.999 31.7746 250-2,000 50.16 152 89.1 gallic acid 2.605 169-125 negative 0.998 nd 62.5-2,000 9 54.6 98.9 5-o-caffeoylquinic acid 5.526 353-191 negative 0.999 47.4291 250-2,000 64.68 196 86.2 4-hydroxyibenzoic acid 6.531 137-93.1 negative 0.999 49.9302 62.5-2,000 2.376 7.2 100.7 catechin 6.660 288.9-245.1 negative 0.999 31.4229 62.5-2,000 2.57 7.8 100 epicatechin 6.666 353-191 positive 0.998 50.7805 62.5-2,000 2.9 8.8 100.6 hesperidin 6.674 611.3-357 positive 0.999 nd 125-2,000 32.67 99 99.5 rutin 6.675 608.9-299.4 negative 0.997 nd 125-2,000 28.5 85 97.8 vanillic acid 6.687 167-151.8 negative 0.998 52.8816 62.5-2,000 2.54 7.7 100 syringic acid 6.703 197.1-181.8 negative 0.999 nd 62.5-2,000 4.22 12.8 100.5 caffeic acid 6.703 178.9-135.1 negative 0.999 1.0283 125-2,000 25.74 78 99.7 luteolin -7-glucoside 6.740 449-286.9 positive 0.997 nd 62.5-1,000 16.5 50 100.6 apigenin-7-o-glucoside 6.808 430.8-267.4 negative 0.998 nd 125-1,000 18.24 55.3 100.8 quercetin-3-glucoside 6.816 432.7-299.5 negative 0.995 nd 62.5-2,000 9.87 29.9 100.1 oleuropein 6.849 539.1-275.1 negative 0.999 nd 62.5-2,000 17.35 52.6 101.9 rosmarinic acid 6.875 358.9-160.7 negative 0.998 27.9655 62.5-2,000 15.9 48.2 100.6 p-coumaric acid 6.919 163-119 negative 0.999 10.2367 62.5-2,000 3 9.1 100.3 4hydroxybenzaldehyd 6.929 121-92 negative 0.999 10.79 31.25-2,000 1.91 5.7 99.9 trans-ferulic acid 6.968 193.1-133.9 negative 0.998 51.0571 31.25-2,000 7.26 22.3 100.3 gentisic acid 7.243 153-109 negative 0.999 14.1623 250-2,000 44.55 135 99.9 protocatechuic acid 7.243 152.9-108.9 negative 0.999 30.2216 62.5-2,000 15.44 46.8 100.4 quercetin 7.306 300.7-150.9 negative 0.997 nd 62.5-1,000 14.85 45 98.8 apigenin 7.555 269-117 negative 0.999 nd 125-2,000 17.82 54 101.2 naringenin 7.588 270.9-119.1 negative 0.999 nd 125-2,000 24.37 73.8 101.2 trans-cinnamic acid 7.591 148.8-104.8 negative 0.999 26.1761 62.5-2,000 13.59 41.2 102 kaempferol 7.613 284.9-116.9 negative 0.998 nd 62.5-2,000 12.39 37.5 101.1 rt – retention time, fc – final concentration, nd – not detected, r2 – coefficient of determination, rsd –relative standard deviation, lod/loq (μg.l-1) – limit of detection/quantification, dw – dry weight. 7 nova biotechnol chim (2022) 21(1): e978 2 table 4. effects of the aqueous extract of rubia tinctorum on the relative weight of organs and relative body weight in rats during acute toxicity study. group relative organ weight [%] relative body weight. [%] heart liver spleen kidney lung temoin er 0.31 ± 0.02 0.376 ± 0.04 3.16 ± 0.44 3.606 ± 0.39 0.32 ± 0.03 0.35 ± 0.06 0.63 ± 0.04 0.608 ± 0.04 0.633 ± 0.03 0.733 ± 0.12 4.88 13.16 the data present the mean value of three replicates ± sd. results were analysed by standard t-test, treated group were compared to the control group, p>0.05. er – group treated with aqueous extract of rubia tinctorum. these results are in agreement with those of marhoume et al. (2019) who found that the butanolic extract had no toxic effects on treated rats. moreover, it was consistent with the study of karim et al. (2010), reporting that the tolerated dose of aqueous extract of rubia tinctorum was up to 10 g.kg-1 body weight in albino mice, and no mortality and toxic symptoms were observed. table 5. effects of rubia tinctorum aqueous extract on haematological parameter in rats during 14 d of oral acute toxicity study. haematological parameters control group treated group (2,000 mg. ml-1) wbc /103 μl-1 lym /103 μl-1 rbc/ 106 μl-1 hb / g.dl-1 ccmh /g. dl-1 plt /103 μl-1 tcmh / pg pcv / % 12.2466 ± 1.30 7.965 ± 0.47 9.263 ± 0.30 17.06 ± 1.19 33.9 ± 0.78 625.33 ± 108.7 18.433 ± 0.83 54.33 ± 3.05 9.43 ± 1.09 6.3433 ± 1.62 7.26 ± 0.68 14.166 ± 1.15 33.9 ± 0.78 652 ± 37 19.5 ± 0.26 57.333 ± 0.57 the data presents the mean value of three replicates ± sd. results were analysed by standard t-test, the treated group was compared to the control group, p>0.05. rbc – red blood cells; wbc – white blood cells; hb – hemoglobin; lmy – lymphocytes; plt – platelet count; ccmh – mean corpuscular hemoglobin concentration in red blood cells; tcmh – mean corpuscular hemoglobin content in hematite; pcv – packed cell volume. table 6. effects of rubia tinctorum aqueous extract on biochemical parameter in rats during 14 d of oral acute toxicity study. biochemical parameter control group treated group (2,000 mg. ml-1) uree crea tgo tgp cholesterol triglyceride 0.56 ± 0.01 4.76 ± 0.30 66.69 ± 20.81 32.3 ± 3.39 0.66 ± 0.05 0.4 ± 0.06 0.386 ± 0.03 4.7 ± 0.26 54.58 ± 13.65 32.2 ± 6.89 0.48 ± 0.10 0.423 ± 0.14 the data presents the mean value of three replicates ± sd. results were analysed by standard t-test, treated group were compared to the control group; p>0.05. crea – creatinine; tgp – glutamate-pyruvate transaminase; tgo – aspartateamino transferase. anti-anaemic activity the effect of acute administration of root aqueous extract on haematological parameters is shown in tables 7, 8, and 9. the haematological parameters were measured before the treatment (d0), after phenylhydrazine-induced anemia (d2), and up to 15 days. as stated in table 8, the treatment with phenylhydrazine decreased rbc, hb, ht, mch, mcv levels and increased mchc level compared to the control group. table 7. estimation of haematological parameter before induction of the anaemia. hematological parameter gr(i) gr(ii) gr(iii) gr (iv) gr(v) rbc /103 μl-1 8.26 ± 0.3** 7 ± 0.03*** 8.76 ± 0.25*** 8.23 ± 0.078*** 7.84 ± 0.03*** hb / g.dl-1 17.6 ± 1.19** 15.7 ± 0.43*** 17.9 ± 0.31 16.3 ± 0.11*** 16.3 ± 0.06*** ht / % 45.83 ± 2.30*** 43.28 ± 0.22*** 50.96 ± 0.14*** 47.99 ± 0.35*** 46.38 ± 0.03 ccmh /g. dl-1 34.3 ± 0.72*** 33.8 ± 0.02*** 35.2 ± 0.32*** 33.8 ± 0. 27*** 35.1 ± 2.23 *** mch /fl 17.7 ± 0.64*** 19.5 ± 0.02 35.2 ± 0.32*** 33.8 ± 0. 27*** 35.1 ± 2.23 *** pcv / % 40.59 ± 0.12*** 43.28 ± 0.03*** 50.96 ± 0.71 47.99 ± 0.001*** 46.38 ± 0.04*** *p0.05; **p0.01; ***p0.001, significantly different from the control group. results were analyzed by anova test. values expressed are means ± sd (n = 3). rbc – red blood cells; hb – hemoglobin; pcv– packed cell volume; ht – haematocrit; ccmh – mean corpuscular hemoglobin concentration; mch – mean corpuscular hemoglobin. 8 nova biotechnol chim (2022) 21(2): e978 2 table 8. estimation of haematological parameter after induction of anaemia. hematological parameter gr(i) gr(ii) gr(iii) gr(iv) gr(v) rbc /103 μl-1 8.32 ± 0.51* 4.25 ± 0.035*** 4.66 ± 0.04*** 4.72 ± 0.21*** 4.63 ± 0.02*** hb g.dl 1 17.78 ± 0.26* 11.35 ± 0.35*** 11.1 ± 0.21 12.05 ± 0.15*** 12.85 ± 0.07*** ht / % 45.88 ± 0.33*** 35.98 ± 2.26*** 38.01 ± 0.33*** 36.58 ± 0.35 38.12 ± 0.02 ccmh /g. dl 1 33.9 ± 0.47*** 39.85 ± 0.49 40 ± 0.21*** 52.8 ± 3.45*** 37.35 ± 3.04*** mch /fl 18.81 ± 0.51*** 17.8 ± 1.41*** 18.6 ± 0.49 17.4 ± 0.3*** 18.15 ± 0.07 pcv / % 41.33 ± 2.4*** 33.48 ± 5.79*** 39.25 ± 2.16 32.03 ± 5.86*** 42.62 ± 3.55 *p0.05; **p0.01; ***p0.001, significantly different from the control group. results were analyzed by anova test. values expressed are means ± sd (n = 3). rbc – red blood cells; hb – hemoglobin; pcv– packed cell volume; ht – haematocrit; ccmh – mean corpuscular hemoglobin concentration; mch – mean corpuscular hemoglobin. however, iron and root aqueous extract administration significantly raised the levels of rbc, ht, hb, mch, and pcv (p<0.001) (table 9). indeed, iron treatment exhibited strong improvement in the rbc levels (from 4.66 ± 0.04 103 μl-1 to 6.96 ± 1.25 103 μl-1). similarly, the aqueous extract enhanced rbc levels in a dosedependent manner, by 69.82 and 71.67 % for gr iv and gr v, respectively. moreover, an increase of hemoglobin (hb) and hematocrit (ht) in treated groups (iii, iv, and v) was noted as compared to the anemic rat group (gr ii). our results revealed that mchc increased considerably in all groups after treatment with phenylhydrazine. however, aqueous extract and iron resulted in a decrease of mchc level from 38.01 ± 0.33, 32.09 ± 1.88, 38.12 ± 0.02 g. dl-1 to 30.55 ± 2.89, 31.3 ± 0.92, 32.86 ± 0.65 g.dl-1 in gr iii, gr iv and gr v, respectively. our results are similar to those of pandey et al. (2016) who reported a significant reduction in haematological parameters in phz-injected rats compared to a control group. this result is related to the toxicity of phenylhydrazine. indeed, the administration of phenylhydrazine into the bloodstream causes oxidative stress in erythrocytes, membrane disruption, and embrittlement as well as hemolysis which triggers events such as premature ageing of erythrocytes, resulting in the lack of hemoglobin and circulating erythrocyte (ogbe et al. 2010; marhoume et al. 2019). table 9. estimation of haematological parameter after treatment. haematological parameter gr(i) gr(ii) gr(iii) gr (iv) gr(v) rbc /103 μl-1 8.66 ± 0.03* 6.07 ± 0.30*** 6.96 ±1.25*** 6. 76 ± 0.35*** 6.46 ± 0.17*** hb / g.dl-1 17.89 ± 0.41** 11.95 ± 0.21*** 13.25 ± 0.77* 12.66 ± 0.69*** 12.9 ± 0.52*** ht / % 46.02 ± 1.45** 35.28 ± 0.86*** 39.6 ± 1.27* 32.09 ± 1.88*** 38.13 ± 1.70*** ccmh g.dl-1 33.97 ± 0.85*** 32.2 ± 0.28*** 30.55 ± 2.89*** 31.3 ± 0.92** 32.86 ± 0.65 mch / fl-1 18.98 ± 0.10*** 19.15 ± 0.07* 18.35 ± 1.90** 18.43 ± 0.61*** 20.63 ± 2.08** pcv/ % 41.56 ± 0.78*** 33.28 ± 0.86*** 42.6 ± 1.27 34.09 ± 2.30*** 43.13 ± 1.70** *p0.05; **p0.01; ***p0.001, significantly different from the control group. results were analyzed by anova test. values expressed are means ± sd (n = 3). rbc – red blood cells; hb – hemoglobin; pcv– packed cell volume; ht – haematocrit; ccmh – mean corpuscular hemoglobin concentration; mch – mean corpuscular hemoglobin. as displayed in table 9, the anaemia was restored by daily oral administration of rubia tinctorum aqueous extract (200, 400 mg /kg/body weight) and iron for 15 days. to the best of our knowledge, this is the first study revealing the anti-anaemic activity of the rubia tinctorum plant, thus rendering the comparison difficult. there are several possible explanations for this finding. the first one is that it might be due to its composition of polyphenols. as indicated in table 2, most of the compounds detected and identified have been previously reported in the literature for their antioxidant and/or anti-inflammatory bioactivity. ogbe et al. (2010) reported that secondary metabolites like flavonoids and alkaloids repair free radical damage to red blood cells and protect them from oxidative stress. anthocyanins have been used in strengthening kidney function, 9 nova biotechnol chim (2022) 21(2): e978 2 treating anaemia, promoting blood circulation, and eliminating blood stasis in traditional chinese medicine (sari et al. 2019). moreover, innih et al. (2020) reported that the aqueous leaf extract of spondias mombin caused stimulation of the lymphoid follicle at different degrees, from mild to moderate and increased the number of red blood cells in the red pulp. koriem et al. (2018) observed that oral administration of 5-o-caffeoylquinic acid for two-week protects against anaemia and mineral disturbances in 4-tert-octylphenol toxicity by enhancing oxidative stress and apoptosis in rats. furthermore, the return of haematological indices in the treated group to normal ranges are not necessarily related to the reported phytochemical constituents (ohadoma 2016), which brings us to mineral elements. in fact, musyoka et al. (2016) reported that copper and iron have synergetic effects promoting hematopoiesis. moerever, sheth et al. (2021) pointed out in their study that the antianaemic activity of raktavardhak kadha can be attributed to its iron content and it may prevent hemolytic anaemia induced by phenylhydrazine. thus, it is not surprising to find a positive antianaemic effect in our study, as iron is the most abundant element in our plant. besides, the antianaemic effect of rubia tinctorum could be due to the presence of vitamins such as ascorbic acid or organic acids. many authors have demonstrated that vitamin b6, vitamin b12, vitamin c, vitamin e, and folic acid play an important role in the erythropoietic mechanism, especially in the presence of iron, copper, and other elements such as cobalt (musyoka et al. 2016). a study performed by zhang et al. (2020) on the effects of malic and citric acids on growth performance, antioxidant capacity, hematology, and immune response of carassius auratus gibelio showed that the appropriate addition of citric acid and malic acid to the diet regulated hematological parameters and the expression of immune-related genes and improved the antioxidant capacity of carassius auratus gibelio fish. similarly, in a previous study (salovaara et al. 2002), it was suggested that organic acids improve iron absorption. the researchers studied the effect of tartaric, malic, succinic, citric and oxalic, and fumaric acids on fe (ii) and fe (iii) uptake in the human epithelial cell line caco-2. the results showed that tartaric, malic, succinic, and fumaric acids increased the uptake of fe (ii) and fe (iii), and citric and oxalic acids increased the fe (iii) uptake. based on these results, it can be stated that the presence of organic acids in our plant may be one of the factors that contributed to the antianaemic activity. furthermore, tang et al. (2013) found that citric acid and l-malic acid have protective effects on myocardial ischemia/reperfusion injury, which may be associated with their anti-inflammatory, antiplatelet aggregation, and direct protective effects on cardiomyocytes. in summary, these findings showed remarkable antianaemic effects. it constitutes a scientific basis justifying the traditional use of rubia tinctorum in anaemic disease. on the other hand, in future studies, it will be better to measure also the ferritin and serum vitamin levels which are important markers in anaemia diagnostic. conclusion this is the first report on the antianaemic activity of the aqueous extract obtained from the roots of rubia tinctorum. based on these results, it can be concluded that the ld50 of the aqueous extract of rubia tinctorum roots was much above 2,000 mg.kg-1 and that oral administration of this extract up to 400 mg.kg-1 is safe for nutrious and therapeutic uses. furthermore, it can be suggested that the root of rubia tinctorum l. has an antianaemic effect, inhibiting the hemolysis of red blood cells. the antianaemic activity may be due to the composition of the extract contents including polyphenols, iron, and other non-identified molecules. therefore, rubia tinctorum extract could be a promising treatment for anaemia. further studies are required to understand the mechanism involved in the anti-anaemic action of rubia tinctorum and to identify active constituents of the plant. acknowledgments the authors would like to thank the university mustapha stambouli of mascara (department of biology and technology), mr. ahmed belaouni, mr. yahia khelef, ms. 10 nova biotechnol chim (2022) 21(1): e978 2 faiza fedoul, ms. sara mechaala, ms. aicha boutaleb for their help during this research. conflict of interest the authors declare that they have no conflict of interest. reference aboud as (2010) hplc analysis of rubia tinctorum and its effect of methanol and aqueous extract on bacteria isolated from 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global database on anaemia, who, geneva, switzerland. yilmaz ma, ertas a, yener i, akdeniz m, cakir o, altun m, temel h (2018). a comprehensive lc–ms/ms method validation for the quantitative investigation of 37 fingerprint phytochemicals in achillea species: a detailed examination of a. coarctata and a. monocephala. j. pharm. biomedical. 154: 413-424. zhang l, zhang p, xia c, cheng y, guo x, li y (2020) effects of malic acid and citric acid on growth performance, antioxidant capacity, haematology and immune response of carassius auratus gibelio. aquacult. res. 51: 2766-2776. 12 nova biotechnol chim (2020) 19(2): 223-231 doi: 10.36547/nbc.v19i2.777  corresponding author: catherineadeseko@gmail.com nova biotechnologica et chimica purification and biochemical characterization of polyphenol oxidase from seeds of melon (citrullus colocynthis) david morakinyo sanni, catherine joke adeseko and samuel olufemi bamidele enzyme and biotechnology unit, department of biochemistry, federal university of technology, p.m. b 704, akure, ondo state, nigeria article info article history: received: 17th august 2020 accepted: 20th october 2020 keywords: activity cucurbitaceae molecular weight physico-chemical properties substrates abstract polyphenol oxidase (ppo) is an enzyme that is responsible for the enzymatic browning of fruits and vegetables. this is generally undesired process and need to be prevented in food technology. ppo from seeds of citrullus colocynthis was purified, the physicochemical properties such as effects of ph and temperature, substrate specificity, effects of inhibitors and cations on ppo activity and the kinetic parameters for four substrates namely, catechol, l-dopa, gallic acid and tyrosine, were determined. the purification steps resulted in 41-fold with 10 % yield, and the optima ph and temperature values for ppo from c. colocynthis were found to be ph 7.0 and 60 °c, respectively using catechol as substrate. about 9 % enzyme initial activity was retained after 60 min of incubation at 80 °c, and the apparent molecular weight was determined as 42 kda by partially denaturing sds-page. ppo activity was inhibited by ascorbic acid, sds and certain divalent (ca2+, zn2+, mg2+ and, fe2+) and monovalent (na+) metal. moreover, purified enzyme solution showed diphenolase activity toward catechol, gallic acid, l-dopa and monophenolase activity toward tyrosine, therefore, tyrosinase was identified as the only one ppo in c. colocynthis seeds. this study revealed the use of temperature above 80 °c to inhibit ppo activity during processing and storage of melon seeds.  university of ss. cyril and methodius in trnava introduction cucurbits are edible crops found in the cucurbitaceae family, they are distributed in the tropical and subtropical countries. a variety of plant belonging to this family with a lot o f nutritional benefits is citrullus colocynthis (l.) (benmoumou and madidi 2019). the seeds of c. colocynthis contain proteins, essential oils, mineral, dietary fibre and other nutritionally important components that could be harnessed as alternatives for human diet (kumar et al. 2008). c. colocynthis seeds can be obtained either in shelled or unshelled form in west african markets and are used greatly in cookery. the seed of the c. colocynthis is whitish in color, with oval-flat shape, consisting edible fatty acids such as linoleic and oleic acids (teixeira da silva and hussain 2017). c. colocynthis seeds are part of the condiments used in the preparation of sauces consumed in most african nations, they are consumed crushed or grilled and served to thickening sauces and sometimes make into a cake for its delicacy (benmoumou and madidi 2019). similar to other commercially-available crops, dehulled c. colocynthis is prone to browning due to post-harvest effect. in the course of dehulling and crushing or making into powder, mailto:catherineadeseko@gmail.com nova biotechnol chim (2020) 19(2): 223-231 224 the color changes from whitish to greyish, making it appears dull, and eventually turns brown, thereby loses its acceptability for market value. fruits, vegetables and seeds are susceptible to enzymatic browning, which is typically catalyzed by polyphenol oxidase (ppo) (liu et al. 2007; guven et al. 2017; adeseko et al. 2019). ppos catalyse monoand o-diphenol conversion to o-quinones during ripening, postharvest handling, storage and processing of fruits and seeds (spagna et al. 2005; sélles-marchart et al. 2006). enzymatic browning is caused by the oxidation of phenolic compounds to quinones and their eventual (non-enzyme-catalysed) polymerization to melanin pigments (jiang et al. 2003). the evidence of ppo is proved by the brownish colouration found in the tissue of numbers of fruits and vegetables, which eventually leads to discoloration observed in many plant food materials (yang et al. 2004; núñezdelicado et al. 2005). oxidative browning reactions in many foods of plant origin, generally cause deterioration in food quality by changing structural, nutritional and organoleptic properties and these reactions significantly diminish consumer acceptance, thereby reducing the economic values (dincer et al. 2003). the aim of this study was to isolate and purify ppo from the seeds of c. colocynthis and investigate its intrinsic physicochemical properties, in order to ameliorate the adverse browning often experience during processing and storage of melon seeds, for consumer acceptability enhancement. experimental sample preparation fully matured fruits of c. colocynthis (egusi maga) were purchased from a local market in akure (ondo state, nigeria) and identified at department of crop science and pest, school of agriculture, federal university of technology (akure, ondo state, nigeria). the seeds were removed, air dried for two weeks and later dehulled manually. preparation of crude enzyme extract melon seeds (400 g) of c. colocynthis were thoroughly homogenized in 1.2 l of ice cold 25 mm phosphate buffer (ph 6.8) containing 10 mm ascorbic acid using a warring blender. the homogenate was filtered using four layers of cheese cloth. the filtrate was again filtered using layers of glass wool to remove the floating lipid, followed by centrifugation in a centrifuge at 16,000 rpm for 30 min at 4 °c. the supernatant was kept at -4 °c for 45 min. the lipid layer was separated. the supernatant was stored in a refrigerator and used as crude enzyme for further experiments. determination of protein concentration protein concentration was determined according to the method described by lowry et al. (1951) using bovine serum albumin (bsa) as a standard. determination of ppo activity ppo activity was determined with a spectrophotometer by measuring an increase in absorbance at 420 nm at room temperature using catechol as substrate as described by mayer (2006) with a slight modification. the reaction mixture consisted 0.2 ml freshly prepared enzyme solution and 2.8 ml of 10 mm catechol in 20 mm potassium phosphate buffer (ph 6.8) while the blank contained the buffer and the substrate. one unit (u) of ppo activity was defined as the amount of the enzyme that increased the absorbance by 0.001 per min. purification of ppo from c. colocynthis the crude enzyme (250 ml) was brought to 80 % ammonium sulfate saturation, the precipitate was collected by centrifugation at 16,000 rpm for 10 min. the precipitate was dissolved in 5 ml of 0.1 m potassium phosphate buffer (ph 6.8) and dialyzed with the same buffer at 4 °c overnight. the dialysate was placed on a deae-a50 sephadex column (3.5 × 13 cm). the column was pre-equilibrated with 0.1 m potassium phosphate buffer (ph 6.8) and the protein was eluted using the same buffer (flow rate: 10 ml.h-1). the unbound proteins were eluted by a linear gradient of 0 to 0.5 m nacl in 0.1 m potassium phosphate buffer (ph 6.8). the absorbance of the fraction was nova biotechnol chim (2020) 19(2): 223-231 225 monitored at 280 nm and fractions were assayed for ppo activity at 420 nm using catechol as substrate. the fractions with ppo activity were pooled and concentrated using 4 m sucrose. gel filtration of the concentrated peak fractions exhibiting ppo activity was carried out on a sephadex g-200 column (1.4 × 75 cm; flow rate: 5 ml.h-1) using the same buffer. absorbance of each fraction was taking at 280 nm while fractions exhibited enzyme activity were pooled together, concentrated and an aliquot was used for sdspage. determination of molecular weight of ppo the molecular weight of the purified ppo was determined by sds-page using 10 % gel according to laemmli et al. (1970) with standard protein markers (17 – 103 kda) and were stained with coomassie brilliant blue. determination of c. colocynthis ppo substrate specificity four different substrates (catechol, gallic acid, l-dopa, and tyrosine) at 10 mm concentration were prepared in 0.1 m phosphate buffer (ph 6.8). ppo activity was determined according to the standard assay procedure at corresponding wavelength 420 nm (catechol), 270 nm (gallic acid), 475 nm (l-dopa) and 300 nm (tyrosine). effect of ph on ppo activity in presence and absence of sds the enzyme ph optimum was determined with and without sds according to the method of sanni (2016) using various buffers at 0.1 m ph ranges from 2.0 – 9.0. the reaction mixture contained glycine-naoh buffer (ph 2.0 – 3.0); 0.1 m sodium acetate buffer (ph 4.0 – 5.0); 0.1 m potassium phosphate buffer (ph 6.0 – 7.0) and 0.1 m trishcl buffer (ph 8.0 – 9.0) in the presence and absence of 0.69 mm sds. enzymatic activity was determined according to the standard assay procedure. effect of ph on stability and activity of ppo the ph stability of the purified enzyme was determined according to the method of sanni (2016), by preparing various buffers of ph 2.0 – 9.0 using 0.1 m glycine naoh (ph 2.0 – 3.0), 0.1 m sodium acetate buffer (ph 4.0 – 5.0), 0.1 m potassium phosphate buffer (ph 6.0 – 7.0) and 0.1 m tris-hcl buffer (ph 8.0 – 9.0), and then incubating the purified enzyme with each specified buffer solution for 6 h. the residual activity was determined by drawing 1 ml of aliquot enzyme at one-hour interval subsequently after initial 0-hour activity according to standard assay method. effect of temperature on stability and activity of ppo the effect of temperature on ppo activity was investigated by varying the temperature conditions between 30 – 80 °c. the reacting mixture consisted purified enzyme and catechol was incubated at the stated temperatures while 1 ml of aliquot enzyme was withdrawn at an interval of 10 °c after 10 min of incubation. the activity was determined according to the standard assay procedure. the thermal stability was determined by incubating the enzyme at different temperature conditions: 30 – 80 oc. the initial activity was determined at the 0 min while the residual ppo activity was determined at 10-min interval for each temperature for 1 h according to the standard assay procedure. kinetic parameters of c. colocynthis ppo the kinetic constants, km and vmax, of the purified enzyme was determined using lineweaver-burk plot with catechol, gallic acid, tyrosine, and l-dopa as substrates, at varying concentrations (5 – 40 mm) in 0.1 m potassium phosphate buffer (ph 6.8). effect of inhibitors on c. colocynthis ppo activity ppo activity was determined in the presence of ascorbic acid, edta, urea and sds (5, 10 and 20 mm). the reaction mixture was incubated for 20 min and the change in absorbance was measured by spectrophotometer at 420 nm. control tests for inhibitors plus substrate plus buffer were also run at the same time. nova biotechnol chim (2020) 19(2): 223-231 226 table 1. purification of ppo from c. colocynthis. step total volume [ml] protein concentration [mg.ml-1] total protein [mg] activity [u.ml-1] total activity [u] specific activity [u.mg-1] purification fold yield [%] crude enzyme 1120 7.40 8,288 0.8 896 0.1 1 100 [nh4]2so4 precipitation 85 6.50 553 2.0 170 0.3 2.8 19 deae sephadex a-50 40 2.75 100 2.8 112 1.1 10 13 sephadex g-200 15 1.25 18.8 5.7 85.5 4.6 41 10 total protein (mg) = protein concentration (mg.ml-1) × total volume (ml); total activity (u) = activity in the fraction (u.ml-1) × total volume (ml); specific activity (u.mg-1) = total activity (u)/total protein (mg); yield (%) = (total activity of purified step/total activity of the crude) × 100 purification fold = (specific activity of purified step/specific activity of the crude). effect of cations on ppo activity the effect of cations on ppo activity was determined using cu2+, mg2+, fe2+ and zn2+ salts at concentration of 5, 10, and 20 mm respectively dissolved in catechol solution containing 0.1 mm phosphate buffer (ph 6.8) for 10 min. purified enzyme solution, 0.1 ml was added with 2.9 ml of the mixture of each metal ion and buffer solution were incubated for 20 min while the enzyme activity was determined according to the standard assay procedure. results and discussion isolation and purification of c. colocynthis ppo ppo from c. colocynthis was purified using ammonium sulfate precipitation, ion-exchange and size exclusion chromatography. the specific activity of the purified enzyme was 4.6 u.mg-1; a 41-fold purification of the enzyme was achieved fig. 1. sds-page of the purified ppo fraction after gel filtration. lane a is the molecular weight of the purified ppo from c. colocynthis and lane b represents molecular weight of protein markers. with 10 % yield. the summary of the purification procedure is given in table 1. page in the presence of sds produced a single protein band as represented in fig. 1. the subunit molecular weight of the purified enzyme was estimated as 42 kda by sds-page, this result is consistent with a previous report from marques et al. (1995), whose studies on apple ppo for the native (42 kda) and proteolyzed (27 kda) forms detected under partially denaturing conditions were found to have molecular weights of 64 and 42 kda, respectively. studies on broad bean ppo also showed that the 45 kda was obtained as the molecular weight under partially denaturing conditions (cary et al. 1992; robinson and dry 1992). in general, molecular weights of ppos vary significantly from source. the isoforms of ppos from many plant sources were reported to range in molecular mass from 32 to over 200 kda, mostly within the range of 35 – 70 kda (flurkey 1986; sherman et al. 1991; steffens et al. 1994; fraignier et al. 1995; van gelder et al. 1997; yang et al. 2000). physicochemical and kinetic parameters of ppo the ph optimum was observed at neutral ph 7.0 while about 14.3 – 28.6 % relative activity was observed at acidic region, ph 2.0 – 5.0 (fig. 2). the ph optimum of ppo from plants also varies depending on the plant source. yoruk and marshall (2003) reported that ph optimum varies widely with plant source but is generally in the range of 4.0 – 8.0. the influence of ph in presence of sds on ppo activity is summarized in fig. 3, the observed deactivation of c. colocynthis ppo by low nova biotechnol chim (2020) 19(2): 223-231 227 fig. 2. effect of ph on the activity of purified ppo. data represent mean ± std (n = 3). concentration of sds with ph is in agreement with the generally reported activation of ppo by extreme low concentration of sds (escribano et al. 1997). kenten (1957) has reported the activation of crude bean leaf ppo by sds below 1 mm sds. though some authors (moore and flurkey 1990; jimenez and garcia-carmona 1996; escribano et al. 1997; laveda et al. 2000) revealed from their experiments, the joint effects of ph and sds on ppo activity that the detergent causes a shift in ph optimum of the enzyme from low to higher ph values but their reports is at variance with the same optimum ph observed in the presence and absence of sds in this study. however, this behavior of a shift in ph does not seem ubiquitous as similar ph optimum profiles with and without sds were obtained for latent potato leaf ppo (sanchez-ferrer et al. 1993). subjecting c. colocynthis seeds to acidic medium could possibly control its browning effect owing to inhibition of ppo activity at this said ph, thereby increasing its quality. table 2. stability of ph of ppo from c. colocynthis. ph residual activity [%] 2 17.4±2.5 3 23.2±4.4 4 33.2±8.7 5 43.5±2.5 6 46.0±9.1 7 69.6±8.6 8 37.9±5.0 9 17.4±5.0 data represent the mean ± standard deviation of replicate readings (n = 3). fig. 3. effect of ph on the activity of purified ppo in presence (diamonds) and absence (squares) of sds. data represent mean ± std (n = 3). the results of effect of ph on the stability of the purified ppo is presented in table 2; there was a drastic reduction in enzyme activity at acidic phs. the enzyme was able to retain about 17.0, 23.2, 33.2, 43.5, 46.0, and 69.6 % residual activity for ph 2 – 7 respectively. however, at ph 7.0, a high percentage relative activity of about 70 % was observed, while about 37 and 17 % residual activities were observed for ph 8.0 and 9.0, respectively. the influence of temperature on ppo activity and stability are presented in fig. 4 and table 3 respectively. however, an optimum temperature at 60 °c was achieved using catechol as substrate. there was a gradual increase in activity of the enzyme with increase in temperature from 30 – 50 °c given 33 – 63.9 % relative activities but almost complete deactivation of ppo was observed at 80 °c. the observed temperature here compared well with the reported 60 °c as temperature optimum for strawberry (serradell et al. 2000) and cucumber ppos (miller et al. 1990). table 3. thermostability of ppo after 60 min of incubation. temperature [°c] residual activity [%] 30 61.9±3.5 40 56.5±2.8 50 55.1±2.3 60 51.2±1.7 70 42.1±1.3 80 9.1±0.9 the experiment was repeated three times, and each value is given as the mean ± standard deviation. nova biotechnol chim (2020) 19(2): 223-231 228 fig. 4. effect of temperature on the activity of ppo. the results were expressed as percentage relative to the maximum activity at 60 °c taken as 100 %. data represent mean ± std (n = 3). yang et al. (2000) had earlier ascertained that ppo from different plant sources has optima temperatures ranging from 30 – 60 °c. nevertheless, at temperatures above 60 °c, a decrease in ppo activity was observed with 42 and 9.1 % residual activity at 70 and 80, respectively. an optimum temperature, 60 observed in this study is relatively higher than those from other plant sources such as apple (zhou et al. 1993), banana (yang et al. 2000) and mango and corn tassel (robinson et al. 1993; guven et al. 2016) with a temperature optimum of 30 °c; cocoa bean (lee et al. 1991) and 45 °c; sunflower (raymond et al. 1993). variations in temperature may also alter the solubility of oxygen, one of the substrates required for ppo to perform its catalytic activity (wang et al. 2007). the thermal stability of ppo from c. colocynthis is presented in table 3. about 62, 56, 55, and 54 % residual activity were observed at 30, 40, 50, and 60 °c respectively after 60 min of incubation. however, about 42 and 9.1 % residual activity were measured at 70 and 80 °c, respectively. the ppo of this was moderately stable with about 50 % table 4. kinetic parameters of ppo from c. colocynthis. substrate km [mm] vmax [u.min-1] vmax/km [u.mm-1] catechol 5.40 1.51 3.0 ×10-1 gallic acid 6.38 0.78 1.2 ×10-1 l-dopa 7.48 0.67 0.9 ×10-1 tyrosine 7.48 0.54 0.7 ×10-1 the experiment was repeated three times, and each value is given as the mean ± standard deviation. activity recorded after 60 min of incubation at 60 °c. these results compared favourably with the earlier studies of valero et al. (1988), who reported a complete inactivation of grape ppo at 75 °c after 15 min of incubation. thermal stability of plant ppos is influenced by the nature of phenolic substrate used during determination (park and luh 1985). the results of the kinetic parameters of c. colocynthis ppo are summarized in table 4, the km values of ppo using catechol, gallic acid, l-dopa and tyrosine as substrates were: 5.04, 6.38, 7.48 and 7.89 mm respectively while the values for vmax were: 1.51, 0.78, 0.67 and 0.54 u.min-1, respectively. therefore, catechol having the low km with the highest value for vmax is the best substrate for c. colocynthis ppo. the activity of the purified ppo c. colocynthis using four different substrates was in order of: catechol > gallic acid > l-dopa > tyrosine as shown in table 5. the level of ppo activity towards phenolic substrates varies widely in the plant kingdom (sherman et al. 1995). however, these differences may be due to the nature of the side chains, number of hydroxyl groups and their position in the benzene ring of the substrates (oktay et al. 1995; mueller et al. 1996). inhibition of c. colocynthis ppo activity the effect of cations and inhibitors on ppo activity is presented in table 6. fe2+, zn2+, mg2+ and, ca2+ were found to inhibit the activity of the enzyme while cu2+ increases the enzyme activity at all concentrations investigated. however, there was increase in percentage inhibition as the concentration of the metal ions increases. ascorbic acid, edta, sds, and urea were observed to inhibit the activity of ppo from melon seeds. table 5. substrate specificity of ppo from c. colocynthis. substrate relative activity [%] catechol 100±0 gallic acid 74.1±9.9 l-dopa 33.3±4.9 tyrosine 22.2±5.6 the experiment was repeated three times, and each value is given as the mean ± standard deviation. nova biotechnol chim (2020) 19(2): 223-231 229 table 6. effect of cations on ppo activity. cation relative activity [%] 5 mm 10 mm 20 mm cuso4 106±5.6 114±3.3 117±5.5 feso4 36.1±2.0 44.4±0.8 66.7±2.6 znso4 66.7±2.0 56.6±2.9 33.3±2.6 mgso4 25.2±3.9 50.0±5.7 69.4±2.5 nacl 44.4±5.6 13.9±1.7 6.68±2.46 cacl2 38.9±2.9 27.8±4.8 11.1±4.2 the experiment was repeated three times, and each value is given as the mean ± standard deviation. there was increase in the percentage inhibition as the concentration of the inhibitors increases (table 7). sds was a stronger inhibitor with 58 % reduction in enzymatic activity at 5 mm and over 90 % reduction with increasing concentration above 10 mm. edta and ascorbic acid are strong inhibitors of ppo from c. colocynthis. earlier studies on ppo have revealed sds as a potent inhibitor of tyrosinase activity and it was suggested that this compound might cause inhibition by forming complexes with copper atoms in the active site (kong et al. 2000). sds with a concentration of 5 mm and above inhibited the ppo activity of c. colocynthis (liu et al. 2004). however, ppo from c. colocynthis showed 83.3 % inhibition in the presence of urea at 5 mm concentration, this is in agreement with the earlier studies of liu et al. (2004) and endo et al. (2003). in this study, the presence of zn2+, mg2+ and, fe2+ showed no activating effect on ppo activity but an increase in enzyme activity was observed in the presence of cu2+ at all concentrations investigated. the result is consistent with the earlier reports of kong et al. (2000), liu et al. (2004), dalfard et al. (2006) and whose studies on plant ppos confirmed increase in enzyme activity in the presence of copper ion. conclusion this study revealed optimum temperature of the purified ppo from c. colocynthis for its enzyme activity to be 60, while there was almost total enzyme deactivation at 80 °c after 60 min of incubation. the enzyme molecular weight was 42 kda. nevertheless, the enzyme oxidized tyrosine, therefore, tyrosinase was identified as the only one ppo in c. colocynthis seed. table 7. effect of inhibitors on ppo activity. inhibitor relative activity [%] 5 mm 10 mm 20 mm ascorbic acid 79.2±4.0 37.2±5.7 20.8±1.3 edta 75.8±6.7 54.2±3.3 8.3±3.4 sds 41.7±6.8 29.3±2.4 12.0±3.3 urea 83.3±9.7 53.3±5.3 37.5±1.9 the experiment was repeated three times, and each value is given as the mean ± standard deviation. the enzyme was deactivated at acidic ph and temperature above 70 °c. therefore, subjecting processed melon seeds to temperatures above 80 °c could inhibit the browning effect of ppos in c. colocynthis and its products. conflict of interest the authors declare no conflict of interest. references adeseko cj, sanni dm, salawu so, kade ij, fatoki ht (2019) hplc-uv standard phenolic constituents of african bush mango (irvingia gabonensis) and molecular docking on polyphenol oxidases. j. appl. life sci. int. 22: 1-11. benmoumou a, el madidi s (2019) variability analysis of the seeds and oil yields of several accessions of citrullus colocynthis (l.) collected in morocco. int. j. food sci. agric. 3: 287-291. cary jw, alan ar, flurkey wh (1992) cloning and characterization of cdnas coding for vicia faba polyphenol oxidase. plant mol. biol. 20: 245-253. dalfard ab, khajeh k, soudi mr, naderi-manesh h, ranjbar b, sajedi rh (2006) isolation and biochemical characterization of laccase and tyrosinase activities in anovel melanogenic soil bacterium. enzyme microb. technol. 39: 1409-1416. dincer b, colak a, aydin n, kadioglu a, güner s (2003) characterization of polyphenol oxidase from medlar fruits (mespilus germanica l. rosaceae). food chem. 77: 1-7. endo k, hayashi y, hibi k, hosono t, beppu t, ueda k (2003) enzymological characterization of epoa, a laccase-like phenol oxidase produced by streptomyces griseus. j. biochem. 133: 671-677. escribano j, cabanes j, garcia-carmona f (1997) characterization of latent polyphenol oxidase in table beet: effect of sodium dodecyl sulphate. j. sci. food agric. 73: 34-38. flurkey wh (1986) polyphenol oxidase in higher plants. plant physiol. 81: 614-618. fraignier m, marques l, fleuriet a, macheix j (1995) nova biotechnol chim (2020) 19(2): 223-231 230 biochemical and immunochemical characteristics of polyphenol oxidase from different fruits of prunus. j. agric. food chem. 43: 2375-2380. guven rg, aslan n, guven k, bekler fm, acer o (2016) purification and characterization of polyphenol oxidase from corn tassel. cell. mol. biol. 62: 6-11. guven rg, guven k, bekler fm, acer o, alkan h, dogru m (2017) purification and characterization of polyphenol oxidase from purslane. food chem. 37: 356-362. jiang ym, yao ih, lichter a, li jr (2003) postharvest biology and handling of litchi fruit. j. food agric. environ. 2: 76-81. jimenez m, garcia-carmona f (1996) effect of sodium dodecyl sulphate on polyphenol oxidase. phytochem. j. 42: 1503-1509. kenten rh (1957) latent phenolase in extracts of broad-bean (vicia faba l.) leaves. activation by acid and alkali. biochem. j. 67: 300-307. kong kh, hong mp, choi ss, kim yt, cho sh (2000) purification and characterization of a highly stable tyrosinase from thermomicrobium roseum. j. biotechnol. appl. biochem. 31: 113-118. kumar s, kumar d, kamal m, saroha singh n, vashishta b (2008) antioxidant and free radical scavenging potential of citrullus colocynthis (l.) schrad. methanolic fruit extract. acta pharm. 58: 215-220. laemeli uk (1970) cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature. 227. 680-685. laveda f, núñez-delicado e, garcia-carmona f, sanchezferrer a (2000) reversible sodium dodecyl sulphate activation of latent peach polyphenol oxidase by cyclodextrins. arch. biochem. biophys. 379: 1-6. lee pm, lee k, karim mia (1991) biochemical studies of cocoa bean polyphenol oxidase. j. sci. food agric. 55: 251-260. liu l, cao s, xie b, sun z, li x, miao w (2007) characterization of polyphenol oxidase from litchi pericarp using (-) epicatechin as substrate. j. agric. food chem. 55: 7140-7143. liu n, zhang ty, wang j, huang yp, ou jh, shen p (2004). a heat inducible tyrosinase with distinct properties from bacillus thuringiensis. lett. appl. microbiol. 39: 407-412. lowry hn, rosenbrough j, farr al, randall rj (1951) protein measurement with the folin phenol reagent. j. biol. chem. 193: 265-275. marques l, fleuriet a, macheix j (1995) characterization of multiple forms of polyphenol oxidase from apple fruit. plant physiol. biochem. 33: 193-200. mayer am (2006) polyphenol oxidase in plants and fungi: going places? 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(colocynthis): biotechnological perspectives. emir. j. food agric. 29: 83-90. nova biotechnol chim (2020) 19(2): 223-231 231 valero e, varon r garcia-carmona f (1988) characterization of polyphenol oxidase from airen grapes. j. food sci. 53: 1482-1485. van gelder cwg, flurkey wh, wichers hj (1997) sequence and structural features of plant and fungal tyrosinases. phytochemistry 45: 1309-1323. wang y, jiang h (2007) purification and characterization of manduca sexta serpin-6: a serine proteinase inhibitor that selectively inhibits polyphenol oxidase-activating proteinase-3. insect biochem. mol. biol. 34: 387-395. yang c, fujita s, ashrafuzzaman m, nakamura n, hayashi n (2000) purification and characterization of polyphenol oxidase from banana (musa sapientum l.) pulp. j. agric. food chem. 48: 2732-2735. yang rj, li sq, zhang qh (2004) effects of pulsed electric fields on the activity of enzymes in aqueous solution. j. food sci. 69: 241-248. yoruk r, marshall mr (2003) physiochemical properties and function of plant polyphenol oxidase: a review. j. food biochem. 27: 361-422. zhou p, smith nl, lee cy (1993) potential purification and some properties of monroe apple peel polyphenol oxidase. j. agric. food chem. 41: 532-536. nova biotechnol chim (2019) 18(2): 94-101 doi: 10.2478/nbec-2019-0012  corresponding author: suta7@uniba.sk nova biotechnologica et chimica targeting transgene to seed resulted in high rate of morphological abnormalities of camelina transformants dominika šutá1,, ildikó matušíková2 and alžbeta blehová1 1 department of plant physiology, faculty of natural sciences, comenius university in bratislava, ilkovičova 6, mlynská dolina, bratislava 84215, slovak republic 2 department of ecochemistry and radioecology, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava 91701, slovak republic article info article history: received: 18th august 2019 accepted: 10th october 2019 keywords: camelina sativa embryo developmental deformation floral dip transformation red fluorescent protein abstract false flax (camelina sativa l.) is currently under-exploited but highly promising oilseed crop. combining camelina’s attractive agronomic traits with its unprecedented ease for genetic engineering makes it an ideal plant chassis for biotechnology applications, in particular synthetic biology strategies. for targeted expression of transgene particularly to seeds requires identification and application of seed specific promoters. in the present study two cultivars of camelina, namely zuzana and smilowska, were used for transformation at early flowering stage using the floral dip method. the plants were inoculated with agrobacterium bearing a construct for expression of red fluorescent protein (rfp) under the control of the seed specific cruciferin promoter cruc from arabidopsis. transgenic seeds and plants were identified on the basis of red fluorescence (rfp) and kanamycin resistance. relatively high transformation efficiency of 8 % was achieved particularly for the cultivar zuzana. however, many of regenerants exerted developmental deformations such as lack of shoot apical meristem, deformed or absent cotyledons, etc. furthermore, the activity of the cruc promoter was still active also in true leaves rendering this promoter as inappropriate for seed targeting of the transgene. nevertheless, genetic transformation remains a tool for direct modulation of pathways for oil synthesis in oilseed crops.  university of ss. cyril and methodius in trnava introduction false flax, camelina sativa (l.), is an ancient crop of the brassicaceae cultivated in europe as an important oilseed crop for many centuries before it was displaced by higher-yielding crops, such as canola, soybean or sunflower. like other brassicaceae, camelina’s seeds accumulate starch during the early stage of embryo development (na et al. 2018), but later it largely diminishes when seeds mature. it is proposed that the turnover of starch during seed development is functionally linked with cell division (andriotis et al. 2010). camelina seeds store oil and protein as major carbon and nitrogen reserves (gesch 2014). the camelina oil yields and fatty acid composition depends on cultivars, soil composition but also environmental conditions (gesch 2014; ševčík et al. 2018; obeng et al. 2019). the proportion of polyunsaturated fatty acids (pufas) in seeds, for example, decreases with temperatures (obour et al. 2017). since higher content of unsaturated fatty acid in the oil is critical for its uses as jet fuel and biodiesel (kim et al. 2015), the timing bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:23 utc mailto:suta7@uniba.sk nova biotechnol chim (2019) 18(2): 94-101 95 of camelina planting to ensure flowering and seed filling, periods of adequate soil water supplyand favourable growing temperature are important for camelina production. further, camelina oil is rich in omega-3 fatty acids but poor in minor lipid components such as tocopherols and phytosterols compared to the other conventional oilseeds (even most fish oils), rendering it suitable for food applications as health-promoting lipids (budin et al. 1995; belayneh et al. 2018). although camelina has many attractive agronomic features (e.g. short lifecycle, remarkable adaptation to a wide range of climatic conditions, etc.), its relatively low oil yield (compared to other oilseed plants) represents a real limitation to it using in chemical and/or pharmaceutical industries (faure and tepfer 2015). improving oil yield is therefore a priority for the development of this crop for large-scale use. desired properties of the oil can be achieved by the use of various transformation techniques. camelina can be successfully transformed using both the floral dip and floral vacuum infiltration methods (lu and kang 2008; liu et al. 2012). recently, two c. sativa cultivars were successfully transformed also with agrobacterium tumefaciens, for which the shoot tips with apical meristems appeared the best target tissue (sitther et al. 2018). fully developed, intact transgenic camelina plants can be obtained within 6 – 8 weeks (lu and kang 2008; sitther et al. 2018). expression of transgenes can result in production of seed oil with attractive physicochemical properties such as reduced viscosity and freezing point, and thus suitable for preparation of biodegradable lubricants, emulsifiers, plasticizers, and second-generation biofuels (liu et al. 2015; bansal and durrett 2016; haslam et al. 2016). recently zhu et al. (2018) suggested that the maize master regulator, zmlec1, driven by a downstream seed-specific promoter, is a promising target for increasing oil yield in transgenic camelina seeds. further, camelina plants transformed with eadact gene from euonymus alatus synthesize large amounts of sn-3-acetyl triacylglycerols (acetyl-tags), primarily in the endosperm and mature embryo cells (durrett et al. 2010; hu et al. 2017; bansal et al. 2018; chhikara et al. 2018). modifying the composition of camelina seed oil opens new possibilities for diversifying oilseed production, for example towards the accumulation of bioactive compounds (e.g. terpenes) used in food additives, cosmetics, drugs, and for other bio-based products. transformation of camelina per se does not appear as demanding, although failure of the applied method has also been reported (lu and kang 2008). moreover, targeted expression of transgene particularly to seeds can be problematic. model organisms are being widely screened for identifying promoters that are tissue or developmental stage-specific to maximize the effect of transgene expression without affecting other tissues during growth and development (jeong et al. 2014; polóniová et al. 2015). the development of promoter systems expressed specifically in seeds or in particular constituents or tissues/compartments of seeds is indispensable not only for the mentioned reasons, but also to control the nourishment of the embryo, dispersal to a new location, and dormancy under unfavourable conditions (jeong et al. 2014). camelina has previously been transformed with seed-specific phaseolin promoter (lu and kang 2008). in this work, we tested the suitability of the cruciferin (cruc) promoter to drive transgene expression in camelina seeds. this strong promoter from arabidopsis thaliana is embryo-specific with activity that starts between mid-globular and early heart embryo stages (becerra et al. 2006), but is inactive in other (vegetative) tissues like leaves or flowers (mietkiewska et al. 2000). we show that the transformation of camelina can be hampered, while potential reasons are discussed. we used two camelina cultivars zuzana and smilowska, which are promising for breeding in slovakia. experimental bacteria the bacterial strain of agrobacterium tumefaciens strain lba4404 was used, carrying a binary vector pev1 with the rfp reporter and selectable nptii genes (moravčíková et al. 2008). the former gene was under the control of the cruciferin promoter sequence (cruc), while the latter one by the nos promoter. bacteria were cultivated on lb medium, (10 g.l-1 peptone, 6 g.l-1 nacl, 6 g.l-1 yeast bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:23 utc nova biotechnol chim (2019) 18(2): 94-101 96 extract; ph 5.8). bacterial suspension (300 μl) and appropriate antibiotics (100 mg.l-1 of kanamycin and 25 mg.l-1 of rifampicin) were added into a liquid lb medium and cultivated for overnight on a shaker (shaker dos-20l; 220 rpm) in the dark at 28 °c. the agrobacterium cells were separated from medium by centrifugation at 2,500 rpm for 2 min and the pellet was suspended in the infiltration medium (od600 ~ 1.0), consisting of half-strength ms salts and 0.025 % (v/v) silwet l-77. floral dip method of camelina sativa transformation and selection two c. sativa cultivars zuzana (czech republic) and smilowska (poland) were transformed via floral dip infiltration using the above-mentioned strain of agrobacterium tumefaciens. one hundred c. sativa seeds planted in 15 × 35 cm pot filled with soil were cultivated in a growth chamber under controlled environmental conditions (16/8 h long-day photoperiod with the light intensity 100 μmol.m-2s-1 and stable temperature 24 °c). plants at early flowering stage were dipped into agrobacterium cell suspension for 15 seconds and covered with plastic dark film and placed in the dark for 24 h. these t0 plants were then moved to the growth chamber and cultivated (16/8 h long-day photoperiod; 100 μmol.m-2.s-1 with stable temperature 24 °c) until mature seeds were harvested. one hundred seeds from t0 plants were sterilized with 4.7 % (w/v) sodium hypochlorite solution for 15 min and then washed three to five times with sterile distilled h2o. washed seeds were sown on petri dishes with a half-strength ms medium (ph 5.8) supplemented with 10 g.l-1 sucrose, 7 g.l-1 agar and 150 mg.l-1 kanamycin. the antibiotics concentration to be applied was determined based on preliminary screening of its (obviously negative) effect on non-transgenic germinating seedlings after 7 days growth. the seeds were cultivated in a growth chamber with a 16/8 h light/dark cycle for germination and seedling growth. transgenic plants were examined for red fluorescence under a stereomicroscope m 165 fc (leica, germany) after 7 – 14 d cultivation. results and discussion like other brassicaceae plants, camelina’s seeds accumulate non-polar lipids, presence of which was detected in many oil bodies, primarily in cotyledons (fig. 1). oil content and properties render this species extremely attractive for food, but relatively cheaper agronomic costs and the possibility to modify the properties of its oil makes it promising for the biofuel industry as well. genetic transformation approach has been successful in achievement of the desirable camelina traits (for review see aznar-moreno and durrett 2017), while specific promoters are important for seed-targeted modification to occur. we transformed two camelina cultivars zuzana and smilowska with agrobacteria using floral dip method. seeds were collected from 100 plants. a total of 100 seeds from each cultivar were let to germinate on media with kanamycin and the number of resistant individuals was scored. a b hy r co fig. 1. longitudinal sections of camelina seeds observed by stereomicroscope (m 165 fc leica, germany) (a) and confocal laser microscopy fluorescence image (fv1200 ix83, olympus, japan) of cotyledons showing oil bodies stained with bodipy tm 493/503 according to martinčová et al. (2019) (b). co —cotyledons; hy — hypocotyl; r — radicle. the bar represents a = 500 µm; b = 50 µm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:23 utc nova biotechnol chim (2019) 18(2): 94-101 97 table 1. overview of the camelina transformation. camelina cultivar seeds of transformed plants totala rfpb regenerantsc te [%]d zuzana 100 11 8 8 smilowska 100 6 2 2 a total number of seeds from t0 plants screened; b number of seeds expressing red fluorescence; c number of potential transgenic regenerants identified on the basis of kanamycin resistance; d transformation efficiency (te) expressed as a number of kanamycin resistant regenerants obtained as percentage of the seeds used. the data showed that two cultivars markedly differ in transformation efficiency; efficiency of 8 % was achieved for the cv. zuzana but only 2 % for the cv. smilowska (table 1). the former value is much higher than the transformation efficiency of 0.8 % obtained using the same transformation method by liu et al. (2012), and also than the efficiency of 1.3 % obtained using vacuum infiltration by lu and kang (2008). noteworthy, the later authors failed to obtain transformed c. sativa (cv. celine) plants using floral dip technique. previously, efficient transformation of two c. sativa cultivars (pl650159 and pl650161) has been described by sitther et al. (2018), while the authors concluded that shoot tips with apical meristems were the best target tissues for agrobacteriummediated transformation of c. sativa plants in vitro. as easily scorable reporter gene we used the red fluorescing dsrfp, which has previously been used to transform camelina under control of camv promoter (lu and kang 2008), and had shown to remain active in desiccated mature seeds with no obstruct excitation or emission of the fluorescence by the light-colored seed coat. we could not detect fluorescence in any intact camelina seed at all, probably due to week transgene expression. in contrast, strong fluorescence was typical for germinating embryos (fig. 2 and 3). however, not all red-fluorescing embryos survived on selection media in later developmental stage, pointing on insufficient tolerance to antibiotics (e.g. due to chimeric nature), or incomplete transgene (boszorádová et al. 2019). the phenomenon of chimerism, for example, frequently occurs a b c d e f fig. 2. selection of rfp+ c. sativa embryos after 7 days of cultivation (a – d) and regeneration of rfp+ potential transgenic plants (e – f) after 14 days of cultivation. the bar represents a, b = 1 cm; c, d = 250 µm; e, f = 1 cm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:23 utc nova biotechnol chim (2019) 18(2): 94-101 98 to a different extent in all systems where a recombination event has to take place in multicellular structures, e.g. during development from calli (moravčíková et al. 2008). surprisingly, red fluorescence of surviving transformants was observed also later during development and growth, especially in the regenerants of the ana (fig. 2 and 3). this suggests that the cruc promoter does not stop activation of the rfp as expected. ectopic expression of this promoter has been reported previously in rapeseed (boszorádová et al. 2019) and tobacco (moravčíková et al. 2008), but shifted activation of a heterologous promoter in time has also been observed by others (luo et al. 2007; polóniová et al. 2015). given the critical requirement of transgene expression solely in the seeds of camelina, the cruc appears inappropriate for use in oil biotechnology. the transgenic (in each case still rfp positive) seedlings have green expanded cotyledons and green true leaves. in contrast, control and nontransgenic seedlings had white cotyledons and they were unable to produce true leaves in the given (cultivation) conditions (fig. 3). the surprising result of our transformation efforts was ~30 % of aberrant phenotypes seedlings (fig. 3 and 4). the observed phenotypic and developmental abnormalities included reduced or partially disrupted shoot apical meristems, and deformed or absent cotyledons (fig. 4). these defects likely hamper the completion of embryo development and/or seed germination. according to schwartz et al. (1994), defects in arabidopsis embryo development appear in the globular stage of embryogenesis, while the abnormalities in the suspensor function were detected soon after at the heart stage. altered development of the embryo in mutant seeds leads indirectly to the proliferation of suspensor cells and expression of properties characteristic of the embryo proper. in this context is also important that storage protein and lipid bodies, which normally accumulate only in the arabidopsis embryo, were present in both structures (schwartz et al. 1994). this is consistent with the work recently published by zhu et al. (2018), who achieved more than 20 % increased oil content in the transgenic camelina seeds, but they did not observe any adverse effects on growth and development. furthermore, the changes in the composition of oil do not affect the germination of camelina seeds, and therefore these oils can be used by plants as an energy source during the embryo development, seed germination, in regulation processes of seedlings growth and development, and even during the cultivation in the field (liu et al. 2015). previously, tissue-specific expression of aspartate aminotransferase in brassica napus produced transformants with aberrant phenotype, such as abnormal leaf morphology and photosynthesis as well as stunted growth (wu et al. 2002; shu et al. 2002; ahlert et al. 2003; mcallister et al. 2016). ahlert et al. (2003) speculated that the position of transgene insertion may have played a key role in the phenomenon. a b c d e f fig. 3. generation of transgenic camelina sativa plants. seeds of transformed plants were plated on media with 150 mg.l-1 kanamycin (a, d) and their transgenic nature was identified on the basis of red fluorescence (rfp+) and resistance to antibiotics. non-transgenic camelina seedlings (b) had white cotyledons and showed very low or no rfp activity (c). red fluorescence indicated transgene expression in transgenic camelina plants regenerated on ms media with kanamycin (f). the bars at a – f = 1 cm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:23 utc https://www.sciencedirect.com/topics/earth-and-planetary-sciences/leaf-morphology nova biotechnol chim (2019) 18(2): 94-101 99 since aberrant transformants are undesired sideproducts of many experiments, they are rarely discussed in detail and their reasons are not further explored. there are many quite well described reasons for changes at dna level (ranging from point mutations and methylation differences to transposon induction, gene amplification, chromosomal aberrations and ploidy level changes; kaeppler et al. 2000; jain 2001; bregitzer et al. 2002; boszorádová et al. 2011, 2014). numerous aspects of plant transformation are associated with the stress of the exposed tissues. for example, the use of antibiotics – especially at higher concentrations like in our study – may induce epigenetic and/or genetic changes of the plant genome (bardini et al. 2003; madlung and comai 2004). the agrobacterium infection itself, and also insertions of small fragments of transgenic dna can also be a potential source of genome-wide mutations. as these would usually be missed by southern blot and even by polymorphism analysis techniques, the numbers of genome-wide mutations in plants transformed by agrobacterium (and possibly also by particle bombardment) are probably underestimated (makarevitch et al. 2003). importantly, the presence of strong transgene promoters, including the commonly used cauliflower mosaic virus (camv) promoter and also the cruc promoter used in this study, may also result in mis-expression (especially overexpression) of endogenous genes (moravčíková et al. 2008; boszorádová et al. 2019) at a distance of up to 12 kbp (ichikawa et al. 2003). transcriptional read-through and improper mrna processing (protein translation/folding) can also occur when the nos terminator is used (rang et al. 2005). conclusions since the current plant transformation methods are mutagenic, efficient production of safe transgenic plants for biotechnology would require more data on the frequency and molecular basis of a b c d e f fig. 4. red fluorescence in large number of embryos with developmental deformations in development, such as deformed (a – d) or absent cotyledons (e – f). the bar represents a, b = 250 µm; c – f = 1 cm. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:23 utc nova biotechnol chim (2019) 18(2): 94-101 100 transformation-induced mutations (wilson et al. 2006). those presently available describe aberrant arabidopsis plants created by agrobacteriummediated transformation, while those from other species (including important crop plants) describe at most a few transgenic individuals with incompletely analysed genome changes. in our system, both strong (ectopically active) promoter and terminator combined with high antibiotics concentrations could be responsible for aberrant regenerants. though formation of aberrant plants has not been reported as consequence of neither rfp production not cruc promoter activity, for camelina the cruc did not prove to be promising for seed-directed genome modification. acknowledgement this work was supported by the slovak research and development agency under the contract no. apvv-160051. we sincerely thank to mgr. peter kaštier phd. for his 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eur. food res. technol. 220: 438-443. schwartz bw, yeung ec, meinke dw (1994) disruption of morphogenesis and transformation of the suspensor in abnormal suspensor mutants of arabidopsis. development 120: 3235-3245. shu q, cui h, ye g, wu d, xia y, gao m, altosaar i (2002) agronomic and morphological characterization of agrobacterium-transformed bt rice plants. euphytica. 127: 345-352. sitther v, tabatabai b, enitan b, dhekney s (2018) agrobacterium-mediated transformation of camelina sativa for production of transgenic plants. j. biol. methods. 5: e83. ševčík p, joríková ľ, hozlár p, hájeková e, ondrejíčková p (2018) the effect of growing conditions on camelina (camelina sativa l.) oil content – sustainable feedstock for biofuels production. in 6th international conference on chemical technology. 16.-18.4. 2018 mikulov, czech republic, p. 244-247. wilson ak, latham jr, steinbrecher ra (2006) transformation-induced mutations in transgenic plants: analysis and biosafety implications. biotechnol. genet. eng. 23: 209-238. wu dx, shu qy, wang zh, cui hr, xia yw (2002) quality variations in transgenic rice with a synthetic cry1ab gene from bacillus thuringiensis. plant breed. 121: 198-202. zhu y, xie l, chen gq, lee my, loque d, scheller hv (2018) a transgene design for enhancing oil content in arabidopsis and camelina seeds. biotechnol. biofuels. 11: 46. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:23 utc nova biotechnol chim (2019) 18(2): 124-132 doi: 10.2478/nbec-2019-0015 corresponding author: timotej.jankech@gmail.com nova biotechnologica et chimica determination of methylxanthines in tea samples by hplc method timotej jankech1,, mária maliarová1 and nicholas martinka2,3 1department of chemistry, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava 91701, slovak republic 2department of clinical biochemistry, alexander winter hospital, winterova 66, piešťany 92101, slovak republic 3department of laboratory investigation methods, trnava university in trnava, hornopotočná 23, trnava 91843, slovak republic article info article history: received: 24th july 2019 accepted: 20th november 2019 keywords: food hplc methylxanthines tea abstract methylxanthines such as caffeine, theophylline, theobromine are significant and widespread psychoactive substances. we developed the isocratic method with optimum composition of the mobile phase 90 % water: 10 % acetonitrile and confirmed repeatability of retention times and peak areas. the developed hplc method was applied to determine the content of methylxanthines in selected types of black and green teas available on the market. of the black teas (tea bags), the highest concentration of theobromine was found in ceylon tea (18.98 mg.l-1). the highest concentration of caffeine was in a cup of earl gray tea (254.09 mg.l-1). among loose black teas, the highest content of both theobromine and caffeine was found in pu erh superior tea, where the theobromine content was 24.62 mg.l-1 and the caffeine content was 520.67 mg.l-1. of green powder teas, highest caffeine content (306.46 mg.l-1) was in shizuoka matcha premium and the highest content of theobromine (8.45 mg.l-1) was found in gaba midori. from the loose green tea, the highest concentration of theobromine (12.85 mg.l-1) was in lung ching west lake. the highest caffeine content (484.85 mg.l-1) was in gyokuro shizuoka premium tea. in both types of teas the amount of theobromine and caffeine was quantified, but the presence of theophylline was not proven. data on contents of these metabolites in tea products are highly informative for consumers. university of ss. cyril and methodius in trnava introduction several mild psychostimulants are derived from methylxanthines, including widely used and highly popular caffeine, theobromine and theophylline (xia et al. 2013). the structures of these compounds (fig. 1) with several health benefits are derived from the purine base xanthine and are obtained from plant secondary metabolism. caffeine is the most routinely ingested bioactive substance throughout the world. it is a natural alkaloid found in more than 60 plants including coffee beans, tea leaves, cola nuts, and cocoa pods. its concentration varies depending on the type of product, agronomic and environmental factors, as well as processing (de mejia and ramirez-mares 2014). caffeine possess a number of effects: improves performance during sleep deprivation, in shift workers it leads to fewer mistakes caused by tiredness, moderate doses in athletics can improve sprit, endurance or tolerance and team sports performance. however, high dose of caffeine has adverse effect on health (mufakkar et al. 2015). theophylline, also known as dimethylxanthine, is naturally found in tea and cocoa beans. for several decades, theophylline has been used as a therapeutic agent of chronic airway disease (asthma) with a narrow serum therapeutic range bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:30 utc mailto:timotej.jankech@gmail.com nova biotechnol chim (2019) 18(2): 124-132 125 (jafari et al. 2011). the main drawbacks of this active pharmaceutical ingredient (api) are related to its narrow therapeutic index and several side effects, i.e. nausea, headache, dizziness and vomiting. increasing evidence shows that it has significant anti-inflammatory effects in chronic obstructive pulmonary disease at lower plasma concentrations (zhu et al. 2015). fig. 1. structures of the methylxanthines analysed in this study (gibson and fowler 2014). theobromine is a bitter alkaloid of the cacao plant, providing specific flavour in chocolate as well as in a number of other foods (xia et al. 2013). although, chemically is very similar to other methylxanthines, such as caffeine and theophylline, theobromine stimulates the central nervous system to a lesser extent than these other methylxanthines. ingestion of theobromine has demonstrated health benefits, including protection of the enamel surface of teeth, cough suppression and cardiovascular protection, increasing hdl and decreasing ldl cholesterol concentrations (rodriguez et al. 2015). different analytical techniques and sample preparation protocols have been proposed for the simultaneous determination of the methylxanthines in foods and biological fluids. analytical techniques routinely used for the analysis of these components include highperformance liquid chromatography (hplc) or gas chromatography-mass spectrometry (gc-ms) (xia et al. 2013). an overview of published papers dealing with determination of methylxanthines in various matrices is provided in table 1. the aim of this study was the simultaneous determination of methylxanthines (caffeine, theobromine and theophylline) on chromatographic column luna polar c18, which is suitable for separating polar analytes, such as methylxanthines. the versatile c18 ligand provides hydrophobic interactions while the polar modified surface provides enhanced retention of polar analytes and aqueous stability. the method was applied to analyze the content of methylxanthines in different types of commercially available tea, namely black and green loose teas as well as powdered green teas. in literature, data on the amount of methylxanthines are mostly associated with the type of tea (black-green). experimental chemicals and reagents hplc-grade acetonitrile was purchased from central chem (banská bystrica, slovakia). table 1. overview of published papers dealing with determination of methylxanthines in various matrices (tzanavaras et al. 2010). sample column mobile phase analysis time [min] detection beverages, tea, coffee rp-c18 (100 mm×3.2 mm i.d.×3 μm) meoh:buffer (ph = 3.5) (10:90, v/v) 16 amperometric cocoa nova-pak c18 (150 mm×3.9 mm i.d.×4 μm) meoh:h2o (20:80, v/v) 10 uv at 274 nm coffee, tea lichrospher 100 rp-18 (244 mm×4.4 mm i.d.×5 μm) h2o:etoh:ach (75:24:1, v/v/v) 10 uv at 273 nm human urine supelcosil lc-18-db (250 mm×2.1 mm i.d.×5 μm) meoh:buffer (ph = 5.0) (20:80, v/v) 10 uv at 280 nm human serum kromasil ods c18 (150 mm×4.6 mm i.d.×5 μm) meoh:buffer (ph = 4.5) (30:70, v/v) 12 uv at 270 nm beverages, chocolate bondesil c18 (150 mm×4.0 mm i.d.×5 μm) h2o:me(et)oh:ach (75:20:5, v/v/v) 12 uv at 273 nm compound substituent r′ r″ r‴ caffeine ch3 ch3 ch3 theobromine ch3 ch3 h theophylline h ch3 ch3 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:30 utc nova biotechnol chim (2019) 18(2): 124-132 126 table 2. an overview of the processing of tested tea samples. type product name weight volume [ml] leaching time [min] water temperature* [°c] [g] black teas a earl grey 2 250 4 100 a darjeeling 2 250 4 100 a english breakfast 2 250 4 100 a english tea no.1 2 250 4 100 a ceylon 2 250 4 100 a decaffeinated 2 250 4 100 b pu erh superior 5 200 2 100 b darjeeling gopaldhara wonder tea muscatel 5 200 1 100 b darjeeling gopaldhara wonder gold 5 200 1 100 green teas c shizuoka matcha premium 1 100 70 – 80** c japan kate-benifuki 1 100 80** c gaba midori 1 100 70 – 80** b zhu cha 5 200 2 70 – 80 b lung ching west lake 5 200 2 70 – 80 b houjicha shizuoka 5 200 2 70 – 80 b gyokuro shizuoka premium 5 200 2 60 – 65 b sencha shizuoka superior 5 200 2 70 – 80 b nepal silver needles 5 200 2 85 – 90 tea type: a – bag, b – loose, c – powder; * ultrapure water; ** boiled water, cooled down to the required temperature. standards of caffeine (≥ 99 % purity), theobromine (≥ 98 % purity) and theophylline (≥ 99 % purity) were purchased from sigma-aldrich (darmstadt, germany). ultrapure water (0.055 μs.cm−1) was prepared from distilled water using the ultrapure water (type 1), simplicity uv device. apparatus the analysis was performed using waters hplc instrumentation, which consist of a dual solvent pump (binary hplc pump 1525), thermostat (column heater 1500 series), autosampler (autosampler 2707) and pda detector (photodiode array detector 2998). chromatograph was equipped with a column luna polar c18 100 å, 100 × 4.6 mm, 3 µm (phenomenex). the whole system was monitored by empower 2 software. the column temperature was 35 °c, flow rate was set at 1 ml.min-1 and injection volume was 10 μl. the detection of methylxanthines was performed at 273 nm and uv scan 210 – 400 nm. green and black tea samples preparation all green and black tea samples were prepared according to the preparing instructions for individual teas. weights of both green and black tea samples, leaching time and water temperature were different depending on tea as shown in the table 2. the samples were cooled down to room temperature. subsequently, the samples were centrifuged for 5 minutes at 12.0 g. supernatants were diluted as needed to keep analytes concentration within the linear range. the prepared sample infusions as well as the diluted sample infusions were analysed by hplc-pda using the mobile phase: 10 % acetonitrile : 90 % ultra-pure water. all samples were prepared and diluted in ultrapure water. three samples were prepared from each type of tea. from each sample, triplicate portions were taken for analysis by hplc. results and discussion dependence of retention factors on mobile phase composition the methylxanthines studied (theobromine, theophylline and caffeine) are structurally very similar, therefore it was necessary to choose the proper composition of the mobile phase for their separation. the retention factor is a qualitative bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:30 utc nova biotechnol chim (2019) 18(2): 124-132 127 fig. 2. dependence of retention factors on content of methanol in the mobile phase [%] is shown for theobromine (orange line), theophylline (blue line) and caffeine (red line). parameter for characterizing the chromatographic properties of the separated substances. retention factors of analytes were observed in two types of mobile phases: the first was water (a): methanol (b), and the second water (a) : acetonitrile (b). the dependence of analyte retention factors on the percentage of methanol in the mobile phase is presented on the fig. 2. fig. 4. oxo-enol tautomerism of theobromine (a). chromatogram of separation of standards of methylxanthines (the mobile phase 80 % water : 20 % methanol) with the split peak of theobromine (b). fig. 3. dependence of retention factor on content of acetonitrile in the mobile phase [%] is shown for theobromine (orange line), theophylline (blue line) and caffeine (red line). at the first measurement, when the mobile phase contained 100 % of component b, all three methylxanthines eluted together at the same time. gradually, we reduced the volume percentage of component b in the mobile phase to 10 %. with each reduction the time was prolonged, and at 10 % methanol, caffeine eluted ~ 30 min after the analysis started. the optimal resolution of the individual analytes was achieved when the mobile phase was 80 % water: 20 % methanol and the analysis time was acceptable (10 min). however, at this composition of mobile phase separation was not appropriate because the theobromine peak diffused. this could be caused by the oxo-enol tautomerism provided by theobromine (fig. 3a). chromatogram that depicts such behaviour of theobromine is shown on the fig. 3b. furthermore, as in the previous case, we carried out a series of measurements of the standards solutions (theobromine, theophylline, caffeine) in the acetonitrile/water mobile phase. the dependence of the retention factor on the percentage of acetonitrile in the mobile phase is shown on the fig. 4. since acetonitrile has a higher eluting power than methanol, the individual analysis times were shorter. we found that the retention time is the same for all components from 100 % acn to 40 % acn in the mobile phase. optimal separation was achieved when the mobile phase composed 10 % of acn, because under these conditions resolution between theobromine a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:30 utc nova biotechnol chim (2019) 18(2): 124-132 128 table 3. basic analytical parameters of the hplc method. analyte retention time [min] concentration range [µg.ml-1] linear regression r2 theobromine 2.59 0.506 – 8.096 y = 33915x + 4095.2 0.996 theophylline 3.66 2.504 – 40.064 y = 32042x + 951.93 0.998 caffeine 6.60 5.038 – 100.76 y = 29444x − 32335 0.999 and theophylline was 1.68 and resolution between theophylline and caffeine was 3.16, what we consider adequate. total analysis time was 8 min. the developed hplc method was validated in terms of linearity and within-day precision (repeatability). linearity was evaluated in range of 5 – 100 μg.ml-1 for caffeine, 2.5 – 40 μg.ml-1 for theophylline and 0.5 – 8 μg.ml-1 for theobromine. peak areas was used for signals evaluation. the obtained regression equations (n = 6) for all analytes as well as coefficients of determination are given in table 3. in order to evaluate the repeatability of the method (within-day precision) mixtures theobromine and caffeine was injected at two concentration levels for five replicates each. we determined the content of caffeine and theobromine in tea samples, thus we did not perform the method repeatability for theophylline. results are shown in table 4. relative standard deviations for retention times were less than 1 %; only in a single case this value was above 1 %. relative standard deviations for the areas of theobromine and caffeine, except of a single concentration of theobromine (1.21 μg.ml-1), were up to 5 %. same table also shows the relative standard deviations of averaged retention times and peak areas of individual analytes. determination of methylxanthines in selected tea samples prepared tea extracts were analysed by developed hplc method and the peaks of the methylxanthines were subsequently identified by comparison of uv spectra and retention times with corresponding data of the standards. table 5 provides an overview of the contents of theobromine and caffeine in individual bag teas. concentrations are given corresponding to a single table 4. within-day precision of developed hplc method. analyte and its concentration [μg.ml-1] tr [min] tr* ± sd [min] rsd [%] y [μv/sec] y* ± sd [μv.sec-1] rsd [%] theobromine (1.21) 2.593 2.589±0.003 0.130 41910 45059±3852 8.549 2.587 41531 2.586 48569 2.588 43509 2.593 49778 theobromine (2.48) 2.599 2.591±0.006 0.236 92921 88171±4275 4.848 2.589 84447 2.582 85976 2.591 92704 2.592 84809 caffeine (8.80) 6.610 6.586±0.042 0.651 293843 291375±2163 0.742 6.510 293068 6.603 291558 6.609 288819 6.600 289588 caffeine (45.71) 6.658 6.629±0.074 1.113 1389757 1378153±6765 0.491 6.663 1373970 6.667 1374120 6.659 1374350 6.497 1378568 tr – retention time; sd – standard deviation; rsd – relative standard deviation; y – peak area. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:30 utc nova biotechnol chim (2019) 18(2): 124-132 129 table 5. theobromine and caffeine contents in bag black teas. tea product name theobromine caffeine c [mg/250 ml] ± sd c [mg.l-1] ± sd c [mg/250 ml] ± sd c [mg.l-1] ± sd earl grey 4.62±0.20 18.46±0.79 63.52±4.27 254.09±17.07 darjeeling 2.63±0.18 10.52±0.73 62.05±1.76 248.19±7.03 ceylon tea 4.74±0.14 18.98±0.56 56.97±2.56 227.90±10.24 english breakfast 3.34±0.24 13.36±0.98 53.13±3.55 212.51±14.21 english tea no.1 3.12±0.37 12.49±1.50 52.68±1.15 210.72±4.58 decaffeinated tea 3.32±0.14 13.26±0.55 2.94±0.08 11.76±0.34 cup of tea, prepared according to the instructions of the producer (mostly in mg/250 ml). as shown in table 5. theobromine content in all bag black teas ranges from 3 – 5 mg/250 ml, while the highest concentration was found in ceylon tea (4.74 mg/250 ml). caffeine content (excluding decaffeinated tea), varied in the range from 50 to 65 mg/250 ml with highest concentration in earl grey tea (63.52 mg/250 ml). the second group of black teas analyzed was loose black teas. table 6 shows a summary of their theobromine and caffeine contents. concentrations are again presented as for single cup in mg/200 ml, for individual samples prepared according to the instructions for preparation. the highest content of both theobromine and caffeine can be found in pu erh superior tea (table 6), where the fig. 5. chromatographic separation of methylxanthines in black tea sample english breakfast (blue, solid line) and standards of methylxanthines (orange, dotted line). theobromine concentration was 4.92 mg/200 ml and of the caffeine it was 104.13 mg /200 ml. the lowest caffeine content (71.08 mg/200 ml) was measured in darjeeling gopaldhara wonder gold tea. the lowest theobromine content (1.51 mg/200 ml) was in darjeeling gopaldhara wonder tea muscatel. an example of a chromatographic separation of methylxanthines in black tea samples is shown in fig. 5. as it in evident from tables 5 and 6, there is a clear difference between bag and loose tea, especially in terms of caffeine content. the visual comparison of contents of theobromine and caffeine in black teas are shown in fig. 6, where teas are ranked by decreasing caffeine or theobromine content. abbood and aldiab (2017) analysed 3 samples of black tea using rp-hplc/uv system, but determined only caffeine. in the black tea samples, the indicated amounts of caffeine were 16.5 mg.g-1; 18.3 mg.g-1; 33.4 mg.g-1, which are comparable to those we found in loose black teas listed in table 6. similarly, the rp-hplc/uv method was used by bae et al. (2015) to determine the caffeine content of a black tea sample. the detected amount of caffeine was 20.09 mg.g-1, which is also comparable with values reported in our study in table 6 (bae et al. 2015). we divided green teas according to their character to powdered and loose green teas to powdered and loose green teas. the table 7 shows the contents of theobromine and caffeine in powdered green tea teas. all three green tea powders contained table 6. theobromine and caffeine content in loose black teas. name theobromine caffeine c [mg/200 ml] ± sd c [mg.l-1] ± sd c [mg/200 ml] ± sd c [mg.l-1] ± sd pu erh superior 4.92±0,31 24.62±1.54 104.13±1.13 520.67±5.63 darjeeling gopaldhara wonder tea muscatel 1.51±0,26 7.54±1.32 76.87±1.39 384.33±6.96 darjeeling gopaldhara wonder gold 3.32±0,18 16.62±0.89 71.08±7.41 355.38±37.03 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:30 utc nova biotechnol chim (2019) 18(2): 124-132 130 fig. 6. content of caffeine (a) and theobromine (b) in individual black teas. relatively small amounts of theobromine, where the theobromine concentrations ranged from 0.20 to 0.90 mg/100 ml. the highest concentration of theobromine (0.85 mg/100 ml) was found only in gaba midori. the highest content of caffeine (30.65 mg/100 ml) was found in shizuoka matcha premium tea. the second group of green teas was loose green teas. the calculated concentrations for this group of teas is summarized in table 7. an example of a chromatographic separation of methylxanthines in green tea sample is shown in fig. 7. the highest concentration of theobromine was found in lung ching west lake tea and it was 2.57 mg/200 ml. fig. 7. chromatographic separation of methylxanthines in green tea sample gyokuro shizuoka premium (solid line) and standards of methylxanthines (dotted line). the lowest concentration of theobromine was found in nepal silver needles and it was 0.56 mg/200 ml. the highest caffeine content (96.87 mg/200 ml) was found in gyokuro shizuoka premium tea. by contrast, the lowest caffeine content (44.00 mg/200 ml) was found in zhu cha tea. contents of caffeine and theobromine in green teas are shown in fig. 8. in both, teas are ranked by decreasing content of methylxanthines. grujić-letić et al. (2016) quantified caffeine in different tea samples by hplc/dad. the caffeine content of samples was: 12.63 mg.g-1 in black tea, 10.13 mg.g-1 in green tea and 9.81mg.g-1 in white tea. table 7 shows that comparing to our analysis, the resulting caffeine content reported by authors is relatively low. on the other hand, caffeine contents in white tea coincide (caffeine content, which we found in the white tea sample – nepal silver needles was 9.74 mg.g-1) (grujić-letić et al. 2016). in other study, srdjenovic et al. (2008) applied hplc/uv method with detection at 273 nm to determine caffeine, theobromine and theophylline in a green tea sample; these authors reported 12.63 mg.g-1 caffeine content, 0.32 mg.g-1 theobromine content, and theophylline was below the limit of detection. the content of caffeine was comparable to some loose green teas (table 7). the theobromine content reported by the authors was relatively table 7. theobromine and caffeine content in powdered green teas. theobromine caffeine name c [mg/100 ml] ± sd c [mg.l-1] ± sd c [mg/100 ml] ± sd c [mg.l-1] ± sd shizuoka matcha premium 0.33±0.04 3.34±0.37 30.65±0.68 306.46±6.83 gaba midori 0.85±0.28 8.45±2.79 25.87±8.55 258.65±85.53 japan kate-benifuki 0.29±0.02 2.88±0.17 23.16±0.56 231.55±5.63 a u a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:30 utc nova biotechnol chim (2019) 18(2): 124-132 131 fig. 8. content of caffeine (a) and theobromine (b) in green teas. low and more comparable to powdered green teas (the values in table 7). similarly to srdjenovic et al. (2008) we failed to detect theophylline in the tested tea samples. reports on similar analyses of individual tea types in more detail are rather scarce in literature. the authors of various works that are available only mention whether it is black, green or white tea, in relation to given metabolites contents. furthermore, the many of authors in their works mainly looked at the content of caffeine as a major component in various matrices. for these reasons, comparison of data on individual methylxanthines in different types of teas is difficult and is restricted to the tea group only (black, green and white). conclusions this paper reports on development of hplc method for simultaneous determination of methylxanthines in selected biological matrices. we worked only with isocratic elution, where we searched for appropriate type and composition of the mobile phase. we identified the mobile phase consisting of water : acetonitrile better than mixture of water and methanol because the obtained peaks were narrower and more symmetric. in both types of tested teas (black and green) the presence of theobromine and caffeine was quantified. presence of theophylline was not detected since it generally occurs in teas at very low concentrations and therefore its values were below our limit of detection. from a general comparison of theobromine content in green and black teas, we can conclude that theobromine content is higher in black teas. concentration values of caffeine in green and black teas are overlapping. the content of theobromine and caffeine and differences between green and black teas are probably related to fermentation process apply ing to black tea preparation and on prolongation of enzymatic activity of xanthine methylases which produce caffeine form theophylline and theobromine. it means primary content will be depending on condition of fermentation process in black teas. secondary content of methylxanthines found in tea is highly dependent on the sample preparation, especially leaching time and water temperature. acknowledgement authors thanks to mgr. milica jahelková from tea trade – čajový dom for providing of tea samples. this work was supported by the slovak research and development agency under the contracts no. appvv-17-113 and apvv-16-0173. conflict of interest the authors declare that they have no conflict of interest. references abbood a, aldiab d (2017) hplc determination of caffeine in some beverages and pharmaceutical dosage forms available in syrian market. j. chem. pharm. sci. 10: 11741179. bae ik, ham hm, jeong mh, kim dh, kim hj (2015) simultaneous determination of 15 phenolic compounds and caffeine in teas and mate using rp-hplc/uv detection: method development and optimization of extraction process. food chem. 172: 469-475. de mejia eg, ramirez-mares mv (2014) impact of caffeine and coffee on our health. trends endocrinol. metab. 25: 489-492. a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:30 utc nova biotechnol chim (2019) 18(2): 124-132 132 gibson cm, fowler pw (2014) aromaticity of caffeine, xanthine and the dimethyl xanthines. tetrahedron lett. 55: 2078-2081. grujić-letić n, rakić b, šefer e, milanović m, vujić i, milić n (2016) quantitative determination of caffeine in different matrices. mac. pharm. bull. 62: 77-84. jafari mt, rezaei b, javaheri m (2011) a new method based on electrospray ionisation ion mobility spectrometry (esiims) for simultaneous determination of caffeine and theophylline. food chem. 126: 1964-1970. mufakkar m, fatima k, faheem h, ahmad s, khan mh (2015) estimation of caffeine in different brands of energy drinks by ultra-violet spectroscopy. int. j. sci. res. pub. 5: 1-3. rodriguez a, costa-bauza a, saez-torres c, rodrigo d, grases f (2015) hplc method for urinary theobromine determination: effect of consumption of cocoa products on theobromine urinary excretion in children. clin. biochem. 48: 1138-1143. srdjenovic b, djordjevic-milic v, grujic n, injac r, lepojevic z (2008) simultaneous hplc determination of caffeine, theobromine, and theophylline in food, drinks and herbal products. j. chromatogr. sci. 46: 144-149. tzanavaras dp, zacharisa ck, themelisa dg (2010) rapid determination of methylxanthines in real samples by highperformance liquid chromatography using the new fastgradient® narrow-bore monolithic column. talanta 81: 1494-1501. xia z, ni y, kokot s (2013) simultaneous determination of caffeine, theophylline and theobromine in food samples by a kinetic spectrophotometric method. food chem. 141: 4087-4093. zhu b, haghi m, nguyen a, goud m, yeung s, young pm, traini d (2015) delivery of theophylline as dry powder for inhalation. asian j. pharm. sci. 10: 520-527. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:30 utc nova biotechnol chim (2021) 20(1): e890 doi: 10.36547/nbc.890 1 nova biotechnologica et chimica phytotoxic effects of cdte quantum dots on root meristems of allium cepa l. oleksandr smirnov 1, 2, , mariia kovalenko 1 , leila-аnastasiia karpets 1, 2 , volodymyr dzhagan 3, 4 , olga kapush 3 , veronika dzhagan 1 , yevheniia konotop 1 , nataliya taran 1 1 department of plant biology, educational and scientific centre “institute of biology and medicine”, taras shevchenko national university of kyiv, 64/13 volodymyrska str., kyiv 01601, ukraine 2 institute of plant physiology and genetics, national academy of sciences of ukraine, 31/17, vasylkivska st., kyiv, 03022, ukraine 3 v. lashkaryov institute of semiconductors physics, national academy of sciences of ukraine, 41, pr. nauki, kyiv 03028, ukraine 4 physics department, taras shevchenko national university of kyiv, 64/13 volodymyrska str., kyiv 01601, ukraine  corresponding author: plantaphys@gmail.com article info article history: received: 3 rd march 2021 accepted: 25 th may 2021 keywords: allium-test cdte quantum dots cell viability cytotoxicity mitotic index tolerance index abstract the effect of solutions of cadmium telluride quantum dots (cdte qd) as powerful cytotoxic effectors was investigated using a standard allium cepa l. test system. the diameters of synthesized cdte qd derived from the optical data varied within 3 – 4 nm. toxicity of experimental solutions of cdte qd at the organism level were evaluated by measuring biomass growth of onion roots and cytotoxic influence was estimated based on proliferative activity of root meristem cells. all of cdte qd experimental solutions in concentration 10 µm significantly inhibited the growth of allium cepa l. roots, proliferative activity, and total dehydrogenase activity. relation between the qds size and their phytotoxicity was not found. however, the highest inhibiting impact was for qds solution with a nanocrystals size of 3.5 nm. their ability to penetrate into cells and interact with their intracellular components may cause inhibiting mitosis without fixed clastogenic and aneugenic effects. solutions of cdte qd at a given concentration can be considered as potent cytostatic agents for plant cells with antimitotic properties.  university of ss. cyril and methodius in trnava introduction the extraordinary properties of semiconductor quantum dots (qds) have resulted in their widespread application in various fields, from light-emitting and photovoltaic to chemical sensing and biomedical applications (kargozar et al. 2020; shu et al. 2020; wang et al. 2020). qds of cdbased ii-vi compounds, such as cds, cdse, cdte, are the most developed and understood in terms of both the synthesis and optical properties (yadav et al. 2020). their bright (with photoluminescence quantum yield up to 90 %) coloured emission can be tuned over the whole visible spectrum by varying the qd size or/and composition (i.e. forming allow qds, e.g. cdsexte1-x). however, the toxicity of qds remains a concern and delays their commercialization (jang et al. 2018). an efficient way to reduce the cd toxicity and simultaneously boost their optical properties is covering qd with a shell of more environmentally mailto:plantaphys@gmail.com nova biotechnol chim (2021) 20(1): e890 2 friendly material, e.g. zns, additionally stabilized by bio-compatible polymers (raevskaya et al. 2007). the most recent trend in making fluorescent qds more bio-friendly is synthesizing compounds not containing very toxic metals (cd, hg, or pb), for instance ag-in-s (raevskaya et al. 2018), cuin-se (lox et al. 2018) or cu-zn-sn-s (stroyuk et al. 2018). the latter materials allow tuning of the light absorption and photoluminescence (pl) in the visible and near ir spectral ranges, while zno qds are a good alternative for the uv range (panasiuk et al. 2014). unfortunately, an inherent drawback of all the above cd-free qds is the large spectral width of the pl band, 100 – 200 nm, which does not allow as fine color tunability as in the case of cd-based qds. moreover, recent studies show toxicity also of qds consisting of rather non-toxic elements (kays et al. 2020). therefore, intense research of ii-vi qds with advanced optical properties continues (yeshchenko et al. 2020), currently focused mostly on developing various functionalization of the nc surface and reducing toxicity (borovaya et al. 2016; filali et al. 2019; garmanchuk et al. 2019; lima et al. 2019). of special interest are water-soluble qds synthesized directly in aqueous solutions using bio-compatible stabilizers (ligands) (raievska et al. 2020). better understanding of the behaviour of qds in various biological environments is needed, including their impact on human health, plants, etc. since the possibility of accumulation, uptake, and trophic transfer is long been known for cd ions (bardáčová et al. 2017), allocation of cd-based qds has also been studied (hu et al. 2010; majumdar et al. 2019; sun et al. 2021). from the second half of the 20 th century in ecotoxicological research, the allium-test using various colorants is extensively used for toxicity investigation of various environmental pollutants due to its low cost and high sensitivity (fiskesjö, 1997; leme and marin-morales 2009). studies on the effects of cd ions using allium-test have shown toxicity on macroscopic – growth responses and microscopic levels – decreasing of proliferative activity of root meristems with various clastogenic and aneugenic effects (seth et al. 2008; zou et al. 2012; arya and mukherjee 2014). previously in our investigation, allium-test has also been used for the assessment of toxic impact of stabilized and non-stabilized nanoparticles of essential or biocide metals and their oxides, e.g. cu, c2o, cu2o, fe, fe2o3, fe3o4, zn, zno, mn, mn3o4, mn2o7, ag, ag2o (konotop et al. 2014; 2019). the aim of this work was to investigate toxicity of experimental solutions of cdte qd at levels of individual cells and entire organism using the standard allium cepa l. test system. experimental nanomaterials all chemicals used were of analytical grade or of the highest purity available. milli-q water was used as a solvent. cdi2, naoh, and thioglycolic acid (tga ≥90 %) were purchased from himlaborreactive (ukraine). cdte qds were synthesized by means of colloidal chemistry, following the protocol described by us previously (kapush et al. 2019). briefly, the low-temperature colloidal synthesis was performed in the reactor with complete mixing and in the presence of tga as a stabilizer. deionized water was used as the dispersion medium. cdi2 was dissolved in water and tga was added under stirring, followed by adjusting the ph to 11 by dropwise addition of naoh solution. h2te gas was passed through the solution using argon as a carrier gas. the cdte precursors formed at this stage were subsequently converted into cdte qds after heating the solution at 100 °c. within the given synthesis route (kapush et al. 2019), the ncs of different size can be obtained by varying the magnitude of the electrical current flowing through the electrochemical cell in which the synthesis of the ncs took place. in particular, by changing the current from 0.1 to 0.5 a, a series of samples with the average nc size of 3 nm to 4 nm could be obtained, as estimated from the photoluminescence spectra in fig. 1. photoluminescence pl spectra were measured from colloidal nc solutions in a standard 10  10 mm quartz cuvette using shimadzu rf-1501 fluorimeter. nova biotechnol chim (2021) 20(1): e890 3 plant material and growth conditions the phytotoxicity of water solutions of cadmium telluride quantum dots were studied using the standard test object allium cepa l. (fiskesjö 1985). for each option, 10 equal onion bulbs, cv. goliant, were cultivated on experimental solutions of cdte qd for 5 days at 25 °c. control plants grew in distilled water. experimental solutions were prepared by dilution with distilled water 100 times to achieve concentration of 10 µm of cdte qd. analysis of growth response, genotoxic and cytotoxic effects at the end of exposure time, fresh weight and length of roots per bulb were measured. growth response was estimated according to the tolerance index (ti, %) calculated as the ratio of the fresh length or weight of roots of the experimental group to the same parameter of the control group (wilkins 1978). onion roots were fixed in clark solution (ethanol : acetic acid = 3 : 1) and after maceration in 1m hcl at the temperature 60 °c throughout 15 min were stained with 1 % (w/v) aqueous solution of toluidine blue. the temporary oppressed slides with root’s tips were prepared and further analyzed under a light microscope (primostar, carl zeiss) at 400-fold magnification. different phases of mitosis in cells of the root apical meristem were counted to calculate mitotic index, for evaluation of genotoxic effects of the experimental solutions. the mitotic index (mi, %) was presented as number of cells in the state of division to the total number of cells observed (3,000) (eq. 1): мі = (p+м+а+т)/(і+p+м+а+т) × 100 % (1) where p, m, a, t, i are quantity of cells in pro-, meta-, ana-, telo-, and interphase of mitosis respectively (konotop et al. 2019). cytotoxicity assessment was providing by following the protocol of majumdar et al. (2017) with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) which is based on potential of dehydrogenases of living cells to reduce mtt. for cell viability (cv) investigation, 10 mg of fresh root meristem was transferred to a microcentrifuge tube and 1 ml of 0.1 % (w/v) mtt aqueous solution was added into each tube and incubated 4 h in the dark. after incubation, the mtt solution was discarded and the root meristems were homogenized in 0.5 ml 2n koh and 0.5 ml 99.99 % dmso solution in microcentrifuge tubes. after the color change observed in the solution, the content was centrifuged at 5000g at room temperature for 5 min. the clear supernatant was transferred to 1 ml spectrophotometry cuvette and the absorbance was determined at 570 nm. the optical density (o.d.) value was calculated for 1 mg of fresh tissue. cv was obtained by the formula (eq. 2) and expressed as a percentage (%): cv = (o.d. of control set – o.d. of cdte qds set/o.d. of control set) × 100 % (2) statistical analysis each experiment was performed at least triplicate. the data were subjected to analysis of variance (anova) with subsequent student's t-test or duncan's multiple range test. data are expressed as means of replicates ± standard deviation (bars) and were considered reliable at a significance level of p<0.01. results and discussion cdte qd solutions analysis the cdte qds used in this study were of several different sizes determined from the spectral position of the maximum of their pl emission, following the generally accepted calibration curves (kamal et al. 2019). the pl spectra of the qds solution are shown in fig. 1a, while fig. 1b shows normalized spectra, for a better visibility of the different spectral position of their maxima. the qd diameters derived from the optical data (3 – 4 nm) are attributed to the crystalline part of the qd as schematically shown in inset to the fig. 1a. an important parameter for biological applications and investigations using qds is their hydrodynamic size, which includes the ligand shell and most often also the layer of solvent molecules. in our case, nova biotechnol chim (2021) 20(1): e890 2 when the thioglycolic acid was used as the used as the stabilizer and water as solvent, the hydrodyna fig. 1. photoluminescence spectra of the cdte qds used in this work (measured in parental solutions). absolute pl intensity is in (a) and (b) shows spectra with normalized intensity for better visibility of different peaks position for different samples (indicated on both figures). the inset to (a) schematically shows the qd stabilized by the ligand (tga). hereinafter: #1 – 5 are numbers of qd experimental solutions. mic diameter of the qds was expected to be larger by not more than 1 nm compared to the one determined from the pl spectra, i.e. 4 – 5 nm, for the series of the samples used in this work. the validity of the above estimation of the hydrodynamic qd size was confirmed by dynamic light scattering investigations of other samples pre pared by the same synthesis approach. growth response the obtained results testify that the experimental solutions of cdte qds vastly influence the growth of the roots of allium cepa l. (fig. 2). fig. 2. growth response of allium cepa l. on 5 th day of cultivation on cdte qd experimental solutions (#1 – 5 are numbers of qd experimental solutions). morphometric indices of onion roots under the cultivation on experimental solutions are presented in fig. 3. all solutions of cdte qds inhibited the roots growth more than by 50 %, index of tolerance (ti) calculated by length (fig. 3a) showed less obvious toxicity influence of experimental solutions than ti calculated by mass (fig 3b). ti of plants grown on the first, fourth and fifth solutions (1, 4, 5) of cdte qds did not differ significantly among themselves. 4 nova biotechnol chim (2021) 20(1): e890 5 the cdte qd aqueous solutions used in this study were chosen because these cd-based semiconductor nanocrystals in contact with aqueous media have been shown to leach cd ions, fig. 3. tolerance index (ti) of allium cepa l. under cultivation on cdte qd experimental solutions. ti (%) is calculated by length (a) and by mass (b) of roots of onion bulbs. hereinafter: control comprises 100 %; means followed by the same letters were not significantly different at p<0.01 according to the duncan’s multiple range test (#1 – 5 are numbers of qd experimental solutions). which are known for its high toxicity (el rasafi et al. 2020). qds are fluorescent unique semiconductor nanocomposites that are being widely developed for their strictly size-dependent physicochemical, optical and photophysical traits (rocha et al. 2017). these traits have made qds a suitable option for applications in various mainstream market products, including lightemitting devices such as displays of smartphones, computers and television (hobson 2009). furthermore, it has perspectives in biomedical research, mainly as more affective labels for multitarget simultaneous multicolor imaging of tissues and cells compared with other molecular biotechnologies and traditional fluorescent materials (wang et al. 2020). despite the fact that cd-based qds are causing agents of reactive oxygen species (ros) in plant and animal cells and free radical damage cellular compartments, their mechanisms of phytotoxicity are still being studied (michalet et al. 2005; banerjee et al. 2021). the root growth did not depend on the size of qd in the culture medium. similarly to modlitbová et al. (2018), bulbs of allium cepa in control variant displayed significantly greater total length of the root system in comparison to the bulbs exposed to 10 μm of all cdte qd experimental solutions. toxicity of cdte qd was connected with leaching of free cd ions from cdte qds (modlitbová et al. 2018). furthermore, these authors claim that matching of the manifestation of toxicity between cdcl2 and cdte qds showed no significant differ rences in growth response at both tested concentrations and both terms of exposure. cytotoxicity and genotoxicity to understand the causes of growth responses of plant under the impact of cdte qds, microscopic investigation of a. cepa root meristem cells was conducted (fig. 4). fig. 4. root meristem cells of control variant allium cepa l. in the different phases of mitosis on the 5 th day of cultivation (magnification 400-fold): 1 – interphase, 2 – prophase, 3 – metaphase, 4 – anaphase, 5 – telophase. the effect of cdte qds solutions on the root cells division is shown in fig. 5. the phytotoxic effect was noted for all experimental solutions – mitotic index of root meristem cells significantly differed from the control. all solutions of cdte qds inhibited proliferative activity of root meristems more than by 50 %. the most reduced proliferative nova biotechnol chim (2021) 20(1): e890 6 activity of cells of root apical meristem by 78 % was observed in plants grown on the third solution of cdte qds that accorded with ti of the same experimental option. first, second, fourth and fifth solutions (1, 2, 4, 5) of cdte qds vastly inhibited the mitosis in root meristem cells by 51 %, 58 %, 59 % and 49 % respectively (fig. 5). fig. 5. proliferative activities of cells of allium cepa l. root meristems (vertical axis is mi, %) under cultivation on cdte qd experimental solutions (#1 – 5 are numbers of qd experimental solutions). cdte qds capable of releasing free cadmium ions, leading to the disruption of dna replication, chromosomal aberrations, gene mutation and the generation of ros (el rasafi et al. 2020). su et al. (2010), on the model of human embryonic kidney cells, shown the higher level of cytotoxicity of cdte qds than that of cd ions and that the cytotoxicity was not only because of free cd ions releasing. cytotoxicity was found to be closely depended on the size of quantum dots, with smaller (2 nm) diameter qds demonstrating more toxic influence than larger (5 nm) qds (lovrić et al. 2005). chen et al. (2014) demonstrated the potential toxic effects of qds on inducing ros generation, pro-oxidant scavengers’ activity and dna laddering in root and shoot tissues of wheat plants. investigation of root cells death or meristem cells viability based on method that shown (nad(p)h) dependent dehydrogenase enzyme attendant in living cells is capable of reducing tetrazolium dye mtt. this enzyme activity takes place when the mitochondria are active therefore, the rate of reduction can directly relate to the number of viable cells. the metabolically inactive (dead) cells do not show this ability (majumdar et al. (2017). analysis of mtt-test results shown that first, second, fourth and fifth solutions of cdte qds (1, 2, 4, 5 in fig. 6) inhibited total dehydrogenase activity of root meristem cells approximately by 25 – 30 %. fig. 6. the percentage of cell viability of root meristems of allium cepa l. under cultivation on cdte qd experimental solutions (#1 – 5 are numbers of qd experimental solutions). the most reduced total dehydrogenase activity of cells of root apical meristem (52 %) was observed in plants grown on the third solution of cdte qds (1) that accorded with both ti and mi traits of the same experimental option. since experimental solutions of cdte qd affected growth parameters and proliferative features less than dehydrogenase activity of the root meristem, a relatively high cell viability rate could be explained by involving the cdte qds as cytostatic and no cytocidal effectors in the cell cycle of animal cells (zalgevičienė et al. 2012; garmanchuk et al. 2019). on the other hand, genotoxic influence of cd ions was manifested in the form of various clastogenic and aneugenic breaches in anaphase and telophase steps of mitosis in root meristem cells of allium cepa l. and glycine max l. (seth et al. 2008; zou et al. 2012; arya and mukherjee 2014), disrupting the organization of cytoskeleton elements (gzyl et al. 2015). conclusion recent review (pagano et al. 2018) suggested that the extent to which quantum dots affect plant growth and development and their potential toxic effects may depend on qds size, structure and stabilization characteristics. nevertheless, even the shell presence around the qd core does not prevent the penetration and accumulation of qds in plant nova biotechnol chim (2021) 20(1): e890 7 tissues (modlitbová et al. 2018). due to their size, the qds used in our investigation could be absorbed by the onion root. however, links between the qds size and their phytotoxicity were not found. all tested solutions with nanoparticles with a size of 3 – 4 nm inhibited root growth by more than 50 %, as evidenced by the decrease in ti by length, ti by mass and mi. the most unfavorable conditions for onion plants were in a solution containing qds with a size of 3.5 nm with maximum absorption at 540 nm. the study did not detect genotoxic effects of qds, although other studies have shown mitosis damages caused by cd ions (arya and mukherjee 2014). the inhibition of plant growth could be explained by the cytostatic effect of qds, but the mechanism remains unknown. we suggest future phytotoxicological investigation and prolonged ecotoxicology tests with qds in environmentally relevant conditions to expand our knowledge about possible long-term effects on living organisms. acknowledgement the study was supported partially by fundamental topic of plant biology department of taras shevchenko national university of kyiv 16кф036-07 “solving problematic issues of diversity and stress tolerance of representatives of ukrainian flora and mycobiota under global climate changes” (2016-2020 academic years). conflict of interest authors declare that they have no conflict of interest. references arya sk, mukherjee a (2014) sensitivity of allium cepa and vicia 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dextrangraft-pnipam/au nanoparticle/cdte quantum dots hybrid nanosystem. plasmonics. 75. zalgevičienė v, kulvietis v, bulotienė d, didžiapetrienė j, rotomskis r (2012) the effect of nanoparticles in rats during critical periods of pregnancy. medicina (kaunas). 48: 256-264. zou j, yue j, jiang w, liu d (2012) effects of cadmium stress on root tip cells and some physiological indexes in allium cepa var. agrogarum l. acta biol. cracov. s. botanica. 54: 129-141. 8 nova biotechnol chim (2020) 19(1): 37-51 doi 10.36547/nbc.v19i1.576  corresponding author: toluwase.fatoki@fuoye.edu.ng, fheztbioinformatics@gmail.com nova biotechnologica et chimica in silico study of anticarcinogenic potential of the selenoprotein bthd from drosophila melanogaster. identifying the anticancer peptide crsur from the conserved region toluwase hezekiah fatoki1,2,, omodele ibraheem1, amos olalekan abolaji3 and david morakinyo sanni4 1translational bioinformatics unit, department of biochemistry, federal university oye, pmb 373 oye-ekiti, ekiti state, nigeria 2fhezt bioinformatics laboratory, ifaki-ekiti, ekiti state, nigeria 3department of biochemistry, faculty of basic medical sciences, college of medicine, university of ibadan, oyo state, nigeria 4department of biochemistry, federal university of technology akure, pmb 704, akure, ondo state, nigeria article info article history: received: 28th february 2020 accepted: 5th may 2020 keywords: anticancer peptide drosophila melanogaster mechanism design nf-kappa-b signaling selenoprotein bthd abstract drosophila melanogaster is used as a model system in biomedical studies. selenoprotein is the major biological form of selenium in eukaryotes. selenoproteins are generally involved in catabolic pathways in bacteria and archaea, whereas it participates in anabolic and antioxidant processes in eukaryotic. in this study, anticancer potential of selenoprotein bthd of d. melanogaster was investigated using bioinformatics methods. results showed that selenoprotein bthd of d. melanogaster may have dual properties as evident by its orthology with selenoprotein h (selh) of homo sapiens and conserved domain of fructokinase-like protein 2 of vitis vinifera. these dual properties were also revealed in the phylogenetic analysis, while further structural modeling showed that selenoprotein bthd possibly exists as homotetramer in the native functional structure. the anticancer property of selenoprotein bthd was proposed to be by synergy of antioxidant or redox activities of thioredoxin and glutathione reductase (tgr) domain and the signaling function of fructokinase-like protein 2 domain both in golgi apparatus and cytoplasm, through energy deprivation. the anticancer peptide crsur was identified from conserved region of selenoprotein bthd, of which its cyclic form showed potential anticancer properties predictively through e3 ubiquitin-protein ligase regulating nf-kappa-b signaling by unleashing cells for spontaneous formation of the ripoptosome.  university of ss. cyril and methodius in trnava introduction the genomic sequence of d. melanogaster is about 115229998 bp and contains 13329 annotated genes (adams et al. 2000). drosophila possess genes systems which regulate nutrient uptake, storage and metabolism that are critical to survival and have been found conserved almost in all eukaryotes including humans, brown alga, zebrafish, mouse, escherichia coli, and caenorhabditis elegans (allocca et al. 2018; hatfield et al. 2014). d. melanogaster has been used as a model system for toxicological studies and diseases mechanism such as neurological mailto:toluwase.fatoki@fuoye.edu.ng mailto:fheztbioinformatics@gmail.com nova biotechnol chim (2020) 19(1): 37-51 38 disorders, developmental disorders, metabolic and storage disorders, cancer and cardiovascular disease (bier 2005; abolaji et al. 2014; saraiva et al. 2018). the major biological form of selenium in eukaryotes is selenocysteine (sec) and its mostly found in the active site of selenoproteins. selenocysteine is called the 21st amino acid which has chemical structure differs from cysteine only by the presence of selenium in place of the sulfur atom. sec is co-translationally inserted into a polypeptide chain in response to in-frame uga codons directed by the sec insertion sequence element, a stem-loop structure in the untranslated regions (3-utrs) of selenoprotein mrnas (hatfield et al. 2014). the human genome contains 25 selenoprotein genes (kryukov et al. 2003) and they are involved in a variety of functions, most notably redox homeostasis. larger selenoproteomes can be found in aquatic organisms such as zebrafish, and brown alga (lobanov et al. 2007; hatfield et al. 2014). selenoproteins are generally involved in catabolic pathways in bacteria and archaea, whereas eukaryotic selenoproteins participate rather in anabolic and antioxidant processes (herbette et al. 2007). selenoproteins participate in thyroid hormone metabolism, muscle formation, selenocysteine synthesis and in sperm maturation (rederstorff et al. 2006). according to gladyshev et al. (2016), selenoproteins without known functions include selenof (selenoprotein f, 15-kda selenoprotein, sep15), selenoh (selenoprotein h, selh, c11orf31), selenok (selenoprotein k, selk), selenom (selenoprotein m, selm), selenoo (selenoprotein o, selo), selenot (selenoprotein t, selt), and others. human selenoprotein enzymes with known functions such as thioredoxin reductases (tr1), glutathione peroxidases (sep15 and gpx2) are important cellular redox-regulators needed by both normal and cancer cells, which result in antiand protumorigenic effects at a tissue-specific cellular level (hatfield et al. 2014). in liver tri exhibits anticancer protein and in lung tr1 is a pro-cancer protein and a prime candidate for cancer therapy (yoo et al. 2006; carlson et al. 2012). the gpx1 polymorphisms are associated with cancer risk (zhuo and diamond 2009). it remains to be elucidated whether these antiand pro-tumorigenic effects are tumor stage or grade-dependent. the known selenoproteins in d. melanogaster are dsps2, dselk (former called dselg) and dselh (also known as dselm or bthd) (hirosawatakamori et al. 2000; castellano et al. 2001). dselk has one cysteine homolog and dselh has two (castellano et al. 2001; martin-romero et al. 2001). dselh appears to belong to a class of selenoproteins widely distributed across the phylogenetic spectrum, as dselh was found in zebrafish, human and mouse expressed sequence tag (est) databases (dickiy et al. 2007; novoselov et al. 2007). the ability of dselh to reverse the toxic effects of glutathione depletion in schneider cells was suggested to reflect a glutathione sparing effect via increased activity of an alternative anti-oxidant pathway, which restores the perturbed anti-oxidant-pro-oxidant balance (morozova et al. 2003). disruption of selenophosphate synthetase expression has recently been shown to modulate the ras/mapk signalling cascade in flies (morey et al. 2001). ser/thr kinase domain is one of the core kinase cascades in d. melanogaster and mammals (yin and zhang 2011). in d. melanogaster, ser/thr kinase domain is found in the core kinases of hippo signaling pathway, as well as in the fourjointed and discs overgrown of upstream regulatory components (yin and zhang 2011). defects in the core kinases and some of the upstream components of the pathway lead to robust organ overgrowth and link to numerous cancers (pan 2010; zhang et al. 2009). the hippo signaling pathway limits organ size by regulating cell proliferation and apoptosis. in this study, anticancer potential of selenoprotein bthd of d. melanogaster was investigated based on the available genomic data and publications. the gene product with anticancer properties in an insect could be useful in the development of biologic agent for human cancer therapy. experimental drosophila melanogaster selenoproteins the selenoprotein was searched from d. melanogaster database on ensembl genome browser v97 (http://www.ensembl.org). the genes http://www.ensembl.org/ nova biotechnol chim (2020) 19(1): 37-51 39 obtained from ensembl were looked up in the d. melanogaster database (www.flybase.org). the protein sequences and information were obtained from uniprot database (www.uniprot.org). the protein sequence of bthd of d. melanogaster was used to query homo sapiens database on the blastp server of ncbi (camacho et al. 2009). conserved protein domain human selenoproteins tri, sep15 and gpx2 has been reported to possessed antiand protumorigenic effect (hatfield et al. 2014). the sequence of these proteins was obtained from uniprot. the conserved protein domain of all the selenoproteins of d. melanogaster and four selenoproteins of homo sapiens were investigated using the protein sequences on the cdd server (https://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb .cgi) of ncbi (marchler-bauer et al. 2017). the protein sequence of bthd of d. melanogaster was used to query database of eight plants in the taxonomy of mesangiospermae identified from cdd results, on the blastp server of ncbi (camacho et al. 2009). phylogenetics analysis the protein sequence of selenoproteins of d. melanogaster and h. sapiens obtained from the previous steps, were used for phylogenetics study. multiple sequence alignment on clustalo server (www.ebi.ac.uk/tools/msa/clustalo) was carried out and phylogenetic tree was constructed. the tree data was visualized at www.phylo.io. structural modeling of selenoprotein bthd the three-dimensional structure of selenoprotein bthd of d. melanogaster was modelled on swissmodel server (www.swissmodel.ch) using protein sequence (camacho et al. 2009; remmert et al. 2012; waterhouse et al. 2018). integration of anticarcinogenic mechanism of selenoprotein bthd the d. melanogaster pathways associated with growth of tumor were obtained from www.kegg.jp and available information in the scientific literatures was mined. these data were integrated with the key results of this study and anticarcinogenic mechanism of selenoprotein bthd of d. melanogaster was proposed. in silico prediction anticancer peptides of selenoprotein bthd and their physicochemical properties the prediction of anticancer peptides in selenoprotein bthd was performed on anticp server protein scan (https://webs.iiitd.edu.in/raghava/anticp/submit_pro t.php), and generated the fragments amino acids residues of length of 5 with minimum support vector machine (svm) score of 1.15, and predict their anticancer property along with all table 1. details of selenoprotein genes, and proteins of d. melanogaster integrated from ensembl, flybase and uniprot. gene name chromosome location no. of transcript human orthologs [species\gene symbol] best transcript name and id length [nt] uniprot id length [amino acids] subcellular location bthd chr. x: 13,612,13113,613,228 1 hsap\selenoh bthd-ra, fbtr0073806 977 q9vyb0 249 cytoplasm selg chr. x: 11,887,28411,888,24 1 hsap\selenok selg-ra, fbtr0073570 827 q7z2c4 110 golgi apparatus selr chr. 3r: 10,863,35510,868,642 8 hsap\msrb3 selr-re, fbtr0082295 1156 q8ink9, d3dmp0 208, 192 nucleus, cytoplasm, cytoskeleton selt chr. 2l: 5,010,9225,12,127 3 hsap\selenot selt-ra, fbtr0079000 960 m9pcl4, q9vmv6 198 integral component of membrane, endomembrane system http://www.flybase.org/ http://www.uniprot.org/ https://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi https://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi http://www.ebi.ac.uk/tools/msa/clustalo http://www.phylo.io/ http://www.swissmodel.ch/ http://www.kegg.jp/ https://webs.iiitd.edu.in/raghava/anticp/submit_prot.php https://webs.iiitd.edu.in/raghava/anticp/submit_prot.php nova biotechnol chim (2020) 19(1): 37-51 40 fig. 1. model structures of selenoprotein bthd of d. melanogaster in homotetramer oligomeric state. the important physico-chemical properties like hydrophobicity, charge, pi etc. the cell-penetrating efficacy of peptides were predicted on cppred-rf server (http://server.malab.cn/cppred-rf/index.jsp) (wei et al. 2017). in silico target prediction and pharmacokinetics of anticancer peptides of selenoprotein bthd the structure and smiles (simplified molecular input line entry specification) format of selenocysteine-containing peptide (in cysteine form) was obtained from pepsmi server (https://www.novoprolabs.com/tools/convertpeptide-to-smiles-string) and cysteine residue was edited to selenocysteine on https://pubchem.ncbi.nlm.nih.gov/edit3/index.html. in silico prediction of target was done on swisstargetprediction server, where homo sapiens was selected as target organism (diana et al. 2019). the active peptides (peptides with predicted target) were then subjected to in silico absorption, distribution, metabolism, and excretion (adme) screening on swissadme server at default parameters. (diana et al. 2017). results the search on the ensembl genome browser showed four genes of selenoproteins present in the d. melanogaster genome. the gene products, orthology in h. sapiens and subcellular location were obtained from flybase and uniprot databases, as shown in table 1. each of the four selenoproteins in d. melanogaster has one ortholog in human. the homology alignment of bthd protein sequence (uniprot id: q9vyb0) by blastp confirmed 41.38 % similarity with human selh (c11orf31) selenoprotein (uniprot id: q8izq5) which is located in the golgi apparatus and cytoplasm. the conserved domain of d. melanogaster and h. sapiens are summarized in table 2. the conserved domain of selenoprotein bthd was found to be exceptional with no similarity in h. sapiens unlike the other three. the conserved protein domain of bthd was classified as kinase which belong to pln02967 superfamily of fructokinase-like protein 2 (ec 2.7.1.4), and belong to protein clusters conserved in taxonomy of mesangiospermae in eukaryotic plant. this study is the first to discover and report the plant-like properties of bthd. further homology alignment of the bthd protein sequence against eight plants in the pln02967 superfamily of taxonomy mesangiospermae, only showed 33.33 % similarity to an uncharacterized selenoprotein h (uniprot id: d7su28) of vitis vinifera. moreover, human selh contained domain architecture which is similar to that selt of d. melanogaster and has been noted as thioredoxin and glutathione reductase (tgr domain). this domain is homodimeric, fadcontaining member of the pyridine nucleotide disulfide oxidoreductase family. table 3 shows the summary of homology analysis of bthd protein m1 m2 m3 http://server.malab.cn/cppred-rf/index.jsp https://www.novoprolabs.com/tools/convert-peptide-to-smiles-string https://www.novoprolabs.com/tools/convert-peptide-to-smiles-string https://pubchem.ncbi.nlm.nih.gov/edit3/index.html nova biotechnol chim (2020) 19(1): 37-51 41 nova biotechnol chim (2020) 19(1): 37-51 42 nova biotechnol chim (2020) 19(1): 37-51 43 fig. 2. multiple sequence alignment of all selenoproteins of d. melanogaster, four selenoproteins of h. sapiens and a selenoprotein of v. vinifera. sequence of d. melanogaster against h. sapiens and v. vinifera integrated from ensembl browser v97, blastp of ncbi and uniprot. the structural model parameter of bthd selenoprotein on swissmodel server is shown in table 4. the structure was modeled as homotetramer based on the percentage similarities to three different template proteins; 2ojl (crystal structure of q7waf1_borpa from bordetella parapertussis), 2obk (crystal structure of the putative se binding protein from pseudomonas fluorescens), and 2oka (crystal structure of q9hyq7_pseae from pseudomonas aeruginosa) as shown in fig. 1. this structure can be further elucidated through x-ray crystallography. the result in fig. 2 showed the position of conserved amino acid residues among selenoproteins (d7su28_ vitvi, bthd_drome and selh_human). the phylogenetic tree confirmed the evolutionary relatedness of bthd gene of d. melanogaster, selh gene of h. sapiens and d7su28 gene of v. vinifera (fig. 3). the integral mechanism of anticarcinogenic property of selenoprotein bthd is shown in fig. 4. this mechanism was based on the glycolytic energy stress induced by hepatocytes depletion of atp by fructose and its impact of ampk1, hippo signaling and notch1 signaling pathways; the role of trehalose to regulate glucose metabolism in stress condition; the ability of isomaltose to regulate adenylate biosynthesis; and the capacity v. vinefera to provide fructose and phytochemicals such as resveratrol. based on the mechanism proposed in this study, we have hypothesized that amino acid sequences ehcrsur and gaprrga from selenoprotein bthd as well as ehckqcn and ekprrgc from v. vinefera (grape) could be the key bioactive peptides that can alone and synergistically modulate this mechanism and impact the required antioxidant effect. this was validated by the results of anticancer peptides from selenoprotein bthd shown in table 5, where selenocysteine-containing peptide crsur has svm score of 1.37 and high nova biotechnol chim (2020) 19(1): 37-51 44 fig. 3. the phylogenetic tree of all selenoproteins of d. melanogaster, four selenoproteins of h. sapiens and a selenoprotein of v. vinifera. cell-penetrating uptake efficiency, while rrgaf has svm score of 1.28 and high cell-penetrating uptake efficiency. the structure of the crsur peptide in linear and cyclic (head-to-tail bond) is shown in table 6. the predicted targets of crsur peptide from selenoprotein bthd show that this selenocysteine-containing peptide have anticancer and antiviral or antineuronal properties based on the biological processes by the target genes obtained such as furin, integrin beta3, andbaculoviral iap repeat-containing protein (table 7). the result of pharmacokinetics of crsur (table 8) indicate that gastrointestinal route will not be good for the administration of bioactive peptide. discussion selenoprotein bthd (bthd) has been reported to possesses antioxidant potential (castellano et al. 2001). selenoprotein t (selt) is a thioredoxindisulfide reductase (ec 1.8.1.9) that belongs to the selwth family and selt subfamily. selenoprotein r (selr or msrb) is a peptidemethionine (r)-s-oxide reductase (ec 1.8.4.12) which belongs to the msrb met sulfoxide reductase family (kryukov et al. 2003). the thioredoxin and glutathione reductase (tgr domain) is homodimeric, fad-containing member of the pyridine nucleotide disulfide oxidoreductase family which contains a c-terminal motif cyssecys-gly, where secys is selenocysteine encoded by tga which is a stop codon in some sequence). (sun et al. 2001 tigr02174 domain is a member of the superfamily cl01407 together with related to pfam10262, a domain found in both bacteria and animals selenoproteins selt, selw, and selh (dickiy et al. 2007). taxonomy of mesangiospermae belongs to eukaryotic plant and its pln02967 superfamily was similar to an uncharacterized selenoprotein h (uniprot id: d7su28) of v. vinifera. some studies have reported that grape (v. vinifera) extracts showed cytotoxicity towards cultured cells as well as inhibited tumor growth in animal models (shrotriya et al. 2012; sun et al. 2012). different molecular mechanisms have been proposed for these protective effects of grape extracts, such as inhibition of enzymes playing an essential role in cell proliferation (e.g. human topoisomerase i) and inhibition of angiogenesis (agarwal et al. 2004; stagos et al. 2005). the result of a double-blinded randomized crossover human trial showed that dietary supplementation of grape seed extract at a dose of 600 mg/day for 4 weeks can decrease of oxidative stress and enhance glutathione (gsh)/oxidized glutathione (gssg) and total antioxidant status (kar et al. 2009). the anticancer effects of whole black grape (seeds included) extract have been reported in the cancerous colon tissues of humans by inhibition in dna turnover enzymes (durak et al. 2005). an in silico study has reported the molecular targets for the key bioactive components present in grape such as resveratrol, piceatannol, and scirpusin a (fatoki et al. 2018a). nova biotechnol chim (2020) 19(1): 37-51 45 fig. 4. integrated anticarcinogenic mechanism of selenoprotein bthd. comprehensive review of anticancer properties of grape can be found in another publication (zhou and raffoul 2012). in d. melanogaster, three forms of hexokinases (hex a, b and c) were found by agar gel electrophoresis (madhavan et al. 1972). hex a and hex b have been mapped on the same structural gene on the x chromosome (voelker et al. 1978) while hex c on the second chromosome (jelnes 1971). however, whether these three hexokinases were either aldose or ketose was not investigated. the chromosomal location of bthd is nova biotechnol chim (2020) 19(1): 37-51 46 table 6. structure of the peptide crsur linear and head-to-tail bond. type smiles structure linear n[c@@h](cs)c(=o)n[c@@h](cccnc( =n)n)c(=o)n[c@@h](co)c(=o)n[c@ @h](c[se])c(=o)n[c@@h](cccnc(=n) n)c(=o)o head-to-tail bond n1[c@@h](cs)c(=o)n[c@@h](cccnc (=n)n)c(=o)n[c@@h](co)c(=o)n[c@ @h](c[se])c(=o)n[c@@h](cccnc(=n) n)c1=o chromosome x: 13,612,131-13,613,228 (table 1). fructokinase (also known as ketohexokinase; khk), which catalyzes the phosphorylation of fructose to fructose 1-phosphate, was identified by maldi-tof ms and found expressed at extremely low rates in the renal tumor tissues (hwa et al. 2006). a study has shown that fructoseinduced atp depletion in human, rat and mouse hepatocytes cause full protected against tumor necrosis factor-alpha (tnf-α)-induced cytotoxicity, whereas hepatic tumor cell lines showed increased hexokinase ii (hkii) expression which inhibited fructose-mediated cytoprotection (speicher et al. 2010). study has shown that trehalose do not stimulate rapid increases in blood glucose and excessive secretion of insulin and gastric inhibitory polypeptide (gip) promoting fat accumulation (yoshizane et al. 2017). it has been demonstrated that trehalose-6-phosphate (t6p) inhibits yeast hexokinase 2 (hkii) activity, thus it is likely that this metabolite regulates glycolysis by modulating the flow of phosphorylated sugars towards this pathway (blázquez et al. 1993; nova biotechnol chim (2020) 19(1): 37-51 47 table 7. predicted targets of crsur peptide from selenoprotein bthd. s/n targets uniprot id crsur peptide [% probability] linear head-to-tail bond 1 furin p09958 60 45 2 proprotein convertase subtilisin/kexin type 4, 5, 6 q6uw60, q92824, p29122 60 45 3 complement factor b ba fragment p00751 55 4 complement c2 p06681 55 5 neurotensin receptor type 1, 2 p30989, o95665 50 6 wd repeat-containing protein 5, 5b p61964, q86vz2 40 45 7 coagulation factor vii, ixa heavy chain p08709, p00740 40 40 8 factor x light chain p00742 40 40 9 complex (integrin beta-3) p08514/p05106 35 50 10 e3 ubiquitin-protein ligase xiap p98170 – 45 11 baculoviral iap repeat-containing protein 2, 3, 8 q13490, q13489, q96p09 – 45 probability on target was computed based on a cross-validation. they may therefore not represent the actual probability of success for any new molecule. thevelein et al. 1995). t6p plays a critical role as a sensing molecule that promotes sugar fermentation and glucose repression in yeast (vicente et al. 2018). thus, trehalose diet by cancer patient may be necessary to trigger energy stress which will in turn open up cancer cell to the pathway of selenoprotein bthd. isomaltose will be needed to mitigate against the diabetes signaling molecules that are associated with cancer cell proliferation. isomaltose inhibit adenylosuccinate lyase which is a more proximal enzyme in the adenylosuccinate biosynthesis pathway, lowers s-amp levels and impairs glucosestimulated insulin secretion (fatoki et al. 2018b), and may help to reduce the risks associated with obesity and type 2 diabetes (van can et al. 2012). previous studies haves established that phosphoglycerate kinase 1 (pgk1) and pyruvate kinase m2 (pkm2) the only two atp-generating glycolytic enzymes, which function as protein kinases and play active roles in tumor development (li et al. 2016a). another study has shown that hepatocellular carcinoma (hcc) cells reduce the fructose metabolism rate, and involved a switch in expression from fructokinase c (khk-c) to fructokinase a (khk-a) (li et al. 2016b), and in the process khk-a enhanced nucleic acid synthesis for tumorigenesis (li et al. 2016c), and also enhanced p62’s aggregation with kelchlike ech-associated protein 1 (keap1) and nuclear factor erythroid 2-related factor 2 (nrf2) activation (xu et al. 2019). nrf2 is located in the cytoplasm and guided by keap1, but under oxidative stress nrf2 moves to the nucleus, where it binds the antioxidant response element (are) and drives the expression of several downstream genes such as γ-glutamyl cysteine synthetase modifier subunit (gclm), glutamate cysteine ligase catalytic subunit (gclc), heme oxygenase-1 (ho-1) and nad(p)h quinine oxidoreductase-1 (nqo1) (shibata et al. 2008; singh et al. 2008; ding et al. 2010; kansanen et al. 2013). a novel peptide activator of a key antioxidant gene transcription pathwayin the hippocampus, which disrupted the nrf2–keap1 interaction in global cerebral ischemia model has been reported (tu et al. 2015). the hippo signaling pathway plays a crucial role in cell proliferation, apoptosis, differentiation, and development. transcriptional co-activators yes-associated protein 1 (yap) and ww domaincontaining transcription regulator protein 1 (taz), are major effectors of the hippo signaling pathway (bae et al. 2017). they function as transcription factors along with tead (tea domain family member) in the nucleus, which increases expression of such target genes as ctgf, cyr61, axl, and survivin (bae et al. 2017). study has shown that phosphofructokinase 1 (pfk1) mediates glucose-induced yapand taz-tead interactions (enzo et al. 2015). energy stress, as induced by culturing cells in glucose-free conditions, results in inhibition of yap activity in mouse hepatocytes in vivo as demonstrated by starvation/re-feeding experiments (wang et al. 2015). in recent time, small peptides having anticancer properties have emerged as a potential nova biotechnol chim (2020) 19(1): 37-51 48 table 8. pharmacokinetics of crsur peptide from selenoprotein bthd. s/n parameters crsur peptide linear head-to-tail bond 1 molecular weight [g.mol-1] 669.64 651.62 2 heavy atoms (ha) 41 40 3 molar refractivity 151.53 167.52 4 total polar surface area (å2) 362.55 328.33 5 lipophilicity consensus logp -3.96 -3.73 6 water solubility esol class highly soluble very soluble 7 gastrointestinal absorption low low 8 blood brain barrier (bbb) permeant no no 9 p-glycoprotein substrate no no 10 cytochrome p450s inhibitor no no 11 skin permeation log kp [cm.s-1] -15.29 -12.84 12 lipinski violation 3 3 13 bioavailability score 0.17 0.17 14 synthetic accessibility 5.62 6.40 alternative approach for cancer therapy (thundimadathil et al. 2012). anticancer peptides (acps) are small (5 – 30 amino acids) peptides, often derived from antimicrobial peptides (amps) and are cationic in nature (tyagi et al. 2013), while cell-penetrating peptides (cpps) are small peptides that have unique inherent ability to directly enter cells without significantly damaging the cell membrane (wei et al. 2017). in this study, the selenocysteine-containing peptide crsur was identified and further investigated among all peptides obtained. the results show that cyclic peptide crsur will be a good anticancer agent through e3 ubiquitin-protein ligase regulating nfkappa-b signalling by unleashes cell for spontaneous formation of the ripoptosome, a large multi-protein complex that has the capability to kill cancer cells in a caspase-dependent and caspaseindependent manner (bertrand et al. 2011). conclusions low calories diets can cause significant reduction in tumor incidence and tumor growth, and the mechanistic links between diet and cancer which has remain poorly understood (warr et al. 2018), has been unravelled for application in human health through this in-silico study. we have shown that selenoprotein bthd can be good antioxidant supplement alone or together with the whole fruit juice of v. vinifera, not only against cancer but also virus infection and for overall human health (moghadaszadeh and beggs 2006). thus, it is possible to build on the understanding of the antioxidant/anticancer potential of bthd by investigating new synthetic peptides from the conserved regions. further study will be to 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signaling pathway and organ size control. fly 3: 68-73. zhou k, raffoul jj (2012). potential anticancer properties of grape antioxidants. j. oncol. 2012: 803294. zhuo p, diamond am (2009) molecular mechanisms by which selenoproteins affect cancer risk and progression. biochim. biophys. acta 1790: 15461554. nova biotechnol chim (2020) 19(1): 109-115 doi 10.36547/nbc.v19i1.583  corresponding author: juraj.cernak@upjs.sk nova biotechnologica et chimica hydrogen atom disorder in the crystal structure of o-phenylenediacetic acid lenka krešáková, juraj kuchár and juraj černák department of inorganic chemistry, institute of chemistry, faculty of sciences, p. j. šafárik university in košice, moyzesova 11, 04154 košice, slovak republic article info article history: received: 28th february 2020 accepted: 5th may 2020 keywords: o-phenylenediacetic acid crystal structure hydrogen atom disorder hydrogen bonded dimer abstract h2opda (1) (o-phenylenediacetic acid) was prepared in the form of single crystals. it exhibits molecular crystal structure. the main feature of the supramolecular structure of 1 is formation of hydrogen bonded dimers via a pair of o-ho hydrogen bonds. the hydrogen atoms in cooh groups are disordered in 0.67(2):0.33(2) ratio. the conformation of the free acid is characterized by cis arrangement of the carboxylate groups with respect to the plane of the aromatic ring. the powder diffraction pattern of the bulk sample corroborated the phase identity of the commercial material with that of the studied single crystal.  university of ss. cyril and methodius in trnava introduction o-phenylenediacetic acid (h2opda) is a dicarboxylic acid which, after partial or full deprotonation, can act, in the form of hopda(1-) or opda(2-) anions as counter anion in various salts (imai et al. 2007; garcía-zarracino et al. 2008) as well as o-donor anionic ligand in complexes. within the complexes variable coordination fashions were reported, like terminal hopda(1-) o-donor ligand in [co(hopda)2(pym)2] (pym = hpyrazol-4-yl)methane) (sengupta et al. 2012), bridging o,o´-donor ligand in [cu(opda)(pipm)]4h2o (pipm = (piperazine-1,4diyl)bis[(pyridin-4-yl)methanone) (czarnecki et al. 2019) or bis(chelate) bridging ligand in [cd(opda)(bimb)] (bimb = benzimidazol-1ylmethyl)benzene) (yu et al. 2015) to mention few examples. although the crystal structures containing {o-c6h4(ch2-coo)2} structural fragment are rather numerous, surprisingly, upon the search in csd (groom et al. 2016) the crystal structure of the acid alone was not reported up to now. on the other hand, crystal structures of its isomers, namely m-phenylenediacetic acid, h2mpda (ref-code hofbou) and p-phenylenediacetic acid, h2ppda (rigxov) were already reported (fun et al. 2007; zhu 2008). it is familiar that carboxylate groups, under certain condition, are able to form polymeric-like catemer chains within crystals (d’ascenzo and auffinger 2015). thus comprehensive structure determination and classification of these supramolecular motifs should be of interest in pharmaceutical industry, biomolecular sciences, or crystal engineering. similar motifs occur in the form of amino acids or motifs involving ligands bearing carboxylate groups (modec and klančar 2012). following our experiments with the aim to prepare single crystals of magnetically active ce(iii) complex with opda(2-) ligand we have unexpectedly obtained single crystals of the free acid. here we report identification, crystal, molecular and supramolecular structure of h2opda. mailto:juraj.cernak@upjs.sk nova biotechnol chim (2020) 19(1): 109-115 110 experimental materials cerium(iii) nitrate, ce(no3)3, naoh, h2pda and ethanol (99.8 %) were purchased from commercial sources and used as received. synthesis of single crystals of h2pda to the colorless aqueous solution of ce(no3)3 (200 mg, 0.46 mmol, 10 ml of h2o) was added a water (15 ml) solution of h2opda (134 mg, 0.69 mmol). the formed solution with ph adjusted to ph = 3 using 0.1 mol.l-1 aqueous solution of naoh was refluxed 90 min yielding yellow microcrystalline powder which was separated by filtration and the light yellow filtrate was left aside. after one week separated light yellow crystals (lk9) which were separated by filtration, rinsed quickly by small amounts of ethanol and diethylether and dried on air. ir (cm–1): 3065 (w), 3027 (w), 2977 (w), 2939 (w), 2654 (br), 2548 (br), 1682 (s), 1605 (w), 1493 (m), 1453 (m), 1436 (w), 1416 (m), 1394 (m), 1341 (m), 1314 (w), 1284 (w), 1252 (br), 1209 (m), 1191 (w), 1155 (s), 1095 (w), 1050 (w), 910 (br), 812 (m), 758 (m), 742 (s), 701 (s), 667 (m), 650 (m), 626 (m), 543 (m), 500 (m), 445 (m), 413 (w). 1h nmr: [ppm] 3.6 s(4h), 7.21 s(4h-ar), 12.15 s(2h-oh) 13cnmr: [ppm] 39.5 (2xch2), 126.9 (c4,c5), 130.6 (c3,c6), 134.09 (c1,c2), 172.4 (cooh) physical measurements the ir were recorded on a nicolet 6700 ft-ir spectrophotometer from thermo scientific equipped with a diamond crystal smart orbit™ in the range 4000 – 400 cm-1. nmr data were collected at room temperature on a varian vnmrs spectrometer operating at 599.87 mhz for 1h, and 150.84 mhz for 13c. spectra were recorded in dmso-d6 and the chemical shifts were referenced to the residual solvent signal (1h nmr 2.50 ppm, 13c nmr 39.5 ppm). the 2d gcosy, ghsqc and ghmbc (optimized for a long range coupling of 8 hz) methods were employed. x–ray crystallography x–ray experiments were carried out on a fourcircle -axis xcalibur2 diffractometer equipped with a ccd detector sapphire2 (oxford diffraction). the crysalis software package (oxford diffraction 2003) was used for data collection and reduction. analytical absorption corrections using crystal faces were applied (clark and reid 1995). the crystal structure of h2opda was solved by direct methods and further refined using the shelxtl and shelxl programs (sheldrick 2015), respectively, incorporated in the wingx program package (farrugia 2012). hydrogen atoms of the aromatic ring and methylene groups were placed in the calculated positions and allowed to ride on the parent atoms with isotropic thermal parameters tied with the parent atoms (u(h) = 1.2u(ch2)). hydrogen atoms h11 and h31 of the cooh groups involving o1 and o3 atoms, resp. were easily found in the difference map and were included in the refinement first as idealized oh groups riding on the parent o atom; their isotropic thermal parameters were tied with the parent o atoms (u(h) = 1.5u(o)). during last cycles of the refinement it was found that the differences in c-o(h) and c=o distances are rather small (see discussion) and, in addition, in the difference map were present small maxima at the positions attributable to hydrogen atoms near (c=)o2 and (c=)o4 oxygen atoms. these findings indicated possible disorder, i.e. partial protonation of both oxygen atoms within the respective cooh groups. the adoption of this model allowed to refine occupancy factors for both orientations of protonation which converged to ratio 0.67(2):0.33(2) with refinement of the positions of hydrogen atoms; the o-h distances were restrained to 0.82(1) å. it should be noted that the refinement program did not indicate splitting of the corresponding oxygen atoms. the structural figures were drawn using the diamond software (brandenburg 2008). some geometric parameters were calculated using the parst program (nardelli 1999). program mercury csd 4.2.0 development (macrae et al. 2006) was used for comparison of molecular structures. the hirshfeld surface was calculated using the program crystalexplorer17 (turner et al. nova biotechnol chim (2020) 19(1): 109-115 111 table 1. crystal data and structure refinement for h2opda. empirical formula c10 h10 o4 formula weight 194.18 temperature [k] 291(2) wavelength [å] 0.71073 å crystal system monoclinic space group p21/n unit cell dimensions [å, ] a = 14.5276(15) b = 4.8274(3) c = 14.8183(13)  =118.058(13) volume [å3] 917.08(17) z 4 density (calculated) [g.cm-3] 1.406 absorption coefficient [mm-1] 0.110 crystal dimensions [mm3] 0.867 x 0.193 x 0.057  range for data collection [] 3.116 to 26.493 index ranges –9  h  17, –5  k  5, –17  l  12 reflections coll./ obs. 3078/1709 absorption correction analytical tmin/tmax 0.942 / 0.994 goodness-of-fit on f2 (all/obs.) 1.035/1.034 final r indices [i>2(i)] r1 = 0.0349, wr2 = 0.0802 r indices (all data) r1 = 0.0508, wr2 = 0.0900 largest diff. peak and hole [e å–3] 0.160 and –0.145 2017). crystal data and final parameters of the structure refinement are summarized in table 1, while selected geometric parameters are given in table 2. possible hydrogen bonds are gathered in table 3. results and discussion synthesis and spectroscopic characterization single crystals of the h2opda suitable for single crystal study were obtained unexpectedly by crystallization from the reaction mixture containing cerium nitrate, naoh and h2opda. we note that our effort to prepare single crystals by simple recrystallization of the commercially obtained h2opda from various solvents were unsuccessful; the “best” results were obtained by recrystallization from acetone but the tiny dendritic needles were not suitable for single crystal study. the obtained single crystals of h2opda were checked by ft-ir, 1h and 13c nmr spectroscopies; the observed absorption bands and positions of the resonance pics are gathered in the experimental part. crystal structure the crystal structure of h2opda is molecular (fig. 1) and the unit cell contains 4 molecules of h2opda.there are three points concerning the structural results which merit some comments. the first issue concerns the c-o(h) and c=o bond distances. in h2opda the mean experimental c-o(h) and c=o bond distances are 1.277 å and 1.248 å, resp. (table 2). the search in csd (groom et al. 2016) has shown that these values are from the ranges found for corresponding bond table 2. selected geometric parameters [å, ] for h2opda. c8-o1 1.2824(17) c10-o3 1.2695(18) c8-o2 1.2473(17) c10-o4 1.2477(19) c7-c8 1.496(2) c9-c10 1.507(2) c1-c7 1.5123(19) c2-c9 1.516(2) o1-c8-o2 123.51(13) o3-c10-o4 123.50(14) nova biotechnol chim (2020) 19(1): 109-115 112 table 3. hydrogen bonds [å, º] in h2opda; only the thus with higher occupied hydrogen atoms are shown. d-h...a d(d-h) d(h∙∙∙a) d(d∙∙∙a) <(dha) o1-h11∙∙∙o4i 0.827(10) 1.884(11) 2.7053(15) 172(3) o3-h31∙∙∙o2i 0.819(10) 1.841(10) 2.6598(15) 177(3) symmetry code: i: 1 x, 1 y, 1 z. distances in structures containing {c6-ch2-cooh} structural fragment (c6 is an aromatic ring with any substituent; organic structures, 198 hits), e.g. similar values of 1.279 å and 1.247 å were found in (2,3-dihydro-1h-benzo[b]cyclopenta[d]furan-8-yl)acetic acid (widyam) (sun et al. 2018). on the other hand, the mean values of 1.31(2) and 1.22(2) å for c-o(h) and c=o bonds, resp., in carboxyl-syn structural fragment were reported (d’ascenzo and auffinger 2015). results of our refinement (see the experimental part) suggest that the rather small observed differences between the c-o(h) and c=o bonds can be explained by hydrogen atom disorder within both cooh groups. analogous hydrogen atom disorder associated with variation of the corresponding c-o(h) and c=o bonds studied by means of neutron diffraction was reported for benzoic acid (wilson et al. 1996). the second point worth of discussion is the conformation of the h2opda molecule, more specifically, the positions of the carboxylate groups with respect to the plane of the aromatic ring. while c7 and c9 methylene carbon atoms lie almost in the plane of the aromatic ring (the fig. 1. thermal ellipsoid plot of the molecular structure of h2opda along with atoms numbering scheme. the thermal ellipsoids are drawn on 30 % probability level. hydrogen atoms within the carboxylate groups are disordered (h11/h21 and h31/h41) with occupation factors in the 0.67(2):0.33(2) ratio. distances of these c7 and c9 atoms from the aromatic plane are 0.0055(16) å (c7) and 0.0140(16) å (c9), the c8 and c10 atoms of the carboxylate groups are both above the plane formed by the aromatic ring (cis arrangement). these positions of the carboxylate groups can be expressed by torsion angles c6-c1-c7-c8 and c1-c2-c9-c10 with values of 94.61(17) and 75.75(18)°, respectively. we note that the planes formed by carboxylate groups deviate only slightly from co-planarity as can be seen from the value of 10.74(17)° for the angle between the two planes formed by the respective {coo} groups. it may be interesting to compare the conformation of the h2opda molecule, more precisely the positions of the carboxylate groups with respect to the plane of the aromatic ring, with the corresponding structural entities in potassium salt, k(hopda) (garcía-zarracino et al. 2008) and four reported salts containing organic cations (imai et al. 2007; modec and klačnar 2012; liu et al. 2016; lin et al. 2017). in the potassium salt, like in the free acid, both coo and cooh groups are placed in cis arrangement with respect to the aromatic plane, like in the free acid, and the fig. 2. comparison of the molecular structures of h2opda molecule (green) and hopda(-) anion in k(hopda) (red) (left), and h2opda molecule (green) and opda(2-) anion in (dpenh2)(opda)ch3oh (red) (right). the structures were superimposed in such a way that the aromatic rings are overlapped as close as possible. nova biotechnol chim (2020) 19(1): 109-115 113 fig. 3. view on the supramolecular hydrogen bonded dimers of h2opda. only hydrogen atoms with higher s.o.f.s are shown for clarity. r1 and r2 are hydrogen bonded rings, both exhibit descriptor . symmetry code: i: 1 x, 1 y, 1 z. corresponding torsion angles of 97.3(2) and 72.1(3)° are comparable with those found in h2opda; the only difference is the slight rotation of both coo groups around the c-c single bond (see fig. 2, left). on the other hand, in all four salts with organic cations, the two acetate groups are oriented in the space oppositely, i.e. one is situated above and the second below the plane of the aromatic ring (trans arrangement). as an example we can mention (dpenh2)(opda)ch3oh (dpenh2 2+ = (1r,2r)-1,2diphenylethylenediammonium dication) (imai et al. 2007) in which the corresponding torsion angles are 97.83 and -83.12°; the structure overlay of the anion opda(2-) in this salt with h2opda is shown on fig. 2 (right). it seems the {opda} structural fragment due to its flexibility as to the rotation around methylene group is enough flexible to form in the crystalline state conformation leading to maximization of the lattice energy. the third point to mention is the formed supramolecular structure. two molecules of h2opda, the asymmetric one and its congener by center of symmetry, are linked by a pair of relatively strong hydrogen bonds of the o-h···o type yielding supramolecular dimers (h2opda)2 (table 3, fig. 3); the hydrogen bonded ring systems can be described using descriptor according to graph set analysis (bernstein et al. 1995). we note that no stacking interactions (the shortest distance between the centers of gravity of the aromatic ring is 4.7856(5) å) nor car-h···o weak hydrogen bonds were found, so the formed supramolecular dimers interact only by weak intermolecular interactions of van der waals nature. the shortest contact is the distance of 2.60 å between (c7-)h7a hydrogen atom from methylene group and o2ii (ii: x, 1 + y, z) atom from carboxylate group (c7o2 distance is 3.4884(4) å). accordingly, the calculated hirshfeld surface of the supramolecular dimer is featureless and the very weak pink colored circles correspond to the above mentioned contacts (fig. 4, upper). fig. 4. upper: hirshfeld surface of the hydrogen bonded dimers [h2opda]2. lower: hirshfeld surface of the h2opda molecule. the hydrogen bonded congener molecule (ball model) is also shown. hydrogen bonds leading to dimerization are shown as green dashed lines. nova biotechnol chim (2020) 19(1): 109-115 114 fig. 5. packing diagram of h2opda. on the fig. 4 (lower) is shown the hirshfeld surface of the h2opda molecule alone clearly displaying the red spots which reflect the formation of o-h···o type hydrogen bonds. the fig. 5 shows the packing of the supramolecular dimers. conclusions single crystals of h2opda crystallized from the reaction mixture formed of cerium(iii) nitrate, naoh and h2opda. their chemical identity was corroborated by spectroscopic methods. single crystal x-ray structure analysis confirmed the molecular structure of the free acid with cis arrangement of the two acetate groups with respect to the aromatic plane. results of the structure analysis indicate hydrogen atom disorder in ratio 33 and 67 % within the carboxylate groups. two h2opda molecules are linked by a pair of oh···o type hydrogen bonds yielding supramolecular dimers, which are linked only by weak intermolecular interactions. supporting information crystallographic data for the compound h2opda has been deposited with the cambridge crystallographic data centre, ccdc 1981160. copies of the information may be obtained free of charge from the director, ccdc, 12 union road, cambridge, cb21ez, uk (fax: +44–1223–336033; e-mail: deposit@ccdc.cam.ac.uk or www: http://www.ccdc.cam.ac.uk). acknowledgement this work was supported by the slovak grant agencies apvv-18-0016 and vega 1/0063/17. we thank dr. vilková for measuring the nmr spectra. conflict of interest the authors declare that they have no conflict of interest. references d’ascenzo l, auffinger p (2015) a comprehensive classification and nomenclature of carboxyl-carboxyl(ate) supramolecular motifs and related catemers: implications for biomolecular systems. acta crystallogr. b struct. sci. cryst. eng. mater. 71: 164-175. 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vandhana kumar2, sheenal aashna2, shipaldika verma2, zafiar tasmeen naaz2, a. a. mohamed hatha4 1school of agriculture, geography, environment, ocean and natural sciences, the university of the south pacific, suva, fiji 2department of science, school of science and technology, the university of fiji, lautoka, fiji 3department of fisheries, college of agriculture, fisheries and forestry, fiji national university, koronivia, fiji 4department of marine biology, microbiology and biochemistry, cochin university of science and technology, kerala, india  corresponding author: ajaymsp1@gmail.com article info article history: received: 18th january 2021 accepted: 21st may 2021 keywords: aerobic plate count batissa violacea biofilter bivalve coliform depuration abstract the effectiveness of the freshwater bivalve batissa violacea depuration was tested in closed and open water circulatory system over a 48 h period. the closed circulatory system included a sand biofilter. microbial levels were assessed every 4 h using total aerobic plate count (tpc) for heterotrophs and most probable number (mpn) for coliforms. tpc and coliform loads in bivalve tissue reduced rapidly to low and undetectable levels in a closed circulatory system while open system showed a slower reduction. both tpc and coliform loads remained above detectable levels throughout the depuration period. closed system showed similar patterns of logarithmic reduction of tpc and coliforms in all cases with r2>0.95 and p<0.001. similar results were observed for tank water however, reduction of tpc and coliforms were slower. biofilm formation was observed in the interior walls of the aquarium tanks over 48 h in all cases. physicochemical parameters did not show any significant change. the reduction in tpc and coliform load in b. violacea suggests that biofilter in a closed water circulatory system is a simple, cost-effective, water conserving and effective way to significantly reduce the spoilage and coliform bacterial load that is accumulated in the clams.  university of ss. cyril and methodius in trnava introduction pacific island countries (pics) have a large proportion of populations residing near natural water bodies including the coastal and river systems (andrew et al. 2019). these populations generally have heavy socio-economic and subsistence dependence on these coastal resources from these sources including fiji (waqalevu 2015; andrew et al. 2019). bivalves are known as a rich source of protein and are highly perishable in untreated form which hinders the process of transport and storage (frazier and westhoff 2000; udoh et al. 2017). shellfish-borne bacterial and viral diseases are one of the major concerns for public health (botana 2014). bivalves accumulate heavy metal toxins and various pathogens from fecally contaminated natural water during their filter feeding behaviour (hatha et al. 2005a; botana 2014; waqalevu 2015). therefore, inappropriate disposal of raw and partially treated sewage into the natural water mailto:ajaymsp1@gmail.com nova biotechnol chim (2021) 20(2): e824 2 bodies is a major factor contributing to the increasing incidence of shellfish-borne illness (hatha et al. 2005a). escherichia coli is considered as a common fecal indicator bacterium (coliform) which is used to measure microbial contamination of bivalve molluscs (murchie et al. 2005) and is directly related to the degree of anthropogenic impact. since specific pathogen detection is always time-consuming and costly, e. coli is used as a surrogate organism, the numbers of which provide an estimate of degree of pollution and possible presence of pathogens that it contains (oliveira et al. 2011). batissa violacea or kai, as known locally, is a freshwater bivalve that serves as a popular food item in fiji (richards et al. 1994). fishermen around rewa river in fiji catch bivalves (waqalevu 2015) for subsistence consumption or to sell it in supermarkets (frozen and without shells) and in live condition in local market and roadside stalls. during sale, the bivalves are normally stored at ambient temperature that is conducive for proliferation of pathogenic and mesophilic bacteria (hatha et al. 2005a; waqalevu 2015). they are generally less processed and are mostly known to be consumed raw (oliveira et al. 2011). b. violacea is a filter feeder targeting suspended matter including sediments, detritus, diatoms and algae (mayor et al. 2018). filter feeding exposes the bivalve to high levels of varied bacterial flora including pathogenic and non-pathogenic bacteria (hatha et al. 2005a; waqalevu 2015; oranusi et al. 2018). depuration is a technique used worldwide within the shellfish industry. depuration of bivalves typically takes place for 48 h and has proven to reduce the number of bacterial load in shellfish significantly (amadi 2016; waqalevu 2015). during the depuration, shellfish continue to filter feed in clean running water, and previously ingested microorganisms are eventually expelled and entrapped in a thread like faeces coated with layer of mucous. when bivalve molluscs are held in clean water in tanks for several hours it maximizes the expulsion of intestinal contents. waqalevu (2015) used a uv filter system for depuration of b. violacea from rewa river of fiji over a three-day period. reduction of coliform numbers were observed however, coliforms still showed significant presence in the bivalve and tank water even after 72 h of depuration. also, the depuration system was water intensive and not as economical for local use. a more efficient and economical depuration system is required for b. violacea. hatha et al. (2005a) investigated the gut contents of b. violacea from rewa river and found a varied flora of spoilage and pathogenic bacterial load. a suitable technique is required to reduce bacterial load from the bivalves to make it safe for consumption. both waqalevu (2015) and hatha et al. (2005a) recommended an effective and economical depuration system for b. violacea in fiji. an economical depuration system based on recirculating water through a biofilter could greatly conserve water and achieve depuration of the bivalve clams to safe levels of bacterial load for human consumption. this study was aimed at: (1) determining the effect of depuration on total aerobic bacteria and coliform levels in b. violacea tissue; (2) comparing the effectiveness of closed water circulatory system having a biofilter set-up with an open water system having continuous flow of water on total aerobic bacteria and coliform load in b. violacea tissue over a 48 h depuration period. experimental sample collection and site bivalve samples were collected from a sandy freshwater river ecosystem called rewa river, located in the south-eastern region of the island of viti levu in fiji (178°31'14.3"e and 178°31'14.5"e longitude; 17°59'12.2"s and 18°00'51.0"s latitude) (fig. 1). samples were hand collected by divers from depths of 1 to 3 m using polythene bags. a water sample was taken at the site by dipping a sterile airtight glass bottle about 1 m below the surface and holding it faced opposite the current flow for 1 min. this sample was taken back to the lab for ph, salinity, dissolved oxygen (do) and bacteriological analysis. water temperature was taken of the river at the collection site using alcohol bulb thermometer (thomas scientific 1173h09). sediment samples were taken aseptically by scooping out from the river bottom into sterile polythene bags. more than 400 live clams were nova biotechnol chim (2021) 20(2): e824 3 collected by scooping them with hand from the sediment at the bottom of the river and placed in sterile polythene bags. a sample of two clams was immediately taken in sterile polyethylene bag and fig. 1. location of the sample collection site in the southeastern region of the island of viti levu in fiji. rewa river watershed is shown. sample collection area within rewa river (baulevu) is marked in black box (dotted line). inset shows fiji and its location within the south pacific region. transferred into a cooler with ice for total aerobic plate count (tpc) and coliform count. the time interval for transporting of kai from site to depuration tanks was approximately 30 min. more than 400 clams were transported to the laboratory in live condition maintained at around 20 oc in a sterile container. upon reaching the lab the clams were transferred into two aquarium tanks (made of silica glass) in equal numbers (200 bivalves each). the time between collection of samples and transferring the bivalves in the aquarium tanks was approximately 1 h. the first tank was maintained with closed water circulatory system with a biofilter set-up while the second tank was maintained under open water system without a biofilter. both set-ups are shown in fig. 2. experimental set-up and sampling bivalves were placed in the tanks on a mesh platform below 20 cm depth from the water surface and at least 20 cm from the bottom surface (fig. 2). after placing the bivalves in each aquarium tank, two bivalves were removed and placed in a homogenizing sterile bag. this was noted as sampling at zero h. tap water (chlorinated) was used for the depuration tanks. the temperature of water in the aquarium tank was taken before placing the clams inside the aquarium tank. tank water and swab sample of the aquarium tank were taken. 10 ml of tank water was taken from below the water surface aseptically in sterile vials. a water sample was also taken prior to introduction of bivalves in the tanks. for swab samples, a sterile cotton swab was taken and dipped in a tube of nova biotechnol chim (2021) 20(2): e824 4 sterile distilled water (sdw). excess water was driven out by pressing against the walls of the tube. the inside walls of the aquarium tank were swabbed in a square of ~5 cm × 5 cm (25 cm2). the tip of the swab was then cut off in a 10 ml dilution blank of sdw. bivalve, water, swab samples and temperature were taken at every 4 h interval for up to 48 h. ph, salinity and, do were also measured every 4 h. water, bivalve and swab samples were analyzed for tpc and coliform load. fig. 2. experimental set-up, (i) closed water circulatory system; (ii) open water system; (a) bivalves; (b) wire mesh; (c) tap water; (d) water pump; (e) water inlet; (f) sponge; (g) sediment (sand) layer; (h) perforated pipe; (i) water outlet back in the tank; (j) water (tap water) inlet source; (k) water outlet leading out of the tank. arrows show water pathway. bacteriological analysis bivalve shells were thoroughly washed with sdw. the shells were aseptically opened with a sterile scalpel and the meat was transferred to sterile homogenizing bags. the meat from two bivalves was weighed and diluted 10 fold accordingly with sdw. for example, if the weight of meat from two bivalves was 10 g, 90 ml of sterile distilled water was poured into the bag. the meat was homogenized using a digester for 5 min. each sample was serially diluted up to 10-5 using 9 ml sdw blanks. for tank and swab samples, 1ml of the water was taken and serially diluted up to 10-5 using 9 ml sdw blanks. 1ml and 0.1 ml of each dilution was plated using pour plate technique (clark 1967) in standard plate count agar and incubated at 37 oc for 48 h. for each sample, three replicates were done. after incubation, the colonies were counted and the tpc load of the undiluted sample was calculated. tpc load was expressed as colony forming units per gram of sample (cfu.g-1) for bivalve samples and colony forming units per ml of sample (cfu.ml-1) for tank water and swab samples. for coliform analysis, 10-2 dilution of each sample was used. standard most probable number (mpn) method using 3 tube dilution (west 1989) was used for coliform enumeration. 1 ml of the 10-2 dilution was pipetted out into 99 ml of sdw giving a 10-4 dilution. 3 × 10 ml volumes of this dilution were transferred into 3 tubes of 10 ml sterile double strength lactose broth. 3 × 1 ml and 3 × 0.1 ml volumes of the samples were transferred to 10 ml sterile single strength lactose broth. each tube contained an inverted durham’s tube. the tubes were incubated at 37 oc for 24 – 48 h. the inverted durham’s tubes were checked for gas production. the numbers of positive tubes were recorded in each sample and referred to mpn table to find out the mpn index of coliform load. physicochemical analysis of water temperature, salinity, do and the ph measurements were taken at every 4 h interval for 48 h. ph and temperature were measured using nova biotechnol chim (2021) 20(2): e824 5 digital ph electrode with temperature sensor (ph meter, hanna instruments hi2002), salinity was measured using digital conductivity probe (ec meter, hanna instruments hi2030), do was measured using digital polarographic dissolved oxygen probe (do meter, hanna instruments hi2040). all measurements were performed according to the manufacturer's instructions manual. the whole experimental set-up and procedure was repeated three times (three trials) from samples collection to physicochemical analysis of water and results were presented as mean of multiple replicates within each trial. statistical analysis least squares regression models with polynomial functions were used to determine the trends of bacterial load reduction patterns. models at p<0.05 were used to develop suitable trend reproduction models. models were subjected to t-tests, f-tests, and selections for best fit models were made using akaike information criterion (aic) (akaike 1981), r2 and p-values. anova was used for comparison of bacterial load and load reduction patterns. for this study all statistical analysis was performed using the “r” software, version 3.6.1 (the r core development team 2019). results bacteriological analysis text bacteriological analysis of water and sediment sample of the site showed high level of tpc (cfu.ml-1) and coliform load (mpn.ml-1) for each of the three experimental trials (fig. 3). tpc and coliform loads were much higher for trial 3 compared to trials 1 and 2. also, tpc and coliform load of sediment was significantly higher than water from the site for all three trials (p<0.001). other parameters of sampling site are shown in table 1. analysis of the bivalves showed high initial loads detectable levels and slightly above fao standard requirement of <300 cfu.100 g-1 (lee et al. 2008) for all three trials throughout the test. fig. 3. total plate count bacteria (tpc) (cfu.ml-1) and coliform load (mpn.ml-1) of water and sediment samples obtained from the sampling site in rewa river, rewa, fiji. the graphs show (a) tpc and (b) coliform load for three experiment trials (trial 1, trial 2, trial 3) from the site of bivalve sample collections. each bar is shown as mean ± se of three replicates. of tpc and coliform at 0 h (fig. 4) for each of the three trials. for closed circulatory system, both tpc and coliform loads reduced rapidly to low and undetectable levels within 16 h which persisted for up to the 48 h mark. in all closed system trials fecal coliform load reached below fao standard requirement of <300 cfu.100 g-1 (lee et al. 2008). open system showed slower reduction of both tpc and coliform loads. bacterial loads remained above both, the tpc and coliform load reduction trends for closed system were significantly different (p<0.05) from open system in all cases. all three trials for closed system showed similar patterns of growth reduction. in all three cases, the growth reduction of tpc and coliforms in the bivalve and tank water can be determined using the equations as outlined below with r2>0.95 and p<0.001. the growth reduction trend of tpc in b. violacea over nova biotechnol chim (2021) 20(2): e824 6 the depuration period for closed system follows the general trend represented by eq. 1: (1) where t is the population of tpc in the bivalve, t is the depuration time, a is the tpc population at depuration time t of zero (0) h and ε is an undefined error term. the growth reduction trend of coliforms in b. violacea over the depuration period for closed system follows the general trend represented by eq. 2: (2) where c is the population of coliforms in the bivalve and b is the coliform population at depuration time t of zero (0) h. table 1. physical and bacteriological parameters of the sampling site in rewa river. values are shown as mean ± se of 5 replicates. parameter value water temperature [oc] 27.40 ± 1.42 ph 6.94 ± 0.24 salinity [ppt] 0.03 ± 0.01 dissolved oxygen [mg.l-1] 7.06 ± 0.04 turbidity [m] 1.24 ± 0.34 total plate count [cfu.ml-1] – bivalve 9.95 × 106 ± 9.81 × 102 coliform load [mpn.ml-1] – bivalve 904.34 ± 18.5 fig. 4. variation in bacterial load of bivalve (batissa violacea) over a 48 h depuration period for three different trials. (a) total plate count bacteria (tpc) (cfu.g-1); (b) coliform load (mpn.ml-1). (i) closed water circulatory system; (ii) open water system. each point is shown as mean ± se of three replicates. prior to introduction of bivalves, aquarium tank water samples showed negligible tpc and undetectable coliform load for all three trials (fig. 5). water samples taken 4 h after introduction of bivalves showed high level of both tpc and coliform load. this could have been due to the release of microbes in mucous threads into the water by the clams. for closed circulatory system, nova biotechnol chim (2021) 20(2): e824 7 tpc reduced to very low levels within 28 h while coliforms reduced to undetectable levels in 24 h. the microbial populations remained at these levels up to the 48 h period. for the open system, tpc continued to increase for up to 20 h followed by reduction in number beyond this point. coliforms increased in number from 4 to 8 h followed by reduction. tpc remained above detectable levels at 48 h. coliforms reduced significantly but remained above detectable levels for trial 3 and reached undetectable levels for trials 1 and 2. the difference may be due to the initial higher coliform load for trial 3. for closed circulatory system, the population reduction pattern for tpc and coliforms were similar for all three trials. the growth reduction trend of tpc in tank water for closed system over the depuration period from the 4 h mark follows the general trend represented by eq. 3: (3) where w is the population of tpc in tank water and d is the tpc population at depuration time t of zero (4) h. the growth reduction trend of coliforms in tank water for closed system over the depuration period from the 4 h mark follows the general trend represented by eq. 4: (4) where f is the population of coliforms in tank water and g is the coliform population at depuration time t of zero (0) h. table 2 shows statistical information for the fitness of eqs. 1 – 4 with actual values. fig. 5. variation in bacterial load of tank water over a 48 h bivalve depuration period for three different trials. (a) total plate count bacteria (tpc) (cfu.ml-1); (b) coliform load (mpn.ml-1). (i) closed water circulatory system; (ii) open water system. each point is shown as mean ± se of three replicates. bacteriological analysis of the swab samples showed that at zero h there was undetectable tpc and coliform count for all three trials. as shown in fig. 6, both the tpc and coliform load increased through progression of the depuration period in all cases. tpc and coliform loads reached higher values for open system compared to closed circulatory system. these results confirmed the formation of biofilm in the interior tank walls of the glass aquarium over the 48 h depuration period. nova biotechnol chim (2021) 20(2): e824 8 table 2. statistical parameters for equations fitness to the trajectories for different experimental trials. equation fit r2 p-value df t-value f statistic aic equation 1 trial 1 0.977 1.10 × 10-09 10 21.41 458.30 30.57 trial 2 0.957 5.20 × 10-07 10 11.28 127.30 32.55 trial 3 0.960 1.69 × 10-08 10 16.17 261.50 72.84 equation 2 trial 1 0.987 5.51 × 10-11 10 29.02 842.10 73.85 trial 2 0.975 1.46 × 10-09 10 20.81 433.10 70.09 trial 3 0.954 8.35 × 10-08 10 13.70 187.60 133.92 equation 3 trial 1 0.996 1.17 × 10-13 10 53.90 2905.00 13.02 trial 2 0.951 4.28 × 10-08 10 14.69 215.70 38.91 trial 3 0.968 5.18 × 10-09 10 18.27 333.80 89.28 equation 4 trial 1 0.974 4.13 × 10-10 10 27.38 687.30 71.53 trial 2 0.964 4.36 × 10-09 10 24.28 383.40 65.23 trial 3 0.967 8.35 × 10-09 10 25.70 407.36 67.25 fig. 6. variation in bacterial load of the biofilm developed on tank interior wall over a 48 h bivalve depuration period for three different trials. (a) total plate count bacteria (tpc) (cfu.ml-1); (b) coliform load (mpn.ml-1). (i) closed water circulatory system; (ii) open water system. each point is shown as mean ± se of three replicates. the trajectory of physiochemical parameters of aquarium tank water for the three experimental trials over the depuration period of 48 h is shown in fig. 7. temperature varied within a difference of 1 – 3 oc but remained relatively constant. for the open system, a slight dip in temperature was observed between 20 and 36 h range. this may be due to the cooling of the sourced tap water at late night and morning hours. dissolved oxygen remained fairly constant over the depuration period except for a dip at the 8 h mark for closed circulatory system. this was observed for all three experimental trials. this may be due to the use of oxygen by high microbe load upon first nova biotechnol chim (2021) 20(2): e824 9 introduction followed by recovery which may have been due to the mixing of oxygen from dripping water from the pump and tap. the ph value did not show any significant change and salinity remained at 0 throughout the experiment. fig. 7. variation in physicochemical properties of aquarium tank water over the 48 h depuration period for three different trials. (a) temperature (oc); (b) dissolved oxygen (mg.l-1); (c) ph; (d) salinity (ppt). (i) closed water circulatory system; (ii) open water circulatory system. each point is shown as mean ± se of three replicates. nova biotechnol chim (2021) 20(2): e824 10 discussion b. violacea is a filter feeder exposed to high bacterial load in the aquatic environment (mayor et al. 2018). as such, high bacterial load is expected in the gut contents of b. violacea as shown by baker (2016). elevated levels of pathogens and spoilage bacteria in bivalves pose a health hazard for consumers. while spoilage bacteria do not necessarily pose a direct health risk, they increase the rate of food spoilage which can affect consumer health. tpc of b. violacea from rewa river has been shown to contain bacterial community from predominantly micrococcus and bacillus genera, as well as aeromonas, pseudomonas, vibrio, acinetobacter, streptococcus and alcaligenes (hatha et al. 2005a). aeromonas and vibrio contain potential pathogen species of importance in seafood (hatha et al. 2005b; vivekanandan et al. 2005). depuration of bivalves is necessary to ensure that bacterial load in b. violacea reaches low and acceptable levels for consumption (hatha et al. 2005a; lee et al. 2008; waqalevu 2015). this work was aimed at determining the effectiveness of depuration in closed and open water systems for reduction bacterial load in b. violacea to safe levels for consumption. the set-up was aimed to be both economical and effective to be feasible at both industrial and small scale levels. the results showed that using a sand biofilter in closed water circulatory system was highly effective in reducing tpc and coliform load in b. violacea to undetectable levels within 16 h of depuration. the biofilter seemed to have trapped the microbes over time. this system proved to be much better in comparison to the open circulatory system where tpc and coliform loads reduced at much slower rate and remained above detectable levels throughout the depuration period. similar results were observed in the dore and lees (1995) where open system of depuration took much longer to reduce tpc and coliform loads to acceptable levels in comparison to closed water system for oysters (crassostrea gigas) and mussels (mytilus edulis). b. violacea with high initial bacterial load had a much slower reduction trend in open water depuration system. for closed circulatory system, initial bacterial load in b. violacea had little impact on bacterial reduction trend in contrast to open water system. previous works have shown depuration of bivalves to be effective in reducing tpc and coliform loads to undetectable and safe levels for human consumption (schweimanns and felbeck 1985; dore and lees 1995; sreenath and hatha 2005). waqalevu (2015) used a closed system of b. violacea depuration with a uv filter system in a 5,000 l tank. although reduction of coliforms was observed in the bivalves, significant numbers were still present even at 72 h of depuration. also, coliform load in tank water initially reduced followed by an increase over the depuration period. the depuration set up was also highly water intensive and costlier than the simple closed water depuration system designed in this study. for closed water circulatory system, the growth reduction of tpc and coliforms in bivalve and aquarium water follows an exponential reduction trend in all cases as represented by eqs. 1 – 4. eqs. 1 and 2 can be used to determine the depuration period for reduction tpc and coliform load in b. violacea to safe levels for human consumption and/or in food industries and market areas where the bivalve is sold or packaged fresh. the initial bacterial load needs to be known for the equations. eqs. 3 and 4 can be used to estimate when the aquarium water will have low bacterial load and is safe for release into the environment. significant loads of coliforms were present in the bivalves as well as site water and sediment samples in all cases. this indicates organic pollution from household sources including faecal pollution (martinez and oliveira 2010) in the shellfish growing waters. waqalevu (2015) showed the presence of e. coli and enterobacter in rewa river water, indicating anthropogenic fecal pollution. bivalves tend to bioaccumulate pathogenic microorganisms within their tissues over time (martinez and oliveira 2010). while there are stringent standards in developed countries for shellfish growing waters, such regulatory controls are non-existent/not followed in many of the developing countries. large number of fishermen harvest shellfish from the rich shellfish beds of natural waters and sell it for human consumption without proper depuration. development of a simple, cost-effective and water conserving nova biotechnol chim (2021) 20(2): e824 11 depuration system would be of great use to local fishermen and even for domestic use. in this study depuration using a biofilter in a closed water circulatory system was effective in reducing coliform and tpc load in the tissues of b. violacea to low and safe levels in a short period of time. the reduction in the tpc and coliform load in the aquarium tank water with biofilter probably had been due to the biofilter system. when bacteria and faecal waste laden mucus from shellfish passed through white sand in the biofilter, they could have been trapped while allowing water to pass through back into the tank. formation of biofilm on the interior tank walls was observed in all cases. both tpc and coliform levels increased in approximately a linear pattern over the depuration period. biofilms formation is a survival strategy for microorganisms (prakash et al. 2003; johnson 2008; kumar et al. 2017). the aquarium walls were made of silica glass, which provides a suitable substrate for microorganisms which would likely be the reason for biofilm formation. however, the biofilm did not affect the bacterial load in the bivalve or tank water samples. from a practical point of view, in an industrial setup, open system has an advantage over closed system. in closed system, the sand in the biofilter needs to be replaced and the tank walls cleaned between each batch. in open system only tank walls need to be cleaned between batches. this saves some time. however, the overall advantage is higher for closed system in terms of the economic cost and conservation of water. the closed water circulatory system with biofilter assembly conserves a lot of water as it does not have additional water input or output. it uses a fixed amount of water which circulates with assistance of a water pump. water being the most precious resource, there is a pressing need to conserve the same in all the systems that use water. closed system uses sand as a biofilter where the mucus laden with microorganisms get trapped. this can be later treated to remove the bacterial load. this is another advantage over the open water system where there is a continuous flow of water and microorganisms released by the bivalves get released into the environment. the collection and treatment of this large volume of water will not be cost effective or feasible. the limitation of the closed water circulatory system with biofilter setup is that it is ineffective in preventing biofilm formation. however, the flaking off of the biofilm associated bacteria will take much more duration that the required depuration time. hence the reintroduction of the biofilm associated bacterial load does not pose a threat to the efficacy of closed water system evaluated in this study. with every depuration cycle, there is a need to clean the depuration tank as not doing so may affect depuration time and flaking off of the biofilm can cause recontamination of the bivalves. conclusion the reduction in tpc and coliform load in freshwater clams (b. violacea) suggests that the biofilter in a closed water circulatory system is a simple, cheap and effective way to significantly reduce the bacterial load that is accumulated in the clams. it can be concluded that proper use of the closed water circulatory system can reduce the risk or bacterial related health problems from consumption of b. violacea. aseptic methods and proper handling need to be used to prevent postdepuration contamination of the bivalves. hatha et al. (2005a) recommended usage of ice made from potable water for packing b. violacea to control post-harvest proliferation of pathogens and spoilage bacteria. proper sanitation and cooking will also help in reducing the risks from pathogens. depuration has negligible effect on the taste of b. violacea (waqalevu 2015) and does not pose any threat of losing its taste popularity among consumers. although depuration is also known to reduce metal toxicity from shellfish (anacleto et al. 2015), this was not assessed in the current study. the two set-ups tested here have only been tested against bacterial loads. it should be noted that there are other forms of health risks from bivalve consumption such as heavy metal, toxins and viruses which have not been tested here. further work is needed in these areas. acknowledgments this research was supported by the university of fiji through availability of materials. there was no funding received. the university of fiji is acknowledged for providing the resources nova biotechnol chim (2021) 20(2): e824 10 and administrative support necessary for carrying out the write-up of the project. we thank mr. roneel prasad of the university of fiji for providing laboratory support to facilitate the experiments without which this work would not be possible. conflict of interest the authors declare that they have no conflict of interest. references akaike h (1981) likelihood of a model and information criteria. j. econom. 16: 3-14. amadi l (2016) mortality and microbial diversity of raw, processed and storage of mangrove oysters (crassostrea gasar). int. j. environ. res. public health. 3: 7-13. anacleto p, maulvault al, nunes ml, carvalho ml, rosa r, marques a (2015) effects of depuration on metal levels and health status of bivalve molluscs. food control 47: 493-501. andrew nl, bright p, de la rua l, teoh sj, vickers m (2019) coastal proximity of populations in 22 pacific island countries and territories. plos one 14: 1-15. baker g (2016) food 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pathogens and public health indicator organisms in shellfish. in austin b and austin da (eds.), methods for the microbiological examination of fish and shellfish. ellis horwood, chichester. pp. 273-308. 12 nova biotechnol chim (2021) 20(1): e748 doi: 10.36547/nbc.748 1 nova biotechnologica et chimica antioxidant and hypoglycemic activities of various solvent fractions of methanol extract of terminalia alata heyne ex roth trunk-bark quang vinh nguyen 1,, pham van hung 2 , anh dzung nguyen 1 1 institute of biotechnology and environment, tay nguyen university, 567 le duan rd., buon ma thuot city, 630000, dak lak province, vietnam 2 international university, vietnam national university in ho chi minh city, quarter 6, linh trung ward, thu duc district, ho chi minh city, vietnam  corresponding author: vinh12b@gmail.com article info article history: received: 7 th november 2020 accepted: 16 th february 2021 keywords: α-amylase and α-glucosidase inhibitors anti-hyperglycemic dpph radical scavenging terminalia alata abstract the antioxidant and hypoglycemic capacities of various solvent fractions from the trunk-bark methanol extract of terminalia alata heyne ex roth (t. alata) were investigated. the dpph radical scavenging assay was used to evaluate the antioxidant activity, and the methods for determination of digestive enzymes inhibitory activity and fasting blood glucose reduction capacity in diabetic rats were used to determine the hypoglycemic activity of the extract fractions. the results indicated that higher total phenolics content was measured with increasing polarity of extraction solvent and dpph radical scavenging activity coincided with phenolics content. ethyl acetate fraction (eaf), n-butanol fraction (bf), and water fraction (wf) were obtained. they possessed α-amylase inhibition with the ic50 values of 0.056 ± 0.001, 0.138 ± 0.005, and 0.022 ± 0.001 mg.ml -1 , respectively, which were lower than acarbose (ic50 = 0.154 ± 0.02 mg.ml -1 ). in contrast, ic50 values of αglucosidase inhibitory activities of these fractions were higher than those of acarbose. in addition, these fractions also lowered fasting blood glucose concentrations in streptozotocin induced diabetic rats at a dose of 200 mg.kg -1 bw (body weight) without inducing body weight loss, which was not observed when treating with acarbose. the eaf and wf of trunk-bark of t. alata are recommended as potent sources for further research on antioxidant and hypoglycemic activities.  university of ss. cyril and methodius in trnava introduction genus terminalia comprises more than 250 species (cock 2015), while several of them have been used as traditional medicines in asian countries including vietnam. recently, bioactivities of terminalia species have been reported by numerous authors. solvent extracts of leaves and trunk-bark of t. nigrovenulosa showed strong antioxidant and antimicrobial activities (nguyen and eun 2011; 2013). moreover, methanol extract of t. chebula fruits has antioxidant (lee et al. 2007), antimicrobial (malekzadeh et al. 2001), antidiabetic (gao et al. 2007), and anticancer (sabu and ramadasan 2002; saleem et al. 2002) activities. terminalia alata heyne ex roth (t. atala) belonging to the combretaceae family is widely grown in deciduous forests in the southern part of vietnam. t. alata plants have been used as mailto:vinh12b@gmail.com nova biotechnol chim (2021) 20(1): e748 4 traditional remedies for anti-diarrhea in the treatment of chronic dysentery, sore throat, laryngitis, and hemorrhoids. nguyen et al. (2016) indicated that the methanol extract of t. alata trunk bark possessed antioxidant efficiency and hypoglycemic capacity in streptozocine induced rat model. however, effectiveness of different solvents on the solubility of bioactive compounds and their corresponding bioactivities has not yet been studied. several reports mentioned that the bioactive components in the plants concentrated on different solvent fractions depending on plant species. khled khoudja et al. (2014) indicated that water fraction (wf) of t. algeriensis was the most effective fraction amongst these investigated solvent fractions, whereas the highest antioxidant activity of combretum erythrophyllum (burch.) was found in ethyl acetate fraction (eaf) (fanyana et al. 2017). report of emran et al. (2015) indicated that chloroform fraction (cf) expressed the highest clot lysis activity. therefore, this research aims to investigate the antioxidant and hypoglycemic activities of different solvent fractions of t. alata to reveal their potency for the highest bioactivities. experimental chemicals 2,2-diphenyl-2-picrylhydrazyl hydrate, gallic acid, folin-ciocalteu reagent, potassium ferricyanide, sodium nitrite, aluminium chloride, streptozotocin, pancreatic α-amylase, rat intestinal acetone powder, p-nitrophenyl-α-d-glucopyranoside, and dinitrosalicylic (dns) acid were purchased from sigmaaldrich (st. louis, mo, usa). other chemicals and reagents reached the analytical grade. plant samples the collection of the trunk bark of t. alata was located in yok don national park, dak lak province, vietnam. after cutting out from the tree, the barks were dried at ambient temperature with active ventilation to obtain the moisture of 6 %, ground by grinder using a cutting mill (lmm010w, lihom inc., seoul, korea) and then stored at -30 ºc in polyethylene (pe) bags or directly use. the dried trunk-bark powder was extracted three times with methanol by maceration method as reported by nguyen et al. (2016). briefly, the extraction was carried out using a ratio of trunkbark/methanol of 1 : 10 (w/v), shaking at room temperature for 24 h and then filtering through filter paper (no. 1). the residue was then extracted twice more with methanol as described above. the extracts were combined and concentrated at 60 ºc under vacuum using a rotary evaporator (ika, germany) to obtain the crude extract. the crude extract was then dissolved in distilled water at a ratio of crude extract/distilled water of 1 : 5 (w/v) for fractionation. the aqueous solution was further partitioned with different solvents as described by nguyen et al. (2015). briefly, partition with hexane obtained the hexane fraction (hf) and the aqueous fraction was sequentially partitioned with chloroform, ethyl acetate an n-butanol. the chloroform (cf), ethyl acetate (eaf), and nbutanol (bf) fractions were filtered and dried by evaporation under vacuum. the final aqueous fraction (wf) was filtered and dried by freezing dryer. animal the 5-week old wistar albino rats weighed 20 – 25 g were used for in vivo hypoglycemic activity experiments. the rats were obtained from the institute of vaccines and medical biologicals (ivac), nha trang, vietnam, kept in the plastic cage houses in 12 h light/12 h dark cycle room at 25 ± 2 ºc and fed with a standard rodent diet and water ad libitum before testing. the experiments were carried out under the regulation of the institutional animal care and use committee at the tay nguyen institute of hygiene and epidemiology, dak lak province, vietnam. total phenolic content of extracts was determined using folin–ciocalteu method with slight modification by nguyen et al. (2016). dpph radical scavenging activity was followed by nguyen et al. (2016). rat intestinal α-glucosidase inhibitory activity was performed by the method of kwon et al. (2006) 2 nova biotechnol chim (2021) 20(1): e748 3 with slight modification by nguyen et al. (2016), using acarbose as the positive control. the inhibition of porcine pancreatic α-amylase activity was carried out by the method of kwon et al. (2006) with slight modification described by nguyen et al. (2016). acarbose is the α-glucosidase inhibitor and was used as the positive control. acute oral toxicity and glucose level in normal rat studies acute oral toxicity study was carried out as a slightly modified method of organization for economic co-operation and development (oecd 2001) guidelines 423 (acute toxic classic method) and described details by nguyen et al. (2016). briefly, fractions with the concentrations of (100, 200 and 300 mg.kg -1 body weight) were daily oral administration of investigated fractions, animals (6 rats/each group) were observed individually at least once during the first 30 min, periodically during the first 24 h, with special attention given during the first 4 h, and daily afterward, they were fed with extracts and observed for a total of 8 days for toxicity and fasting blood glucose levels determination by blood glucose meter (bionime gm110, bionime, swiss). streptozotocin-induced diabetic rats ice-cold aqueous streptozotocin (150 mg.kg -1 body weight) was used to treat male wistar albino rats intraperitoneally (nguyen et al. 2016). evaluation of extracts on streptozotocin-induced diabetic rats the experiments were described by nguyen et al. (2016) which were carried out by single and multidose experiments. blood glucose meter (bionime gm110, bionime, switzerland) was used for measurement of fasting glucose concentrations. acarbose has been used as medicine for controlling postprandial blood glucose. therefore, it was used as positive control in compared to the efficiency of extracts in controlling postprandial blood glucose. saline was used for dissolving extract fractions. therefore, it is used as a negative control. data analysis all experiments were carried out in triplicates and results were shown as the mean values with standard deviations. the significant statistic for each experiment was determined using the analysis of the variance test (anova) and the lsd (least significant difference). the post-test was used for the comparison of the mean values. differences were considered significant at p < 0.05. results total phenolics contents and dpph inhibitory activity of t. alata extract fractions table 1 shows total phenolics contents measured in the fractions from t. alata. the highest values of total phenolics were obtained in more polar solvents phenolics, namely in water fraction (wf), followed by n-butanol fraction (bf), and ethyl acetate fraction (eaf). in contrast, they were almost absent in hexane fraction (hf). the inhibitory activities of fractions against dpph are compared to a positive control (ascorbic acid) via ic50 values in table 1. table 1. total phenolics contents and dpph inhibitory activity of the fractions from t. alata bark. samples/control total phenolics content [mg gae.g -1 dry extracts] dpph radical scavenging activity ic50 [mg.ml -1 ] hf cf 246.329 ± 5.530 a 3.063 ± 0.023 a eaf 589.613 ± 1.937 b 0.290 ± 0.010 b bf 704.694 ± 0.927 c 0.237 ± 0.006 c wf 773.349 ± 9.151 d 0.241 ± 0.003 c ascorbic acid 0.240 ± 0.010 c different letters indicate a significant difference amongst the values with a row at p<0.05, (-) no detection, hf, cf, eaf, bf and wf are hexane, chloroform, ethyl acetate, n-butanol and water extract fractions, respectively. alpha-amylase and alpha-glucosidase inhibitory activities of fractions the ic50 values of α-amylase and α-glucosidase inhibitory activities of different fractions are shown in table 2. the results indicate that both the enzymes were inhibited by most of the extract nova biotechnol chim (2021) 20(1): e748 4 fractions at the investigated concentrations, except for the hf. the strongest inhibitory activity against α-amylase was found in the wf, followed by bf and eaf with the ic50 values 0.022 and 0.056 mg.ml -1 , respectively. these fractions had higher α-amylase inhibitory activity than acarbose (the positive control) (table 2). in contrast, the αglucosidase inhibitory activity of methanol extract and fractions was lower than acarbose (table 2). amongst these fractions, bf and eaf possessed the strongest α-glucosidase inhibitory effects, followed by wf (table 2). moreover, all three types of extract fractions possessed much higher inhibitory activities against both enzymes compared to that of the methanol extract. this suggests that fractionation of crude extract could concentrate the active compounds in solvent fractions. table 2. effect of fractions of t. alata on α-amylase and αglucosidase activities. sample/controls α-glucosidase inhibition ic50 (mg.ml -1 ) α-amylase inhibition ic50 (mg.ml -1 ) methanol extract > 4 a 0.13 ± 0.01 c hf cf 3.787 ± 0.087 b > 20 a eaf 1.330 ± 0.133 d 0.056 ± 0.001 d bf 1.251 ± 0.059 d 0.138 ± 0.005 c wf 1.729 ± 0.025 c 0.022 ± 0.001 e acarbose 0.492 ± 0.02 e 0.154 ± 0.02 b different letters indicate a significant difference amongst the values with a row at p<0.05, (-) no detection, hf, cf, eaf, bf and wf are hexane, chloroform, ethyl acetate, n-butanol and water extract fractions, respectively. acute toxicity study the normal rats were daily fed with the fractions of terminalia atala by oral intubation at a dose of 100, 200 and 300 mg.kg -1 body weight and observed for eight days. the results in table 3 indicated that it is non-toxic at an oral dose up to 300 mg.kg -1 body weight in all rats being oral administration of fractions for eight days of experiment except for chloroform fraction (cf). the rats treated with cf at a dose of 300 mg.kg -1 bw died after 8 days of treatment. therefore, 200 mg.kg -1 bw was used as the safe dose for investigation of anti-hypoglycemic activity of all fractions. effect of single dose fractions on fasting blood glucose concentrations in diabetic rats the effects of the safe dose of all fractions or acarbose addition on blood glucose concentrations in streptozotocin induced diabetic rats were examined (table 4). the fasting blood glucose concentrations of streptozotocin-induced diabetic rat models used in this study ranged from 10 to 17 mmol.l -1 . in the single dose evaluation, the acarbose treatment showed the maximum effect within 2 – 4 h and slightly increased after 6 – 8 h. on the other hand, the eaf and wf had significant effect within 2 – 8 h and 4 – 8 h, respectively. the results in table 4 suggested that the blood glucose concentrations in diabetic rats treated with individual fractions were slowly reduced and kept constant for longer time as compared to acarbose (positive control). in this respect, the eaf was found to possess the highest effect on the reduction of glucose concentration in diabetic rats, followed by wf (table 4). the glucose concentration decreased in rats treated with eaf after 2, 4, 6 and 8 h, respectively. the strongest drop of glucose concentration in rats (52 %) was found upon treatment with wf for 4 h, while the glucose concentration remained constant until 8 h. for comparison, treatment with acarbose resulted in decreased glucose concentration to 49.66 % after 2 h with further decrease after 8 h. effect of multiple dose fractions on fasting blood glucose concentrations in diabetic rats the changes in glucose concentration in diabetic rats treated with the fractions were shown in table 5. generally, the daily oral treatment with fractions at the investigation dose significantly decreased blood glucose concentrations in diabetic rats compared to the corresponding control group. the significant reduction in postprandial blood glucose concentration by the fractions and acarbose was observed after the 8 th day and remained unchanged until the 12 th day. in contrast, there was significantly increased the glucose concentration in diabetic rats treated with saline (control rats) under the same conditions. as shown in table 5, the elevated glucose concentrations in diabetic rats were about 24.92 % and 51.71 % after treatment nova biotechnol chim (2021) 20(1): e748 3 with saline for 8 and 12 days, respectively. acarbose exerted a stronger effect on reduction in postprandial blood glucose concentration in diabetic rats than the fractions. as a result, both single and multiple doses of fractions showed the effective hypo-glycemic activity in streptozotocininduced rats. the results (fig. 1) indicated the table 3. effect of administration of fractions of t. alata on blood glucose level and status of normal rats. stt samples/ controls oral dose [mg.kg -1 body weight] blood glucose concentration [mmol.ml -1 ] day 0 day 4 th day 8 th 1 cf 100 8.23 ± 1.06 7.73 ± 0.81 7.97 ± 0.94 200 9.20 ± 0.95 8.53 ± 1.10 8.37 ± 0.39 300 8.83 ± 1.10 a 7.07 ± 0.55 b died 2 eaf 100 7.33 ± 0.46 6.83 ± 0.47 6.80 ± 0.46 200 7.33± 0.10 7.07 ± 0.36 6.80 ± 0.45 300 8.93 ± 0.49 8.60 ± 0.36 8.17 ± 0.45 3 bf 100 8.67 ± 0.21 7.43 ± 0.81 7.53 ± 0.81 200 8.40 ± 0.36 7.77 ± 0.89 7.43 ± 0.32 300 8.00 ± 0.53 8.27 ± 0.29 7.77 ± 0.45 4 wf 100 9.03 ± 1.00 8.50 ± 0.69 8.20 ± 0.10 200 7.53 ± 0.58 7.87 ± 0.58 7.30 ± 0.44 300 8.13 ± 0.25 7.87 ± 0.58 7.67 ± 0.49 5 saline 9.00 ± 1.81 8.50 ± 0.35 7.73 ± 0.66 different letters indicate a significant difference amongst the values with a row at p<0.05, cf, eaf, bf and wf are hexane, chloroform, ethyl acetate, n-butanol and water extract fractions, respectively. table 4. fasting blood glucose concentrations in diabetic rats (single dose) treated with fractions at a dose of 200 mg.kg -1 body weight. samples/ controls blood glucose concentration [mmol.l -1 ] 0 h 2 h 4 h 6 h 8 h cf 12.18 ± 0.98 b 12.56 ± 0.70 b 12.83 ± 1.25 a 14.76 ± 1.04 a 14.19 ± 0.98 a eaf 11.53 ± 1.10 a 8.27 ± 1.75 b 6.80 ± 1.65 b 7.87 ± 2.39 b 7.63 ± 1.53 b bf 12.07 ± 0.35 a 10.77 ± 0.74 a 11.73 ± 1.45 a 12.00 ± 0.72 a 9.13 ±1.17 a wf 14.23 ± 1.50 a 10.90 ± 4.66 a 6.83 ± 0.25 b 7.00 ± 0.95 b 7.17 ± 2.06 b positive control (acarbose) 14.10 ± 1.93 a 7.10 ± 0.95 b 8.17 ± 1.52 b 9.10 ± 0.62 b 12.23 ± 2.06 a negative control (saline) 13.40 ± 2.0 a 12.30 ± 1.9 a 13.60 ± 2.1 a 15.40 ± 1.8 b 16.50 ± 1.0 b different letters indicate a significant difference in time at p<0.05, cf, eaf, bf and wf are hexane, chloroform, ethyl acetate, n-butanol and water extract fractions, respectively. table 5. effect of fractions on blood glucose concentrations in diabetic rats as treatment days (multiple dose). different letters indicate a significant difference in time at p<0.05, eaf, bf and wf are hexane, chloroform, ethyl acetate, n-butanol and water extract fractions, respectively. change of body weight of diabetic rats treated with multiple doses of the individual fraction or acarbose (positive control) during 12 days. the body weight of diabetic rats treated with saline (negative control) was slightly increased until the 8 days (7 %) and then trended to reduce after 12 days. in contrast, the body weight of the acarbosetreated diabetic rats significantly decreased after 8 days of treatment (13 %) and 16 % after 12 days of treatment. however, the diabetic rat groups treated samples / controls concentration of blood glucose [mmol.l -1 ] day 0 day 4 th day 8 th day 12 th eaf 13.43 ± 2.14 a 11.53 ± 4.40 a 8.67 ± 0.75 b 8.00 ± 0.20 b bf 13.03 ± 1.61 a 11.80 ± 1.25 a 8.80 ± 1.56 b 8.40 ± 1.15 b wf 13.70 ± 1.80 a 10.53 ± 0.61 a 9.80 ± 0.60 b 8.60 ± 0.70 b positive control (acarbose) 13.20 ± 3.21 a 10.00 ± 5.90 a 6.90 ± 3.75 b 5.93 ± 2.27 b diabetic rats (saline) 10.70 ± 0.50 b 10.70 ± 1.22 b 13.37 ± 2.42 ab 16.23 ± 3.19 a normal mouse (saline) 7.43 ± 0.32 a 7.53 ± 0.46 a 7.10 ± 0.66 a 6.90 ± 0.79 a 5 nova biotechnol chim (2021) 20(1): e748 6 with fractions had no significant change in body weight during treatment duration (fig. 1). discussion the research related to the development of new compounds for prevention of diseases has been recently increased, particularly regarding the role of flavonoids and phenolic acids as antioxidants (lopez-velez et al. 2003). furthermore, the phenolic compounds possess several biological activities that play important roles in disease prevention, including antioxidant and antidiabetic activities (kunyanga et al. 2012). according to a report of vats et al. (2002), the hypoglycemic activity of most medicinal plants was due to the action of phytochemicals which might reduce the activity of starch hydrolyzing enzymes in track to retard the sharply raising in postprandial blood glucose concentration in diabetic patients. moreover, phytochemicals proved that they could promote insulin secretion and/or improve effective glucose transport and metabolism in muscle (gray 1995). therefore, a number of evaluations of medicinal plants extracts as well as isolated components from the plants as natural sources for inhibition of starch hydrolyzing enzyme and control the postprandial blood glucose concentration in diabetic rats have been done so far. nguyen et al. (2016) showed that the crude extract of t. atala possessed a relatively high effect on both starch hydrolyzing enzymes inhibition and hypoglycemic activity in diabetic rats. several reports indicated that active compounds with different polarity might be present in the plant, and there was a relationship between amounts of phenolic compounds containing in extracts and biological capacity (khled khoudja et al. 2014; emran et al. 2015; fanyana et al. 2017). abarcavargas et al. (2016) indicated that the phenolics compounds appeared to be dependent on the type of solvent used, its polarity index, and the solubility of phenolics compounds in the extraction solvent. the finding in this study showed that fractions which contained higher content of total phenolics also possessed a stronger dpph radical scavenging activity (table 1). the results in this study are in accordance with many reports (piljac-žegarac et al. 2005; nguyen and eun 2011; kunyanga et al. 2012; chai and wong 2012). however, the direct correlation between the inhibition capacity against α-glucosidase activity and total phenolics contents was not observed in this study. eaf and bf containing medium content of total phenolics possessed the strongest α-glucosidase inhibitory effect, followed by wf (table 2). however, the highest inhibition of α-amylase activity was found in wf containing the highest total phenolics fig. 1. effects of fractions on body weight of diabetic rats (multiple doses). different letters above the bars for the same fraction-treatment or saline-treatment or acarbosetreatment indicate statistically significant differences at p<0.05. b a b b a a a a b -20 -15 -10 -5 0 5 10 0 day after 4 days after 8 days after 12 days th e c ha ng e o f b od y w eig ht (% ) f. etac f. but f. wat acarbose (positive control) saline (negative control) eaf, b a b b a a a a b -20 -15 -10 -5 0 5 10 0 day after 4 days after 8 days after 12 days t he c ha ng e of b od y w ei gh t (% ) f. etac f. but f. wat acarbose (positive control) saline (negative control) wf, b a b b a a a a b -20 -15 -10 -5 0 5 10 0 day after 4 days after 8 days after 12 days th e c ha ng e o f b od y w eig ht (% ) f. etac f. but f. wat acarbose (positive control) saline (negative control) bf, b a b b a a a a b -20 -15 -10 -5 0 5 10 0 day after 4 days after 8 days after 12 days th e ch an ge o f b od y w ei gh t ( % ) f. etac f. but f. wat acarbose (positive control) saline (negative control)saline (negative control), b a b b a a a a b -20 -15 -10 -5 0 5 10 0 day after 4 days after 8 days after 12 days t he c ha ng e of b od y w ei gh t ( % ) f. etac f. but f. wat acarbose (positive control) saline (negative control) acarbose (positive control). eaf, bf and wf are hexane, chloroform, ethyl acetate, n-butanol and water extract fractions, respectively. nova biotechnol chim (2021) 20(1): e748 7 content compared to eaf and bf. results could be explained that the enzyme inhibitory activity of fractions depended on both the total number of phenolic compounds and the structure characteristics of individual phenolics in the extracts (pulido and saura-calixto 2000; gálvez et al. 2005; kwon et al. 2006). hyperglycemia is a phenomenon that occurred in type 2 diabetes patients that the glucose concentration in blood suddenly increases due to the hydrolysis of starch by pancreatic α-amylase and intestinal αglucosidase to glucose that is easily absorbed by the small intestine (gray 1995). therefore, the efficient method to control type 2 diabetes is the utilization of intestinal α-glucosidase and pancreatic α-amylase inhibitors due to their potent therapy to control hyperglycemia (krentz and bailey 2005). the extracts of several terminalia species possessed anti-hyperglycemic activity. nguyen et al. (2016) presented that methanol extract of t. alata possessed α-amylase and αglucosidase inhibitory activities as well as antihyperglycemic activity in diabetic rats. as the results presented by sabu and kuttan (2002), the methanol extracts of t. chebula and t. belerica at a dose of 100 mg.kg -1 body weight decreased 12.7 % and 13.1 % of glucose in diabetic rats after 4 hours treatment, respectively. the results of our previous study also indicated that the crude extracts of t. belirica and t. alata at a dose of 200 mg.kg -1 bw reduced 45.40 % and 46.01 % of glucose in diabetic rats after 4-h treatment, respectively (nguyen et al. 2016), similar to reduction rate of glucose in diabetic rats treated with wf and eaf at the same conditions in this study. moreover, similar to the crude extract, the fractions also had no significant effect on glucose concentration in normal rats at a dose of 200 mg.kg -1 body weight because a homeostasis in normal rats could control the normal regulatory mechanisms involved in carbohydrate metabolism (vats et al. 2002). in contrast, the fractions showed hypoglycemic activity in diabetic rats in both single and multiple doses (table 5). the significant reduction in blood glucose concentration in diabetic rats was found after the 8-day treatment with all fractions and acarbose (table 5) and kept constant until the 12 th day. table 4 expresses that the fractions which possessed high α-amylase inhibitory effect represented a strong effect on lowering blood glucose concentration in diabetic rats. thus, αamylase inhibitors could be considered as the effective drug for post-prandial hyperglycemia treatment because of the inhibition of gluconeogenesis and reduction of glucose absorption (youn et al. 2004). conclusions the current study found that ethyl acetate and water extract fractions from t. alata contained relatively high amount of phenolic contents. these fractions showed relatively high antioxidant activity and inhibitory activities against starchhydrolyzing enzymes. especially, these fractions significantly reduced blood glucose concentration in diabetic rats, which could be used for postprandial hyperglycemia treatment. further research on isolation and identification of the individual active compounds from ethyl acetate and water extract fractions as well as evaluation of their in vivo antioxidant activities and lowering blood glucose concentration in diabetic rats should be carried out in the future. acknowledgements this research is funded by vietnam national foundation for science and technology development (nafosted) under the grant number 106.99-2020.17. conflict of interest the authors declare that they have no conflict of interest. references abarca-vargas r, malacara cfp, petricevich vl (2016) characterization of chemical compounds with antioxidant and cytotoxic activities in bougainvillea x buttiana holttum and standl, (var. rose) extracts. antioxidants. 5: 45. chai tt, wong fc (2012) whole-plant 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husheem m, härkönen p, pihlaja k (2002) inhibition of cancer cell growth by crude extract and the phenolics of terminalia chebula retz fruit. j. ethnopharmacol. 81: 327-36. vats v, grover jk, rathi ss (2002) evaluation of anti hyperglycemic and hypoglycemic effect of trigonella foenum-graecum linn, ocimum sanctum linn and pterocarpus marsupium linn in normal and alloxanized diabetic rats. j. ethnopharmacol. 79: 95-100. youn jy, park hy and cho kh (2004) anti-hyperglycemic activity of commelina communis l.: inhibition of alphaglucosidase. diabetes res. clin. pract. 66s: 149-55. nova biotechnol chim (2020) 19(2): 124-137 doi: 10.36547/nbc.v19i2.765  corresponding author: daniela.chmelova@ucm.sk nova biotechnologica et chimica recombinant dna technology as a tool for improving production of polyhydroxyalkanoates by the natural producers daniela chmelová, barbora legerská and miroslav ondrejovič department of biotechnology, faculty of natural sciences, university of ss. cyril and methodius in trnava, nám. j. herdu 2, trnava, sk917 01, slovak republic article info article history: received: 12th october 2020 accepted: 24th november 2020 keywords: aeromonas bacillus cupriavidus halomonas polyhydroxyalkanoates pseudomonas recombinant abbreviations: crispr – clustered regularly interspaced short palindromic repeats hph – high pressure homogenization mscl – mechanosensitive channels of large conductance pha – polyhydroxyalkanoate tca – tricarboxylic acid cycle abstract polyhydroxyalkanoates (phas) are a group of the biodegradable polyesters, and represent an alternative to conventionally used petroleum-based plastics resistant to biodegradation. the production is not cost-competitive compared to conventional plastics, although, there are several bacterial producers capable of pha accumulating up to 80 % of their cells dry weight using low-cost substrates. pha production can be improved by transferring specific enzymes or entire metabolic pathways from the most efficient producers to other natural producers. therefore, the review is focused on genetic modification of bacterial producers, namely the genera cupriavidus, pseudomonas, halomonas, aeromonas and bacillus, for efficient industrial production of phas. recombinant pha producer can use non-traditional substrates like agro-industrial wastes, namely whey, lignocellulose or glycerol. it is possible to influence the shape and size of the producer's cell by over-expression or knockout of selected genes or to affect its preference for a specific component of a culture medium by modulation of a producer's basal metabolism. the costs of pha production still be reduced by simplifying the downstream process by enzymatic hydrolysis of selected parts of the cell or blocking the protective mechanisms of the cell against its autolysis caused by the ionic strength of the solution.  university of ss. cyril and methodius in trnava introduction polyhydroxyalkanoates (phas) as the microbial storage biopolymers are usually accumulated in response to the absence of a source of macroelements as oxygen, nitrogen, phosphorus, sulfur, iron or magnesium with an excess of carbon. cupriavidus, pseudomonas, aeromonas, bacillus, alcaligenes, enterobacter and certain cyanobacteria and halophilic bacteria produce phas under these conditions (leong et al. 2014). although some producers, namely alcaligenes eutrophus, azotobacter vinelandii uwd, alcaligenes latus or recombinant escherichia coli, are able to constantly accumulate phas without nutrient-limited conditions), but usually accumulate less pha (keshavarz and roy 2010). the producers usually generate short-chain length (scl) phas composed of monomers containing 3 to 5 carbon atoms (c. necator, a. latus), mainly polyhydroxybutyrate (phb) or polyhydroxyvalerate (phv), or medium-chain length (mcl) phas composed of monomers consisting of 6 to 14 carbon atoms (aeromonas caviae, pseudomonas putida) such as polyhydroxyhexanoate (phh), polyhydroxyoctanoate (pho), polyhydroxydecanoate (phd) or polyhydroxydodecanoate (phdd). pha producers rarely accumulate longmailto:daniela.chmelova@ucm.sk nova biotechnol chim (2020) 19(2): 124-137 125 chain length (lcl) phas composed of monomers containing more than 15 carbon atoms (zhang et al. 2006; tan et al. 2014b). the material properties of phas are similar to those of conventional synthetic petroleum-based plastics and therefore, represent a suitable alternative made from renewable resources (tian et al. 2005; berezina and martelli 2014; troschl et al. 2017). moreover, these biopolymers are fully biodegradable into carbon dioxide and water by microorganisms from a variety of artificial and natural environment, including wastewater, soil, rivers, lakes, seas, and even hot springs and polar regions (chen et al. 2018; favaro et al. 2019; tan et al. 2020). despite the above-mentioned advantages, the cost of pha production remains high compared to traditional plastics (koller et al. 2013). the aim of the research is therefore to find a fast and cheap way to produce phas. researchers have previously focused on finding a suitable and low-priced substrate for the production of specific phas, including the use of by-products derived from a production process as a source of carbon and energy. current research is concerned with the metabolic improvement of selected species, either in terms of influencing metabolic pathways leading to pha accumulation, expanding the range of substrates used by the producers or facilitating isolation of phas from the cell. in this review, we summarize the latest knowledge about the possibilities of increasing pha production by genetically modified pha producers of the genera cupriavidus, pseudomonas, halomonas, aeromonas and bacillus. genetically modified pha producers genetic modifications for inexpensive carbon sources utilization selection of carbon source is one of the important factors that influence the price of a pha product. it must be cheap and available in large quantities (choi and lee 1997). by-products from various industrial or agricultural productions are strong candidates for this purpose (koller et al. 2005; solaiman et al. 2006). however, a number of phaaccumulating bacterial producers cannot use them as a substrate, but this can be changed through genetic engineering. whey high in lactose, proteins and lipids as a byproduct of milk processing is available in large quantities, but only some strains from the group of pha producers can utilize it, namely hydrogenophaga pseudoflava dsm1034 (koller et al. 2007), methylobacterium sp. zp24 (yellore and desai 1998) and thermus thermophilus hb8 (pantazaki et al. 2009). therefore, the induction of the lacz gene coding for β-galactosidase was one of the first attempts to expand the range of substrates for pha production. povolo et al. (2010) disrupted the phaz1 (pha depolymerase) gene of the c. necator wild-type strain by the lacz gene replacement with the aim of producing phas in a medium with lactose as the sole carbon source. in contrast to the wild-type c. necator, the recombinant strain was able to grow and produce phb in a medium with lactose and whey. phb accumulation represented 30 % of cell biomass after 48 h in a hydrolyzed whey permeate composed mainly of glucose and galactose, and 22 % of cell biomass after 48 h in a nonhydrolyzed permeate high in lactose. the yield of phb probably reduced due to non-optimal cultivation conditions. however, due to the low yields of phas, research is focused on finding the native pha-accumulating producers containing lactose-degrading enzyme (favaro et al. 2019). molasses is another agricultural by-product of sugarcane or sugar beet processing in the sugar industry. the material contains sucrose usable as the sole carbon source for a pha producer with the sucrose-degrading enzyme system. if this is not the case, then a gene encoding β-fructofuranosidase, an enzyme that cleaves sucrose to glucose and fructose, can be inserted into the genome of a pha producer. park et al. (2015) inserted this gene into the c. necator wild-type strain to produce high content of phb (73.2 wt %) from molasses. the conversion of byproducts from oil processing has also been achieved with genetically modified strains. an example is the utilization of glycerol as a carbon source for pha production. cardozo et al. (2019) inserted the bmgd gene encoding glycerol3-phosphate dehydrogenase into the bacillus megaterium pha producer, and observed nova biotechnol chim (2020) 19(2): 124-137 126 a higher biomass yield and pha content compared to the wild-type producer. lignocellulose is one of the promising widely available and renewable sources of carbon and energy. hydrolysates of various plants have previously been used as the substrates for pha production. in hydrolysates, the target substrate for pha production was glucose as a hydrolytic product released from cellulose. then, research focused on the utilization of hemicellulose degradation products by incorporating genes required for the consumption of arabinose (lu et al. 2013) or xylose (liu et al. 2014) into c. necator. although these carbohydrates can be used as substrates, lignocellulosic material is generally difficult to use in fermentation processes due to the complicated structure and compounds with a potentially toxic effect on bacterial growth. phenolic compounds linked to lignocellulose via ester bonds pose a challenge for bioproduct conversion. they are released from lignin as well as hemicelluloses and significantly inhibited the growth of a wide range of microorganisms (graf and altenbuchner 2014; bouarab-chibane et al. 2019). currently, attempts to assimilate phenols from lignocellulose to valuable products are known. zhou et al. (2020) focused on the use of ferulic acid for pha production. although the pha producer of p. putida kt2440 has natural ability to convert ferulic acid to mcl-pha, the organism's low tolerance to ferulic acid and its toxicity complicate fermentation process. zhou et al. (2020) modified the genome of p. putida kt2440 using a crispr/cas9 (clustered regularly interspaced short palindromic repeats) genomeediting strategy and via this method, nine genes involved in ferulic acid-to pha bioconversion were integrated into the p. putida genome. the recombinant strain increased the production of pha to 270 mg.l-1 associated with the utilization of 20 mg.l-1 ferulic acid from a medium. genetic modifications for production of pha in co-cultivation system co-cultivation represents an interesting strategy for the simultaneous cultivation of two or more organisms. the metabolic product of the first producer becomes a substrate for another in order to produce the desired metabolite and also utilize a by-product (goldford et al. 2018; fedeson et al. 2020). this concept occurs naturally in the environment, meaning various types of bacteria in communities form a network of interspecies interactions. therefore, the strategy appears to be a suitable alternative for the utilization of difficultto-use carbon sources for pha producers. the co-cultivation system for pha production usually consists of two bacterial strains (fedeson et al. 2020; hobmeier et al. 2020; liu et al. 2020). hobmeier et al. (2020) tested the possibility of mcl-pha production in a co-culture of synechococcus elongatus cscb with p. putida. s. elongatus fixes co2 and metabolizes it to sucrose at high salt concentrations and then p. putida metabolizes sucrose and accumulates mcl-pha in the cytoplasm. however, s. elongatus requires a nitrate-containing medium for efficient sucrose production and p. putida can convert nitrate to ammonium via nitrite that uses as a nitrogen source for its growth. this is a challenge because the pha production by p. putida is more profitable under nitrogen-limited conditions. the recombinant strain of p. putida with a deletion of the nast gene lost the ability to convert nitrate as a nitrogen source, but the ability to grow in a medium with ammonium was not affected. the mutant was able to produce up to 8.8-fold higher pha yield (14.8 %) in the co-culture compared to the wildtype strain. co-cultivation system consisting of recombinant bacterial species of s. elongatus and p. putida was also used in the work of fedeson et al. (2020). the aim was the simultaneous production of mclphas and the bioremediation of toxic 2,4-dinitrotoluene (2,4-dnt). the recombinant sucrose-uptake p. putida (hobmeier et al. 2020) was further modified by insertion of the dnta and dntb genes from burkholderia sp. r34 allowing the degradation of 2,4-dnt to form nontoxic metabolites of nitrite, pyruvate and propionylcoa. this co-cultivation resulted in the degradation of 2,4-dnt associated with the production of mcl-pha. although, a lower mcl-pha yield compared to the results of the other studies (yang et al. 2019; hobmeier et al. 2020) was observed, the advantage is the combination of biopolymer production and bioremediation thus extending the possibility of using co-cultivation strategies. nova biotechnol chim (2020) 19(2): 124-137 127 liu et al. (2020) verified the possibility of coculture system consisting of recombinant e. coli and p. putida strains. the production of mcl-pha was stimulated by the presence of acetate produced by recombinant e. coli. moreover, e. coli was modified by knockout of the ptsg, manz, atpfh and envr genes resulting in the preferred carbon source being xylose (not glucose) and p. putida had an amplified acetate assimilation pathway by overexpression of acetyl-coa synthetase and acetate kinase-phosphotransacetylase (yang et al. 2019). the growth of p. putida was ensured by glucose as a carbon source and mcl-pha production by acetate produced by the recombinant e. coli strain. the highest mcl-pha yield was observed by using a 20 g.l-1 carbon source (glucose : xylose, 1 : 1; w/w), namely 0.541 g.l-1, which is 3.9-fold higher compared to p. putida monoculture. morphology engineering for improving pha production morphology engineering is an interesting approach to increase the efficiency of pha production without interfering the metabolic pathways of pha production. this type of engineering affects the morphology of the cell and allows the formation of larger pha granules, which should also facilitate their isolation. a number of genes are responsible for the shape of the cells, and by modifying them, it is possible to change the shape of the cell from rods to fibers or to form large from small spheres. for example, the mincd gene is responsible for cell length, the ftsz gene for the production of cell division protein and the mreb gene for cytoskeleton protein production (jiang and chen 2016; elhadi et al. 2016; zhao et al. 2019). over-expression of the mreb gene led to the formation of larger cells compared to the control (wu et al. 2016), but over-expression of this gene associated with knockout of the mincd gene increased mcl-pha production in p. mendocina nku (zhao et al. 2019). however, this strategy was more successful in the process of producing phb by the recombinant e. coli strain (elhadi et al. 2016). tan et al. (2014a) observed that the inducible over-expression of the mincd gene during the stationary growth phase of halomonas td08 elongated cell shape, leading to 1.2-fold higher accumulation of phb in the cells. another advantage of modifying the cell shape was the formation of intertwined fiber network during the production of phb. this network allowed the self-flocculation and precipitation of cells, simplifying the subsequent downstream processing. jiang et al. (2017) developed a temperaturesensitive plasmid expression system containing the mreb or ftsz genes and introduced this construct into h. campaniensis cells. recombinants are able to grow at 30 °c and their growth is comparable to the wild-type strain. morphological changes such as cell size expansion or cell shape elongation did not appear until the temperature was raised to 37 °c and the temperature-sensitive plasmid was no longer replicated in the recombinants. this led to an increase in phb yield, which reached up to 80 % cell dry weight. the results of the above-mentioned research show a new interesting strategy for the industrial pha production by combining the expression and/or knockout of various metabolic and morphological genes. it is possible to influence not only the cell morphology but also the morphology of the pha granules themselves. the phap1 gene is responsible for regulating the size and number of pha granules. knockout of this gene resulted in the production of pha granules up to 10 μm accumulated in the intracellular space of recombinant halomonas bluephagenesis td01 cells. the resulting size was limited only by the size of the cell itself (shen et al. 2019). genetic modifications for improving pha biosynthesis phb synthesis is one of the most studied metabolic pathways of phas. it consists of three basic enzymatic steps including enzymes such as phaa (β-ketothiolase), phab (nadph-dependent acetoacetyl-coa reductase) and phac (pha synthase) (fig. 1). phaa catalyzes the condensation of two acetyl-coa molecules to acetoacetyl-coa that is converted to (r)-3-hb-coa ((r)-3hydroxybutyryl-coa) by phab. in the final step, phac polymerizes (r)-3-hb-coa into phb. phac belongs to the key enzymes for pha biosynthesis and catalyzes the polymerization of hydroxyacylnova biotechnol chim (2020) 19(2): 124-137 128 coa thioesters. the other pha biosynthetic pathways differ mainly in the enzymes forming various hydroxyacyl-coa thioesters for final polymerization by phac. β-oxidation of fatty acids is an alternative pha biosynthetic pathway for the formation of hydroxyacyl thioesters (fig. 1). the products of this pathway are mainly mcl-phas due to the length of formed precursors. the key enzymes are fade (acyl-coa dehydrogenase) and phaj (enoyl-coa hydratase). fade catalyzes the reaction of acylcoa to enoyl-coa and phaj uses enoyl-coa as a substrate. some phas can be synthesized from (r)-3-hydroxyacyl-coa obtained from 3-ketoacylcoa using fabg (3-ketoacyl-coa reductase) (philip et al. 2007). the third pha biosynthetic pathway involves two important enzymes, namely phag (3-hydroxyacylacp-coa transferase) and fabd (malonyl-acp transacylase) (fig. 1). the substrates are converted to (r)-3-hydroxyacyl-acp and then to (r)-3hydroxyacyl-coa by phag and thus to pha by phac (philip et al. 2007; laycock et al. 2013). the involvement of above-mentioned enzymes in pha biosynthesis associated with important metabolic pathways such as calvin, ribulose monophosphate (rump) or krebs cycle, glycolysis as well as pathways for biosynthesis and degradation of amino acids and fatty acids (lu et al. 2009). bacteria can accumulate phas composed of different monomers depending on the carbon source used (noda et al. 2005), with phac playing a key role in this process. the phac enzymes with different specificity can use variable precursors for pha synthesis. phac can be classified into one of four classes (i – iv) based on primary structure, substrate specificity, and subunit composition (rehm 2003; pötter and steinbüchel 2005). recently, tan et al. (2020) discovered a new phac isolated from the antarctic janthinobacterium isolate, which could be included in the new fifth class. over-expression of the phac gene generally leads to higher yields of phas. akdoğan and çelik (2020) over-expressed the native phac gene in b. megaterium, resulting in increased biomass yield and pha content compared to the wild-type strain in both batch and fed-batch cultivation processes. the use of the phac gene isolated from other pha producers may lead to the incorporation of the new hydroxyacyl-coa thioester monomers to the growing pha chain, and moreover, knockout of the original phac gene and incorporation of the new phac gene can cause the formation of a completely new pha. after insertion of the phac gene from pseudomonas sp. 61-3 into c. necator, the mutant was able to accumulate copolymer p(3hb-co-3ha) with a relatively high 3hb content (matsusaki et al. 2000a; matsusaki et al. 2000b; matsumoto et al. 2001). cha et al. (2020) tested the effect of four different phac genes from thiocapsa pfennigii, c. necator h16, chromobacterium violaceum, and paracoccus denitrificans on pha production by p. putida em42. knockout of the phac1 and phac2 native genes in all four mutants resulted in the production of p(3hv-co-4hv) copolymer instead of the naturally produced mcl-pha consisting of 3hd and 3ho. the most common ratio of the accumulated scl-pha was 60 : 40 (w/w) for 3hv and 4hv, respectively and 3hb content was only about 1 %. the mutant with the phac gene from p. denitrificans produced a copolymer with a ratio of 3hv : 4hv 96 : 4 (w/w). this synthase showed lower substrate specificity for 4hv compared to other synthases. insertion of the phac gene from another producer may be accompanied by a negative effect on the type of produced pha. the aeromonas hydrophila mutant with the phac gene from pseudomonas stutzeri 1317 accumulated pha consisting of c4-c12 monomers with a dominant amount of 3hhx instead of p(3hv-co-4hh) (liu et al. 2011). tan et al. (2020) used the c. necator mutant with the phac genes from antarctic janthinobacterium isolates. no accumulation of pha was observed in the first case and in the second case, the mutant accumulated only 18.7 wt % of p(3hb). the higher phb content was determined in biomass growing at 20 °c compared to 30 °c. the key factor influencing the choice of synthase source is both the specificity of the enzyme and the differences of cultivation conditions between the pha producer and the phac source. the other important enzymes for phb biosynthesis are phaa and phab responsible for the preparation of precursors for the synthesis of phb chain (philip et al. 2007). if phaa and phab expression is too nova biotechnol chim (2020) 19(2): 124-137 129 fig. 1. metabolic pathways of pha production (modified from choi et al. 2020). legend: 3ha-coa – 3-hydroxyacyl-coa; 3hb – 3-hydroxybutyrate; 3hp – 3-hydroxypropionate; 3hv – 3-hydroxyvalerate; 4hbd – 4-hydroxybutyrate dehydrogenase; aldd – aldehyde dehydratase; bktb – β-ketothiolase; cat2 – 4-hydroxybutyryl-coa transferase; coa – coenzyme a; dhab – glycerol dehydratase; dhat – 1,3-propanediol dehydrogenase; faba – 3-hydroxyl-acp dehydrase; fabb – β-ketoacyl-acp synthase i; fabd – malonyl-coa:acp transacylase; fabf – β-ketoacyl-acp synthase ii; fabg – 3-ketoacyl-acp reductase; fabh – β-ketoacyl-acp synthase iii; fabi – enoyl-acp reductase; fada – 3-ketoacyl-coa thiolase; fadb – 3-hydroxyacylcoa dehydrogenase; fadd – acetyl-coa synthetase; fade – acyl-coa dehydrogenase; hada – isocaprenoyl-coa:2hic coatransferase; pcs' – propionyl-coa synthetase; pct – propionyl-coa transferase; pdup – propionaldehyde dehydrogenase; phaa – β-ketothiolase; phab – acetoacetylcoa reductase; phac – polyhydroxyalkanoate synthase; phad – phapolyhydroxyalkanoate depolymerase; phag – 3-hydroxyacyl-acp-coa transferase; phaj – enoyl-coa hydratase; prpe – propionyl-coa synthetase; sbm – methylmalonyl-coa mutase; sucd – succinate semialdehyde dehydrogenase; ygfg – methylmalonyl-coa decarboxylase. low and there are no other sources of metabolic precursors for pha synthesis, over-expression of the phac gene does not lead to an increase in biopolymer accumulation (liu et al. 2011). the activity of phaa and phab should be checked before modifying the producer's phac gene. overnova biotechnol chim (2020) 19(2): 124-137 130 expression of the phaa and phab genes in the a. hydrophila mutant caused 85 % increase in copolymer accumulation compared to the wildtype strain. knockout of the acetic acid pathway was also beneficial for this strain resulting in further 47 % higher accumulation of pha copolymer due to the prevention of acetic acid formation (liu et al. 2011). unlike liu et al. (2011), shi et al. (2020) did not reduce acetic acid production by deleting certain genes in the acetic acid pathway, but used this acid as a carbon source to produce hbt in the a. hydrophila mutant with over-expression of the phaa and phab genes. moreover, overexpression of enzymes involved in the acetic acid pathway caused further 6-fold higher phb content compared to that produced by control strain without this modification. the high activity of phaa and phab caused the accumulation of a sufficient intracellular concentration of 3-hydroxybutyryl-coa, a key intermediate for phb synthesis by phac. subsequent overexpression of phac led to further increase in pha accumulation to 19.82 % of cell biomass. this is the first study to report the production of phb by engineered a. hydrophila in the acetatepropionate medium. phab uses nadh or nadph to catalyze its reaction. the most of phb-accumulating bacteria use nadph dependent phab (ling et al. 2018). nadph is a typical cofactor for the redox reaction of anabolism. selected species of bacteria such as allochromatium vinosum, azotobacter beijierinckii and h. bluephagenesis have nadh dependent phab. nadh plays a key role in respiration or anaerobic fermentation processes at high or low oxygen concentrations, respectively (sauer et al. 2004). nadph-dependent phabs are dependent on the availability of a reduced form of nadph, which is consumed in any anabolic process in the cell and therefore, a significant accumulation of pha occurs under essential element-limited conditions (n, p, s, fe or mg) with an excess of carbon (steinbüchel 1991). at the same time, nadh is used exclusively within respiration or fermentation. ling et al. (2018) demonstrated that h. bluephagenesis produced higher concentrations of nadh under oxygen-limited conditions. blocking the etf operon limited electron transfer from nadh, a lack of oxygen increased the nadh/nad+ ratio, resulting in an increase phb content to 90 % of dry weight. phb production under oxygen-limiting conditions represents a significant reduction in energy consumption due to the possibility of carrying out fermentation with a lower agitation rate and less aeration (ling et al. 2018). the problem of nadh excess inhibiting pyruvate metabolism and the tricarboxylic acid (tca) cycle was solved by adding acetic acid to the culture medium. nadh-dependent phab consumes nadh during the reduction of acetic acid to 3hb-coa. moreover, the addition of the acetic acid increased both the biomass yield and the phb content. production of new copolymers can be achieved by replacing the phab gene with genes involved in other metabolic pathways of pha production. jung et al. (2019) prepared the recombinant c. necator strain by deletion of the phab1, phab2 and phab3 genes and over-expressing a synthetic operon consisting of phac2 from rhodococcus aetherivorans, phaa and phaj from pseudomonas aeruginosa. the recombinant strain produced the terpolymer p(3hb-co-3hv-co-3hhx) (42 % of dry weight) in a medium containing volatile fatty acids such as propionate and butyrate instead of p(3hb). yu et al. (2020) deleted the native phac gene in h. bluephagenesis and expressed the phac and phaj genes from a. hydrophila and endogenous fadd (acyl-coa synthetase). the mutant produced copolymer p(3hb-co-3hhx) with the high content of 3hh (35 %). fade and phaj play a key role in β-oxidation of fatty acids. although fade represents a ratelimiting step in β-oxidation of fatty acids, it has received little attention (eggers and steinbüchel 2013; magdouli et al. 2015). phaj is one of the more studied enzymes because its insertion or modification makes it possible to obtain scl-mcl copolymers using a recombinant c. necator strain with more advantageous physical properties, such as flexibility and elasticity, compared to commonly produced phb (noda et al. 2005). flores-sánchez et al. (2020) constructed a recombinant c. necator with an inserted phaj1 gene from p. putida kt2440 and a fade gene from e. coli k12. activation of the fade gene caused the higher biomass yield and pha accumulation than the original strain. the phaj1 gene from p. putida was selected because of its high affinity for 3hhx nova biotechnol chim (2020) 19(2): 124-137 131 and 3ho substrates. its activation led to the implementation of 3ho and 3hd into the formed scl-mcl copolymer. this result supports the hypothesis that phac from c. necator can incorporate also mcl monomers into the pha chain if suitable metabolic precursors are available. deletion of phag and phaz genes involved in β-oxidation of fatty acids causes the production of mcl-pha with an increased content of 3hd or 3hdd monomers. zhao et al. (2020) prepared the p. mendocina nk-01 mutant with deleted phag and phaz genes. deletion increased the amount of 3hd (94.7 mol %) and 3hdd (68.7 mol %) in a medium containing sodium decanoate and sodium dodecanoate. fadd allows efficient conversion of lipids to pha through fatty acid metabolism (fig. 1). liu et al. (2011) observed that co-expression of the vgb (vitreoscilla hemoglobin gene) and fadd (escherichia coli) genes in a. hydrophila 4ak4 led to the production of homopolyester p(3hv) (47.74 %). the vitreoscilla hemoglobin (vhb) is an important oxygen-binding protein. vhb is induced at low concentration of oxygen in wild-type producers, effectively binding the oxygen and supplies it to the terminal respiratory oxidases, leading to respiration under oxygen-deficient conditions (webster 1988). this mechanism affects the production of pha by bacteria such as e. coli, a. hydrophila and p. putida (liu et al. 2011; duarte de oliveira et al. 2020). tang et al. (2020) expressed the vgb gene in c. necator together with the knockout of the ldh gene (l-lactate dehydrogenase). the mutant showed higher productivity and phb content and lower production of fermentation by-products compared to the wildtype strain in low oxygen culture medium. ouyang et al. (2018) expressed low-oxygen inducible vhb in pha producers of h. bluephagenesis td01 and h. campaniensis ls21. both mutants achieved more intensive biomass growth and higher phb production. however, duarte de oliveira et al. (2020) observed that the production of vhb did not mitigate the negative effect of oxygen limitation. the efficiency of vhb engineering must be assessed for each producer. there is a possible explanation for these different results. while duarte de oliveira et al. (2020) focused on the producers of mcl-pha derived from precursors of β-oxidation of fatty acids, tang et al. (2020) worked with the producers of scl-pha derived from precursors of glycolysis. duarte de oliveira et al. (2020) also observed the lower biomass yield and pha content compared to the wild-type strain. these results suggest that an incorrectly selected metabolic modification may cause an increase in the metabolic load of the cell, resulting in a decrease in pha production. 3-hydroxyacyls are commonly produced, but it is also possible to enrich the biopolymer with 4-hydroxyacyls by inserting the orfz gene (4hbtransferase) responsible for the conversion of 4hb to 4hb-coa. the h. bluephagenesis mutant with the orfz gene from clostridium kluyveri with the t7-like expression system (mmp1) produced copolymer p(3hb-co-4hb) and the total content of the copolymer was 63 % of dry weight (chen et al. 2017). shen et al. (2018) were also able to increase the accumulation rate of this copolymer up to 80 % by using a more suitable promoter. genetic modifications to avoid pha degradation the intracellular activity of pha depolymerase represents another challenge in pha production. the enzyme is activated in the absence of a carbon source and degrades the accumulated pha granules in the cell, resulting in the release of carbon and energy that the producer can use for its growth (steinbüchel and hein 2001). these enzymes are present on the surface of pha granules (rehm 2003) and, therefore the inactivation of intracellular pha depolymerase by genetic engineering appears to be necessary to increase pha recovery. povolo et al. (2015) deleted the phaz1 gene encoding pha depolymerase in the c. necator wild-type strain and their results suggest an increase in phb accumulation during bacterial growth. however, knockout of the phaz3 gene had no statistically significant effect on phb accumulation, suggesting that the phaz3 gene is unlikely to play a role in phb degradation (povolo et al. 2010). kobayashi and kondo (2019) also observed 2.9-fold higher phb production by rhodobacter sphaeroides hj mutant with knockout of phaz. moreover, knockout of this gene associated with over-expression of phb biosynthesis genes (phaa3, phab2 and phac1) caused 3.9-fold higher phb production compared nova biotechnol chim (2020) 19(2): 124-137 132 to the wild-type strain. similar results have been reported by other authors focusing on the inactivation of mcl-pha depolymerases (de eugenio et al. 2007; cai et al. 2008). it should be noted that knockout of pha depolymerase is not always associated with increased pha recovery. zhou et al. (2020) observed that the incorporation of the phac1 gene in p. putida ktc9n13 associated with disruption of the phaz gene did not cause a significant increase in phb accumulation. also, vo et al. (2015) did not observe a significant effect on increasing pha production by damaging the phaz gene in p. putida kt2440. deletion of this gene caused 1.2-fold lower mcl-pha production than wild-type strain. salvachúa et al. (2020) did not observe a significant effect on pha production by disruption of the phaz gene in p. putida kt2440. different results of these studies could be due to various cultivation conditions and genetic variability (difference of genotypes, deletion of the selected phaz gene) (zhou et al. 2020). genome reduction for improving pha production genome reduction is a process in which appropriate deletion of selected genome fragments can increase producer growth, broaden the substrate specificity, improve energy metabolism, and increase the product yield and the genome stability (fehér et al. 2007; chi et al. 2019). the process eliminates a large proportion of the genome which is not important for the growth and production of pha but consumes a lot of energy and carbon (wang et al. 2018). the p. mendocina nk-01 mutant with 14 large fragments removed and 7.7 % genome reduction accumulated 11-fold higher mcl-pha content than the wild-type strain (fan et al. 2020). deletion of 6 genes involved in the β-oxidation of fatty acids of p. medocina caused an increase of the dominant 3hd monomer to 90.01 mol % and total mcl-pha content to 58.6 mol %. moreover, this modification increased the cell shape due to the higher accumulation of pha granules in the intracellular space (zhao et al. 2020). p. putida kt2440 with 4.12 % reduction of the total genome size increased the amount of mcl-pha up to 1.5-fold compared to the p. putida wild-type. knockout of gcd gene (glucose dehydrogenase gene) in the p. putida mutant caused a further 22.17 % increase in pha content (liang et al. 2020). wang et al. (2018) deleted genes involved in the production of flagella and pili in the p. putida kt2442 genome. the genome reduction by 1.2 % resulted in accelerated mutant growth, no flagella production, reduced biofilm production and 45.6 % higher pha production than the original strain. genetic modifications for simplify pha isolation down-stream processes affect the total cost of pha production. the choice of isolation and purification steps is also related to the application of the biopolymer, because some steps may alter pha properties or add undesirable biomass-derived compounds such as lipoproteins or toxins to the final product (koller et al. 2013). modification of the producer's genome by molecular biology tools may facilitate the process of isolating pha granules from the cell (borrero-de acuña et al. 2017; poblete-castro et al. 2020) or reduce the number of isolation steps (boynton et al. 1999; rodríguez gamero et al. 2018). in recent years, the goal is to develop a programmable cell lysis system that allows the isolation of intracellular metabolites by autolysis of the production organism (hajnal et al. 2016; tamekou lacmata et al. 2017; borrero-de acuña et al. 2017). hori et al. (2002) used the phage holin-endolysin system in b. megaterium to release pha from cells. ultimately, approximately two-thirds of the accumulated pha was released from the cells after induction of lysis by xylose. cell lysis lasted approximately 30 h. martinez et al. (2011) used a similar system inducible by 3-methyl benzoate in p. putida kt2440. it allowed the disruption of approximately 86 % of the cells within 15 h and facilitated the subsequent extraction of pha. borrero-de acuña et al. (2017) used toluic acid as the inductor in the phage holin-endolysin system. although the use of the toluic acid inducer in p. putida kt2440 reduced the biomass by 25 % during cultivation, mcl-pha content was maintained at 30 %. after lysis, 95 % of the bacterial cells were disrupted and approximately 75 % of the total biopolymer was recovered during 16 h. hajnal et al. (2016) tested the implementation of the lambda nova biotechnol chim (2020) 19(2): 124-137 133 lysis system works in h. campaniensis. spontaneous autolysis was observed after centrifugation of the cells and prolonged passive standing. the use of the lytic system has some disadvantages such as relatively long lysis time (15 – 30 h) and low amount of polymer released (75 86 %) (hori et al. 2002; martinez et al. 2011; borrero-de acuña et al. 2017). another possibility is to modify the mechanisms responsible for maintaining cell wall integrity. oprf (porin f) is a protein responsible for the permeability and integrity of the cell wall (chevalier et al. 2017), opre (porin e) increases the diffusion of small ions (jaouen et al. 2006), mscl (mechanosensitive channels of large conductance) acts as a safety valve opening pores in sudden hypo-osmotic shock, what is the last protection of the cell against their lysis due to osmotic shock (colombo et al. 2003). poblete-castro et al. (2020) modified this system in p. putida by over-expressing the oprf and opre, and deleting mscl. due to hypertonic and hypotonic treatment, 95 % of the cells were lysed within 3 h, with 94 % mcl-pha yield. high-pressure homogenization (hph) is one of the most widely used methods for cell lysis and is also one of the environmentally acceptable methods. however, its use releases large amounts of dna, which increases the viscosity of the solution and complicates subsequent purification procedures (ling et al. 1998; tamer et al. 1998; van wegen et al. 1998). the decrease of viscosity is usually achieved by expensive processes, such as heat treatment or the addition of hypochlorite or commercially available nucleases (koller et al. 2013). however, it is possible to insert the nuclease gene into a natural producer of pha to reduce the viscosity and the cost of the downstream process. this method was also used by boynton et al. (1999), which inserted the nuc gene encoding a nuclease from staphylococcus aureus into natural pha producers such as c. necator and p. putida. no negative effect on pha growth and production was observed for both pha producers. it was recorded the lower viscosity of the solution compared to wild-type strains treated with a commercial nuclease. the highest nuclease activity was observed for p. putida. rodríguez gamero et al. (2018) inserted the nuc gene into two native pha producers, c. necator dsm 545 and delftia acidovorans dsm 39. the gene was expressed in both producers, and the inserted gene did not affect the amount of produced biomass. in both cases, a higher amount of phb was reported. the nuclease activity was higher in the c. necator mutant; the recombinant was used in the next experiments. the lower viscosity of the solution after hph was observed compared to the original strain. conclusion genetic engineering of native pha producers appears to be a suitable way to reduce the cost of pha production. molecular biology techniques can be used to broaden the range of substrates suitable for the production of phas, such as whey, molasses or crude glycerol. morphology engineering as a new strategy for the construction of efficient microbial cell factories makes it possible to obtain larger and easier isolatable pha granules from modified cells. overexpression of selected genes involved in the pha synthesis or knockout of the genes for specific metabolic pathways are associated with several advantages, such as increased pha production, expansion of the range of produced biopolymers with different material properties adaptable to the intended purpose. genetic modification also provides various ways to increase the number of monomers for pha synthesis. the co-cultivation of two or more recombinant bacterial species appears to be promising due to cost reduction as well as the utilization of by-products. the overall resulting cost of phas can be reduced by a suitable isolation method through the implementation of selected self-destruction mechanisms based on the lytic system of bacteriophages or by porins. a key aspect of pha production is use of by-products that presents an alternative to products primarily intended for human consumption. production of phas can be introduced as a part of future biorefinery concept. acknowledgements this work was supported by the slovak research and development agency under the contract no. appv-18-0420. nova biotechnol chim (2020) 19(2): 124-137 134 conflict of interest the authors declare that there is no conflict of interest. references akdoğan m, çelik e (2020) enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) biopolymer by recombinant bacillus megaterium in fedbatch bioreactors. bioprocess biosyst. eng. 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n (2020) development of a crispr/cas9n-based tool for metabolic engineering of pseudomonas putida for ferulic acid-to polyhydroxyalkanoate bioconversion. commun. biol. 3: 98. nova biotechnol chim (20xx) xx(x): e916 doi: 10.36547/nbc.916 1 nova biotechnologica et chimica chemical profile, antioxidant and photoprotective activities of essential oil and crude extracts of algerian thymus serpyllum nariman madouni1, , boumediene meddah1, tir touil aicha1, chawki bensouici2, yavuz s cakmak3, alessandra piras4, danilo falconieri4, pascal sonnet5 1bioconversion, microbiological engineering and health safety, snv faculty, mustapha stambouli university sidi said, mascara 29000, algeria 2centre de recherche en biotechnologie, ali mendjli, nouvelle ville uv 0.3, constantine bp e73, algeria 3department of biotechnology and molecular biology, faculty of science and letters, aksaray university, aksaray 68100, turkey 4department of chemical and geological sciences, university of caligari, citadella universitaria, sp 8, monserrato-sestu km 0.700, monserrato (ca) 09042, italy 5agir laboratory: agents infectieux, résistance et chimiothérapie, ea4294 ufr de pharmacie, université de picardie jules verne, amiens 80037, france  corresponding author: nariman.madouni@univ-mascara.dz article info article history: received: 20th march 2021 accepted: 16th august 2021 keywords: antioxidant essential oil gc/ms hplc phenolic extracts thymus serpyllum abstract thymus serpyllum l. is an aromatic and medicinal plant widely used in algerian folk medicine. it was collected from the mascara region in the north-west of algeria and studied with the aim to provide more knowledges and update about chemical composition, antioxidant and skin-protective activities of essential oil, ethanolic and infusion extracts. the chemical analysis of investigated t. serpyllum essential oil (eo) was performed for the first time in this research work. it was carried out by gc/ms for identifying of 25 components where the dominated compounds were carvacrol (66 %) and γ–terpinene (11.5 %). ethanolic and infusion extracts were analyzed using hplc/dad detector type chromatography and revealed that benzoic acid and rosmarinic acid were found as the major compounds. the antioxidant activity was determined using the dpph, galvinoxyl radical (gor), cuprac, reducing power, and o-phenanthroline methods. all extracts showed a significant antioxidant capacity with different mechanisms. however, ethanol and infusion extracts showed stronger capacity than eo. moreover, the photo-protective (skin-protective) activity of t. serpyllum extracts was explored for the first time in our study. extracts exhibited high values of sun protective factors (spf) with 38.34 ± 2.29 and 38.82 ± 2.23 for ethanol and infusion extract, respectively. these results suggest a potential use of thymus serpyllum as a source of bioactive compounds with antioxidant and skin-protective properties.  university of ss. cyril and methodius in trnava introduction a permanent exposure to various stimulus and aggressors can generate the production of reactive oxygen species (ros) and reactive nitrogen species (rns) with higher concentrations. these reactive species are produced by a variety of biochemical processes (e.g. production of energy atp, biosynthetic and detoxification reactions) and can be neutralized by molecules called “antioxidants” (martins et al. 2015; yang et al. 2018). an excess of production with a low level of mailto:nariman.madouni@univ-mascara.dz nova biotechnol chim (20xx) xx(x): e916 2 antioxidants can lead to the appearance of almost all pathologies such as inflammations, cancer, alzheimer, aging and skin diseases (kindl et al. 2015). therefore, some studies are in quest for natural products that can be applied orally or topically to ameliorate skin reactions, reverse and block uv radiations (korać and khambholja 2011). in food processing, one of the main problems is lipid oxidation that can occur especially during storage and distribution, leading to the development of disagreeable flavors and potential occurrence of toxic substances (shahidi and ho 2007). as a preventive strategy the antioxidants are remarkably used as additives. therefore, an increasing interest in natural antioxidants provides aromatic and medicinal plants as an alternative (zehiroglu and sarikaya 2019). among these plants, those belonging to thyme genus comprising of 215 species, mainly prevalent in mediterranean regions, north africa, south europe, and asia (jarić et al. 2015). thymus serpyllum l. known as wild thyme and called “zaatar” in regional language, grows naturally in the mascara mountains. since antique times, infusions and decoctions of wild thyme were used in folk medicine. fresh and dry leaves are used in mediterranean kitchen as flavoring and preservative food agent (nikolić et al. 2014). based on the bioactivity of wild thyme reported in literature and no such investigations were performed about t. serpyllum of the mascara region, the aim of this study was to explore the chemical profile and to determinate different mechanisms of antioxidant potential of eo, ethanol and infusion extracts. further a skin-protective activity was investigated to estimate the sun protection factor (spf). experimental plant material and reagents the aerial parts of investigated thymus serpyllum l. were gathered during the flowering stage (may – july 2019) in the north-west of algeria (mascara region). botanical identification was achieved by prof. benhassaini hachemi and the voucher specimen (e01/lbv/udl/2019) has been deposited at the herbarium. the collected material (leaves and flowers) was dried in shade at a temperature 28 – 30 °c for 10 days, then grinded into fine powder. the chemical products and reagents used in all experiments were: 1,1-diphenyl-2-picrylhydrazyl (dpph), neocuproine, 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (trolox), trichloroacetic acid (tca), 1,10-phenanthroline, potassium ferricyanide, ethanol, methanol, acetone (all were obtained from sigma-aldrich gmbh, sternheim, germany. the iron (iii) chloride (fecl3), ammonium acetate, and copper (ii) chloride (cucl2) were obtained from biochem chemopharma (cosne-cours-sur-loire, france). magnetic stirrer and clevenger were used for preparation of crude extracts and essential oil respectively. antioxidant and photoprotective activities were carried out on a 96-well microplate reader enspire multimode plate reader (perkinelmer, inc., walthman, usa). extracts preparation essential oil (eo) was isolated by hydro-distillation using clevenger apparatus for 3 h. the yield (%) was given as weight of eo on weight of 100 g plant powder (w/w). in order to prepare an ethanolic extract, 20 g of plant powder was extracted by 200 ml of ethanol for 24 h under stirring in the dark. taking in consideration that wild thyme is traditionally used as a tea infusion; 200 ml of boiling distilled water is poured over 20 g of plant powder. the mixture was left for 30 min with occasional agitation. extracts were filtered and evaporated under reduced pressure until dryness. the obtained eo, ethanol and infusion extracts were stored in dark glass bottles in the freezer (-20 °c) until characterizations (gc/ms, hplc) and antioxidant evaluations. gc/ms analysis extracted eo was analyzed using gas chromatography (gc) and gas chromatographymass spectrometry (gc/ms). analytical gas chromatography-flame ionization detector (gc/fid) was performed using an agilent chromatograph fitted with the agilent 7683b autosampler (agilent technologies, inc., santa nova biotechnol chim (20xx) xx(x): e916 2 clara, usa) (1 : 20 split ratio), fused silica column and dual flame ionization detectors (fid). the operation conditions were as follows: volume of 1 µl sample (diluted in n-hexane 1 : 100, w/w) was injected and helium was used as a carrier gas. eo analysis was carried out by gc/ms system consisted of a gas chromatograph (agilent model 6890n, santa clara, ca) with hpl capillary column and connected to a quadruple mass spectrometer detector. the operating conditions for gc analysis were the same as described above for gc/fid. the ms conditions were as follows: ionization energy voltage 70 ev; quadruple temperature 150 °c; interface temperature 205 °c and mass spectra scan at a rate of 5 scan/s. identification of sample compounds was performed by making comparison of their mass spectra with nist 02 data and adams mass spectra libraries. premised on gc/fid peak areas without fid response factor correction, the percentage of each component was estimated. hplc characterization phenolic characterization was achieved following the method described by caponio et al. (1999). with minor modifications qualitative and quantitative evaluation were performed using the hplc instrument hp-agilent 1292 infinity on c18 column with dad diode array detector. extracts were prepared in a concentration of 20 mg.ml-1 and the injected volume into the column was 10 µl. the mobile phase used was a 3 % acetic acid (v/v) in (a) water and (b) methanol. elution gradient was used as follows: 93 % a/ 7 % b for 0.1 min, 72 % a/ 28 % b for 20 min, 75 % a/ 25 % b for 8 min, 70 % a/ 30 % b for 7 min and 15 min a similar gradient was 67 % a/ 33 % b for 10 min, 58 % a/ 42 % b for 2 min, 50 % a/ 50 % b for 8 min, 30 % a/ 70 % b for 3 min, 20 % a/ 80 % b for 2 min and 100 % b in 5 min till the end of the run abdulqadir et al. (2018). detection of eluates was carried out at 278 nm and retention times of analyzed phenolic compounds were compared with the following available pure standards: gallic acid, (+)-catechin, chlorogenic acid, caffeic acid, hydroxybenzoic acid, epicatechin, syringic acid, coumaric acid, transferrulic acid, sinapic acid, benzoic acid, hesperidin, rosmarinic acid, cinnamic acid and quercetin. the quantitative analysis was determined using external calibration curve of each standard and the results were expressed as µg.g-1 of extract. dpph free radical scavenging assay the antioxidant effect of extracts in scavenging dpph (2, 2-diphenyl-1-picrylhydrazyl) free radical was evaluated according to el aanachi et al. (2020). about 40µl of sample ranging at different concentrations was added to160 µl dpph methanol solution (0.1 mm). after 30 min of incubation, absorbances were read at 517 nm. the antioxidant standard used is trolox. the results were expressed as 50 % inhibition concentration (ic50) and mentioned as means ± sd data. galvinoxyl free radical scavenging activity (gor) gor scavenging assay was determined according to the described method of shi et al. (2001). a volume of 40 μl of sample at different concentrations was added to 160 μl of methanolic solution of galvinoxyl (0.1 mm). after 120 min incubation, the absorbance was evaluated at 428 nm. methanolic galvinoxyl solution was used as a control and trolox as a standard. reducing power assay the reducing power of tested samples was performed according to gali and bedjou (2019), with minor modifications. 10 µl of sample was mixed with 40 μl of phosphate buffer 0.2 m (ph 6.6), 50 μl of 1 % k3fe(cn)6 and then the plate was incubated at 50 °c for 20 min. 50 μl of 10 % tca (trichloroacetic acid), 40 μl h2o and 10 μl of 0.1 % fecl3 (ferric-chloride) were added. the absorbance of the resulting mixture was measured at 700 nm and results were expressed as absorbance a0.50 μg.ml -1 which represented the concentration producing 0.5 absorbance. cuprac assay the cupric reducing antioxidant ability was performed according to the cuprac method of apak et al. (2004). in each well of the microplate a 3 nova biotechnol chim (20xx) xx(x): e916 2 mixture was constituted with 40 µl of sample, 60 µl acnh4 (ammonium acetate) buffer, ph 7.50, 50 µl neocuproine and 50 µl of cucl2·2h2o (copper ii chloride solution). the absorbance was measured at 450 nm after 60 min of incubation. results had been given as absorbance a0.5 μg.ml -1. phenanthroline assay the evaluation of phenantroline activity was carried out in accordance with the method described by szydlowska-czerniaka et al. (2008). briefly, into 96 well round-bottomed plate 10 μl of extract at various concentrations were placed, then 30 μl of o-phenanthroline (0.5 %), 50 μl of fecl3 (0.2 %), and 110 μl of methanol were added. after 20 min of incubation in the dark at 30 °c, the absorbance was measured at 510 nm. the values of the measurements have been expressed as a0.5 µg.ml -1. photoprotective (skin-protective) activity the photoprotective activity was achieved following mansur et al. (1986) and el aanachi et al. (2021). the samples were prepared at a concentration of 2 mg.ml-1 diluted in absolute ethanol and then analyzed at wavelengths from 290 to 320 nm (uv) with spans of 5 nm, using ethanol as a blank. spf was calculated using the formula (eq. 1): spfspectrophotometric=cfх (1) where: ee(λ) is the erythemal effect spectrum. i(λ) is solar intensity spectrum. abs(λ) is absorbance. cf is the correction factor (= 10). ee(λ) and i(λ) are steady values. statistical analysis experimental tests were achieved in triplicate (n = 3) and results were given as means with standard deviation (mean ± sd). values of ic50 and a0.5 for in vitro antioxidant activities were estimated by linear regression analysis and statistical significances were established at p≤0.05 using variance analysis anova one-way and tukey test (multiple range comparison). results chemical composition of essential oil the chemical profile of t. serpyllum eo from the mascara region was described for the first time in this research and the chromatogram is presented in (fig. 1). the obtained eo was light yellow in color, had a strong smell and yielded at 5.66 % (w/w). fig. 1. chromatogram of thymus serpyllum essential oil. identified compounds with relative amounts and retention times are illustrated in table 1 where, twenty-five compounds were found. carvacrol was detected as the main component at 66 % of the essential oil. the γ-terpinene was detected at (11.5 %), thymol (7.5 %) and p-cymen (3.9 %). in lower percentages, other components were present as αpinene, linalool, myrcene, and α-thujene. determination of phenolic profile ethanolic and infusion extracts of t. serpyllum were analyzed by hplc-dad detector type. the investigation of the phenolic pattern was performed using 15 phenolic standards. results are in table 2 and were expressed in µg.g-1 of extract. rosmarinic acid was the main component of infusion (17,250 µg.g-1), followed by benzoic acid (5,200 µg.g-1) 4 nova biotechnol chim (20xx) xx(x): e916 2 and hydroxybenzoic acid (1,020 µg.g-1). besides the abundant compounds in ethanolic extract were benzoic acid and hydroxybenzoic (2,450 µg.g-1, 1,340 µg.g-1). syringic acid, coumaric acid, and sinapic acid were detected (50 µg.g-1, 60 µg.g-1, and 70 µg.g-1, respectively) in the ethanolic extract and not identified in the infusion. quercetin was not found in both extracts. antioxidant activities for the anti-radical scavenging ability test, the dpph and galvinoxyl free radicals were used. according to results of the dpph scavenging assay mentioned in table 3, the ethanolic and infusion extracts proved that both presents an excellent scavenging activity with an ic50 values of (25.12 ± 0.23 and 29.14 ± 0.41 µg.ml-1, respectively). the eo have shown the lowest radical scavenging activity with ic50 (>100 µg.ml -1). these results are less than antioxidant standard trolox (5.12 ± 0.21 µg.ml-1). for gor free radical assay, the results are indicated in table 3, where ethanolic and infusion extract revealed a similar activity with values of ic50 (24.56 ± 1.69 and 25.13 ± 0.82 µg.ml-1). the results of the ferric reducing power presented in table 3 showed that the ethanolic extract exhibited the lower a0.5 (31.70 ± 2.31 µg.ml-1) in meaning, the highest activity in comparison with infusion extract and eo (33.04 ± 1.84 and >100 µg.ml-1, respectively). analysis data of cuprac assay mentioned in table 3 indicated that ethanolic extract showed the best reducing copper transition metals with an a0.5 (17.83 ± 0.54 µg.ml-1) close moderately to trolox (8.69 ± 0.14 µg.ml-1), and ascorbic acid (8.31 ± 0.15 µg.ml-1). infusion and eo presented a0.5 values of (23.25 ± 0.46 and 37.30 ± 2.20 µg.ml-1) respectively. table 1. chemical composition in (%) of essential oil from thymus serpyllum by gc/ms with retention indices on hp-5ms capillary column. notes: total identified = 99.9%; mh: monoterpenes hydrocarbons = 22.5 %; mo: oxygenated monoterpenes = 75.4 %; sh: sesquiterpene hydrocarbons = 1.6 %; so: sesquiterpene oxygenated = 0.1 %; phpr: phenylpropanoids = 0.3%; tr: retention time; ri: retention indices. peak tr [sec] tr [min] ri [log. kovats] compound formula class area [%] 1 303 5.0422 929 α-thujene c10h16 mh 1.0 2 313 5.2171 937 α-pinene c10h16 mh 2.0 3 335 5.5843 952 camphene c10h16 mh 0.1 4 378 6.3056 979 β-pinene c10h16 mh 0.1 5 400 6.6685 991 myrcene c10h16 mh 1.1 6 425 7.0838 1005 γ-phellandrene c10h16 mh 0.2 7 436 7.2630 1012 iso-sylvestrene c10h16 mh 0.1 8 448 7.4597 1018 α-terpinene c10h16 mh 1.7 9 463 7.7089 1027 p-cymene c10h14 mh 3.9 10 471 7.8444 1031 limonene c10h16 mh 0.6 11 509 8.4827 1050 (e)-β-ocimene c10h16 mh 0.1 12 532 8.8717 1062 γ-terpinene c10h16 mh 11.5 13 594 9.9034 1089 terpinolene c10h16 mh 0.1 14 61 10.3187 1099 linalool c10h18 o mo 2.0 15 771 12.8542 1167 borneol c10h18 o mo 0.2 16 801 13.3439 1178 terpinene-4-ol c10h18 o mo 0.2 17 966 16.1023 1246 carvacrol methyl ether c10h16 o phpr 0.3 18 1090 18.1701 1293 thymol c10h14 o mo 7.0 19 1121 18.6903 1305 carvacrol c10h14 o mo 66.0 20 1376 22.9395 1407 α-gurjunene c15h24 sh 0.1 21 1399 23.3242 1418 (e)-caryophylene c15h24 sh 1.1 22 1446 24.1023 1438 aromadendrene c15h24 sh 0.1 23 1582 26.3668 1493 viridiflorene c15h24 sh 0.2 24 1650 27.5034 1522 δ-cadinene c15h24 sh 0.1 25 1772 29.5318 1572 spathulenol c15h24 o so 0.1 5 nova biotechnol chim (20xx) xx(x): e916 4 table 2. qualitative and quantitative phenolic composition of t. serpyllum. nd: not determined as seen in the table 3 for the phenanthroline assay, the eo expressed the best antioxidant capacity with value of (12.77 ± 1.19 µg.ml-1) followed by ethanol extract a0.5 (13.40 ± 0.73 µg.ml -1) and infusion with (21.42 ± 0.61 µg.ml-1). table 3. antioxidant activity of t. serpyllum extracts. extracts dpph ic50[µg.ml-1] gor ic50[µg.ml-1] reducing power a0.5[µg.ml-1] cuprac a0.5[µg.ml-1] phenanthroline a0.5[µg.ml-1] ethanolic extract infusion extract essential oil trolox ascorbic acid 25.12 ± 0.23b 29.14 ± 0.41a >100 5.12 ± 0.21c 4.39 ± 0.01d 24.56 ± 1.69a 25.13 ± 0.82a >100 4.31 ± 0.05b 5.02 ± 0.01b 31.70 ± 2.31a 33.04 ± 1.84a >200 5.25 ± 0.20b 3.62 ± 0.29b 17.83 ± 0.54c 23.25 ± 0.46b 37.30 ± 2.20a 8.69 ± 0.14d 8.31 ± 0.15d 13.40 ± 0.73b 24.27 ± 5.40a 12.77 ± 1.19b 5.21 ± 0.27c 3.08 ± 0.02c notes: a0.5 and ic50 are mentioned as concentrations at 0.5 absorbances and concentration making 50 % inhibitions percentages respectively. values of a0.5 and ic50 are indicated as means ± sd of three tests and the values stated in the unchanged column with different superscripts (a, b, c, d) presents significant differences at (p<0.05). (>100) indicates that values of ic50 and a0.5 are higher than 100 µg.ml -1. the photo-protective (skin-protective) activity the dermoprotective activity of t. serpyllum extracts is investigated for the first time by measuring of the sun protection factor (spf). the spf values for wild thyme extracts are presented in table 4. infusion and ethanolic extracts showed similar values with spf (38.82 ± 2.23 and 38.34 ± 2.29, respectively). eo exhibited a weak activity with (4.81 ± 0.25). table 4. spf values of t. serpyllum extracts. extracts spf ethanolic infusion essential oil 38.34 ± 2.29 38.82 ± 2.23 4.81 ± 0.25 values of spf are indicated as means ± sd of three tests. discussion essential oils extracted from aromatic and medicinal plants are constituted of various volatile and lipophilic compounds, obtained from various chemical classes (turek and stintzing 2013). phenolic compounds ethanol extract [µg.g-1] infusion extract [µg.g-1] gallic acid (+)-catechin chlorogenic acid caffeic acid hydroxybenzoic acid epicatechin syringic acid coumaric acid trans-ferrulic acid sinapic acid benzoic acid hesperidin rosmarinic acid cinnamic acid quercetin 80 630 60 100 1340 130 50 60 250 70 2450 300 480 10 nd 80 680 170 20 1020 630 nd nd 110 nd 5200 610 17250 190 nd 6 nova biotechnol chim (20xx) xx(x): e916 6 the yield of extracted essential oil by hydrodistillation from t. serpyllum was in accordance to the european pharmacopoeia (2010) (a minimum of 0.3 % (w/w)) as mentioned by (wesołowska et al. 2012). reported yields from poland and jordan were 2.5 % and 1.05 %, respectively, according to studies of abu-darwish et al. (2009) and wesolowska et al. (2014). comparing these results with ours, the content (5.66 %) of extracted eo was higher. the chemical composition after analysis by gc/ms revealed that carvacrol was the main component. thus, it agreed with those reported by kirillov et al. (2016). however, the amount of carvacrol detected (66 %) was higher in comparison with other studies. significant differences were observed about a major component of t. serpyllum where it was found p-cymene in the italian wild thyme (d’auria and racioppi 2015). the difference in percentages of carvacrol, γ-terpinene, and p-cymene in t. serpyllum can be affected by climatic factors following reports of wesołowska et al. (2012). the results of the gc/ms analysis showed a wide variation in main components and their amount from those mentioned in other works. according to banaeva et al. (1998), these variations can be attributed to the geographical source, collecting season, climatic conditions and extraction methods. polyphenol compounds such as phenolic acids, flavonoids, tannins, and coumarins can be extracted from plants using several techniques and different solvents (perron and brumaghim 2009). the chromatographic analysis in the conducted study detected the rosmarinic acid, hydrobenzoic acid, and benzoic acid as the main components. our results are similar to those of janiak et al. (2017) who have reported that rosmarinic acid is the main component in aqueous extract of wild thyme. furthermore, it was found with higher amounts than their results. quercetin was not found in both extracts which is in conformity with miron et al. (2011) studies. according to jovanović et al. (2016) the concentration of phenolic compounds and their structure can influence their bioactive properties. studies reported by jovanović et al. (2019) confirmed that the phenolic pattern of thyme species can be influenced by variability of experimental extraction methods including solvent type (polarity, ratio and mixture), temperature also bound and free phenolic extracts. evaluation of antioxidant ability for crude extracts must be performed using more than one method due to the complexity of the antioxidant process (aruoma 2003). for the anti-radical scavenging ability test, dpph and galvinoxyl free radicals were used. the gor and dpph radical scavenging power were evaluated in term of hydrogen atom and/or electron donating capacity and determined by ic50 where low values represent high antioxidant capacities. the infusion presented a high amount of rosmarinic acid in the hplc analysis than ethanol extract but slightly low antioxidant potential. this can be explained by reports of kulišić et al. (2006) who mentioned that the power of compounds in water infusions may decrease a little due to their dilution and combination with other substances in the mixture of infusion. the ethanolic and infusion extracts showed the best free radical scavenging activity using the galvinoxyl free radical than dpph. these results are consistent with works of tirzitis et al. (2010) who revealed that galvinoxyl is more reactive against phenolics and tightly allied to physiological oxygen radical action than dpph. the cuprac and reducing power antioxidant assays were assessed in order to associate properties of t. serpyllum bioactive compounds with their antioxidant potential (iron binding capacity). obtained results about the reducing power of iron indicated the ability of t. serpyllum polyphenols to act as electron donors. eghbaliferiz (2016) mentioned that polyphenols are able to form a stable complex with transition metals. regarding the cuprac, the antioxidant activity is determined on measurement of absorbance for a yellow-orange complex (copper (i)-neocuproine) obtained by reduction of copper (ii) into copper (i) in the presence of antioxidant compounds at 450 nm. this method was developed by apak et al. (2004; 2007) who reported that this reaction can estimate lipophilic and hydrophilic antioxidants (αtocopherol and β-carotene) at the same time. kϋlcϋ et al. (2019) reported a lower cupric reducing activity than our results for ethanolic extract of turkish t. serpyllum. basing on our results and according to apak et al. (2004) the cuprac reagent was sensitive against thiol-kind oxidants 7 nova biotechnol chim (20xx) xx(x): e916 6 and entails faster kinetics than reducing power method. furthermore, kim and choe (2018) stated that the metal chelating activity depended on structure, location and number of hydroxylgroups, ph, and the concentration of polyphenols. evaluation of antioxidant activity with phenanthroline method is based on the iron chelating ability of extracts by reacting with formed complex ferrous-o-phenanthroline and reducing the fe (iii) to fe (ii) (berker et al. 2007). this method was used for extracts and edible oils as reported by szydłowska-czerniak et al. (2008) but as far as we could possibly know that was not tested for essential oils. depending on our results, essential oil revealed the higher reducing ability and was quite similar to ethanolic extract. this can be explained by its richness of phenols (carvacrol 66 % and thymol 7.5 % as mentioned above in gc/ms results). these results can be in conformity with christodouleas et al. (2014) who reported that lipophilic extracts contributed more than 90 % to the total reducing of whole oil using phenanthroline-fe method. the sunburn, skin inflammation and skin cancer may appear as a result of regular exposure to uv radiation. these uv rays can promote photochemical reactions that induce the generation of free radicals such as o2 , hoo-, and oh· (batista et al. 2021). some studies are in quest for natural sunscreen products to reverse and block these uv radiations. the photo-protective capacity of plant extracts is investigated by measuring the sun protection factor (spf). the levels of protection were mentioned by schalka et al. (2011) where sfp values were minimum (2-12), moderate (12-30), high (30-50), and maximum at ˃50. spf values of ethanolic and infusion extracts indicates high skin-protective activity due to their polyphenols that can absorb at wavelength between 280nm and 320nm. these results can make the t. serpyllum extracts as potential skin-protective agents that can be used as additives in dietary and in the production of sunscreens for better photoprotection. conclusion thymus serpyllum extracts were evaluated for their chemical profile, antioxidant, and skin-protective activities. experimental results of antioxidant assays (dpph, gor, cuprac, reducing power, and phenanthroline) revealed that both of ethanol and infusion extracts showed high antioxidant activities. although the richness of infusion extract with phenolic acids was mainly by rosmarinic acid, the active compounds of infusion can be slightly affected by dilution and their reaction with other compounds in mixture and this has been proven by other studies. in this study, we reported for the first time the good potential of t. serpyllum as a photoprotective agent which can offer an initiation point for therapeutic research to use these extracts in a formulation of dermo-protective products for skin disorders. basing on our results, it can be concluded that t. serpyllum from the mascara region represented an effective source of antioxidant components that may be used as an alternative to synthetic antioxidants for treatment of pathologies associated with free radical damages and in food industries as additives to retard food deterioration and to upgrade the storage length of food products. acknowledgments the authors are grateful to prof. benhassaini hachemi (laboratory of plant biodiversity, conservation and enhancement, faculty of natural and life science, university of djillali liabes sidi bel abbes, algeria) for identification of plant material of thymus serpyllum l. conflict of interest the authors declare 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sarikaya sbo (2019) the importance of antioxidants and place in today’s scientific and technological studies. food sci. technol. 56: 4757-4774. 10 nova biotechnol chim (2020) 19(2): 165-174 doi: 10.36547/nbc.v19i2.636  corresponding author: nworie.sunday@ebsu.edu.ng nova biotechnologica et chimica synthesis of biochar conjugated schiff base composites and their enhanced antimicrobial activity against five pathogenic organisms felix sunday nworie, frank ik. nwabue, wilberforce oti, confidence obasi, chinwe ejim and blessing nwafor department of industrial chemistry, ebonyi state university, abakaliki, nigeria article info article history: received: 30th july 2020 accepted: 23rd november 2020 keywords: antimicrobial/biocidal evaluation biochar pathogens plantain peel rice husk schiff base abstract the search for antimicrobial drug of high bio-efficacy not prone to multiple microbial resistance has been on the rise in recent time. this study focused on the preparation, characterization and antimicrobial evaluation of schiff functionalized hno3 activated plantain peel and rice husk biochar against five pathogenic bacteria. the activated rice husk and plantain peel biochar were characterized using braunauer-emmett-teller, x-ray diffraction and fourier transform infra-red spectroscopy. based on the result, the activated rice husk and plantain peel biochar were amorphous and crystalline respectively with pore surface area and pore size for activated rice husk and plantain peel biochar as 9.369 and 27.32 (m2.g-1) and 8.790 and 16.65 (cc.g-1), respectively. the fourier transform infra-red spectroscopy result indicated bonds, such as –oh, c=o and –n-h, where actual chelation and electrostatic attraction mechanism are prevalent responsible for antimicrobial potency. the cell bioactivity was hampered due to permeation of the biocidal agents, cell membrane disruption and generation of reactive oxygen species enhanced by conjugation of schiff base based biochar to the microbial cell wall leading to death of microbes. the ease of schiff base release and reusability of the nanocomposite favors the product as an efficient, low cost, effective and promising nanocomposite for decontamination of bacteria infested media.  university of ss. cyril and methodius in trnava introduction contamination of water by microbial agents is a serious global challenge threatening human health and aquatic safety. current disinfection method though reliable for efficient containment of microbial infection but the by product, which is seemingly deleterious calls for concern. similarly, the emergence of antimicrobial resistance among pathogenic organisms which makes treatment difficult for some life-threatening infections have been on the increase prompting serious concern on the subject. currently, it is predicted that if the trend of antimicrobial resistance remains unabated, more people are likely to die of drug-resistant infections than cancer by 2050. proliferation of pathogenic organisms is common in all areas of human endeavour, such as food and other production industries, soils and aqueous media. in most african agrarian villages and municipalities nigeria inclusive, illicit deposition and disposal of waste has become the bane. not only that these wastes are improperly and indiscriminately deposited, there is no efficient and effective means of management, control or conversion into useful products. most mailto:nworie.sunday@ebsu.edu.ng nova biotechnol chim (2020) 19(2): 165-174 166 agricultural by-products are termed waste simply because it is not useful to the locality or there is no further use of such product and they are of serious environmental concern (tang et al. 2020). most of the wastes have been noted to be useful to man and other animals after conversion to other form (chen et al. 2019). specifically, plantain peel waste has been noted to have medicinal properties as bioactive compounds, such as flavonoids, tannins, alkaloids, glycosides, and terpenoids are present and exert pharmacological effect as an antioxidant, antidiabetic, anti-inflammatory and antibiotic substance (okareh et al. 2015). some bioactive agents (norepinephrine, dopamine, and serotonin) were also discovered by researchers in the peels and pulp with the norepinephrine and dopamine relevant in elevating blood pressure whereas serotonin impairs gastric secretion and stimulates the smooth muscle of the intestines (asuquo and udobi 2016). several studies have noted that peels of plantain and rice husk exhibit antifungal and antibiotic properties (kala et al. 2015; asuquo and udobi 2016; banji and adebayo 2020). abakaliki, nigeria is known for rice production and the management of the rice husk a byproduct of the milling exercise has been a problem for several decades. the rice husk has led to wide fires attacking vegetation and increasing the emission of carbon (iv) oxide into the atmosphere. environmental pollution is increased if the rice husk is burnt to generate energy (used as fuel) rendering the process environmentally unfriendly and a search for alternative friendly and environmentally benign and palatable method of managing the waste becomes imminent. biochar a carbon-rich product obtained when biomass materials, such as wood, manure or leaves is heated in a container with little or no available air (oxygen) is currently under study by numerous researchers because of their wide applications in different areas of life (nworie et al. 2019). consequently, biochar generally produced under thermal decomposition of organic material under limited supply of oxygen (o2) and at relatively low temperature (< 700 °c) is germane in soil management to regulate the fertility of the soil and avert effect of climate change, carbon sequestration to keep the environment benign reducing or eliminating greenhouse gases, immobilization of pollutants and metal adsorption through adsorption of the pollutants and metal ions on the porous surfaces and exposed active functional groups by activation, and other miscellaneous applications (ghorbani and eisazadeh 2015). several studies have indicated that biochar is effective and efficient not only in soil amendment but also in sequestration of carbon, pollutant removal from soil and aqueous media and other miscellaneous application (ghorbani and eisazadeh 2015). recently, it was reported that conversion of biomass into biochar not only result in creating renewable energy (synthetic gas and bio oil) but has succeeded in limiting the amount of co2 in the atmosphere which as well is a means of environmental cleaning and purification (zhao et al. 2018; chen et al. 2020). to increase the functionality, effectiveness, efficiency and modify electronic property as well as molecular size of the biochar, there is need for functionalization of the biochar using oxygen or nitrogen containing specie. a schiff base, imine, aldimine or azomethine is a compound with the general structure r2c = nr' serve as ligands which stabilize central metal ions in low oxidation state as they form co-ordination complexes with the metal ions useful in many applications as catalysts, antimicrobial agents and other miscellaneous applications (nworie 2018). bis(salicylidene)ethylenediamine(salen) and bis(acetylacetonato) ethylenediamine (acacen) are both popular chelating tetradentate schiff bases very important in homogeneous catalysis and co-ordination chemistry. their incorporation or functionalization into biochar for antimicrobial evaluation is yet unknown but the presence of n2o2 atoms in their moiety is believed to provide enough oxygen and nitrogen functionalities imparting multilateral properties to the composite. functionalization of biochar surface is currently a promising and emerging area of research due to increased effectiveness, permeability, ease of release, recyclability, efficiency, stability and multiple action related properties of the composite material. this research is imminent especially now that multi-resistant bacterium or the super bugs are on the increase with scientists exploring several avenues to avert or arrest this misnomer that have led to loss nova biotechnol chim (2020) 19(2): 165-174 167 of money and delay in handling diagnosed ailments. this study is aimed at (i) developing a low cost, eco-friendly biocidal agent using biochar and schiff bases (salen and acacen), (ii) characterizing the activated biochar, (iii) regeneration/reusability of the used biocidal agent. experimental equipment braunauer-emmett-teller (bet) was measured using micromeritics asap 2020 system. x-ray diffraction(xrd) pattern was obtained on a bruker® d8 discover diffractometer, equipped with a lynx eye detector, under cu-ka radiation (l ¼ 1.5405 å). with data collected in the range of 2θ =10 to 100°, scanning rate at 0.010°.min-1, 192 s per step and samples placed on a zero background silicon wafer slide. cary 630 agilent technologies, usa was used for fourier transform infra-red spectroscopy (ftir) analysis. biochar sample collection, preparation and activation plantain peels was collected from abakaliki meatmarket while rice husk was collected from abakaliki rice mill cleaned and kept in dried polyethene bags. the samples (rice husk and plantain peels) were washed with distilled water, cleaned of soil and other impurities, sun dried to reduce moisture content for 7 days and then oven dried at 60 °c for 6 h. the samples were carbonized following the procedure reported elsewhere (nworie et al. 2019) at 550 and 580 °c for plantain peels and rice husk respectively in a n2 environment. the biochar formed were collected and activated using 0.1 m hno3 for 1 h to yield nitric acid activated plantain peel biochar (appb) and nitric acid activated rice husk biochar (arhb) and products severally washed with deionized water to neutral ph. the formed products were then oven dried at 110 °c for 12 h and kept to be used when desired. synthesis of bis(salicylidene)ethylenediamine (salen) ethylenediamine (22.475 g) was carefully added with stirring to 91.336 g of salicyladehyde in a 500 cm3 beaker. at initial stage, a yellow hot mixture was formed which translated to golden yellow cake on crystallization (nworie and nwabue 2017). recrystallized of the product was effected with carbon tetrachloride to yield golden yellow crystals (64.45 % yield, m.p 121±1 °c). synthesis of bis(acetylacetonato)ethylenediamine (acacen) ethylenediamine (22.475 g) was gradually added with stirring to acetylacetone (74.881 g) in a 250 cm3 beaker with the two liquids previously chilled in ice salt mixture for 30 min. the exothermic process was followed with formation of a whitish crystal which on addition of excess ethylenediamine gave deep golden yellow color. crystallization was effected by stirring leading to a cream colored cake and the product recrystallized from carbon tetrachloride air dried (45.29 % yield, m.p 98±1 °c). impregnation of biochar immobilization of the activated biochar was done by measuring 0.5 g of the activated biochar samples (plantain peel and rice husk) into two separate 250 cm3 conical flask. this was followed by the addition of 5 cm3 of 0.5 % salen prepared by dissolving 5 g in 100 cm3 absolute ethanol and 5 cm3 of 0.5 % acacen prepared by dissolving 5 g in 100 cm3 of distilled water. the mixture was stirred for 3 h, filtered, washed to remove unreacted salen and acacen and oven dried at 60 °c for 1 h. the formed product between salen or acacen and activated plantain peel biochar regarded as salen or acacen functionalized activated plantain peel (s-appb or a-appb) and between salen or acacen and activated rice husk biochar known as salen or acacen functionalized activated rice husk biochar (s-arhb or a-arhb) were kept for further antimicrobial evaluation. nova biotechnol chim (2020) 19(2): 165-174 168 antimicrobial susceptibility/screening test the antimicrobial activity of test samples against common pathogenic microbes, such as staphylococcus aureus, pseudomonas aeruginosa, aspergillus sp., candida sp. and escherichia coli were evaluated using kirby bauer susceptibility test method (nworie et al. 2018). the culture of the test bacteria were grown in nutrient broth (l-s biotech) and adjusted to 0.5 mcfarland turbidity standards with the inoculums aseptically swabbed on the surface of mueller hinton agar plates using sterile swab sticks. commercially available single antibiotic disc (tm media, laboratory chemicals, cas 7177 48-2, code 3007) impregnated with ampiclox were aseptically placed on the mueller hinton agar plates as control. both the samples and the control were incubated at 37 °c for 18 – 24 h and the diameters of inhibition zone for the four separate determinations were recorded. the biocidal activities of the functionalized biochar were executed in a sterile agar plates with single plates used for a particular bacterial strain. in the experiment, 50 μl of 2 μg.l-1 test samples in agar medium was added to the first plate, followed by separate additions after four serial dilutions. consequently, 50 μl of the prepared bacterial cultures were separately added to the plates as previously stated and the plates incubated at 37 °c. the diameter of inhibition and comparison to standard were recorded. the minimum inhibitory concentration (mic) was evaluated using the agar well dilution method by varying the concentrations of test samples between 5 – 20 μg.l-1 and test solution in agar medium incubated at 37 °c. 20 40 60 80 100 2(deg) j in te n s it y (a .u ) k m fig 1: xrd diffractogram of j, k and m in te n si ty ( a .u ) 20 40 60 80 100 2(deg) j in te ns ity (a .u ) k m fig 1: xrd diffractogram of j, k and m fig.1. x-ray diffraction pattern of activated plantain peel biochar. the diameter of inhibition was recorded. the level of significance at p < 0.05 was statistically determined. regeneration and reusability of the used biochar and composites was effected using different hcl concentrations and varied time of equilibrations and four biocidal-acid decimation cycle. results and discussion the xrd (cu-ka radiation) patterns of arhb and appb are shown in figs. 1 and 2, respectively. the xrd diffraction pattern of arhb showed one single strong peak at 2θ = 18 – 30°, two well resolved broad peaks and a less resolved broad peak at 2θ = 70 – 80° which is characteristic of amorphous silica phase arising from mesoporous structure of nanocomposite observed in biochar carbonized below 600 °c (pulido-novicio et al. 2001; zhao et al. 2011). a peak for potassium oxide is usually found at 2θ = 29 – 31 (jindapon et al. 2015). some authors (yang et al. 2019) shown that biochar surfaces conjugated with metal oxides exert increased biocidal activity. the xrd analysis indicated peaks of metal oxides which enhanced oxygen functionalities, increased reactive oxygen species, permeability of the biochar and composites leading to cell death. the xrd diffraction pattern of appb indicated sharp peak at 2θ = 40.53° with observed decomposition of some species after biochar formation at 2θ = 24.24 and 31.31° (jindapon et al. 2015). noticeable ordered crystallinity of the nanocomposite was observed and scherrer equation (eq. 1) (zhao et al. 2018) was applied to determine the average crystallite size as 14.56 nm indicating the mesoporosity of the nanocomposite (nworie et al. 2019): fig. 2. x-ray diffraction pattern of activated rice husk biochar. nova biotechnol chim (2020) 19(2): 165-174 169 fig. 3. nitrogen adsorption/desorption bet plot of activated plantain peel biochar. (1) where k is scherer constant; β is the full width at half maximum; is the wavelength; and θ is bragg angle. table 1. bet surface area and pore size of activated biochar. sample pore surface area [m2.g-1] pore size [cc.g-1] activated rice husk biochar 9.369 27.32 activated plantain peels biochar 8.790 16.65 brunauer-emmett-teller (bet): surface area and pore size analysis it has been noted (alothman 2012) that in any bet analysis, the effective area where capturing of micro-organism can occur under controlled experimental conditions for mesoporous, macroporous and nonporous solid is the probe accessible region. the nitrogen adsorption/desorption isotherms of the activated illustrated by the bet isotherm plot are shown in figs. 3 and 4 for arhb and appb respectively. clear analysis of the isotherm indicated that it is mesoporous and similar to a type iv iupac classical isotherm classification (alothman 2012). in arhb and appb, the hysteresis loop was observed over the pressure range of 0.9 – 1.0 consistent of type hi common when the pore size is beyond a known critical width wider than 4 nm observable in networks of ink bottle pores (alothman 2012). there was observed shift in the stpg-1 for the isotherm of arhb and appb fig. 4. nitrogen adsorption/desorption bet plot of activated rice husk biochar. upwards by 8 and 15 cm3 respectively. the surface area (m2.g-1) and pore size (cc.g-1) was 8.790 and 16.69 for appb respectively and 9.369 and 27.32 for arhb respectively. it could be observed from the bet analysis presented in table 1 that arhb has greater pore surface area and pore size than the appb. the large surface area per gram of a sample of the biochar showed increased ability for micro-organisms destruction as they get denatured on interaction with biochar (nworie and nwabue 2017). ftir analysis of activated biochar samples the ftir result of the appb is shown in fig. 5. a broad band representing hydroxyl group (-oh) was observed at 3,421 cm-1 for appb attributed to o-h stretching vibration due to intra and intermolecular hydrogen bonding (kezerle et al. 2018; nworie et al. 2020). the bands observed at 2,855 and 2,922 cm-1 is due to –ch2 groups (banerjee and chattopadhyaya 2017; zhang et al. 2019). the band observed at 1,744 cm-1 could be that of the carbonyl group (c=o). spectra band peaks observed at 1,461 cm-1 was assigned to c=c phenol ring stretching vibration (kezerle et al. 2018). the band observed at 1,371 cm-1 was assigned to c-n stretching of the amine group (banerjee and chattopadhyaya 2017; kezerle et al. 2018). the band that extends from 857 to 763 cm-1 was due to ester vibration of monosubstituted aromatic rings (kezerle et al. 2018). the ftir spectra bands of arhb is shown in fig. 6. a broad band representing hydroxyl group (-oh) was observed at 3,246 cm-1 and is due to intra and intermolecular hydrogen bonding nova biotechnol chim (2020) 19(2): 165-174 170 (kezerle et al. 2018). the bands observed at 2,922 cm-1 could be that of –ch2 groups (banerjee and chattopadhyaya 2017; zhang et al. 2019; nworie et al. 2020). the band observed at 1908 cm-1 was attributed to the carbonyl group (c=o). the peaks observed at 1,595 cm-i could be that of c=c phenol ring stretching vibration from lignin (kezerle et al. 2018). the intense band of 1,054.8 cm-i observed between 2,000 – 400 cm-i region was assigned to c-o, c=c or c-c-o stretching vibration of ether group of cellulose, hemicellulose or lignin (banerjee and chattopadhyaya 2017; kezerle et al. 2018; nworie et al. 2019). the band that extends from 790 to 790.2 cm-1 was assigned to ester vibration of monosubstituted aromatic rings (kezerle et al. 2018). antimicrobial evaluation of naked and schiff base/biochar composites there was variation in the level of microbial inhibition of the various samples derived from activated rice husk and plantain peels biochar and their schiff base impregnated counterparts. the effect was observed against isolates of escherichia coli, staphylococcus, pseudomonas, aspergillus sp. and candida sp. (figs. 7 and 8). the inhibition potential of rice husk and plantain peels biochar immobilized with salen and acacen indicates that they are capped with the same bioactive compounds (flavonoids, tannins, phlobatannins, alkaloids, glycosides and terpenoids) initially present in the plants and are majorly responsible for their medicinal properties. these phytochemicals have been reported to exert multiple biological and pharmacological effects (antibacterial, antihypertensive, antidiabetic and anti-inflammatory activities and their bioactivity are responsible for the definite physiological effects exerted on the human body (asuquo and udobi 2016; banji and adebayo 2020). the susceptibility test against escherichia coli showed that among the various samples, that salen immobilized plantain peel biochar has the highest inhibition value (fig. 7). generally, there was observed increase fig. 5. ftir spectra of activated plantain peel biochar. fig. 6. ftir spectra of activated rice husk biochar. nova biotechnol chim (2020) 19(2): 165-174 171 in zone of inhibition for the salen schiff base immobilized biochar compared to the naked biochar counterpart (figs. 7 and 8). this observation could be explained by chelation theory as chelation increases the lipophilicity and denaturation of the cell wall of the micro-organism (nworie et al. 2018). similarly, the bioactivity increased due to ligation of the micro-organism cell wall bonds with the –oh group of the immobilized biomaterial and exchange of ions at the –c=o interfaces of the impregnated biochar and bacterial cell well thereby increasing denaturation followed by cell death (nworie and nwabue 2017). there is perceived free radical generation when the schiff base (salen or acacen) impregnated biochar was intercalatively co-ordinated to the cell of the micro-organism enhancing cell wall permeation, magnifying intracellular atp leakage and quantitatively disrupting the activity functionality of dna and rna of the cells inducing cell death a confirmation of increased bactericidal activity the antimicrobial activity of salen immobilized. rice husk and plantain peel biochar shows that inhibitory diameter of 15 and 12 mm respectively was recorded against staphylococcus isolate. quite low unexpected inhibition value of 4 mm and 5 mm (fig. 7) against pseudomonas was observed with salen immobilized rice husk and plantain peel biochar respectively. this could be due to reverse mechanism indicating that the process is not controlled by chelation but by overtone theory (hsueh et al. 2017; nworie et al. 2018). the inhibition value of aspergillus sp. was 16 mm for immobilized plantain peel biochar whereas a very low value of inhibition was observed with all the biochar with candida sp. the low inhibition could be attributed to the resistance of this organism and the relative reluctance of the biochar and immobilized biochar counterpart to cross over the cell membrane thereby decreasing the bioactivity. generally, it could be observed that the rice husk biochar inhibited the growth of the selected microbes better than the plantain peels biochar. this could be attributed to the greater pore surface area and pore size of the rice husk biochar as supported by the bet analysis. exceptional degree of inhibition of aspergillus sp. (19 mm) was observed with the a-arhb which was better than the control drug (ampiclox, 18 mm) further confirming that acacen or salen fig. 7. biocidal evaluation of naked biochar/salen biochar composites. (arhb = light blue, appb = brown, a-arhb = silvery white, a-appb = yellow, control = deep blue). data are given for controls, arhb, appb, a-arhb and a-appb. antimicrobial tests were done with mean ± sd (n = 4). values of the activated biochar and composites were presented at p ≤ 0.05 level of significance. fig. 8. biocidal evaluation of naked biochar/acacen biochar composites. (arhb = light blue, appb = brown, a-arhb = silvery white, a-appb = yellow, control = deep blue). data are given for controls, arhb, appb, a-arhb and a-appb. antimicrobial tests were done with mean ± sd (n = 4). values of the activated biochar and composites were presented at p ≤ 0.05 level of significance. nova biotechnol chim (2020) 19(2): 165-174 172 table 2. mic of activated biochar, salen and acacen composites. biochar/ composites e. coli staphylococcus pseudomonas aspergillus candida arhb 15±0.01 19±0.28 16±0.31 15±0.01 14±0.24 appb 17±0.05 20±0.42 18±0.65 16±0.01 13±0.11 s-arhb 10±0.45 12±0.07 9±0.75 11±0.02 17±0.03 s-appb 11±0.50 14±0.97 10±0.64 12±0.05 16±0.09 a-arhb 9±0.02 13±0.35 7±0.46 11±0.25 17±0.28 a-appb 12±0.04 13±0.36 9±0.08 13±0.56 18±0.44 data are given for arhb, appb, s-arhb and s-appb. antimicrobial tests were done with mean ± sd (n = 4). values of mic of the activated biochar and composites were presented at p ≤ 0.05 level of significance. functionalized arhb is a better anti-bactericidal agent. similar studies on the use of copper (i) oxide immobilized biochar shows that the functionalized nanocomposites presents itself as better biocidal agent than the naked nanoparticle biochar (yang et al. 2019). the xrd diffractogram further supported the higher inhibitory diameter of rice husk biochar and its impregnated counterpart as it is amorphous as against plantain peel which is crystalline. biochar in the amorphous state provides excellent and wider surface area for decimation of microbes (yang et al. 2019). considering the inhibition diameter of the activated biochar and composites, there was significant difference in the inhibition of escherichia coli, staphylococcus and aspergillus between the positive control and the activated biochar and composites at p ≤ 0.05. however, a clear investigation of the inhibition of pseudomonas and candidiasis revealed a slight difference at p ≤ 0.05. effect of naked biochar/composites concentration on the inhibition of microbes the minimum inhibitory concentration of test samples were determined by dispersing the naked biochar and composites in agar medium and on solidification, the various microbe cultures were inoculated on the agar-composite medium, incubated for 24 h in the temperature of 37 °c. from table 2 it could be observed that the minimum inhibitory concentration (mic) of naked biochar are higher than those of the composites indicating that the composites are better bactericidal agent. some authors (yang et al. 2019) who evaluated the mic of biochar modified with metal oxides observed that naked biochar have higher mic compared with functionalized biochar, an indication of the composites nanostructures, which enhanced microbial cytoplasmic penetration and increased ros generation leading to cell death. from table 2, there is no significant difference in the mic for the inhibition of the microbes between the naked biochar and schiff base composites at p ≤ 0.05. the values as recorded in table 2 clearly indicated higher microbial action of the composites which could have resulted from the multiplex action of the increased functionalities. consequently, it is advocated that for increased antimicrobial result with biochar, immobilization or functionalization of the surface is necessary. mechanism of biocidal action of schiff base/biochar composites the surface of the biochar and functionalized nanocomposite is basically positively charged whereas that of the bacterial membrane is negatively charged inducing electrostatic interaction catalyzed by increased surface area. the electrostatic interaction disrupts film, cell membrane structure and permeability and induces damage to bacterial proteins (gao et al. 2017). increased oxidative stress manifests with lysis, membrane instability, cell wall denaturation and death. intercalation of functional groups especially sulfur containing groups to mesosomes inhibits dna replication, cellular respiration, cell division and suffocation. biochar on interacting with the microbial cell wall instigates the production of reactive oxygen species (ros), such as superoxide anions (o2 -), hydroxyl radicals (oh-) and hydrogen peroxide (h2o2) which are highly deleterious to microbial species. the ros basically disintegrates biologically important species present in the micro-organisms, such as amino acids, nova biotechnol chim (2020) 19(2): 165-174 173 proteins, lipids, carbohydrates and nucleic acids. functionalization with introduction of oxygen and nitrogen functionalities leads to alteration in electronic properties and molecular size thereby inducing ros production and multiplication (varlamov 2005; nworie et al. 2016; nworie 2016; kilonzo et al. 2018). consequently, the electrondonor-acceptor characteristics common among multifunctional materials also enhances ros production and multiplication through fenton-type reaction (yang et al. 2019). the high biocidal property of the nanocomposite is as a consequence of the positively charged surface of salen and acacen functionalized biochar containing oxygen and nitrogen containing group functionalities derived from the schiff bases which electrostatically interacts with the negatively charged bacterial cell wall. this interaction leads to cell wall denaturation and death. the higher biocidal efficiency of the functionalized biochar could be as a result of the characteristic nano-size (from xrd and bet measurement), toxicity and shape. the biocidal mechanism of biochar and functionalized nanocomposite can be said to involve destruction and malfunctioning of cell well induced by metallic oxides on the biochar surface (xrd analysis implicated some metallic oxides, such as potassium oxide as a peak in the biochar), disturbance in electron transport and energy generation, production of ros especially by incorporation of oxygen or nitrogen containing groups, inactivation of cell enzymes and inhibition of dna replication. regeneration/reusability of used biochar and schiff base composites the fate of the used biochar and composites is important to prevent environmental contamination and also to determine the economy of the process. the regeneration and reusability ensures eco-friendliness and economic nature of used sorbents which are of high industrial importance. in the regeneration experiment, different acid (hcl) concentrations (1, 0.5, 0.1 and 0.01 m) were used for the treatment of used biochar/ composites. the used biochar/composites were equilibrated in 5 cm3 acid solution for varied time of 5, 10, 15 and 20 min. maximum microbial decimation from the used sorbent was observed at 1 m hcl and 10 min shaking and reusability up to four times obtained with only 15 % decrease in effectiveness and efficiency. conclusion this study evaluated the bactericidal activities of biochar derived from plantain peel and rice husk and their schiff base functionalized counterpart. the results of the antimicrobial screening against five pathogenic microbes showed that the biochar and their schiff base impregnated counterpart are very effective against escherichia coli, staphylococcus, pseudomonas, aspergillus and candida sp. the generation of ros on the surface of the microbes in contact with schiff base functionalized activated biochar can be the main mechanism for antimicrobial potency or death of microbes. evaluating the structure and size of the activated biochar revealed that amorphous biochar inhibited microbial growth than the crystalline biochar. similarly, the schiff base functionalized biochar are better biocidal agent than the naked biochar. the study indicated that at that there is no significant difference in the inhibition of microbes by naked and functionalized biochar at p ≤ 0.05 level of significance. regeneration and reusability of used biochar and composites indicated the fabricated materials as efficient, low cost and environmentally friendly. acknowledgement the authors are 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under the influence of cerium larisa m. cheban1, , alexander b. shcherbakov2, nadiya m. zholobak2, 3, mikhailo m. marchenko3 1department of biochemistry and biotechnology, chernivtsi national university named after y. fedkovych, kotsiubynsky 2 str., chernivtsi 58012, ukraine 2danylo zabolotny institute of microbiology and virology of the nasu, acad. zabolotny 154str., kyiv 03680, ukraine 3kyiv national university of technologies and design, nemyrovycha-danchenka 2 str., kyiv 01011, ukraine  corresponding author: l.cheban@chnu.edu.ua article info article history: received: 29th april 2021 accepted: 7th januar 2022 keywords: acutodesmus dimorphus cerium dioxide nanoparticles cerium ions desmodesmus armatus productivity abstract the effect of various concentrations (1 μm – 100 mm) of citrate-stabilized cerium dioxide nanoparticles or cerium ions (3+) on the biomass production of two species of unicellular green algae desmodesmus armatus (chod.) hegew and acutodesmus dimorphus (turpin) tsarenko was studied, the amount of chlorophyll, proteins and lipids in the algae biomass was determined. it was shown that at the concentrations of 0.01 m to 0.1 m nanoparticles and cerium salt have pronounced toxic effects on the algal cultures, manifested by a sharp increase in the level of lipids in the biomass combined with the decrease in chlorophyll and protein. at lower concentrations, cerium dioxide nanoparticles stimulate algae biomass accumulation, probably due to a change in key metabolic pathways, accompanied by an increase in the accumulation of carbohydrates in the biomass. for cerium salt, these effects are less pronounced. thus, depending on the concentration of the objects used, it is possible to obtain an increase in the food biomass production enriched with lipids or carbohydrates as appropriate to the biotechnological objectives. introduction of interest for the study are the group of rare earth elements, lanthanides, named from the first representative of the group. this group includes cerium, which is characterized by oxygen nonstoichiometry and relatively low toxicity (ivanov et al. 2009; shcherbakov et al. 2020). cerium (iii) salts are used as antineoplastic, antiemetic, antiviral, bacteriostatic and bactericidal agents (jakupec et al. 2005; zholobak et al. 2010; charbgoo et al. 2017). this chemical element has attracted the attention of scientists as an inorganic antioxidant, capable of effectively protecting living systems against oxidative stress. despite the low incidence and concentration of lanthanides in soils, these elements can have a stimulating effect on the growth and development of organisms (chen et al. 2001; goecke et al. 2015; kang et al. 2020). lanthanides are known to significantly improve light energy absorption, energy transfer, electron transfer rate and phosphorylation rate, thus stimulating the accumulation of organic matter in plant cells (huang et al. 2008; gong et al. 2011; goecke et al. 2015). it is also believed that lanthanides can be used as substitutes for macronutrients (zeng et al. 2000; chen et al. 2001; hong et al. 2002). mailto:l.cheban@chnu.edu.ua nova biotechnol chim (2022) 21(2): e954 2 the ability of lanthanides, including cerium, to partially substitute ca in plant cells under cadeficient conditions is already known (hong et al. 2002; chao et al. 2009). a similar compensation mechanism also works when plant cells are deprived of magnesium or manganese (yin et al. 2009; yuguan et al. 2009а, 2009b; zhou et al. 2011). however, the biological behaviour of these elements is based on the principle of analogy with base metal ions: their properties are similar, but still not identical. also, the complex mechanism of the correlation between the response of plant organisms to exposure to lanthanides and their amount in the nutrient medium is well established. the effects of even low concentrations of these elements on microalgae cells have also been shown to depend on the food security and the possible stress state of the latter (laszló et al. 2001; goecke et al. 2015; řezanka et al. 2016). on the other hand, there is information on the toxic effects of cecl3 salt (chen et al. 2001; tai et al. 2010; kosak et al. 2018). the use of nanoparticles in the form of nanoacquahelates stabilized with carboxylic acids, particularly citric acid, is promising. carboxylated nanoparticles have the ability to easily penetrate the cytoplasmic membrane and then dissociate from the ligands, which accounts for their high biological activity. the toxicity of nanoacetylates is thought to be much lower than that of the corresponding inorganic salts (borisevich et al. 2012). algae, as promising producers of organic raw materials for various needs, are convenient for studying the effects of nanopreparations (jreije et al. 2020). despite considerable interest in the prospects of using cerium on biological objects of different levels of organization, its effects on microalgae cultures remain understudied. we have shown the potential of two species of green algae, desmodesmus armatus (chod.) hegew and acutodesmus dimorphus (turpin) tsarenko, as feed organisms in industrial aquaculture (cheban and grynko 2017; cheban et al. 2018). the biomass of genera desmodesmus and acutodesmus microalgae, due to their small size and rather high content of amino acids, proteins, polyunsaturated fatty acids, and carotenoids, is used as an alternative complete feed in aquaculture for feeding zooplankton and fish. at the same time, there is a need for techniques that can increase the productivity of these algal cultures both in overall biomass and in major nutrients. however, the mechanism of factors contributing to the growth of microalgae is not fully studied. little is known about the mechanism of the positive effect of lanthanides on the cells of higher plants and whether it will be similar in the cells of algae, either. the choice of optimal concentrations that can cause a stimulating effect is also a problem. the aim of this work was to comparatively study the effect of nanocerium and cerium salt on the main indicators of growth activity and productivity of two green algae of the family scenedesmaceae – d. armatus (chod.) hegew and a. dimorphus (turpin) tsarenko. experimental the characteristics of the test-objects as test-objects cerium chloride salt (sigma, usa) and citrate-stabilized sol contained cerium dioxide nanoparticles (3 – 4 nm particle size, 1 : 1 stabilizer-to-cdn ratios) were used. a citrate solution of equimolar concentration was used as a control. citrate-stabilized aqueous sol of ceo2 was prepared by the method proposed by ivanov et al. (2010). briefly, 2.0 g of citric acid was mixed with 25 ml of a 4 m aqueous cerium (iii) chloride solution). the resulting solution was rapidly poured, under stirring, into 100 ml of a 3 m aqueous ammonia solution, and then exposed for 2 h at ambient conditions and further boiled for 4 h. then, the solution was cooled to room temperature and purified from precursors and by-products by sedimentation and further re-dispersion. according to tem data, the average ceo2 particle size in this sol was about 3 – 4 nm (fig. 1). nova biotechnol chim (2022) 21(2): e954 3 fig. 1. photos from (a) – tem microscopy and histogram of size distribution and (b) – high-resolution transmission electron microscopy (hrtem) used cerium nanoparticles. biological material and cultivation conditions the studies were performed on algological pure monocultures of green algae d. armatus (chod.) hegew and a. dimorphus (turpin) tsarenko (ibash-a), obtained from the collection of the kholodny institute of botany of the national academy of sciences of ukraine, for which we express our gratitude to it. algae were pre-cultivated under conditions of cumulative culture to the exponential growth phase in zender and gorham’s modification of the fitzgerald's medium n 11 (zolotareva et al. 2008). all manipulations related to the sowing of algae cultures were performed in a laminar box under sterile conditions. the inoculum-to-nutrient medium ratio was 1 : 10. cultivation was performed in a climatic room with photoperiod of 16 hours provided by fluorescent lamps at a light intensity of 2,500 – 4,000 lux and temperature of 24 ± 2 ºc. to create optimum conditions for studying the effects of cerium salt and cerium nanoparticles, the algal biomass was concentrated and washed of nutrient medium residues in sterile distilled water. algae cells were isolated from the culture medium by centrifugation at 3,500 g for 15 min. using the heraeus biofuge stratos benchtop centrifuge (heraeus holding gmbh, hanau, germany). the final cell concentration was 2.6 × 105 cells.ml-1. washed algae cells were incubated in a solution of distilled h2o with the addition of the appropriate concentration of cerium compounds. nano-cerium sol, citrate, or cerium ions (iii) were added to the hydrated cells at the concentrations of 0.001 mm, 0.01 mm, 0.1 mm, 1 mm, 10 mm and 100 mm. after 5-day exposure, the amount of algal biomass, cellular chlorophyll а, protein and lipid content were assayed. the amount of biomass was determined from culture density using an optical index at 750 nm at agilent carywin uv 60 (agilent technologies, inc., santa clara, usa). the transition from the optical density (od) units (d750) to the value of absolutely dry biomass (adb) was carried out through the empirical coefficient k: аdb = k × d750 (hevorhyz et al. 2008). the coefficient k (k = g od unit per liter) for both cultures was determined experimentally in three independent repeats. following the cultivation, the entire biomass was separated by centrifugation at 3,500 g and 4 ºc for 15 min and, if necessary, frozen at 20 ºc. centrifuged microalgae biomass was disintegrated on an usdn-2t. in the hydrated cells, the total proteins were determined by the lowry (lowry et al. 1951) method and the total lipids were assessed (knight et al. 1972). the pigments were extracted from the hydrated microalgae cells with chloroform-methanol 2 : 1 and centrifuged at 1,500 g until the discoloration of the extract (macías-sánchez et al. 2008). pigment spectra were measured in the combined supernatant. the pigment concentrations were calculated by formulae using ods at wavelengths corresponding to the absorption maxima (640 – 660 nm) of chlorophyll а. data analysis statistical treatment of data obtained was a b nova biotechnol chim (2022) 21(2): e954 2 performed using a biostat 2009 professional 5.8.1 software in accordance with recommendations (gtantz et al. 1998; gubler and genkin 1973). all values were measured in six-fold repeatability. results are presented as the median and the interval between the first and third quartiles. a validity check for the null hypothesis was performed using non-parametric wilcoxon-mann-whitney criteria. the difference between control and experimental groups was judged to be statistically significant at p < 0.05. results biomass accumulation is the first and indicative criterion for assessing the effect of compounds on algal cultures. the table 1 showed the results of a. dimorphus and d. armatus biomass accumulation in the presence of cecl3 solution, citrate or cdn in the cultivation medium. compared to the intact culture medium, the presence of cdn in the culture medium at a concentration of 0.001 – 10.0 mm resulted in a statistically significant increase of the a. dimorphus biomass by 20 – 75 %. presence of cdn in d. armatus culture medium resulted in a 25 – 50 % increase of algae biomass in a significantly narrower concentration range of 0.01 – 1.0 mm. it should be noted that the increase in biomass in concentration 10 – 100 mm of cdn or citrate can be attributed to citrate, a stabilizer in cdn, as an organic substrate, then lower concentrations of 0.001 – 1.0 mm were not caused by the presence of citrate, but rather by cdn. it is interesting to note that the introduction of 0.001 mm citrate into the culture medium statistically significantly inhibited the accumulation of biomass (growth) of the culture compared to the culture grown on a standard medium. the use of cdn stabilized with citrate (m : m) in a similar concentration on the contrary – increased the biomass yield by 20 – 30 %. at the same concentration, cerium salt did not affect accumulation of a. dimorphus biomass (table 1). table 1. cdn, citrate, or cecl3 effect on the biomass accumulation in tested green algae. the test objects concentration [mm] a. dimorphus d. armatus cdn citrate cecl3 cdn citrate cecl3 0 9.7 [9.3 10.0] 11.4 [10.7 11.6] 0.001 12.6* [12.1 12.6] 7.6** [6.4 8.0] 9.5 [9.3 9.8] 12.0** [11.9 12.1] 10.2 [9.6 10.7] 10.7 [10.6 10.8] 0.01 14.2* [14.0 -14.3] 9.0 [8.1 10.0] 11.0* [10.2 11.9] 14.5* [14.1 14.8] 10.7 [10.6 10.8] 10.7 [10.1 11.8] 0.1 15.2** [14.7 15.8] 10.5* [10.3 11.0] 12.28** [11.1 12.6] 15.1* [14.3 15.8] 11.3 [11.0 11.8] 12.1 [11.3 12.6] 1 16.9** [16.5 17.8] 11.0* [10.3 11.6] 14.5** [14.0 15.1] 20.0* [19.5 20.7] 11.5 [11.1 12.0] 13.9* [13.2 14.0] 10 12.0* [11.9 12.1] 13.6** [13.4 14.1] 11.5* [11.2 11.8] 12.8** [12.7 13.2] 12.9** [12.9 13.0] 11.9 [10.9 12.9] 100 11.1* [10.5 11.2] 11.0* [11.0 11.2] 9.9 [9.7 10.6] 12.0 [11.2 12.4] 11.0 [10.4 11.7] 11.0 [10.9 11.1] notes: * – p ˂ 0.05; ** – p ˂ 0.01, n = 6, results presented as the median and the interval between the first and third quartiles [q1-q3]. the maximum quantity of biomass of both cultures was observed when cdn was applied in an amount of 1 mm. both algocultures showed a similar reactive response to exposure to all factors, some numerical differences being due to the species specifics of the algocultures. thus, in a. dimorphus culture, the amount of biomass increased by 60 % upon cdn exposure, while in d. armatus culture it increased by 45 %. the results obtained clearly correlate with the amount of chlorophyll a in a. dimorphus and d. armatus cells (table 2). 4 nova biotechnol chim (2022) 21(2): e954 3 table 2. cdn, citrate, or cecl3 effect on сhlorophyll a concentration in green algae cells. the test objects concentration [mm] a. dimorphus d. armatus cdn citrate cecl3 cdn citrate cecl3 0 10.5 [10.2 11.0] 10.3 [10.0 10.6] 0.001 11.0 [10.9 11.7] 10.1 [8.8 11.2] 10.1 [9.7 10.6] 9.9 [9.7 10.1] 10.0 [9.7 10.4] 9.9 [9.3 10.5] 0.01 10.4 [10.0 11.0] 10.3 [9.6 11.1] 10.4 [10.1 10.9] 10.3 [10.0 10.6] 9.9 [9.7 10.1] 10.6 [10.0 11.0] 0.1 10.0 [9.5 11.5] 11.1 [10.7 11.4] 10.6 [10.3 10.9] 10.7 [10.1 10.9] 9.9 [9.2 10.5] 9.9 [9.4 10.0] 1 10.2 [9.8 10.6] 10.9 [10.1 11.6] 10.5 [10.4 11.1] 9.7 [9.5 10.4] 9.4 [9.2 10.6] 10.0 [9.7 10.6] 10 11.0 [10.7 11.2] 10.6 [10.4 10.9] 6.7** [6.5 7.0] 9.9 [9.2 10.1] 9.0* [8.8 9.8] 8.7** [8.1 8.9] 100 10.6 [10.3 11.3] 10.8 [10.4 11.0] 6.1** [5.7 6.8] 10.4 [10.0 10.9] 9.5* [8.7 9.9] 7.9** [7.3 8.0] notes: * – p ˂ 0.05; ** – p ˂ 0.01, n = 6, results presented as the median and the interval between the first and third quartiles [q1-q3]. its amount does not significantly differ from the control values, except for the concentration variants in which the growth activity of both algocultures is inhibited (10 – 100 mm). but even under these conditions, a decrease in the amount of chlorophyll a was noted when exposed to cecl3, while in the presence of cdn, on the contrary, the negative effect of high concentrations of cerium ions on algocultures is compensated. despite the positive effect of cerium both in ionic form and in the form of nanoparticles on the growth activity of a. dimorphus and d. armatus, a biochemical analysis showed some negative effect of cecl3 on the process of protein accumulation in the algal biomass (table 3). table 3. the effect of cdn, citrate or cecl3 on the protein content (% of biomass) in the biomass of tested green algae. the test objects concentration [mm] a. dimorphus d. armatus cdn citrate cecl3 cdn citrate cecl3 0 49.9 [48.6 50.9] 56.7 [56.0 57.5] 0.001 50.0 [46.1 51.2] 50.9 [49.5 51.5] 33.9* [33.3 34.8] 53.5* [53.3 54.6] 52.4* [51.9 53.6] 46.0** [45.5 46.8] 0.01 50.9 [49.3 51.4] 50.7 [49.6 51.8] 34.1* [33.1 35.0] 54.5* [53.4 55.7] 54.6* [53.5 55.2] 46.5** [46.0 47.5] 0.1 49.6 [49.2 50.7] 50.0 [48.3 51.0] 33.5* [32.8 34.7] 58.0* [57.8 58.1] 54.4* [53.5 55.3] 44.3** [43.9 44.8] 1 44.5** [42.7 45.7] 47.3 [47.0 48.6] 32.5* [31.3 33.5] 54.5* [52.5 55.3] 53.6* [52.4 54.7] 43.5** [42.3 44.0] 10 37.0** [35.1 38.0] 45.0* [44.1 45.8] 25.3* [24.1 26.0] 52.8** [52.2 53.1] 50.5** [50.0 51.6] 40.0** [39.9 42.1] 100 28.5* [27.9 30.5] 43.6* [43.1 44.7] 18.1* [17.5 22.1] 50.5** [49.6 51.7] 50.9** [50.3 51.1] 40.0** [38.9 41.6] notes: * – p ˂ 0.05; ** – p ˂ 0.01, n = 6, results presented as the median and the interval between the first and third quartiles [q1-q3]. thus, a rather low content of total protein in cells was noted in the presence of cdn ≥ 1 mm. the use of nanocerium in a smaller amount does not reliably reduce the protein content in algal cells. the use of cecl3 in all cases is accompanied by a decrease in the amount of protein in a. dimorphus cells by 25 – 55 %, and in d. armatus cells by 10 – 25 %. while the amount of protein tended to 5 nova biotechnol chim (2022) 21(2): e954 2 decline, there was an increase in the accumulation of lipids in the biomass of both cultures (table 4). table 4. the effect of cdn, citrate or cecl3 on the lipid content (% of biomass) in the biomass of tested green algae. the test objects concentration, mm a. dimorphus d. armatus cdn citrate cecl3 cdn citrate cecl3 0 15.2 [14.6 15.6] 16.1 [15.8-16.7] 0.001 15.8 [15.5 16.1] 15.3 [14.7 15.9] 16.6* [16.0 17.4] 19.5** [18.7 20.1] 19.6** [19.1 20.7] 29.0** [28.1 29.7] 0.01 16.2 [14.8 16.8] 16.3* [15.9 17.2] 19.0** [18.9 19.5] 18.4* [18.0 18.9] 17.9* [17.2 18.6] 29.8** [29.0 30.0] 0.1 16.1 [15.2 16.3] 16.0* [15.8 16.6] 22.9** [22.8 23.6] 18.2* [17.9 18.7] 17.8 [16.5 18.7] 21.5** [21.0 22.0] 1 18.2* [18.0 18.9] 17.1* [16.8 17.3] 28.4** [27.7 29.5] 16.1 [15.2 16.3] 16.9 [16.3 17.3] 18.9** [18.2 20.0] 10 27.8** [25.7 28.7] 16.4* [15.9 17.0] 31.1** [30.8 31.6] 16.2 [14.8 16.8] 16.0 [15.7 16.6] 16.9* [16.8 17.3] 100 29.8** [28.1 31.3] 17.0* [16.7 17.6] 31.3** [30.2 32.5] 15.8 [15.5 16.1] 15.6 [15.1 16.0] 16.4 [16.0 16.9] notes: * – p ˂ 0.05; ** – p ˂ 0.01, n = 6, results presented as the median and the interval between the first and third quartiles [q1-q3]. there is a clear correlation between the amount of cecl3 in the medium and the increase in the number of total lipids in a. dimorphus and d. armatus cells. it should be noted that judging by changes in the number of total lipids in algal biomass in response to the presence of cerium ions. a. dimorphus culture is more sensitive than that of d. armatus. thus, in the presence of 0.01 mm cecl3 in the culture medium the amount of total lipids in a. dimorphus cells increases by 20 % while in the presence of ≥1 mm of cecl3 this indicator increases by almost 100 %. cdn does not have such a strong impact on lipid accumulation by algae cells, which can be considered as an indirect sign of its lower toxicity. in the presence of 10 – 100 mm of cdn in the culture medium an increase of 80 – 90 % in the number of lipids in a. dimorphus biomass compared to control samples biomass was recorded. thus, the results obtained allowed us to estimate the effect of cdn on cells of two green algae. a. dimorphus and d. armatus. having compared all indicators. we determined the optimal cdn concentrations which when applied to bring an increase of the biomass in both algocultures and optimize this biomass by productivity outputs (fig. 2). a 6 nova biotechnol chim (2022) 21(2): e954 3 fig. 2. the effect of cdn (а) or cecl3 (b) on key indicators of the green algae viability. the data on the content of biomass, chlorophyll a, proteins, and lipids in percent are given, calculated relative to their amount in the control samples of biomass grown on a standard medium. notes: on the abscissa – the concentration of cerium; on the y-axis – the percentage of content of biomass, chlorophyll a, proteins, and lipids in percent, calculated relative to their amount in the control samples of biomass grown on a standard medium (taken as 100 %), n = 6, error bars indicate [q1-q3] of the mediane. it was determined that 0.1 mm of cdn is the optimal amount of the preparation that causes positive changes in cells a. dimorphus and d. armatus. under such conditions, it is possible not only to increase the yield of biomass of algal crops, but also to achieve balanced levels of chlorophyll a, proteins, and lipids. the concentration of 1 mm of cdn can be considered as suboptimal, recommended for use in cases of an urgent need for rapid mass growth of cell mass of green algae a. dimorphus and d. armatus. the use of cecl3 in all analyzed concentrations leads to increased growth activity of algocultures, but also in all cases there is negatively affects the processes of proteins and lipids accumulation. the generalized results are presented in the form of a summary table, which allows to clearly determine, depending on the goal, the dose of cdn or cecl3 (table 5). table 5. the nontoxic (cc0. mm) concentration of cdn and cecl3. criteria acutodesmus dimorphus desmodesmus armatus cdn citrate cecl3 cdn citrate cecl3 chlorophyll a 100.0 100.0 1.0 100.0 1.0 1.0 total proteins 0.1 0.5 ˂0.001 ˂0.001 ˂0.001 ˂0.001 total lipids 0.1 0.001 ˂0.001 ˂0.001 ˂0.001 ˂0.001 biomass increasing (> 20 %) ˂0.001 – 1.0 – 0.1 – 1.0 0.01 – 1.0 – 1.0 discussion like most trace elements, ce depending on the concentration can have both positive and negative effects on the growth and development of algae. it is known that in the aqueous medium np ceo2 undergo redox modifications, form polydisperse aggregates, which lead to a change in their catalytic activity (santschi et al. 2017; gagnon et al. 2018; rozhin et al. 2021). first of all, it should be noted that the sharp increase in the amount of lipids found in d. armatus and a. dimorphus cells is a well-known response of algae cells to stress, in our case to the presence of high concentrations of cerium in the growth medium. a similar increase in the amount b 7 nova biotechnol chim (2022) 21(2): e954 2 of lipids in response to the presence of alkaline earth metals has been described for chlorella vulgaris and scenedesmus obliquus (gorain et al. 2013). dunaliella tertiolecta, and tetraselmis suecica (ghafari et al. 2016). stress activation of metabolic pathways of lipid synthesis by ce3+ ions may be due to inhibition of further conversion of the intermediate product of glycolysis – glyceraldehyde-3-phosphate. in this case, instead of the synthesis of carbohydrates, the synthesis of triacylglycerols (wang et al. 2009; li et al. 2010) will take place, which will be accompanied by an increase in the accumulation of lipids in the cells of microalgae. as a consequence of such processes, the redistribution of the relation between the major macromolecules in the cells of algae, which we found. it is known that high concentrations (≈ 30 µm.l-1) of lanthanides lead to a sharp slowdown in the growth processes of unicellular algae (tai et al. 2010), while low concentrations stimulate these processes (laszló et al. 2001; goecke et al. 2015; řezanka et al. 2016). in our studies it was shown that in the presence of ≤ 1 mm cerium concentration in the medium, there is an intensification of growth processes in cultures d. armatus and a. dimorphus and, as a consequence an increase in the amount of biomass by more than 25 %. however, the effect of any chemical element depends also on the form of its introduction into the incubation medium (chen et al. 2000; tai et al. 2010). cecl3 has a more pronounced toxic effect on algae cultures than cdn. a similar effect was shown by zirconium on the cells of the alga chlorella pyrenoidosa (laszló et al. 2001). lanthanides can also replace certain metals in some physiological processes, as has been demonstrated, in particular, in plants with ca2+ or mg2+ deficiency (huang et al. 2008а; chao et al. 2008; gong et al. 2011) the primary cause of this phenomenon may be the property of lanthanides, whose combinatorial ability is much higher than that of other divalent cations, to interact with a large number of biological macromolecules to form stable complexes (goecke et al. 2015). it is believed that the ability of lanthanides, including cerium, to mimic some biological functions of ca2+, primarily affects the processes of photosynthesis and ion transport in plant cells (wei and zhou 2000; gong et al. 2011). obviously, this property of cerium was decisive in the manifestation of the stimulating effect on the growth processes of green algae cultures d. armatus and a. dimorphus, studied by us. the stimulating effect of ce on photosynthetic organisms is associated with the ability of the latter to accumulate in chloroplasts as well at low concentrations of mg2+.to replace it in the chlorophyll molecule (zhou et al. 2011). the effects we found are most likely related to an increase in carbohydrate production. the mechanism of this process is due to the influence of ce ions on intracellular anabolic pathways in particular, transmebranic mitochondrial transport of pyruvate and oxaloacetate production. it is known that rare earth metals in general, and cerium in particular, significantly improve the process of light energy absorption, energy transfer, electron transfer rate and phosphorylation rate, thus stimulating the accumulation of organic matter in cells (zeng et al. 2000; gong et al. 2011). conclusion compared to cdn, cecl3 solution is more toxic to green algae cells. cdn is 100 – 1,000 times less toxic than cecl3 solution. it is shown that the use of high concentrations of ce is accompanied by a well-known response of algae cells to the action of a stress factor – an increase in total lipids and a decrease in the synthesis of proteins and chlorophyll a. the introduction of cdn into the culture of algae in non-toxic concentrations (cc0 and below) provides a significant increase (25 – 75 %) in the total amount of biomass, whereas with the use of cecl3 solution such an effect is absent. the obtained results allow assessing the mechanism of realization of biological effects of cerium dioxide nanoparticles. conflict of interest the authors declare that they have no conflict of interest. references borisevich vb, kaplunenko vg, kosinov nv, borisevich 8 nova biotechnol chim (2022) 21(2): e954 3 bv, sukhonos vp, khomin nm, teliatnikov av, voloshina na, tkachenko sm, doroshchuk va, korzh av, litvinenko di, kulida ma, kulinich sl, borosevich vb jr, borisevich ib, dimchev va (2012) nanomaterials and nanotechnologies in the veterinary practice. kyiv: vd «avitsena». 512 p. chao l, bofu p, weiqian c, hao h, liang c, xiaoqing l, xiao w, faishu h (2008) influences of calcium deficiency and cerium on growth of spinach plants. biol. trace elem. res. 121: 266-275. chao l, weiqian c, yun l, hao h, liang c, xiaoqing l, fashui h (2009) cerium under calcium deficiency – influence on the antioxidative defense system in spinach plants. plant soil. 323: 285-294. charbgoo f, ahmad mb, darroudi m (2017) cerium oxide nanoparticles: green synthesis and biological applications. int. j. nanomed. 20: 1401-1413. cheban l, grynko o (2017) use of acutodesmus dimorphus (turpin) tsarenko as a fodder organism for daphnia growing. acta biol. univ. daugavp. 17: 141-148. cheban l, grynko o, dorosh i (2018) co-cultivation of daphnia magna (straus) and desmodesmus armatus (chod.) hegew. in recirculating aquaculture system wastewater. fish. aquat. life 26: 57-64. chen wj, gu yh, zhao gw. tao y, luo jp, hu td (2000) effects of rare earth ions on activity of rubpcase in tobacco. plant sci. 152: 145-151. chen wj, tao y, gu yh, zha gw (2001) effect of lanthanide chloride on photosynthesis and dry matter accumulation in tobacco seedlings. biol. trace elem. res. 79: 169-176. gagnon c, bruneau a, turcotte p, pilote m, gagné f (2018) fate of cerium oxide nanoparticles in natural waters and immunotoxicity in exposed rainbow trout. j. nanomed. nanotechnol. 9: 2. ghafari m, rashidi b. haznedaroglu bz (2016) effects of macro and micronutrients on neutral lipid accumulation in oleaginous microalgae. biofuels. 9: 147-156. goecke f, jerez c, zachleder v, figueroa fl, bišová k, řezanka t, vítová m (2015) use of lanthanides to alleviate the effects of metal ion-deficiency in desmodesmus quadricauda (sphaeropleales, chlorophyta). front. microbiol. 6: 1-12. gong x, hong m, wang y, zhou m, cai j, liu c, gong s, hong f (2011) cerium relieves the inhibition of photosynthesis of maize caused by manganese deficiency. biol. trace elem. res. 141: 305-316. gorain pc, bagchi sk, mallick n (2013) effects of calcium magnesium and sodium chloride in enhancing lipid accumulation in two green microalgae. environ. technol. 3: 1887-1894. gtantz s (1998) medical-biological statistics. praktika, moscow. gubler ev, genkin aa (1973) application of non-parametric statistic criteria in medical-biological research. medicine, moscow, 142 p. hevorhyz rh, shchepachyov sh (2008) metodyka yzmerenyia plotnosty suspenzyy nyzshykh fototrofov na dlyne volny sveta 750 nm. sevastopol: otdel byotekhnolohyy y fytoresursov ynbium nan ukraynu. 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biotechnol chim (2020) 19(2): 200-207 doi: 10.36547/nbc.v19i2.775  corresponding author: dimitrismakris@uth.gr nova biotechnologica et chimica stability of salvia fruticosa mill. polyphenols and antioxidant activity in a citrate-based natural deep eutectic solvent spyros grigorakis1,2, abedalghani halahlah1 and dimitris p. makris3, 1department of food science & nutrition, school of environment, university of the aegean, mitr. ioakim street, myrina 81400, greece 2food quality & chemistry of natural products, mediterranean agronomic institute of chania (m. a. i. ch.), international centre for advanced mediterranean agronomic studies (ciheam), p.o. box 85, chania 73100, greece 3green processes & biorefinery group, department of food science & nutrition, school of agricultural sciences, university of thessaly, n. temponera street, karditsa 43100, greece article info article history: received: 10th july 2020 accepted: 14th september 2020 keywords: antioxidant activity deep eutectic solvents polyphenols salvia fruticosa stability abstract in a previous study, it was demonstrated that a novel deep eutectic solvent (des), composed of lactic acid and sodium citrate dibasic at a molar ratio of 15:1 (lascdb15), was a high-performing system with regard to polyphenol extraction from the medicinal plant salvia fruticosa mill. (greek or cretan sage). however, an issue of particular importance that should be addressed is the stability of the extract in this novel liquid since the information available to-date on extract stability in des is rather limited and inconclusive. in this frame, this study was undertaken to generate extracts of s. fruticosa with la-scdb15 (a 77/23 w/w mixture of des with water) and examine their stability in this solvent. s. fruticosa extracts exhibited remarkable stability under both accelerated and long-term conditions, and the antiradical activity and the ferric-reducing power of the extracts were shown to suffer virtually trivial modifications. further analytical examination with liquid chromatography-diode array-tandem mass spectrometry assured that the major polyphenolic phytochemicals occurring in salvia fruticosa extracts underwent non-significant changes and remained practically intact. it was concluded that the neoteric des lascdb15 may provide outstanding stability in polyphenol-containing extracts and its testing on other plant extracts is proposed as a further step towards revealing its stabilizing potential.  university of ss. cyril and methodius in trnava introduction a high number of populations around the globe routinely utilize medicinal and aromatic plants (maps) as folk medical agents, but also food, for centuries. currently, sound scientific data form a solid ground to support the pharmacological and nutritional attributes of maps and substantiate health claims and functionality (anton et al. 2019). the ongoing interest for maps and map-based commodities derives from consumer demands for natural products with functional properties, which has stimulated a large development of neoteric botanical-based ingredients for foods, pharmaceutical and cosmetics (campa and baron 2018; colombo et al. 2020). s. fruticosa mill., otherwise known as s. triloba (greek or cretan sage) is a lamiaceae species widely distributed in the east mediterranean. several bioactivities have been attributed to this medicinal plant, which mailto:dimitrismakris@uth.gr nova biotechnol chim (2020) 19(2): 200-207 201 are mainly due to its polyphenolic substances (exarchou et al. 2015; sarrou et al. 2016). yet, to-date the implementation of green extraction methodologies to produce of polyphenol-enriched extracts from s. fruticosa is very limited. deep eutectic solvents (des) are novel liquids composed of inexpensive, benign and reusable materials, embracing natural compounds, such as organic acids and their salts, polyols, amino acids etc. usually, des are composed of two constituents, one that functions as hydrogen bond acceptor (hba) and another one as hydrogen bond donor (hbd), while des synthesis is of low-cost, simple, and fast. major intrinsic characteristics of des are the tunability of composition to achieve water (im)miscibility, the low vapor pressure, and the absence of flammability, which make des ideal solvents for a range of green processes (espino et al. 2018). by virtue of their peculiar properties, des have currently become the choice of preference for the effective extraction of numerous natural products, but important issues pertaining to extract stability in des are presently poorly addressed. polyphenols are inherently molecules prone to oxidation and/or other structural modifications, and thus the study of polyphenol stability under a given set of conditions is of paramount importance. recently, our group reported the use of a novel des, coded la-scdb15, which displayed very high performance in the extraction of polyphenols from s. fruticosa (grigorakis et al. 2020b). to further appraise the applicability of this solvent in the generation of polyphenolenriched extracts, this study was undertaken to examine the stability of s. fruticosa polyphenols in extracts produced using la-scdb15. stability was assessed by monitoring the antiradical activity and the ferric-reducing power of the extracts, by employing an accelerated and a long-term test, at various temperatures. moreover, to better illustrate the effect of extract storage in la-scdb15, liquid chromatographydiode array-tandem mass spectrometry (lc-dadms/ms) analyses were also performed, to trace changes in major polyphenolic phytochemicals. experimental chemicals all chromatographic analyses were accomplished with solvents of hplc grade. l-lactic acid (80 %) was from fisher scientific (loughborough, uk). sodium citrate dibasic sesquihydrate (> 99 %), rosmarinic acid ( 98 %), gallic acid (97 %), sodium carbonate ( 99.5 %), ascorbic acid ( 99 %), sodium acetate trihydrate ( 99.5 %), luteolin 7-o-glucoside ( 98 %), 2,2-diphenylpicrylhydrazyl (dpph) (95 %) and chlorogenic acid ( 95 %) were from sigmaaldrich (darmstadt, germany). folin-ciacalteu reagent was from merck (darmstadt, germany). 2,4,6-tripyridyl-s-triazine (tptz) (98 %), acetic acid, methanol and iron chloride hexahydrate were from honeywell/fluka (steinheim, germany). the deep eutectic solvent (des), composed of lactic acid (la) and sodium citrate dibasic (scdb) at a molar ratio of 15 : 1, was synthesized as described earlier (grigorakis et al. 2020b). the des was used as aqueous mixture (77/23 w/w). plant material certified s. fruticosa was provided by a botanicals store (chania, greece). the specimen composed of the aerial parts of the plant and it was received in dried form, in hermetically closed plastic packaging. upon receipt, the plant material was pulverized in a table mill (tristar, tilburg, the netherlands), as described previously (grigorakis et al. 2020a) and stored under refrigeration (4 c). preparation of polyphenol extracts extraction of s. fruticosa was performed using the optimized process, as reported elsewhere (grigorakis et al. 2020b). in short, 0.375 g of dried plant material was mixed with 15 ml 77 % (w/w) des/water and ultrasonicated for 15 min in an ultrasonication bath (sonorex bandeline, berlin, germany), at room temperature (23 ± 2 c). the ultrasonication settings were: power, 120 w; frequency, 100 hz; acoustic energy density, nova biotechnol chim (2020) 19(2): 200-207 202 120 w.l-1. after ultrasonication, which was the pretreatment stage, batch stirred tank extraction was carried out at a stirring speed (ss) of 900 rpm, at 80 c, for 150 min. the extract thus obtained was centrifuged for 10 min at 10,000g and the transparent supernatant was used for stability tests. determination of the antiradical activity (aar) the assay was performed with a stoichiometric methodology, using dpph (cevalos-casals and cisneros-zevallos 2003; athanasiadis et al. 2017). the total stoichiometries (nt) of the reaction between dpph and polyphenols in the extract were determined using the following eq. 1: (1) where ctp is the total polyphenol concentration of the extracts (mg.l-1), ε (dpph) = 11,12610-6 μm-1.cm-1, a0 is the a515 at t = 0 and af is the a515 at t = 30 min. results were expressed as μmol dpph per mg total polyphenols. for all measurements, an hp 8452a diode-array spectrophotometer was used. determination of the ferric-reducing power (pr) a previously published protocol was used (karakashov et al. 2015). briefly, volume of 0.05 ml of ferric chloride (4 mm in 0.05 m hcl) was combined with an equal volume of extract and the mixture was heated up at 37 c, in a water bath, for 30 min. after incubation, 0.9 ml of tptz solution (1 mm in 0.05 m hcl) was added and the mixture was allowed to stand at room temperature for another 10 min. the absorbance at 620 nm was recorded using suitable control and results were reported as mm ascorbic acid equivalents (aae). total polyphenol concentration the methodology described elsewhere was implemented (karakashov et al. 2015). in an eppendorf tube of 1.5 ml, volume of 0.02 ml extract, 0.78 ml deionized water and 0.05 ml folin-ciocalteu reagent were mixed and left to react for 2 min, in the dark, at ambient temperature. then, 0.15 ml of 20 % sodium carbonate was added and the mixture was allowed to stand for 60 min. a calibration curve constructed with gallic acid was used for quantification. chromatography a method previously reported was used (grigorakis et al. 2020a). a finniganmat p4000 pump coupled to a uv6000lp diode array detector (thermo scientific, waltham, ma, u.s.a.), and a tsq quantum access lc/ms/ms, equipped with a surveyor pump (thermo scientific, walltham, ma, u.s.a.), and controlled by xcalibur 2.1, tsq 2.1 software, were employed. the column was superspher rp-18, 125 mm  2 mm, 4 μm, kept at 40 c. the injection volume was 10 μl and the eluents used were (a) 2.5 % acetic acid and (b) methanol, operated at a flow rate of 0.3 ml.min-1. the gradient program was as follows: 0 min, 100 % a; 22 min, 65 % a; 32 min, 65 % a; 60 min, 0 % a; 65 min, 0 % a. acquisition of mass spectra was done with negative ionization, employing sheath gas pressure 30 mtorr, auxiliary gas pressure 15 mtorr (2  10-5 bar), collision pressure at 1.5 mtorr (2  10-6 bar) and capillary temperature 300 c. quantification was performed using a calibration curve of luteolin 7-o-glucoside (5 – 1,500 μg.l-1, r2 = 0.9982), chlorogenic acid (50 – 1,500 μg.l-1, r2 = 0.9986) and rosmarinic acid (50 – 3,000 μg.l-1, r2 = 0.9985). all standard solutions were prepared in hplc grade methanol. accelerated stability test a volume of extract (20 ml) was placed in a glass vial and heated up at 50, 60, 70, 80 and 90 c for 240 min, by means of a heating magnetic stirrer (velp scientifica, ny, usa). sampling was accomplished at 30-min intervals to assay antioxidant activity. long-term stability test equal volumes of extract (20 ml) were transferred into glass vials and stored in the fridge (7 c), on the bench (23 ± 2 c) and in a water bath adjusted at 40 c, for 30 days. during this period, nova biotechnol chim (2020) 19(2): 200-207 203 special care was taken to avoid extract contact with light. sampling was carried out at 3-days intervals to determine antioxidant activity. statistical analysis procedures were repeated twice, and determinations were carried out in triplicate. values were given as averages ± standard deviation (sd). distribution analysis was carried out with jmp™ pro 13 (sas, cary, nc, usa). linear correlations were accomplished with sigmaplot™ 12.5 (systat software inc., san jose, ca, usa). results and discussion accelerated stability test the objective of the test was to ascertain whether the antioxidant properties of the extract could be impacted as a result to exposure to a range of temperatures, varying from moderate (50 c) to severe (90 c) heating, for 240 min, and thus to draw conclusions regarding extract stability in la-scdb15. the results of the test are depicted in fig. 1. over the range 50 to 80 c, the extract displayed by almost 17.5 – 21 % higher aar compared to the initial (untreated) sample. however, aar declined to a level equal to the initial one, after heating at 90 c. differences amongst values were shown to be non-significant (p > 0.05), which indicated that increases in temperature did not affect aar to a significant extent. furthermore, no consistent trend was observed in aar as a response to temperature. likewise, pr remained virtually intact since the differences found amongst the initial extract and the extracts treated at 50 to 90 c varied between0 and 3.1 %. this finding strongly suggested that la-scdb15 used to produce the extract, provided exceptional stability. long-term stability test this test was employed to trace fluctuations in both aar and pr during storage of the extract for 30 days, at different storage temperatures. as can be seen in fig. 2, the aar of the extract stored at 7 c exhibited a significant decline by 39.9 %, fig. 1. comparison of aar (upper plot) and pr (lower plot) of s. fruticosa extracts, undergone no treatment (initial) and after treated at 50 – 90 c, for 240 min. from 9.41 (day 0) to 5.66 μmol.dpph.mg-1 tp (day 12). however, aar recovered to 8.91 μmol.dpph.mg-1 tp by the end of the examination period (day 30). on the other hand, the extract stored at ambient conditions (23 c) displayed less intense variations and, after an increase to 9.87 μmol.dpph.mg-1 tp (3.1 %) at day 12, it dropped to 8.10 μmol.dpph.mg-1 tp at day 30. the extract stored at 40 c manifested a different pattern, as its aar increased up to 10.16 μmol.dpph.mg-1 tp at day 15, but it declined to 8.93 μmol.dpph.mg-1 tp at day 30. thus, at the end of the treatment the extracts had practically equal aar, irrespective of the storage temperature. the monitoring of pr revealed a diversified time course than that seen with aar. nova biotechnol chim (2020) 19(2): 200-207 204 fig. 2. monitoring of aar (left plot) and pr (right plot) of s. fruticosa extracts, stored at 7, 23 and 40 c, for 30 days. the pattern observed for the samples stored at 7 and 40 c was almost identical, and at the end of the treatment, the extracts had pr of 11.97 and 11.07 mm aae, respectively. on the contrary, the extract stored at 23 c showed rather large variations between day 18 and day 30, while its final level (day 30) was 12.04 mm aae. the difference amongst pr values at day 30 and the initial extract (9.53 mm aae) was low and statistically non-significant (p > 0.05), which further confirmed the outstanding stability of the extract in la-scdb15. early studies evidenced that the antioxidant activity, as evaluated by aar and pr, might reflect changes associated with polyphenolic composition, such as oxidation (sioumis et al. 2005). on the other hand, examinations on stability of polyphenol-containing extracts in des are particularly limited, but the evidence emerged suggested that des may provide improved stability over conventional solvents. such an effect has been demonstrated for safflower (carthamus tinctorius) pigments in a glucose-choline chloride des (dai et al. 2014) and catharanthus roseus anthocyanins in a lactic acid-glucose des (dai et al. 2016). long-term stability studies on moringa oleifera extracts in a glycerol-sodium acetate des showed that aar displayed a constant decline over a 18-days storage period, at every temperature tested (4, 22 and 50 c), which obeyed pseudo-first order kinetics (karageorgou et al. 2018). at 50 c, where the highest declining rate was found, polyphenols were extensively degraded, and the authors argued that this was the reason for the low aar levels recorded at the end of the treatment. in that case, addition of hydroxypropyl β-cyclodextrin was found to slow down aar drop. similarly, olive leaf (olea europaea) extracts in a glycerol-glycine des showed a decreasing trend in pr, which followed pseudo-zero order kinetics over a period of 20 days (athanasiadis et al. 2018), irrespective of the assay temperature (4, 22 and 50 c). the presence of methyl β-cyclodextrin delayed the progression of the phenomenon, yet at 50 c some principal metabolites suffered extensive degradation and a novel yellow pigment was formed. however, unlike investigations revealing a reduction in either aar or pr, other studies illustrated that there was no specific pattern regarding the evolution of antioxidant activity during storage. monitoring of both aar and pr of onion solid waste extracts produced with a glycerol-sodium propionate des, for a period of 30 days at 22 c, showed that at the end of storage aar was enhanced by 19 %, whereas pr was virtually unaffected (stefou et al. 2019). at the same time, no major changes were observed in the polyphenolic profile of the extracts. on the other hand, olive leaf extracts in a lactic acid-ammonium acetate des, containing β-cyclodextrin, displayed a striking increase in aar by 100 % after 30 days at 22 c, although in this case too, pr was stable and fluctuated within nova biotechnol chim (2020) 19(2): 200-207 205 rt: 0.00 59.98 0 5 10 15 20 25 30 35 40 45 50 55 time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 r e la ti v e a b s o rb a n c e nl: 1.54e5 nm=329.5330.5 pda des77%_ pos_1_5_2 au t (min) day 0 (initial) 1 2 3 rt: 0.00 59.98 0 5 10 15 20 25 30 35 40 45 50 55 time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 r e la ti v e a b s o rb a n c e nl: 1.28e5 nm=329.5330.5 pda fr30_1_5_ pos2 au t (min) day 30 at 4 c rt: 0.00 59.98 0 5 10 15 20 25 30 35 40 45 50 55 time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 r e la ti v e a b s o rb a n c e nl: 1.18e5 nm=329.5330.5 pda rt30_1_5 _pos_2 au t (min) day 30 at 23 c rt: 0.00 59.98 0 5 10 15 20 25 30 35 40 45 50 55 time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 r e la ti v e a b s o rb a n c e nl: 1.18e5 nm=329.5330.5 pda 40c30_1_ 5_pos_2 au t (min) day 30 at 40 c fig. 3. chromatograms of s. fruticosa extracts, obtained at 330 nm, illustrating the effect of extract storage at different temperatures after 30 days, on the major polyphenolic compounds. peak assignment: 1– chlorogenic acid; 2 – luteolin 7-oglucuronide; 3 – rosmarinic acid. narrow limits (chakroun et al. 2020). paradoxically, the major polyphenols in the extract showed a decrease by 4.4 – 42 %. on the basis of the above-mentioned, it could be argued that both aar and pr exhibited very high stability in la-scdb15, which has not been previously encountered for other extracts. polyphenolic composition to shed more light onto the effect of la-scdb15 on the stability of s. fruticosa extracts, liquid chromatography-diode array-tandem mass spectrometry (lc-dad-ms/ms) analyses were undertaken. the scope of this examination was the detection of alterations in the polyphenolic profile of the extracts stored at different temperatures, as well as quantitative changes in major polyphenolic phytochemicals. chlorogenic acid and rosmarinic acid were identified by comparing the retention time and uvvis spectra with those of authentic standards. their identity was also confirmed by their respective pseudo-molecular ions at m/z = 353 and 359. luteolin 7-o-glucuronide was tentatively identified considering the pseudo-molecular ion at m/z = 461 and the aglycone (luteolin) at m/z = 285 (grigorakis et al. 2020a). the traces, recorded at 330 nm, of the extracts stored at various temperatures (7, 23, 40 c), had identical polyphenolic profile and no major differences were seen (fig. 3). this outcome indicated neither extensive decomposition of any of the principal metabolites, nor the formation of any other substance, and evidenced the stability of the extract, irrespective of the storage temperature. to better portray possible changes in chlorogenic acid, luteolin 7-o-glucuronide and rosmarinic acid, brough about during storage, a quantitative investigation was also performed (table 1). storage at 7 c resulted in a by 5.6 % decrease in the sum nova biotechnol chim (2020) 19(2): 200-207 206 table 1. quantitative data on changes occurred on major s. fruticosa polyphenols, after storage of extracts at different temperatures, after 30 days. values given (mg.g-1 dry mass) represent means ± standard deviation. compound storage temperature initial 4 c 23 c 40 c chlorogenic acid 0.085±0.002 0.083±0.006 0.085±0.005 0.082±0.006 luteolin 7-o-glucuronide 5.99±0.17 5.47±0.167 5.25±0.31 5.92±0.04 rosmarinic acid 14.67±0.33 14.05±0.27 13.25±0.33 13.48±0.08 sum 20.75 19.6 18.59 19.48 of compounds, while at 23 and 40 c the corresponding changes were 10.4 and 6.1 %. for all compounds considered, the modifications in their concentration found were limited and statistically non-significant, highlighting once again the extraordinary stability of the extract in la-scdb15. conclusions in the current examination, the stability of polyphenol-containing extract from the medicinal plant s. fruticosa in a novel des termed as la-scdb15, was studied by deploying an accelerated and a long-term stability test. the accelerated test, performed over a range of temperatures varying from 50 to 90 c, provided substantial evidence for exceptional extract stability, since the antioxidant activity remained virtually unaffected upon treatment for 240 min. the long-term test monitored antioxidant activity variations over a period of 30 days at various temperatures and assured that the extract suffered no major alteration in its antioxidant properties. a clear confirmation of stability emerged from lc-dadms/ms analyses, which showed that the major polyphenols occurring in s. fruticosa extracts remained practically intact, irrespective of the storage temperature. the study demonstrated a remarkable stability of polyphenol extracts in the la-scdb15. the use of this neoteric solvent regarding effective extract storage remains to be elucidated for other plant materials too. this will confirm the polyphenol-stabilizing ability of this liquid and enable its wider applicability. conflict of interest the authors declare that they have no conflict of 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development of sodium propionate-based deep eutectic solvents for polyphenol extraction from onion solid wastes. clean technol. environ. policy 21: 1563-1574. nova biotechnol chim (2020) 19(1): 80-88 doi 10.36547/nbc.v19i1.580  corresponding author: jesupemienibukun@gmail.com nova biotechnologica et chimica molecular characterization and evaluation of crude oil remediation potential of some rhizobia isolated from plant root nodules jesupemi mercy enibukun and bolatito esther boboye department of microbiology, federal university of technology, pmb 704, akure, ondo state, nigeria article info article history: received: 6th february 2020 accepted: 2nd may 2020 keywords: bioremediation cajanus cajan crude oil pollution molecular characterization phaseolus vulgaris rhizobia abstract this study aimed to determine the molecular identities and genetic relatedness of rhizobia isolated from pigeon pea and pinto beans, and assess their remediation potential in the presence of 1 %, 3 % and 5 % (w/v) crude oil in minimal medium for 7 days incubation period. standard microbiological and molecular methods which include amplification and purification of 16s rrna, agarose gel electrophoresis, and sequencing. results showed molecular identities of six rhizobia from pigeon peas as bradyrhizobium diazoefficiens usda122, rhizobium leguminosarum wsm2304, b. japonicum n61, r. leguminosarum n741, r. leguminosarum bihib1217, and b. japonicum e109; and three rhizobia obtained from pinto beans were r. leguminosarum n871, b. diazoefficiens usda110 and b. japonicum semia5079. all tested rhizobia (9) showed petroleum degradation ability, as they all grew in the 1, 3 and 5 % (w/v) crude oil minimal medium under laboratory conditions. b. diazoefficiens usda122 showed the highest optical density (od) value of 1.184 ± 0.05 on 7th day at 1 % (w/v) crude oil contamination, while r. leguminosarum n741 has the lowest od value of 0.372 ± 0.02 at 5 % (w/v) crude oil on 7th day. for all the rhizobia, increase occurred throughout incubation period at 1, 3 and 5 % (w/v) except r. leguminosarum n741 and r. leguminosarum bihib1217. in conclusion, the association of r. leguminosarum bihib1217 and r. leguminosarum n871 from pigeon pea and pinto beans respectively, were found most effective in crude oil degradation and thus they are recommended as a promising association for remediation of crude oil spilled soils.  university of ss. cyril and methodius in trnava introduction the ability of humans to change the environment has increased faster than the ability to predict the effect of that change (ikuesan 2015). pollution of the environment is one of the major effects of human technological advancement. pollution can be defined as the introduction of deleterious substance into the environment that endangers human health and other natural resources (onojake 2004). it results when a change in the environment harmfully affects the quality of human life and other essential living things such on animals, microorganisms and plants (ogbogbodo et al. 2005; eremrena and akonye 2013). crude oil is a naturally occurring complex mixture of thousands of hydrocarbons and non-hydrocarbon compounds, including heavy metals. organic chemicals such as hydrocarbons are major constituents of petroleum, which can enter the aquatic environment through natural and anthropogenic sources (daniel and nna 2016). total petroleum hydrocarbon (phc) is a mixture of measurable number of petroleum-based mailto:jesupemienibukun@gmail.com nova biotechnol chim (2020) 19(1): 80-88 81 hydrocarbons found in crude oil in an environmental media (rauckyte et al. 2010). some of the chemicals found in tph are hexane, benzene, toluene, xylene, naphthalene as well as other petroleum products and gasoline components. crude oil contamination constitutes one of the most prevalent sources of environmental pollution due to variation in chemical composition of crude oil and its products degradation in the industrialized world (mandal et al. 2012). crude oil pollution can be defined as the introduction of crude oil or its derivatives with its associated gases into the environment (air, water and land) in quantities that are poisonous or capable of causing immediate physical, chemical and biological damages to the affected ecosystem (lee et al. 2001; inoni 2006; tanee and anyanwu 2007; nyananyo 2008; tanee and akonye 2009). petroleum industry’s effluents, oily sludge and oil spills cause a serious threat to the environment as their constituents are toxic, mutagenic and carcinogenic (nyananyo 2008; wokoma 2014; ataikiru et al. 2018). the remediation of oil contaminated soils has been a major problem in oil producing countries and recently use of plants to clean such soils has been investigated. several methods such as physical and chemical means have been adopted in remediation of hydrocarbon polluted soil or the detoxification of hazardous substances (diplock et al. 2009). however, these methods were not effective in the total recovery of polluted soils. phytoremediation and bioaugmentation engage the process of converting organic waste into an innocuous state by the use of plants and microbes respectively. due to the abilities of certain microbes to mineralize hydrocarbon components into environmentally friendly substances such as carbon dioxide and water, the ability of bacteria in breaking down hydrocarbons has gained growing attention in modern day research (kadali et al. 2012; gkorezis et al. 2016). the degradative activity of bacteria isolated from hydrocarbon polluted soil has been investigated by monitoring the changes in optical density (od) which was directly proportional to the activity (boboye et al. 2010). there is premise that the genome of these organisms, harbour genes or enzymes responsible for the degradation (gkorezis et al. 2016). legumes belong to the family fabaceae or leguminosae. legumes are plants that form symbiotic association with rhizobia in the symbiosis legumes nitrogen fixing root or stem nodules. legumes include cowpea, soybean, pigeon peas, pinto beans, alfalfa, chicken peas, lentils, peanut and lupin beans (ismail et al. 2014). cajanus cajan (l.) which commonly called pigeon pea, red gram, congo pea, or gungo pea, is one of the most common legumes cultivated in the tropics and subtropics, for its edible seed (fig. 1). it is a legume domesticated majorly in the india fig. 1. the plant and seed of (a) pigeon pea (cajanus cajan) and (b) pinto beans (phaseolus vulgaris). a b nova biotechnol chim (2020) 19(1): 80-88 82 and north-eastern africa in over 3,000 years ago (mallikarjuna et al. 2011). pigeon pea is found useful in many areas as growth enhancer, alley crop, and protein and trace nutrient supplement, control of weeds and nematodes as well as medication (odeny 2007). the pinto beans (phaseolus vulgaris) are legumes referred to as common beans (fig. 1), they spread throughout south and central america, europe, africa and asia (rodino et al. 2001). pinto beans are the most highly consumed dried bean in the united states. its protein content is comparable with those in other legumes like cowpea and groundnut which had been used in complementing maize diet rich in mineral and fibre. legumes are known to have an advantage over non-leguminous plants in phytoremediation because of their ability to fix nitrogen and thus, do not have to compete with microorganisms and other plants for limited supplies of available soil nitrogen at oil-contaminated sites (cameron 2003). the root nodule symbiosis established between legumes and rhizobia is an exquisite biological interaction responsible for fixing a significant amount of nitrogen in terrestrial ecosystems. the success of this interaction depends on the recognition of the right partner by the plant within the richest microbial ecosystems on earth, the soil. the economic and ecological importance of legumes is evidenced by the high number of species that are cultivated and commercialized, as well as by their ability to obtain nitrogen from a symbiotic interaction with soil bacteria known as rhizobia (oberai and khanna 2018). plant–microbial interactions will be better understood by considering the organism in its natural environment together with its microbiome (thijs et al. 2016). 16s rrna gene sequencing is a well-utilized method for nucleic acid-based detection and identification of microbes, their taxonomic assignment, phylogenetic analysis and the study of microbial diversity (quast et al. 2013). the objective of this study was to isolate and determine the molecular identities and phylogenetics of some tropical rhizobia from pigeon pea and pinto beans, and assess the crude oil degradative ability of these isolated rhizobia. since nitrogen act as alternative to chemical fertilizers, the study of the crude oil bioremediation potential of rhizobia-legume association will provide strategy for farming on polluted agricultural soil. experimental source of legumes pigeon pea (cajanus cajanus) and pinto beans (phaseolus vulgaris) legumes were sourced from oba market, akure, nigeria. the legumes were identified and authenticated at the department of crop, soil and pest management (csp) of the federal university of technology, akure, nigeria. materials, media and reagents all reagents used in this study were of analytical and microbiological standard. nutrient agar (na) composed of: meat extract (1 g.l-1); peptone (5 g.l-1); yeast extract (2 g.l-1); sodium chloride (8 g.l-1); agar (15 g.l-1); and distilled water (1 l), at ph 6.8. mineral salt medium (msm) composed of (g.l-1): 1 g nacl, 1 g kh2po4, 1 g na2hpo4, 0.5 g nh4nh3, 0.5 g (nh4)2so4, 0.2 g mgso4.7h2o, 0.02 g cacl2.2h2o, 0.002 g fecl3, and 0.002 g mnso4.2h2o in 1.0 litre of distilled water, at ph 7.0 and sterilizes at 121 oc for 15 min by autoclave. modified mineral salt medium (mmsm) consisted of (g.l-1): 4 g kh2po4, 4 g nh4nh3, 0.2 g mgso4.7h2o, and 0.01 g cacl2.2h2o, in 1.0 l of distilled water, at ph 7.0 and sterilized at 121 oc for 15 min by autoclave. msm was used for growth of isolated mutants that is used in this experiment while mmsm was used to encourage oil emulsion process which leads to oil degradation. isolation of rhizobium from legumes and cultivation of test bacteria surface sterilization of the seeds were done with 3.5 % (w/v) sodium hypochlorite solution for 15 min and rinsed in sterile distilled water. the seeds were transferred into sterile sandy soil in different sterilized plant jar, regularly wet with mmsm and allowed to germinate at 28 ± 2 oc (daytime) for 90 days in a locally-made greenhouse facility. the root nodules of the germinated plants were used respectively. the root aggregates were nova biotechnol chim (2020) 19(1): 80-88 83 collected and sterilized using 3.5 % (v/v) hypochlorite solution, sterile root was crushed with tweezers, then the root nodule extracts were grown on nutrient agar, incubated at room temperature (28 oc) for 24 hours. bacterial colonies were gram stained by method of fawole and oso (2007); the bacterial cells that retained the purple stain from the crystal-violet staining were referred to as gram positive and those that showed pink from the safranin staining were referred to as gram negative. each was cultured in nutrient broth overnight at 30 oc until log phase (optical density at 600 nm) and used for molecular characterization. identification and molecular characterization of bacterial isolates total dna was extracted from 2 ml of each grown bacterium. the cells were pelleted by centrifuging at 12,000 rpm for 2 min. the pellet was mixed with tris-edta (te) buffer and treated with sodium dodecyl sulphate for 10 min. isopropanol (600 µl) was added and centrifuged at 10,000 rpm for 10 min and decanted. the pellet formed was dissolved in 0.1 ml of a te buffer. the dna was quantified using nanodrop machine at 260 nm and 280 nm. the 16s rrna gene was amplified using primers: (i) 5ʼ agagtttgatcctggctcag 3ʼ and (ii) 5ʼ gacgggcrgtgwgtrca 3ʼ. the polymerase chain reaction (pcr) mix contained 10x buffer, 100 mm dntps, 2.52 m mgcl2, 2u taq dna polymerase, 1.0 μl of each primers, 2.0 μl of the extracted dna and sterilized distilled water to make a final volume of 25 ml. the mixture was subjected to amplification on pcr machine with the following parameters: initial 94 °c 3 min, followed by 25 cycles run in a thermal cycler, each comprising of 1 min at 94 °c, 1 min at 94 °c and 1.5 min at 94 °c. the final denaturation was at 94 °c for 10 min. about 2.0 μl of the pcr product was subjected to 1.0 % (w/v) agarose-gel electrophoresis at 90 v for about 30 min, and the resulted gel was observed under uv transilluminator and photographed. 1 µl of the amplified dna was sequenced on the applied biosystems genetic analyzer at facility in international institute of tropical agriculture (iita), ibadan nigeria. the dna sequence of the pcr product of the 16s rrna gene of each bacterium was subjected to homology analysis using blastn program of the national center for biotechnology information (ncbi). preparation of bacterial inoculum nine rhizobia that grew equal to and above 0.600 optical density in the presence of crude oil at the end of the 7 days evaluation experiment on crude oil degradative capability were used. the rhizobia were inoculated into 5 ml nutrient broth and incubated at 28 oc for 24 h. it was centrifuged at 4,000 rpm for 5 min. the cells were re-suspended in 5 ml sterile distilled water. this constituted the bacterial inoculum. evaluation of crude oil degradative capability of the test bacteria the mineral salt medium (bushnell-hass) was prepared. the medium was allowed to cool and crude oil was sterilized using 0.45 μm millipore filter and added to make final crude oil concentration of 1 %, 3 % and 5 % volumes of 1, 3 and 5 ml. uninoculated msm broth containing the crude oil served as the control. the broth was inoculated with each rhizobium species and incubated at 28 ± 2 °c for 7 days. culture tubes were agitated at 4,000 rpm daily using shaking incubator to provide adequate oxygen for the bacteria to grow. during the incubation the optical density of each cultured rhizobium was read daily with spectrophotometer at 600 nm (boboye et al. 2010). statistical analysis the data were taken in triplicates and expressed as mean ± standard deviation (sd) using microsoft excel. results the concentration of dna from rhizobia samples ranged between 186.6 to 277.6 ng.µl-1. the 260/280 ranged between 1.81 to 1.97, and indicated the quality of dna of all the rhizobia were good. the 16s rrna fragment was amplified by pcr on dna from rhizobia. gel separation nova biotechnol chim (2020) 19(1): 80-88 84 fig. 2. the pcr products amplified on 16s rrna gene of rhizobia isolated from pigeon pea (isolates a1-a6) and pinto bean (isolates b1-b3). lane m represents the dna ladder. confirmed the presence of a 1500 bp fragment (fig. 2). arrangement of the nucleotides in the pcr product from the dna extracted from each of the rhizobia isolated from pigeon pea and pinto beans. the number of each base in the total nucleotides in each rhizobium varied from one test bacterium to the other with total nucleotides ranging from a1 to a6 (403 to 551) bases for bacterium isolated from pigeon pea. the molecular identities of the bacteria isolated from pigeon pea and pinto beans are shown in table 1. among the bacteria isolated from pigeon pea were three species bradyrhizobium and three species of rhizobium. in pigeon pea, the percentage identity was lowest for isolate coded a6 (96 %) and highest (100 %) for bacteria a1, a, a3, and a5. the molecular identities of the bacteria associated with pinto beans were two species of bradyrhizobium and one species of rhizobium. the percentage identity for the bacteria isolated was the lowest for isolate b2 (99 %) and highest (100 %) for b1 and b3. the phylogenetic tree (fig. 3) shows the fig. 3. a phylogenetic tree of the crude oil degrading rhizobia identified by molecular analysis. relatedness of the bacteria isolated from pigeon pea and pinto beans. the phylogenetic tree of the isolate has three clades. from the first clade, there are three strains of rhizobium leguminisarum (wsm2304, n871, and n741) which are closely related. in the second clade bradyrhizobium japonicum e109 is closely related to bradyrhizobium diazoefficiens usda 110 than rhizobium leguminisarum bihb 1217 but shares the same ancestors. bradyrhizobium diazoefficiens usda 122 and bradyrhizobium japonicum semia5079 are closely related but shares a distance relationship with the other identified rhizobia bradyrhizobium japonicum n61 in the third clade. we studied the ability of the test rhizobia to grow in the presence of crude oil, supplied to growth media at concentrations 1 %, 3 % and 5 % (v/v). the od values were taken as measure of growth thus crude oil degradation. our data showed that crude oil differently affected the growth of individual isolates from pigeon bean (fig. 4) as well as from pinto beans (fig. 5). generally, table 1. molecular identities of bacteria associated with pigeon pea and pinto beans. rhizobia code identified bacteria identity [%] accession number a1 bradyrhizobium diazoefficiens usda122 100 cp013127.1 a2 rhizobium leguminosarum wsm2304 100 cp01193.1 a3 bradyrhizobium japonicum n61 100 cp017637.1 a4 rhizobium leguminisarum n741 99 cp013595.1 a5 rhizobium leguminosarum bihib 1217 100 cp022665.1 a6 bradyrhizobium japonicum e109 96 cp010313.1 b1 rhizobium leguminisarum n871 100 cp013590.1 b2 bradyrhizobium diazoefficiens usda 110 99 cp011360.1 b3 bradyrhizobium japonicum semia5079 100 cp007569.1 a1-a6 represent bacteria isolate from pigeon pea. b1-b3 represent bacteria isolate from pinto beans https://www.ncbi.nlm.nih.gov/nucleotide/1109364050?report=genbank&log$=nucltop&blast_rank=2&rid=yu3w0tku015 https://www.ncbi.nlm.nih.gov/nucleotide/1037415903?report=genbank&log$=nucltop&blast_rank=4&rid=yu43gcnc014 https://www.ncbi.nlm.nih.gov/nucleotide/1229052029?report=genbank&log$=nucltop&blast_rank=2&rid=yu43gcnc014 https://www.ncbi.nlm.nih.gov/nucleotide/736032532?report=genbank&log$=nucltop&blast_rank=4&rid=yu3w0tku015 https://www.ncbi.nlm.nih.gov/nucleotide/1037409780?report=genbank&log$=nucltop&blast_rank=5&rid=yu43gcnc014 https://www.ncbi.nlm.nih.gov/nucleotide/1027477049?report=genbank&log$=nucltop&blast_rank=3&rid=yu3w0tku015 https://www.ncbi.nlm.nih.gov/nucleotide/627779227?report=genbank&log$=nucltop&blast_rank=5&rid=yu3w0tku015 nova biotechnol chim (2020) 19(1): 80-88 85 fig. 4. growth of test rhizobia isolated from pigeon pea in presence of crude oil in the medium (1 – 5 % w/v). data represent means ± sd (n = 3). a = b. diazoefficiens usda122; b = r. leguminosarum wsm2304; c = b. japonicum n61; d = r. leguminosarum n741; e = r. leguminosarum bihib1217; f = b. japonicum e109. addition of 1 % (w/v) crude oil promoted rhizobia growth the most, followed by 3 % and 5 % oil concentration (fig. 4 and 5). the effect of oil was significant at p < 0.05. best growth was observed for b. diazoefficiens usda122 at 1 % (w/v) crude oil contamination (od 1.184 ± 0.05 on 7th day). in contrast, most inhibited was the growth of r. leguminosarum n741 (od 0.372 ± 0.02 on 7th day) by 5 % (w/v) crude oil in growth media. relatively flat growth curve was obtained for the rhizobia isolate b. diazoefficiens usda122 (fig. 4a). of the rhizobia isolated from pigeon pea plants, b. japonicum e109 had the highest growth rate (od 0.899 ± 0.06 on 7th day), while the lowest one was obtained for b. diazoefficiens usda122 on day 6 (fig. 4a and f). moreover, among the rhizobia isolated from pinto beans plants, b. japonicum semia5079 had the highest od (1.107 ± 0.05 on 7th day) when compared to the od obtained for r. leguminosarum n871 (fig. 5a and c). the increase in optical density of each test bacteria in the presence of crude oil indicates utilization of the oil for growth and degradation of the test hydrocarbon. the results of this study showed that each test bacteria could survive and utilize crude oil at the percentages investigated. discussion in bacteria, 16s rdna forms core conserved region with about 1,500 base pair. the region is employed for bacteria identification. this correlated with data obtained by mohamed et al. (2014) that 16s rrna region is involved in bacterial identification. studies have shown that endophytic bacteria have a better capacity to enhance petroleum hydrocarbon (phc) phytoremediation than rhizosphere or soil bacteria (weyens et al. 2010; yousaf et al. 2011). cultivable endophytic bacteria have been isolated from various plants species which include maize (gutierrez-zamora and martinez-romero 2001), wheat (larran et al. 2002), sugar cane (loiret et al. 2004), and mimosa pudica (pandey et al. 2005). a molecular characterization study on endophytic nova biotechnol chim (2020) 19(1): 80-88 86 fig. 5. growth of test rhizobia isolated from pinto beans in presence of crude oil in the medium (1 – 5 % v/v). data represent means ± sd (n = 3). a = r. leguminosarum n871; b = b. diazoefficiens usda110; c = b. japonicum semia5079. bacteria isolated from leguminous plants nodules and roots cultivated in the rhizosphere soil of acacia reported the presence of ensifer meliloti (sinorhizobium meliloti) in nodules of chickpea and common bean as 40.9 %, rhizobium sp. in nodules and roots of common bean and lentil as 31.8 %, and enterobacter sp. isolated in roots of faba bean, common bean, chickpea and lentil as 27.3 % (taoufiq et al. 2018). the increasing in optical density of each test bacteria in the presence of crude oil indicates the survival and utilization of the oil for growth and degradation of the test hydrocarbon. the bacteria have strains that utilized the crude oil as a sole source of carbon and energy since there was no other source of carbon and energy. this rhizobium responded to crude oil and showed increased optical density at the end of the seventh day. this means that the genes encoding each rhizobium were not inhibited by the crude oil hydrocarbons. these microorganisms are likely to possess enzymatic capacity to degrade the crude oil (olukunle and boboye 2013). thus, increase in the hydrocarbon utilizing bacterial population conforms to the data from the work done by lawson et al. (2012) and ataikiru et al. (2018). lawson et al. (2012) have reported that introduction of phc into the soil encourages high microbial biomass. joshi and pandey (2011) also suggested that the percentage of petroleum utilizing bacteria in a particular environment appears to be an index of the presence of hydrocarbons in the environment. the bacteria might have broken the oil down into simple carbon compounds that are used to make the sugars, fat and proteins needed for growth and energy production ultimately the by-product become carbon dioxide and water (okerentugba and ezeronye 2003). this finding agrees with the report of salam et al. (2011) that both gram-negative and positive bacteria have been implicated in the mineralization of hydrocarbon pollutant. the dominance of gramnegative bacteria in the samples agrees with that of kaplan and kilts (2004), that gram-positive bacteria, when detected in bioremediation, are never diverse or dominant. the high moisture content of the oils might have provided the moisture necessary for bioactivity thus supporting the growth and survival of these microbes. reduction in od of the oil medium at different incubation times could be due to possible disappearance of the crude oil in the medium due to exhaustion of the hydrocarbon which served as the carbon source for various metabolic activities. the difference in oil degradation ability of the rhizobia could be attributed to the difference in their genetic abilities in utilizing hydrocarbons as substrate vis-à-vis absorption, oxidation and environmental factors. similar suggestion was made by majid et al. (2008) and onuoha et al. (2014) when they studied spent oil degradation with some bacteria. rhizobia used in this research could play a vital role in plant nutrient cycling and replenishment in the soil. these microbes are also important for hydrocarbon degradation in soil. their role includes the degradation and biotransformation of petroleum compounds into simple harmless compounds (oluyege et al. 2011). biodegradation by microbes is the key removal process of hydrocarbons which is controlled by physicochemistry, environmental conditions, nova biotechnol chim (2020) 19(1): 80-88 87 bioavailability and the presence of catabolically active microbes (stroud et al. 2007). one of the major challenges with phytoremediation is that it takes long time. large-scale clean-up approach was applied to tackle the deepwater horizon oil spill in the usa, by direct delivery of a dispersant that boosted the natural biodegradation of the oil (atlas and hazen 2011). also, studies have shown that a field-scale process of contaminated soil bioremediation is possible (pelaez et al. 2013; pizarro-tobías et al. 2015). conclusion in conclusion, this work has impacted the understanding and effectiveness of the crude oil degradative abilities of the isolated and tropical rhizobia species using their compactible legumes to degrade crude oil in soil. the use of plantassociated bacteria such as rhizobia to degrade toxic synthetic organic compounds in environment may provide an efficient, economic and sustainable bioremediation technology. this laboratory scale research study can also be applied on a large-scale study thus, are environmentally friendly and have been observed to promote the biodegradation of hydrocarbons. the results of this study showed that each test bacteria could survive and utilize crude oil at the percentages investigated. thus, they will be useful to fixed nitrogen when added to (bioaugmentation and bio-stimulation strategies) the root of the pigeon peas and pinto beans planted in a crude oil polluted soil. conflict of interest the authors declare that they have no conflict of interest. references ataikiru tl, philip o, chinedu c (2018) bioremediation of bonny light crude oil polluted soil by bioaugmentation using yeast isolates (candida adriatica zim 2468 and candida taoyuanica mya-4700). int. j. environ. res. public health 5: 52-61. atlas rm, hazen tc (2011) oil biodegradation and bioremediation: a tale of the two worst spills in u.s. history. environ. sci. technol. 45: 6709-6715. boboye b, olukunle of, adetuyi fc (2010) degradative activity of bacteria isolated from hydrocarbon-polluted site in ilaje, ondo state, nigeria. afr. j. microbiol. res. 4: 2484-2491. cameron ag (2003). lab agnote no. c38, lab agnote, new york, 77 p. daniel ie, nna pj (2016) total petroleum hydrocarbon concentration in surface water of cross river estuary, niger delta, nigeria. asian j. environ. ecol. 1: 1-7. diplock ee, mardlin dp, killham ks, paton gi (2009) predicting bioremediation of hydrocarbons: laboratory to field scale. environ. pollut. 157: 1831-1840. eremrena, po, akonye la (2013) growth and biochemical performance of cassava-manihot esculenta crantz to crude oil polluted soil amended with centrosema pubescens benth and npk. j. appl. sci. environ. manag. 17: 195-201. fawole mo, oso ba (2007) laboratory manual of microbiology, spectrum book limited ibadan, nigeria, pp. 15-22. gkorezis p, daghio m, franzetti a, van hamme jd, sillen w, vangronsveld j (2016) the interaction between plants and bacteria in the remediation of petroleum hydrocarbons: an environmental perspective. front. microbiol. 7: 1836. gutierrez-zamora m, martinez-romero e. 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animesh-fet@sust.edu nova biotechnologica et chimica growth and quality parameters of tea (camellia sinensis) mediated by arbuscular mycorrhizal fungi animesh sarkar1,, md. musfiqur rahman1, jayanto kumar sarkar2, mahabub alam1 and md. h. rashid3 1department of food engineering and tea technology, shahjalal university of science and technology, sylhet 3114, bangladesh 2department of environmental science and technology, saitama university, 255 shimo-okubo, sakura-ku, saitama 338-8570, japan 3department of agronomy, bangladesh agricultural university, mymensingh 2202, bangladesh article info article history: received: 14th april 2020 accepted: 16th september 2020 keywords: antioxidant arbuscular mycorrhizal fungi camellia sinensis chlorophyll iaa polyphenol abstract arbuscular mycorrhizal fungi (amf) inoculation not only increases the growth but also improves the quality of many commercial plants. tea (camellia sinensis) plants were grown on different growth medium (with and without amf inoculation) and the chemical properties of the leaves were assayed and compared. the growth media were sterilized soil with amf, sterilized soil, natural soil inoculated with amf, natural soil, and natural soil in natural condition with amf. the highest root colonization (23 %) was found in tea plants grown on natural soil with amf, whereas no colonization was found in the sterilized soil treatment. the highest level of leaf chlorophyll-a (2.74±0.06 μg.ml-1), chlorophyll-b (1.77±0.03 μg.ml-1) and carotenoid (0.35±0.01 μg.ml-1) contents were found in tea plants grown on natural soil under natural condition with amf. the highest polyphenol concentration (64.46 mg.l-1) was found in natural soil inoculated with amf whereas the lowest (38.09 mg.l-1) was recorded in sterilized soil. the highest contents of tannin (30.34 mg.ml-1) and reducing sugar (46.61 mg.l-1) were recorded in plants grown on natural soil under natural condition with amf and the lowest values (21.22 mg.ml-1, 33.16 mg.l-1, respectively) in sterilized soil treatment. though antioxidant properties (% scavenging effect) did not differed due to treatments, the highest iaa (indole-3-acetic acid) concentration (3.16 μg.ml-1) was recorded in tea plants grown on natural soil under natural condition with amf. the study concludes that amf inoculation improves the quality of tea leaves.  university of ss. cyril and methodius in trnava introduction tea (camellia sinensis) plants grow well on acidic soils. these soils show low values of ph, high al concentration, and negligible availability of nutrients. low quantity of p is one of the major limiting factors for the productivity of tea plants due to low natural content and high p fixation capacity of acidic soil. plants grown on acidic soils often encounter relatively severe mineral stress; such effects include toxic (al and mn) to deficiency symptoms (singh et al. 2010). excess al is especially damaging to root growth and overall development for plants growing in acidic soils (miyasaka et al. 1991). amf-root symbiosis may help alleviate some of the problems mailto:animeshsarkarbau@gmail.com mailto:animesh-fet@sust.edu nova biotechnol chim (2020) 19(2): 232-239 233 that the plants encounter when grown in acidic soils (li et al. 1991) including al and/or mn toxicity. mycorrhizal fungi functions in promoting plant growth across floodplain chronosequences were described by sarkar et al. (2015b). but their functions in promoting plant growth are unknown across hilly region having low nutrients content. amf can provide nutrient to plants in those regions. response to mycorrhizal inoculation is linked with the level of soil fertility and it is well know that p is the most influential element in amf development and efficiency (sarkar et al. 2016). in p-deficient soils, the yields of agricultural and field crops under field and greenhouse conditions were observed to be highly dependent on their mycorrhizal state (sarkar et al. 2015a). there is a diversification of arbuscular mycorrhizal fungi in tea plants in uttarakhand state, india (gupta and sharma 2010). we found seasonal colonization of arbuscular mycorrhiza fungi in the roots of camellia sinensis (tea) in different tea gardens of india (sharma et al. 2013). amf increases supply of plant nutrients by obtaining sources of nutrients which would usually not be accessible to plants (tarafdar and marschner 1994). some ericoid fungi have the ability to break down phenolic compounds in soils that can interfere with absorption of nutrients (bending and read 1997). mycorrhizas can cause changes in root architecture, vascular tissue, etc. to develop in size (hetrick et al. 1994). such alliances between fungus-plants boost plant growth and enhance root production. arbuscular mycorrhiza (am) plays a vital role on enhancing the plant growth and yield by increasing the supply of phosphorus to the host plant. the mycorrhizal beneficiaries can include increased yield, accumulation of nutrients and/or reproductive success (lewis and koide 1990). amf have a great effect on growth and nutrients assimilation of coarse and low nutrient soil (sarkar et al. 2015a). fungal hyphae can play a significant role in nutrient cycling through the acquisition of nutrients from saprophytic fungi (lindahl et al. 2007). microbial action in the rhizosphere is a main consideration that decides the accessibility of supplements to plants and has an essential impact on plant wellbeing and efficiency. the impact of amf inoculation on the growth and development of many crops and forest species are well documented. however, reports on the effects of amf on growth and quality of tea are very scanty. the goal of this study was to evaluate the role of arbuscular mycorrhizal fungi in the growth and quality of tea. experimental soil and plant propagate collection soil sample was collected from 0.61 m depth from the top of a tillah (small mountain) located at the shahjalal university of science and technology (sust) campus (24° 55’ 31.89”n and 91° 50’ 12.34”e) of bangladesh. plant propagates of tea variety (bt2) was collected from malnicherra tea estate, sylhet. commercial biofertilized having spore of arbuscular mycorrhizal fungus named ‘serakinkon’ was bought from central glass company, tokyo, japan. soils collected from different points on tillah were composited to make a homogenous soil pool and the required amount of soil form the composite bulk was used for the experiment. the properties of the collected soils were homogenous and there were no significant differences between the individual soil samples and the composite soil in terms of soil particle size small stone like, ph, percentage of organic-matter content, total carbon (tc), total nitrogen (tn), total phosphorus (tp), available p concentrations, sulphur (s), potassium (k). the soil used for experiment had an average ph of 4.09 (highly acidic), organic-matter content 0.67 % (very low), available p 2.93 μg.g-1 soil, tn 0.039 % (very low), and sulphur 12.83 μg.g-1. soil that was used had low nutrient compare to optimum nutritional level having very low n, p, k, s and organic matter (om) contents, and highly acidic. experimental procedure and design the experiment was conducted in the form of pot culture in the experimental tea garden at shahjalal university of science and technology (sust) campus. the experiment consisted of 5 treatments viz., (i) sterilized soil inoculated with mycorrhiza (ss+am) in pot condition; (ii) sterilized soil (ss) in pot condition; (iii) natural soil inoculated with nova biotechnol chim (2020) 19(2): 232-239 234 mycorrhiza (ns+am) in pot condition; (iv) natural soil (ns) in pot condition; and (v) natural soil cultivated in ground soil inoculated with mycorrhiza (nss+am). the pots were placed in experimental tea garden and each treatment had three replications (repeated in space). each pot had diameter of 3.66 m and contained 6 kg of soil. for sterilizing, soil was autoclaved twice at 121 °c for 30 min to eliminate any indigenous amf. we used a commercial inoculant, serakinkon powder, which consists of 50 gigaspora margarita becker & hall spores per gram powder to inoculate the soils of the respective treatments (higo et al. 2010). twenty grams of serakinkon powder/kg soil was evenly mixed with soil before using in a pot. pots were watered regularly by tap water. after 105 day’s tea leaf are harvested. determination amf colonization in roots roots were collected from plants and washing was done by tap water. the roots were cut into 1 cm in size and boiled in 2.5 % (w/v) potassium hydroxide at 90 °c for 30 min followed by thoroughly washing with tap water. the root samples were acidified with 1 % (v/v) hcl for 24 h and then bleached in 10 % (v/v) hydrogen peroxide for 60 min. the bleached roots were boiled in 0.5 % (w/v) aniline blue at 90 °c for 30 min. finally, the roots were de-stained in glycerol and observed under microscope. the total number of colonization counted were divided by the number of view fields investigated (newman et al. 1981; plenchette et al. 1983; dodd and jeffries 1986). estimation of chlorophyll and carotenoids chlorophyll-a, chlorophyll-b and carotenoids contents of tea leaves were calculated using the method stated by wellburn (1994). tea leaves were weighed and grounded with a mortar and paste with a sufficient quantity of prechilled methanol (100 %). finely ground sample was filtered and made up to 50 ml in a standard volumetric measuring flask using methanol and further diluted five times with methanol. absorbance of the diluted methanolic extract was read at 470, 653 and 666 nm using a uv-visible spectrophotometer (model-t60u, pg instruments limited, uk) against methanol as blank. determination of iaa leaves and roots of were cut off (1 g) using a scalpel, frozen immediately in a refrigerator and lyophilized. after homogenization, the lyophilized sample was subjected to cool extraction at –20 °c using 7.5 ml of beileski modified methanol/water/acetic acid mixture (75:20:5, v/v) for 12 h. the solvent used allows the enzymatic degradation of phytohormones to be blocked without extracting large quantities of lipids. solid was separated by centrifugation for 15 min at 8,000 rpm and extracted second time for 30 min. one ml of supernatants mixed with one drop of orthophosphoric acid and 2 ml of salkowski’s reagent (50 ml, 35 % (v/v) perchloric acid and 1 ml, 0.5 m ferric chloride). we measured iaa content by spectrophotometric method at 530 nm (lwin et al. 2012). estimation of polyphenols and reducing sugars tea leaves comprising two leaves and an apical bud (approx. 1 g) were ground well with addition of 100 % ethanol. the contents were filtered and the filtrate was made up to 50 ml with 100 % ethyl alcohol. the alcoholic extract was used for estimation of polyphenols and reducing sugars. an aliquot of 1 ml alcoholic extract was taken in a volumetric flask and diluted to 50 ml with distilled water. two milliliters of diluted extract was mixed with 4 ml of 1:1 folin-ciocalteu reagent, 1 ml water and 2 ml of 35 % (w/v) sodium carbonate. the contents were further mixed with distilled water to make 10 ml solution, and the mixture was vigorously shaken and allowed to stand still for 30 min. using uv-visible spectrophotometer, the absorbance of the formed blue color was taken at 700 nm against the blank reagent. the quantity of polyphenols present in tea leaves was measured using the standard calibration curve derived from known gallic acid concentrations (10 to 50 mg.l-1), and the results were expressed as the equivalent percentage of gallic acid (dev-choudhury and goswami 1983). nova biotechnol chim (2020) 19(2): 232-239 235 reducing sugar content was estimated following the methods suggested by hedge et al. (1962). an aliquot of 2 ml of the alcoholic extract was diluted to 10 ml with distilled water. the diluent (1 ml) was taken in a test tube and incubated with 4 ml of ice cold acidified anthrone reagent (0.2 %, w/v in concentrated sulphuric acid). the contents were then incubated for 8 min in a boiling water bath and cooled down under running water to ambient temperature. the absorbance of the green color formed was taken in a uvvisible spectrophotometer at 630 nm against the reagent blank. percent reducing sugars (dextrose equivalents) present in tea samples was computed using the standard calibration curve where known concentrations of (+) dextrose were used to derive calibration curve. determination of total tannin content the tannins were determined using the folinciocalteu phenol reagent as reported by amorim et al. (2008). the sample extract (0.2 ml) was mixed with 8.3 ml of distilled water and 0.5 ml of folin-ciocalteu phenol reagent, held for 5 min at ambient temperature. after that 1 ml of sodium carbonate solution of 35 % was introduced. the mixture was well stirred, left for 20 min at ambient temperature and absorbance was taken at 725 nm. instead of sample, the blank was prepared with distilled water. determination of antioxidant properties fresh and infested tea leaves were collected and dried in a drier for 6 h at 35 °c and powdered. the leaf powders were extracted using 100 % methanol with continuous swirling for 1 h at room temperature. extracts were filtered with whatman filter paper while methanol was evaporated via rotary evaporator and finally, 50 mg.ml-1 concentration was made. the scavenging effects of samples for 1,1-diphenyl-2-picrylhydrazyl (dpph) radical were monitored according to the method of the previous report by yen and chen (1995). briefly, samples of different concentrations (50 mg.ml-1, 10 mg.ml-1, 2 mg.ml-1, 0.4 mg.ml-1 and 0.08 mg.ml-1) were taken and the absorbance of each concentration of test sample was measured at 517 nm. 2.0 ml aliquot of test sample (in methanol) was added to 2.0 ml of 0.16 mm dpph methanolic solution. the mixture was vortexed for 1 min and then left to stand at room temperature for 30 min in the dark, and its absorbance was read at 517 nm. the ability to scavenge the dpph radical was calculated using the following equation (eq. 1): statistical analysis the results were analyzed statistically using the spss (version 16.0) program (ibm, usa) to determine the average value and standard deviation of the mean. differences with p-values < 0.05 were considered statistically significant. results amf colonization in roots among 5 treatments, colonization (%) was maximum in natural soil inoculated with mycorrhiza (23 %). in sterilized soil (ss), no mycorrhizal colonization was found while 13 % colonization was found in ss+am treatments. colonization was less than 5 % in natural soil (ns) while it was 7.5 % in natural soil inoculated with am (table 1). table 1. amf colonization in tea root under different treatments. treatment† colonization [%] ss+am 13.0 ss 0.0 ns+am 23.0 ns < 5.0 nss+am 7.5 †sterilized soil inoculated with mycorrhiza (ss+am) in pot condition; sterilized soil (ss) in pot condition; natural soil inoculated with mycorrhiza (ns+am) in pot condition; natural soil (ns) in pot condition, and natural soil under natural condition inoculated with mycorrhiza (nss+am). chlorophyll and carotenoids contents in leaves photosynthetic pigments, such as chlorophyll and (1) nova biotechnol chim (2020) 19(2): 232-239 236 table 2. chlorophyll and carotenoids contents in tea leaves under different treatments. treatments† chlorophyll a [μg.ml-1] chlorophyll b [μg.ml-1] carotenoids [μg.ml-1] ss+am 1.53±0.01 1.36±0.02 0.21±0.01 ss 0.85±0.05 0.68±0.03 0.12±0.06 ns+am 1.30±0.05 1.29±0.02 0.10±0.01 ns 0.63±0.01 0.71±0.01 0.04±0.00 nss+am 2.74±0.06 1.77±0.03 0.35±0.01 †sterilized soil inoculated with mycorrhiza (ss+am) in pot condition; sterilized soil (ss) in pot condition; natural soil inoculated with mycorrhiza (ns+am) in pot condition; natural soil (ns) in pot condition, and natural soil under natural condition inoculated with mycorrhiza (nss+am). carotenoids, of different treatment were evaluated (table 2). the highest amount of chlorophyll a and b was found in leaves of tea plant growing under treatment nss+am, while the lowest amount of the these pigments were estimated in tea plants grown under ns and ss treatments, respectively. the highest value of carotenoid content was found in tea plants growing under treatment nss+am, however, plants growing under natural condition (ns) produced the lowest carotenoid content in leaves. iaa concentration amf treatment had significant effect on iaa concentrations in tissues of tea plant (fig. 1). the highest amount of iaa was found in treatment ns+am (3.16 μg.ml-1) and the lowest amount was fig. 1. iaa concentrations in tissues of tea plant under different treatments. sterilized soil inoculated with mycorrhiza (ss+am) in pot condition; sterilized soil (ss) in pot condition; natural soil inoculated with mycorrhiza (ns+am) in pot condition; natural soil (ns) in pot condition, and natural soil under natural condition inoculated with mycorrhiza (nss+am). error bars indicate ± sd of the mean. found in treatment ss (1.23 μg.ml-1) (fig. 1). polyphenol content in leaves polyphenol contents in leaves of tea grown under different treatment were determined (fig. 2). it was significantly affected by amf treatment. the highest concentration of polyphenol was found on ns+am (64.46 mg.l-1) followed by nss+am (59.32 mg.l-1). the lowest concentration of the same was found in ss (38.09 mg.l-1) (fig. 2). tannin content in leaves tannin concentration in tea leaf was significantly affected by amf treatment (fig. 3). the highest concentration of tannin found in nss+am fig. 2. polyphenol concentrations in tea leaves grown under different treatments. sterilized soil inoculated with mycorrhiza (ss+am) in pot condition; sterilized soil (ss) in pot condition; natural soil inoculated with mycorrhiza (ns+am) in pot condition; natural soil (ns) in pot condition, and natural soil under natural condition inoculated with mycorrhiza (nss+am). error bars indicate ± sd of the mean. nova biotechnol chim (2020) 19(2): 232-239 237 fig. 3. tannin concentrations in tea leaves grown under different treatments. sterilized soil inoculated with mycorrhiza (ss+am) in pot condition; sterilized soil (ss) in pot condition; natural soil inoculated with mycorrhiza (ns+am) in pot condition; natural soil (ns) in pot condition, and natural soil under natural condition inoculated with mycorrhiza (nss+am). error bars indicate ± sd of the mean. (30.34±0.51 mg.ml-1) and the lowest concentration was found in ss+am (21.22±0.54 mg.ml-1). reducing sugars in leaves amount of reducing sugars in examined tea leaves ranged from 33.16 ppm to 46.61 ppm. it was significantly affected by amf inoculation. we got the highest reducing sugar level in ns+am (46.61±0.77 mg.l-1). lowest amount among was found in ss (33.16±0.65 mg.l-1) (fig. 4). antioxidant properties of leaves the scavenging effect of tea leaves on dpph radicals varied with different treatments ranged fig. 4. reducing sugar concentrations in tea leaves grown under different treatments. sterilized soil inoculated with mycorrhiza (ss+am) in pot condition; sterilized soil (ss) in pot condition; natural soil inoculated with mycorrhiza (ns+am) in pot condition; natural soil (ns) in pot condition, and natural soil under natural condition inoculated with mycorrhiza (nss+am). error bars indicate ± sd of the mean. from 89.04 % to 94.97 % in 0.2 mg.ml-1; 95.33 % to 97.75 % in 0.4 mg.ml-1 and 98.91 % to 99.78 % in 0.6 mg.ml-1 (table 3). maximum amount was found in ns+am treatment in most of the cases though minimum amount was varied. discussion effects of amf on colonization and growth influencing parameters mycorrhizal colonization differs from 0 to 90 % in different plants (khan 1978) and mycorrhizal colony exists in soil naturally (liu et al. 2016). in the present experiment, mycorrhizal colonization increased in tea plants with the inoculation of am fungi in pot condition but the colonization level table 3. antioxidant properties of tea leaves grown under different treatments. treatments† scavenging effect [%] of different concentrations of tea leaf ‡ 0.2 mg.ml-1 0.4 mg.ml-1 0.6 mg.ml-1 ss+am 89.04±1.29 95.33±2.21 98.91±1.78 ss 94.94±1.35 97.75±0.05 98.69±0.08 ns+am 94.97±0.71 97.75±0.19 98.87±0.02 ns 88.91±4.95 96.30±0.11 98.06±0.07 nss+am 93.14±2.11 96.78±0.06 99.78±1.21 †sterilized soil inoculated with mycorrhiza (ss+am) in pot condition; sterilized soil (ss) in pot condition; natural soil inoculated with mycorrhiza (ns+am) in pot condition; natural soil (ns) in pot condition, and natural soil under natural condition inoculated with mycorrhiza (nss+am); scavenging effect on ‡1,1-diphenyl-2-picrylhydrazyl (dpph) radical. nova biotechnol chim (2020) 19(2): 232-239 238 was comparatively low from expected level in natural condition. one of the possible cause was insufficient time of plant growth. to get average 60 – 80 % of colonization it requires six to eight month in natural condition (sharma et al. 2013). but in artificial application mycorrhizal colony varies within 23 – 28 % (sarkar et al. 2015a) and response can be found in 90 – 120 days after plantation in pot condition though it depends on plants, nutritional status, types of mycorrhiza etc. the present experimental result shows that tea plants inoculated with am fungi had better growth. in chlorophyll test, the concentration of chlorophyll was higher in treatment nss+am as it was inoculated with mycorrhiza. at the same time, indigenous am fungi strains were existed which also performed in colony development. previous study documented the same result (sarkar et al. 2015a). in addition, mycorrhiza uptake nutrients such as phosphorus, sulphur, nitrogen from soil so the concentration of chlorophyll in amf inoculation was greater than uninoculated plant while phosphorus is responsible for change in chlorophyll concentration in leaves (zuccarini 2007). iaa is an important parameter in leaves and roots that indicates the growth hormone auxin present in leaves and roots. higher amount of iaa indicates high rate of growth in plant. among the treatments, we found that ns+am had higher amount of iaa. both amf commercial and indigenous helps to achieve higher amount of auxin. natural soil (ns) and natural soil in soil condition (nss+am) also show interesting results in this way. but ss shows lower result that indicates poor growth rate. this results are in agreement with previous works where amf was reported to change iaa concentration in tissue and affect growth and quality of tea leaves (kornberg and krebs 1957). effect of amf on tea quality parameter polyphenol content was increased with the inoculation of amf and the response was higher in pot condition. depending on the application of amf, the concentration of polyphenol changes as amf uptake nitrogen, phosphorus like nutrient from soil (chabeli et al. 2008) which can support the present experimental results. tannin is important parameter for color development of tea. tannin content in nss+am treatment showed the highest value where sterilized sample showed lower result as initially present mycorrhiza and other important essential microorganism were killed by sterilization. we know that amf uptakes organic nitrogen from soil (whiteside et al. 2012) that is responsible for change in tannin concentration in leaves (hoflandzijlstra and berendse 2009). in the case of reducing sugar, the natural soil treatment inoculated with mycorrhiza showed higher rate of reducing sugar content. the amount of reducing sugar changed by the effect of mycorrhizal application or nonapplication. the amf applied treatment contained much amount of reducing sugar compare to other treatments (singh et al. 2010). but for antioxidant content, only a little differences shown by inoculated or non-inoculated treatments. conclusion the study was carried out to evaluate the effect of amf on quality parameters of tea (camellia sinensis). tea plants with amf application had higher root colonization than the non-inoculated plants. the amf colonization significantly and positively affected most of the quality parameters of the tea leaves. polyphenol content and antioxidant properties of tea leaves can be increased by applying arbuscular mycorrhizal fungi. amf application also increased the leaf pigment content. these results imply that application of amf will be much effective for tea cultivation and the load of chemical fertilizers can be reduced by its application. acknowledgements the authors would like to thank the technical staff of food engineering and tea technology laboratory, shahjalal university of science and technology university, bangladesh for their contribution to this work. funding the authors are thankful to ministry of science and technology, bangladesh (sl. 90) for financial support. nova biotechnol chim (2020) 19(2): 232-239 239 conflict of interest the authors declare that they have no conflict of interest. references amorim el, nascimento je, monteiro jm, peixoto sobrinho t, araújo ta, albuquerque up (2008) a simple and accurate procedure for the determination of tannin and flavonoid levels and some applications in ethnobotany and ethnopharmacology. funct. ecosys. commun. 2: 88-94. bending gd, read dj (1997) lignin and soluble phenolic degradation by ectomycorrhizal and ericoid mycorrhizal fungi. mycol. res. 101: 1348-1354. chabeli p, mudau fn, mashela p, soundy p (2008) effects of nitrogen, phosphorus and potassium nutrition on seasonal tannin content of bush tea (athrixia phyliciodes dc.). s. afr. j. plant soil. 25: 79-83. dev-choudhury m, goswami m (1983) a rapid method for determination of total polyphenolic matters in tea camellia sinensis l. two bud. 30: 59-61. dodd j, jeffries p (1986) early development of vesiculararbuscular mycorrhizas in autumn-sown cereals. soil biol. biochem. 18: 149-154. gupta rk, sharma c (2010) diversity of arbuscular mycorrhizal fungi in camellia sinensis in uttarakhand state, india. afr. j. biotechnol. 9. hedge j, hofreiter b, whistler r (1962) carbohydrate chemistry. 17. in whistler rl, be miller jn (eds.), academic press, new york. hetrick b, wilson g, figge d (1994) the influence of mycorrhizal symbiosis and fertilizer amendments on establishment of vegetation in heavy metal mine spoil. environ. pollut. 86: 171-179. higo m, isobe k, kang dj, ujiie k, drijber ra, ishii r (2010) inoculation with arbuscular mycorrhizal fungi or crop rotation with mycorrhizal plants improves the growth of maize in limed acid sulfate soil. plant prod. sci. 13: 74-79. hofland-zijlstra jd, berendse f (2009) the effect of nutrient supply and light intensity on tannins and mycorrhizal colonisation in dutch heathland ecosystems. plant ecol. 201: 661-675. khan a (1978) vesicular‐arbuscular mycorrhizas in plants colonizing black wastes from bituminous coal mining in the illawarra region of new south wales. new phytol. 81: 53-63. kornberg h, krebs eh (1957) synthesis of cell constituents from c2-units by a modified tricarboxylic acid cycle. nature 179: 988-991. lewis j, koide r (1990) phosphorus supply, mycorrhizal infection and plant offspring vigour. funct. ecol. 4: 695-702. li xl, marschner h, george e (1991) acquisition of phosphorus and copper by va-mycorrhizal hyphae and rootto-shoot transport in white clover. plant soil 136: 49-57. lindahl bd, ihrmark k, boberg j, trumbore se, högberg p, stenlid j, finlay rd (2007) 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growth and quality parameters of tea [camellia sinensis (l.) o. kuntze] through inoculation with arbuscular mycorrhizal fungi in an acid soil. biol. fertil. soils. 46: 427-433. tarafdar j, marschner h (1994) phosphatase activity in the rhizosphere and hyphosphere of va mycorrhizal wheat supplied with inorganic and organic phosphorus. soil biol. biochem. 26: 387-395. wellburn ar (1994) the spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. j. plant physiol. 144: 307-313. whiteside md, digman ma, gratton e, treseder kk (2012) organic nitrogen uptake by arbuscular mycorrhizal fungi in a boreal forest. soil biol. biochem. 55: 7-13. yen g-c, chen h-y (1995) antioxidant activity of various tea extracts in relation to their antimutagenicity. j. agric. food chem. 43: 27-32. zuccarini p (2007) mycorrhizal infection ameliorates chlorophyll content and nutrient uptake of lettuce exposedto saline irrigation. plant soil environ. 53: 281287. nova biotechnol chim (2019) 18(2): 166-178 doi: 10.2478/nbec-2019-0019  corresponding author: karimmoussaceb@yahoo.fr nova biotechnologica et chimica treatment and remediation by the stabilization/solidification process based on hydraulic binders of soil contaminated by heavy metals rachid sahnoune and karim moussaceb laboratoire de technologie des matériaux et de génie des procédés (ltmgp), faculté de technologie, université a/mira bejaia, route targa-ouzemour, bejaia 06000, algeria article info article history: received: 21st may 2019 accepted: 20th november 2019 keywords: contamination heavy metals hydraulic binders remediation soils stabilization/solidification abstract nature and the environment are affected by various human industrial and/or urban discharges. remediation for this problem requires first and foremost an in-depth analysis and an overall characterization of the intrinsic properties of the pollutionreceiving environments. secondly it is necessary to predict in these environments the behavior of dangerous chemical species (here particularly heavy metals) in the long term. this study focuses mainly on a detailed characterization of 4 soil samples sampled in vicinity of wild dump-boulimat located 15 km west of the city of bejaia-algeria. the samples were characterised by atomic absorption spectrometry, x-ray diffraction, fluorescence x and infrared spectroscopy. the data showed high concentrations of metallic elements especially zn (2,651.8 mg.kg-1) and ni (163.44 mg.kg-1) in the soil samples. for their remediation, the stabilization/solidification (s/s) process with hydraulic binders appeared promising in reducing the polluting power of metal. this approach has considerably reduced the content of pollutants; 98 % removal was obtained for ni and 99 % for zn. the xrd analysis technique revealed the occurrence or absence of metallic elements in the crystallized phases.  university of ss. cyril and methodius in trnava introduction for a long time, waste and landfills have posed a serious problem leading to various environmental issues that industries have created, and developing several diseases including respiratory, dermatological or other. human is the main driver of technological and scientific development through these innovations that make life simplier and easier, however, he is the main author of pollution generation at the scale industrial (mining, steel, etc.) or domestic (civil) such as household waste, hospital waste (stuart et al. 1998; sefouhi et al. 2010; prasadini et al. 2014). wastes (solid, liquid, gas) produced are released into the environment in the form of landfills, which are mostly uncontrolled, and in the presence of water, air and in contact with the receiving environment, becomes an active source of pollution developing complex and toxic elements with high geochemical mobility (sefouhi et al. 2010), which are easy to transport and transfer (prasadini et al. 2014), contaminating all the environment of proximity (air, soil, waters), thus influencing the life of the individual by the development of several diseases (cutaneous, pulmonary, neurological, etc.) causing an imbalance in the chain of life (stuart et al. 1998; sefouhi et al. 2010; prasadini et al. 2014; belebchouche et al. 2014). a range of tests and experiments have been conducted in recent years on the analysis and characterization of waste, leaching and modeling bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc mailto:karimmoussaceb@yahoo.fr nova biotechnol chim (2019) 18(2): 166-178 167 of long-term waste behavior (bozkurt et al. 2000). accordingly, this work has two objectives. the first is characterization of the site hosting the wild discharge of the region of boulimat-bejaia, which opens with all the types of rejections, by determining the physico-chemical parameters (ph, conductivity, moisture), amount of material and particle sizes (granulometry) (qiao et al. 2007; chikhi et al. 2012), composition and chemical speciation (xrd, fx, ftir, tclp, aas) (windt et al. 2007) soils that define the nature and the intrinsic parameters of the discharges (planel et al. 2004; laforest et al. 2007), the second objective is to study of the evolution of the soil stability by the addition of hydraulic binders (zampori et al. 2009; drouiche et al. 2019). the s/s method is an innovative method, which has been proven in the field by its treatment of more than 57 toxic elements (gollmann et al. 2010; du et al. 2010; moussaceb et al. 2012; moussaceb et al. 2013) by: (i) chemical stabilization of toxic element by adsorption and/or ion exchange which is the consequence of the formation of chemical bonds between the waste and the hydraulic binder (fuessle et al. 2004; moussaceb et al. 2012; epa 2014), (ii) solidification by the transformation of waste into a solid monolith for the purpose of trapping contaminating species inside the cementitious matrices (shi et al. 2004; moussaceb et al. 2012; epa 2014) thus reducing the mobility of toxic chemical species (epa 2014) that focus primarily on the addition of a hydraulic binder (portland cement) to contaminated soil (moussaceb et al. 2012). according to (laurent et al. 2007) a study of s/s on samples of contaminated soil performed for 28 days to 1 year made the soil non-dangerous. further (rémillard 2012), reported that the decontamination of the soil contaminated with cd, cr, cu, pb, zn (shannon, municipality, canada) with s/s showed very good trapping results even after a long leaching period. tests conducted by soliditech. inc. on a site contaminated by pb and pcb in the region morganville, new jersey (usa) demonstrates a good application potential of the method (bisson et al. 2012). preliminary results of soil samples from the site examined in this work showed a high zinc and nickel content, thus presenting a site overload by these two elements. the soil samples treated by the s/s process for a 28-day curing period, showed retention rates in zn/ni metals of up to 99 % in the cementitious matrices. experimental for an eventual physico-chemical and mechanical analysis of our soil samples, an arsenal of standardized methods (fx, xrd, ftir, aas) as well as standardized tests (granulometry, humidity, electrical conductivity, ph, tclp) were used to achieve our objectives, namely: (i) determination of the intrinsic properties of the contaminated soil samples, (ii) improvement of the trapping of heavy metals inside the cementitious matrices (segolene 1992). sampling sampling was carried out at the boulimat-bejaia landfill site after shovelling and evacuation of the first two centimeters with a stainless shovel, four soil samples to a depth of up to 40 cm were carried out according to the method of targeted sampling according to iso 1993 and epa 1991. the sampling points are shown in fig. 1 and their coordinates in table 1 (pellet et al. 1993; liliane et al. 2007; bisson et al. 2012). table 1. location of soil sampling points with the decimal degree system (dds). sample index latitude (n) ° x longitude(e) ° y e (i) 36.789016 5.007027 e (ii) 36.789283 5.007847 e (iii) 36.789030 5.008666 e (iv) 36.788747 5.009202 determination of selected physical parameters the soil samples were treated according to iso 11464, which consists of drying in the open air for 15 days before proceeding to physico-chemical analyzes (pellet et al. 1993; ministère du développement durable, de l’environnement et des parcs du quebec 2010; bisson et al. 2012). sieving granulometry determined the particle size distribution of soil by fractionation on the shakers sv008 sieving machine according to afnor nf bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc nova biotechnol chim (2019) 18(2): 166-178 168 fig. 1. sampling points of the boulimat-bejaia landfill, the number 1-4 correspond to samplings sites of the samples ei-eiv (table 1). x31-101 (violaine 2007; bisson et al. 2012). residual moisture measurement determined the dry mass lost of a soil sample dried at 103±3 °c whose mass is constant at 40 °c, according to afnor nf x31-102 (segolene 1992; marie et al. 2012). ph (h2o) of soils was determined according to afnor x 31-103 recommends with the hanna ph211 device (segolene 1992; liliane 2007; kebir 2012). electrical conductivity was determined by the dissolution of a soil sample in water according to the solid/liquid ratio, and which is measured with the apparatus hanna ec215, according to afnor nf x 31-113 (segolene 1992). 𝑆/𝐿 = 1/5 …………… (1) mineralogical composition – x-ray diffraction (xrd) x-ray diffraction consists in the identification of minerals by electron bombardment in the form of electromagnetic waves, where the atoms follow a specific arrangement according to the crystalline planes following to the bragg law. 𝑛𝜆 = 2𝑑 ∗ 𝑆𝑖𝑛ɵ° ………… (2) where 𝑛 – diffraction order, 𝜆 – x-ray wavelength, d – inter-reticular distance, ɵ° – bragg angle. the indexation of peaks allows the identification of mineralogical phases present by referring to the american society or testing materials (astm) sheets. the diffractograms of our soil samples were obtained on the device after treatment with panalatical x'pert hight score (ministère du développement durable, de l’environnement et des parcs du quebec 2010; marie et al. 2012; eti 2012; moussaceb et al. 2019). fourier transform infra-red (ftir) spectrometry the ftir is used to identify the functional groups existing within our soil samples following a vibrational transition interval between 4000 – 400 cm-1 (mejbri et al. 1995; mounia 2013) with the spectrometer shimadzu iraffinity-1s, samples preparation is made in kbr pellet. chemical composition the simple extraction procedures are methods that run in one step, and those using several methods. these concluded: (i) x-ray fluorescence, which gives the proportions of the major elements as an oxide by bombardment of the sample surface with x photons, performed using the skyray 7000xrf experimental setup. (ii) atomic absorption spectrometry (thermo scientific ice 3000) was used for the quantification of trace elements present in soil samples to be treated and those after acid leaching preparation (toxicity characterisation leaching procedure – tclp) (bisson et al. 2012; drouiche et al. 2019). leaching tests the tclp-epa 1311 test is a method used to evaluate the mobility of inorganic species in order to evaluate whether an industrial residue is considered as a dangerous leachable material or not. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc nova biotechnol chim (2019) 18(2): 166-178 169 it is carried out by dissolving a soil sample in a solution containing 2.572 g of sodium hydroxide and 5.7 ml of acetic acid with a solid/liquid ratio: 𝑆/𝐿 = 1/20.....................… (3) where the leachate recovered after filtration was analyzed for heavy metals by atomic absorption spectroscopy (aas) (lassin 2002; liliane 2007; moussaceb et al. 2019). soil samples processing once the soil samples have been characterized, an operation of treatment was carried out, and for this purpose mixtures of several variation were established by addition of contaminated soil (between 0 and 40 %) and cement. the cement used in this study is portland cement cem i from ain el kbira, algeria. table 2 shows the formulations made in accordance with standard nf p 98-230-3 (bozkurt et al. 2000; prasadini et al. 2014). the constituents were introduced into a mixer and mixed dry for 2 min. then they spread on a noncontaminating surface at the thickness of the largest grain and then the water is added by spraying and they are left for 28 days at a ambient temperature (20±3 °c) to undergo characterization tests, namely: xrd, fx, tclp (berger 2009). the realization of the mixtures soil/ cement is based on the report (bouzeroura et al. 2018). 𝑤/(𝑐 + 𝑠) = 0.5…………… (4) with: w – mixing water [ml], c – portland cement [g], s – contaminated soil [g]. the quantities of cement are fixed at 450 g and those of the soil are varied from 0 to 40 %. the quantities used are given in table 2. table 2. mixtures formulations with quantity of cement (mc), water (w) and soil (s). mc+s [g] mc [g] w [ml] s [g] (s/c) ×100 [%] w/(s+c) 495 450 247.5 45 10 0.5 540 450 270 90 20 0.5 585 450 292.5 135 30 0.5 630 450 315 180 40 0.5 table 3. granulometry parameters of soil samples (jerome et al. 2012). laws and parameters unit ei eii eiii eiv d5 particles diameter at 5 % mm 0.09 0.13 0.10 0.07 d10 particles diameter at 10 % mm 0.18 0.27 0.23 0.11 d16 particles diameter at 16 % mm 0.33 0.45 0.39 0.18 d25 particles diameter at 25 % mm 0.61 0.76 0.67 0.34 d30 particles diameter at 30 % mm 0.91 0.98 0.87 0.48 d50 particles diameter at 50 % mm 1.48 1.75 1.45 1.23 d60 particles diameter at 60 % mm 1.88 1.97 1.79 1.62 d75 particles diameter at 75 % mm 2.52 2.5 2.45 2.32 d84 particles diameter at 84 % mm 2.98 3.02 2.93 2.84 d90 particles diameter at 90 % mm 3.30 3.34 3.29 3.24 d95 particles diameter at 95 % mm 3.65 3.66 3.63 3.57 curvature coefficient 𝑪𝒄 = 𝑫𝟑𝟎 𝟐 𝑫𝟔𝟎⁄ ∗ 𝑫𝟏𝟎 c te 2.44 1.80 1.84 1.29 uniformity coefficient 𝑪𝒖 = 𝑫𝟔𝟎 𝑫𝟏𝟎⁄ c te 10.44 7.29 7.78 14.72 sorting index (trask) 𝑺𝒐 = √ 𝑫𝟕𝟓 𝑫𝟐𝟓 cte 2.03 1.83 1.91 2.61 asymmetric trask coefficient 𝑺𝒌 = 𝑫𝟐𝟓 ∗ 𝑫𝟕𝟓 𝑫𝟓𝟎 𝟐 cte 0.70 0.63 0.78 0.52 inter-quartile 𝜟 = √ 𝑫𝟖𝟒 𝑫𝟏𝟔 ⁄ cte 3.00 2.59 2.74 3.97 angularity coefficient (kurtosis) k = (𝑫𝟕𝟓−𝑫𝟐𝟓) 𝟐∗(𝑫𝟗𝟎−𝑫𝟏𝟎) cte 0.30 0.29 0.29 0.31 porosity p = 0.13 + 0.21 (𝐷50+0.002) 0.21 mm -1 0.32 1.75 2.11 2.54 dispersion index (folk/ward) d = (𝐷84−𝐷16) 4 + (𝐷95+𝐷5) 6.6 mm 1.20 1.17 1.17 1.19 asymmetric index of krumbein ski = (𝑫𝟕𝟓+𝑫𝟐𝟓)−(𝟐∗𝑫𝟓𝟎) 𝟐 mm 0.085 0.085 0.11 0.10 bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc nova biotechnol chim (2019) 18(2): 166-178 170 results and discussion characterisation of sampled, non-treated soil granulometry distribution was rather similar for tested soil samples (for ei shown in fig. 2a) from values of individual parameters (table 3) it can be seen that the values of the coefficients recorded are as follows: curvature coefficient ranged between 1.29 – 2.44, uniformity coefficient between 7.29 – 14.72, sorting index (trask) between 1.83 – 2.61, asymmetric trask coefficient between 0.52 – 0.78, inter-quartile between 2.59 – 3.97, angularity coefficient (kurtosis) between 0.29 – 0.31, porosity in range of 0.31 – 0.33, dispersion index (folk/ward) between 1.16 – 1.20, asymmetric index of krumbein in range of 0.085 – 0.11. fig. 2. characterisation of non-treated soil samples. granulometry distribution of soil sample ei and the grain diameters at percentage between 5 % to 95 % corresponding to d5 to d95 (a). weight distribution of the same soil sample according to sieves series in range 0.063 – 4 mm, depending on the afnor nf x31-101 standard (b) corresponding data are given in table 3. these data point on a varied grain size, a spread and poorly graded distribution, and a ranking of the grain size on the coarse fraction side with a random arrangement of poorly sorted spheres. (blott and pye 2001; jerome et al. 2012). thus, our samples represent a spreading and well graded particle size. the results in fig. 2b show the weight distribution of sample ei. highly similar pattern was observed for the weight distribution of eii-eiv samples (data not shown). it is concluded that our soil samples have the same weight proportions with a high mass presence at mean diameters (2 and 1.5 mm). these represent respectively the intervals 2 – 1 mm and d > 2mm, and which vary between 32.34 % – 39.57 % and 25.4 % – 32.25 %, respectively. according to the standard nf p18-560 and according to the classifications of strakhov and wentworth (jerome et al. 2012), it is concluded that our soil is coarse sand-type. the main properties of soil samples are shown in table 4. the data show that the moisture content varies between 1.11 % and 3.10 %. these values are strongly linked to actual climatological factors during the moment of sampling just after the summer period of 2016/2017, as well as the presence of waste that makes the soil hydrophobic to water. according to souhila (2005) the loss of mass in the soil does not only refer to water but also to the evaporation of volatile organic matter likely to intervene at 60 °c. the ph of soil samples varies between 8.05 and 8.25 (table 4), suggesting that the soil is an alkaline soil (marie 2009), favoured by the dominant geological formation of the study site which is calcareoussedimentary formation. according to souhila (2005) basic ph values have been found in the geological state of the studied soil and which is alkaline. furthermore, kebir (2012) reports that the basic ph of soil samples of the landfill refers to biological activity in field. table 4. physical parameters of soil samples according to afnor nf x31 standards. sample moisture [%] ph (h2o) conductivity [ms.cm-1] e i 3.05 8.20 689 e ii 2.47 8.05 1173 e iii 3.10 8.10 958 e iv 1.11 8.25 519 a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc nova biotechnol chim (2019) 18(2): 166-178 171 fig. 3. infrared spectrum of soil samples from localities ei-eiv (fig. 1) before treatment. the values of conductivities range from 519 to 1,173 ms.cm-1, which is classified as slightly saline soil according to duran scale (marie 2009). these values indicate a strong interaction between the salts present in the soil and the mineral character of the study site. fourier transform infrared spectroscopy (ftir) was applied to identify functional groups. the analysed soil samples have a high siliceous rock content, which has a vibrational domain at 2,100 cm-1 and 1986 cm-1 and then at 1,124 cm-1 and 1162 cm1. for calcareous rock, there are strong transition at 2506 cm-1, 1,790 cm-1 and 1,400 cm-1, whereas the vibrational plugs of the intermolecular water appear at 3,408 cm-1, 3,364 cm-1, 3,396 cm-1 and 3,378 cm-1 for samples ei, eii, eiii and eiv, respectively. the latter reappear at 1,637 cm-1 and 1,634 cm-1 for eii and eiii, respectively. similar results were found from an ftir characterization study on soil samples from southern brazil (dick 2003). table 5 summarizes the overall results for each soil samples. the infrared spectrum from soil samples is presented in fig. 3 and corresponding functional groups are characterised in table 5. the results of x-ray fluorescence showed that soil samples ei, eii and eiii have high proportions of sio2, ranging from 45.80 % to 50.37 %, and al2o3 ranging from 13.77 % to 15.09 % (table 6). a low cao content of 11.67 % to 18.39 % was recorded as well. on the other hand, the soil sample iv has a high proportion of cao, which is of the order of 48.39 % and low levels of sio2 and al2o3 (19.06 % and 7.27 %, respectively). the presence of metal elements in small proportions in the form of traces has been observed. these results can be explained by the fact that the soil samples ei, eii and eiii were taken in places with a high presence of silica and alumina, resulting from the rejections of the discharge in question, which consist of glasses and aluminum, and their provenance from the mother rock of the region which is rich in silicates and aluminates. on the other hand, the results obtained in the soil sample eiv are confirmed by the fact that the sampling was carried out on the eastern periphery of the landfill and close to a limestone bed (aggregate quarry). the metallic trace elements found in these soil samples returned to the nature of the waste bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc nova biotechnol chim (2019) 18(2): 166-178 172 table 5. infrared spectroscopy of the functional groups from soils samples using shimadzu iraffinity-1s (foil et al. 1952; maglione et al. 1975). soil sample wavelength [cm-1] identification band ei eii eiii eiv nd 3626 3623 3626 vibration si-o-h of clay 3408 3363 3396 3378 vibration oh of h2o nd nd nd 2506 calcite nd 2108 2111 2101 vibration si-o of quartz 1986 1998 1990 2003 vibration si-o of quartz 1798 1790 1796 1796 calcite nd 1637 1634 nd vibration oh of h2o 1407 1422 1406 1410 calcite 1125 nd nd 1163 si-o of quartz 1035 983 1002 1009 vibration of si-o of alumino-silicate 872 871 874 875 vibration of co3 of caco3 nd 776 776 798 vibration of co3 of caco3 714 nd 710 713 vibration of co3 of caco3 nd 693 695 nd calcite/quartz 663 nd nd nd nd 607 nd nd nd nd 517 526 522 526 vibration of si-o-al 419 447 449 465 vibration of si-o-si discharged into the landfill (household, hospital, urban and industrial waste, etc.). according to antonine (2012) the high abundance of caco3 content refers to the presence of carbonates in rocks of study site and for metallic minerals in the form of hydroxide or sulphide. the chemical compositions of analysed soil samples are shown in table 6. the leachates obtained by dissolving (tclp test) were subjected to a chemical analysis, by aas, to determine the contents of the metallic elements. it is found that the contents of the soil samples (table 6) perfectly meet the afnor nf u44-041 (1985) (segolene 1992) standard except for [ni] > 50 mg.kg-1 in the soil sample ei and [zn] > 300 mg.kg-1 in the soil sample eiii. the results shown in table 6 can be justified by acid rain, which frequently affects the region, thus promoting the migration of heavy metals through leachates that percolate from the landfill to the surrounding soil. according to kebir (2012) the persistence of these metallic elements concentrations refers to the origin of the waste as well as to the industrial effluents discharged into the landfill. according to the xrd results, the soil has a similarity in initial mineralogical composition, which is a clay soil consisting mainly of quartz and calcite, with heterogeneity in the secondary phases that show contamination with heavy metals. examples include dwornikite and retgersite for ei and wurtzite, hopeite and hetaerolite for eiii. the diffractograms of soil samples ei and eiii are shown in fig. 4, while the corresponding mineralogical phases are summarized in table 7. table 6. chemical composition of the analysed soil samples from localities e-eiv. soil sample ei eii eiii eiv compound content [%]a sio2 50.37 47.07 45.80 19.6 al2o3 15.9 13.77 14.9 7.27 fe2o3 4.74 5.2 5.14 4.17 cao 11.67 16.20 18.39 48.39 mgo <0.05 nd nd 0.79 na2o <0.05 <0.05 <0.05 <0.05 k2o 2.7 2.2 2.4 1.7 tio2 0.48 0.46 0.48 0.23 mno 0.09 0.20 0.13 0.19 p2o5 0.28 0.42 0.35 0.76 so3 0.09 0.15 0.05 0.021 nio 0.0028 0.0035 0.0026 <0.005 zno 0.0088 0.0071 0.0056 0.019 pbo 0.0031 0.0028 0.0057 0.0123 metal content [mg.kg-1]b zn 9.388 4.194 2651.8 20.78 ni 163.44 38.3 0.22 <ld pb 0.4 <ld 3.4 2 a data obtained by x-ray fluorescence. b according to afnor nf u44-041 standard, after tclp leaching test. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc nova biotechnol chim (2019) 18(2): 166-178 173 table 7. mineralogical composition of ei sample using x’pert high score. mineral phases chemical composition references pattern soil sample ei quartz sio2 01-078-1252 calcite caco3 01-083-1762 urea co(nh2)2 01-086-2276 muscovite kal3si3o10(oh)2 01-075-0948 despujolsite ca3mn(so4)2(oh)6(h2o)3 01-072-0388 tridymite sio2 01-075-1323 dwornikite (ni, fe)so4.h2o 00-035-0558 argentopyrite agfe2s3 00-038-0459 domeykite cu3as 00-044-1475 manganotapiolite (mn, fe)ta2 00-049-1870 retgersite niso4(h2o)6 01-075-0937 kyanite al2sio5 01-076-0170 hatrurite ca3sio5 01-086-0402 soil sample eiii hetaerolite mn2o4zn 00-024-1133 rostite al(so4)(oh).5h2o 00-041-1382 quartz sio2 01-078-1252 calcite caco3 01-083-0578 hopeite zn3(po4)2.4h2o 00-001-0975 halloysite al2si2o5(oh)4 00-003-0184 sarcopside fe3(po4)2 00-039-0341 anthophyllite mg7(si8o22(oh)2) 01-075-0909 collinsite ca2mg(po4)2.2h2o 00-026-1063 clinzoisite ca2al3(sio4) (si2o7)(o, oh)2 00-021-0128 chalcocite cu2s 00-023-0961 wurtzite zns 01-072-0162 reinhardraunsite ca5(sio4)2(oh)2 00-029-0380 linthian saponite na0,2(mg,li)3si4o10(oh)2.4h2o 00-025-1385 quartz sio2 01-078-1252 calcite caco3 01-083-1762 urea co(nh2)2 01-086-2276 muscovite kal3si3o10(oh)2 01-075-0948 despujolsite ca3mn(so4)2(oh)6(h2o)3 01-072-0388 tridymite sio2 01-075-1323 dwornikite (ni, fe)so4.h2o 00-035-0558 argentopyrite agfe2s3 00-038-0459 domeykite cu3as 00-044-1475 manganotapiolite (mn, fe)ta2 00-049-1870 retgersite niso4(h2o)6 01-075-0937 kyanite al2sio5 01-076-0170 hatrurite ca3sio5 01-086-0402 it is concluded that physico-chemical characterization study of the project site samples, which all the results obtained; converge towards the same conclusion suggest contamination of the site by zinc and nickel, posing a great danger to the environment, fauna and flora as well as to human health. characterization of s/s soil samples decontamination of the contaminated sites is impe rative, therefore a soil process has been proposed, namely remediation by s/s process with hydraulic binders. after such treatment, ftir analysis revealed the disappearance of several vibrational bungs and the appearance of others such as ettringite at 3,400 cm-1 and c-s-h at 1,400 cm-1 and 1,000 cm1 (fig. 5). further, the bung of calcite was detected. similar results were found in a study reported by catherine (2003). the functional groups corresponding to data in fig. 5 are given in table 8. bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc nova biotechnol chim (2019) 18(2): 166-178 174 20 30 40 50 60 70 0 200 400 600 800 1000 1200 1400 12 1213 10 1 2 8 4 9 2 11 5 2 12 10 13 1; 7 1 311 6 4 c o u n t s o f s a m p l e e i [ % ] 2 t h é t a [  ° ] a 20 30 40 50 60 70 0 200 400 600 800 1000 1200 96 14 3 4 129713 34 12 43 4 7 11 10 4 7 9 1 5 4 3 8 2 2 6c o n u t s o f s a m p l e e i i i [ % ] 2 t h é t a [ ° ] b fig. 4. x-ray diffraction of non-treated soil samples ei (a) and eiii (b) after treatment with panalatical x'pert hight score. 4000 3500 3000 2500 2000 1500 1000 500 40 50 60 70 80 90 100 2102,45 3398,17 448,32 516,97 713,76 870,9 1003,62 t r a n s m i t t a n c e o f s a m p l e e i s / s 4 5 0 / 1 8 0 [ % ] w a v e l e n g t h [ c m -1 ] 1401,79 a 4000 3500 3000 2500 2000 1500 1000 500 0 50 60 70 80 90 100 110 463,14 518,43 713,14 873,39 1000 1410,25 2120,24 t r a n s m i t t a n c e o f s a m p l e e i i i s / s 4 5 0 / 1 8 0 [ % ] w a v e l e n g t h [ c m -1 ] 3400 b fig. 5. infrared spectra of the stabilized / solidified soil sample eis/s450/180 (a) and eiiis/s450/180 (b). 10 20 30 40 50 60 70 0 2000 4000 6000 8000 10000 172 4 85 10 86 12 14 73 3 109 13 11 16 1 15 c o u n t s o f s a m p l e e i s / s 4 5 0 / 1 8 0 [ % ] 2 t h e t a [ ° ] a 20 30 40 50 60 70 0 200 400 600 800 14 15 9 3 5 5 7 7 16 1131 11 8 46 26 12 4 10 5 c o u n t s o f s a m p l e e i i i s / s 4 5 0 / 1 8 0 [ % ] 2 t h e t a [  ° ] b fig. 6. x-ray diffraction of soil samples eis/s (450/180) (a) and eiiis/s (450/180) (b) after treatment with panalatical x'pert hight score. table 8. infrared spectroscopy of the functional groups from stabilized/solidified samples soils using shimadzu iraffinity-1s. wavelengths for each samples (cm-1) eis/s eiiis/s identification band 3398 3400 hydrated ettringite 2103 2120 vibration oh (h2o) 1402 1410 c.s.h 1004 1000 c.s.h 871 873 ettringite 714 713 vibration of co3 (caco3) 517 518 gibbsite 448 463 gibbsite/silicate the x-ray fluorescence results obtained on the ei and eiii samples of the s/s soil are recorded in fig. 6 and table 9. the results obtained show that cao content in soil samples ei and eiii decreased due to remediation process from 11.67 % and 18.39 % (before s/s) to 46.08 % and 55.84 % (respectively), due to the accumulation of cao proportions present in the soil and the cement after the hydration phenomenon on the other hand, content of both sio2 and al2o3 decreased after s/s; a b a b a b bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc nova biotechnol chim (2019) 18(2): 166-178 175 table 9. chemical composition from soil sample eis/s450/180 in oxidation form by the x-ray fluorescence. sample (es/s) eis/s eiiis/s 450/0 450/45 450/90 450/135 450/180 450/0 450/45 450/90 450/135 450/180 cement/soila sio2 14.62 12.82 17.47 17.83 16,82 14.62 15.26 16.55 19.41 21.23 al2o3 2.4 3.9 2.62 2.76 2.5 2.4 2.6 2.5 2.9 3.49 fe2o3 2.31 3.9 2.86 2.94 2.71 2.31 2.58 2.74 2.97 3.26 cao 53.00 46.08 48.13 48.26 51.35 53.00 51.86 49.49 48.86 46.06 mgo 1.36 1.55 1.5 1.65 1.7 1.36 1.25 1.38 1.58 <ld k2o 0.52 0.67 0.58 0.59 0.58 0.52 0.55 0.56 0.67 0.77 tio2 0.20 0.37 0.27 0.31 0.25 0.20 0.23 0.25 0.3 0.34 mno 0.04 0.04 0.048 0.044 0.041 0.04 0.05 0.05 0.05 0.06 p2o5 1.5 0.98 0.95 1.6 1.4 1.5 1.3 0.93 0.98 0.82 so3 <ld 0.12 0.014 0.007 <ld <ld <ld <ld <ld 0.17 nio <ld <ld <ld <ld <ld <ld <ld <ld <ld <ld zno <ld <ld <ld <ld <ld <ld <ld <ld <ld <ld pbo <ld <ld <ld <ld <ld <ld <ld <ld <ld <ld metal contentb ni [mg.kg-1] nd 0.17 0.32 0.42 1.69 nd nd nd nd nd retentions rate [%] nd 97.68 97.82 98.09 94.25 nd nd nd nd nd zn [mg.kg-1] nd nd nd nd nd nd 0.21 9.1 23.73 30.70 retentions rate [%] nd nd nd nd nd nd 99.82 96.22 93.37 93.56 a data obtained by x-ray fluorescence. b according to afnor nf u44-041 standard, after tclp leaching. the sio2 content dropped from 50.37 % and 45.80 % of the two samples to 12.82 % and 15.26 % after s/s, respectively. this decrease results from incorporation of sio2 from quartz. as for al2o3 the values of 15.09 % and 14.09 % of non-treated ei and eiii soil samples decrease to 3.09 % and 2.06 % after s/s, respectively. this decrease is due to growth of hydration phases of cement and more specifically c.s.h and ettringite for al2o3. the presence of so3 in our s/s materials refers to the formation of the cement phase (ettringite) as well as the presence of sulphur in the soil and cement. the variation of the values of si, al, s and ca refer to the hydration reaction between the materials of soil and those of hydraulic binder; they are mobilised to form hydrates of cements after s/s (saussaye et al. 2016). on the other hand, the absence of the metallic oxides in the samples before s/s can be explained by the mechanism of substitution of elements with those of cement and which made it possible to trap chemically and physically the metallic elements inside cement matrix. s/s of soil samples resulted in strong retention of the contents of ni and zn; their content increased to 98.09 % for (ni) and 99.82 % for (zn) in the soil samples eiii and ei, respectively (table 9). x-ray diffraction results obtained on s/s treated sample ei (450/180) and eiii (450/180) are given in fig. 6. the soil sample ei exerted disappearance of certain mineralogical phases such as retgersite (niso4 (h2o)6), but the appearance of other mineralogical phases such as sodium nickel sulfite hydroxide hydrate (nani (so3)2 oh h2o). for soil sample eiii the disappearance of certain mineralogical phases such as hopeite (zn3 (po4)2 4h2o) and the appearance of other phases, such as descloizite (pb (zn, cu) vo4 (oh)) was observed in (table 10). compared with the work of berger (2009) on substitutions of metallic trace elements with the phases of cement (natural or synthetic, total or partial) we found that for the two phases found after s/s, there is a probability of substitution of (ni) from sodium nickel sulfite hydroxide hydrate with (al) of ettringite for sample ei, and (pb, zn) from descloizite with (ca) of ettringite for sample eiii (table 11). conclusion this study analyzed the influence of the addition of hydraulic binder to the contaminated soil as well as of the proportional addition from cement trapping of heavy metals in cementitious matrices. our results showed that granulometric characteristic of soil bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc nova biotechnol chim (2019) 18(2): 166-178 176 table 10. mineralogical composition of soil samples eis/s (450/180) and eiiis/s (450/180) using x’pert high score. mineral phase chemical composition reference pattern soil sample ei sophiite zn2(seo3)cl2 00-045-1463 galaxite mnal2o4 00-001-1302 fayalite fe2sio4 01-071-1669 ferrocarpholite (fe0, 76 mg0, 24)2al4(si4o12) (oh) 8 01-081-1503 nickelbischofite nicl2.6h2o 00-025-1044 ettringite ca6al2(so4)3 (oh)12.25h2o 00-009-0414 ammonium nickel chromium oxide hydrate (nh4)2ni(cro4)2.6h2o 00-021-0705 nickelboussingaultite (nh4)2ni(so4)2.6h2o 00-031-0062 nickel sulfite hydrate niso3.2h2o 00-034-0315 potassium nickel selenate hydrate k2ni(seo4)2.6h2o 00-035-0774 sodium nickel sulfite hydroxide hydrate nani2oh(so3)2.h2o 00-037-0016 fersilicite fesi 00-038-1397 nickel trans-[dichlorobis(2-oxopropyldiphenylphosphine)] c30h30cl2nio2p2 00-040-1756 brucite mg(oh)2 01-082-2455 clinohedrite cazn(sio4) (h2o) 01-083-2269 bementite mn6, 927(si6o15) (oh) 8 01-082-1236 cummingtonite mg7si8o22(oh)2 01-072-0114 soil sample eiii calcite caco3 01-083-0578 tachyhydrite camg2cl6.12h2o 00-034-0788 burbankite (na2ca)(sr2ca)(co3)5 00-026-1374 anilite cu7s4 00-024-0058 descloizite pb(zn, cu)vo4(oh) 00-041-1369 reinhardbraunsite ca5(sio4)2((oh)f) 01-075-1709 tobermorite (cao)x sio2 zh2o 00-006-0005 brushite capo3(oh).2h2o 00-009-0077 zinc malonate dihydrate c3h2o4zn.2h2o 00-024-1990 zinc selenate hydrate znseo4.h2o 00-026-0004 pyrophyllite al2si4o10(oh)2 01-074-1037 quartz sio2 01-083-2473 ettringite ca6al2 (so4)3(oh)12·26h2o 00-041-1451 palmierite k2pb(so4)2 00-020-0902 calcium zinc aluminium oxide ca3al4zno10 00-049-0280 muscovite kal3si3o10(oh)2 01-071-1049 table 11. mineralogical composition from the probability of substitution phases in selected soil samples. phase type chemical formula soil sample ei s/s (450/180) sodium nickel sulfite hydroxyde hydrate (na ni2 oh (so3)2 h2o) clinohedrite (cazn)(sio4)(h2o) soil sample eiiis/s (450/180) descloizite (pb (zn, cu) vo4 (oh)) calcium zinc aluminium oxide (ca3 al4 zn o10) samples increased because the mineral composition of the limestone rocks dominates on the site, alkaline ph which defines the nature of the sedimentary rocks, and causes very high soil conductivities. we identified the profiles of present major elements as well as metallic elements in the mineralogical structure of soil samples. the high concentration of ni and zn detected in the soil surrounding the landfill probably results from leaching with acid rain that caused a migration of trace metallic elements from waste to soil. the analyses and characterizations of our s/s samples revealed new functional groups belonging to cement phases such as ettringite and c.s.h. further, x-ray fluorescence results suggest that the metallic trace elements disappear from soils bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc nova biotechnol chim (2019) 18(2): 166-178 177 because they are likely trapped inside the cementitious matrices. elevated proportion of cao and decreased contents of sio2 and al2o3 in the soil samples might results from cement interaction with the soil during its hydration. aas confirmed ni and zn retention rates of up to 98 %. x-ray diffraction analyses revealed disappearance of certain mineralogical phases with metallic trace elements after the treatment. on the other hand, hydrated cement phases were identified, such as ettringite, portlandite and c.s.h in the form of tobermorite with presence of metallic trace elements. substitution occurred between the metallic trace elements and the elements of the hydrated cement. it is concluded that the s/s process using hydraulic binders represents a promising method in the treatment of contaminated soils. conflict of interest the authors declare that they have no conflict of interest. references antonine p (2012) caractérisation multi-échelles des phases porteuses des polluants métalliques zn et pb dans un sédiment mis en dépôt de l’analyse de terrain au rayonnement synchrotron. university of 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bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc nova biotechnol chim (2019) 18(2): 166-178 178 maglione g, carn m (1975) spectres infrarouges des matériaux salins et des silicates néoformés dans le bassin tchadien. cahiers orstom, ser. géologie 7: 3-9. marie l (2012) caractérisation physico-chimique d’un sédiment marin traité aux liants hydrauliques – évaluation de la mobilité potentielle des polluants inorganiques. national institut of applied sciences of lyon, france, p. 100-121. marie m (2009) accélération de ciment au laitier par du ciment sulfo-alumineux. national institut of applied sciences of lyon, france, p. 98-99. mejbri r, matejka g, lafrance p, mazet m (1995) fractionnement et caractérisation de la matière organique des lixiviats de décharge d’ordures ménagères. revue des sciences de l’eau 8: 217-236. ministére du déveleppement durable, de l’environnement et des parcs du quebec guide d’échantillonnage a des fins d’analyses 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(2010) quebec. centre d’exprtise en analyse environnementale du quebec, p. 41-44. mounia c (2013) analyse et spéciation des métaux dans un oued en zone minière cas de l’oued essouk. university de constantine 1, algeria, 49 p. moussaceb k, ait-mokhtar a, merabet d (2012) influence of leaching conditions on the release kinetics of lead, chromium and nickel from solidified/stabilized cementitious materials. environ. technol. 33: 2681-2690. moussaceb k, belebchouche c, ait-mokhtar a, merabet d (2013) evaluation of the impact of ni, cr and pb contained in effluents of an industrial unit by the process of stabilization/solidification using hydraulic binders. int. j. environ. res. 7: 485-494. moussaceb k, bellache d, ait-mokhtar a (2019) influence of the leaching tests on the release of heavy metals from cementitious materials obtained by the solidification of petroleum sludge wastes. j. solid waste technol. manag. 45: 139-152. pellet m, laville-timsit l (1993) echantillonnage de sols pour caractérisation d’une pollution : guide méthodologique, brgm-france, 16-17: 64-65. planel d, sercombe j, le bescop p, adenot f, torrenti jm (2006) long-term performance of cement paste during combined calcium leaching-sulfate attack: kinetics and size effect. cement concrete res. 36: 137-143. prasadini p, lakshmi gs (2014) environmental science birm 301 study material. college of agriculture, rajendranagar, p. 4-7. rémillard j (2012) influence de l’altération physique sur les caractéristiques physico-chimiques de monolithes de sols contaminés traités par stabilisation/solidification au ciment. ecole de technologie supérieure, university of quebec, p. 25-26. qiao x, poon cs, cheeseman cr (2007) investigation into stabilization/solidification performance of portland cements through cement clinker phases. j. hazard mater. 238-243. saussaye l, leleyter l, baraud f, boutouil m (2016) trace and major elements stabilisation in soils treated with hydraulic binders. eur. j. environ. civ. eng. 22: 755-769. sefouhi l, kalla m, aouragh l (2010) trends and problems of municipal solid waste management in batna city and ň prospects for a sustainable development. int. j. sustain. water environ. syst. 1: 15-20. segolene r (1992) arrêté du 18 décembre 1992, stockage de certains déchets industriels spéciaux ultimes et stabilisés pour les installations existantes, 5620. shi c, spence r (2004) designing of cement-based formula for solidification/stabilization of hazardous, radioactive and mixed wastes. crit. rev. environ. sci. tech. 34: 391417. souhila k (2005) décomposition des matières organiques et stabilisation des métaux lourds dans les sédiments de dragage. institut national des sciences appliquées de lyon, france, 74. stuart me, klinck bl (1998) a catalogue of leachate quality for selected landfills from newly industrialised countries, british geological survey, united kingdom, p. 1-4. violaine m (2007) modifications de la composition de déchets métallifères, miniers et industriels, stabilisés par liants hydrauliques et soumis a des tests de lixiviation, university of limoge, 79 p. windt dl, badreddine r (2007) modelling of long-term dynamic leaching tests applied to solidified/stabilized waste. j. waste manag. 27: 1638-1647. zampori l, gallo sp, dotelli g (2009) long-term leaching test of organo-contaminated cement-clay pastes. j. hazard. mater. 170: 1041-1049. . bereitgestellt von slovenská poľnohospodárska knižnica | heruntergeladen 28.02.20 12:42 utc nova biotechnol chim (2021) 21(1): e815 doi: 10.36547/nbc.815 1 nova biotechnologica et chimica enhancing oilseeds cold pressing techniques based on hazop analysis for prickly pear seeds, opuntia ficus-indica (l.) mill. kamilia alhamdaoui1, , chouaib benqlilou2, haiat essalmani1 1department of biotechnology, university abdel malek essaadi tangier, faculty of sciences and techniques of tangier, ancienne route de l’aéroport, km 10, ziaten. bp416. tangier, morocco 2department of industrial process engineering,energy systems and thermal processes, rabat school of mines, av. ahmed cherkaoui, bp753, agdal, rabat, morocco  corresponding author: alhamdaoui.kamilia@gmail.com article info article history: received: 12th january 2021 accepted: 18th march 2021 keywords: cold press specification hardness hazop analysis oilseed tocopherol yield abstract the main goal of the present study was to determine optimal conditions for the extraction of oil with high added value by cold pressing technique. namely the feeding conditions of the opuntia ficus-indica (l.) mill. (ofi) seeds, as well as the definition of the manipulable variables of the kern-kraft 20 press were specified. these specifications combine hazop (hazard and operability) analysis and experience plan to find out correlation between yield and oil quality with the pressure, temperature, nozzle diameter, residence time, pressing speed while considering high hardness level of the seed under study. the seed oil extracted according to the proposed approach was highly unsaturated where linoleic acid is the main fatty acids (60.42 % of total fatty acids), followed by oleic acid (21.65 %), palmitic acid (12.24 %), and stearic acid (3.88 %), respectively. furthermore, it is worth mentioning that beta-sitosterol and gamma-tocopherol were the principal components of sterols and tocopherols representing 67.56 % of total sterols and 330 mg.kg-1 of total tocopherols. the proposed approach applied to prickly pear seeds had preserved the tocopherol fraction (796.70 mg.kg-1 of total tocopherols) about two times more than other coldpressed seed oils approaches. based on trial-and-error conditions, no additional operating problem even has been reported with a seed of high level of hardness. seed should be introduced with a humidity level of 10 % without grinding. moreover, no heating should be supplied and an optimal pressing speed of 30 and a nozzle diameter of 15 mm.  university of ss. cyril and methodius in trnava introduction the oilseed industry allows the production of oil as a high value-added product with specified quality that meets the requirements of downstream health industries, namely cosmetics, pharmaceuticals and nutraceuticals. in recent years, a growing demand has been observed which consequently promotes extractive techniques under the concept of biorefining (laurent et al. 2011). these industries extract vegetable oils through various techniques, the most commonly cost-efficient are based on solvent extraction and cold pressing. although the latter has a reduced yield compared to the former one, it is more appropriate for health-related purposes since it is solvent free and preserves oil quality in terms of fatty acids and the levels of bioactive elements such as: tocopherol, squalen, and sterol (koski et al. 2002; bail et al. 2008). however, during the cold pressing method if the mailto:alhamdaoui.kamilia@gmail.com nova biotechnol chim (2021) 20(1): e815 2 press set points conditions are not well specified and seed properties (such as hardness, oil content, moisture content, granulometry, etc.) are not taken into account, serious problems could emerge in terms of yield. for instance, significant oil loss in the cake, quality deterioration, productivity loss, production cost could be caused. other problems such as operability risks could occur, such as operating loss, mechanical equipment failure and occupational risks. in the literature there are some guidelines that indicate the appropriate conditions for oil seed extraction (schaufler and schaufler et al. 2013). nevertheless, less research has been done on seed hardness, whose approach has been mainly based on trial and error procedure. in order to achieve adequate cold pressing and produce high-quality oil without operating issues, a cold pressing method is carried out on prickly pear (opuntia ficus-indica (l.) mill.) seeds, using hazop analysis to study the operational and hazardous conditions related to the cold pressing process. prickly pear seeds are considered a raw-material for cosmetic oil production. they contain valuable oil. however, they present a relatively high level of hardness. consequently, these seeds are chosen as reference to study seeds leading to low yield as well as operability risks. the adopted approach combines hazop analysis and experience plan, so it is possible to specify the optimal conditions for oilseed extraction based on cold pressing. indeed, hazop as the method of qualitative approach allows to identify and record hazards based on formalized and systematic thinking through a multidisciplinary team examining potential intent deviations (kletz 1999; reniers et al. 2005). hazop significantly reduces the number of experiments to carry out in order to find out the best correlation between yield and quality of the extracted oil and the manipulated variables of the cold pressing, namely pressure, temperature, nozzle diameter, residence time, pressing speed. to assess the oil quality, the gas chromatography (gc) and high-performance liquid chromatography (hplc) were performed to determine the composition of fatty acids, sterols and tocopherols, respectively. experimental oil extraction by cold pressing method seeds of the prickly pear (opuntia ficus-indica (l.) mill. (ofi) with high hardness are the most used seeds for oil extraction in morocco. samples from the aït baamrane region in southern morocco were obtained and their properties were studied (weight, humidity, grain size, and others). seed oil was extracted by the cold pressing method (fig. 1) at a laboratory scale. it is a technological mechanical process of extracting oil from the seeds by applying the required pressure according to seed characteristics. the cold pressing was performed using a screw press kern & kraft press (kk-20, germany) with capacity of 20 kg.h-1 of seed, equipped with engine of 2.0 kw, a speed variator, various nozzle diameter as well as a heating system for temperature control. fig. 1. scheme of the cold pressing extraction of oil from ofi seeds. oil yield the seed oil content was determined using the soxhlet method according to iso 659 (1998). seeds were grinded using a blender into a fine and homogeneous powder. twenty grams of powder was subjected to extraction with hexane (100 ml) at 40 60 °c in the soxhlet extractor. after evaporation of nova biotechnol chim (2021) 20(1): e815 3 the solvent under reduced pressure, using a rotary evaporator at 40 – 60 °c, the oil content was calculated and expressed as a percentage of seed dry matter according to the following formula (eq. 1): 𝑌(%) = 100 · 𝑊𝑒 𝑊𝑡 (1) we : weight of fat extracted (g) wt : weight of test sample (seed powder) (g) after determining seed oil content, the total yield extracted by cold pressing was calculated according to the formula (eq. 2): 𝑌𝑖𝑒𝑙𝑑 = 100 · 𝑂𝑖𝑙 𝑦𝑖𝑒𝑙𝑑 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑒𝑑 𝑏𝑦 𝑐𝑜𝑙𝑑 𝑝𝑟𝑒𝑠𝑠𝑖𝑛𝑔 𝑆𝑒𝑒𝑑𝑜𝑖𝑙 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (2) fatty acid composition the fatty acid profile was determined using method iso 5508 (1990). before the analysis, fatty acids were converted to fatty acid methyl esters by shaking a solution of 60 mg of oil and 3 ml of hexane with 0.3 ml of 2 n methanolic potassium hydroxide. fatty acids were analyzed by gas chromatography gc (agilent g-97-04a) equipped with flame ionization detector (fid) and a columnar capillary filled with a stationary phase (70 % cyanopropyl polysilphenylene, length: 60 m, internal diameter: 0.25 mm, film thickness: 0.250 μm). the carrier gas was hydrogen, and the total gas flow rate was 1.6 ml.min-1. the initial column temperature and the final temperature were 140 °c and 200 °c, respectively. the injector and detector temperature were 280 °c. fatty acids were identified by comparison of their retention times with standard mixtures. sterols composition the sterol fraction was determined using the method iso 6799 (1991) by gc (agilent 19091j-436 hp-5, 5%-phenylmethylsiloxane). after the extraction and isolation of the unsaponifiable, the gas chromatographic analyzes were carried out using a column (length: 60 m, nominal diameter: 250 μm, nominal film thickness 0.25 μm) with a helium carrier gas (flow 1.5 ml.min-1). the column temperature was isothermal at 270 °c, injector and ms detector temperature were 300 °c. the sterols quantification was based on the comparison of the spectra with those in the literature. tocopherols composition tocopherols were identified based on the international standard iso 9936 (2006), using hplc shimadzu lc-10ad vp 230v ce normal phase isooctane/isopropanol (99 : 1, v/v). solution of 2 g of oil in 25 ml of isooctane (flow rate of 1.2 ml.min1) was directly used for the hplc, using octadecylsilane silica (c18) column as stationary phase (length : 250 mm, internal diameter : 4.6 mm, thickness of the film : 5 μm) with the fluorometer detector set at 292 nm, to ensure a high selectivity and to differentiate many forms of isomers antioxidants. tocopherols were identified by comparing their retention times with those of authentic standards. quality indices (acidity, peroxide value) in order to check the quality and appropriate conservation of the obtained seed oil, the acidity (a) (expressed as percentage of oleic acid) and the peroxide value (pv) (expressed as milliequivalents of active oxygen per kg of oil) were measured. acidity (a) is the amount of free fatty acids expressed as a percentage of oleic acid. the acidity of the prickly pear seed oil was determined according to the method iso 660 (1996). an amount of 10 g of prickly pear oil was added to 50 ml of neutralized ethanol/diethyl ether mixture (1 : 1, v/v) in the presence of phenolphthalein as indicator, then titrated with potassium hydroxide solution naoh (0.1 m). acidity was calculated as follows (eq. 3): 𝐴 (%) = 𝑉 × 𝐶 × 𝑀 10 × 𝑚 (3) v: naoh volume (ml); c: naoh concentration (mol.l-1); m: oleic acid molar mass (282 g.mol-1) m: oil mass in grams (g). peroxide value (pv) is defined as the number of mili-equivalents of oxygen per kilogram of fat. it is determined following the method iso 3960 (1977). nova biotechnol chim (2021) 20(1): e815 4 an amount of 5 g of prickly pear oil was added to 10 ml of chloroform, 15 ml of acetic acid, mixed all with 1 ml of saturated potassium iodide. the mixture was left in darkness, then 75 ml of distilled water was added. the mixture was titrated with sodium thiosulphate (0.01 n), using starch solution as indicator. the peroxide value was calculated as follows (eq. 4): (4) vx : sodium thiosulfate volume (ml) for the titration; v0: sodium thiosulfate volume (ml) for the blank; n: sodium thiosulfate normality; m: oil mass in grams (g). hazop method during the preliminary experiments, various incidents during the cold press oil extraction of prickly pear seeds were observed due to their high hardness. it caused serious problems in yield (significant oil loss in the cake, quality deterioration, etc.) and operability (operating loss, mechanical equipment failure, occupational risks). for this purpose, the hazop method was adopted to determine operational and risk conditions during start-up, steady-state operation, and press shut down. the system was subdivided mainly into three subsystems as shown in the table 1. table 1. the proposed nodes for hazop analysis application. in each node the process parameters were identified (table 3) considering the experiment design of the in situ oil extraction, the feedback from oilseed industrial operators and the literature data. (fra̧czek et al. 2005; lacoste et al. 2005; altuntaş and yıldız 2007; soetaredjo et al. 2008; van hoed et al. 2010; matthäus and özcan 2011; nitièma-yefanova et al. 2012; chougui et al. 2013; schaufler and schaufler et al. 2013; rabrenović et al. 2014; fuentes-bargues et al. 2016). the study was performed following the steps of the hazop process (fig. 2). indeed, the process parameters associated with guidewords (table 2) were depicted in order to unveil the most significant deviations that could involve risks or dangers in each subsystem. eighteen significant deviations were generated (table 3). table 2. standard guidewords and their generic meanings. fig. 2. hazop process according to iso 31.010 (2011).  2 0 ( ) 1000meqo xkg v v n pv m     sub system node raw material conditioning & feeding i: seed supply conditions and press loading production (steady state operation) ii: seed pressing for oil extraction and cake recovery purposes conditioning and storage of the obtained oil iii: conservation of the extracted oil guidewords interpretation no (not, none) none of the design intent is achieved more (more of, higher) quantitative increase in a parameter less (less of, lower) quantitative decrease in a parameter as well as (more than) an additional activity occurs reverse logical opposite of the design intention occurs other than (other) complete substitution another activity takes place or an unusual activity occurs or uncommon condition exists 4 nova biotechnol chim (2021) 20(1): e815 table 3. identification of deviations leading to risks / hazards. deviation (parameters/keywords) more of less of not reverse more than other node i seed moisture d1 d2 drying / humidification d3 seed granulometry d4 d5 seed hardness d6 seed cleanness d7 node ii press loading d8 d9 d10 screw speed d11 heating temperature d12 press pressure: nozzle choice d13 d14 preheating the press d15 node iii cleaning the press after use d16 oil storage under cold conditions d17 opacity of the oil storage bottles d18 it prevents efficient operation. 5 nova biotechnol chim (2021) 20(1): e815 6 table 4. summary table of risk causes associated with deviations. deviation d1 d2 d3 d4 d5 d6 cause the seeds are stored in a humid place. the seeds are stored in an airconditioned and sunny space. the seed dryer or humidifier is not operative. the pressed seeds are very thin or have been preliminary grinded. grinding large seeds is not done properly to achieve the desired particle size. seeds that are intrinsically hard or too dry (under extreme conditions of temperature and pressure). consequence oil yield loss: seeds become mellow and the oil is strongly related to the water. moisture content is often the culprit of a bad pressing. oil yield loss: the seeds get harder and the oil is caught inside the dried cells. oil yield loss: the pre-drying considerably increased the hardness of seeds. this automatically generates an interrupt, blocking the press head. zero yield: because of the fineness of seeds. seeds came out through pores dedicated to the oil evacuation. productivity loss: a significant amount of the oil came out the cake. machine breakdown and productivity loss: the screw cannot grind the seed that remains caught inside the press. congestion did not let the screw react to advance the material even if the revolutions per minute increased to high enough values. nova biotechnol chim (2021) 20(1): e815 table 4. summary table of risk causes associated with deviations. continued. deviation d7 d8 d9 d10 d11 d12 cause seeds are not washed or contain a significant amount of dust at start-up, the press is generally loaded. a total loading of press is required to ensure the desired pressure. during steady-state operation the press is not loaded globally. seeds with other elements are introduced (metal parts, plastics, paper, etc.) the revolutions per minute applied to the motor speed controller are high. the temperature could increase if the heat is supplied via the press heating ring, or if the pressure inside the press increases, it directly increases the temperature consequence quality loss: dust containing undesirable elements decreases the oil quality. same consequence of d6 productivity loss: there is not enough pressure inside the press and a significant amount of oil in the outgoing cake. machine breakdown and quality loss: hard objects break the screw and plastic elements can significantly damage the oil quality. machine breakdown and productivity loss: short residence time in the press involving high oil content in the cakes it is also possible to have overcrowding between the hopper and the screw because the transfer of material is more important than the pressing speed. quality loss: the cold pressing seed should be maintained at a temperature below 50 °c 7 nova biotechnol chim (2021) 20(1): e815 6 table 4. summary table of risk causes associated with deviations. continued. deviation d14/d13 d15 d16 d17 d18 cause the incompatibility between the nozzle diameter and the seed hardness. preheating the head press is not carried out for a sufficient period and with the required temperature. postpone the machine cleaning. oil storage at high temperature. oil storage in a transparent bottle. consequence productivity loss and operating loss: if the nozzle is too small, the seeds at the exit of the press are retained and the extraction of the cake will not be executed, we notice that the solid seeds start to come out of the oil outlet pores. however, if the nozzle is too big, there will not be enough pressure, which will probably cause an oil loss in the cake. operating loss and oil yield loss: the mechanical gear of the drive parts at the press head does not take place properly and can easily cause mechanical stress that will cause a crack under the applied force. moreover, this quenching will increase the seed hardness. machine breakdown and operating loss: cleaning and maintenance must be carried out immediately after pressing because the delay caused a very strong bond between the seeds and the metal of the press head. forced opening probably will break the press head. quality loss: some fatty acids are more fragile than others, especially unsaturated fatty acids which have a high rate of oxidation under certain conditions (heat, oxidation catalyst, etc.) they degrade which produce a change in the properties of the vegetable oil (smell of rancidity, color change). this change is toxic for the organism. quality loss: many factors accelerate oxidation: oxygen, light (uv), contact with prooxidizing metals (iron or copper), the presence of pigments such as chlorophyll, the presence of enzymes (lipases ...), and the heat that will alter the oil quality and its richness of the vitamin e. 8 nova biotechnol chim (2021) 20(1): e815 table 5. corrective measures and nominal values to set up for an adequate cold pressing. deviation d1/d2 d3 d4/d5 d6 d7 d8/d9 d10 measure humidity control (measure the seed moisture content) definition of storage conditions. the seeds must be dried before pressing. sieving to determine the seed size, to choose the right nozzle diameter. if the seeds are relatively hard, it is necessary to ensure that their moisture is in the standards (10% by weight). it would be preferable to work: without heating the press head, with the larger 15 mm diameter nozzle and with a slow residence time to avoid rapid accumulations at the press crusher barrel. it would be necessary to initially introduce a small amount of seed into the loading tank and leave the machine in normal operation and wait for the appearance of the first cake to come out and then increase the load gradually to reach a full load. washing the seeds before use is essential to remove undesirable particles, and it is always followed via air blowing, it makes the pressing much easier. provide a loading protocol based on the recommendations and measures presented previously (deviation d6). it is necessary to completely fill the seed press to cause a sufficient pressure and consequently to increase the yield. sorting out the raw material is necessary to avoid any undesirable particles during loading. nominal value average seed moisture content is an average of the order of 10 %. seeds humidity control the seed size must be compatible with the used nozzle the water content has a significant effect on the seed hardness. however, using a hardness test could predict grain hardness based on particle size analysis. towards zero dust fulfilling the press in a steady state. pure raw material 100 % 9 nova biotechnol chim (2021) 20(1): e815 6 table 5. corrective measures and nominal values to set up for an adequate cold pressing. continued. deviation d11 d12 d13/d14 d15 d16 d17 d18 measure reducing the screw speed increases the residence time, ensuring more time for pressing, thus increasing the efficiency of the extraction. it is not necessary to press seeds by heat in order to preserve the maximum of the antioxidant elements ensuring good quality of oil (the heating created during the pressing process alone could reach a high temperature). choose the appropriate nozzle according to the seed hardness. preheat the press head via the heating ring for a period of 30 min at a temperature of 100 °c, and at the same time rotate the screw at 30 50 rpm. the rotation of the screw has a major role in the mechanics, the dilation and the uniformity of the temperature throughout the pressing chamber. it is recommended to activate the screw rotation with heat before the press loading. clean the mechanical parts of the press head after use. store the oils in a fresh place and avoid excess heat. preferably a refrigerator in a firm bottle to minimize the oxidation phenomenon. using an opaque bottle to avoid sunlight and ultraviolet light that affect the oil quality. it is also important to clean the outer edges and the screw thread of the bottle with a dry tissue as a precaution to avoid the internal humidity of the bottle. nominal value 30 rpm pressing without temperature or at lower degree. (t < 50 °c) nozzle diameter 15 mm (for prickly pear seeds) preheat the press head for 30 min at a temperature around 100 °c and a press speed 30-50 rpm. immediately. fresh place (t < 20 °c). opaque bottle with an inner aluminium wall is covered with a cling film which prevents the direct contact of the oil with the metal and avoids oxidation. 10 https://www.linguee.fr/anglais-francais/traduction/ultraviolet%2blight.html https://www.linguee.fr/anglais-francais/traduction/ultraviolet%2blight.html https://www.linguee.fr/anglais-francais/traduction/ultraviolet%2blight.html https://www.linguee.fr/anglais-francais/traduction/ultraviolet%2blight.html https://www.linguee.fr/anglais-francais/traduction/ultraviolet%2blight.html https://www.linguee.fr/anglais-francais/traduction/ultraviolet%2blight.html https://www.linguee.fr/anglais-francais/traduction/ultraviolet%2blight.html nova biotechnol chim (2021) 20(1): e815 11 besides, the results of the risks identified by the hazop analysis are depicted in table 4, which are more specifically “operability risks” that affect the press performance, the product quality and the economic operation of the unit. therefore, hazop study forms a useful guide to the maintenance personnel, providing measurements – recommendation and nominal values listed in table 5, to achieve adequate cold pressing. results and discussion the optimal conditions of cold pressing method the cold-pressing operability risks have been identified based on hazop analysis. each process parameters have been studied in order to determine the optimal pressing conditions. the experience plan has been reduced, according to hazop results, instead of performing experiments on all the manipulable variables of the press, the seeds were pressed based on the screw speed variable. the press performance was more effective and without any operating problems when the pressing is based on the corrective measures and nominal values mentioned below (table 6 and 7). table 6. raw material storage and feeding press conditions. seed properties nominal values seed moisture average of the order of 10 %. seed hardness high hardens level seed granulometry the grain size must be compatible with the used nozzle, without grinding the seeds. seed cleanness towards zero dust table 7. optimal conditions of prickly pear seeds cold pressing. parameters nominal value corrective measures screw speed 30 rpm reducing the speed to 30 rpm increases the residence time thus ensuring more time for the pressing to take place and thus increasing the extraction efficiency. temperature pressing without heat pressing without heat or at lower temperature (t < 50 °c). the heat created during the pressing process is enough. it is not necessary to press seeds by heat in order to preserve the maximum of the antioxidant elements, ensuring good quality of oil. press loading total filling of the press (100 %) load the seeds with small quantities (wrists of the hand) until the appearance of the first cake (10 – 15 s) then completely fill the press. it is necessary to completely fill the seed press to cause a sufficient pressure and consequently to increase the yield. nozzle diameter 15 mm the grain size must be compatible with the used nozzle. preheating the press preheat the press head for 30 min at a temperature around 100 °c and a press speed 30 – 50 rpm. the rotation of the screw has a major role in the mechanics, the dilation and the uniformity of the temperature throughout the pressing chamber. it is recommended to activate the screw rotation before the press loading with heat. cleaning the press after use press after use immediately non-cleaning causes a very strong bond between the seeds and the metal of the press head. a forced opening probably will break the press head. oil storage under cold conditions fresh place (t < 20 °c) opaque bottle with an inner aluminium wall is covered with a cling film which prevents the direct contact of the oil with the metal and avoids oxidation. oil yield the cold-press extraction process was carried out 6 times, without heat changing only the pressing speed and considering hazop analysis results mentioned previously. the results obtained in table 8 show that the highest oil yield is 4.85 % on a dry basis when the pressing is at speed of 30 rpm and the lowest oil yield is 3.13 % when the pressing speed is slower at 15 rpm, also the yield decreased to 4.17 % when the nova biotechnol chim (2021) 20(1): e815 12 14 16 speed increased above 30 rpm. when the screw speed is slower than 30 rpm, it provides more time (long residence time) for the seed pressing, but at the same time the press clogs more, which leads to decrease the seeds flow loading and minimize yield. indeed, when the screw turns faster, more material is moving through the press and this provides less time for the oil to migrate out and be separated from the meal. so, in both cases, a lower yield is obtained. as a result, the optimum pressing speed is 30 rpm, at this speed, the press oil extraction rate is higher and a lower amount of oil left in the meal. table 8. variation of oil extracted yield with pressing speed. the obtained cold-pressed seed oil yield (4.85 %, on a dry basis) is lower compared to the seed residual oil content (7 %) extracted by hexane. significant amount of oil remains stored in the cake produced during cold pressing. according to the data, a comparative study (mouden et al. 2012) that was carried out on opuntia ficus indica (l.) mill. seeds (origin from aït baamrane), through two different extraction methods, one by cold pressing and the other by solvent, the maximum oil obtained was (5.90 %) following the cold pressing method and (7.79 %) following a solvent extraction method. in other studies, as taoufik et al. (2015) and el hachimi et al. (2015) results, the seed oil yield of the same studied variety (ofi originate from aït baamrane, morocco) by the same extraction process (solvent extraction) was (7.6 – 8.74 %), respectively. the difference between the yield could be attributed to the cultivar, geographical origin of fruits, degree of maturity and the storage conditions but also to the extraction protocols and analytic assays (brahmi et al. 2020, el hachimi et al. 2015). in our case, the cold pressing yield was influenced by seed properties and its storage conditions, press feeding, and operating conditions. oil characterisation the extracted seed oil has some interesting organoleptic properties, namely a greenish yellow color with a strong characteristic odor. according to ramadan and mörsel (2003) study, ofi seed oil contains a lower amount of carotenoids (b-carotene) compared to pulp oil. carotenoids may be the source of the light-yellow hues of seed oil, while the pulp oil is characterised by dark orange hues. fatty acid composition the gc results (table 9) showed that linoleic acid is the dominant fatty acid, with high content that reaches up to (60.42 %) followed by oleic acid (21.65 %), palmitic acid (12.24 %) and stearic acid (3.88 %), respectively. apparently, the extracted oil is highly unsaturated. table 9. fatty acid compositions of prickly pear seed oil. fatty acids concentration [%] palmitic acid (c16:0) 12.24 palmitoleic acid (c16:1 n-7) 0.70 margaric acid (c17:0) 0.05 heptadecanoic acid (c17:1) 0.02 stearic acid (c18:0) 3.88 oleic acid (c18:1 n-9) 21.65 linoleic acid (c18:2 n-6) 60.42 α-linolenic acid (c18:3 n-3) 0.22 arachidic acid (c20:0) 0.33 paullinic acid (c20:1 n-7) 0.35 sfa : c16:0+c18:0 16.12 pufa: c18: 60.42 pufa/sfa: c18:2/ (c16:0+c18:0) 3.74 sfa = saturated fatty acids; pufa = polyunsaturated fatty acids; pufa/sfa = unsaturation ratio. our results are in good agreement with taoufik et al. (2015) results, linoleic acid is the dominant fatty acid (61.5 %) followed by oleic acid (21.3 %), palmitic acid (11.9 %) and stearic acid (3.3 %), respectively. there is no difference between the fatty acid concentration of our cold-pressed oil and the fatty acid concentration of the solvent extracted oil from the same ofi, and from the same geographical origin (aït baamrane, morocco). according to brahmi et al. (2020) results, the algerian cold-pressed seed oil (ofi) contains test screw speed [rpm] oil content [%] dry basis 1 15 3.13 2 20 3.78 3 25 4.07 4 30 4.85 5 35 4.57 6 40 4.17 12 nova biotechnol chim (2021) 20(1): e815 13 15 linoleic acid as the main compound represents (55,81%), followed by oleic acid (12.99 %), palmitic acid (10.8 %) and stearic acid (2.97 %), respectively. the cold-pressed seed oil of brahmi et al. (2020) has a lower concentration of fatty acids compared to our results and taoufik et al. (2015) results. therefore, we conclude the extraction method type does not affect the concentration of fatty acids, the geographical origin of the fruit does. indeed, ramadan and mörsel (2003) explained the difference between the content of fatty acids which is due to different locations of the cactus cultivation, the content of individual fatty acids differed significantly. also, it is influenced by geographic and climatic differences (el finti et al. 2013). sterols composition the sterol fraction of ofi seed oil is defined by betasitosterol as the main component, representing 67.56 % of the total sterols, followed by campesterol (11.87 %) and δ5-avenasterol (4.62%). other compounds (stigmasterol, δ7-avenasterol, cholesterol) were found with a lower percentage (table 10). table 10. sterol composition of prickly pear seed oil. sterols concentration [%] cholesterol 1.03 β-sitosterol 67.56 campesterol 11.87 δ5avenasterol 4.62 δ7-avenasterol 2.72 stigmasterol 2.47 the results are in strong alignment with taoufik et al. (2015), ramadan and mörsel (2003). the betasitosterol represented in their studies 75 % and 72 % of total sterols fraction of ofi seed oil, respectively. the contents of δ5and δ7-avenasterols were higher in cold-pressed oil in comparison with their contents in both solvent extracted oils (4.8 %, 3.1% ̶ 0.9 %, 0.53 %, respectively). phytosterols, including beta-sitosterol, campesterol, stigmasterol, and others, are known for having many benefits for human health such as lowering of blood cholesterol. for several decades, it has been appreciated the consumption of plant sterols and stanols that lead to favorable shifts in circulating lipid levels (jones et al. 2000). tocopherols composition the cold-pressed oil contained the gammatocopherol as the major dominant fat-soluble vitamin representing 97.56 % of total tocopherols. this significant content exceeds the gamma-tocopherol content in the ofi solvent extracted oil (taoufik et al. 2015), that was 89.1 % from total tocopherols. regarding the alpha-tocopherol content that represented 1.5 %, it was similar. however, betatocopherol was not detected in our oil analysis (table 11). table 11. tocopherols composition of prickly pear seed oil. (nd – not detected) tocopherols [mg.kg-1] [%] alpha 12.65 1.58 beta nd nd gama 777.30 97.56 delta 6.74 0.84 total tocopherols [mg.kg-1] 796.70 nd according to ramadan and mörsel (2003), gammatocopherol was the dominant compound in amount of 330 mg.kg-1, followed by alpha-tocopherol (56 mg.kg-1). our experiment revealed that gammatocopherol content was significantly higher (777.30 mg.kg-1), followed by alpha-tocopherol that represents (12.65 mg.kg-1). amount of total tocopherols was 796.70 mg.kg-1 that was also much more than referred ramadan and mörsel (2003) (403 mg.kg-1 ). assessment of prickly pear seed oil yield and quality showed that almost 70 % of high-quality oil has been extracted that contains about double of the total tocopherols in comparison to results of ramadan and mörsel (2003). this is due to the methodology followed, which considered the seed properties and its storage conditions, as well as the press feeding and operating conditions. this approach was less devastating and improved the quality of cold-pressed seed oil to 49.4 % compared to results from other studies (table 12). pressing without heat enhanced the quality of prickly pears seed oil, preserved its richness in tocopherols sensitive to light (ultraviolet radiation) and heat. in agreement with bostyn et al. (2008), it was clearly explained that tocopherols 13 nova biotechnol chim (2021) 20(1): e815 12 14 16 degradation is due to the major factor which is the temperature, especially above 100 °c. according to rueda et al. (2016) multivariate factor analysis revealed that tocopherols, particularly the gammatocopherol, was the strongest variable influencing the discrimination of different oils. furthermore, gamma-tocopherol helps reduce inflammation, guards against certain cancers, and protects against radiation exposure (gysin et al. 2002; stone et al. 2004). the gamma-tocopherol, as other tocopherols, also helps moisturize the skin while fighting against wrinkle-forming free radicals (nagata et al. 2010). prickly pear oil also contains a high level of antioxidants and has antibacterial properties. some healthful compounds reduce skin inflammation, as well as prevent skin damage and acne (koubaa et al. 2017). ofi seed oil is a suitable and safe carrier for delivering other nutrients that cannot be directly applied to the skin, including vitamin a. ofi seed oil has valuable applications in cosmetic applications (alzahabi et al. 2019). ofi seed oil is highly unsaturated, rich in antioxidants therefore, it could also be added to foods as natural antioxidant additive and fatty acids supplement. table 12. assessment of prickly pear seed oil yield and quality. reference 1 reference 2 vitamin e concentration [mg.kg-1] 796.7 403 yield [%] 4.85 9.8 oil density [kg.m3] 0.9 optimization [%] 49.4 1cold-pressed prickly pear seed oil extracted from seeds originated from aït baamrane, morocco 2prickly pear seed oil extracted by solvent, originate from berlin, germany (ramadan and mörsel 2003). acidity observed acidity of ofi seed oil was 0.8 %. it is a lower value in relation to the standard value approved by codex alimentarius (1999) (6 % for cold-pressed oil) and also in comparison with studies brahmi et al. 2020 (2.12 %) and özcan et al. 2011 (1.41 %). these high values might be due to the chemical reactions in seeds during oil harvesting, handling or processing. oil exposed to inappropriate storage conditions deteriorates its quality in different ways, but most often by hydrolysis or oxidation (kandji 2001). according to the previous data, ofi seed oil is pure and can be considered virgin oil. peroxide value the pv could be considered an indicator of oils and fats quality and stability (zahir et al. 2017). the pv of ofi seed oil is 9.6 meq o2.kg -1 an acceptable peroxide value at the standard value of 15 meq of peroxides.kg -1 of oil for a cold-pressed oil specified by the codex alimentarius (1999). different cultivars of prickly pear and different extraction methods revealed pv in range from 9.50 to 18.02 meq o2.kg -1 (south african prickly pear, solvent extracted, dewit et al. 2017), 12 meq o2.kg 1 (algerian prickly pear, cold-pressed, brahmi et al. 2020), and 1.63 meq o2.kg -1 (turkish prickly pear, solvent extracted, özcan et al. 2011). the pv could be affected by the oil oxidation during the extraction and conservation conditions, decreasing of certain antioxidant substances present in oil such as carotenoids, vitamins a and e, and squalenes (zahir et al. 2017). conclusion the present study established a risk prevention platform to achieve adequate cold pressing performance for the oil industry. the methodology and correct measurements resulting from the proposed approach could be taken into account for the cold pressing of prickly pear seeds, but also other oilseeds with high hardness of seeds. the seed oil yield was influenced by properties of seeds, seed storage conditions, press feeding, and operating conditions. presented methodology had no effect on the fatty acid and sterol contents, but affected oil quality in terms of tocopherols content. oil had high quality, was rich in essential fatty acids and sterols, very high contents of tocopherols, chemically free from organic solvents, with lower value of acidity and peroxide value. this indicated its purity and stability. acknowledgements this work would not have been possible without the financial support of cnrst (morocco) program of excellence scholarships for research. 14 nova 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history: received: 26th february 2021 accepted: 11th december 2021 keywords: bacterial metagenome analysis high-performance sequencing kombucha taxonomic diversity abstract the aim of the present work was to analyze the taxonomic diversity in the kombucha bacterial consortium during long-term cultivation in the north (arctic) of the european part of russia. the high-performance sequencing showed that 99.1 % of the bacterial fraction of the fresh 7-d culture was proteobacteria of mostly cellulose-producing genera with the order acetobacterales being dominant and represented by the komagataeibacter (87.3 %) and gluconobacter (6.3 %). aging of the kombucha bacterial consortium from 20 to 90-d cultivation lead to the reduction of number of cellulose-producing bacteria and intense growth of cellulolytic bacteria including clostridiales, bacteroidetes, actinomyces. also the acidophilic microorganisms have been detected. the long-term growth of the kombucha bacterial consortium can be considered as a succession of microbial communities. introduction kombucha is a mutualistic symbiotic community of bacteria and yeast known for several centuries in the areas of europe, asia and africa. the liquid brew obtained as a result of the microbial community development is consumed as a healthy drink of domestic production (matei et al. 2018; villarreal-soto et al. 2018; ivanišová et al. 2019; may et al. 2019; khaleil et al. 2021). the second product of the microbiome activity, bacterial cellulose, has been intensively studied due to its valuable properties and potential applications (campano et al. 2016; jozala et al. 2016). the drink quality depends mostly on starting nutrient medium and the taxonomic diversity of the microbiome. recent molecular and genetic approaches to the taxonomic study allow an accurate quantitative analysis of the species diversity (chirak et al. 2013). culture-independent approaches, such as high-performance sequencing, eliminate the difficulties associated with cultivation, and possible contamination with noncultivated species. in contrast to controlled laboratory conditions, domestic conditions do not ensure the stability of the culture, and the household geographical location affects the formation of unique kombucha communities. microbial consortiums formed in geographically remote territories differ significantly in taxonomic diversity (safak et al. 2002; ramadani et al. 2010; marsh et al. 2014). for example, significant differences have been described for kombucha samples from 32 mailto:k.bolotova@narfu.ru nova biotechnol chim (2022) 21(1): e884 2 households in germany (mayser et al. 1995). the kombucha microbiome forms in specific conditions of a particular geographical area, thus, the influence of environmental factors is also significant. it is important to note that previously taxonomic changes in the kombucha consortium were only registered during cultivation for less than 21 days (marsh et al. 2014; chakravorty et al. 2016). our preliminary studies on kombucha longterm cultivation have shown that near the 90th day the redistribution of dominant species took place. thus, changes in the trophic structure and diversity of the community, accompanied by a change in the dominant species, i.e. the succession of the microbiome is of great interest. the aim of this work was to study changes in the taxonomic diversity of the bacterial fraction of kombucha community during microbiome cultivation up to 90 days in households in the north (arctic) of the european part of russia. experimental kombucha preparation and sampling five samples of the kombucha consortium were cultivated at five different households (russia, arkhangelsk region). the starting kombucha inoculum was the same for all household samples. it was divided into five equal portions and placed in different households. the microbiome cultivation was carried out at 20 – 25 °c in 3 l glass vessel for 7 d (household a), 10 d (household b), 15 d (household c), 20 d (household d) and 90 d (household e) on a semi-synthetic medium. the media contained sucrose as the main c-source and black tea extract. black tea infusion was prepared by adding of dry black tea leaves to hot water at the concentration of 10 – 12 g.l-1. after steeping for 15 min infusion, the infusion was filtered and cooled to room temperature and then 20 g.l-1 of sucrose was added to the infusion. cultivation was carried out under static conditions by the periodic method. samples of the kombucha consortium harvested in cellulosic matrix were separated from the cultural medium and frozen to preserve the genetic material. dna isolation, pcr reaction and highperformance sequencing of the bacterial community a nucleospin soil kit (macherey-nagel, germany) was used for dna isolation from kombucha samples according to the protocol. the cell membranes were destroyed by milling of 100 – 200 mg of a sample with 0.1 mm diameter ceramic balls in a lysing buffer twice for 20 min at 6,000 rpm using a precellys 24 homogenizer (bertin technologies, france). the taxonomic composition of the community was determined by high-performance sequencing based on the analysis of amplicon libraries of ribosomal operon fragments. universal primers f515/r806 for a variable section of the 16sррнкv3-v4 gene (gtgccagcmgccgcggtaa/ggactacvsg ggtatctaat) specific to a wide range of microorganisms, including bacteria and archaea were used (bates et al. 2010). pcr reaction was carried out in 15 µl mixture containing 0.5 – 1 unit of q5® high-fidelity dna polymerase (new englang biolabs, usa), 5 pm of forward and reverse primers, 10 ng of dna matrix, and 2 nm of each dntp (lifetechnologies). the mixture was denatured at 94 °c for 1 min, followed by 35 cycles for 30 seconds at 94 °c, for 30 seconds at 50 °c, and for 30 seconds at 72 °c. the final elongation was carried out at 72 °c for 3 min. pcr products were purified using ampurexp (beckman coulter, usa) following the illumina recommended protocol. the libraries were prepared in accordance with the miseq reagent kit preparation guide (illumina inc. san diego, usa). the libraries were sequenced in accordance with the instructions on an illumina miseq device (illumina inc. san diego, usa) with miseq ® reagent kit v3 reagents with a double-sided reading. there were received at least 10,000 reads for each library. data obtained from sample sequencing were processed using trimomatic (bolger et al. 2014) and qiime (caporaso et al. 2010) software packages (available at: http://quiime.sourceforge.net/). the procedures for denoising and chimeras deleting were carried out using the dada2 r package as described in (callahan et al. 2016) to obtain a table http://quiime.sourceforge.net/ nova biotechnol chim (2022) 21(1): e884 3 of frequencies of unique sequences (asv, amplicon sequence variant). primary bioinformatic processing of the obtained data was performed with the results presented as a diagram of bacteria distribution. the results were processed using the microsoft office excel software package. scanning electron microscopy analyses morphology of the bacterial cellulose matrix was visualized using the high-resolution scanning microscope sem sigma vp zeiss (germany) equipped within-lens detector at 10 kv accelerating voltage. prior to the microscopic study samples of bacterial cellulose were subjected to lyophilization using labconco equipment (freezone 2.5 l, usa). results and discussion the microscopy of the 7-d biofilm sample showed the bacteria and yeasts microbial cells entrapped in the cellulose matrix (fig. 1). the cellulose-producing bacteria in the kombucha microbiome provide the cellulose biosynthesis and structuration of consortium matrix (fig. 1b) (bolotova et al. 2016). the formation of a polysaccharide matrix may involve bacteria that produce non-cellulose exopolysaccharides, such as lactic acid bacteria. yeasts convert media sucrose to ethanol and carbon dioxide in the process of alcoholic fermentation, while bacteria provide assimilation of ethanol by converting it to acetic acid (campano et al. 2016). fig. 1a represents yeast budding forms and rod-shaped bacterial cells in the structure of the cellulose matrix. fig. 1. morphology of bacteria and yeasts in the cellulose nanofibrous network (a) and spatial localization of cellulosesynthases and cellulose fibrils at the surface of the cellulose-producing cell (b). bars: a – 1 µm; b – 200 nm. metagenomic studies of the bacterial fraction showed that the content of unclassified bacteria varied in the studied samples from 0 to 13.8 % (table 1). the following bacterial phyla were found in all kombucha samples, regardless of the cultivation period: actinobacteria (0.3 – 27.7 %), firmicutes (0.3 – 23.3 %) and proteobacteria (22.1 – 99.1 %). the bacteroidetes were absent at early stages of culture growth (7-d culture); as the cultivation proceeded the bacteroidetes biomass increased and it reached 8.3 % as the community ages. at the initial cultivation stages (7-d culture), kombucha microbiome bacteria consisted of 99.1 % protobacteria including members of cellulose-producing species. the phylum was represented by the alphaproteobacteria (93.6 %) and gammaproteobacteria (5.5 %) classes. the order acetobacterales is the dominant one in the class alphaproteobacteria and is represented by the genera komagataeibacter (87.3 %) and gluconobacter (6.3 %). the number of lactobacillales members also varied depending on the cultivation time from 0.2 – 2.5 % (7 – 20 d) to 14 % (90 d) (data are not presented in table 1). the lactobacillales is included in table 1 as the representatives of phylum firmicutes. nova biotechnol chim (2022) 21(1): e884 2 table 1. composition of the kombucha bacterial microbiome. phylum percentage in the bacterial microbiome [%] 7 d (hous.а) 10 d (hous.b) 15 d (hous.c) 20 d (hous.d) 90 d (hous.e) proteobacteria 99.1 86.1 97.4 95.1 22.1 actinobacteriа 0.3 2.4 0.5 2.0 27.7 firmicutes 0.3 5.8 1.5 1.3 23.3 bacteroidetes – 3.8 0.5 0.7 8.3 patescibacteria – 1.1 – – 0.3 fusobacteria – 0.5 – – 0.6 verrucomicrobia – – – – 1.1 epsilonbacteraeota – – – – 0.7 planctomycetes – – – 0.1 0.5 cyanobacteria – – – – 0.6 acidobacteria – – – 0.1 0.4 deinococcus-thermus – – 0.1 – 0.1 spirochaetes – – – – 0.2 chlamydiae – – – – 0.1 synergistetes – – – – 0.1 archaea: thaumarchaeota – – – – 0.1 unclassified 0.3 0.3 – 0.7 13.8 earlier metagenomic studies of kombucha microbial communities (jayabalan et al. 2014; reva et al. 2015) also revealed the dominance of two acidobacterial genera, komagataeibacter (gluconacetobacter) and gluconobacter. the composition of the community has been shown to depend on the growing conditions (reva et al. 2015). although, the bacterial community was relatively stable, in some cases it included additionally lactobacillus. similar studies (coton et al. 2017) have found that the dominant bacterial species belonged to the families acetobacteraceae and, to a lesser extent, to the lactobacteriaceae. the gluconacetobacter species were isolated from the commercial kombucha kombu australia and were used to produce cellulose (nguyen et al. 2008). in most cases, the gluconacetobacter bacteria composed more than 85.0 % of the bacterial community, while the acetobacter members were represented in a small amount (2.0 %) (marsh et al. 2014). lactobacillus and some other subdominant species were also found in kombucha consortium in previous works (marsh et al. 2014; coton et al. 2017). therefore, literature data show the relative homogeneity of the kombucha bacterial community. our results obtained for households in the north of the european part of russia for the microbiome cultured up to 20 d correspond to previous works. in all the samples for cultivation period of 7 – 20 d, proteobacteria specifically acetic acid bacteria, dominated. the accumulation of acetic acid and ethanol in the kombucha microbiome prevents the development of foreign microflora. in addition, ethanol can reduce the growth rate and aging of the bacterial population in the microbial community. with an increase in the cultivation time up to 90-d, the composition of the microbiome changed and three divisions dominated in almost equal proportions: actinobacteria (27.7 %), firmicutes (23.3 %) and proteobacteria (22.1 %). as the microbiome ages micrococcales, corynebacteriales, pasteurellales and betaproteobacteriales (fam. burkholderiaceae), sphingomonadales developed in it (fig. 2). the content of acetic acid bacteria, the key species for the formation of cellulose and creating optimal conditions for the development of the microbiome, was reduced to 1 % by 90 d. the dominant genera of the aged culture (fig. 2) included lactic acid bacteria of the genus lactobacillus (6.3 %) and some streptococcus species. acetobacteria and lactobacteria were shown to be in symbiotic relationship; lactic acid bacteria metabolites, xylitol and acetic acid, promoted the growth of acetic acid bacteria (yang et al. 2010). during prolonged kombucha fermentation organic acids, such as gluconic, lactic, succinic, etc. are accumulated in the media (chen et al. 2000; greenwalt et al. 2000). in 90-d culture minor acid-producing genera were detected, specifically cutibacterium or 4 nova biotechnol chim (2022) 21(1): e884 2 propionibacterium (7.6 %) and veillonella (1.1 %), which are involved in the synthesis of propionates and acetates, as well as corynebacteria (2.0 %), facultative anaerobes with redox-enzymatic metabolism, participating in the synthesis of glutamic acid (fig. 2). futhermore microbiome, bacterial cellulose became a substrate for microbial enzymes. among the detected genera clostridiales (3.6 %) in phylum firmicutes, bacteroidetes and actinomyces (1.9 %) in phylum actinobacteria represented the most active cellulose destructors observed at the early stages of development of the microbial community and not detected in a 90-dayold culture. fig. 2. percent of actinobacteria, firmicutes, and proteobacteria phylum that predominate in the 90-day-old culture community, % of the bacterial microbiome (genera that are less than 1 % present in the community are not shown in the diagram). the substrate depletion, the accumulation of acidic metabolites, and the formation of local anaerobic zones in the cellulose matrix during long-term cultivation lead to the development of microorganisms that are completely uncharacteristic of the kombucha symbiosis. unique genera were identified in arctic region kombucha. those were nitrogen fixers of the rhizobiaceae family, ensifer (0.6 %) and rhizobium (0.1 %), which are able to assimilate air nitrogen when the nutrient medium is depleted. previously, members of the family acetobacteraceae isolated from kombucha also demonstrated the ability to nitrogen fixation (dutta et al. 2010). cyanobacteria or blue-green algae, representing the division of photosynthetic gramnegative bacteria, were identified in an amount of 0.5 %. archaea of the nitrososphaeraceae family, ammonia-oxidizing chemolithotrophs, were also found in the sample. their adaptation in the environment can be associated with autolytic processes and proteolysis accompanied by ammonification. thus, the development of kombucha bacterial consortium over time can be considered as a succession of microbial communities resulted in changes in its composition. during kombucha fermentation, acetic acid bacteria assimilated sugars and produced exopolysaccharides and acidic metabolites. then microorganisms that assimilate organic compounds produced in autolytic processes and enzymatic hydrolysis of biomass, use specific metabolic schemes, adapted and developed. hydrolytic bacteria provided cellulose destruction. monomers and metabolites were consumed by dissipotrophs. anaerobic hydrolytics and dissipotrophs constitute the primary group of anaerobes. the primary hydrogen-producing anaerobes as well as secondary anaerobes consuming unfermentable compounds were detected. conclusion the taxonomic diversity was analysed for kombucha samples harvested in households located in northern european russia. high5 nova biotechnol chim (2022) 21(1): e884 2 performance sequencing revealed qualitative and quantitative differences in the composition of the microbiome during long-term cultivation. the microbiome cultivated up to 20 d showed a relative uniformity of the bacterial community. during the initial period (7-d culture), cellulose-producing proteobacteria represented 99.1 % of the consortium bacteria. the dominant genera were komagataeibacter (87.3 %) and gluconobacter (6.3 %). when the microbiome aged (90-d cultivation), the number and variety of cellulosesynthesizing bacterial species significantly decreased. bacterial cellulose became a carbon source for the microbial community. microorganisms secreting cellulolytic enzymes, clostridiales, members of bacteroidetes and actinomyces, started to grow. along with acetic acid bacteria, acid-producing propionibacterium, veillonella, corynebacterium adapted and developed. the nitrogen-fixing genera of the rhizobiaceae family, archaea of the nitrososphaeraceae, as well as cyanobacteria were identified in 90-d culture of the arctic region kombucha microbiome. acknowledgments the reported study was funded by rfbr according to the research project № 18-33-00855 “supermolecular organization of cellulosic microfibrils derived from plant and bacterial cellulose”, northern (arctic) federal university, 2019. the research involved scientific equipment from the shared use of equipment center “arktika” (narfu) and the core centrum “genomic technologies, proteomics and cell biology” in arriam. conflict of interest the authors declare that they have no conflict of interest. references bates st, berg-lyons d, caporaso jg, walters wa, knight r, fierer n (2011) examining the global distribution of dominant archaeal populations in soil. isme j. 5: 908917. 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biotechnologica et chimica effect of chitosan membranes against gram-negative bacteria isolated from cutaneous ulcers jaime lópez-cervantes 1 , ana a. escárcega-galaz 1 , dalia i. sánchez-machado 1, , benjamín ramírez-wong 2 , ramssel d. valenzuela-rojo 1 , olga n. campas-baypoli 1 , ernesto u. cantú-soto 1 , carlos u. mendivil-gastelum 1 1 instituto tecnologico de sonora, ciudad obregon, mx-85000 sonora, mexico 2 universidad de sonora, hermosillo, mx-83000, sonora, mexico  corresponding author: dalia.sanchez@itson.edu.mx article info article history: received: 23 rd december 2020 accepted: 7 th may 2021 keywords: antimicrobial agent chitosan escherichia coli gram-negative skin ulcers in vitro membranes antibiotic antibiotic sensitivity abstract the objective of this study was to evaluate the in vitro effectiveness of membranes developed with pure chitosan and chitosan in a mixture with glycerol-honey against gram-negative bacteria isolated from skin ulcers. the membranes were prepared by the solvent evaporation technique. the identification and antibiotic sensitivity of microorganisms were determined in microplates, and in vitro tests were developed by the agar diffusion technique. the most frequently isolated microorganism was escherichia coli with 43.75 % prevalence. all membranes showed antimicrobial effects by direct contact against proteus mirabilis, escherichia coli, enterobacter aerogenes, pseudomonas aeruginosa, klebsiella pneumoniae and morganella morganii. antibiograms showed that most of these microorganisms are multiresistant to antibiotics. all of this suggests that chitosan-based membranes are a safe alternative for the treatment of infected cutaneous ulcers compared to traditional antibiotics. the outcomes of this study confirm that membranes made of a biodegradable polymer, such as chitosan have activity against multidrug-resistant gram-negative bacteria that grow in infected skin ulcers.  university of ss. cyril and methodius in trnava introduction chronic wounds cause severe financial problems due to the medical attention they require. these types of wounds are characterized by slow healing, or they may never heal, which generates physical and emotional stress to the patient. the degree of infection always depends on the species and concentration of the microorganism, as well as the response of the host (akuzy et al. 2018). according to data reported by davies et al. (2007), chronic wounds on the skin affect approximately 3 % of people over 60 years of age; additionally, these types of wounds maintain polymicrobial biota. the microbial biota found in chronic wounds includes staphylococcus, streptococcus, pseudomonas and coliforms. it is also possible to find anaerobic bacteria because conditions are generated for them to proliferate through the combination of necrotic tissue and low levels of oxygen. for gentili et al. (2012), the presence of 10 5 or more cells per gram of tissue is a fundamental indicator to confirm that healing will be slow. goswami et al. (2017) reported more frequent microorganisms to be staphylococcus aureus, pseudomonas aerumailto:dalia.sanchez@itson.edu.mx nova biotechnol chim (2021) 21(1): e806 2 ginosa, staphylococcus epidermidis, and candida albicans. specifically, s. aureus and p. aeruginosa are the cause of nosocomial infections with an incidence of 20 – 40 % and 5 – 15 %, respectively (mama et al. 2014). particularly, p. aeruginosa is an opportunistic gram-negative pathogen capable of causing severe infections in ulcers, in addition to being resistant to multiple antibiotics (myhrman et al. 2013). microorganisms of clinical origin develop various mechanisms of resistance to antibiotics due to prolonged therapies. therefore, the selection of the type and dose of antibiotic is essential to prevent the spread of resistance between microorganisms and promote the healing of ulcers (cunha et al. 2018). chitosan is a natural polymer obtained by deacetylation of chitin; it is composed of dglucosamine and n-acetyl-d-glucosamine (balti et al. 2017). its biological applications are due to its biocompatibility, biodegradability, and its nontoxicity (babushkina et al. 2015; el-malek et al. 2017). the amine groups of chitosan provide the antimicrobial capacity (ashrafi et al. 2018) and hemostatic properties. due to its chemical properties, chitosan has the ability to form films, hydrogels, and fibers (kiroshka et al. 2014; khalil et al. 2015). its potential application in the area of medicine is due to its affinity to aminoglycosides of human tissues (babushkina et al. 2015). additionally, it is characterized as being hypoallergenic (koryagin et al. 2006). the mechanism of action of chitosan is that it alters the cell surface and its permeability, causing the leakage of intracellular substances. this is attributed to the fact that the positive charges of the amino group of chitosan interact with the negative charges of the surface of microorganisms (demir et al. 2010; uranga et al. 2019). according to dragostin et al. (2016), when applying dressings based on chitosan for wound healing, chitosan is degraded into n-acetyl glucosamine, which is used to accelerate the re-epithelialization process. the strategy of using pure chitosan and its topical combinations as an alternative to antibiotics has been proposed to health institutions. in reference to the above, the aim of this work was to evaluate the in vitro effectiveness of films developed with pure chitosan and in mixture with glycerol-honey against gram-negative bacteria isolated from cutaneous ulcers. honey was incorporated into chitosan films as an adjuvant agent because it has been used to eliminate infections, is a healing agent in skin ulcers (alam et al. 2014) and is an antiinflammatory agent (meo et al. 2017). according to basualdo et al. (2007), honey promotes the formation of granulation tissue, the growth of the epithelium and healing. in addition, honey has antimicrobial activity against s. aureus, p. aeruginosa and k. pneumoniae (el-malek et al. 2017). experimental physicochemical characteristics of chitosan in this research we worked with chitosan obtained in our laboratory (sánchez-duarte et al. 2012). the humidity and ash content of the chitosan were 92.42 ± 0.07 % and 0.37 ± 0.02 %, respectively. its molecular weight was 119.48 kda and the degree of deacetylation was 84.59 ± 0.87 %. additionally, the functional groups characteristic of chitosan were identified by fourier transform infrared spectroscopy (ftir). preparation of chitosan membranes the membranes were prepared using the solvent evaporation technique. six different formulations were developed, three were of pure chitosan at 1, 2 and 3 % in acetic acid at 1 % (w/v), another of 2 % chitosan with 0.2325 ± 0.01 g of glycerin and the other two formulations were 2 % chitosan and honey (95 : 5 v/v), glycerin was added to one of them. the honey was diluted in water (80 : 20 v/v). all the mixtures were homogenized until all the components were fully incorporated. specifically, 10 ml of each of the formulations were measured independently and a plastic mold was poured and subsequently dried at 40 °c for 24 h. finally, the membranes were detached from the molds and stored in sterile plastic bags. sample collection the study was descriptive and cross-sectional. the samples were taken from infected ulcers of patients hospitalized by a doctor specialized in epidemiology. specifically, the sampling was done nova biotechnol chim (2021) 21(1): e806 3 in the ulcer center with sterile cotton tipped applicator and was introduced in a transport device stuart (copan transystem r , brescia, italy) to keep the isolate in good condition. once all the samples were collected, they were transported in a hermetically sealed container to a laboratory certified in microbiological analysis and were analyzed in a period no longer than one hour. all patients involved in the trial confirmed their participation with informed consent and the research protocol was approved by the institutional ethics committee. isolation, identification and antibiotic sensitivity to favor the isolation and identification of microorganisms, samples were seeded by crossstreaking on macconkey agar (bd bioxon, cuautitlán izcalli, estado de méxico, méxico) for gram-negative bacteria and on mannitol salt agar (bd bioxon, cuautitlán izcalli, estado de mexico, mexico) for gram-positive bacteria, and the plates were incubated at 37 °c for 24 h. for fungi, the samples were seeded on biggy agar (bd bioxon, cuautitlán izcalli, estado de mexico, mexico) by sowing and sweeping, and the plates were incubated at 30 °c for 24 h. subsequently, the identification of microorganisms was performed by the broth microdilution technique. the isolated colonies were taken with a prompt tm inoculation rod and placed in prompt tm inoculation bottles until reaching a concentration of 0.08 on the macfarland standard. the microplates were inoculated with 100 µl in each well and incubated at 37 °c for 24 h. for the gram-negative bacteria, the siemens type 44 microplates were used (b1017-305) and for gram-positive type 33 (b1017-211). finally, the plates for the identification of the microorganisms were read using labpro command center software (microscan  labprotm), which indicates the percentage of certainty corresponding to the microorganism identified. antibiotic sensitivity was measured by the minimum inhibitory concentration (mic) according to the clsi (clinical and laboratory standards institute 2014) criteria. the antibiotics studied were ampicillin, amikacin, ampicillin/sulbactam, cefuroxime, cefotetan, ticarcillin/clavulanate, piperacillin/tazobactam, cefazolin, cefotaxime, ceftazidime, imipenem, ceftriaxone, moxifloxacin, cefepime, gentamicin, trimetropin/sulfa-methoxazole, levofloxacin, ciprofloxacin, cefota-xime/clavulanate, ceftazidime/clavulanate, tobra-mycin, meropenem and aztreonam. one combination for gram-negative (klebsiella pneumoniae) is shown, of which the identification was confirmed by biochemical tests and the sensitivity and resistance to antibiotics were evaluated simultaneously. in vitro assays of chitosan membranes against gram-negative bacteria the in vitro tests were determined by the kirbybauer agar diffusion technique. specifically, for each isolate a dilution in broth was made in an inoculation bottle prompt tm , and with a sterile swab, the boxes were inoculated in mueller hinton agar in seeding by sweeping. each membrane was divided into disks with a diameter of 16.4 ± 0.10 mm. three discs were placed with a sterile clamp on the inoculated medium. in addition, two discs of whatman no. 1 paper impregnated independently with 1 % acetic acid and 0.9 % sodium chloride were placed as blanks. the plates were incubated at 37 ° c for 24 h, and finally, the increase in area and the inhibition halo generated by each membrane was measured. results phenotypic identification of microorganisms in skin ulcers in total, 23 ulcers of infected patients were sampled. gram-negative and gram-positive bacteria were found with 51.61 % and 29.03 % prevalence, respectively. in addition, it was possible to isolate candida albicans with a prevalence of 19.35 %. previous studies related to gram-positive bacteria were published, particularly with regard to s. aureus (escárcega-galaz et al. 2017). specifically, this work focused on gram-negative bacteria. from all of the ulcers sampled, 16 gramnegative strains were isolated. among them, proteus mirabilis, escherichia coli, enterobacter aerogenes, pseudomona aeruginosa, klebsiella pneumoniae and morganella morganii were nova biotechnol chim (2021) 21(1): e806 2 identified. table 1 shows a comparison of the microorganisms isolated in this investigation with respect to that reported by other authors. table 1. comparative analysis of incidences of gramnegative bacteria in skin ulcer. antibiotic sensitivity of gram-negative bacteria bacterial resistance to the 23 antibiotics studied is presented in table 2 (si-2). regardless of the phenotype, it was found that all isolated gramnegative bacteria were resistant to ampicillin (cmi ˃16 μg.ml -1 ), ampicillin/sulbactam (cmi ˃16/8 μg.ml -1 cefuroxime (cmi ˃16 μg.ml -1 ), cefazolin (cmi ˃16 μg.ml -1 ), cefotaxime (cmi ˃32 μg.ml 1 ), moxifloxacin (cmi ˃4 μg.ml -1 ), cefepime (cmi ˃16 μg.ml -1 ) and ciprofloxacin (cmi ˃2 μg.ml -1 ). however, this group of bacteria showed sensitivity to cefotetan (mic <16 μg.ml -1 ), piperacillin/tazobactam (mic <8 μg.ml -1 ), imipenem (mic <4 μg.ml -1 ), cefotaxime/clavulanate (mic <0.5/4 μg.ml -1 ), ceftazidime/k clavulanate (mic <0.25/4 μg.ml -1 ) and meropenem (mic <4 μg.ml -1 ). in vitro assays of chitosan membranes against gram-negative bacteria to determine the potential antimicrobial activity in a clinical setting, chitosan membranes were tested against gram-negative bacteria isolated from ulcers of hospitalized patients. in vitro tests with chitosan-based membranes were adverse for all microorganisms. for all bacterial isolates, the only effect was found by direct contact without the formation of the inhibition halo. membranes tend to modify their size in relation to the concentration of chitosan. table 3 shows the percentage of increase in area observed for each bacterium. as the concentration of chitosan in the membrane increased, its percentage increasing in area also increased. fig. 1 shows an inhibition assay with escherichia coli in chitosan membranes formulated with glycerol and honey. fig. 1. in vitro microbiological assay with e. coli in the chitosan 1 % (a), chitosan 2 % + glycerol (b) and chitosan 2 % + honey (c) membranes. microorganisms this work mama et al. (2014) kateel et al. (2018) malik et al. 2013) proteus mirabilis 12.50 1.5 escherichia coli 43.75 20 34.69 27.8 enterobacter aerogenes 6.25 pseudomonas aeruginosa 12.50 8 30.61 15.6 klebsiella pneumoniae 18.75 10 5.8 morganella morganii 6.25 0.7 klebsiella species 12.63 proteus species 5.10 klebsiella oxytoca 7 proteus vulgaris 3.5 4 nova biotechnol chim (2021) 21(1): e806 3 table 2. antibiogram of the 16 isolated skin ulcers. ud 003 ud 004 ud 005 ud 006 ud 008 ud 009 ud 010 ud 011 ud 013 ud 016 ud 017 ud 019 ud 022 ud 025 ud 028 ud 029 antibiotic p. mirabilis e. coli e. coli e. coli e. coli e. coli e. aerogenes p. aeruginosa p. mirabilis k. pneumoniae m. morganii k. pneumoniae e. coli k. pneumoniae p. aeruginosa e. coli ampicillin r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 amikacin r˃32 r˃32 r˃32 r˃32 r<4 r<4 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 s<4 s<4 r˃32 ampicillin/sulbactam r˃16/8 r˃16/8 r˃16/8 r˃16/8 r˃16/8 r˃16/8 r˃16/8 r˃16/8 r˃16/8 r˃16/8 r˃16/8 r˃16/8 r˃16/8 r˃16/8 r˃16/8 r˃16/8 cefuroxime r˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 cefotetan r˃32 s<16 r˃32 r˃32 s<16 s<16 r˃32 r˃32 r˃32 r˃32 s<16 s<16 r˃32 s<16 r˃32 r˃32 ticarcycline/k clavulanate r˃64 r˃64 r˃64 r˃64 r˃64 r˃64 s<16 r˃64 r˃64 r˃64 r˃64 r˃64 r˃64 s<16 r˃64 r˃64 piperacillin/tazobactam r˃64 r˃64 r˃64 r˃64 s<8 s<8 s<8 r˃64 r˃64 s<8 r˃64 s<8 r˃64 s<8 s<8 r˃64 cefazolin r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 cefotaxime r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 ceftazidime r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 s<2 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 imipenem r˃8 s<4 s<4 s<8 s<4 s<4 s<4 r˃8 r˃8 r˃8 s<4 s<4 s<4 s<4 r˃8 s<4 ceftriaxone r˃32 s<8 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 r˃32 moxifloxacin r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 cefepime r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 gentamicin r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 s<1 s<1 s<1 r˃8 trimethoprim/sulfamethoxazole r˃2/38 r˃2/38 r˃2/38 r˃2/38 r˃2/38 s<2/38 r˃2/38 r˃2/38 r˃2/38 r˃2/38 r˃2/38 r˃2/38 r˃2/38 r˃2/38 r˃2/38 r˃2/38 levofloxacin r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 s<2 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 r˃4 ciprofloxacin r˃2 r˃2 r˃2 r˃2 r˃2 r˃2 r˃2 r˃2 r˃2 r˃2 r˃2 r˃2 r˃2 r˃2 r˃2 r˃2 cefotaxime/k clavulanate r˃4/4 r˃4/4 r˃4/4 r˃4/4 s<0.5/4 s<0.5/4 s<0.5/4 r˃4/4 r˃4/4 r˃4/4 s<0.5/4 s<0.5/4 r˃4/4 s<0.5/4 r˃4/4 r˃4/4 ceftazidime/k clavulanate r˃2/2 r˃2/2 r˃2/4 r˃2/4 s<0.25/4 s<0.25/4 s<0.25/4 r˃2/4 r˃2/4 r˃2/4 s<0.25/4 s<0.25/4 r˃2/4 s<0.25/4 r˃2/4 r˃2/4 tobramycin r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 r˃8 s<1 s<1 r˃8 r˃8 meropenem s<4 s<4 s<4 s<4 s<4 s<4 s<4 r˃8 r˃8 r˃8 s<4 s<4 s<4 s<4 s<4 r˃8 aztreonam r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 r ˃16 s<8 r ˃16 5 nova biotechnol chim (2021) 21(1): e806 10 table 3. area increase [%] of chitosan membranes. membrane p. mirabilis e. coli p. aeruginosa k. pneumoniae e. aerogenes m. morganii ch 1 % 31.9 ± 20.6ª 28.0 ± 21.1ª 30.86 ± 19.3ª 26.1 ± 14.1ª 27.6 ± 5.9ª 32.8 ± 8.8 b ch 2 % 36.6 ± 24.9 ab 45.4 ± 55.5 bc 36.00 ± 18.4ª 30.7 ± 20.7 ab 46.8 ± 56.2 ab 39.2 ± 4.5 d ch 3 % 64.6 ± 63.5 c 54.4 ± 27.4 c 39.07 ± 35.7ª 44.8 ± 36.9 ab 58.0 ± 40.6 ab 54.7 ± 19.8 bc ch 2 % + g 23.2 ± 5.2ª 34.0 ± 17.8 ab 26.10 ± 14.3ª 29.2 ± 14.7 ab 23.6 ± 14.8ª 30.7 ± 5.0 a ch 2 % + h 32.4 ± 12.1 ab 35.2 ± 12.4 ab 34.52 ± 2.5ª 54.9 ± 52.5 c 30.8 ± 40.9 ab 46.9 ± 0.0 cd ch 2 % + h + g 51.4 ± 6.3 bc 46.0 ± 14.9 bc 37.99 ± 4.1 a 35.7 ± 38.9 ab 64.9 ± 18.4 c 76.0 ± 18.1 e initial area: 213.82 mm 2 (n = 6). discussion mama et al. (2014) reported that 87.4 % of 150 samples were positive for pathogens, of which 53 % were gram-negative bacteria and 47 % gram-positive. other authors report that the prevalence for p. aeruginosa is in the range of 20 – 30 % in ulcers (schmidtchen et al. 2003). today, a large number of microorganisms have developed resistance to antibiotics, which is why antibacterial drugs are required as new alternatives (babushkin et al. 2015; el-malek et al. 2017). mama et al. (2014) reported that most isolates are resistant to ampicillin and cephalexin with 96 % and 92.4 %, respectively. on the other hand, malik et al. (2013) reported that resistance to antibiotics among microorganisms is very variable and illustrated it for p. aeruginosa (63.7 %), p. mirabilis (57.5 %), m. morganii (57.5 %), e. faecalis (55.2 %), acinetobacter sp. (51.9 %), p. vulgaris (50.3 %), e. coli (45.9 %), k. pneumoniae (44.8 %), s. aureus (44.3 %), k. oxytoca (42.9 %), and coryneform sp. (37.1 %). the antibiotics that they evaluated included penicillins, cephalosporins, monobactams, carbapenems, aminoglycosides, chloramphenicols, quinolones, and fluoroquinolones, β-lactam inhibitors, macrolides, lincosamides, and glycol-peptide. finally, aspiroz et al. (2017) sampled two patients with ulcers in the leg and mentioned that pseudomonas aeruginosa tends to present a greater recurrence of multi-resistance to antibiotics. the use of antibiotics is only recommended when the patient presents symptoms of infection. some broadspectrum medications used for ulcer infection are ampicillin/sulbactam, ticarcillin/clavulanate, coamoxiclav, clindamycin, quinolone, and cephalosporin. the antimicrobials that have been used to combat the infections are silver sulfadiazine, fusidic acid, metronidazole, sodium chloride, chlorhexidine, and iodine povidone (howell-jones et al. 2005). likewise, when the membranes of chitosan-glycerol are in contact with moisture, they decrease in size due to the membranes being plasticized. chitosanhoney-bee membranes greatly increase in size by absorbing moisture from the culture medium because honey has the characteristic of being hygroscopic (sasikala et al. 2013). through statistical analysis, it was found that there was a significant difference between treatments for each of the microorganisms isolated. while for p. aeruginosa, there was no significant difference between the membranes. akyzu et al. (2018) reported zones of inhibition of 14.56 ± 0.81 mm for p. microbilis and 14.56 ± 0.92 mm for p. aeruginosa, the zone of inhibition for each microorganism increased by approximately 45 %. on the other hand, michalska-sionkowska et al. (2018) performed antimicrobial assays with 1 % chitosan membranes combined with collagen-gentamicin against e. coli, staphylococcus aureus and pseudomonas aeruginosa; the zones of inhibition were approximately 25 – 30 mm. as reported by balti et al. (2017), chitosan does not have the ability to diffuse through agar due to its molecular weight, which is why it does not form halos of inhibition. for chitosan to possess the antimicrobial activity it must be solubilized at a low ph in organic acids (simunek et al. 2010). in grampositive bacteria, chitosan has a strong bactericidal 6 nova biotechnol chim (2021) 21(1): e806 3 effect however, in gram-negative bacteria inhibition is considered slower and depends mainly on the molecular weight, degree of deacetylation, concentration and ph of chitosan (mrázek et al. 2010). the activity of chitosan against gram-negative bacteria is because the molecule tends to become polycationic below its pka (ph 6.3) (campana et al. 2017). in addition, the amino group of c2 can interact with the anionic components of negative surfaces, such as lipopolysaccharides and proteins, modifying their cellular permeability. chitosan at concentrations of 0.1 mg.ml -1 can bind to the negatively charged bacterial surface causing alteration of the cell membrane and leakage of intracellular components. when this polymer is found at high concentrations (2 and 5 mg.ml -1 ), it tends to coat the bacterial surface, limiting the release of intracellular components and mass transfer (khalil et al. 2015). bee honey has the potential to fight bacterial infections that have generated resistance to antibiotics and cannot be fought with traditional antibiotics (kwakman et al. 2011). when the honey is at a ph of 3.1 – 4.5, the glucose is found as gluconolactone, which provides an environment that favors the activity of the fibroblasts and the healing of wounds. together, the presence of hydrogen peroxide stimulates the generation of fibroblasts and angiogenesis (alam et al. 2014). in conclusion, the incidence of infected skin ulcers requires safe therapies that promote their healing. one answer to this problem is the use of chitosan membranes. these membranes have confirmed their activity by contact against gram-negative bacteria isolated from ulcers and that are resistant to antibiotics. therefore, chitosan membranes are a viable alternative as antimicrobial agents for the treatment of infections without the risks involved with antibiotics. acknowledgment escárcega-galaz gratefully acknowledges the conacyt by ph.d. scholarship: 417707. this research was funded by the instituto tecnologico de sonora with the project profapi (2021-0011) and by the national council for science and technology (pdcpn2014: 248160). conflict of interest the authors declare that they have no conflict of interest. ethical disclosures protection of human and animal subjects: the research protocol was approved by the institutional ethics committee. all patients involved in the trial confirmed their participation with informed consent. confidentiality of data: the authors declare that no patient data appears in this article. right to privacy and informed consent: the authors declare that no patient data appears in 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extracellular intracellular silver nanoparticles monascus purpureus eg pigments abstract pigments play an important role in the pharmaceutical industry as well as in the food industry. biological synthesis of pigments has attained more revenue for easy extraction, high growth rate and high yield. the production of pigments by monascus purpureus eg was investigated in several static batch cultures, the most suitable medium for its yellow, orange, and red extracellular pigment production was yeast glucose medium (yg), while malt extract medium (me) had maximum production for its intracellular pigment. the effect of some physical factors on growth and pigment production was studied. direct illumination inhibited growth and pigment production. antimicrobial activities of pigments were observed against selected gram-negative (g-ve) and gram-positive (g+ve) bacteria. the antibacterial effects of red pigment on gand g+ were highly effective compared to yellow and orange pigments. the extracted pigment was used for the reduction of the aqueous silver nitrate into silver nanoparticles (agnps). the biosynthesized agnps were structurally characterized using uv-vis spectra which showed absorption peaks at 437, 453 and 447 nm for ph values 3.5, 6.5 and 9.5, respectively. the optimum ph for the maximum synthesis of nanoparticles was 6.5. it showed no nitrate reductase activity, but the synthesized agnps exhibited strong antimicrobial activity against g+ and gbacteria. introduction monascus purpureus belongs to the phylum ascomycota, monascaceae family is purplish-red in shading. for quite a while, it has been customarily significant in china, thailand, and japan for the creation of red rice wine, red soybeans, cheddar, and ang-kak rice. it was discovered to create safe regular colours and other remedially significant auxiliary metabolites, including citrinin and statins (liu et al. 2020). it is not just utilized as a food colorant, seasoning specialist, and additive; however, it is likewise generally applied in clinical purposes to bring down blood cholesterol, hostile to diabetes, mitigating and to forestall osteoporosis (arunachalam and narmadhapriya 2011). it has the capacity as a saccharification specialist and ethanol maker. the past examination by takeshita et al. (2016) indicated that a mix of m. purpureus and saccharomyces cerevisiae k7 could create ethanol in mixed drinks. the shade of a food substance is essential to mirror its newness and security. it is likewise a pointer of good tasteful and sensorial qualities (malik et al. 2012). mailto:aelfallal55@gmail.com nova biotechnol chim (2022) 21(1): e979 2 ongoing years, the food business has zeroed in on creating characteristic colours from plants and microbial sources to defeat the utilization of manufactured shades that are possibly dangerous to human well-being and the climate. regular colours delivered by microorganisms have acquired significance due to their low water solvency and the shaky idea of plant inferred shades against warmth and light (sharmila et al. 2013). the main trait of m. purpureus is its capacity to integrate colours from polyketide chromophores and β-keto acids by esterification. the shades created by it are grouped into in any event six sorts of shades dependent on shading: (1) red colour (rubropunctamine and monascorubramine); (2) orange colour (rubropunctatin and monascorubrin) and (3) yellow colour (monascin and ankaflavin) (fig. 1) (guo et al. 2016). the structure of colours created by monascus species relies upon elements, for example, the sort of substrate and nitrogen source, ph, and temperature (haque et al. 2016). yellow colour monascin, c21h26o5 ankaflavin, c23h30o5 orange colour rubropunctatin, c21h22o5 monascorubrin, c23h26o5 red colour rubropunctamine, c21h23o4 monascorubramine, c23h26o4 fig. 1. six major monascus pigments chemical structures. current exploration found that monascus shades (mps) had a scope of organic exercises, for example, cell reinforcement, anticancer properties, anti-cholesterols, and invulnerable enhancer metabolites (park et al. 2005; el-sayed et al. 2020). as the monascus can produce lovastatin alongside with function as a colour it is also a cholesterol-lowering specialist by stifling cholesterol amalgamation using hindrance of hmg-coa reductase compound, which intervenes the biosynthesis of cholesterol in the liver and is known as a strong inhibitor simvastatin (seenivasan et al. 2020). in this manner, the presence of lovastatin in bioproducts of a life form is an extra preferred position making m. purpureus significant in clinical and drug ventures by agreeably working with different mixes or independently to animate body cells in the therapy of alzheimer's infection or malignant growth, enlistment of apoptotic cell demise, a decrease of aggravation, hindrance of tumorigenesis and viral replication (choe et al. 2020). the present study aims to evaluate the best possible fermentative conditions for maximum production of bio-pigment, its antimicrobial effects and study m. purpureus pigments as a reducing and capping agent for the rapid synthesis of silver nanoparticles (agnps). experimental microbial cultures m. purpureus eg was obtained from the microbiology lab (mayada el-fawal), botany and microbiology department, faculty of science, damietta university, egypt. it was maintained on potato dextrose agar (pda) medium at 30 ºc and preserved at 4 ºc, with sub-culturing every four nova biotechnol chim (2022) 21(1): e979 3 weeks. the bacterial strains (bacillus subtilis and escherichia coli) were kindly provided by prof. mohamed i. abou-dobara (professor of microbiology, botany and microbiology department, faculty of science, damietta university, egypt). chemicals silver nitrate was purchased from panreac quimica s.l.u (barcelona, spain). the other chemicals and solvents were purchased from sigma aldrich (saint louis, usa) and el nasr pharmaceutical chemicals company (oubour, qalyubi, egypt). cultivation media yeast glucose medium (yg) (chen and tseng 1989), modified lin’s medium (ml) (lee et al. 2001), sabouraud dextrose medium (sd) (jacobs and gerstein 1960), malt extract medium (me) (thom and church 1926), czapex-dox medium (waksman 1967) and potato dextrose medium (pd) (beever and bollard 1970) were the basic fermentation media. nutrient agar medium was used in the study of antimicrobial activity tests. evaluation of different media, physical parameters on growth and pigment production three different solid culture media potato dextrose agar (pda), malt extract agar (mea) and sabouraud dextrose agar (sda) were prepared then sterilized by autoclaving, inoculated centrally on petri dishes containing those media with a 6mm. agar disc cut, with sterilized cork-borer from the edge of 7-day old m. purpureus culture growing on pda agar and incubated for 7 days at 20, 30, and 50 ºc and then after incubation period the most suitable degree of temperature for growth and pigment production was determined. the effect of dark and light was performed in petri dishes and flasks. m. purpureus culture was covered (dark conditions) with aluminium foil paper for comparison with the other culture that was continuously cultured under light for 7 days at 30 ºc. pd, sd, me, yg, ml broth media were sterilized at 121 ºc for 15 min then were inoculated with a 6mm agar disc of m. purpureus and incubated for 7 days at 30 ºc under static conditions (memmert incubator, memmert gmbh + co. kg, schwabach, germany). after incubation, the mycelia were separated from the broth by using pre-dried and weighed whatman filter no. 1 then was dried in an oven at 80 ºc and weighed to determine biomass. extracellular pigments analysis estimation in static culture was performed using culture filtrate. the pigment mycelia were separated from broth by filtration. the cell-free culture filtrate was then centrifuged (xiangshui fada medical apparatus factory, model 800, xiangshui fada medical treatment machinery factory, xiangshui, china.) at 7,511×g for 15 min, and the supernatant was decanted. the filtrate was diluted with distilled water, and the extracellular pigment concentration was determined using a uvvisible spectrophotometer (model uv1100, shanghai yoke instrument company co., ltd., shanghai, china.) at wavelengths of 400, 460 and 500 nm for yellow, orange, and red pigments, respectively. distilled water was used as blank control. the amounts of extracellular pigments were obtained via multiplying the sample absorbance by the applied dilution (yang et al. 2019). intracellular pigments analysis the mycelia were washed twice by distilled water and collected, crushed, and soaked in 10 ml of ethanol aqueous solution (70 % v/v; ph = 2) for 2 h in a horizontal shaking incubator (incubator wis10 shaking incubator top-door orbital motion benchtop shaker incubator, model wis-10, witeg labortechnik gmbh, wertheim, germany) at 0.4×g to extract the intracellular pigments. the ethanol extract of intracellular pigments was centrifuged at 7,511×g for 15 min to separate the mycelia, after which the supernatant was collected. the corresponding ethanol aqueous solution was subjected to intracellular pigment concentration measurement using a uv-visible spectrophotometer (shanghai yoke instrument company, model uv1100, china) at wavelengths of 400, 460 and 500 nm for yellow, orange, and red nova biotechnol chim (2022) 21(1): e979 3 pigments, respectively. ethanol (70 % v/v, cut-off wavelength; 210) was also used as blank control. the amounts of intracellular pigments were obtained by multiplying the sample absorbance by the applied dilution (yang et al. 2019). antimicrobial activity of pigments extracted pigments were tested for their antimicrobial activity against two types of bacteria by using the agar well diffusion method. the tested microbial strains were e. coli and b. subtilis. standardized suspension of each of the tested strains (108 cfu.ml-1) was swabbed uniformly into the sterilized culture medium (nutrient agar medium), mixed well, poured to petri plates and made discs then placed filtrate of both culture media in each disc. the plates were incubated for 24 h at 37 ºc, the inhibition zones were measured. silver nanoparticles biosynthesis using monascus red pigment the extracellular monascus red pigment-mediated synthesis of agnps was executed as follows, 100 µl (1 mg.ml-1 concentration) of red pigments solution was added into 2.5 ml silver nitrate (agno3) (2.0 × 10 -4 m) solution (ph 7.0 ± 0.2) in clean glass tubes. then, the tube was vigorously shaken for proper mixing of the reactant and kept under high-intensity sunlight and observed for the visual colour change of silver salt solution. simultaneously, the controls of silver nitrate (2.5 ml silver nitrate without pigments) and pigments (100 µl pigments in water) were separately run at the same condition (koli et al. 2018). nitrate reductase activity nitrate reductase activity of the fungus was assayed by obtaining two tubes, the first one was mycelia of the fungus with 10 ml of agno3 and the second one was the control which consisted of 10 ml of supernatant of the fungal filtrate and 10 ml of water then incubated in shaking incubator (benchtop shaker incubator, model wis-10, korea) in the dark conditions for 5 – 7 days until the developed brown colour appeared and then measured by uv-vis; spectrophotometer at the wavelength of 540 nm. characterization of agnps the formation of brown or yellow-orange color after reaction of monascus pigments with whitish color bulk agno3 solution in the presence of sunlight was the primary indication of conversion of silver nitrate into nanosized particles. uv-vis spectrophotometric analysis the extracellular red monascus pigment-mediated synthesis of agnps was confirmed by scanning of brown agnps solution on uv/vis/nir spectrophotometer (model v-630, jasco international co., ltd., hachioji-shi, japan) in the range of 300 – 700 nm. effect of ph on agnps synthesis the optimum concentration of agno3 (1 mm) was added to the fungal filtrate and the ph of the reaction mixture was adjusted to 3.5, 6.5, and 9.5 (el-baz et al. 2016), using 1 m hno3 and 1 m naoh solutions to obtain the optimum conditions for the maximum synthesis of nanoparticles. antimicrobial activity of agnps at different ph values biosynthesized agnps at different ph values 3.5, 6.5, and 9.5 were tested for their antimicrobial activity against two types of bacteria: e. coli and b. subtilis by also using the agar well diffusion method and after incubation for 24 h at 37 ºc, the inhibition zones were measured. statistical analysis the data were statistically analyzed using spss software version 18. all values in the experiments were expressed as the mean ± standard deviation (sd). results and discussion 4 nova biotechnol chim (2022) 21(1): e979 3 morphological characters of m. purpureus on different media different morphological characteristic of m. purpureus was shown when grown on different solid culture media such as pda, mea and sda. in the early stages of mycelial growth on malt and sabouraud media the mycelium was white and rapidly turned to rich orange with small aerial development and in the case of pda medium it changed into red color and each colony has whitish edges (fig. 2). fig. 2. growth of m. purpureus eg on different solid culture media (a) pda, (b) mea and (c) sda, (d) dorsal view showing pigment production on sda. based on microscopic examination we observe that is the formation of cleistothecium that contains up to 120 or more oval ascospores which means that m. purpureus preferred sexual reproduction (patrovsky et al. 2019). effect of temperature on m. purpureus growth and pigment production the results showed that m. purpureus grew in two tested temperatures (20 ºc and 30 ºc) and was not able to grow at 50 ºc. the maximum growth and pigmentation were observed at 30 ºc (fig. 3). it was shown less growth and pigmentation at 20 ºc. the activity of enzymes for red pigment production was optimum at 30 ºc, but it stopped at 50 ºc. it can be concluded that temperature plays a vital role in the metabolism of cells, as it affects the growth and pigment yield. carvalho et al. (2005) also reported a shift in absorbance maxima of pigment yield at different incubation temperatures. fig. 3. effect of temperature on growth and pigment production of m. purpureus (a) 30 ºc, (b) 20 ºc and (c) 50 ºc. effect dark and light conditions on m. purpureus growth and pigment production in this study, it is observed that the production of pigment increased by the incubation in total darkness and there was a reduction in growth and pigment yield under continuous illumination. these assays were performed on different culture mediums (fig. 4). there was an increase in the biomass of m. purpureus in total darkness and a clear reduction in continuous illumination and these observations disagree with babitha et al. (2008) who reported that when considering the growth of the organism, direct illumination favoured the growth but under total darkness, there was a reduction in the biomass. the wavelength dependence of this response was investigated for red pigment production, and it had shown that light inhibits the pigment yield as in the case of total darkness the absorbance was 2.209 and under continuous illumination was 1.371. colonies with more growth were observed in petri dishes covered with aluminium foil, and linear extension was determined (table 1). 5 nova biotechnol chim (2022) 21(1): e979 3 table 1. the diameter of m. purpureus radial growth (mean ± sd) on different culture media in dark conditions and the presence of light. culture media diameter [mm] dark light mea 90 ± 0.2 50.8 ± 0.2 sda 70 ± 0.5 50.1 ± 0.4 pda 70 ± 0.1 30.6 ± 0.2 light has a depression influence on spores and aerial mycelium production, which was similar to zhang et al. (2020) results. the negative effect of light can be reversed by applying darkness exposure. miyake et al. (2005) studied the effect of different conditions of illumination, using the blue, green, and red light and he found that the red light showed little effect on the growth and pigment production. fig. 4. the effect of light; (i) and dark; (ii) conditions on monascus culture morphology and pigment production while growth on (a) mea, (b) pda and (c) sda. the colonies grown under the direct illumination resulted in less pigment production and that may be according to the presence of photoreceptors that make a response to dark and light like these receptors that found in neurospora crassa as reported by ballario and macino (1997). effect of different liquid culture media on growth and pigment production the results showed that m. purpureus grew rapidly on all tested culture media, but the texture of mycelium and color was dependent on media type (fig. 5). we observed that pd and me were the best media to promote fungal growth after incubation for 7 days at 30 ºc. the highest dry weight was achieved when m. purpureus grew on pd medium (fig. 6). these results indicated that this fungus has a complex system of the enzyme (lytic enzymes) that enables it to grow on different substrates and we can say that it preferred growth on complex culture mediums. fig. 5. batch fermentation profile showing pigment production and biomass of m. purpureus while growth on (a) me, (b) pd, (c) yg and (d) sd broth media. fig. 6. the effect of different culture media on m. purpureus mycelial dry weight (g). m. purpureus grow on the pd medium better than ml medium followed by sd, yg and ml broth media, respectively (highly significant = * p<0.05, n = 3). the current study focused on determining intracellular and extracellular yellow, orange, and red pigments in different culture media. fig. 7 showed that the yg broth medium was the best culture medium for maximum production of extracellular yellow, orange, and red pigments of 1.591, 1.912, and 2.391, respectively. this current result was similar to pisareva and kujumdzieva 6 nova biotechnol chim (2022) 21(1): e979 3 (2010) and lee et al. (2001) who concluded that glucose as the sole carbon source was suitable for producing high pigment yields. the mechanism for high pigment yield may be related to the high expression levels of certain proteins in the polyketide synthesis pathway and the high concentration of primary metabolism-generated molecules as substrates for polyketide synthesis (huang et al. 2018). me broth medium had the maximum production of intracellular yellow, orange, and red pigment of 1.508, 1.869 and 2.377, respectively. our finding is in agreement with huawei et al. (2019) who reported that maltose was the most suitable carbon source responsible for high intracellular monascus pigments. patrovsky et al. (2019) studied the influence of initial ph and nitrogen source on the production of monascus pigments during submerged liquid cultivation and reported that red pigments are made by the reaction of orange pigments with compounds containing an amino group, and the reaction is followed by the formation of a mixture of red pigment. on the other hand, talapragada et al. (2017) revealed that specific amino acids at 1% influence the type and amount of extracellular or intracellular bio-products and fresh biomass produced by m. purpureus under submerged static conditions. pengnoi et al. (2017) reported that pigment production in this fungus can be achieved by solidstate fermentation technique using agricultural products other than rice. cornmeal supplemented with 8 % (w/w) glucose was a suitable substrate for pigment production. so, it can be concluded that the composition of the medium influences the color and amount of the produced pigment, as proved by shi et al. (2015). fig. 7. estimation of the produced pigments by m. purpureus. antimicrobial activity of monascus pigments m. purpureus showed antimicrobial activity against the g+ve b. subtilis and the g-ve e. coli but we observed that the influence was various with the different culture media. table 2 showed that pigments extracted from yg medium filtrate were more effective as an antimicrobial agent against the tested bacteria. it was noticed that g+ve bacterium has been more sensitive to the action of m. purpureus pigment with an inhibition zone of 20.63 mm compared with the g-ve bacterium; the inhibition zone was 10.6 mm (fig. 8). 7 nova biotechnol chim (2022) 21(1): e979 3 table 2. antibacterial activity of different m. purpureus culture filtrates. tested bacteria zone of inhibition (mean ± sd, mm) yga sda mea mla pda b. subtilis 20.63 ± 0.03 -ve 20.06 ± 0.06 -ve 20.46 ± 0.06 e. coli 10.6 ± 0 -ve 10.16 ± 0 -ve 10.5 ± 0 where -ve resembles no inhibition zone formation. these results disagree with the results by talapragada et al. (2017) that reported that the antimicrobial action of extract containing m. purpureus pigment had shown negative results against e. coli which may be attributed to the type of the media or tested strain. fig. 8. antimicrobial effect of m. purpureus pigments and agnps on e. coli (a) and b. subtilis (b) where (1): agnps, (2): sd, (3): malt, (4): pd, (5): yg and (6): ml culture filtrate. abdel ghany et al. (2015) reported that culture filtrate of monascus strain had antibacterial action against bacillus spp. other research finds that antimicrobial activity is significantly determined by cell growth conditions and culture medium compositions (narsing et al. 2017). the fungus monascus can produce pigments, lovastatin (monacolin k), citrinin, dimerumic acid and c-aminobutyric acid, usually in the stationary growth phase. some of these minor pigments might be intermediates or degradation products of the main pigments, and a possible relationship between monascusones a and b and the major yellow pigment, monascin, has been proposed (jongrungruangchok et al. 2004). lovastatin is a typical secondary metabolite that is produced in the stationary growth phase, and its production is subject to glucose repression. m. pilosus was induced to produce lovastatin in a liquid medium using a mixed substrate of maltose: glycerol (1 : 7) and peptone as the nitrogen source (miyake et al. 2006). orange monascus pigment has a weak antimicrobial activity, whereas the red pigment has little or no activity. a change in the oxygen part of orange pigment to a nitrogenous compound (amino acid) causes a color change to red (saisadan et al. 2016). lovastatin is a lactone that is readily hydrolyzed in vivo to the corresponding βhydroxyacid and strong inhibitor of hmg-coa reductase, a hepatic microsomal enzyme that catalyzes the conversion of hmg-coa (3-hydroxy3-methylglutaryl-coenzyme a) to mevalonate, an early rate-limiting step in cholesterol biosynthesis (moghadasian 1999). therapeutic properties of culture filtrate of monascus spp. could be due to monascidin a, which is a secondary metabolite produced by monascus spp. (talapragada et al. 2017). pigments colours of m. purpureus differed after extraction from the different culture media as shown in fig. 9. 8 nova biotechnol chim (2022) 21(1): e979 3 fig. 9. pigments extracts from m. purpureus were obtained after 7 days from static liquid cultivation at 30 ºc, in different culture media (a) sd, (b) me, (c) pd, (d) yg and (e) ml broth media. antimicrobial activity of agnps the results showed that there was visually yellowish-brown agnps as soon as the silver nitrate solution was added to the m. purpureus supernatant and then exposed to the sunlight (fig. 10). in addition, m. purpureus filtrate showed no nitrate reductase activity which indicates that pigment is the main reducing power, and this result agrees with el-batal et al. (2015) who reported that the high red pigment content of the m. purpureus culture filtrate could be responsible for the synthesis and stabilization of the agnps. fig. 10. agnps synthesis in the dark and light conditions. (a) m. purpureus showed no nitrate reductase activity, (b) showing agnps synthesis in the light whereas its pigments were reducing power. monascus red pigment-mediated agnps synthesis was evaluated for their antimicrobial activity against two pathogens, including one g+ve (b. subtilis) and one g-ve (e. coli) bacteria. the agnps inhibited the growth of the tested pathogens but showed higher antimicrobial activity towards b. subtilis compared to e. coli (fig. 11). b. subtilis was more susceptible to agnps showing the higher zone of inhibition 6 ± 0.1 mm at 10 µg.ml-1 agnps while e. coli with an inhibition zone of 1 mm and this was attributed to the effect of the nanoparticles. the interpretation of the reduction of the antibacterial activity by agnps instead of increasing with the pigments that m. purpureus pigments reducing power was decreased after nanoparticles formation (el-baz et al. 2016). in this work, we also studied the effect of agnps that was synthesized at different ph values on bacterial growth and results showed that synthesized agnps at ph6.5 was more effective compared to the agnps that was synthesized at the other ph values and had higher distribution on the g+ve bacteria. feng et al. (2000) reported different mechanisms of the antimicrobial action of nanomaterials such as penetrating the cell wall and plasma membrane ending with damaging dna molecules. others suggested that nanomaterials might interact with thiol groups in proteins, which induces the inactivation of microbial proteins. in the presented study, negatively charged m. purpureus eg crude metabolite compounds have an opposite charge with gram-positive bacteria, in that way killing them more easily than gram-negative bacteria due to the electrostatic attraction. koli et al. (2018) reported that the zone of inhibition increased by increasing the concentration of agnps and they determined the concentration of agnps which inhibit the 50 % growth of e. coli bacteria. the ic50 concentrations of agnps were observed to be 8.53 ± 0.6 µg.ml-1. 9 nova biotechnol chim (2022) 21(1): e979 3 fig. 11. antibacterial effect of biosynthesized agnps at different ph values; (1) ph 6.5, (2) ph 9.5 and (3) ph 3.5. the effect of ph values, on the agnps synthesis, could be illustrated in fig. 12. the uv-vis absorbance spectra of the different reaction peaks were 437, 453 and 447 nm for ph values 3.5, 6.5 and 9.5, respectively. the height of the uv-vis spectrum and peak area, obtained from the reaction and carried out at ph 6.5, are considerably higher than those obtained at other ph values (fig. 13) and indicates higher nanoparticle productivity. the obtained results, in the current study, are correlated with those obtained by kim et al. (2002); jaidev and narasimha (2010); el-zahed et al. (2019) and el-zahed et al. (2021) studies that also showed the influence of agnps against g-ve and g+ve bacteria in addition, they added that there was a remarkable antifungal effect of agnps on candida albicans and s. cerevisiae. this was in accordance with the results obtained by sneha et al. (2010), who reported that low acidic and alkaline ph values decreased the agnps synthesis as silver tends to precipitate at alkaline ph, which could be due to the formation of the silver hydroxide. similarly, el-baz et al. (2016) recorded ph 6.5 as the best for silver nanoparticles biosynthesis. lately, the antimicrobial activity of negatively charged oxygen compounds (pigments) had been reported as the bioactive compounds in the biological control (abdel ghany et al. 2015). in the present study, the antimicrobial action of agnps has been associated with m. purpureus eg crude metabolite compounds chemical properties and their versatility such as deacetylation degree and molecular weight. this deacetylation action decreases the oxygen negative charge of pigments in addition to the positive charge of the silver ions that led to the decrease in the antimicrobial action of the bound agnps with m. purpureus eg crude metabolite compounds (lin et al. 2019). moreover, el-baz et al. (2016) suggested that no nitrate reductase was detected by m. purpureus, whereas its pigments reducing power was decreased after nanoparticles formation indicating its role in the agnps biosynthesis. it was previously reported by broder and koehlerthat (1980) that the extracellular pigments were combined with the nitrogenous components of the medium. also, the higher polarity of the red pigments was known to enhance their binding to water-soluble nitrogencontaining organic compounds, which has been proposed as the solubilization mechanism. on the other hand, recent research regarding the use of fungi has generally investigated potential redox systems using silver nitrate as the source of silver ions and m. purpureus extract having a 0.65 ± 0.03 reducing activity (equivalent cysteine, mm) (vahabi et al. 2011). so, a further study of the synthesized agnps should be taken into account to study and improve the antimicrobial efficacy of agnps to have a negative surface charge throughout capping them with negatively charged proteins. the negative charge of the nanometal capping agent might increase the microbicidal effect of the m. purpureus eg crude metabolite compounds. fig. 12. changing in the reaction colour before and after ag+ reduction into agnps by the action of m. purpureus culture filtrate at different ph values (b) 3.5, (c) 6.5 and (d) 9.5 in comparing with (a) negative control. 10 nova biotechnol chim (2022) 21(1): e979 3 fig. 13. uv-visible spectra of the produced agnps atdifferent ph values. conclusions this study examined the effects of dark and light conditions on the growth and pigment yield of m. purpureus eg which revealed that the incubation in the total darkness was the most effective in induction of the pigment production and the colonies grew under the direct illumination resulted in less pigment production. this result postulates that there is an existence of photoreceptors responsive to dark and light in this fungus. the growth at 30 ºc gave the highest yield pigment. it can also be concluded that yg and malt extract broth media were the most favourable to produce extracellular and intracellular pigment of m. purpureus, respectively. these conditions might be beneficial in the production of monascus pigments for use in the food industry. m. purpureus showed antimicrobial activity against grampositive and negative bacterial strains, so its secondary metabolites appeared to have a great potential in medicinal uses. finally, the synthesis of biogenic agnps was considered easy, rapid, safe, and did not need any additional toxic reducing agent as extracellular red pigments are used as a reducing and natural capping agent. moreover, the agnps had shown antibacterial activity against g+ve and g-ve bacterial strains which might be used in the biomedical application and environmental field. conflict of interest the authors declare that they have no conflict of interest. references abdel ghany t, shater a, negm m, al abboud m, 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1 nova biotechnologica et chimica effects of diterpene alkaloids on lipid peroxidation in mitochondria dilnoza kh. muratova 1, , nurali a. ergashev 1 , jobir j. sobirov 2 , utkir kh. kurbanov 3 , muzaffar i. asrarov 1 1 institute of biophysics and biochemistry, national university of uzbekistan named after mirzo ulugbek, almazar district, student town 174, tashkent 100174, uzbekistan 2 tashkent state agrarian university, tashkent region, kibray district, universitet street 2-house, tashkent 100140, uzbekistan 3 acad. s. yunusov institute of the chemistry of plant substances academy of sciences of uzbekistan, m. ulugbek street 77, tashkent 100170, uzbekistan  corresponding author: dil_muratova@mail.ru article info article history: received: 29 th january 2021 accepted: 28 th june 2021 keywords: alkaloids fe 2+ /ascorbate lipid peroxidation malondialdehyde mitochondria abstract antioxidant activity of the alkaloids songorine, napelline, and 1-o-benzoylnapelline has been studied in rat liver mitochondria by lipid peroxidation. songorine, napelline, and 1-о-benzoylnapelline alkaloids had a protective effect on mitochondria, reduced the damaging effect of fe 2+ /ascorbate and releasing of malondialdehyde (mda) into the secondary products of peroxidation. the effect of alkaloids songorine, napelline, and 1-o-benzoylnapelline on the processes of mda formation in rat liver mitochondria in vitro has been studied. the alkaloids napelline and songorine at 200 μm of concentration inhibited the formation of mda by 54 and 44 %, respectively, and 1-o-benzoylnapelline at this concentration inhibited by 95 %. obtained data revealed that 1-о-benzoylnapelline more strongly inhibited the formation of mda compared to songorine and napelline.  university of ss. cyril and methodius in trnava introduction mitochondria play a central role in the energy metabolism of a cell. oxidative disbalance in mitochondria develops against the background of oncogenic, neurodegenerative, cardiovascular, and other diseases (figueira et al. 2013). one of the mechanisms of mitochondrial disorders is the intensification of lipid peroxidation (lpo). activation of lpo is often one of the trigger mechanisms of a number of diseases and is also an aggravating factor in many pathological conditions (lobo et al. 2010). the lpo process leads to damage of the structural organization of cell membranes, changes in membrane permeability, decreasing of membrane potential, uncoupling of oxidative phosphorylation and hydrolysis of atp, decreasing in the rate of electron transfer along the respiratory chain. free radical chain oxidation of unsaturated fatty acids is known to play an important role in the normal functioning of cells as well as in pathological processes and in the pathogenesis of various types of liver damage (lobo et al. 2010). lipid peroxidation product (mda) characterizes the state of the antioxidant system in the organism. the study of the role of lpo in the regulation of the most important cell functions is of interest for a number of reasons. the effect of lipid peroxidation on mitochondrion functions is carried both at the level of the direct effect of lpo products on the mailto:dil_muratova@mail.ru nova biotechnol chim (2021) 20(2): e850 2 lipid matrix of membranes and at the level of various indirect effects. antioxidants are able to neutralize the activity of free radicals, protect the phospholipids of cell membranes from oxidation (kiplimo et al. 2011). the mechanisms of action and antioxidant properties of biologically active substances isolated from plants are extensively studied in order to inhibit the process of lpo caused by oxidative stress, to correct membrane disorders. it is known that plant compounds are the main source of biological material in the production of drugs with antioxidant properties (almeida et al. 2006). the antioxidant properties of aporphine alkaloids and flavonoid have been studied (kiplimo et al. 2011; vetrova et al. 2017). diterpene alkaloids are a promising class of plant substances with a wide spectrum of pharmacological activity. they have antiarrhythmic (dzhakhangirov et al. 1997; shakhidoyatova et al. 2001), antispasmodic (dzhakhangirov et al. 2013), antioxidant (khan et al. 2018), antidepressant (nesterova et al. 2011), antimetastatic (kruczynski et al. 2006), anti-inflammatory (nesterova et al. 2013; marya and khan 2017), and other effects. diterpene alkaloids are preferred over narcotic and non-narcotic analgesics. they affect the accumulation of nerve impulses that are not enough to induce strong arousal, such as morphine, without the side effects of drug dependence, and can be used both for acute (e.g. postoperative) pain and for pathologically debilitating pain. the studied diterpenoid alkaloids and their derivatives have advantages over popular drugs and non-narcotic analgesics. in addition to their effect on central and visceral pain, these alkaloids have myotropic and antispasmodic effects (dzhakhangirov et al. 2013). since the mechanisms of action of diterpene alkaloids on mitochondria have not been previously studied, we aimed to study the effect of these alkaloids on the lpo process in mitochondria. experimental extraction and separation of alkaloids the homogeneity of the substances was tested on plates with silica gel on tlc (fluka analytical, germany) brand in the benzene-ethanol system in ratios of 9 : 1, 20 : 1, the chloroform-methanol system in ratios of 9 : 1 and plates with aluminum oxide in benzene-ethanol 9 : 1. for column chromatography, silica gel of the tlc brand and deactivated aluminum oxide of the "for chromatography" brand were used. ir spectra were taken on a perkin-elmer-2000 instrument; 1 h nmr and 13 c spectra were taken on a jeol instrument (400 and 600 mhz japan) in a cdcl3 solution. extraction of alkaloids of the aconitum karakolicum plant 1.84 kg of air-dry crushed aboveground aconitum karakolicum was exhaustively extracted with 80 % ethanol. the extracts were combined and condensed. the aqueous residue was alkalized with naoh to ph 12 and exhaustively extracted with chloroform. after distilling the solvent, 9.18 g of the total alkaloids were obtained, which was 0.49 % of the plumb line of the dry plant. isolation of alkaloids from aconitum karakolicum the amount of alkaloids was divided on a column with aluminum oxide (300 g). the alkaloids were eluted with cyclohexane, cyclohexane-acetone (1 : 1), chloroform-methanol (5 : 1), (1 : 1), and a 5 % solution of sulfuric acid. the acidic solution was alkalized with naoh to ph 7, 8, 9, 10, 11, 12, respectively, and according to the basicity strength they were exhaustively extracted into 6 fractions with chloroform. from the fraction 4, 0.14 g of songorine was separated using methanol. fractions 3, 5, and uterine solution 4 fractions were separated on the silica gel column, eluding with chloroform, gradually adding methanol, and 0.13 g of songorine, and 0.12 g of napelline were isolated (sultankhodzhaev et al. 1978). alkaloid songorine was also isolated from aconitum monticola by the above methods (nezhevenko et al. 1975). the 1-obenzoylnapelline was obtained by synthesis from these alkaloids (dzhakhangirov et al. 2013; sultankhodzhaev et al. 2017). the structural formulas of alkaloids were drawn by the chemoffice 2002, chemdraw ultra 7.0 software (fig. 1). nova biotechnol chim (2021) 20(2): e850 3 animal treatment in the experiments, white outbred male rats, weighing 180–220 g, were used (obtained from the vivarium of the pharmacology department n ch3 o oh oh ch2 hcl n ch3 oh ho oh ch2 с=о о сн2 он он сн3 нсl n 20 8 6 10 4 19 14 12 15 16 17 18 songorine napelline 1-o-benzoylnapelline fig. 1. structural formulas of diterpene alkaloids studied in this work. of the institute of bioorganic chemistry of the academy of sciences of the republic of uzbekistan, tashkent) in accordance with the ethical rules for working with laboratory animals, according to the “regulations on the bioethics of the use of laboratory animals in scientific research” of institute of biophysics and biochemistry at the national university of uzbekistan named after mirzo ulugbek (protocol dated 22. 02. 2019). the rats were housed under standard laboratory conditions (20–24 °c; natural light regime of sunlight; 65 % humidity, food and water available ad libitum), immobilized with light ether anesthesia and decapitated. isolation of mitochondria mitochondria were isolated from livers by conventional differential centrifugation described by schneider and hageboom (1951). rat liver was homogenized in a medium containing 250 mm sucrose, 10 mm tris (hydroxymethyl) aminomethane hydrochloride (tris-hcl), 1 mm ethylenediamine tetraacetic acid na2-salt (edta), ph 7.4 centrifuged at 1,500  g for 7 min (-2 ° c, -4 °c). mitochondria were sedimented by centrifugation of supernatant at 6,000  g for 15 min (-2° c, -4°c). the final mitochondrial pellet was suspended in a small volume of medium containing 250 mm sucrose, 10 mm tris-hcl, was kept on ice prior to experiments. the mitochondrial protein content was determined by the lowry method modified by peterson (1977). lipid peroxidation as measured by fe 2+ /ascorbate lpo was recorded by inhibition of fe 2+ /ascorbate dependent liver mitochondrial swelling by photometric method in incubation medium contained 125 mm potassium chloride (kcl), 10 mm tris-hcl, ph 7.4, the final amount of protein in the incubation medium was 0.4 mg.ml -1 (schneider et al. 1948). 10 μm ferrous sulfate heptahydrate (feso4) and 200 μm ascorbic acid were added to induce mitochondria swelling. all experiments were conducted at 24–26 °c so that the integrity of the mitochondria was maintained during incubation. the antioxidant activity of the tested compounds was measured by inhibition of fe 2+ /ascorbate-dependent swelling of rat liver mitochondria at 540 nm. the choice of such a methodological approach is due to the fact that a linear correlation relationship between the intensification of lpo processes, induced by the fe 2+ /ascorbic acid system, and swelling of mitochondria was previously established. lpo in the fe 2+ /ascorbate system on the mitochondrial membrane was also assessed by other authors by measuring the swelling of mitochondria under conditions of lpo activation (almeida et al. 2006) which indicated the suitability of using this model as a test system for studying the antioxidant properties of various substances. the intensity of lipid peroxidation in mitochondrial membranes is determined by measuring the concentration of the final product, malondialdehyde (mda). peroxides of unsaturated fatty acids with 2-3 diene bonds, formed during lpo, are nova biotechnol chim (2021) 20(2): e850 2 ultimately converted into mda, which interacts with thiobarbituric acid. induction of nonenzymatic fe 2+ /ascorbate-dependent lpo was performed by adding 10 μm feso4 and 200 μm ascorbic acid to the incubation medium containing 125 mm kcl, 10 mm tris-hcl, ph 7.4. the separation of lpo products was carried out in the presence of thiobarbituric acid (tba). the reaction was stopped by adding 0.220 ml of 70 % (w/v) trichloroacetic acid to incubation medium. thereafter, the mitochondrial suspension was centrifuged at 1,500 × g for 15 min. then, 2 ml of supernatant was taken and poured in 1 ml of 75 % tbа (w/v). 2 ml of h2o and 1 ml of tbа were added to the control solution. the mixture was incubated for 30 min in a water bath. after cooling, a change in optical density was detected at a wavelength of 540 nm (∆540). the amount of formed mda was calculated using the molar extinction coefficient (e = 1.56 × 10 5 m -1 .cm -1 ) (devienne et al. 2007) according to the following formula: nmol mda/mg protein = d/1.56 × 30. the amount of mitochondrial protein was 0.3–0.4 mg per 1 ml of incubation medium. drugs and chemicals the following chemical reagents were used: edta (sandoz, switzerland), tris-hcl (serva, germany), sucrose, feso4, ascorbic acid, kcl, tba, trichloroacetic acid (chemreaktivsnab, russia). all reagents were p.a. grade. data analysis the results were analysed statistically using the origin pro 7.5 (microsoft, usa). the data were evaluated using parametric student’s t-test and are expressed as m ± m. the results that were deemed significant are indicated as follows: * p<0.05 and ** p<0.01. results a number of properties of diterpenoid alkaloids have been identified by our colleagues. they investigated the vasorelaxant effect of these alkaloids (1-o-benzoylnapelline, songorine, zeravshanizine, l-o-benzoylkarakoline, 14-obenzoyltalatisamine) when inhibiting the entry of ca 2+ ions through ca 2+ l and ca 2+ r-channels of the plasma membrane and their release from the sarcoplasmatic reticulum. the effect of these alkaloids on katp channels in smooth muscle cells were also studied, while they protect the rat aorta from damage as a result of hypoxia, showing a vasoprotective effect (mirzayeva et al. 2016; yesimbetov et al. 2019). it was found that the diterpenoid alkaloid songorine (muratova et al. 2021) and 1-o-benzoylnapelline (muratova et al. 2020), similar to diazoxide, activate the mitokatpchannel. napellin-type diterpene alkaloids and their aromatic ester-free derivatives exhibit weak spasmolytic activity, while its derivative 1-obenzoylnapelline has spasmolytic activity (dzhakhangirov et al. 2013). the high antioxidant/antiradical activity of polyphenol compounds has also been studied (gayibov et al. 2019). here was studied the effect of 1-оbenzoylnapellinе, napellinе, and songorinе on lpo processes in the membrane mitochondria in vitro experiments. fe 2+ /ascorbate was used as the lpo inducer. as a result of the studies, it was found that the addition of fe 2+ /ascorbate to the incubation medium increased the rate of swelling of mitochondria by (t–5 min, ∆a540 = 0.330 ± 0.012) 100 % compared to the control. experiments have shown that the addition of fe 2+ /ascorbate to the incubation medium causes swelling of mitochondria in comparison with the control, which indicates lpo and permeabilization of mitochondrial membranes. a study of the effect of 1-o-benzoylnapelline on fe 2+ /ascorbate induced mitochondrial swelling showed a concentration of 0.1 µm inhibited mitochondrial swelling by 19.7 ± 2.9 %. experiments have shown that 1-obenzoylnapellinе had a concentration-dependent inhibitory effect on mitochondrial swelling (fig. 2). adding 1-o-benzoylnapellinе to the incubation medium at a concentration of 0.5 µm for 39.8 ± 2.3 %, 1 µm for 64.9 ± 1.9 %, 5 µm for 79.5 ± 1.7 %, and 10 µm for 96.5 ± 1.2 % prevented the effect of fe 2+ /ascorbate on lpo. experiments have shown that the maximum efficiency was observed at a concentration of 10 µm and the half-maximum inhibitory concentration (ic50) of the fraction on mitochondrial swelling is 0.79 µm. also, similar results were obtained with the action of napelline 4 nova biotechnol chim (2021) 20(2): e850 3 on fe 2+ /ascorbate-induced swelling of mitochondria. napelline also inhibits fe 2+ /ascorbate-induced mitochondrial swelling. adding napelline to the incubation medium at a concentration of 10 µm at 12.4 ± 2.4 %, 25 µm at 30.1 ± 2.4 %, 50 µm at 47.3 ± 2.6 %, 75 µm at 64.5 ± 2.1 %, and 100 µm at 77.1 ± 1.8 prevented the effect of fe 2+ /ascorbate on lipid peroxidation (fig. 3). 0 20 40 60 80 100 * ** ** ** ** и н г и б и р о в а н и е , % пол 0,1 0,5 1 5 10lpo ∆ a 5 4 0 , 1 0 0 % fig. 2. effect of 1-o-benzoylnapelline on mitochondrial swelling induced by lipid peroxidation. the y-axis shows the swelling of mitochondria by fe 2+ /ascorbate and the x-axis shows the concentration of 1-o-benzoylnapellinе (µm) (*p<0.05; **p<0.01; n = 6, error bars indicate ± se of the mean). 0 20 40 60 80 100 ** * ** ** и н ги б и р о в а н и е, % пол 10 25 50 75 100lpo ∆ a 5 4 0 , 1 0 0 % fig. 3. effect of napelline on mitochondrial swelling induced by lipid peroxidation. the y-axis shows the swelling of mitochondria by fe 2+ /ascorbate and the x-axis shows the concentration of napellinе (µm) (*p<0.05; **p<0.01; n = 6, error bars indicate ± se of the mean). subsequently, the effect of songorine on the lpo system induced by fe 2+ /ascorbate has been studied (fig. 4). songorine also prevented the effect of fe 2+ /ascorbate on the swelling of rat liver mitochondria. thus, songorine at concentrations of 10, 25, 50, 75, and 100 µm, respectively, inhibited mitochondrial swelling by 10.8 ± 1.3 %, 23.6 ± 2.5 %, 44.8 ± 2.4 %, 57.5 ± 1.9 %, and 78.0 ± 1.5 % compared with control. when investigating the alkaloid songorine at concentrations of 10, 25, 50, 75, and 100 µm, its inhibitory action on fe 2+ /ascorbate-induced swelling of mitochondria turned out to be concentration-dependent. experiments have shown that the maximum efficiency was observed at a concentration of 100 µm songorinе. the experiments showed that the addition of the fe 2+ /ascorbate system to the incubation medium increased the accumulation of mda in mitochondrial membranes (3.15 nmol.mda.mg.protein -1 ) 100 % relative control. the addition of 1-o-benzoylnapellinе at a concentration of 50 µm prevented the formation of mda on lpo membranes by 33 ± 3.2 %. 0 20 40 60 80 100 ** * ** ** и н г и б и р о в а н и е , % пол 10 25 50 75 100lpo ∆ a 5 4 0 , 1 0 0 % fig. 4. effect of songorine on mitochondrial swelling induced by lipid peroxidation. the y-axis shows the swelling of mitochondria by fe 2+ /ascorbate and the x-axis shows the concentration of songorine (µm) (*p<0.05; **p<0.01; n = 6, error bars indicate ± se of the mean). inhibition of the lpo process was also observed under the action of other concentrations of 1-obenzoylnapelline. thus, the alkaloid 1-obenzoylnapelline at a concentration of 100 µm reduced the accumulation of mda to 60 ± 2.9 %, 150 µm by 79 ± 2.8 %, 200 µm by 95 ± 2.3 % (fig. 5). in the next series of experiments, the effect of napellinе on the lpo system induced by fe 2+ /ascorbate (fig. 5) was studied by adding 50, 100, 150, and 200 µm of napelline alkaloid to the incubation medium. these concentrations of alkaloids reduced the accumulation of mda to 12 ± 3.5 %, 32 ± 3.3 %, 45 ± 3.1 %, and 54 ± 2.7 %. under these conditions, the effect of songorinе on the lpo system induced by fe 2+ /ascorbate was 5 nova biotechnol chim (2021) 20(2): e850 2 studied (fig. 5). the addition of an alkaloid at a concentration of 50 µm by 4 ± 2.6 % and 100 µm by 20 ± 3.6 % prevented the formation of mda on lpo membranes. alkaloid songorinе at a concentration of 150 µm reduced the accumulation of mda to 34 ± 2.7 % and 200 µm by 44 ± 2.9 %. m d a , % 0 50 100 150 200 0 20 40 60 80 100 1-o-benzoylnappelline nappelline songorine concentration of alkaloids, µm fig. 5. the influence of different concentrations of alkaloids, mda accumulation on lpo in mitochondria induced by fe 2+ /ascorbate (n = 5, error bars indicate ± se of the mean). discussion the pharmacological properties of diterpene alkaloids and their derivatives and structural activity relationships have shown that many alkaloids and their derivatives have clear antiarrhythmic activity. the analgesic and antiinflammatory properties of 1-o-benzoylnapelline (sultankhodzhaev et al. 2017) and the increase in the rate of recovery of granulocytic hematopoiesis of napellin have also been studied (zyuz'kov et al. 2013). thus, we have established that the alkaloid 1-obenzoylnapelline acts more effectively on lpo in the mitochondrial membrane than other alkaloids. we found that 1-o-benzoylnapelline acts more effectively than napelline on the mitokatp channel of rat liver mitochondria (muratova et al. 2020). because, 1-o-benzoylnapellinе effectively reduced the damaging effect of fe 2+ /ascorbate and the releasing of mda, due to the introduction of a benzoyl group into the napellinе structure at positions c(1), respectively. it is known that lpo led to a change in the permeability of biomembranes, decreasing in membrane potential, uncoupling of oxidation-phosphorylation, and hydrolysis of atp in mitochondria. the effect of lpo on mitochondria function was realized both at the level of the direct effect of lpo products on the lipid matrix of membranes and various indirect effects. literature data have been shown that alkaloids significantly improve neurobiological outcomes and reduce oxidative stress and neuronal apoptosis. this is associated with a significant decrease in lipid peroxidation, an increase in superoxide dismutase, and a decrease in glutathione levels (ishii et al. 2018; singh et al. 2019). conclusions our results show that the doses used in the lpo study ranged from 495 μg.l -1 to 4.95 mg.l -1 for 1o-benzoylnapelline, from 3.95 mg.l -1 to 39.5 mg.l -1 for the alkaloid songorine, and from 3.59 mg.l -1 to 35.9 mg.l -1 for the alkaloid napelline. it can be concluded that the doses we have studied are close to therapeutic doses. experiments on 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https://pubmed.ncbi.nlm.nih.gov/30536411/ nova biotechnol chim (2021) 20(2): e850 3 (2019) effects of diterpene alkaloids zongorin and 1-obenzoylnapelline on соntractile activity of rat aorta smooth muscle. centr. asia. j. med. 19. 35-44. zyuz’kov gn, zhdanov vv, udut ev, miroshnichenco la, losev ea, simanina ev, chaikovskiy av, suslov ni, povetieva tn, krapivin av, nesterova yv, agafonov vi, minakova my, stavrova la, danilets mg, ligacheva aa, trofimova es, ivanova an, goldberg ve, reikchart dv, dygai, am (2013) mechanisms of napelline action stimulating the regeneration of hemopoietic tissue in cytostatic myelosuppression. bull. exp. biol. med. 155: 431-433. 8 nova biotechnol chim (2021) 20(2): e898 doi: 10.36547/nbc.898 1 nova biotechnologica et chimica molecular identification and technological properties of yeasts isolated from spontaneously fermented cassava waste pulp adelodun l. kolapo1,, raoofat o. salami2, gbemisola o. onipede1 1department of biological sciences, faculty of science, augustine university, ilara-epe, lagos state, nigeria 2department of microbiology, faculty of science, olabisi onabanjo university, ago iwoye, ogun state, nigeria  corresponding author: adelodun.kolapo@augustineuniversity.edu.org article info article history: received: 15th march 2021 accepted: 10th september 2021 keywords: cassava waste pulp livestock feed molecular identification technological properties yeasts abstract the aim of this work was to report on molecular identification and technological properties of the yeast flora isolated from spontaneously fermented cassava waste pulp. this was done with a view of obtaining yeast strains that could be used as a starter culture for the fermentation of cassava waste pulp. molecular identification was based on the nucleotide sequence of the its region of the genomic dna of the yeast isolates while the technological properties evaluated include linamarase (uml1), gelatinase, and haemolytic activity, growth at ph 2.5, and tolerance to 2 % bile salt. all the representative five isolated yeasts were identified as geotrichum silvicola klp04, klp06, klp07, klp08 and geotrichum candidum klp05. the isolates exhibited linamarase activity ranging between 3.3 and 4.2 with strain klp04 having the highest value and strain klp05 the least. none of the isolates demonstrated gelatinase and haemolytic activity except strain geotrichum silvicola klp08 which was partially haemolytic. all the examined yeasts exhibited good growth at ph 2.5, with strain klp08 having the highest viable counts of 4.1 log10cfuml -1 and strain klp04 the least value of 3.5 log10cfuml -1 after 72 h of growth. all the identified yeasts showed strain-specific tolerance to 2 % bile salt with strain klp04 having the highest viable count of 4.3 log10cfuml -1 and strain klp08 the least value of 2.2 log10cfuml -1 at the end of 72 h of incubation. based on all the examined technological properties, geotrichum silvicola klp04 strain had the highest potential to be considered for starter culture for the fermentation of cassava waste pulp. © university of ss. cyril and methodius in trnava introduction the maize grain is a major feed grain and a standard component of livestock diets where it serves as a source of energy (heuzé et al. 2017). for instance, its proportion in monogastric tropical diets could range between 50 and 70 % (pan 1995). however, the high consumption of maize both by the human population and the livestock industries coupled with its low level of production have continuously generated a demand-supply gap with a concomitant price increase. the attendant effect of this scenario on the increased cost of animal feeds has been an age-long one for which cheaper alternative feedstuffs have been developed to replace the expensive conventional ones (salami and odunsi 2003). due to their cheapness, agricultural wastes and by-products of food processing have become the first line of choice as unconventional feed materials in livestock mailto:adelodun.kolapo@augustineuniversity.edu.org nova biotechnol chim (2021) 20(2): e898 2 industries. examples of these materials include sorghum spent grains, wheat offals as well as cassava by-products. according to lukuyu et al. (2014), cassava by-products that have found application in feeding livestock include cassava leaf, cassava leaf meal, cassava leaf protein concentrate, cassava peels, cassava stumps, cassava sievate and cassava pomace/pulp/bagasse/starch residue. cassava meal provides dietary energy to over 500 million people in the world (arc 2014). fao (2001) reported that the global production of cassava in the year 2000 was 172 million tons with africa accounting for 45 %, asia 28 %, and latin america and the caribbean 19 %. nigeria, brazil, thailand, congo (drc), and indonesia are the five top producing countries. current statistics show that nigeria still accounts for 20 % of cassava global production (fao 2020). in contrast to latin america and southeast asia, where the majority of cassava is exported for industrial purposes or animal feed; about 70 to 80 % of cassava produced in nigeria is utilized for human consumption (dada et al. 2010) and only a reported 5 % of cassava was used as livestock feed (apata and babalola 2012). however, in recent times, the industrial potential of cassava to produce starches for textiles, pharmaceutical, food, alcohol, acetone, and dextrin industries are largely being exploited. this current trend is adding another stream of cassava waste to those that have been known to be generated from the processing of cassava tuber for human food. it has been estimated that the supply-demand gap for cassava starch and high-quality cassava flour in nigeria are 290,000 and 485,000 metric tons (mt) per year, respectively (pwc 2020). this corresponds to the respective estimated generation of 870,000 and 485,000 mt of cassava pulp as waste annually. the high cyanogenic glycosides contents as well as low protein content of cassava wastes constitute a restraint to their full exploitation as livestock feed. different processes found to be effective in reducing cyanogenic glycosides include sundrying, ensiling and soaking plus drying (tewe 1992; salami and odunsi 2003). in addition, fermentation of cassava products and by-product with starter microorganisms such as saccharomyces cerevisiae and lactobacillus spp. (oboh 2006; ubalua 2007), trichoderma viride (ezekiel et al. 2010), aspergillus niger and saccharomyces cerevisiae (iyayi and losel 2001), rhizopus oligosporus and aspergillus niger (kolapo et al. 2021), and rhizopus oryzae (vlavonou 1988) resulted in a product with higher protein content, lower cyanogenic glycosides and phytate content. in addition, the inclusion of microorganisms with probiotic potentials to the animal diet is known to promote growth and enhance the performance of livestock (nagpal et al. 2015; arowolo and he 2018). in this regard, some yeasts, such as saccharomyces cerevisiae (alizadeh et al. 2016), selenium yeast and phaffia rhodozyma yeast (shurson 2018) have shown remarkable usefulness. utilization of cassava and its wastes for livestock feeding has long been realized as various reports on their use for feeding poultry (ravindran 1991; salami and odunsi 2003; adeyemo et al. 2014; omede et al. 2017), pigs (iyayi and tewe 1988; unigwe et al. 2014) aquaculture (solomon et al. 1999; okoli 2020) and ruminants (smith 1988; lukuyu et al. 2014; oloruntola et al. 2019) have been documented. in addition, with the increasing demand for cassava starch and high-quality cassava flour in nigeria, many medium and large-scale industries are being attracted to the cassava starch and flour processing sector. in nigeria, the cassava flour industries have been estimated to generate 870,000 and 485,000 mt of cassava waste pulp annually. like previous cassava wastes generated from processing of cassava for human food, appropriate technologies might be deployed to ensure that this new stream of waste (cassava waste pulp) is incorporated into livestock feeding programs. in order to achieve this, the understanding of the yeasts profile and their technological properties of spontaneously fermented cassava waste pulp is necessary. to the best of our knowledge, investigation which focused on molecular identification of the yeast profile of spontaneously fermented cassava waste pulp has not been reported. thus, this study aimed to report on molecular identification and technological properties of the yeast flora isolated from spontaneously fermented cassava waste pulp. this was done with a view of obtaining yeast strains that nova biotechnol chim (2021) 20(2): e898 3 could be used as starter culture for the fermentation of cassava waste pulp. experimental preparation and spontaneous fermentation of cassava waste pulp freshly harvested cassava tubers of the tms 92/0067 variety were obtained from the root and tuber expansion programme of the international institute of tropical agriculture, located in ogere, ogun state, nigeria. the cassava tubers were harvested after 12 months of planting. the tubers were peeled by removing both the bark (outermost thin brown layer) and the cortex of cassava tuber. after peeling, 1 kg of cassava tubers was washed and grated using a mechanical grater to obtain cassava pulp. the pulp was mixed with 0.5 l of clean tap water and stirred vigorously. the resulting suspension was screened using a double layer of cheese cloth to extract the cassava starch. the residual mass was rinsed with excess water for about three times to extract as much starch as possible. the residue left thereafter was cassava waste pulp (cwp). the cwp was divided into three portions. each portion was packed into a salt bag and the mouth tied up and left for 7 days for spontaneous fermentation to take place at the ambient temperature (30 ºc). isolation of microorganisms ten grams (10 g) of fermented cwp from each of the triplicate setups were homogenized in 90 ml of sterile buffered peptone water. the three homogenates were ten-fold serially diluted using the same diluent. aliquots of serially diluted samples were pour-plated on yeast and mould agar supplemented with 100 mg.l-1 chloramphenicol (oxoid, basingstoke hampshire, uk). distinct colonies were streaked on the medium of isolation twice to obtain pure cultures (schwan et al. 2007). the ability of all the yeast isolates to produce linamarase enzyme was evaluated (as described in the subsequent section). pure cultures of yeast isolates with significant linamarase activities were selected and maintained on agar slants at 4 ºc for further molecular characterization studies. preliminary characterization of yeast isolates yeast isolates were assessed macroscopically for colour and appearance. a microscopic examination was done to assess the presence/absence of hyphae and arthrospores. molecular identification of isolates genomic dna extraction from overnight cultures of the isolates grown in luria-bertani broth (acumedia, michigan, usa) was performed by using the zr fungal/bacterial dna miniprep™ kit (zymo research, california, usa) as described by adedeji et al. (2017). fresh cultures were centrifuged at 10,000 × g for 1 min. the microbial cells were lysed by bead beating in a lysis buffer, and the lysate was subsequently centrifuged. the supernatant was passed through a column matrix to allow for dna binding. the bound dna was purified and eluted from the column matrix. agarose gel electrophoresis technique was used to verify the integrity of the eluted dna, while quantification was carried out using qubit 2.0 fluorometer (thermo fisher scientific, massachusetts, usa). pcr amplification of the internal transcribed spacer (its) region of genomic dna of the yeast isolates was done using primers its1: 5′ tccgtaggtgaacctgcgg 3′ and its4: 5′ tcctccgcttattgatatgc 3′ (white et al. 1990). pcr was performed in a total volume of 50 μl containing 30 – 50 ng dna, 100 mm of each primer, 0.05 u.μl-1 taq dna polymerase, 4 mm mgcl2, and 0.4 mm of each dntp. the amplification reaction was performed with a c1000 touch thermal cycler (bio-rad laboratories, california, usa). the thermal cycling condition used was an initial denaturation at 96 °c for 5 min, followed by 30 cycles of denaturation at 96 °c for 45 s, annealing at 56 °c for 30 s and extension at 72 °c for 2 min, followed by a final extension at 72 °c for 5 min and a holding period at 4 °c for infinite time. the pcr amplicons were analysed by electrophoresis in 1 % (w/v) agarose gel with ethidium bromide, 1 kb dna ladders were loaded nova biotechnol chim (2021) 20(2): e898 2 in 5 μl volumes, while 7 μl of the sample was loaded with 2 μl of loading dye. the gel was allowed to run for 2 h at 60 v. gel results were visualized with a chemidoc™ mp system (biorad laboratories, california, usa) to confirm the expected size of the product. the remaining pcr products were purified using nucleospin gel and pcr clean-up kit (macherey-nagel, germany). the sequencing of the purified pcr products was done with prism™ ready reaction dye terminator cycle sequencing kit using the dideoxy chain termination method and electrophoresed with the model abi prism® 3500xl dna sequencer (applied biosystems, foster city, ca, usa) following the manufacturer’s instructions. chromaslite version 2.33 software was used for the analysis of electropherograms, (sense and antisense) resulting from sequencing reaction for good quality sequence assurance. the resulting electropherograms were edited using bioedit sequence alignment editor. after this, the resulting consensus sequences obtained were blast in the ncbi (www.ncbi.nlm.nih.gov) database with the basic alignment search tool (blastn) for homology in order to identify the probable organism in question (dabassa et al. 2019). these sequences were deposited in the genbank (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers mw232914, mw233017, mw233034, mw233050, and ok070746. phylogenetic analysis the phylogenetic analysis was based on the sequence of each species in order to establish relationships among them. the evolutionary history was inferred by using the maximum likelihood method and the hasegawa-kishino-yano model (hasegawa et al. 1985). the tree with the highest log likelihood (-2931.96) was shown. the percentage of trees in which the associated taxa clustered together was shown next to the branches. initial tree(s) for the heuristic search were obtained automatically by applying neighbor-join and bionj algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (mcl) approach, and then selecting the topology with superior log likelihood value. the tree was drawn to scale, with branch lengths measured in the number of substitutions per site. this analysis involved 6 nucleotide sequences. codon positions included were 1st+2nd+3rd+noncoding. there was a total of 485 positions in the final dataset. evolutionary analyses were conducted in mega x (kumar et al. 2018). evaluation of technological properties of isolated yeasts linamarase activity the method described by oyewole (2001) and o’brien et al. (1991) was used to evaluate the ability of the yeast isolates to produce linamarase (ec 3.2.1.21). linamarase activity of the isolates was quantitatively determined as β-glucosidase glycohydrolase. the estimation was carried out by measuring the release of p-nitrophenol from pnitrophenyl-β-d-glucoside using a spectrophotometer. a loop full of 18h culture was added to 6 ml of 10g.l-1 of p-nitrophenyl-β-dglucoside in 50 g.l-1 sodium citrate (ph 6.0) and incubated at 37 ºc for 15 min. three milliliters of 0.1 m sodium carbonate (na2co3) were subsequently added to terminate the reaction. the absorbance was then measured at 420 nm using the jenway model 6305 uv/vis spectrophotometer. a standard curve enabled the conversion of the absorbance obtained to the quantity of pnitrophenol released from p-nitrophenyl-β-dglucoside. one unit of activity was defined as the amount releasing 1 μmol of p-nitrophenol from pnitrophenyl-β-d-glucoside in 1 min under the assay condition. haemolytic and gelatinase activity the yeast isolates were cultured in malt extract broth at 37 °c for 12 – 18 h and then transferred onto blood agar (difco, michigan, usa) plates supplemented with 5 % defibrinated whole sheep blood as described by yoon et al. (2008). after 24 – 48 h, the plates were examined for haemolytic reaction. a partial lysis of red blood cells and greening zone was recorded as α-haemolysis while a clear zone around yeast growth was taken as 4 http://www.ncbi.nlm.nih.gov/ https://www.ncbi.nlm.nih.gov/genbank/ nova biotechnol chim (2021) 20(2): e898 3 β-haemolysis and no lysis was recorded as γhaemolysis. nutrient gelatin medium, consisting of gelatin, peptone, and beef extract was used for the determination of gelatinase activity. 3 ml of gelatin medium that had been previously adjusted to ph 6.8 using 0.1 m naoh was dispensed into test tubes which were then autoclaved at 121 ºc for 15 min. the tubes were allowed to cool in an upright position. for 24 h, yeast cultures were inoculated into the tubes and incubated at 35 ºc for 48 h. a positive result was indicated by partial or complete liquefaction of the inoculated tube at 4 ºc. in a tube with a negative result, the content of the tube remained completely solidified at the end of refrigeration. growth at low ph the ability of the yeast isolates to grow at low ph was evaluated using the method described by conway et al. (1987). fresh culture of yeast strains was inoculated into malt extract broth (1 % v/v) with ph adjusted to 2.5 using 3n hcl. the inoculated broth was then incubated at 37 oc for 72 h. viable cell counts of samples that were aseptically taken at 0, 24, 48, and 72 h were determined. each determination was carried out in triplicates. tolerance to bile salts the ability of the strains to grow in the presence of bile was determined according to the method of conway et al. (1987). the procedure was carried out by inoculating 1% (v/v) fresh culture of selected yeast strains into malt extract broth supplemented with 2% bile salts. this was followed by incubation at 37oc for 72 h. viable cell counts were determined at 0, 24, 48, and 72 h. each determination was carried out in triplicates. statistical analysis data obtained was expressed as means ± standard deviation. analysis of variance was carried out on the data obtained to determine the significance of differences. a two-tailed p-value of less than 0.05 was considered as statistically significant. values that were significantly different were separated using the duncan multiple range test using spss for windows verson 17.0 statistical package. results and discussion a total of nineteen yeast isolates were obtained from the three samples of the investigated naturally fermented cassava waste pulp. out of these, five isolates that demonstrated significant linamarase activity were further studied. the macroscopic appearance of the yeast isolates was creamy white with a glossy surface. microscopic examination of the isolates revealed the presence of true hyphae and arthrospores. the blast of the sequences of yeasts isolated from naturally fermented cassava waste pulp is shown in table 1. the four isolates from this study were identified as geotrichum silvicola while the fifth isolate was identified as geotrichum candidum. fig.1 shows the maximum likelihood phylogenetic tree. it describes the evolutionary relationship between and among strains of organisms based on their character. the marked strains denoted with, klp04, klp05, klp06, klp07 and klp08, are those from the present study while the others are the related/similar strains retrieved from the genbank. as shown in the tree, the strains from this study are not closely related to the strains already deposited in genbank as the degree of relatedness (homology) they shared with the reference strains is less than 70 %, even though they share relatively high similarity (stackebrandt and goebel 1994). a previous report on phenotypic characterization of yeasts presented in some nigerian traditional fermented foods such as burukutu, ogi, and pito confirmed the presence of candida glabrata, debaryomyces hansenii, candida krusei, candida colliculosa, pichia anomala, pichia farinosa, and pichia membranefaciens (alakeji et al. 2015). in a related development, omemu et al. (2007) have isolated saccharomyces cerevisiae, candida krusei, candida tropicalis, geotrichum candidum, geotrichum fermentans, and rhodotorula graminis during the fermentation of maize for ogi production. taxonomic characterization based on morphological, physiological, and biochemical data indicated the presence of rhodotorula glutinis 5 nova biotechnol chim (2021) 20(2): e898 2 (fresenius) f. c. harrison var. glutinis – a linamarin degrading yeast in cassava wastewater treatment lagoons (vasconcellos et al. 2009). previous characterization of yeast ecology of fermented cassava products has documented the presence of different species of the following genera: saccharomyces, candida, hansenula, penicillium, geotrichum, rhodotorula, pichia, and zygosaccharomyces (amoa-awua et al. 1997; oyewole 2001; coulin et al. 2006; schwan et al. 2007). in another development, galactomyces spp was reported to be one of the yeasts involved in the fermentation of sour cassava starch (lacerda et al. 2005). however, geotrichum silvicola sp. nov. has been described as a novel asexual arthroconidial yeast species related to the genus galactomyces (pimenta et al. 2005). recently, geotrichum silvicola has been isolated from spontaneously fermenting motoho, a southern african nonalcoholic sorghum beverage (moodley et al. 2019). the present study is the first to report the presence of geotrichum silvicola in cassava fermented products. table 1. molecular identification of yeasts isolated from spontaneously fermented cassava waste pulp based on the its region of the genomic dna. isolate code identity organisms in genbank with significant alignment with isolate max. score total score query cover identity [%] accession number klp04 geotrichum silvicola geotrichum silvicola cbs 9194 500 500 95 96.43 mw232914 klp05 geotrichum candidum geotrichum candidum aumc10284 472 472 98 93.48 ok070746 klp06 geotrichum silvicola geotrichum silvicola cbs 9194 478 478 95 93.75 mw233017 klp07 geotrichum silvicola geotrichum silvicola cbs 9194 461 461 95 92.90 mw233034 klp08 geotrichum silvicola geotrichum silvicola cbs 9194 572 572 90 97.36 mw233050 fig. 1. the maximum likelihood phylogenetic tree of the sequenced isolates and other reference strains retrieved from genbank (*strains ending with klp 04, klp05, klp06, klp07, and klp08 are isolates from the present study). 6 nova biotechnol chim (2021) 20(2): e898 3 the linamarase activity (uml-1) of the yeast isolated from fermented cassava waste pulp is shown in table 2. the isolates exhibited activity ranging between 3.3 and 4.2 with strain klp04 having the highest value and strain klp05 the least. there is no significance difference among klp06, klp07, and klp08. the value obtained in this report is within the range (0.5 – 5.8) reported by nwokoro and anya (2011) for different fractions of linamarase enzyme of lactobacillus delbrueckii nrrl b-763. various workers have reported that yeasts and lactic acid bacteria are the major microorganisms responsible for the fermentation of cassava (essers et al. 1995; amoaawua et al. 1996; kobawila et al. 2005). the exhibited high linamarase activity of the isolated yeasts in the present study is indicating their capabilities to degrade the cyanogenic glycosides in cassava waste pulp; hence they could be used as starter cultures to produce fermented cassava waste pulp. table 2. linamarase (u.ml-1), gelatinase and haemolytic activity of yeasts isolated from fermented cassava waste pulp. isolate code linamarase activity gelatinase activity haemolytic activity klp04 4.2 ± 0.2a negative γ-haemolysis klp05 3.3 ± 0.1c negative γ-haemolysis klp06 3.7 ± 0.1b negative γ-haemolysis klp07 3.6 ± 0.0b negative γ-haemolysis klp08 3.6 ± 0.1b negative α-haemolysis values are means ± standard deviation (n = 3). within each column, values with different superscripts are significantly different. the gelatinase and haemolytic activities of the isolated yeasts are shown in table 2. none of the isolates demonstrated the two activities except strain klp08, which was partially haemolytic. the results obtained in this study, except for strain klp08, are similar to the findings of franz et al. (2001), mannu et al. (2003) and banwo et al. (2012) on enterococci of food origin. gelatinase genes (gel gene) may be silent, and phenotypes may be negative, even in the presence of gel gene (franz et al. 2001; yoon et al. 2008). most yeast present in food satisfies the important criteria of safety due to long history of safe human consumption in traditional fermented food products. hence, yeasts have the generally regarded as safe (gras) status (fda 2001). all yeast isolates (except strain klp08) in the present study could be tentatively assigned gras status as they exhibited safety potentials. the result of the present study underscores the absolute necessity to establish the safety status of every strain that will be used for food fermentation as intra-species variation was observed for haemolytic activity of the isolated yeasts. the quantitative determination of the effect of different conditions such as ph 2.5 and 2 % bile salt on the growth pattern of the isolated yeasts revealed strain-specific responses. all the examined yeasts exhibited good growth at ph 2.5 after the intervals of 24, 48, and 72 h (fig. 2). at the end of 72 h of growth, strain klp08 showed the highest viable counts of 4.1 log10cfu.ml -1 and strain klp04 the least value of 3.5 log10cfu.ml -1. these results are comparable to the findings of alakeji et al. (2015), who reported highest (6.78 log10cfu.ml -1) and least (2.18 log10cfu.ml -1) viable counts for candida colliculosa pii and pichia membranefaciens ba2 respectively after grew at ph 2.5 for 72 h. in a related development, psomas et al. (2001) and kourelis et al. (2010) reported that strains of saccharomyces cerevisiae, candida albicans and debaryomyces hansenii have survived at ph 3.0. from the results obtained for the influence of 2 % bile salt on the growth pattern of the examined yeast (fig. 3), all the yeasts showed strain-specific tolerance to 2 % bile salt. in this regard, strain klp04 had the highest viable count 4.3 log10cfu.ml -1 and strain klp08 the least value 2.2 log10cfu.ml -1 at the end of 72 h of incubation. debaryomyces hansenii oa3 and candida glabrata spy3 were reported to have the highest (6.25 log10cfu.ml -1) and least (1.88 log10cfu.ml -1) viable counts respectively after grew under 2 % bile salts for 72 h (alakeji et al. 2015). similarly, in-vitro studies have shown that wickerhamomyces anomalus survived excellently in >0.6 % bile salts (garcia-hernadenz et al. 2012; bonatsou et al. 2015). klaenhammer and kullen (1999) had stated that important criteria for the selection of probiotic strains are the maintenance of their cell integrity and retaining of their beneficial metabolic functions during gastrointestinal passage. 7 nova biotechnol chim (2021) 20(2): e898 2 fig. 2. effect of ph 2.5 on the growth of selected yeast strains. fig. 3. effect of 2 % bile salts on the growth of selected yeast strains. in this connection, there is the need for the strains to survive the natural barriers in the intestine, including body temperature, low ph and elevated bile concentration, as czerucka et al. (2007) had stated that the stomach ph at fed state could be as low as 2.5 for 3 h. all the isolated yeasts examined in the present study had impressive growth at the body temperature of 37 ºc, low ph of 2.5 and under 2 % bile salt condition. this may confirm that they are eminently qualified as probable probiotic that could be used for the fermentation of cassava waste pulp for the production of animal feed. however, further consideration of their safety potentials may disqualify strain klp08 on the ground of being partially haemolytic. conclusion based on all the technological properties examined in this study, geotrichum silvicola klp04 strain had the highest potential to be considered for starter culture for the fermentation of cassava waste pulp as it demonstrated the highest linamarase activity and tolerance to 2 % bile salt in addition to having good growth at ph 2.5 and demonstration of gras status. conflict of interest the authors declare that they have no conflict of interest. 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voort and abee 2009). bacillus subtilis can produce biofilms when hot fluids continuously flow over a surface and contaminate dairy products (lindsay et al. 2006). meanwhile, s. putrefaciens biofilm can cause spoilage in fish products and other aquatic food products (bai and rai 2011). besides that, s. putrefaciens can also cause food poisoning by production of neurotoxins, called tetrodotoxin 1 mailto:diana.waturangi@atmajaya.ac.id nova biotechnol chim (2021) 20(2): e919 2 (ttx) (wang et al. 2013). most of biofilms production is initiated through quorum sensing mechanism, where quorum sensing is a communication mechanism between bacteria and regulates gene expression of extracellular molecular signals (hadiwiyono 2009). to overcome this problem, it is important to inhibit bacterial communication. quorum sensing inhibition can be done by degrading extracellular signal molecules using enzymes. one of the bacteria that can produce metabolites which can interfere with quorum sensing process is phyllosphere bacteria. these bacteria are a group of bacteria living on the surface of the plant leaves that have a very large population and have not been much explored. phyllosphere bacteria can produce bioactive compounds that can inhibit the process of quorum sensing. for example, this bacteria can fight phytopathogenic dickeya dadantii by inhibiting of quorum sensing through production of ahl-lactonase as quorum quencher (satwika et al. 2017). the new discoveries can be made regarding phyllosphere bacteria as an anti-biofilm that can be done by interfering quorum sensing or destruction of biofilms that have been formed. the aim of this research was to screen anti-quorum sensing activity and quantification of anti-biofilm activity of phyllosphere bacterial isolates against food spoilage bacteria, such as b. cereus, b. subtilis, and s. putrefaciens. experimental phyllosphere bacterial isolates fifty-four phyllosphere bacterial isolates were recovered from the atma jaya culture collection (catholic university of indonesia, jakarta). these bacteria were isolated during previous study and were recovered from psidium guajava, averrhoa carambola, and anredera cordifolia leaves (juliana 2011; listriani 2011). for isolation of phyllosphere bacteria, the leaves were put into the tubes containing 10 ml of 10 mm of phosphate buffer (ph 8.0). then, the tubes were put into a sonicator for 5 min to release the bacteria from the leaves and homogenized in the buffer. after that, the bacteria suspensions were diluted to phosphate buffer with different serial dilutions (10 -1 , 10 -2 , 10 -3 , and 10 -4 ). a total of 100 μl of the bacterial suspensions were spread on the brain heart infusion agar (bhia) (oxoid ltd., basingstoke, united kingdom) and incubated 28 °c for 48 h. phyllosphere bacterial isolates from previous study were grown into king’s b agar (2 g protease peptone; 1.5 ml glycerol; 0.15 g k2hpo4; 0.15 g mgso4·7h2o; 20 g bacteriological agar; 1 l distilled water) and incubated at 28 °c for 48 h. king’s b is a semiselective media for phyllosphere bacteria containing glycerol as an abundant carbon source (humphrey et al. 2014). in this study, the majority of phyllosphere bacterial isolates were recovered from psidium guajava. from the previous study these bacteria showed rod-shape and gramnegativity. biochemical properties for all the isolates showed as proteus sp. molecular identification was done only for jb 16b that was identified as proteus myxofacies with accession number hq852045 (juliana 2011). food spoilage bacteria the food spoilage bacteria used in this study were bacillus cereus atcc 10876, bacillus subtilis atcc 6633, and shewanella putrefaciens atcc 8071. b. cereus and b. subtilis were inoculated on luria agar (5 g nacl; 5 g yeast extract; 10 g tryptone; 20 g bacteriological agar; 1 l distilled water) (la) and incubated overnight at 37 °c. s. putrefaciens were inoculated on tryptone soya agar (tsa) (oxoid ltd., uk) and incubated overnight at 28 °c. screening for anti-quorum sensing activity screening of anti-quorum sensing activity was done using the overlay agar method described by abudoleh and mahasneh (2017) with some modification. phyllosphere bacteria isolates were inoculated on king’s b media by the straight streak method and then incubated at 28 °c for 48 h. indicator bacteria chromobacterium violaceum was inoculated on luria broth (lb) (5 g nacl; 5 g yeast extract; 10 g tryptone; 1 l distilled water) and incubated at 28 °c for 24 h in an orbital shaker. a total of 100 μl of indicator bacteria from culture (od600 = 0.132) were prepared into 7 ml of semisolid luria agar (0.75 % agar) and poured to nova biotechnol chim (2021) 20(2): e919 3 5 3 overlay on the phyllosphere isolate. then, the media were incubated at 28 °c for 48 h. the positive result of this anti-quorum sensing screening was determined as a clear zone around the phyllosphere isolate which indicates inhibition of violacein pigment production. production of crude extract phyllosphere extraction was done by using a liquidliquid extraction method according to fitriani et al. (2016), with some modifications. phyllosphere bacteria isolates were inoculated into 200 ml of luria broth media and cultivated in the orbital shaker incubator at 28 °c for 48 h, and 120 rpm. then, the culture of the phyllosphere bacterial isolates were transferred into a conical tube and centrifuged at 13,888  g for 15 min. the cell-free supernatant was mixed with the same volume of ethyl acetate and kept in the orbital shaker incubator at 28 °c, 120 rpm, overnight. a solvent layer was taken and evaporated using a rotary evaporator and vacuum oven overnight to obtain the crude extract. after that, 1 % of dimethyl sulfoxide (dmso) were added to obtain a final concentration of stock 20 mg.ml -1 (w/v) and stored at -20 °c to maintain the stability of crude extract. antibacterial activity assay the antibacterial activity test was done using 3 food spoilage bacteria (b. cereus, b. subtilis, and s. putrefaciens) using agar well diffusion method. three pathogenic bacteria were spread onto brain heart infusion agar (bhia) (oxoid ltd., basingstoke, united kingdom) using a sterile cotton bud. then, a sterile cork borer was used to make wells. a total of 100 μl of phyllosphere’s extract were added into each well. streptomycin (10 mg.ml -1 ) was used as a positive control, while 1 % of dmso was used as a negative control. the inoculated media were incubated at 37 °c for 24 h. antibacterial activity was observed by the formation of a clear zone around the well. this assay was performed in triplicates. detection of anti-quorum sensing activity c. violaceum indicator bacteria were used to determine anti-quorum sensing activity using agar well diffusion method. c. violaceum was spread onto bhia media using a sterile cotton bud. sterile cork borer was used to make wells. a total of 100 μl of phyllosphere’s extract were added into each well. streptomycin (10 mg.ml -1 ) was used as a positive control, while 1 % of dmso was used as a negative control. then, the inoculated plates were incubated at 28 °c for 24 h. anti-quorum sensing activity was observed with the formation of clear zones due to the inhibition of violacein pigment production. this assay was performed in triplicates. quantification of antibiofilm activity extracts of phyllosphere bacterial isolates were tested against biofilm formation produced by food spoilage bacteria using 96-well microtiter plates. extracts jb 3b, jb 16b, jb 17b, jb 20b, jb 26b, jb 8f, jb 12f, and ejb 5f were used for this assay. then, three pathogenic bacteria were grown onto brain heart infusion broth (bhib) (oxoid ltd., basingstoke, united kingdom) and incubated at 37 °c for 24 h. for the inhibition assay, 100 μl of each food spoilage bacteria culture (od600 = 0.132) and 100 μl of extract of each phyllosphere were put into 96-well microtiter plates and incubated at 37 °c for 24 h. while for the destruction assay, 100 μl of each pathogenic bacteria culture (od600 = 0.132) were put into 96well microtiter plates and incubated at 37 °c for 24 h. after incubation, 100 μl of the extract of the phyllosphere were put into 96-well microtiter plates and re-incubated at 37 °c for 24 h. then, all of the media were removed, and the adherent cells were rinsed twice by distilled water. cells that had been rinsed were air dried before staining. biofilms were stained with 200 μl of 0.4 % (w/v) of crystal violet solution for 30 min. the dye was removed, and the wells were rinsed 5 times by distilled water and airdried for 5 min. the last step was the addition of 200 µl of ethanol into each well and resuspension to solubilize the crystal violet. optical density was determined using a microplate reader at a wavelength of 595 nm and the percentage of inhibition or destruction was calculated by the formula (eq. 1): nova biotechnol chim (2021) 20(2): e919 2 (1) results screening for anti-quorum sensing activity screening for anti-quorum sensing activity was carried out by overlay agar method. the positive results of this assay were recorded as a clearing zone around the phyllosphere isolates as shown in fig. 1, that indicated the inhibition of violacein pigment production. from this assay, there were 35 out of 54 phyllosphere isolates showing antiquorum sensing activity. fig. 1. screening for anti-quorum sensing activity: positive result from ejb 5f (a) and negative result from jb 40b (b). antibacterial activity assay antibacterial activity assays were done using agar well diffussion method. three food spoilage bacteria were used, including b. cereus, b. subtilis, and s. putrefaciens. the results revealed that the formation of a clearing zone was produced around the well (fig. 2). it was performed that there were 4 (jb 20b, jb 22b, jb 40b, jb 12f) out of 54 phyllosphere isolates with antibacterial activity against b. cereus, while 2 isolates (jb 16b and jb 12f) showed an antibacterial activity against b. subtilis. fig. 2. antibacterial activity assays of phyllosphere bacteria against bacillus cereus (a), bacillus subtilis (b), and shewanella putrefaciens (c). a b a b c 4 nova biotechnol chim (2021) 20(2): e919 3 5 3 detection of anti-quorum sensing activity the crude extracts of 8 out of 54 phyllosphere bacterial isolates gave positive results. antiquorum sensing activity was determined as clearing zones around the well (fig. 3). the isolates that had positive results were jb 3b, jb 16b, jb 17b, jb 20b, jb 26b, jb 5f, jb 12f, and ejb 5f. fig. 3. detection of anti-quorum sensing activity. quantification of antibiofilm activity the crude extracts of phyllosphere bacteria had the ability to produce antibiofilm against biofilm formed by food spoilage bacteria, such as b. cereus, b. subtilis, and s. putrefaciens, either through inhibition or destruction of the mature biofilm formed. all the crude extracts of the phyllosphere could inhibit the formation of biofilms with a value of mean percentage >50 %. for biofilm of b. cereus, 6 crude extracts were tested and the highest inhibition against biofilm of b. cereus was 85 % for jb 17b isolate. the lowest inhibition against biofilm of b. cereus was 59 % for jb 16b isolate. for quantification of antibiofilm against biofilm of b. subtilis, 6 crude extracts were tested. the highest inhibition was 76 % for jb 20b isolates, whereas the lowest inhibition was 38 % for jb 3b isolate. the highest inhibition against biofilm of s. putrefaciens was 94 % for jb 8f isolate and the lowest was 27 % for ejb 5f isolate (table 1). all the phyllosphere isolates have the ability to destruct the formation of biofilm. according to the results of biofilm destruction, a value of the mean percentage was >40 %. the highest destruction against biofilm of b. cereus was 66 % for jb 8f isolate and the lowest was 37 % for jb 3b isolate. the highest destruction against biofilm of b. subtilis was 93 % for jb 17b. the lowest destruction against biofilm of b. subtilis was 66 % for jb 3b isolate. while the highest destruction activity against biofilm of s. putrefaciens was 83 % for jb 8f isolate and the lowest was 39 % for jb 12f isolate (table 1). table 1. biofilm inhibition and destruction against food spoilage bacteria. extracts b. cereus b. subtilis s. putrefaciens % inhibition % destruction % inhibition % destruction % inhibition % destruction jb3b 69 37 38 66 51 66 jb16b 59 43 65 80 jb17b 85 45 74 93 93 78 jb20b 76 75 47 82 jb26b 77 46 56 76 50 66 jb8f 78 66 75 92 94 83 jb12f 36 39 ejb5f 75 30 54 89 27 44 = not be tested because contains antibacterial activity. discussion phyllosphere bacteria is one of the largest microbes habitats on the surfaces of plants. these bacterial communities might be beneficial to plants by producing plant growth hormones as well as production of other bioactive compounds (kim et al. 2012). these bioactive compounds are also capable acting as quorum quencher. our results revealed that during screening of anti-quorum sensing activity, 35 of phyllosphere bacterial isolates out of 54 isolates showed positive results, 5 nova biotechnol chim (2021) 20(2): e919 6 8 where the clearing zones were observed around wells of phyllosphere bacteria. it was indicated as inhibition of violacein pigment production through quorum sensing inhibition of c. violaceum. mechanism of quorum sensing inhibition might be happened through destruction of acyl-homoserine lactone (ahl) molecule which plays an important factor as the autoinducer (abudoleh and mahasneh 2017). the ability of phyllosphere bacteria to inhibit quorum sensing might act as a survival strategy for competition in the extreme and stressful environment due to the fluctuations in physical condition and limited nutrients. phyllosphere bacteria have ahl-degrading activity, such as ahl-lactonase and ahl-acylase. in a previous study phyllosphere bacteria was proven as a potential biocontrol reagent to control plant pathogens such as e. carotovora through signal interference using ahl-lactonase (ma et al. 2013). all of the phyllosphere bacteria in this study were extracted using ethyl acetate and dissolved with dmso to make the concentration of 20 mg.ml -1 . crude extracts of phyllosphere were screened for antibacterial assay, detection of quorum sensing, and quantification of antibiofilm. from antibacterial activity assay, positive results were observed by the formation of clearing zones (younis et al. 2015). there were 4 crude extracts that have antibacterial activity against b. cereus and 2 crude extracts against b. subtilis. the crude extracts that showed antibacterial activity would not be tested for further assay to avoid false positive result in the antibiofilm determination assay. from detection of anti-quorum sensing activity, there were 8 crude extracts that showed positive results. these positive results were observed by the clearing zones formation which indicated pigment inhibition activity by crude extracts of phyllosphere. c. violaceum as an indicator bacteria produces purple pigment, called violacein, the production is initiated by quorum sensing mechanism with ahl acting as an autoinducer (zahin et al. 2010). the main components of violacein production are cvii, ahl, cvir receptor. violacein is synthesized from tryptophan substrate, this substrate is the product of vioabcd operon. cvii synthase converts adenosyl methionine and fatty acids into ahl. in the presence of high concentration of ahl, quorum signal is triggered and ahl binds cvir and control the vioa promoter (stauff and bassler 2011). quorum sensing is not only about pigment production, this mechanism is a way for bacteria to communicate between cells, to produce and respond to extracellular signal molecule at a spesific cell density. besides pigment production, quorum sensing can be used by bacteria for virulence, bioluminescene, sporulation, and biofilm formation (hadiwiyono 2009). biofilm is a bacterial community that attaches to a surface and produces an exopolysaccharide matrix. the first step of biofilm formation is initial attachment stage. on this stage, the bacteria interact and strongly attach. a conditioning film is formed by the adsorption of organic and inorganic nutrients. after this stage, adhered bacteria will produce exopolysaccaride and adhere more firmly. then, the biofilm will mature and obtain a complex structure of biofilm. in the final stage, cells can release and colonize to other surfaces or return to a planktonic state (brackman and coenye 2015). in some bacterial species, the initial attachment stage can use a quorum sensing mechanism. bacteria use signal molecules when cell densities are high to colonize and stick to surfaces (zhang et al. 2009). biofilm can be found in the medical field, environment, or food industry. biofilms in the food industry might be on the food processing equipments or on the food surface. the presence of biofilms on food surfaces can cause spoilage and pathogen microbe contamination. same as on the food surface, several bacteria are known to form biofilms on the food processing with different material, such as stainless steel (ciccio et al. 2015). the quorum sensing inhibition can be used to inhibit or destruct biofilm that formed by food spoilage bacteria. according to the quantification of antibiofilm activity, all of the crude extracts of phyllosphere performed antibiofilm activity, both inhibition and destruction. the highest inhibition against biofilm of b. cereus was 85 % for jb 17b isolate. b. cereus can form biofilms on immersed glass or steel surfaces. the quorum sensing mechanism that induces biofilm formation in b. cereus is regulated by plcr which is activated by papr autoinducer 6 nova biotechnol chim (2021) 20(2): e919 7 peptide (aip) (pomerantsev et al. 2009). the highest inhibition againts biofilm of b. subtilis was 76 % for jb 20b isolate. b. subtilis is an opportunistic pathogen that can contaminate food products by biofilm formation on food processing equipment. the quorum sensing mechanism that induce biofilm is using comx autoinducer and competence sporulation factor (csf) (shank and kolter 2011). other food spoilage bacteria that have been used in this study was s. putrefaciens. this bacteria can be found in aquatic environments which can form biofilm on the surface of food processing. s. putrefaciens can form biofilm on a variety materials, such as stainless steel and glass. biofilm-promoting factor a (bpfa) is the protein contributing to biofilm formation with ai-2 signaling molecules in quorum sensing mechanism (cheng et al. 2016). we found that the highest inhibition against biofilm of s. putrefaciens was 94 % for jb 8f isolate. based on quantification of biofilm inhibition, all of the crude extracts of phyllosphere had a variety percentage of inhibition. it might have happened due to the different mechanism of biofilm formation from each food spoilage bacteria. based on the results of biofilm destruction, the highest biofilm destruction was for jb 8f isolate against biofilm of b. cereus and s. putrefaciens. the biofilm destruction activity against biofilm of b. cereus was 66 % and against biofilm of s. putrefaciens was 83 %. the highest biofilm destruction against biofilm of b. subtilis was 93 % for jb 17b isolate. these results indicated crude extracts of phyllosphere performed bioactive compound like protease, dnase, and another enzyme that can destruct the component of biofilm and potential for antibiofilm agent (ma et al. 2013). in the other study, these 8 crude extracts of phyllosphere had the ability to inhibit and destruct biofilm formation of fish pathogenic bacteria, such as vibrio harveyi, aeromonas hydrophila, and streptococcus agalactiae (nathalia and waturangi 2021). for further study, it is required to characterize the compound from phyllosphere bacteria which have anti-quorum sensing and antibiofilm activity, toxicity assay for phyllosphere extracts, and molecular identification for phyllosphere bacteria for further application. acknowledgements the authors acknowledge research funding support by indonesian ministry of education and culture through competitive national research grant 2019-fundamental research. conflict of interest the authors declare that they have no conflict of interest. references abudoleh sm, mahasneh am (2017) anti-quorum sensing activity of substances isolated from wild berry associated bacteria. avicenna j. med. biotechnol. 9: 23-30. bai aj, rai vr (2011) bacterial quorum sensing and food industry. compr. rev. food sci. food saf. 10: 184-194. brackman g, coenye t (2015) quorum sensing inhibitor as antibiofilm agents. cur. pharm. des. 21: 1-7. cheng yy, wu c, wu jy, jia hl, wang my, wang hy, zou sm, sun rr, jia r, xiao yz (2016) flra represses transcription of the biofilm-associated bpfa operon in shewanella putrefaciens. appl. environ. microbiol. 83: 115. ciccio pd, vergara a, festino ar, paludi d, zanardi e, ghidini s, lanieri a (2015) biofilm formation by staphylococcus aureus on food 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14th january 2021 accepted: 8th march 2021 keywords: anthocyanins butterfly pea extraction phenolic ph abstract an antioxidant compound is the main compound that is used to prevent cell damage from free radicals. these unstable molecules can be produced in the environmental condition such as pollution or lifestyle. one of the antioxidant molecules are anthocyanins, which can be found in the butterfly pea flower. this compound could be obtained from the extraction process. however, extraction conditions such as sample/solvent ratio, extraction time, and ph are the main factors in maximizing the yield. in this research, various factors on anthocyanins and phenolic content in butterfly pea extract were studied to get the optimum extraction condition. extraction of the butterfly pea flower was done using the agitation method with heating and water solvent at 60 °c and various parameters. the sample was a dried butterfly pea flower. various factors in extraction were: sample/solvent ratio, 1 : 20 and 1 : 50 (g.ml-1), extraction time of 90 and 150 min, and ph 1.0 and 7.0. yield is calculated by comparing the extract weight before and after drying. total anthocyanins content and total phenolic content are determined spectrophotometrically. based on the results, the extraction of anthocyanins was affected by the stability of structures at different ph values. the highest total anthocyanins content was 1,206.77 mg.l-1 at sample/solvent ratio 1 : 20, 90 min and ph 1.0 conditions. then, the maximum total phenolic content was 94.04 gae mg.mg-1 sample at the sample/solvent ratio 1 : 50, 90 min and ph 7.0.  university of ss. cyril and methodius in trnava introduction free radical has become a threat to health (phamhuy et al. 2008). this unstable molecule can damage cells causing severe health problems, such as atherosclerosis, arthritis, neurodegenerative disorder, and cancer (dreher & junod 1996; schumacker 2015). according to this health risk, high antioxidant food consumption is needed to ward off the free radical molecules to prevent the risk of severe health problems (pham-huy et al. 2008). the antioxidant can be obtained from vegetables and fruits with red, orange, yellow, and purple colors. butterfly pea flower has a purple color and has been known as a rich antioxidant compound. most people have been using this flower as an herbal food and drink colorant. butterfly pea plant (clitoria ternatea l.) is a plant from the fabaceae family. in indonesia, this plant is commonly called telang. the consumption of butterfly pea flower is believed to relieve pain from mouth sprue and reduce insomnia. the high antioxidant content in this flower promotes various pharmacological activities. according to gollen et mailto:dwi.aditiyarini@staff.ukdw.ac.id nova biotechnol chim (2021) 20(1): e817 2 al. (2018), butterfly pea flower has several pharmacological activities, such as anti-depressant, anti-anxiety, anti-stress, antioxidant, antiinflammation, anti-hyperglycaemia, anti-diabetes, analgesic, cytotoxicity, platelet aggregation inhibitory, and hepatoprotective. various biological activities of this extract are also affected by the content of secondary metabolites. butterfly pea extract contains various phytochemical compounds, such as alkaloids, tannins, glycosides, resins, steroids, saponins, flavonoids, and phenols (manjula et al. 2013). the content of secondary metabolites underlies the potency of the butterfly pea flower as a valuable medicinal plant. the main compound that gives antioxidant activity is anthocyanin. nair et al. (2015) identified 12 phenolic metabolites consisting of nine ternatin anthocyanins and three glycosylated quercetins from the butterfly pea flower. the phenolic compound is also contributed to its antioxidant activity. the phenolic compound has a role to perform the reduction of free radical molecules. however, anthocyanins and phenolic content obtained from the butterfly pea flower is closely affected by the extraction conditions. other factors of the extraction process are ratio sample/solvent and solvent. enhancement of yield occurs when the sample/solvent ratio is increased. according to pham et al. (2019), the maximum sample/solvent ratio was 1 : 25 (g.ml-1) to obtain a high yield. nevertheless, other factors also contribute to this result, such as time extraction, temperature, and extraction method. salacheep et al. (2020) obtained high butterfly pea extract efficiency at 45 min, 40 °c, and sample/solvent ratio 1 : 10 (g.ml-1) using ultrasound-assisted extraction (uae). extraction is also dependent on the solvent choice, which relates to the amount of anthocyanins. andriani and murtisiwi (2018) obtained phenolic content 19.43 ± 1.62 gae (gallic acid equivalents) mg.g-1 sample for ethanol extract of butterfly pea flower. for anthocyanins content, saptarini et al. (2015) achieved high anthocyanins at methanol extract for 145.17 ± 0.81 mg.l-1. ethanol 50 % was also known to obtain higher anthocyanins content than water. methanol and ethanol are suitable solvents for the extraction of anthocyanins. the use of these solvents has proven to produce high content of anthocyanins and phenolic. according to chemat et al. (2012), the manufacturer is currently forced to use a solvent with the absence of risk during the extraction and the safety of solvent traces in the extract. based on that regulation, water is possible to use as an extraction solvent regarding its polar nature. this polarity can be used to extract proteins, sugars, organic acids, and inorganic substances. the stability of anthocyanins also affects the extraction process. based on this reason, a particular ph condition is needed to be considered. saptarini et al. (2015) showed that ph 1.0 gives higher anthocyanins content than ph 2.0. according to wahyuningsih et al. (2017), anthocyanins are more stable in low ph or acid. anthocyanins also give a different color at different ph conditions. this property underlies the potency of anthocyanins from the butterfly pea flower as an indicator in acid-base titration. based on the description above, sample/solvent ratio, ph, and extraction time are factors relating to the obtained extract, specifically for anthocyanins and phenolic compounds. however, the information about the relationship between these factors is still limited. optimum extraction conditions must be known to obtain high yield or secondary metabolites content. therefore, the aim of this study is to assess the effect of different sample/solvent ratio, ph, and extraction time in the extraction of anthocyanins from butterfly pea flower, and to determine the optimal extraction condition of anthocyanins. in this research, we performed the combination of various sample/solvent ratios, time extraction, and ph to extract anthocyanins from butterfly pea flower. extraction is done using agitation with heating methods and water as a solvent. the optimum conditions of extraction are essential information for designing butterfly pea extraction methods on an industrial scale. the relationship of each factor to the stability of anthocyanins can be observed. experimental chemicals and equipments sodium acetate, hydrochloric acid fuming 37 %, potassium chloride, sodium carbonate anhydrous nova biotechnol chim (2021) 20(1): e817 3 were from merck (darmstadt, germany). the gallic acid (>99 %) was from sigma aldrich (saint louis, usa). the solvents were methanol, ethyl acetate, ethanol (96 %) and aquadest. the folinciocalteau reagent was from merck (darmstadt, germany). the ph value was monitored using ph meter (starter 300, ohaus). total anthocyanins content and total phenolic content was determined using uv-vis spectrophotometer (genesys 10s, thermo scientific). extraction of butterfly pea flowers the dried butterfly pea flower was taken from kalasan, sleman, yogyakarta special region. samples were prepared in powder form. ten grams of powdered butterfly pea flowers was prepared and added with aquadest, then heated on a magnetic hotplate stirrer (heidolph, 520 w, 220 v) at 50 °c. the extraction was done in various ph, sample/solvent ratio, and extraction time. acid condition, ph 1.0, was performed by the addition of 1 m hcl to the sample. variation of the extraction condition was shown in table 1. then the extracts were filtered with whatman no. filter paper no. 1. the liquid-liquid extraction method was performed using ethyl acetate. the aqueous extract was evaporated at 50 °c and ethylacetate extract at 40 °c with 6,000 rpm for each extract. furthermore, the butterfly flower's liquid extract was heated using an oven (mammert, schwabach, germany) to remove the remaining solvent. butterfly pea flower's liquid extract was weighed before and after being heated in the oven to calculate the yield value. this extraction was performed once. table 1. extraction conditions of butterfly pea flower. sample code extraction conditions sample/solvent [g.ml-1] extraction time [min] ph 1: 20 (a) 1: 50 (b) 90 (x) 150 (y) 1.0 (1) neutral (7) ax1    ax7    ay1    ay7    bx1    bx7    by1    by7    determination of total anthocyanins content measurement of the total anthocyanins content was carried out as previously described by giusti and wrolstad (2001). butterfly pea flower extract was diluted using 0.025 m kcl buffer ph 1.0 and 0.4 m ch3oona, ph 4.5. anthocyanins content was measured using uv-vis spectrophotometer at a wavelength (λ) 510 nm and 700 nm. the blank was an aquadest. total anthocyanins content was calculated as follows (eq. 1): anthocyanins level = (1) where a = (a510 nm – a700 nm) ph 1 (a510 nm – a700 nm) ph 4.5; mw = molecular weight of cyanidin-3glucoside (g.mol-1); df = dilution factor; l = cuvette width (cm); ε = absorptivity concentration of cyanidin-3-glucoside solution (l.mol-1.cm-1). preparation of gallic acid calibration curves and determination of total phenolic content (tpc) measurement of total phenolic content (tpc) refers to marjoni et al. (2015). weighed 0.2 g of gallic acid and dissolved in 100 ml of methanol. then, gallic acid was prepared in various concentrations 200, 266, 400, 534, and 800 µg.ml-1. from the series of concentrations of the gallic acid solution, 0.2 ml were taken and added with 1 ml of folin-ciocalteau reagent, then homogenized and allowed to stand for 8 min in a dark room. then, 2 ml of sodium carbonate 7.5 % was added, shaken, and left to stand in a dark room for 120 min. furthermore, the absorption measurement was carried out with a wavelength of 765 nm with aquadest as a blank. the absorbance values of each concentration were used to create a standard curve. this curve was created by plotting the gallic acid concentration as horizontal axis and the absorbance as a vertical axis. the equation from the linear regression was used to measure the phenolics content of the sample in gallic acid equivalents (gae) mg.ml-1. in sample preparation for determination of tpc, the butterfly pea flower extract was carried out with a 200x dilution factor. then, 0.2 ml solution was taken and added with 1 ml of folin-ciocalteau reagent, then homogenized and allowed to stand for nova biotechnol chim (2021) 20(1): e817 2 8 min in a dark room. 2 ml of na2co3 7.5 % was added, shaken, and left to stand in a dark room for 120 min. furthermore, the absorbance was carried out at a wavelength of 765 nm with aquadest as a blank. total phenolics content was calculated as follows (eq. 2): tpc = (2) where c = concentration of phenolic compound from linear regression equation (gae mg.ml-1); v = volume of extract used (ml); m = weight of sample used (mg). data verification for each assay was carried out by repeating the measurement three times from the same samples in each treatment (n = 3). then, the standard deviation was calculated, followed by the determination of standard error of mean. results and discussion extraction of anthocyanins from butterfly pea flower extraction is the primary beginning of isolation, purification, identification, and exploration of active compounds from a specific plant. several extraction methods are commonly used to isolate anthocyanins, such as maceration, agitation, ultrasonic-assisted extraction (uae), and microwave-assisted extraction (mae). the yield of the active compound obtained from the extraction is influenced by several factors, such as solvent, extraction time, size of samples, and sample source. based on these reasons, the optimization of the extraction condition is needed to increase the obtained yield. methanol and ethanol are general solvents in many extraction processes regarding their low toxicity and polarity. apart from those solvents, water can also be used to extract several secondary metabolites regarding the polarity. butterfly pea flower is known to contain a high antioxidant compound, anthocyanins. based on its structures, anthocyanins consist of a hydrophobic hydrocarbon part that is easily dissolved in an organic solvent and a polyphenolic functional group dissolved in a polar solvent. based on its polarity, water is one of the solvents that can be used to extract anthocyanins. anthocyanins have different structures in different ph conditions. in neutral ph, anthocyanins produce blueish purple, whereas it gives red extract in an acid condition. according to syahirah et al. (2018), an increase of h+ ion in an acid condition causes the reduction of the ketone group in the quinone ring producing hydroxyl group. it forms a flavylium cation that gives red color (fig.1). fig. 1. changing structures of anthocyanins in different ph condition (wahyuningsih et al. 2017; syahirah et al. 2018). in this research, extraction of anthocyanins was performed in two different ph conditions, acid for ph 1.0 and neutral for ph 7.0. the extraction was done in a simple method, agitation with heating. the result was shown in fig. 2. the red extract was obtained for ph 1.0, whereas ph 7.0 was purple. the highest yield was obtained in by1 extract with sample/solvent ratio 1 : 50, 150 min, and ph 1.0. in general, the yield from the acid condition is higher than the neutral condition. this data showed that acid conditions could help the extraction of anthocyanins. therefore, it increases the yield of extract. acid condition promotes the breakdown of cell vacuole of the plant, and anthocyanins can be easily obtained. it can also be caused by the ionization of anthocyanins in acid conditions forming flavylium cation. this structure improves the solubility of anthocyanins. however, the 4 nova biotechnol chim (2021) 20(1): e817 6 anthocyanins content and phenolic compound must be analysed further. assay repetition is performed to verify the result of these two assays. the error bar cannot be shown in their graph because of its different scale to the tac value. however, the standard error mean of each assay is presented in fig. 4 and fig. 6. we find out that the data are not significantly different based on their standard deviation. standard deviation is not presented, but the maximum standard deviation of tac is 1.30, and for tpc, the maximum standard deviation is 0.66. fig. 2. the yield of butterfly pea flower extract in different sample/solvent ratio, 1 : 20 (a) and 1 : 50 (b), extraction time, 90 min (x) and 150 min (y), and ph condition, acid (1) and neutral (7). total anthocyanins content of extract total anthocyanins content is used to understand the effect of different extraction conditions to get a high amount of anthocyanins from butterfly pea flower. monomeric anthocyanins were measured as cyanidin-3-glycoside using the differential ph method. the result is shown in fig. 3. the highest anthocyanins content is 1,206.77 mg.l-1 at ax1 extract for the following conditions: sample/solvent ratio 1 : 20, extraction time 90 min and ph 1.0. fig. 3. total anthocyanins content of butterfly pea flower extract from different sample/solvent ratio, 1 : 20 (a) and 1 : 50 (b), extraction time, 90 min (x) and 150 min (y), and ph 1.0 (1) and 7.0 (7). standard error of mean (sem) is defined as a measure of dispersion of the sample means compared to population means. sem is calculated by dividing the standard deviation by the square root of sample number. in this study, sem is used to measure how precise the sample mean is. sem of total anthocyanins content from each condition is presented in fig. 4. according to this value, we find out that no significant difference of sample means to population means was significant. the highest of sem, 1.11, is shown in ax1, followed by ax7, bx7, and by7 which have the same sem. then, bx1, ay1, ay7, and by1 give zero sem. it means that there was no different total anthocyanins content between three replicates. the replication of the assay was performed using same samples from the extraction at same time. consequently, the difference of mean value between the replicates is low to zero. data in fig. 3 represents high anthocyanins content in acid conditions compared to the neutral condition. this result corresponds to a yield of extract. low ph increases the obtained anthocyanins content that correlates to the stability of the anthocyanins. some factors, such as ph, temperature, light, and oxygen, also influence the stability of anthocyanins. in low ph, protonation of cyanidin molecule occurs, producing flavylium cation. the flavylium cation structure contains conjugated double bonds, which can perform delocalization of an electron in double bonds, increasing this molecule's stability. different from that, an increase in ph causes deprotonation resulting in a negative ion or anion. inggrid and iskandar (2016) stated that increasing of the ph caused a shift in the anthocyanins structure's equilibrium to an unstable structure. the 5 nova biotechnol chim (2021) 20(1): e817 3 equilibrium of anthocyanins molecules in different ph conditions is shown in fig. 1. this change is followed by the change of color from purple to blue. this characteristic underlies the potential of anthocyanins to be used as a ph indicator. fig. 4. the standard error of mean of total anthocyanins content from each condition. high anthocyanins content can also be affected by solubility of molecules, related to the changes of structures in different ph. in acid conditions, anthocyanins are in ion forms. the ionized form has a high solubility in aqueous solution compared to the molecule form. based on this structure, the highest anthocyanins content is obtained in an acid condition. it also affects the extraction time, which is needed to become shorter. based on data, an extraction time of 90 min produces higher anthocyanins content than 150 min. a similar result was also observed by pham et al. (2019). decreasing anthocyanins content occurred during the increase of extraction time. the highest anthocyanins content was produced at 30 min, then decreased at 45 min extraction time. an anticyclonic decomposition of anthocyanins probably occurs at high-temperature exposure in a long extraction time. based on the sample/solvent ratio factor, the 1 : 20 ratio produced the highest anthocyanins content. an increase in the amount of solvent was not accompanied by an increase in the extract's anthocyanins. a material or sample has a specific characteristic in the absorption of solvent or water. when its absorption capacity reaches the maximum point, the cell will swell and burst simultaneously. it results in the release of pigment from the cell vacuole. if this condition is not fulfilled, the pigment will be detained inside the cell vacuole, causing low yield (see fig. 3 on sample bx1, bx7, by1, and by7). the amount of solvent, which is more than the maximum amount, causes pigment's captivity inside the cell. in the research of pham et al. (2019), lowering anthocyanins content happened when the solvent amount was increased. the highest anthocyanins content is obtained in the sample/solvent ratio 1 : 25 with 121.58 mg.l-1. an increase in extraction time did not have a significant effect on increasing the result. in general, a longer extraction time produces low anthocyanins content. based on these data, the extraction of secondary metabolites, antioxidant compound in this case, must give attention to the equilibrium parameter in sample/solvent ratio, time, and ph. there is an optimum point in each parameter which has been found to maximize the anthocyanin content. higher amount of solvent is not necessarily equivalent to the number of obtained anthocyanins. total phenolic content of extract anthocyanins are secondary metabolites from the flavonoid group. this compound contains a hydroxyl group in its structure, which is positively related to its ability to ward off free radicals. according to that, it is essential to measure the number of phenolic compounds in an extract. the result of phenolic content measurement in this research was shown in fig. 5. the highest total phenolic content was 94.04 gae mg.mg-1 samples from bx7 extract with the following conditions: sample/solvent ratio 1 : 50, extraction time 90 min and ph 7.0. according to the data, the total phenolic content was not proportional to yield and total anthocyanins content. anthocyanin is a derivate single aromatic compound of cyanidin containing functional groups. the number of the hydroxyl groups of each kind of anthocyanins was variated. according to that, the result of total anthocyanins content does not represent total phenolic content. a further assay is needed. 6 nova biotechnol chim (2021) 20(1): e817 6 fig. 5. total phenolic content of butterfly pea flower extract in different sample/solvent ratio, 1 : 20 (a) and 1 : 50 (b), extraction time, 90 min (x) and 150 min (y), and ph condition, 1.0 (1) and 7.0 (7). sem of this assay is presented in fig. 6. low value of sem is low indicates no significant different of total phenolic content mean from three replicates. high sem values for ax7 and ay7 are caused by the difference of measured absorbance that is around 0.01 – 0.02. therefore, the sensitivity of the instrument affects the final result. according to the sample/solvent ratio, the result of total phenolic content varies. total phenolic content at 90 min (x) is higher than 150 min (y). the extraction condition influences this result. in this study, extraction is performed using water and heating. longer extraction time with heating can promote hydrolysis and oxidation of anthocyanins. it results in decreasing in total phenolic content in the extract. based on the medium's acid level, the total phenolic content in ph 1.0 is lower than the neutral condition. the acid condition can help the cell's breakdown, causing pigment release from the vacuole cell. according to the structure of anthocyanins, an acid condition or low ph will increase the hydroxyl group's number by forming flavylium ion. however, the result is different from that theory. padmaja and srinivasulu (2016) obtained the highest total phenolic content of butterfly pea extract at ph 7.2. decreasing phenolic content occurs at high ph from ph 2.0, but increases at ph 7.2. extraction of the phenolic compound from mulberry (morus nigra l.) also results in the highest total phenolic compound at ph 7.0 (espada-bellido et al. 2018). according to that, optimum phenolic content obtains at the neutral condition. anthocyanins are phenolic compounds, but the availability in the plant is only around <10 %. an increase in the total phenolic compound at neutral condition is probably coming from the degradation of other compounds. the product of degradation contributes to the phenolic compound's composition (espada-bellido et al. 2018). fig. 6. the standard error of the mean of total phenolic content from each extraction condition. in this study, we find that the various parameters must be considered to optimize the extraction of anthocyanins and phenolic compounds from plant. these parameters are the sample/solvent ratio, time, and ph. however, the solvents in this process must be considered, in specific for phenolic compounds. extraction in an acid condition using water as a solvent and heating can promote the hydrolysis of anthocyanins or other secondary metabolites. conclusion extraction of the butterfly pea flower is a key process to obtain high quantity and quality of product for scale-up production. the process is affected by the ratio sample/solvent, extraction time, and ph. in this study, the optimum extraction condition producing the highest total anthocyanins content is at sample/solvent ratio 1 : 20 (g.ml-1), 90 min and ph 1.0. the stability of anthocyanin was reached at acid condition, but is not always proportional to total phenolic content. the highest total phenolic content is produced at the sample/solvent ratio 1 : 50 (g.ml-1), 90 min, and neutral condition (ph 7.0). the degradation of other compounds during the extraction contributes to the total phenolic 7 nova biotechnol chim (2021) 20(1): e817 3 measurement. further analysis for antioxidant activity and active compounds is needed and will be performed in the subsequent studies. acknowledgments researchers thank the faculty of biotechnology, duta wacana christian university, for supporting this research financially. we also thank academic staffs who have the support and help this research. conflict of interest the authors declare that they have no conflict of interest. references andriani d, murtisiwi l (2018) penetapan kadar fenolik total ekstrak etanol bunga telang (clitoria ternatea l.) dengan spektrofotometri uv-vis. cendekia j. pharm. 2: 32-38. chemat f, vian ma, cravotto g (2012) green extraction of natural products: concept and principles. int. j. mol. sci. 13: 8615-8627. dreher d, junod af (1996) role of oxygen 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vietnam. iop conference series: materials science and engineering. 544(1). purwaniati arif ar, yuliantini a (2020) analisis kadar antosianin total pada sediaan bunga telang (clitoria ternatea) dengan metode ph differensial menggunakan spektrofotometri visible. j. far. vii(1): 18-23. salacheep s, kasemsiri p, pongsa u, okhawilai m, chindaprasirt p, hiziroglu s (2020) optimization of ultrasound-assisted extraction of anthocyanins and bioactive compounds from butterfly pea petals using taguchi method and grey relational analysis. j. food sci. technol. 57: 3720-3730. saptarini nm, suryasaputra d, nurmalia h (2015) application of butterfly pea (clitoria ternatea linn.) extract as an indicator of acid-base titration. j. chem. pharm. res. 7: 275-280. schumacker pt (2015) reactive oxygen species in cancer: a dance with the devil. cancer cell. 27: 156-157. syahirah ln, lutfi my, atika a, hafiz rm, zulhelmi oa, adzhan mo, khor py (2018) a comparative analysis of clitoria ternatea linn. (butterfly pea) flower extract as natural liquid ph indicator and natural ph paper. dhaka university journal of pharmaceutical sciences. 17: 97103. wahyuningsih s, wulandari l, wartono mw, munawaroh h, ramelan ah (2017) the effect of ph and color stability of anthocyanins on food colorant. iop conference series: materials science and engineering, 193(1). 8 nova biotechnol chim (2021) 20(2): e958 doi: 10.36547/nbc.958 1 nova biotechnologica et chimica typical moroccan goat lactic acid bacteria and their assay as starters linda zaaraoui 1,2,, abdellah bouksaim 1 , maha elhamdani 1,2 , aouatif benali 1 , mohammed oukassou 3 , khadija ounine 2 and mohammed bouksaim 1 1 laboratory of food technology, inra, rcar-rabat, p.o.box 6570, rabat institutes, rabat 10101, morocco 2 ibntofail university, faculty of sciences, p.o.box 242, kénitra, morocco 3 therapeutic physiology laboratory, institute of agronomy and veterinary hassan ii, p.o.box 6202 madinat al irfane, rabat, morocco  corresponding author: zaraoui.linda@gmail.com article info article history: received: 1 st september 2020 accepted: 5 th may 2020 keywords: adjunct culture adjunct starter cultures goat’s milk lactobacillus moroccan dairy products abstract the knowledge of lactic acid bacteria of raw milk and the main factors affecting their variability are particularly important issues for the control of cheese processing and the bioconservation of farm raw milk food products. the present research study concerned the isolation and identification of twenty strains of the lactobacillus genus from goat milk originating from the oulmes region, using the api 50 ch system. all isolates found represented five species: lactobacillus plantarum (43.75 %), lactobacillus brevis (37.75 %), lactobacillus pentosus (6.25 %), lactobacillus salivarus (6.25 %), and lactobacillus acidophilus (6.25 %). according to biochemical activities, the majority of the strains displayed weak acidification and autolysis activities in milk. in contrast, they showed high extracellular proteolytic activity. all isolates produced exopolysaccharides and most of them could metabolize citrate. the absence of hemolytic activity may suggest the use of these isolates as adjunct starters in the food fermentation process.  university of ss. cyril and methodius in trnava introduction lactic acid bacteria are food-grade microorganisms that play a vital role in the fermentation of animal and plant raw materials. their ability to ferment carbohydrates and to a lesser degree, degrade proteins and lipids, leads to the synthesis of a wide range of compounds, such as organic acids, peptides, antimicrobial and aromatic compounds, and exopolysaccharides. these compounds may contribute to the organoleptic, technological, and nutritional characteristics of fermented foods (mozzi et al. 2010; ray et al. 2014). the discovery of the action of lactic bacteria on milk was probably accidental, but their use was perpetuated in the form of natural leavens (chammas et al. 2006; zamfir et al. 2006). over the past fifteen years, considerable interest has developed around the use of lactic cultures with beneficial effects on health or "probiotics" (bifidobacterium, lactobacillus). the probiotics are living microorganisms which, when administered in adequate quantities, are beneficial to the health of the host (fao / who 2001). this definition could not specify the nature of the benefit, and as a result, many health benefits were attributed to lactic acid bacteria with probiotic potential. they include anticancer, anti-diabetic, anti-obesity, anti-diarrheal, mailto:zaraoui.linda@gmail.com nova biotechnol chim (2021) 20(2): e958 immune, anti-allergic, anti-oxidant, antimicrobial, and microbial-balanced activities of the colon (teitelbaum and walker 2002; park et al. 2016). the objective of the present study is to identify strains of lactobacilli isolated from goat milk of the oulmes region and to test some technological aptitudes of these strains. experimental sample collection the samples of goat milk originated from the ait ichou region situated in a rural mountainous region called oulmes city belongs to the province of khemisset, rabat-salé-region kenitra in morocco. the raw goat milk samples were immediately cooled and delivered to the microbiology laboratory in an isotherm container, being analyzed in arrival. isolation and purification of lactic acid bacteria to carry out this operation, ten milliliters of each raw milk samples were aseptically added into 90 ml of sterile 0.9 % nacl solution and mixed thoroughly. serial dilutions (10 – 1 to 10 – 8) were performed and 1 ml aliquots of appropriate dilution were directly inoculated in triplicate on the following media for lactic acid bacteria such as the man rogosa and sharpe (mrs) (fluka, sigma-aldrich) and m17 (oxoid) agar media. after incubation in petri plates for 24 h at 15 ºc, 32 ºc, 38 ºc, and 45 ºc, representative strains of lactic acid bacteria were obtained from m17 and mrs plates of highest sample dilutions. colonies were either randomly picked up or when the plate contained less than 10 colonies (leisner et al. 1997). the purity of the isolates was checked by streaking again to fresh agar plates, followed by macroscopic and microscopic examinations. a total of one hundred purified isolates were collected whose twenty strains occurring in pairs or chains of size baton gram-positive and catalasenegative were selected for further studies. for the conservation, the strains of lactic acid bacteria were stored at -80 °c in mrs broth supplemented with 15 % (v/v) glycerol until use. and their regeneration was monitored by an overnight incubation under 37 °c in mrs broth. api systems the bacteria gram-positive, catalase-negative was the subject of biochemical identification using the biomérieux api system using api 50 ch gallery with api 50 chl medium (biomerieux, marcy star, france) (ghanbari et al. 2009). technological characteristics to perform these bacteria technological characteristics, the following parameters are tested under the optimum conditions. acidifying activity to evaluate the acidifying power, the concerning 20 strains were initially grown in mrs broth at 37 °c for 24 h. and there are inoculated at a level of 1 % in reconstituted sterile skim milk solution (10 % w/v) (fluka, sigma-aldrich). then, the ph was measured (ph 211 precision ph meter, hanna instruments inc., italy) after 2, 4, 6, and 24 h incubation at 37 °c. the acidification rate was calculated as (eq. 1): δph = phf (finalvalue) ph0 (initialvalue) (1) the experiments were carried out in duplicate. proteolytic activity to determine the proteolytic activity, the isolates were subcultured twice in reconstituted skim milk (10 % w/v), containing yeast extract (0.3 % w/v), for 24 h at 37 °c and using 1 % (v/v) inoculum. final growth was performed in skim milk (10 % w/v) for 24 h at 37 °c (1 % v/v inoculum). the proteolytic activity was determined by the quantity of free amino acids released according to the method of church et al. (1983). results were expressed as glycine equivalents (mm) according to a standard curve, prepared using pure glycine in the range of 0 – 10 mm. 2 nova biotechnol chim (2021) 20(2): e958 3 autolytic activity for determining the important autolytic activity factor, the overnight cultures were centrifuged (5000 × g for 15 min at 4 °c). the cell pellet was washed twice using potassium phosphate buffer (10 mm, ph 7.0) and then suspended in potassium phosphate buffer (10 mm, ph 5.5). the obtained cell suspension was subjected to one cycle of freezing (-20 °c for 22 h) and thawing, then incubated at 45 °c for 2 h. the autolytic activity was determined as the percentage decrease in the absorbance at 650 nm at different time intervals as described by boutrou et al. (1998), which was defined as follows (eq. 2): autolytic activity % = (a0 – at) × 100/a0 (2) where a0 = initial absorbance and at = absorbance measured after t hours of incubation. citrate metabolism it is noticed in the technological microorganism’s field that bacteria citrate utilization in the presence of carbohydrates was studied on the special agar medium as described by kempler and mckay (kmk agar) (kempler and mckay 1980). the blue bacteria colonies and/or large blue center colonies were considered citrate positive. exopolysaccharides (eps) production it is known that the production of the exopolysaccharide was evaluated as reported by mora et al. (2002). for this special and important production, overnight cultures were streaked on the surface of plates containing ruthenium red milk (10 % skim milk powder, 1 % sucrose, 0.5 % yeast extract, 0.08 g 1-1 ruthenium red, 1.5 % agar). after incubation at 37 °c for 24 h, non-ropy isolates gave red colonies due to the staining of the bacterial cell wall, while ropy isolates appeared as white colonies. hemolytic activity in this step of the present research work, the hemolytic activity was performed as described by maragkoudakis et al. (2009). the strains were examined for signs of β-haemolysis (clear zones around colonies), α-haemolysis (green zones around colonies) or γ-haemolysis (no clear zones around colonies), and the results are illustrated and interpreted as shown in the part of the results and the discussion. antibacterial activity determination this parameter can play an important role in food preservation. so, to evaluate the antibacterial activity of identified strains, the test was monitored against some known pathogenic bacteria by the good diffusion assay using cell culture or cell supernatant (du toit et al. 1998; jamaly et al. 2011). fresh overnight mrs cultures were centrifuged at 8,000 × g for 10 min, and the cellfree supernatants were used directly or after being filtred aseptically (0.22 μm pore size; serva, heidelberg, germany), neutralized with 1 mol.l -1 naoh (ph 6.5 – 7) and treated with catalase (0.5 mg.ml -1 ) (sigma-aldrich). the lactobacillus cells were diluted with mrs and used for their antimicrobial activity. it is noticed that the indicator pathogenic strains included listeria innocua (lmhae-li 107), staphylococcus aureus (lmhae-sa 105), pseudomonas aeruginosa (atcc 29753), klebsiella pneumonia (cip 53153), micrococcus luteus (atcc15957), and escherichia coli (atcc54127), were tested. they were grown overnight in lb broth (sigma-aldrich) at ph 7.0 and diluted with sterile phosphatebuffered saline (pbs) (ph 7.2). after dilution, the pathogenic strains were mixed with 5 ml of lb soft agar (0.7 %, w/v) to reach a final concentration of 104 cfu.ml -1 , this medium was poured into petri plates prepared in advance with 10 ml of basal agar containing 2 % (w/v) agar. wells of 5 mm diameter were performed using the top of a pasteur pipette and were filled with approximately 50 µl of 108 cfu.ml -1 of identified strains in 0.1 % saline peptone, of cell-free treated supernatant and cellfree untreated supernatant. the plates were then stored at 4 °c for 4 h to allow the radial diffusion of any antimicrobial compound. following incubation at 37 °c for 24 h, the plates were monitored for the appearance of clear zones of inhibition. each test was performed in triplicate. nova biotechnol chim (2021) 20(2): e958 results and discussion api 50 chl systems identification for bacteria selection and identification, the biochemical identification by api 50 ch system is carried out after incubation of the galleries at 37 °c for 24 h. the results are read directly on the seeded gallery. the profiles obtained were analyzed by apilab software, in collaboration with the microbiology laboratory of the national center for scientific and technical research (cnrst). it should be specified here that in the case of inaccurate identifications or unidentified profiles in the api database; the isolate is thus considered as unidentified. table 1. results of bacteria identification by api 50 ch. twenty isolates under study and identified by api 50 ch showed a variety of lactobacillus species. five different species were identified between the 20 isolates (table 1). however, four bacterial species have not been identified; these are isolates 1; 6; 12, and 13. in light of the present results, a dominance of lb has observed lb. plantarum with a percentage of 43.75 %, followed by lb. brevis with a percentage of 37.5 %. the remainder were distributed among lb. acidophilus; lb. pentosus, and lb. salivarus with a percentage of 6.25 % each. first of all the presence of lb. plantarum as the majority species in goat milk samples from oulmes may be explained by a natural selection due to the region biotic environment, giving that these species are considered as the habitual host of plants (zadi karam et al. 2006). also, the two species lb. plantarum and lb. brevis are widely studied, have proved to possess probiotic potential (jamaly et al. 2011; guidone et al. 2014; jia et al. 2017) and are worth exploiting to improve fermented milk products, such as yogurt, cheese, etc. also, the marketed systems are with limited databases, the manufacturer of the gallery can only do their updates. a bacterium absent from the repertoire of identification galleries will not be recognized. in general, the error percentage in the galleries is a function of several parameters that may be due to the experimenter, the api system itself, or a mutation of the microorganism in identification. also, it has been reported that api identification is only about 65 % reliable (soto et al. 1994; ouadghiri et al. 2005), hence the importance of using genotypic techniques. however, the api system remains a useful means for an initial microorganism’s identification. however, it's coupling with other tests such as immunological and molecular could be, a fortiori, interesting to deepen the scientific and technological knowledge that concern the lactic bacteria in morocco. these bacteria can also play an important role in the transformation and valorization of agricultural products of economic and social interest (olives, milk, etc.). acidifying power the results of acidifying activity in milk at 37 °c are shown in fig. 1. the initial ph of the milk was 6.59. all isolates showed low acidifying activity after 2 h (δph2), 4 h (δph4), and 6 h (δph6) incubation, with values ranging from 0.02 to 0.37, from 0.07 to 0.53, and from 0.07 to 0.63 ph units, respectively. as regards their ability to reduce the ph of skim milk in 24 h (δph24), the values of the acidification activity of bacteria isolates studied ranged from 1.75 ± 0.03 to 0.35 ± 0.01 ph units. strains identification api 50 chl [% i.d] 1 lb. sp 2 lb. brevis 99.7 3 lb. pentosus 87.6 4 lb. plantarum 98.8 5 lb. plantarum 86.9 6 lb. sp 7 lb. plantarum 99.4 8 lb. salivarus 94.9 9 lb. plantarum 99.9 10 lb. brevis 99.9 11 lb. plantarum 99.8 12 lb. sp 13 lb. sp 14 lb. brevis 99.5 15 lb. acidophilus 98.7 16 lb. brevis 99.8 17 lb. plantarum 94.4 18 lb. plantarum 99.6 19 lb. brevis 99.7 20 lb. brevis 99.1 4 nova biotechnol chim (2021) 20(2): e958 3 fig. 1. the ph decreasing in reconstituted skim milk after 2 h (δph2), 4 h (δph4), 6 h (δph6), and 24 h (δph24) of incubation at 37 °c, respectively. values are mean ± standard deviation (n = 3). it was noted that lb. brevis16 showed the highest acidification activity (1.75 ± 0.01 ph units), while lb. plantarum11 showed the lowest one (0.35 ± 0.01 units ph). after 24 h none of the lactobacillus strains can be characterized, as fast as they did not reach a ph of the milk below 5.3 after 6 h in optimal growth temperature (beresford et al. 2001). these results are not consistent with those reported by buket et al. (2012) who observed that l. plantarum had a fast acidifying capacity. so, it is known that a rapid decrease in ph during the initial step of cheese preparation is essential for coagulation and for the prevention or reduction of the growth of adventitious microbiota they could not be used as starter organisms. however, they may be useful as adjunct cultures depending on their other important properties associated with industrial needs. proteolytic activity in this phase of the research, all species showed proteolytic activity evaluated at values greater than 1 mm glycine. indees, isolates lb. brevis2, lb. plantarum9, lb. plantarum7, and lb. pentosus3 have shown its highest activity with values (3.8 mm gly, 4.7 mm gly, 9.4, and 7.75 mm gly, respectively). in previous studies (el-ghaish et al. 2010; kholif et al. 2011; moslehishad et al. 2013) also reported that lb. plantarum had the highest proteolytic activity among the tested strains. thus, the values obtained for the lactobacillus strains tested are superior when compared to those reported by herreros et al. (2003) and ballesteros et al. (2006). it should be noted that species exhibiting high acidifying activity do not necessarily have the highest proteolytic activity, the same observation was previously reported by requena et al. (1991), fortina et al. (1998); durluozkaya et al. (2001). this proteolytic activity is one of the most important desirable criteria for complementary cultures as it may be responsible for aroma production, flavor enhancement, cell growth, and inhibitory activity enhancement of the 5 nova biotechnol chim (2021) 20(2): e958 6 fermented final product (yvon et al. 2006; donkor et al. 2007). autolytic activity cell autolysis allows to release of intracellular enzymes, including peptidases that can contributes to maturation and contribute to the development of cheese flavors (fitzsimons et al. 2001; collins et al. 2003; lortal et al. 2005). nevertheless, the degree of autolysis is straining dependent (wilkinson et al. 1994; el-soda et al. 2000). all of the strains tested in the present study showed variable autolytic activities (table 2). in general, they exhibited a low autolysis rate as described by ayad et al. (2004): 0.78 and 17.04 %, respectively. this result is not in agreement with those published by boutrou et al. (1998); wainrichter et al. (2001); salima et al. (2009); dako et al. (1995) and elsoda et al. (1995), who observed higher autolysis (superior of 50 %) for lb. plantarum and lb. brevis. nevertheless, these values are compatible with the potential role of the strains as adjunct cultures (franciosi et al. 2009; jamaly et al. 2010). citrate metabolism based on the results obtained in this study, the lactobacillus strains studied under the present research conditions showed a difference in their ability to use citrate (table 2). of the 16 lactobacillus strains studied just one strain lb. plantarum18 was found to be negative citrate. several studies have shown that some species of non-starter lactic acid bacteria (nslb) isolated from milk and cheese, like lb. plantarum, possess the ability to use citrate as the only carbon source and thereby metabolize it by producing different aromatic compounds such as acetate, lactate, acetone (palles et al. 1998; adesulu-dahunsi et al. 2017). table 2. summary of the results of the biochemical tests of lactobacillus isolates. a presented values are means of duplicate determinations ± sd. exopolysaccharides (eps) production and hemolytic activity the eps produced by lactic acid bacteria are used as thickeners or viscosifiers, stabilizing or emulsifying agents, and as gelling and waterbinding agents or texturizers. it appears that all lactobacillus were able to produce eps as shown in table 2. these cultures will be used as adjuncts cultures for their ability to improve the texture of “rayeb” milk (marshall and rawson 1999). stabilize the yogurt gel and decrease its tendency to strains metabolism of citrate eps production autolysis a [%] proteolysis a [mm gly] lb. brevis2 + + 2.90 ± 0.5 3.79 ± 0.27 lb. brevis10 + + 1.76 ± 0.6 1.10 ± 0.09 lb. brevis14 + + 2.96 ± 0.82 1.48 ± 0.25 lb. brevis16 + + 2.71 ± 0.2 1.25 ± 0.04 lb. brevis19 + + 1.32 ± 0.55 1.82 ± 0.16 lb. brevis20 + + 2.06 ± 0.7 1.22 ± 0.15 lb. plantarum4 + + 3.40 ± 0.5 1.17±0.14 lb. plantarum5 + + 17.04 ± 0.63 1.36±0.14 lb. plantarum7 + + 3.23 ± 0.68 9.39±0.94 lb. plantarum9 + + 4.19 ± 0.89 4.63±0.11 lb. plantarum11 + + 2.33 ± 0.59 1.17±0.34 lb. plantarum17 + + 0.77 ± 0.15 1.30 ± 0.12 lb. plantarum18 + 3.01± 0.12 1.91 ± 0.03 lb. salivarus8 + + 3.58 ± 0.57 2.14 ± 0.05 lb. acidophilus15 + + 5.89 ± 0.89 1.40 ± 0.09 lb. pentosus3 + + 4.07 ± 0.1 7.75 ± 0.36 nova biotechnol chim (2021) 20(2): e958 7 syneresis (parente et al. 2017). and to product drinking yogurt, cheese, fermented cream, and milk based desserts, (cerning 1995; crescenzi 1995). they may also be involved in prebiotic, probiotic, and biological activities, as well as having potential application in the food industry (harutoshi 2013; shiby et al. 2013; silva et al. 2019). none of the strains tested produced hemolysis when tested on sheep blood. the absence of such activity should be a criterion for selecting strains to be used as a starter or adjunct cultures in dairy products (giraffa 1995). antibacterial activity determination lactobacillus species had strong antibacterial against many bacterial pathogens (jamaly et al. 2011; eid et al. 2016) as well as perform essential roles in the preservation of food dairy product for human consumption (mufandaedza et al. 2006; batdorj et al. 2007). according to the results observed in the present work (table 3), four isolates: lb. plantarum5, lb. plantarum9, lb. brevis14, and lb. plantarum17 have no activity against the six pathogens; and none of lactobacillus strains study showed bacteriocin activity spectrum against the indicator organisms since no inhibition was observed when treated supernatants (ph 6.5) was tested. it was the same results found by (jamaly et al. 2011). while for the untreated supernatant it was observed that only the supernatant of the isolates lb. pentosus lb. salivarus8, lb. acidophilus15, lb. brevis19, and lb. brevis20 had antibacterial activity against klebsiella pneumonia cip 53153. however, we notice that the majority of the cells of table3. the antimicrobial activity determination of lactobacillus against pathogenic bacteria. strains listeria innocua lmhae-li 107 staphylococcus aureus lmhae-sa 105 pseudomonas aeruginosa atcc 29753 klebsiella pneumonia cip 53153 escherichia coli atcc54127 micrococcus luteus atcc15957 s st c s st c s st c s st c s st c s st c lb. brevis 2 ++ lb. pentosus3 ++ ++ lb. plantarum4 ++ lb. plantarum5 lb. plantarum7 ++ lb. salivarus8 + ++ lb. plantarum9 lb. brevis10 ++ lb. plantarum11 + lb. brevis14 lb. acidophilus15 + ++ ++ + lb. brevis16 ++ lb. plantarum17 lb. plantarum18 ++ lb. brevis19 + ++ ++ ++ lb. brevis20 ++ ++ + note: no inhibition; + inhibition zone between 2 and 6 mm; ++ inhibition zone larger than 6 mm. c: cells of strains in fresh mrs broth; s: cell-free supernatant; ts: cell-free supernatant adjusted to ph 6.5 – 7 and treated with catalase. results are averages of three experiments. lactobacillus had an effective anti-pathogenic biological activity. the lactobacillus lb. brevis19 presents an antibacterial activity against listeria innocua lmhae-li 107. while lb. acidophilus15 presents an antibacterial activity against pseudomonas aeruginosa atcc 29753. when lb. acidophilus15, lb. brevis19, and lb. brevis20 showed activity on micrococcus luteus atcc15957. for all the lactobacillus isolates, except of lb. plantarum5, lb. plantarum9, lb. plantarum17, and lb. brevis14, they showed activity on klebsiella pneumonia cip 53153. finally, none of the bacteria studied has activity on staphylococcus aureus lmhae-sa 105 and escherichia coli atcc54127. nova biotechnol chim (2021) 20(2): e958 8 conclusion presented research concerned to the lactic acid bacteria isolated from local raw goat milk from the oulmes region. it showed a special and important diversity of bacteria that may be due to the biotope of this area. so, lactobacillus plantarum (43.75 %); lactobacillus brevis (37.75 %); lactobacillus pentosus (6.25 %); lactobacillus salivarus (6.25 %); lactobacillus acidophilus (6.25 %) were identified. this bacteria isolated from 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(2006). bactéries lactiques du lait de chamelle d’algérie: mise en évidence de souches de lactococcus résistantes au sel. tropicultura, 24: 153-156. 10 https://pubmed.ncbi.nlm.nih.gov/?term=mishra+hn&cauthor_id=23391015 nova biotechnol chim (2022) 21(1): e991 doi: 10.36547/nbc.991 1 nova biotechnologica et chimica development of microbial consortium for degradation of organic kitchen waste lemnaro jamir1,2, paavan singhal4, soniya goyal4, robinka khajuria1,5, gurseen rakhra3, chubasenla aochen2, mahiti gupta1,4, 1school of biotechnology and biosciences, lovely professional university, phagwara, punjab 144411, india 2division of biotechnology, icar-rc neh region, umiam, meghalaya 793103, india 3department of nutrition & dietetics, faculty of allied health sciences, manav rachna international institute of research & studies, faridabad 121004, india 4department of biotechnology, maharishi markandeshwar university, ambala 133207, india 5department of biotechnology, harlal institute of management and technology, noida 201310, india  corresponding author: mahitigupta@gmail.com article info article history: received: 28th may 2021 accepted: 30th december 2021 keywords: degradation kitchen waste microbial consortium organic abstract increasing population and urbanization has led to an exponential increase in organic waste. this waste if not treated properly may lead to pollution and health hazards. so, a study was conducted to develop an efficient bacterial consortium for the degradation of organic kitchen waste. different bacterial strains were obtained from mtcc, initially grown on specific media and were screened for their enzymatic activities. eight bacterial strains viz. bacillus subtilus (mtcc 441), b. amyloliquefaciens (mtcc 7913), b. megaterium (mtcc 8510), lactobacillus delbrueckii (mtcc 2365), l. plantarum (mtcc 1407), l. rhamnosus (mtcc 1423), pseudomonas aeruginosa (mtcc 4673) and staphylococcus aureus (mtcc 740) were tested for the production of lipase, amylase, protease and cellulase. all the bacterial strains exhibited enzyme activities except b. megaterium. so, combinations of these strains were used to make consortia on basis of pathogenicity. two different microbial consortiums were prepared with consortia 1 comprising b. subtilis, l. plantarum, l. rhamnosus, l. delbrueckii, b. amyloliquefaciens and consortia 2 comprising p. aeruginosa, s. aureus, l. delbrueckii, l. rhamnosus, b. subtilus. consortium 1 displayed the highest amylase and protease activity as compared to consortium 2, and therefore selected for monitoring the composting parameters of the organic kitchen wastes. the microbial consortium showed significant outcome for composting parameters such as moisture content (65 – 58 %), temperature (25 – 29 ºc), ph (5.5 – 5.7), organic carbon (41 – 36 %) and decrease in mass (25 %) from 25 to 35 d of composting organic kitchen waste. the c/n ratio was calculated at 38/1 which falls within the optimum composting period. developing a microbial consortium significantly reduces the degradation time and thereby the composting process. this microbial consortium is environmentally safe and can therefore, be used to effectively improve the management of organic waste at both the community and industrial level. introduction the accelerated industrialization and urbanization in developing countries generate billion tons of domestic waste annually (singh et al. 2011). this waste is not treated properly due to poor state of collection and transportation. also, the domestic mailto:mahitigupta@gmail.com nova biotechnol chim (2022) 21(1): e991 2 waste collected from various homes is not properly sorted (meng et al. 2021). this has led to various health issues due to its improper disposal in developing countries like china and india among others (li et al. 2020). in india, 68.8 million tons of municipal solid waste is generated per year at a per capita waste generation rate of 500 grams/person/day (gupta and arora 2016). kashyap et al. (2003) stated that about 135.5 million tons/year of municipal solid waste generated in india consists of 30 – 40 % of food waste. the waste produced in india consists of about 50 % organic content as compared to 30 % in the developed countries (joshi and ahmed 2016). carbohydrates, lipids, proteins, and traces of inorganic compounds are the chief chemical components of food waste. according to the available data, asia alone is estimated to have spent about 25 billion us dollars per annum on solid waste management in 1990 and is projected to rise up to 50 billion us dollars per annum by 2025 (hoornweg and thomas 1999). while effective disposal methods such as gasification, landfills etc. are very efficient, these methods tend to have negative impact on the surrounding environment posing risks to public health. the biological handling of such wastes is highlighted to be the best cost-effective method with less adverse impact on the environment (coker et al. 2006). biological treatment of such waste is also referred to as composting. in composting, microorganisms such as bacteria and fungi break down complex organic compounds into simpler stabilized organic matter called compost under aerobic conditions and release carbon dioxide, water, and minerals. the process destroys pathogens and weeds because of the heat release (tweib et al. 2011), converts nitrogen from unstable ammonia to stable organic forms, reduces the volume of waste and improves the physico-chemical properties of the waste. it also makes waste easier to handle and transport, and often allows for higher application rates because of the more stable slow release of n in compost (fauziah et al. 2009). in normal systems, composting has the potential to protect the soil against erosion, reduce soil compatibility, enhance soil water retention and decrease soil acidity, enhance biological and soil biochemical functions, and therefore create an all-encompassing soil ecological symmetry (cao et al. 2021). in these systems, the organic degradation of compost is supported by several coexisting organic microorganisms, and the overall performance of this process is the combined effects of the activity of individual microorganisms with its biochemical functions, which in turn create an all-encompassing soil ecological symmetry (pfotzer and schuler 1997; nevens et al. 2003). synergistic associations are also established among other non-cellulolytic microbial species, and from this association are relied upon to expand the substrate degradation rates (lynd et al. 2002). the synergetic association promotes the decomposition process of kitchen waste, which in turn paves the way for efficient degradation of kitchen waste (ke et al. 2021). rather than utilizing single strains, utilizing microbial consortia, which is an assortment of microorganisms, for the biodegradation of organic waste enables one to exploit the microbial collaborations and making ideal utilization of their intricate regulatory systems (wongwilaiwalin et al. 2010). the bacterial cultures used therefore must be compatible to make a successful microbial consortium, where it can concomitantly produce all the enzymes required for the degradation of kitchen wastes. this study was therefore undertaken to evaluate and develop a microbial consortium that can concomitantly degrade different kitchen wastes under natural conditions and to optimize the degradation conditions. experimental procurement of culture and maintenance of culture bacterial strains viz. bacillus subtilus (mtcc 441), bacillus megaterium (mtcc 8510), bacillus amyloliquefaciens (mtcc 7913), lactobacillus delbrueckii (mtcc 2365), lactobacillus plantarum (mtcc 1407), lactobacillus rhamnosus (mtcc 1423), pseudomonas aeruginosa (mtcc 4673), staphylococcus aureus (mtcc 740) were obtained from school of bioengineering and bioscience, lovely professional university, punjab. the bacterial strains of these microorganisms were inoculated in nutrient broth and incubated at 37 oc for 24 h. the revived nova biotechnol chim (2022) 21(1): e991 3 cultures were maintained on nutrient agar plates and slants and stored at 4 oc for further use. preliminary screening for metabolic characteristics the selected strains were individually inoculated by streaking on selective media such as starch (2 % starch), skim milk, cmc (congo red) supplemented agar and nutrient agar plates with 1 % tributyrin to isolate amylase, protease, cellulose, and lipase producers, respectively. the inoculated plates were then incubated overnight at 37 oc and checked for a zone of clearing around each bacterial isolate. for starch agar, the zone of clearing was observed after flooding the plates with iodine. preparation of bacterial consortium overnight grown bacterial strains were inoculated in 20 ml of nutrient broth to prepare the microbial consortia. two consortia was prepared – consortium 1 containing non-pathogenic microorganism (b. subtilus, b. amyloliquefaciens, lactobacillus delbrueckii, l. plantarum, l. rhamnosus) and consortium 2 containing pathogenic as well as non-pathogenic microorganisms (b. subtilus, b. amyloliquefaciens, l. delbrueckii, l. plantarum, p. aeruginosa, s. aureus). the bacterial strains were inoculated on nutrient agar plates and cultivated overnight. enzymatic assays of selected bacterial cultures and consortium the microbial consortia and the selected bacterial cultures were inoculated for 24 h at 37 oc, and centrifuged at 10,000 rpm for 10 min, respectively, and the supernatants were used for protease and amylase assays. protease assay protease assay was done according to the method of sarkar et al. (2011). to 1 ml of extract, 1 ml of 1 % casein (substrate) was added and incubated at 60 oc for 10 min. to this 0.5 ml of trichloroacetic acid (20 % w/v) was added to stop the reaction. the mixture was allowed to stand for 30 min at rt and then filtered. one ml of the filtrate was then mixed with 5 ml of 0.5m na2co3 solution. for development of color, 0.5 ml of folin-ciocalteau reagent was added and kept in dark for 15 min. absorbance was taken at 660 nm against tyrosine as standard. one unit of protease activity is calculated as the amount of enzyme required to liberate 1 g tyrosine per millilitre in 1 min. amylase assay amylase activity was measured according to sarkar et al. (2011). to 1 ml of extract, 1 ml of 1 % starch (substrate) and 1 ml of citrate-phosphate buffer (ph 6.0) was added and incubated at 50 °c for 30 min. the reaction was ceased by adding 2 ml of 3,5-dinitrosalicylic acid (dnsa) reagent and kept in boiling water bath for 10 min. absorbance was read at 540 nm against glucose as the standard. one unit of amylase activity is defined as the amount of enzyme which releases 1 μm of reducing sugar as glucose per minute, under the assay conditions (u.ml-1). evaluation of kitchen waste degradation the consortium capable of concomitantly producing both proteases and amylases was selected for laboratory trials. three kilograms each of small heaps of kitchen wastes was collected from different canteens located in the campus of lovely professional university (phagwara, punjab, india). each heap was inoculated with 5 % of selected consortium by evenly mixing the inoculum with the wastes, and observations for changes in ph, temperature, weight loss, organic carbon, and moisture content were taken at 1, 10, 20 and 35 d, respectively. ph, temperature, and moisture content the ph of the organic waste of both the control and consortia treated organic waste was checked at 1, 10, 20, and 35 d of composting, respectively. temperature was also noted for the same using a thermometer at 1, 10, 20 and 35 d of composting, respectively. similarly, for moisture content 10 g of each sample was weighed and dried for 24 h in the oven at 100 – 110 oc at 1, 10, 20, and 35 d of nova biotechnol chim (2022) 21(1): e991 2 composting, respectively, and moisture content was determined by calculating the weight difference. organic carbon organic carbon was estimated by rapid titration method of walkley and black (1934). to 1 g of oven-dried waste, 20 ml of 0.1 n k2cr2o7 was added. to this, 20 ml of concentrated h2so4 was added and homogenized by gentle swirling and allowed to stand for 30 min. after completion of the reaction, 250 ml distilled water and 10 ml of 85 % orthophosphoric acid (h3po4) was added with 1 ml of diphenylamine indicator. the mixture was back titrated against 0.5 n ferrous ammonium sulphate with diphenylamine indicator, and the developing reddish brown color was taken as the endpoint. a blank is run without the sample and the final value was derived as per the calculation of khan (2016). carbon, hydrogen, nitrogen and sulphur (chns) analysis chns content in the composting kitchen waste inoculated with selected consortium 1 was analysed with chns-o elemental analyser (thermo scientifictm flashsmarttm, waltham, usa). statistical analysis the assays and experiments were carried out in triplicates, and mean values and standard error were calculated using spss 21.0 software for windows. results and discussion preliminary screening for metabolic characteristics of bacterial strains seven bacterial cultures were selected which produced the desired enzymes required to degrade organic kitchen wastes. the strains of s. aureus, b. subtilus, p. aeruginosa, l. plantarum, l. delbrueckii, l. rhamnosus, and b. amyloliquefaciens showing the hydrolysis zones are presented in fig. 1 and table 1. all the strains showed positive results for enzyme production except for b. megaterium which did not exhibit lipase and cellulase activities. table 1. enzyme production by different bacterial strains. + indicates enzyme activity and indicates no enzyme activity. enzymatic assays of selected bacterial cultures and consortium consortium 1 exhibited higher amylase activity (17.66 u.ml-1) as compared to consortium 2 and individual bacterial strains which are statistically different from each other (p < 0.05) (fig. 2a). bacterial strains in consortium 1 especially b. subtilis, b. amyloliquefaciens, and l. plantarum show appreciable levels of amylase activities; their cumulative action in consortium 1 positively and significantly increased the level of starch degradation in the kitchen waste. studies have shown that the genus bacillus yields a large variety of extracellular enzymes of which amylases and proteases are of major industrial importance. microorganisms amylase lipase protease cellulase b. subtilis + + + + b.megaterium + + p. aeruginosa + + + + s. aureus + + + + l. plantarum + + + + l. rhamnosus + + + + l.delbrueckii + + + + b.amyloliquefaciens + + + + 4 nova biotechnol chim (2022) 21(1): e991 3 amylase activity on 1 % starch agar protease activity on skim milk agar lipase activity on nutrient agar with 1 % tributyrin cellulase activity on cmc (congo red) supplemented agar staphylococcus aureus bacillus subtilus pseudomonas aeruginosa lactobacillus plantarum lactobacillus delbrueckii lactobacillus rhamnosus bacillus amyloliquefaciens fig. 1. bacterial strains showing zones of hydrolysis on plates of starch agar, skim milk agar, nutrient agar of 1 % tributyrin, and cmc (congo red) agar. mesophile bacillus sp. has showed to offer highly alkaline and thermostable α-amylases (saxena et al. 2007; naidu et al. 2013; awasthi et al. 2018). similarly, for protease assay while both consortia exhibited considerable protease activities, consortium 1 produced a higher activity at 20.71 u.ml-1 (fig. 2b) that was significantly higher from consortium 2 and individual bacterial strains (p < 0.05). among individual bacterial strains l. delbrueckii, displayed the highest protease activity. 5 nova biotechnol chim (2022) 21(1): e991 2 fig. 2. enzyme activities of consortia and specific microbes (a) amylase, (b) protease. data presented are the mean ± standard error of three replicates. means with different superscript letters are significant by tukey’s post hoc test (p < 0.05). studies by kliche et al. (2017) observed that the lactobacillus genus, specifically l. delbrueckii and l. helveticus, produce proteases that can hydrolyze αand ß-casein, while its amylase activity was comparatively the least. also, bacterial strains showing higher protein degrading activity include b. subtilis and l. plantarum. these bacteria also indicated higher amylase activities (lim et al. 2019; chen et al. 2020). given that consortium 1 presented enhanced concomitant activities of both amylase and protease in comparison to consortium 2 and the individual bacterial strains, respectively, consortium 1, which consist of non-pathogenic bacterial species, was thus taken for further evaluation on degradation of kitchen waste. evaluation of degradation of kitchen waste – ph generally, during composting period, ph is affected due to changes in the chemical composition of the waste matter. initially, ph falls below neutral due to the formation of organic compounds by the microbes and the formation of ammonium (palaniveloo et al. 2020). studies have shown that from the 2nd week of composting the ph values increased because of the elimination of carbon dioxide and the decomposition of proteins in the waste (saad et al. 2013). the initial ph value of both control and consortium-treated waste was 4.0. the ph of consortium-treated kitchen waste increased to 5.3 and 5.7 on day 20 and day 35, respectively, whereas the ph value in control increased slightly to 4.7 and 5.0 on day 20 and day 35, respectively. compost microorganisms are reported to operate best under neutral to acidic conditions, with ph in the range of 5.5 to 8.0 (mehta et al. 2014; palaniveloo et al. 2020). temperature during composting, the change in temperature has a major influence on the efficiency of the composting process. it is an important parameter to monitor composting efficiency, because it affects not only the biological reaction rates and the dynamic population of microbes, but also the physico-chemical characteristics of composts (chang et al. 2019). from an initial temperature of 20 oc in both consortium-treated kitchen waste and in control was observed, where a linear increase is observed up to day 20. control sample registered temperature increase up to 25 oc while consortia 1 exhibited a higher temperature of 29 oc. a gradual decreasing trend towards the end of the composting process at day 35 was noted. similar temperature variations were observed in various studies of compost of plant residues, pineapple leaves (pl) wastes, mixed vegetables and fruit waste with 6 nova biotechnol chim (2022) 21(1): e991 3 ambient temperature 24 to 30 oc; 25 to 32.5 oc and 35 to 45 oc (gautam et al. 2010; cheng et al. 2013; abu-zahra et al. 2014). more linear changes in temperature were observed in consortia 1 as compared to control. moisture content the moisture content of the consortium-inoculated wastes demonstrated a gradual decline from an initial 74.3 % to 58 % at day 35; in control only slight decrease in the moisture content was observed with increase in composting time and 68 % was recorded at day 35 fig. 3c. according to jain et al. (2019), moisture content has a central influence on the biodegradation of organic matter where it affects the microbial activity, as well as the physical structure, in the composting process. proper moisture content in the composting process is one of the key factors for the success of composting (makan et al. 2013). many researchers have investigated the range of optimum moisture content for composting process to be within 50 % and 70 % (iqbal et al. 2015; wu et al. 2015). thus, the reduction in moisture content was significant in the kitchen waste treated consortium 1 as compared to control. weight loss weight loss is another factor for determining the process of composting (pan et al. 2012). from the data represented in fig. 3d during the laboratory trail the weight of the kitchen waste with consortia 1 reduced from 447 g to about 333 g as compared to control reduced from 470 g to about 400 g. weight loss of 25 % was observed (tiquia et al. 2002). microbial metabolic activity was the primary reason for kitchen waste mass loss. microbes decomposed organic components into micro-molecular organics such as glucose and produced and emitted large amounts of co2 (zhang et al. 2013; yeh et al. 2020). a drastic change in weight loss was observed when kitchen waste was inoculated with microbial consortia1 as compared to control. fig. 3. various parameter of degradation of kitchen waste and consortium 1: a – change in ph; b – change in temperature; c – moisture content; d – weight loss. 7 nova biotechnol chim (2022) 21(1): e991 2 organic carbon the c : n ratio is one of the most important indicator of the composting process where the occurrence of carbon during composting assists microbes during the decomposition process, while nitrogen helps build microbial cell structures (saad et al. 2013). according to rastogi et al. (2020), the optimum composting process befalls through a range of c : n ratio 30 – 40 : 1. also, jain et al. (2019) found that c : n ratio lower than 20 can also be effective for composting process. the initial carbon content of kitchen wastes was 73 % on both the control and consortia 1 treated waste, respectively. the carbon content showed a sharp decrease in consortia 1 treated waste up to 41 % on the 20th d of degradation and 36 % on 35th d of degradation, in contrast the control showed slight decreased of 67 % on the 20st d of degradation and only 64 % up to 35th d of degradation (fig. 4) thus the reduction in organic carbon content was significant in the kitchen waste by consortium 1 as compared to control. fig. 4. organic carbon content of consortium 1 and control during degradation. the sample of kitchen waste treated with consortia1 showed the presence of 38 % carbon content, 1 % nitrogen, and 6 % hydrogen (fig. 5). the c/n ratio in the current study is calculated at 38. c : n ratio of consortium 1 at 18 d was 39 : 1.2 which indicates an efficient degradation process. while the optimum composting process occurs in the c/n ratio of 30 to 40, other studies have also reported that c/n ratio lower than 20 can also be effective for composting process (cao et al. 2021). fig 5. chns analysis of consortia 1. conclusion in this study bacterial strains were selected and screened based on their enzymatic activity to create a microbial consortium to degrade organic kitchen waste. two bacterial consortia were prepared, one containing non-pathogenic bacterial species (b. subtilus, b. amyloliquefaciens, l. plantarum, l. rhamnosus, and l. delbrueckii) (consortia 1) and the other a mixture of pathogenic and nonpathogenic bacterial species p. aeruginosa, s. aureus, l. delbrueckii, l. rhamnosus, b. subtilus (consortia 2). consortia 1 showed better enzymatic production as compared to consortia 2 and consortia 1 was thus taken into consideration as the main consortia for degradation of kitchen waste. bacillus sp. can interact with each other and form a stable micro-ecology system to improve the degradation rate of organic matters, increasing the maturity of kitchen waste and reducing toxin products of the reaction. the bacterial strains used were non-pathogenic and thus will not be harmful to the environment and showed positive results for enzyme production required to degrade the kitchen waste. thus, degradation of organic kitchen wastes by the bacterial consortia is extremely significant. 8 nova biotechnol chim (2022) 21(1): e991 3 the utilization of microbial consortium created through characteristic choice or change of the execution of these microorganisms in natural kitchen waste degradation for treatment of organic kitchen waste in the near future might be the best option. the utilization of microbial consortium created through characteristic choice or change of the execution of these microorganisms in natural kitchen waste degradation in the near future might be the best option. consortia of non-pathogenic bacteria are also an efficient and viable option for an environment and health safety method of waste management. this study warrants further metabolic studies of each microorganism working in synchronization for efficiently degrading the kitchen waste. these consortiums can be scaled to industrial level in fermentors for bulk municipal waste management in the near future. the biogas released and the by products can be used as manure and for other purposes. acknowledgements this work was supported by lovely professional university, phagwara and icar-rc neh region, umiam. authors thank both institutes for providing funds and support to conduct this work. authors also thank panjab university, chandigarh for their analysis of carbon nitrogen ratio. conflict of interest the authors declare that they have no conflict of interest in the publication. references abu-zahra tr., ta'any ra, arabiyyat ar (2014) changes in compost physical and chemical properties during aerobic decomposition. int. j. curr. microbiol. appl. sci. 3: 479486. awasthi 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author: roman.boca@ucm.sk nova biotechnologica et chimica thermodynamics and cooperativeness of the spin crossover roman boča department of chemistry, faculty of natural sciences, university of ss cyril and methodius in trnava, trnava, sk917 01, slovak republic article info article history: received: 17th july 2020 accepted: 11th september 2020 keywords: cooperativeness spin crossover statistical analysis thermodynamic parameters abstract spin transition – a passage from the low-spin electronic state to the high-spin one of fe(iii) and fe(ii) complexes is assessed from several points of view: theoretical modelling, magnetic susceptibility data, and calorimetric measurements. the concept of the cooperativeness in the solid state is discussed in detail. thermodynamic parameters are mutually correlated for a set of analogous fe(iii) complexes by using modern statistical methods.  university of ss. cyril and methodius in trnava introduction thermally driven passage from the low-spin electronic state to the high-spin one is usually termed the spin crossover though also spin transition, spin conversion, and spin equilibrium is frequently used in this content and confused in their meaning. this phenomenon can be considered as a kind of unimolecular reaction where the conversion from l (low-spin) to h (high-spin) states is an process driven by entropy. for such a case s > 0 and h ~ kbt > 0 hold true so that there exists a critical temperature given by the ratio t1/2 = h/s; above t1/2 the change in gibbs t e n e rg y 0 g  t 1/2 1/t ln k 0 hs ls t 1/2 fig. 1. schematic representation of the spin crossover as entropy driven unimolecular reaction. energy alters to g < 0 so that the conversion progresses spontaneously. thermal development of the high-spin mole fraction xh can be used in monitoring conversion degree; the equilibrium constant is expressed as k = xh/(1 – xh). in an ideal case the lnk vs 1/t dependence is a straight line (fig. 1). the spin conversion is often explained using the orbital diagram, as presented in fig. 2 for mononuclear fe(ii) and fe(iii) complexes. electrons promoted from the non-bonding orbitals t2g into the antibonding orbitals eg cause a softening of the adiabatic potential e = f(r) (force constants k(h) < k(l) with its minimum lying at higher high-spin 5t2g eg t2g low-spin 1a1g eg t2g high-spin 6a1g eg t2g low-spin 2t2g eg t2g fe(ii) fe(iii) fig. 2. orbital diagram showing a difference between the low-spin and high-spin complexes fe(ii) and fe(iii) in an octahedral geometry. mailto:roman.boca@ucm.sk nova biotechnol chim (2020) 19(2): 138-153 139 ls hs distance e n e rg y r l rh k h k l fig. 3. interrelation of the adiabatic potentials for the lowspin and high-spin states. metal-ligand distances, r0(h) > r0(l) (fig. 3). the electronic contribution to the transition entropy is given by the spin multiplicities as follows sel = rln[(2sh+1)/(2sl+1)]; this amounts to 9.1 and 13.4 j k-1 mol-1 for fe(iii) and fe(ii) complexes, respectively. a softer adiabatic potential for the hs state implies denser vibrational energy levels which enhances the vibrational contribution to the transition entropy: svib(h) > svib(l). generalized crystal field theory in explaining the spin crossover phenomenon often an orbital picture is utilized. however, this simple approach abstracts from the mutual repulsion of energy and also the spin-orbit coupling. therefore, there is a need of a more sophisticated approach to the spin crossover by using quantumchemical calculations. herein the generalized crystal field theory (gcf) has been applied for such a purpose with numerical outputs (boča 2006). the interelectron repulsion is involved by considering the set of atomic terms labelled by the orbital and spin quantum numbers, i.e. , , , , l s l s m m . a passage to the complex belonging to a point group g requires considering a set of crystal-field (cf) terms , , , , s s m   (like 2t2g, 6a1g, etc.) where g is the component of the multidimensional irreducible representation g. the spin-orbit interaction (soi) splits the cfterms into a set of crystal-field multiplets , ,    ; they need be classified using the irreducible representations of the respective double group (1 through 8 for o’ in bethe notation). in terms of the gcf, a theoretical modelling has been done for fe(iii) [and fe(ii)] systems by involving the interelectron repulsion via racah parameters b = 1,122 [897] cm-1 and c = 4.2 b, crystal-field poles for individual ligands f4(l), spin orbit interaction with the coupling constant  = 460 [400] cm-1, orbital-zeeman and spin-zeeman interactions (boča and herchel 2015). the overall interaction matrix (eq. 1): ee cf soi oz sz 4 diagonalization { ( , ) ( ) ( ) ( ) ( )} ( ) i v b c v f v v b v b e b      (1) is diagonalized and the calculated zeeman levels ei(b) form the partition function (eq. 2): b ( , ) exp[ ( ) / ] i i z b t e b k t  (2) finally, the formulae of the statistical thermodynamics can be utilized in order to calculate magnetization and magnetic susceptibility (eq. 3 and 4): mol ln ( , ) t f z m b t rt b b                 (3) 0 mol mol 0 ( , ) ( ) b m b t t b          (4) since the mapping of ( ) i k e b proceeds for discrete field values bk, numerical derivatives are required that, in fact, are provided after a parabolic fit (eq. 5). (0) (1) ( 2) 2 , , , ( ; ) δ δ k m k m k m k k m k z b t c c b c b   (5) effective magnetic moment constructed from the magnetic susceptibility displays a thermal development that strongly depends upon the crystal field strengths – see fig. 4 for an octahedral fe(iii) system. in a narrow interval of the crystal-field strengths the spin crossover occurs: for the pole strength f4 = 17,700 cm -1, the ground state is highspin 6a1g but for f4 = 18,200 cm -1 it is low-spin 2t2g. a delicate situation exists for the intermediate crystal field (fig. 5): with f4 = 18,000 cm -1 the ground cf-term is high-spin 6a1g so that the spin crossover would not apply. however, the close-lying excited cf-term 2t2g(×6) is split due to the spin-orbit interaction by a rather high nova biotechnol chim (2020) 19(2): 138-153 140 t/k 0 50 100 150 200 250 300 350 400  e ff /  b 0 1 2 3 4 5 6 7 17500 17700 17800 17900 17950 18000 18100 18200 18500 f 4 /cm  14 000 fe(iii)6a 1g 2 t 2g fig. 4. calculated temperature evolution of the effective magnetic moment for octahedral fe(iii) systems having various crystal-field strengths f4 = 6(dq). amount (soi = 690 cm -1) that overcomes the interterm gap (0 = 425 cm -1). consequently, the ground crystal-field multiplet is the component 7(×2) ← 2t2g that it is doubly degenerate: g(7) = 2. thus, the change of the electronic entropy (eq. 6): 1 7 δ ln[ (a ) / (γ )] ln[6 / 2] 0 g s r g g r   (6) is positive so that the spin crossover develops according to the bold curve drawn in fig. 4. for fe(ii) systems the situation is different in several aspects (fig. 6). with a weaker crystal field strength f4 = 12,800 cm -1 (10dq = 7,680 cm-1), the effective magnetic moment corresponds to the high-spin state with a typical course passing through a round maximum on heating. with a bit higher crystal field of f4 = 12,870 cm -1, the ground state is low-spin and on heating the effective magnetic moment increases from zero to the value eff ~ 5.0 b at the room temperature. a typical spin-crossover behaviour proceeds at f4 = 12,900 cm -1. for f4 = 13,000 cm -1 e n e rg y / c m   -400 -200 0 200 400 600 800  8  8  7  7 2 t 2g (x6)  0  soi 6 a 1g (x6) e n e rg y / c m   -200 0 200 400 600 800  3  5  4  5 5 t 2g (x15)  4 1  0  soi 1 a 1g (x1) fig. 5. left – energies of the lowest crystal-field terms and crystal-field multiplets for f4 = 18,000 cm -1 in fe(iii); right – for f4 = 12,900 cm -1 in fe(ii); calculations involve soi. t/k 0 50 100 150 200 250 300 350 400  ef f/  b 0 1 2 3 4 5 6 12800 12850 12860 12870 12900 12950 13000 13100 14000 f 4 /cm  14 000 fe(ii) 5 t 2g 1 a 1g fig. 6. calculated temperature evolution of the effective magnetic moment for octahedral fe(ii) complexes having various crystal-field strengths f4 = 6(dq). the spin conversion is apparently incomplete (it continues to the completeness at the much higher temperature). with f4 = 14,000 cm -1 t he spin transition occurs far above the room temperature and only a “temperature-independent paramagnetism” is visible until t = 400 k. the excited crystal-field term 5t2g is split due to the spin-orbit interaction into three groups of the crystal-field multiplets 5, {3 + 4} and {1 + 4 + 5}. this means that four groups of energy levels are involved in the spin crossover of an octahedral fe(ii) system (fig. 6). in octahedral systems the electron repulsion and the crystal field strength are interrelated by the tanabesugano (ts) diagrams where the term energy (not involving soi) is plotted versus dq/b parameter ( = 10dq = (10/6)f4) – fig. 7. these diagrams are helpful in identifying the critical ratio when the high-spin complex turns to the low-spin one. for fe(ii) systems, the crossover of the terms 5t2g ↔ 1a1g exists at the ratio dq/b = 2.38; this implies dq = 2,135 cm-1 and f4 = 12,809 cm -1. the last value matches the “observed” on-set of the spin transition. analogously for fe(iii), the crossover 6a1g ↔ 2t2g appears at dq/b = 2.70 giving rise dq = 3,029 cm-1 and f4 = 18,176 cm -1; this again matches the region in which the spin crossover is observed. the octahedral geometry, however, is rather hypothetical for real spin crossover systems and at least tetragonal and/or trigonal distortions would be more close to the reality. a two-dimensional map of the lowest crystal field terms is presented in fig. 8 and it can be considered as a generalized nova biotechnol chim (2020) 19(2): 138-153 141 dq/b 0.0 0.5 1.0 1.5 2.0 2.5 3.0 e /b 0 10 20 30 40 50 60 2 t 2g 6 a 1g 6 a 1g 2 t 2g 4 t 1g 4 t 2g 4 a 1g dq/b 0.0 0.5 1.0 1.5 2.0 2.5 3.0 e /b 0 10 20 30 40 50 60 1 a 1g 1 t 1g 3 t 1g 3 t 2g 5 t 2g5 t 2g 5 e g 1 a 1g fig. 7. tanabe-sugano diagrams for octahedral fe(iii) and fe(ii) complexes (experimental b and c parameters were used in calculations). f 4 (xy) 5000 10000 15000 20000 f 4 (z ) 5000 10000 15000 20000 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 eg a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 eg a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 eg eg a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 eg eg a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 eg eg a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 b2 t2 eg a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 b2 b2 b2 t2 f 4 (xy) 5000 10000 15000 20000 5000 10000 15000 20000 t1 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 eg t1 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 eg eg t1 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 eg eg eg t1 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 eg eg eg eg t1 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 eg eg eg eg eg t1 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 eg eg eg eg eg eg t1 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 a2 eg eg eg eg eg eg eg eg t1 a2 a2 a2 a2 a2 a2 a2 a2 eg eg eg eg eg eg eg eg eg eg eg t1 a2 a2 a2 a2 a2 a2 eg eg eg eg eg eg eg eg eg eg eg eg eg t1 a2 a2 a2 a2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t1 a2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg b2 t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg b2 b2 b2 t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg b2 b2 b2 b2 t2 eg eg eg eg eg eg eg eg eg eg eg eg eg b2 b2 b2 b2 b2 b2 t2 eg eg eg eg eg eg eg eg eg eg eg eg b2 b2 b2 b2 b2 b2 b2 t2 eg eg eg eg eg eg eg eg eg eg b2 b2 b2 b2 b2 b2 b2 b2 eg t2 eg eg eg eg eg eg eg eg eg b2 b2 b2 b2 b2 b2 b2 a1 eg eg t2 f 4 (xy) 5000 10000 15000 20000 5000 10000 15000 20000 t1 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t1 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t1 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t1 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t1 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t1 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t1 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t1 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t1 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t1 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t1 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t2 eg eg eg eg eg a1 eg eg eg eg eg eg eg eg eg eg eg eg eg t2 eg eg eg eg a1 eg eg eg eg eg eg eg eg eg eg eg eg eg eg t2 eg eg a1 b2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t2 eg b2 b2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t2 b2 b2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t2 b2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t2 f 4 (xy) 5000 10000 15000 20000 f 4 (z ) 5000 10000 15000 20000 t2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 eg t2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 eg eg t2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 a1 eg eg eg t2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 a1 a1 eg eg eg eg t2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 a1 a1 a1 eg eg eg eg eg t2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 a1 a1 a1 eg eg eg eg eg eg t2 b2 b2 b2 b2 b2 b2 b2 b2 a1 a1 a1 a1 eg eg eg eg eg eg eg t2 b2 b2 b2 b2 b2 b2 a1 a1 a1 a1 a1 eg eg eg eg eg eg eg eg t2 b2 b2 b2 b2 a1 a1 a1 a1 a1 a1 eg eg eg eg eg eg eg eg eg t2 b2 b2 b2 a1 a1 a1 a1 a1 a1 eg eg eg eg eg eg eg eg eg eg t2 b2 a1 a1 a1 a1 a1 a1 a1 eg eg eg eg eg eg eg eg eg eg eg a1 a1 a1 a1 a1 a1 a1 a1 eg eg eg eg eg eg eg eg eg eg eg a1 a1 a1 a1 a1 a1 a1 a1 eg eg eg eg eg eg eg eg eg eg a1 a1 a1 a1 a1 a1 a1 a1 a1 eg eg eg eg eg eg eg eg eg eg a1 a1 a1 a1 a1 a1 a1 a1 a1 eg eg eg eg eg eg eg eg eg eg a1 a1 a1 a1 a1 a1 a1 a1 a1 eg eg eg eg eg eg eg eg eg a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 eg eg eg eg eg eg eg eg eg a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 eg eg eg eg eg eg eg eg a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 a1 f 4 (xy) 5000 10000 15000 20000 5000 10000 15000 20000 t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg a1 a1 a1 a1 eg t2 eg eg eg eg eg eg eg eg eg eg eg eg eg a1 a1 a1 a1 eg eg t2 eg eg eg eg eg eg eg eg eg eg eg eg a1 a1 a1 b2 eg eg eg t2 eg eg eg eg eg eg eg eg eg eg a1 a1 a1 b2 b2 eg eg eg eg t2 eg eg eg eg eg eg eg eg eg a1 a1 b2 b2 b2 eg eg eg eg eg t2 eg eg eg eg eg eg eg eg a1 a1 b2 b2 b2 eg eg eg eg eg eg t2 eg eg eg eg eg eg a1 a1 b2 b2 b2 b2 eg eg eg eg eg eg eg t2 eg eg eg eg eg a1 b2 b2 b2 b2 b2 eg eg eg eg eg eg eg eg t2 eg eg eg a1 b2 b2 b2 b2 b2 b2 eg eg eg eg eg eg eg eg eg t2 eg eg a1 b2 b2 b2 b2 b2 b2 eg eg eg eg eg eg eg eg eg eg t2 eg b2 b2 b2 b2 b2 b2 eg eg eg eg eg eg eg eg eg eg eg eg t2 b2 b2 b2 b2 b2 b2 eg eg eg eg eg eg eg eg eg eg eg eg eg t2 b2 b2 b2 b2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t2 b2 b2 b2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t2 b2 b2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg a2 t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg a2 a2 a2 t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg a2 a2 a2 a2 a2 t2 f 4 (xy) 5000 10000 15000 20000 5000 10000 15000 20000 t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg b2 t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg b2 b2 t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg b2 b2 b2 t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg eg b2 b2 b2 b2 t2 eg eg eg eg eg eg eg eg eg eg eg eg eg eg b2 b2 b2 b2 b2 t2 eg eg eg eg eg eg eg eg eg eg eg eg eg b2 b2 b2 b2 b2 b2 t2 eg eg eg eg eg eg eg eg eg eg eg eg b2 b2 b2 b2 b2 b2 b2 t2 eg eg eg eg eg eg eg eg eg eg eg b2 b2 b2 b2 b2 b2 b2 b2 t2 eg eg eg eg eg eg eg eg eg eg b2 b2 b2 b2 b2 b2 b2 b2 b2 t2 eg eg eg eg eg eg eg eg eg b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 t2 eg eg eg eg eg eg eg eg b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 t2 eg eg eg eg eg eg eg b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 a1 eg t2 eg eg eg eg eg eg b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 eg eg eg t2 eg eg eg eg eg b2 b2 b2 b2 b2 b2 b2 b2 b2 a1 eg eg eg eg t2 eg eg eg eg b2 b2 b2 b2 b2 b2 b2 b2 a1 a1 eg eg eg eg eg t2 eg eg eg b2 b2 b2 b2 b2 b2 b2 a1 a1 eg eg eg eg eg eg eg t2 eg eg b2 b2 b2 a2 a2 a2 a2 a1 a1 eg eg eg eg eg eg b2 b2 t2 eg a2 a2 a2 a2 a2 a2 a2 a1 eg eg eg eg eg eg eg b2 b2 b2 t2 fig. 8. generalized tanabe-sugano diagrams showing three lowest energy levels for equ ax 4 2 [fel l ] bipyramidal complex: top – d6, bottom – d5 system; left panel – ground state, centre (right) – first (second) excited state or a component of the degenerate state. solid line passes through the octahedral arrangement and separates the elongated and compressed tetragonal bipyramid. nova biotechnol chim (2020) 19(2): 138-153 142 ts-diagram (soi not involved) allowing a bordering of the spin crossover. when the pole strengths f4(z) = f4(xy), the diagram collapses to the ts one. in the ts diagram the crossover point refers to a situation when the ls and the hs state are accidentally degenerate. according to fig. 3, the metal-ligand distances obey rl < rh. in fe(ii) complexes, these typically are rl ~1.96 2.00 å and rhl= rh rl ~ 0.16 0.21 å. this shift manifest itself into the ligand field strengths owing to the relationship r= (10dql/10dqh) = (rh/rl) 5. this ratio amounts to r ~ (2.2/2.0)5 = 1.6 so that on passing from hs to ls, the ligand strength increases approximately by a factor of 2 and vice versa. in the ts diagram of iron(ii) spin crossover compounds 10dqh will be situated at the left and 10dql to the right of the crossover point. it must be emphasized that the above modelling refers to electronic factors only and they completely ignore the important contribution of the molecular vibrations to the spin crossover. also, vertical excitations in electronic transitions are assumed instead of the adiabatic ones. the spin crossover can appear also for mn(iii) systems (sl = 0 to sh = 2 transition) as well as co(ii) complexes (sl = 1/2 to sh = 3/2 crossover). master equation for description of the spin crossover, a number of different theoretical models have been developed so far (wajnflasz 1970; bari and sivardiére 1972; slitcher and drickamer 1972; sorai and seki 1974; zimmermann and könig 1977; rao et al. 1981; spiering et al. 1982; adler et al. 1987; bousseksou et al. 1995; cantin et al. 1999; boča et al. 2003). their attempt is to simulate a development of the high-spin mole fraction xh under the thermal propagation: xh = f(t). one of these models is the regular solution & domain model based upon general principles of thermodynamics. the first step in the regular solution model is consideration of mixing entropy smix; this results from the distribution of the ls and hs molecules within the system of n molecules that is simplified by exploiting the stirling formula for factorials (eq. 7): mix b b ! ln ( )![(1 ) ]! { ln (1 ) ln(1 )} n s k xn x n k n x x x x        (7) here, we utilized that xn molecules are in the hs state and (1 – x)n in the ls one. let us consider domains of like spin and of uniform size. then the number of molecules per domain is /n n d and the mixing entropy alters to (eq. 8): mix b b ! ln ( )![(1 ) ]! { ln (1 ) ln(1 )} d s k xd x d k d x x x x        (8) the molar mixing entropy becomes expressed as follows (eq. 9): mix ( / ){ ln (1 ) ln(1 )}s r n x x x x     (9) where a b r n k is the ideal gas constant. the second contribution in the play is intermolecular interactions with energy int e constituted as follows (eq. 10): 2 2 int hh lh ll 2 0 1 2 2 (1 ) (1 )e e x e x x e x j j x j x         (10) where hh lh ll , ,e e e are the interaction energies between hs-hs, ls-hs and ls-ls pairs, respectively. to this end, a rearrangement offers (eq. 11): 0 ll 1 lh ll 2 ll hh lh 2( ) 2 j e j e e j e e e           (11) the molar gibbs energy adopts the form (eq. 12): h l mix int (1 ) x g xg x g ts e     (12) where l g and h g refer to the molar gibbs energies for the ls and hs units, respectively. the equilibrium condition requires (eq. 13): h l , 1 2 ( / ) ln 1 2 0 x t p g x g g r n t x x j j x                   (13) which yields the equation for the mole fraction of the high-spin species (eq. 14):    1 1 2 1 exp (δ δ 2 ) /x n h t s j j x rt       (14) an alternative expression is (eq. 15): nova biotechnol chim (2020) 19(2): 138-153 143 1/[1 ( )]x f x  (15) with the factor (eq. 16):  1 2( ) exp [(δ ) δ 2 ] /f x h j t s j x n rt    (16) such an implicit equation requires solution by an iterative procedure. the entropic term contains two contributions: electronic and vibrational (eq. 17). the partition function of a set of m = 3n – 6 harmonic oscillators (m = 15 for a hexacoordinate complex) is l, b vib,l 1 l, b l, b 1 1 l, b exp( / 2 ) 1 exp( / ) 1 exp ( / 2 ) 1 exp( / ) m i i i mm i i i i h k t z h k t h k t h k t                       and analogously for the hs state. then, in the approximation of an averaged (einstein) modes l h and h h the entropic term becomes (eq. 18): el,h vib,h el vib el,l vib,l h l b l h b δ δ δ ln 2 1 1 exp( / ) ln 2 1 1 exp( / ) m z z s s s r z z s h k t r s h k t                           (18) and the enthalpic one is (eq. 19): 0 hs ls δ δ ( )/ 2h e m h h    (19) now the eqs. 15 – 16 need be solved by an iterative process starting with a trial set of parameters n, h + j1, lh , hh and j2 for each temperature point. finally, the equilibrium constant is expressed as (eq. 20): h h 1 2 h ln ln 1 [( ) 2 ] / x k x h j t s j x n rt          (20) the effect of the individual parameters to the conversion curve and/or equilibrium constant is presented in fig. 9. the condition for the equilibrium (eq. 13), defines the transition temperature t1/2 at which the highspin and low-spin mole fractions are equal, or h 0.5x  (eq. 21): 1 / 2 h l 1 2 0 0 t t g g j j       (21) t/k 0 100 200 300 x h s 0.0 0.5 1.0 (1/t)/k  0.00 0.01 0.02 ln k -5 0 5 t/k 0 100 200 300 x h s 0.0 0.5 1.0 (1/t)/k  0.00 0.01 0.02 ln k 0 5 a) b) c) d) fig. 9. conversion curves modelled by the master equation: a) change of the enthalpy h/r = 100 k (short dashed), 500 k (long dashed) and 1000 k (solid) for fixed t1/2 = h/s = 150 k and j = 0; b) effect of the domain size n = 1 (solid), 5 (long dashed), 50 (short dashed) for fixed h/r = 500 k and t1/2 = 150 k – steepness of the transition; c) effect of the solid-state cooperativeness j/r = 0 (solid), 100 k (long dashed), 300 k (short dashed) for fixed h/r = 500 k and s/r = 5 – deviations from the linearity in the van’t hoff plot; d) effect of molecular vibrations hs b /h k  140 k (solid), 160 k (dashed), 180 k (dot-dashed) for fixed ls hs 1.5  , e e hs ls / 5g g  , and 0/kb = 600 k. the transition temperature (eq. 22): 1/2 1 2 ( ) /t h j j s     (22) includes also two cooperativity factors; a cancellation of 1 2 w j j  can be assumed in the theoretical model. one can overcome the two-body interactions (koudriavtsev 1999) by considering there-body ones producing a third-order term 3 3 h j x in eq. 10; this yields an additional term 1/2 3 (3 / 4) /t j s   in eq. 22. cooperativeness within the regular solution model, the interaction term involves the solid-state cooperativity factor – cooperativeness  through the expression (eq. 23): int (1 )e x x  (23) (17) nova biotechnol chim (2020) 19(2): 138-153 144 this formula originates in the intercentre interaction (eq. 24): 2 int 0 1 2 2 ll lh ll ll hh lh 2( ) ( 2 ) e j j x j x e e e x e e e x          (24) which yields the relationship valid for the regular solution&domain model (eq. 25): int 1 2 1 2 2 2 ( ) (1 2 ) (1 2 ) e j j x j j j x x w x            (25) the remainder (eq. 26): 1 2 hh ll w j j e e    (26) can be absorbed into the effective parameter of the site formation, or it is omitted (eq. 27): eff 1 2 δh j j    (27) then the factor entering the iteration process (eq. 14) relaxes to (eq. 28):  eff( ) exp [ δ (1 2 )] /f x t s x n rt     (28) finally, the cooperativeness becomes expressed in the form (eq. 29): 2 lh ll hh 2j e e e      (29) this expresses a tendency for molecules of one type to interact effectively with molecules of the same spin. parameter distribution model was outlined because behaviour of the solid-state samples is non-ideal: a reduction of the cooperativeness can be described using a statistical distribution. high cooperativeness leads to the thermal hysteresis a rectangular shape of the hysteresis loop. observed profile of the conversion curves, however, is often distorted with marked deviations from the rectangular towards angled shape (boča et al. 2001). the parameter distribution model considers the optimum (maximum) cooperativeness j2 that drops as follows (eq. 30): 2, 2i i j n j (30) (29), where i is the grid point (e.g. 1/100 of the optimum value opt 1n  ). to this end the equation (eq. 31): 1/[1 ( )] i i x f x  (31) contains the factor (eq. 32):  1 2,( ) exp [(δ ) δ 2 ] /i if x h j t s j x n rt    (32) (31) this equation need be solved by an iterative procedure for the trial set of parameters, for selected temperature, and for each mesh point. the iteration procedure starts with (0) 0 i x   in the heating direction, and (0) 1 i x   on the cooling path. the averaged value is given by the formula (eq. 33): mesh mesh h 1 1 / i i i i i x w x w                  (33) (32), using the weights obeying the gaussian distribution (eq. 34): 2 opt exp[ ( ) / ] i i w n n    (34) t/k 350 400 450 x h s 0.0 0.5 1.0 t/k 350 400 450 0.0 0.5 1.0 a) b) t/k 350 400 450  e ff / b 0 1 2 3 4 5 t/k 350 400 450 0 1 2 3 4 5 a) b) fig. 10. parameter distribution model of the spin crossover. distribution model width: d = 0.00001 (abrupt step) and 0.1 (gradual step). used parameters: d0/kb = 2144 k, j/kb = 452 k, reff = 205 and gh = 2.0. modelled using the program mif&fit (boča 2016). the width of the distribution leads to the following effects (fig. 10): nova biotechnol chim (2020) 19(2): 138-153 145 1) 0  causes a sharp distribution so that the model collapses to the abrupt step on heating and cooling, respectively. the conversion curve displays a hysteresis loop possessing the rectangular walls. 2) increased d causes that the hysteresis loop has angled walls and a decreased width. 3) with increasing d the conversion curve is less complete and visibly smoother. thermal hysteresis originates in existence of two minima of the gibbs energy at different temperatures so that on the heating/cooling the system falls into one of them. cooperativeness as the chemical/physical hardness let us reconsider pair-wise interactions among solid-state particles (eq. 35): 2 2 int h ll h lh h h hh h ( ) (1 ) 2 (1 )e x e x e x x e x     (35) which can be rearranged into the form of a taylor expansion (eq. 36): 2 int h 0 1 h 2 h ( )e x j j x j x   (36) the coefficients of the taylor series are eq. 37 – 39 (see fig. 11): 0 ll j e (37) int 1 lh ll h 2 2 0 e j e e x            (38) 2 int 2 ll hh lh 2 h 1 2 0 2 e j e e e x             (39) where m – the chemical potential that equals to minus absolute electronegativity (sen and jorgensen 1987);  – the pearson’s chemical hardness (sen 1993; pearson 2005). the cooperativeness j is then expressed through an excess of the interaction energy (eq. 40): 2 lh hh ll / 2 ( ) / 2 0j j e e e      (40) so that it interrelates to the chemical hardness. another cooperative contribution is (eq. 41): 1 2 hh ll 0w j j e e     (41) and it eventually can be neglected. recall some additional definitions according to pearson (pearson 2005). ell = j0 (ehh + ell)/2 u = j 1 /2 ehh elh w = j 1 + j 2 j = j 2 /2 fig. 11. interrelations of the interaction parameters. a) electronic chemical potential (eq. 42) – derivative of the energy with the number of electrons at the constant potential generated by a system of nuclei: e n         (42) b) absolute electronegativity expressed (eq. 43) as an average of the ionization energy ei and electron affinity eeg: i eg 2 e e       (43) c) chemical hardness (eq. 44): 2 2 1 2 e n         , i eg 2 e e    (44) d) electronegativity shift for two reactants owing to a transfer of n electrons from 2 to 1 (eq. 45 and 46): o 1 1 1 2 n     (45) o 2 2 2 2 n     (46) d) electronegativity (chemical potential) equalization 1 2   yields (eq. 47): o o 1 2 1 2 1 2 ( ) n         (47) electrons move from the site of lower electronegativity to the site of higher electronegativity (eq. 48). this causes an energy lowering: nova biotechnol chim (2020) 19(2): 138-153 146 o o 1 2 1 2 1 4 ( ) e          (48) e) bulk modulus b and compressibility  of a solid is (eq. 49): 1 t p b v v          , [pa] (49) f) physical hardness becomes (eq. 50): 02 ,t v v bv h n n           , [j mol-1] (50) where v0 – molar volume, n – number of particles. g) fluctuations in the number of particles for a grand canonical ensemble (eq. 51): 2 2 0, 1 1 ( ) t v n n n n v kt v h              (51) an ensemble can be crystals of identical volume but with varying numbers of component atoms, i.e. crystals which are physically soft (inverse of hard) possess large fluctuations in n. this set of equations represents a good starting point for investigation of the physical and chemical nature of the solid-state cooperativeness. calorimetry vs. magnetometry the classical thermodynamics deals with the volume work d dw p v  and defines two heat capacities (eq. 52 and 53): 2( , ) ln v v v v u s v z c r t t t t                       (52) (51) 2 ( , ) ln ln ln p p v t p e s p c t z z r t t t t v                                (53) (52), where z is the partition function. in the case of the magnetic work 0 d dw h m again two kinds of the heat capacities are distinguished (eq. 54, 55): 2( , ) ln m m m m u s m z c r t t t t                       (54) 2 ( , ) ln ln ln h h m t h e s h c t z z r t t t t m                                (55) (here, enthalpy is denoted as e, not to be confused with the magnetic field strength h.) for the solid state, the appropriate formula for the molar excessheat capacity measured in the zero field is (eq. 56): ex 2 ln p m m m z c c r t t t                (56) this allows a comparison of the experimental heat capacity (measured using adiabatic or differential scanning calorimeters) with that reconstructed by the theoretical model of the spin crossover (e.g. regular solution&domain model). by substituting the partition function (eq. 57): l l l h h l l l h 0 l h 1 2 2 ( 1) [1 exp( / )] ( 1) 1 exp( / ) 1 ( 1) 1 exp( / ) exp{ [δ ( ) / 2 ( ) (2 1)] / m m s s z h kt s s h kt s s h kt h h m j j j x kt                                   (57) (56) into eq. 56, explicit expression for the heat capacity along with its fortran code is obtained by exploiting capabilities of the mathematica package (wass 1999). the heat capacity and/or its weighted function then become a combination of the underlying lattice vibration functions (taken as polynomials) and the excess-heat capacity (eq. 58 and 59): 2 3 h l l l l 2 3 ex h h h h h (1 )( ) ( ) p p c x a b t c t d t x a b t c t d t c           (58) 2 3 h l l l l 2 3 ex h h h h h ( / ) (1 )( ) ( ) ( / ) p p c t x a b t c t d t x a b t c t d t c t           (59) where only some polynomial terms need be considered. three complexes under the investigation are characterized as follows (fig. 12). complex 1 – [fe(2-pic)3]cl2·meoh is a non-cooperative system; its thermodynamic data were scanned by the adiabatic calorimetry (nakamoto et al. 2001). nova biotechnol chim (2020) 19(2): 138-153 147 t/k 0 50 100 150 200 250 300 350 c p /( j k -1 m o l1 ) 0 100 200 300 400 500 600 700 t/k 50 100 150 200 250 300 350 x h s 0.0 0.5 1.0 (1/t)/k -1 0.004 0.006 0.008 ln k -6 -3 0 3 6 1 t/k 120 150 180 210 240 (c p /t )/ (j k  2 m o l 1 ) 4 6 8 t/k 50 100 150 200 250 300 350 x h s 0.0 0.5 1.0 (1/t)/k -1 0.004 0.006 0.008 ln k -6 -3 0 3 6 100 150 200 c p /( j k -1 m o l1 ) 500 1000 1500 2 t/k 0 50 100 150 200 250 300 c p /( j k -1 m o l1 ) 0 100 200 300 400 500 600 t/k 50 100 150 200 250 300 350 x h s 0.0 0.5 1.0 (1/t)/k -1 0.004 0.006 0.008 ln k -6 -3 0 3 6 0 50 100 150 200 250 300 0 2000 4000 6000 3 fig. 12. experimental (open symbols) and fitted (full lines) thermodynamic functions for 1, 2, and 3. complex 2 – [fe(pybzim)3](clo4)2 belongs to a medium-cooperative system. there is no structural change during the spin crossover as documented by a continuous increase of the lattice parameters. dsc technique has been applied for it (boča et al. 2003). complex 3 – [fe(phen)2(ncs)2] is a strongly cooperative system. the effective magnetic moment increases abruptly near the transition temperature t1/2 and the structural changes accompany the spin transition. the thermodynamic data were collected by the adiabatic calorimetry (sorai and seki 1974). the data fitting by the eq. 56 – 57 gave the set of the spin crossover parameters which are presented in table 1. table 1. parameters of the spin crossover for complexes 1 through 3 from fitting the heat capacity.a parameter b cooperativeness 1 – small 2 – medium 3 – high site format. energy (eff/kb) /k 1370 351 1039 entropic parameters reff = 7545 l /h hc = 454 cm-1 h l / 1.5 h h    reff = 357 cooperativeness (j/kb) /k 20 136 182 h /kj mol-1 11.39 [8.88] 2.92 [3.04] 8.64 [8.60] s /j k-1 mol-1 74.2 [59.5] 19.0 [21.0] 48.9 [48.8] 1/2 /t h s   /k 153 [149] 153 [145] 177 [176] a values in square brackets are the direct calorimetric determination. b simplifications: eff lns r r  , 2 / 2j j  , eff 0 l h 1 2 ( ) / 2m h h j j        . having the experimental heat capacity curve and its temperature-weighted function at the disposal, the numerical integration offers the enthalpy (eq. 60) and entropy (eq. 61) of the spin transition: p max min p d d t t p p t t h c t c t     (60) p max min p ( / ) d ( / ) d t t p p t t s c t t c t t     (61) the measured cp and cp/t data need be corrected for the underlying lattice vibrations, for instance, by subtracting polynomial functions applied below tmin and/or above tmax and the integration limit contains the peak value of tp (fig. 13). the integration can be improved by utilizing the conversion curve xh vs t known from the measurements of the magnetic susceptibility. this enables a construction of the smooth baseline between cmin for the ls and cmax for the hs; the excess enthalpy δ ( )h t (eq. 62) associated nova biotechnol chim (2020) 19(2): 138-153 148 t 3 /k 3 3e+6 6e+6 9e+6 0.5 1.0 1.5 t/k 120 140 160 180 200 c p /( k j k  1 m o l 1 ) 0.5 1.0 1.5 t/k 120 140 160 180 200 ( c p )' /( k j k -1 m o l1 ) 0.0 0.1 0.2 t/k 120 140 160 180 200 (c p / t )/ (j k   m o l 1 ) 5 6 7 t 2 /k 2 20000 40000 5 6 7 120 140 160 180 200 ( c p / t )' /( j k  2 m o l 1 ) 0.0 0.4 0.8 with the spin crossover at the given temperature is: min min 3 ls ls hs max min δ ( ) [ ( )]d ( )d t p t t t h t c a b t t x c c t        (62) the excess entropy at the given temperature δ ( )s t can be evaluated in an analogous way. a direct fitting of the magnetic susceptibility data (t) is possible by considering three contributions in the form of a curie-weiss law, namely for the low-spin species, high-spin system, and eventual paramagnetic impurity (eq. 63, 64, and 65): 2 l 0 l l l l l ( 1) / 3( )c g s s t     (63) 2 h 0 h h h h h ( 1) / 3( )c g s s t     (64) 2 pi 0 pi pi pi pi pi ( 1) / 3( )c g s s t     (65) where the reduced curie constant consists of the physical constants, 2 0 a 0 b b /c n k  . for fe(ii) centres (sl = 0, sh = 2, spi = 5/2) the appropriate set of magnetic parameters consists of l, gh, h, gpi = 2, pi, and pi. for fe(iii) centres (sl = 1/2, sh = 5/2) the active set is gl, l, al, gh = 2.0 and h. some of these parameters can be fixed or omitted in order to avoid an overparametrization. in addition, there are four parameters of the spin crossover that enter evaluation of the conversion curve xh(t), i.e. eff, j, lh and hh (the last again can be fixed). then the susceptibility is balanced as follows (eq. 66): h pi l h h pi pi ( ) (1 )t x x x x        (66) and the equilibrium constant (eq. 67) is: h h pi / (1 )k x x x   (67) the enthalpy of the spin transition (eq. 68) is calculated as a temperature–independent quantity: 0 a eff eff b δ ( / )h n r k   (68) which absorbs the site formation energy, zero-point vibration correction, and eventually the cooperativeness parameters (eq. 69): eff 0 h l 1 2 δ ( ) / 2 ( )e h h m j j       (69) the entropy of the transition (eq. 70) is a temperature-dependent quantity which at the transition temperature is: 1/2 l b 1/2h l h b 1/2 1 exp( / )2 1 (δ ) ln 2 1 1 exp( / ) m t h k ts s r s h k t                (70) this can be approximated through an effective degeneracy ratio (eq. 71): fig. 13. heat capacity analysis for [fe(pybzim)3](clo4)2: a) raw data; b) data for subtraction of underlying lattice vibrations; c) data for numerical integration yielding h and s. a) b) c) nova biotechnol chim (2020) 19(2): 138-153 149 (1/t)/k -1 0.004 0.006 0.008 0.010 ln k -5 0 5 t/k 50 100 150 200 250 300 x h s 0.0 0.5 1.0 susceptibility susceptibility, fitted dsc, fitted fig. 14. comparison of magnetic and calorimetric data for [fe(pybzim)3](clo4)2. empty points – experimental data, lines – fitted. eff δ lns r r (71) where reff is subjected to the fitting procedure. the fitting of the magnetic data and calorimetric data is compared in fig. 14 on the common basis – temperature evolution of the high-spin mole fraction (left) and the van’t hoff plot (right). statistical analysis a number of organic species h2l, acting as pentadentate ligands l2-, has been prepared (fig. 15) by a schiff condensation between the substituted salicylaldehyde (r-sal) and the asymmetric or symmetric triamine (pet or dpt). t/k 0 50 100 150 200 250 300 x h 0.0 0.5 1.0 t/k 0 50 100 150 200 250 300  e ff / b 0 2 4 6 b = 0.1 t t 1/2 fig. 16. magnetic data for [fe(napet)ncs]: left – temperature dependence of the effective magnetic moment, right – a temperature evolution of the calculated high-spin fraction (right); grey circles – experimental data, solid line – fitted. analogously, a set of schiff-base ligands was obtained using the naphthyl skeleton. they were complexed with fe(iii) salts yielding hexacoordinate [feiiilx] complexes. thermal evolution of the effective magnetic moment showing spin crossover is exemplified in fig. 16 along with the fitted curve based upon the regular solution model. in a series of hexacoordinate [feiiil5x] complexes, the transition temperature t1/2 of the spin crossover can be modified by appropriate coligands x (fig. 17) (šalitroš et al. 2009; nemec et al. 2011; krüger et al. 2013, 2015; masárová et al. 2015). this is affected by the enthalpic and entropic terms since t1/2 = h/s holds true. it is expected that n oh n oh nh n oh n oh nh n oh n oh nh n oh n oh nh oet eto n oh cl n oh nh cl n oh meo n oh nh ome n oh n oh nh n oh n oh nh cl cl n oh n oh nh napet 2anapet 1anapet salpet 5cl-salpet 3eto-salpet saldpt 5cl-saldpt 3meo-saldpt fig. 15. sketch of the related pentadentate ligands h2l 5. nova biotechnol chim (2020) 19(2): 138-153 150 fe (iii) n o n o r1 r1 nh x r2 r2 fig. 17. general form of the [fe(r-salpet)x] type complexes; coligands x = cl, n3 , nco, ncs, ncse, and cn. the value of h can be altered by varying the crystal field strength of involved ligands. however, the factors influencing the value of s are more complex as they include electronic (net spin), vibrational, and other contributions. temperature evolution of the effective magnetic moment for related complexes is shown in fig. 18 and 19. it can be concluded that an increase of the crystal field strength 10dq of the coligand x causes a switch of the spin states of complexes t/k 0 50 100 150 200 250 300  e ff / b 0 1 2 3 4 5 6 [fe(saldpt)py](bph 4 ) 2 , sc [fe(5cl-saldpt)py](bph 4 ) 2 , sc [fe(3meo-saldpt)py](bph 4 ) 2 , sc sc sc fig. 19. spin crossover in complexes of [feiii(r-saldpt)py] (bph4)2 type. from the high-spin state (x = cl–, nco–), through the spin crossover (x = ncs–, ncse–), to the lowspin state (x = cn–). in order to unhide latent correlations among thermodynamic parameters influencing the spin crossover, modern multivariate methods were utilized for processing data listed in table 2: pearson correlation (pc), cluster analysis (ca) and the principal component analysis (pca) (augustín and boča 2015). t/k 0 50 100 150 200 250 300  e ff / b 0 1 2 3 4 5 6 [fe(cl-salpet)cl], hs [fe(cl-salpet)nco], hs [fe(cl-salpet)ncs], sc [fe(cl-salpet)ncse], sc [fe(cl-salpet)cn], ls t/k 0 50 100 150 200 250 300 0 1 2 3 4 5 6 [fe(br-salpet)ncs], hs [fe(br-salpet)n 3 ], sc [fe(br-salpet)ncse], sc hs sc ls hs sc  e ff / b sc t/k 0 50 100 150 200 250 300  e ff / b 0 1 2 3 4 5 6 [fe(5br-salpet)ncs], hs [fe(3eto-salpet)ncs], sc * [fe(5cl-salpet)ncs], sc t/k 0 50 100 150 200 250 300 0 1 2 3 4 5 6 -[fe(1anapet)ncs], hs -[fe(1anapet)ncs], sc * [fe(2anapet)ncs], sc [fe(napet)ncs].mecn, sc  e ff / b hs hs sc sc sc sc sc fig. 18. comparison of the spin states in complexes of [feiiil5x] type. top – fixing the schiff-base ligand and varying coligand; bottom – varying the schiff-base ligand and fixing the coligand ncs-. hs – high spin, sc – spin crossover, ls – low spin. for details consult table 2. nova biotechnol chim (2020) 19(2): 138-153 151 table 2. retrieved thermodynamic parameters for spin crossover systems.a compound t1/2 h s j 1h [fe(3eto-salpet)ncs] 84 82 0.9 12 98 2 [fe(5brsalpet)n3]·meoh 142 1.6 11 76 3 [fe(5cl-salpet)ncs] 280 5.8 21 180 4 [fe(5cl-salpet)ncse] 293 6.5 22 197 5 [fe(5br-salpet)ncse] 326 6.0 18 215 6 [fe(salpet)atz] 416 15 37 284 7i,h -[fe(1anapet)ncs] 44 40 1.6 4 82 8 [fe(2anapet)ncs] 114 1.6 14 52 9h [fe(napet)n3]meoh 122 117 1.5 11 99 10 [fe(napet)ncs]mecn 151 1.9 12 87 11 [fe(napet)nco] 155 2.5 16 102 12 [fe(napet)ncse]mec n 170 2.3 13 99 13 [fe(napet)ncs] 186 3.3 18 150 14i [fe(5cl-saldpt)py]bph4 78 0.4 6 63 15 [fe(3meo-saldpt)py] bph4 273 4.5 17 90 16 [fe(saldpt)py]bph4 310 5.4 17 150 a spin crossover between s = 1/2 and s = 5/2; h – spin crossover with hysteresis; i – spin crossover between intermediate spin s = 3/2 and s = 5/2. data of 6 and 7 was omitted in the statistical analysis. units: t1/2/k; h/kj mol-1;s/j k-1 mol-1; (j/kb)/k. taken from (boča et al. 2000; šalitroš et al. 2009; nemec et al. 2011; krüger et al. 2013, 2015; masárová et al. 2015). the results obtained by the cluster analysis are shown in fig. 20. it can be seen that the complexes under study span two clusters: the cluster 1 is formed by complexes for which below-room temperature spin crossover was observed (no. 1, 2, dendrogram ward's method,squared euclidean 0 3 6 9 12 15 18 d is ta n c e 1 2 3 4 589 1 0 1 1 1 2 1 3 1 4 1 5 1 6 fig. 20. dendogram of the cluster analysis (using ward’s method and squared euclidean distance) for complexes numbered according to table 2. 8, 9, 10, 12 and 14). the remaining complexes span the cluster no 2 and they show nearor aboveroom temperature spin transition. the pca analysis yields the biplots of principal components shown in fig. 21. it is seen that the transition temperature t1/2 closely relates to the enthalpy and entropy of the spin transition whereas a correlation with cooperativeness j does not exist. the biplot on the right shows the objects spanning into individual clusters which are well separated between two principal components. finally, the pearson correlation evaluates the correlation coefficients for each pair of variables. it is found that t1/2 – h pair possesses a high correlation coefficient r = 0.97. as already indicated by ca and pca analysis, the correlation of the cooperativeness with the remaining parameters is weak. the transition temperature and the transition entropy are plotted vs the transition enthalpy along with the regression curve. h j biplot -4 -2 0 2 4 component 1 -1.5 -1 -0.5 0 0.5 1 1.5 c o m p o n e n t 2 tc s component 1 c o m p o n e n t 2 plot of pcomp_2 vs pcomp_1 -4 -2 0 2 4 -1.5 -1 -0.5 0 0.5 1 1.5 clustnums 1 2 fig. 21. results of principal component analysis. top – a biplot with a ray diagram, bottom – individual complexes classified by the clusters. nova biotechnol chim (2020) 19(2): 138-153 152 h /kj mol -1 0 1 2 3 4 5 6 7  s / j k -1 m o l1 0 5 10 15 20 25 h /kj mol -1 0 1 2 3 4 5 6 7 t 1 /2 /k 0 50 100 150 200 250 300 350 400 fig. 22. linear correlations t1/2 vs h and s vs h. the confidence intervals (95 %) are drawn as dashed curves. according to fig. 22 the linear relationship is now confirmed. the aspects discussed in the present communication are only a part of the huge story, as documented by a number of alterative models and plethora of experiments about the spin crossover phenomenon. alternative views were comprehensively presented in recent articles (enachescu et al. 2018; nicolazzi et al. 2018; pavlik et al. 2018). conclusions the spin crossover behaviour is based upon a fine tuning of the crystal field strengths balancing the interelectron repulsion. the spin orbit coupling, however, also plays an important role. ideal thermodynamic behaviour, as described by the regular solution&domain model, is further modified by the interactions in the solid state that are considered as the solid-state cooperativeness. the role of the cooperativeness is analogous to the role of the activity in solution models: it allows using the equation for the ideal system but instead of the mole fraction xh, an effective mole fraction j·xh is in the play. the nature of the cooperativeness lies in the electronic (chemical) and nuclear (physical) hardness. thermo-dynamic data about h and s can be obtained not only from the direct calorimetric measurements but also from an appropriate model of the spin crossover adapted to the magnetic data analysis. these parameters are mutually interrelated through the transition temperature t1/2 as proven by the multivariate statistical analysis. acknowledgments grant 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