ЗВІТ З НДР 29-81 ЗА 2007 – 2009 Р Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 19 CHARACTERIST ICS OF THREE YEAST STRAINS FOR WAS TEWA TER T RE A TMEN T *Aleksander SLAVOV1, Zapryana DENKOVA2, Rositsa DENKOVA 3 1Department “Environmental engineering” University of Food Technologies, Plovdiv, Bulgaria, aleksan- der_slavov@yahoo.com 2Department “Organic Chemistry and Microbiology” University of Food Technologies, Plovdiv, Bulgaria, zden- kova@abv.bg 3Department „Biotechnology”, Faculty of Biology, Sofia University „St Kliment Ohridski”, Sofia, Bulgaria, ro- sitsa_denkova@mail.bg *Corresponding author Received 20 March 2012, accepted 27 May 2012 Abstract: The selection of desirable strains with certain activities that have quick adaptivity and effectively remove contaminants from water is of great importance for the technologies for biological wastewater treatment.Three yeast strains are investigated. One of them was isolated from soil, and others are pro- vided by a yeast strains’ collection and denoted as C1 and C2. The identification of the yeast strains is done using the test kit API 20 C Aux (Biomerieux, France) for rapid identification of yeasts. Determination of the enzyme profile is obtained by applying the test kit API ZYM (Biomerieux, France). The three yeast strains accumulate high concentrations of viable cells. They are similar in their ability to oxidize S – containing compounds (elemental S, Na2S2O3) under alkaline conditions lowering the рH of the medium and to exhibit catalase activity. Yeast strain Y exceeds the rest in the expression of lipolytic activity. That is confirmed in the subsequent analysis of its enzyme profile through identification system API ZYM. Through it are found and alpha –, beta – glucosidase and aminopeptidase activities. Keywords: morphological, physiological and biochemical methods, API ZYM, API 20 C Aux, Candi- da famata 1. Introduction One of the most important compounds of the planet is water [1]. The continuous decrease of potable water determines the need for different methods for its purification. Technologies for biological wastewater treatment with the participation of microorganisms, including yeasts, are becoming more and more current. Among the microorfanisms, participating in the wastewater treatment process are representatives of the genera Candida, Pi- chia, Torulaspora, Yarrowia, Geotrichum, Saccharomyces, Cryptococcus, Rhodotoru- la, Trichosporon [2, 3, 4]. The selection of desirable strains with certain activities that have quick adaptivity and effectively remove contaminants from water is of great importance. The aim of this work is the analysis and identification of yeast strains suitable for specific applications in the field of biological wastewater treatment. 2. Materials and methods 2.1. Microorganisms Three yeast strains, marked as Y, C1, C2 are used in this study. Yeast strain Y is isolated from soil and strains C1 and C2 are provided by the microorganism mailto:aleksander_slavov:@yahoo.com mailto:aleksander_slavov:@yahoo.com mailto:zdenkova:@abv.bg mailto:zdenkova:@abv.bg mailto:rositsa_denkova:@mail.bg mailto:rositsa_denkova:@mail.bg Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 20 collection of the Department of Organic Chemistry and Microbiology at the University of Food Technologies, Plovdiv, Bulgaria. 2.2.Nutrient media 2.2.1. Malt extract medium. Composition: malt extract (Kamenitza, Bulgaria) in 1:1 ratio with tap water (vol/vol). pH=6,5 – 7,0. The medium is sterilized for 25 minutes at 121ºC [5]. 2.2.2. Malt extract medium. Composition: malt extract (Kamenitza, Bulgaria) in 1:1 ratio with tap water (vol/vol) + 2 % agar – agar (w/vol). pH=6,5 – 7,0. The medium is sterilized for 25 minutes at 121ºC [5]. 2.2.3. Luria – Bertani glucose medium (LBG). Composition (g/dm3): triptone (Difco) – 10 g, yeast extract – 5 g, NaCl – 10 g, glucose (Scharlau) – 10 g. pH=7,5. The medium is sterilized for 25 minutes at 121ºC. 2.2.4.Soy – caseine broth medium. Com- position (g/dm3): triptone (Difco) – 17g, soy peptone (Scharlau) – 3 g, NaCl – 5 g, K2HPO4 – 2,5 g, glucose (Scharlau) – 2,5 g. pH=7,3 ± 0,2. The medium is sterilized for 25 minutes at 121ºC. 2.2.5.Citrate utilization medium (Simons medium). Composition (g/dm3): Na(NH4)2PO4 – 1,5 g; KH2PO4 – 1 g; MgSO4.7H2O – 0,2 g; Na – citrate – 3 g; alcohol solution of bromthymol blue – 1%; agar – agar – 2%. The medium is sterilized for 25 minutes at 121ºC [6]. 2.2.6.Gelatinase activity medium. Compo- sition (g/dm3): triptone (Difco) – 10 g; yeast extract (Scharlau) – 5 g; NaCl – 10 g; glucose (Scharlau) – 10 g; gelatine (DDR) – 250 g. рН 7,5. Medium is dispensed into tubes and is sterilized for 25 min at 121ºС [6]. 2.2.7. Proteolytic activity medium. Com- position: malt agar medium with 10 % (vol/vol) solution additive (10 cm3 milk/100 cm3 water) of skimmed milk powder (Scharlau) [6]. 2.2.8.Lipolytic activity medium (Tween – 80 compounds hydrolysis). Composition (g/dm3): peptone (Scharlau) – 10 g; NaCl – 5 g; CaCl2 – 0,1 g; Tween – 80 (Merck) – 10 cm3; agar – agar – 20 g. рН 7 – 7,4. The medium is sterilized for 25 minutes at 121ºC [5]. 2.2.9. Sulphur–containing compound oxidation mediua. 2.2.9.1. Starckey broth medium. Composi- tion (g/dm3): elemental S – 10 g; КH2PO4 – 3 g; MgSO4.7H2O – 0,2 g; CaCl2.2H2O – 0,2 g; (NH4)2SO4 – 0,5 g; FeSO4 – traces; indicator – bromocresol purple. рН 8,0. The medium is prepared in two versions: with glucose (5 g/ dm3) and without glu- cose. It is sterilized at Koch apparatus for 30 min on three consecutive days [7]. 2.2.9.2. NCL – broth medium. Composi- tion (g/dm3): elemental S – 10 g; (NH4)2SO4 – 0,2 g; MgSO4.7H2O – 0,5 g; CaCl2.2H2O – 0,25 g; FeSO4 – traces; in- dicator – bromocresol purple. The medium is prepared in two versions: with glucose (5 g/ dm3) and without glucose. It is steri- lized at Koch apparatus for 30 min on three consecutive days [7]. 2.2.9.3. Thiosulphate agar medium. Com- position (g/dm3): Na2S2O3 – 5g; K2HPO4 – 0,1 g; NaHCO3 – 0,2 g; NH4Cl – 0,1 g; agar – agar – 20 g. рН 8,0. The medium is prepared in two versions: with glucose (5 g/ dm3) and without glucose. It is sterilized at Koch apparatus for 30 min on three con- secutive days [7]. 2.2.10. NO3- – reductase activity medium. Composition (g/dm3): peptone (Scharlau) – 5 g; meat extract (Scharlau) – 3 g; KNO3 – Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 21 1 g. рН 7.0. The medium is sterilized for 25 minutes at 121ºC [8]. 2.2.11. NH4+ – citrate medium (for nitrify- ing activity). Composition (mol/dm3): Na – citrate.2Н2О – 9,5.10-3; NH4Cl – 9,35.10-3; KH2PO4 – 1,47.10-3; MgSO4.7H2O – 1,62.10-4; CaCl2 – 1,36.10-7; FeSO4.7H2O as EDTA – complex – 3,6.10-5. The medium is sterilized for 25 minutes at 121ºC [9]. 2.2.12. Blood agar (NCIPD, Bulgaria). Composition (g/dm3): casein hydrolysate – 14 g; NaCl – 5 g; peptone – 4,5 g; yeast extract – 4,5 g; defibrinated sheep blood – 70 cm3; agar – agar – 12,5 g. pH 7,3 ± 0,2. All medium components are sterilized for 15 min at 121ºС. After cooling to 45ºС – 50ºС 70 cm3 defibrinated sheep blood is added aseptically and is poured in sterile Petri dishes [8]. 2.2.13. Gorodkova medium (for yeast spo- rulation). Composition (g/dm3): peptone – 10 g; NaCl – 5 g; месен meat extract – 10 g; glucose – 2,5 g; agar – agar – 20 g. рН 6,5 – 7,0. The medium is sterilized for 25 minutes at 121ºC [5]. 2.2.14. Acetate agar medium (for yeast sporulation). Composition (g/dm3): NaOOCCH3.3H2O – 5 g; agar – agar – 20 g. pH 6,5 – 7,0. The medium is sterilized for 25 minutes at 121ºC [8]. 2.2.15. Minimal agar medium (for yeast sporulation). Composition (g/dm3): agar – agar – 20 g. рН 6,5 – 7,0. The medium is sterilized for 25 minutes at 121ºC [8]. 2.2.16. Rice agar medium (Fluka Anali- tycal) (for pseudomycellium identification). Composition (g/dm3): rice extract powder – 0,7; agar – agar – 20 g. The medium is prepared in two versions – with and without Tween – 80 (Merck) (1 cm3/dm3). рН 5,8 ± 0,2. The medium is sterilized for 25 minutes at 121ºC. 2.2.17. Motility medium (NCIPD, Bulga- ria). Composition (g/dm3): peptone – 10 g, meat extract – 3 g, NaCl – 5 g, agar – agar – 4 g. рН 7,4 ± 0,2. The medium is sterilized for 25 minutes at 121ºC. 2.3.Cultivation and storage of the ana- lyzed microorganisms. Yeasts grow in malt extract medium at 30ºC in thermostat for 48 hours and are stored in a refrigerator at 4ºC for 2 months. 2.4. Analytical methods. 2.4.1. Morphological and cultural me- thods 2.4.1.1. Cellular and colonial morpholo- gy. Description of cellular and colonial morphology of the studied yeast strains is performed by microscopic observation the developed on malt agar single colonies of the studied strains. 2.4.1.2. Identification of pseudomycellium formation ability. A part of the cultural medium of the studied strain, that has been cultivated for 48 hours in a thermostat is taken with flamed and cooled bacteriological loop and spread in touch on agar plates with rice-agar. The inoculated plates are incubated under aerobic conditions for 2-4 days at 23ºC - 28ºC. A microscopic preparation is prepared from the developed colonies of the tested strain, which is examined for the presence of specific pseudomycellium or other specific formations in yeasts. The presence of vegetative cells only determines the result as negative. 2.4.1.3.Identification of spore formation ability. The ability of the yeast to form spores under unfavorable environmental conditions - lack of nutrients - is examined. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 22 Using a flamed and cooled bacteriological loop a part of the pre-developed on malt agar 48 - hour colonies of the studied strain is taken and is stroked on a Petri dish with minimal agar medium Gorodkova agar or acetate agar. Incubation conditions - 20ºC - 25ºC for 7 days. A microscopic preparation is prepared from the biomass and it is stained by the method of Moller. Spores are rubine red and vegetative bodies – blue. 2.4.1.4. Determining the number of viable cells in the development of the cultures in liquid medium. Sterile malt extract is inoculated under aseptic conditions with yeast suspension, 5 cm3 medium is inoculated with 1 cm3 of the inoculum of the studied cultures. After incubation in a thermostat at 30ºC for 48 h, tenfold dilution method is done and malt agar plates are inoculated. Petri dishes are thermostatted at 30ºC for 48 h so that the strain would form single colonies, which are then counted. 2.4.2. Physiological and biochemical me- thods 2.4.2.1. Citrate utilization. Developed in containers with 30 cm3 1:1 malt extract at 30ºC for 48h yeast cultures are centrifuged at 3000 min-1 for 10 min. The supernatant is discarded and the biomass is resuspended in 5 cm3 sterile physiological solution. The yeast inoculum is taken using a sterile bacteriological loop and it is streak-spread on petri dishes with Simons medium. The inoculated plates are incubated at 30ºC for 48h. Positive results are recorded in a case of a colour change of the medium from green to blue. 2.4.2.2. Determination of the gelatinase activity of the tested strains. The preparation method of the yeast inoculum is analogous to that in 5.1. The suspension of the tested strain is taken with a sterile bacteriological loop and is put in point onto a pre-sterilized medium with gelatin. The tubes are incubated for 7 days at room temperature and the presence of crater melting is monitored [6]. 2.4.2.3. Proteolytic activity determination using fusion agar method. The ability of the yeast strains to hydrolyze milk proteins in agar medium with milk additive is investigated. Wells with d = 6 mm are made on Petri dishes with medium for proteolytic activity. After inoculation, the plates are incubated at 30ºC for 48 hours. Results are reported as positive in the case of a formation of a brighter halo around the wells of the plates. A lack of halo is a sign of an inability to utilize milk proteins. 2.4.2.4. Determination of the lipolytic ac- tivity using fusion agar method. Lipolytic activity includes the ability of the analyzed strains to hydrolyze the compounds of Tween - 80. Wells with d = 6 mm are made on the Petri dishes with agar me- dium. After inoculation the Petri dishes are cultivated at 30ºС for 1 - 7 days. Formation of a turbid zone around the wells, due to the precipitation of Ca - salts of the formed free fatty acids, indicates the presence of lipolytic activity. 2.4.2.5. Analysis on the ability of the stu- died strains to oxidize S-containing com- pounds. The analysis of the oxidation of S- containing compounds includes development of the yeast strains on selective media. Tubes with sterile liquid Starkey medium and NCL medium "with" and "without" glucose are inoculated with 1 cm3 yeast suspension, prepared as in 5.1. With a bacteriological loop the same suspension is used to streak plates with thiosulphate media "with" and "without" glucose. The plates and tubes are cultivated in a thermostat at 30ºC for 15 days. Changes in the color of the indicator bromocresol purple (from purple to yellow on Starkey and thiosulphate media; and Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 23 from yellow to dark yellow or purple on NCL – broth) are reported as positive results. 2.4.2.6.Determination of nitrifying activity of the studied strains. Nitrifying activity includes the development of the analyzed yeasts on liquid medium with ammonium salts being the sole nitrogen source. A suspension of the tested yeast strains is prepared, as described in 5.1. 1 cm3 of it is used to inoculate tubes with 5 cm3 of ammonia - acetate medium and the tubes are incubated at 30ºC for 24 hours. If the yeast strain has nitrifying activity, NO3- iones would be formed in the medium. Their presence is determined by test - strips (110,020 Nitrate Test Merckoquant, Merck). Some microorganisms have the ability to reduce NO3- to NO2-. Therefore, the content of NO2- in the medium is determined, using a test - strip (110,022 Nitrate Test Merckoquant, Merck). Results are recorded as positive in the presence of NO3- and NO2- or negative - when NO3- and NO2- are absent in the medium. 2.4.2.7. Determination of the nitrate - reductase activity of the studied strains. Prepared as in 5.1., the suspensions of the studied cultures are used to inoculate medium with KNO3 - 1 cm3 suspension is used to inoculate 5 cm3 of sterile medium for determination of nitrate - reductase activity. Incubation is carried out for 7 hours at 30ºC. The presence of NO2- in the medium is considered as a positive result. Their presence is determined by test - strip (110,022 Nitrate Test Merckoquant, Merck). Result are recorded as positive - in the presence of NO2- or negative - in the absence of NO2- in the medium. 2.4.2.8.Determination of the hemolytic activity of the investigated cultures. Some microorganisms have the ability to utilize blood, breaking down red blood cells. Depending on the mechanism of their hydrolysis there are three types of hemolytic activity. In α - hemolysis iron from hemoglobin is oxidized and the colonies become dark - green. In β - hemolysis the erythrocytes and the hemoglobin in them are degraded, and a bright halo is formed around some of the colonies. γ – hemolysis is observed in the case of no hemoglobin hydrolysis. Using a bacteriological loop inoculum is taken from the suspension of the tested yeast strain, prepared as in 5.1. and it is stroked on blood agar. The inoculated plates are cultured for 48 hours at 30ºC. The presence of hemolytic activity is recorded: in the cases of α - or β – hemolysis, the result is positive, while for γ – hemolysis, it is negative. 2.4.2.9. Determination of the catalase activity of the analysed cultures. Catalase activity is determined by the method described in [6]. 2.4.2.10. Determination of the oxidase activity of the investigated cultures. Oxidase activity test includes an analysis of the suspension of the studied strains for the presence of the enzyme cytochrome - oxidase. In the presence of molecular oxygen, cytochrome - oxidase can reduce the number of organic substances, including reagent NaDi (1 - naphthol + diamine dimetilparaphenylene) with the formation of indophenole blue. Test - strips for oxidase activity (Microbiology Bactident Oxidase 1.13300.0001, Merck) are places in the suspension of yeasts prepared as in 5.1. After 20-60 s the result is compared to a color scale. Positive result are recorded in the case of a color change of the strip from white to blue to blue - violet. 2.4.2.11. Determination of the profile of enzyme activity of the studied cultures. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 24 The determination of the profile of enzyme activity is performed, using the test kit API ZYM (BioMerieux) for semiquantitative determination of the enzyme profile of the studied strain. Fresh 24-hour culture of the tested strain is centrifuged for 15 minutes at 5000 g, the obtained biomass precipitate is washed twice and resuspended in API suspension medium. The API ZYM strips are placed in the incubation boxes and the microtubes are inoculated with the pre- pared cell suspension. The sample is incu- bated for 4 to 4,5 hours at 30° C. After the incubation one drop of reagent A and one drop of reagent B are pipetted into each microtubule. After 5 min staining result is reported according to the colour scheme described in the manufacturer’s instruc- tions. The enzyme activity is determined according to the colour scale from 0 (no enzyme activity) to 5 (maximum enzyme activity). 2.4.2.12. Determination of the biochemi- cal profile of the investigated cultures. The system API 20 C Aux (BioMerieux SA, France) for identification of yeast spe- cies based on the consumption of 19 car- bon sources is used for the determination of the biochemical profile of the tested cultures. Fresh 24-hour culture of the tested strain, developed on malt agar, is resuspended according the instructions of the manufacturer in API C resuspension medium. The honeycomb wells on the bot- tom of the incubation boxes are filled with sterile physiological solution. The API 20 C strips are placed in the incubation boxes and the microtubules are inoculated with the prepared cell suspension. The sample is incubated for 48h to 72h at the optimum temperature for each of the studied strains. Results are recorded according the change in turbidity in comparison to the control (microtubule 0). The results are processed with apiweb® identification software. 3. Results and discussion The ability of the yeast strains to grow in three cultural media is investigated. The results represented in Table 1 show that the best liquid medium for the strains’ development is malt extract (2.1.). The amount of viable cells is determined during cultivation of the investigated yeast strains on malt extract liquid medium. Test results (Table 2) show that each of the three yeast strains accumulate high con- centration viable cells (over 1012 cfu/cm3) for 48 h cultivation at 30°С. Table1 Media for yeast development St ra in Medium LBG (Luria – Bertani with glu- cose) ME (Malt extract) SCB (Soy – Casein broth) Y – Abundand sludge, Uniform turbidity – С1 – Abundand sludge, Uniform turbidity – С2 – Abundand sludge, Uniform turbidity – Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 25 Table 2 Concentration of viable yeast cells, cultivated in malt extract liquid medium Yeast strains Average (cfu/cm3) Y 5,6.1013 C1 1,0.1013 C2 1,4.1012 The colonial characteristics of the re- searched strains are determined after inoculating malt agar medium (2.2) and cultivation for 48 h (Table 3). Cell mor- phology is done with coloured microscope preparations. By inoculating minimal agar media - Go- rodkova agar, acetate agar and rice agar – it is determined, that researched yeast strains don’t form pseudomycellium and chlamidospores. Experiment results, presented in Table 4, show considerable similarities between the investigated yeast strains. Three cultures don’t hydrolyse gelatine, don’t exhibit ni- trifying and nitrate – reductase activities, are catalase – positive and oxidoreductase – negative. All of them are non – motile, without proteolysis activity. They can as- similate citrate, with the exception of yeast strain Y. They cannot hydrolyze hemoglo- bin (γ – hemolysis). All analyzed strains don’t assimilate sul- phur compounds in non – glucose medium at рН < 5 and рН 8. С2 is an exception (NCL broth without glucose). Analogy be- tween strains is also observed in S – con- taining compounds at рН 8 (Na2S2O3 me- dium with glucose). As a difference from С1, Y and С2 are developed in NCL broth with glucose. Yeast strain Y, isolated from soil, exceeds C1 and C2 in its values for the lipolytic activity. Table 3 Colonial characteristics and cell morphology of the investigated yeast strains Strain Colonial characteristics Cell morphology Colony description Visualization Cell description Visualization Y Round colonies with wave – like ends, smooth surface, 4-5 mm in diameter, soft consis- tence, swelled, drop – like with plateau Yeasts, ellipse shaped. With vegetative breeding, with budding, without pseudomy- cellium formation С1 Round colonies with wave – like ends, smooth surface, 4-6 mm in diameter, soft consis- tence, whitish in colour Yeasts, ellipse shaped, with single arrangement and make clusters, with vegetative breeding, with budding, with- out pseudomycellium forma- tion С2 Round colonies with wave – like ends, smooth surface, 4-6 mm in diameter, soft consis- tence, whitish in colour Yeasts, ellipse shaped, with single arrangement and make clusters, with vegetative breeding, with budding, with- out pseudomycellium forma- tion Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 26 Table 4 Biochemical features of the analyzed yeast strains Strain Y С1 С2 Lipolytic activity, mm + 13,7±1,4 – – Nitrifying activity – – – Gelatinase activity – Facultative anaerobes – Facultative anaerobes – Facultative anaerobes Oxidation of S – contain- ing com- pounds Mediums with glu- cose Na2S2O3 + Growth + Growth + Growth Starkey – – Growth – Growth NCL + – + Mediums without glucose Na2S2O3 – Growth – Growth – Growth Starkey – – Growth – NCL – – + Catalase activity + + + Oxidoreductase actividy – – – NO3 - - reductase activity – – – Proteolytic activity 24 h – – – 48 h – – – Citrate utilization – + + Hemolytic activity γ γ γ Motility – – – Aiming at a more complete analysis of the possibilities for application of the yeast strain Y in the field of wastewater treat- ment its enzyme profile is investigated with API ZYM. The performed investigations (Table 5) show the presence of alkaline phosphatase, esterase, esterase-lipase, lipase, leucine – aminopeptidase, valine – aminopeptidase, cysteine – aminopeptidase, acid phospha- tase, phosphohydrolase, alpha – glucosi- dase and beta – glucosidase. The presence of different lipolytic enzymes confirms the results from the biochemical tests for the lipolytic activities, shown in Table 4. The ability of yeast strains to utilize 19 dif- ferent carbon sources, included in the test kit API 20 C Aux for rapid identification of yeasts, is investigated. Pseudomycellium and chlamidospore for- mation is determined using rice agar. The results from the analyses are genera- lized in Table 6. After data processing with the software apiweb® yeast strain Y is identified as Candida famata (synonym Torulopsis candida [10] with reliability 99,9% (Table 7). C1 is identified as Candida famata - 53,8%, Candida lusitaniae – 22,7%, Can- dida guillermondii – 21,6%. C2 is identified as Candida famata - 54,7%, Candida lusitaniae – 23,1%, Candida guil- lermondii – 22%. Regardless of the lower confidential values for the identification of С1 and С2, they are undoubtedly representatives of the genus Candida. Oth- er tests must be conducted in order to determine their species identification. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 27 Table 5 Enzyme profile of the yeast strain Y Enzyme Activity of yeast strain Y 1 Control - 2 Alkaline phosphatase 3 3 Lipase C4 1,5 4 Lipase C8 0,5 5 Lipase C14 0,5 6 Leucine-aminopeptidase 5 7 Valine-aminopeptidase 3,5 8 Cysteine-aminopeptidase 3 9 Trypsin - 10 Chymotrypsin - 11 Acid phosphatase 4 12 Naphthol – AS – BL – phosphohydrolase 1 13 α-galactosidase - 14 β-galactosidase - 15 β-glucuronidase - 16 α-glucosidase 4 17 β-glucosidase 5 18 α-glucoseaminidase - 19 α-manosidase - 20 α-fucosidase - Table 6 Ability of the investigated yeast strains to utilize the 19 carbon sources in API 20 C Aux № Substrate Y С1 С2 1 Control – – – 2 D – glucose + + + 3 Glycerol + + + 4 Calcium 2 – keto – gluconate + + + 5 L – arabinose – – – 6 D – xylose + + + 7 Adonitol + + + 8 Xylitol + + + 9 D – galactose + + + 10 Inositol – + – 11 D – sorbitol + + + 12 Methyl – α – D – glucopyranoside + + + 13 N – acetylglucoseamine + + + 14 D – cellobiose + + + 15 D – lactose (bovine) + – – 16 D – maltose + + + 17 D – saccharose + + + 18 D – trehalose + + + 19 D – melezitose + + + 20 D - raffinose – – – 21 Hyphae/Pseudohyphae – – – Table 7 Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel MareUniversity - Suceava Volume XI, Issue 2 – 2012 28 Strains, identified according to the software apiweb® Strain Species % of reliability Y Candida famata 99,9 C1 Candida famata 53,8 Candida lusitaniae 22,7 Candida guillermondii 21,6 C2 Candida famata 54,7 Candida lusitaniae 23,1 Candida guillermondii 22,0 4. Conclusion. As a result of the conducted experimental analysеs the following more important conclusions can be drawn: 1. A yeast strain is isolated and is identified using the methods of the conventional taxonomy Candida famata (synonym Torulopsis candida [10]); 2. The strain has the ability to utilize S – containing compounds in the presence of glucose, lowering the рН value; 3. It has clearly defined catalase and lipo- lytic activities. The last one is also con- firmed by the presence of wide range of enzymes with such activity. Its beta – and alpha glucosidase activity, leucine –, va- line – and cysteine – aminopeptidase activ- ity, acid – and alkaline phosphatase activi- ty, less significant – naphthol – AS – BL – phosphohydrolase activity and the least is С4 –, С8 – и С14 – lipase activity are also significant. 5. References. [1] PALELA, M., IFRIM, G., BAHRIM, G., The annals of the university Dunarea de Jos of Galati Fascicle VI – food technology, new series, Vol. II (XXXI), 23-30, (2008) [2] LEMMEL, S., HEIMSCH, R., EDWARDS, L., Optimizing the continuous production of Candida utilis and Saccharomycopsis fibuliger on potato processing wastewater. Applied and environmental microbiology, Vol. 37, № 2. 227-232, (1979) [3] YANG, Q., ANGLY, F., WANG, Z., ZHANG, H., Wastewater treatment systems harbor specific and diverse yeast communities. Biochemical engi- neering journal., Vol. 58-59. 168-176, (2011) [4] YANG, Q., YANG, M., ZHANG, S., LV, W., Treatment of wastewater from a monosodium glu- tamate manufacturing plant using successive yeast and activated sludge systems. Process biochemistry, Vol. 40, 2483-2488, (2005) [5] BESHKOV, M., KAROVA, Е., MURGOV, I., Practical handbook of microbiology. Christo G. Danov, (1970) [6] MURGOV, I., DENKOVA, Z., Microbiology. Nova timkompakt. 308, (2004) [7] VIDYALAKSHMI R., SRIDAR, R., Isolation and characterization of sulphur oxidizing bacteria. Journal of culture collections, Vol. 5, 73-77, (2006 – 2007) [8] ATLAS, M., Handbook of microbiological me- dia. Fourth edition. Taylor and Francis Group, (2010) [9] PAPEN, H., VON BERG, R., HINKEL, I., THOENE, B., RENNENBERG, H., Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O и NO production in exponentially growing cultures. Applied and environmental microbiology. 2068-2072 стр., (1989). [10] KREGER VAN RIJ, KREGER-VAN RIJ, N.J.W. (ed), The Yeasts: a taxonomic study. 3rd Edition. Elsevier Science Publishers B.V., Amsterdam, The Netherlands, (1984) 2.1. Microorganisms 2.1. Microorganisms 2.1. Microorganisms 2.2. Nutrient media 2.2.1. Malt extract medium. Composition: malt extract (Kamenitza, Bulgaria) in 1:1 ratio with tap water (vol/vol). pH=6,5 – 7,0. The medium is sterilized for 25 minutes at 121ºC [5]. 2.2.2. Malt extract medium. Composition: malt extract (Kamenitza, Bulgaria) in 1:1 ratio with tap water (vol/vol) + 2 % agar – agar (w/vol). pH=6,5 – 7,0. The medium is sterilized for 25 minutes at 121ºC [5]. 2.2.3. Luria – Bertani glucose medium (LBG). Composition (g/dm3): triptone (Difco) – 10 g, yeast extract – 5 g, NaCl – 10 g, glucose (Scharlau) – 10 g. pH=7,5. The medium is sterilized for 25 minutes at 121ºC. 2.2.4. Soy – caseine broth medium. Composition (g/dm3): triptone (Difco) – 17g, soy peptone (Scharlau) – 3 g, NaCl – 5 g, K2HPO4 – 2,5 g, glucose (Scharlau) – 2,5 g. pH=7,3 ± 0,2. The medium is sterilized for 25 minutes at 121ºC. 2.2.5. Citrate utilization medium (Simons medium). Composition (g/dm3): Na(NH4)2PO4 – 1,5 g; KH2PO4 – 1 g; MgSO4.7H2O – 0,2 g; Na – citrate – 3 g; alcohol solution of bromthymol blue – 1%; agar – agar – 2%. The medium is sterilized for 25 minutes at 121ºC [6]. 2.2.6. Gelatinase activity medium. Composition (g/dm3): triptone (Difco) – 10 g; yeast extract (Scharlau) – 5 g; NaCl – 10 g; glucose (Scharlau) – 10 g; gelatine (DDR) – 250 g. рН 7,5. Medium is dispensed into tubes and is sterilized for 25 min at 121ºС [6]. 2.2.7. Proteolytic activity medium. Composition: malt agar medium with 10 % (vol/vol) solution additive (10 cm3 milk/100 cm3 water) of skimmed milk powder (Scharlau) [6]. 2.2.8. Lipolytic activity medium (Tween – 80 compounds hydrolysis). Composition (g/dm3): peptone (Scharlau) – 10 g; NaCl – 5 g; CaCl2 – 0,1 g; Tween – 80 (Merck) – 10 cm3; agar – agar – 20 g. рН 7 – 7,4. The medium is sterilized for 25 minutes at 121ºC [5]. 2.2.9. Sulphur–containing compound oxidation mediua. 2.2.9.1. Starckey broth medium. Composition (g/dm3): elemental S – 10 g; КH2PO4 – 3 g; MgSO4.7H2O – 0,2 g; CaCl2.2H2O – 0,2 g; (NH4)2SO4 – 0,5 g; FeSO4 – traces; indicator – bromocresol purple. рН 8,0. The medium is prepared in two versions: with glucose (5 g/ dm3) and without glucose. It is sterilized at Koch apparatus for 30 min on three consecutive days [7]. 2.2.9.2. NCL – broth medium. Composition (g/dm3): elemental S – 10 g; (NH4)2SO4 – 0,2 g; MgSO4.7H2O – 0,5 g; CaCl2.2H2O – 0,25 g; FeSO4 – traces; indicator – bromocresol purple. The medium is prepared in two versions: with glucose (5 g/ dm3) and without glucose. It is sterilized at Koch apparatus for 30 min on three consecutive days [7]. 2.2.9.3. Thiosulphate agar medium. Composition (g/dm3): Na2S2O3 – 5g; K2HPO4 – 0,1 g; NaHCO3 – 0,2 g; NH4Cl – 0,1 g; agar – agar – 20 g. рН 8,0. The medium is prepared in two versions: with glucose (5 g/ dm3) and without glucose. It is sterilized at Koch apparatus for 30 min on three consecutive days [7]. 2.2.10. NO3- – reductase activity medium. Composition (g/dm3): peptone (Scharlau) – 5 g; meat extract (Scharlau) – 3 g; KNO3 – 1 g. рН 7.0. The medium is sterilized for 25 minutes at 121ºC [8]. 2.2.11. NH4+ – citrate medium (for nitrifying activity). Composition (mol/dm3): Na – citrate.2Н2О – 9,5.10-3; NH4Cl – 9,35.10-3; KH2PO4 – 1,47.10-3; MgSO4.7H2O – 1,62.10-4; CaCl2 – 1,36.10-7; FeSO4.7H2O as EDTA – complex – 3,6.10-5. The medium is sterilized for 25 minutes at 121ºC [9]. 2.2.12. Blood agar (NCIPD, Bulgaria). Composition (g/dm3): casein hydrolysate – 14 g; NaCl – 5 g; peptone – 4,5 g; yeast extract – 4,5 g; defibrinated sheep blood – 70 cm3; agar – agar – 12,5 g. pH 7,3 ± 0,2. All medium components are sterilized for 15 min at 121ºС. After cooling to 45ºС – 50ºС 70 cm3 defibrinated sheep blood is added aseptically and is poured in sterile Petri dishes [8]. 2.2.13. Gorodkova medium (for yeast sporulation). Composition (g/dm3): peptone – 10 g; NaCl – 5 g; месен meat extract – 10 g; glucose – 2,5 g; agar – agar – 20 g. рН 6,5 – 7,0. The medium is sterilized for 25 minutes at 121ºC [5]. 2.2.14. Acetate agar medium (for yeast sporulation). Composition (g/dm3): NaOOCCH3.3H2O – 5 g; agar – agar – 20 g. pH 6,5 – 7,0. The medium is sterilized for 25 minutes at 121ºC [8]. 2.2.15. Minimal agar medium (for yeast sporulation). Composition (g/dm3): agar – agar – 20 g. рН 6,5 – 7,0. The medium is sterilized for 25 minutes at 121ºC [8]. 2.2.16. Rice agar medium (Fluka Analitycal) (for pseudomycellium identification). Composition (g/dm3): rice extract powder – 0,7; agar – agar – 20 g. The medium is prepared in two versions – with and without Tween – 80 (Merck) (1 cm3/dm3). рН 5,8 ± 0,2. The medium is sterilized for 25 minutes at 121ºC. 2.2.17. Motility medium (NCIPD, Bulgaria). Composition (g/dm3): peptone – 10 g, meat extract – 3 g, NaCl – 5 g, agar – agar – 4 g. рН 7,4 ± 0,2. The medium is sterilized for 25 minutes at 121ºC. 2.3. Cultivation and storage of the analyzed microorganisms. 2.4. Analytical methods. 2.4.1. Morphological and cultural methods 2.4.1.1. Cellular and colonial morphology. Description of cellular and colonial morphology of the studied yeast strains is performed by microscopic observation the developed on malt agar single colonies of the studied strains. 2.4.1.2. Identification of pseudomycellium formation ability. A part of the cultural medium of the studied strain, that has been cultivated for 48 hours in a thermostat is taken with flamed and cooled bacteriological loop and spread in touch on agar plates with rice-agar. The inoculated plates are incubated under aerobic conditions for 2-4 days at 23ºC - 28ºC. A microscopic preparation is prepared from the developed colonies of the tested strain, which is examined for the presence of specific pseudomycellium or other specific formations in yeasts. The presence of vegetative cells only determines the result as negative. 2.4.1.3. Identification of spore formation ability. The ability of the yeast to form spores under unfavorable environmental conditions - lack of nutrients - is examined.Using a flamed and cooled bacteriological loop a part of the pre-developed on malt agar 48 - hour colonies of the studied strain is taken and is stroked on a Petri dish with minimal agar medium Gorodkova agar or acetate agar. Incubation conditions - 20ºC - 25ºC for 7 days. A microscopic preparation is prepared from the biomass and it is stained by the method of Moller. Spores are rubine red and vegetative bodies – blue. 2.4.1.4. Determining the number of viable cells in the development of the cultures in liquid medium. Sterile malt extract is inoculated under aseptic conditions with yeast suspension, 5 cm3 medium is inoculated with 1 cm3 of the inoculum of the studied cultures. After incubation in a thermostat at 30ºC for 48 h, tenfold dilution method is done and malt agar plates are inoculated. Petri dishes are thermostatted at 30ºC for 48 h so that the strain would form single colonies, which are then counted. 2.4.2. Physiological and biochemical methods 2.4.2.1. Citrate utilization. Developed in containers with 30 cm3 1:1 malt extract at 30ºC for 48h yeast cultures are centrifuged at 3000 min-1 for 10 min. The supernatant is discarded and the biomass is resuspended in 5 cm3 sterile physiological solution. The yeast inoculum is taken using a sterile bacteriological loop and it is streak-spread on petri dishes with Simons medium. The inoculated plates are incubated at 30ºC for 48h. Positive results are recorded in a case of a colour change of the medium from green to blue. 2.4.2.2. Determination of the gelatinase activity of the tested strains. 2.4.2.3. Proteolytic activity determination using fusion agar method. The ability of the yeast strains to hydrolyze milk proteins in agar medium with milk additive is investigated. Wells with d = 6 mm are made on Petri dishes with medium for proteolytic activity. After inoculation, the plates are incubated at 30ºC for 48 hours. Results are reported as positive in the case of a formation of a brighter halo around the wells of the plates. A lack of halo is a sign of an inability to utilize milk proteins. 2.4.2.4. Determination of the lipolytic activity using fusion agar method. Lipolytic activity includes the ability of the analyzed strains to hydrolyze the compounds of Tween - 80. Wells with d = 6 mm are made on the Petri dishes with agar medium. After inoculation the Petri dishes are cultivated at 30ºС for 1 - 7 days. Formation of a turbid zone around the wells, due to the precipitation of Ca - salts of the formed free fatty acids, indicates the presence of lipolytic activity. 2.4.2.5. Analysis on the ability of the studied strains to oxidize S-containing compounds. The analysis of the oxidation of S-containing compounds includes development of the yeast strains on selective media. Tubes with sterile liquid Starkey medium and NCL medium "with" and "without" glucose are inoculated with 1 cm3 yeast suspension, prepared as in 5.1. With a bacteriological loop the same suspension is used to streak plates with thiosulphate media "with" and "without" glucose. The plates and tubes are cultivated in a thermostat at 30ºC for 15 days. Changes in the color of the indicator bromocresol purple (from purple to yellow on Starkey and thiosulphate media; and from yellow to dark yellow or purple on NCL – broth) are reported as positive results. 2.4.2.6. Determination of nitrifying activity of the studied strains. Nitrifying activity includes the development of the analyzed yeasts on liquid medium with ammonium salts being the sole nitrogen source. A suspension of the tested yeast strains is prepared, as described in 5.1. 1 cm3 of it is used to inoculate tubes with 5 cm3 of ammonia - acetate medium and the tubes are incubated at 30ºC for 24 hours. If the yeast strain has nitrifying activity, NO3-iones would be formed in the medium. Their presence is determined by test - strips (110,020 Nitrate Test Merckoquant, Merck). Some microorganisms have the ability to reduce NO3- to NO2-. Therefore, the content of NO2- in the medium is determined, using a test - strip (110,022 Nitrate Test Merckoquant, Merck). Results are recorded as positive in the presence of NO3- and NO2- or negative - when NO3- and NO2- are absent in the medium. 2.4.2.7. Determination of the nitrate - reductase activity of the studied strains. Prepared as in 5.1., the suspensions of the studied cultures are used to inoculate medium with KNO3 - 1 cm3 suspension is used to inoculate 5 cm3 of sterile medium for determination of nitrate - reductase activity. Incubation is carried out for 7 hours at 30ºC. The presence of NO2- in the medium is considered as a positive result. Their presence is determined by test - strip (110,022 Nitrate Test Merckoquant, Merck). Result are recorded as positive - in the presence of NO2- or negative - in the absence of NO2- in the medium. 2.4.2.8. Determination of the hemolytic activity of the investigated cultures. Some microorganisms have the ability to utilize blood, breaking down red blood cells. Depending on the mechanism of their hydrolysis there are three types of hemolytic activity. In α - hemolysis iron from hemoglobin is oxidized and the colonies become dark - green. In β - hemolysis the erythrocytes and the hemoglobin in them are degraded, and a bright halo is formed around some of the colonies. γ – hemolysis is observed in the case of no hemoglobin hydrolysis. Using a bacteriological loop inoculum is taken from the suspension of the tested yeast strain, prepared as in 5.1. and it is stroked on blood agar. The inoculated plates are cultured for 48 hours at 30ºC. The presence of hemolytic activity is recorded: in the cases of α - or β –hemolysis, the result is positive, while for γ – hemolysis, it is negative. 2.4.2.9. Determination of the catalase activity of the analysed cultures. Catalase activity is determined by the method described in [6]. 2.4.2.10. Determination of the oxidase activity of the investigated cultures. Oxidase activity test includes an analysis of the suspension of the studied strains for the presence of the enzyme cytochrome - oxidase. In the presence of molecular oxygen, cytochrome - oxidase can reduce the number of organic substances, including reagent NaDi (1 - naphthol + diamine dimetilparaphenylene) with the formation of indophenole blue.Test - strips for oxidase activity (Microbiology Bactident Oxidase 1.13300.0001, Merck) are places in the suspension of yeasts prepared as in 5.1. After 20-60 s the result is compared to a color scale. Positive result are recorded in the case of a color change of the strip from white to blue to blue - violet. 2.4.2.11. Determination of the profile of enzyme activity of the studied cultures. The determination of the profile of enzyme activity is performed, using the test kit API ZYM (BioMerieux) for semiquantitative determination of the enzyme profile of the studied strain. Fresh 24-hour culture of the tested strain is centrifuged for 15 minutes at 5000 g, the obtained biomass precipitate is washed twice and resuspended in API suspension medium. The API ZYM strips are placed in the incubation boxes and the microtubes are inoculated with the prepared cell suspension. The sample is incubated for 4 to 4,5 hours at 30° C. After the incubation one drop of reagent A and one drop of reagent B are pipetted into each microtubule. After 5 min staining result is reported according to the colour scheme described in the manufacturer’s instructions. The enzyme activity is determined according to the colour scale from 0 (no enzyme activity) to 5 (maximum enzyme activity). 2.4.2.12. Determination of the biochemical profile of the investigated cultures. The system API 20 C Aux (BioMerieux SA, France) for identification of yeast species based on the consumption of 19 carbon sources is used for the determination of the biochemical profile of the tested cultures. Fresh 24-hour culture of the tested strain, developed on malt agar, is resuspended according the instructions of the manufacturer in API C resuspension medium. The honeycomb wells on the bottom of the incubation boxes are filled with sterile physiological solution. The API 20 C strips are placed in the incubation boxes and the microtubules are inoculated with the prepared cell suspension. The sample is incubated for 48h to 72h at the optimum temperature for each of the studied strains. Results are recorded according the change in turbidity in comparison to the control (microtubule 0). The results are processed with apiweb® identification software.