Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(1): 29-43, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/cayologia-249 Citation: B. Yilmaz Öztürk (2019) Intracellular and extracellular green synthesis of silver nanoparticles using Desmodesmus sp.: their Antibacte- rial and antifungal effects. Caryologia 72(1): 29-43. doi: 10.13128/cayolo- gia-249 Received: 21th april 2018 Accepted: 20th november 2018 Published: 10th May 2019 Copyright: © 2019 B. Yilmaz Öztürk. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Intracellular and extracellular green synthesis of silver nanoparticles using Desmodesmus sp.: their Antibacterial and antifungal effects Betül Yılmaz Öztürk Eskişehir Osmangazi University Central Research Laboratory Application And Research Center (ARUM), 26480 Eskişehir, Turkey E mail: byozturk@ogu.edu.tr. ORCID: 0000-0002-1817-8240 Abstract. In this study aim was to perform green synthesis of synthesis silver nano- particles (LAC-AgNPs, RAE-AgNPs and BAE-AgNPs) by using Desmodesmus sp., intracellular and extracellular synthesis methods and to compare the obtained prod- ucts with physicochemical characterization techniques. The structural, morphological and optical properties of the synthesized nanoparticles were characterized using UV- Vis spectroscopy, TEM, SEM-EDS, FTIR, DLS and zeta potential. These results clearly show that silver nanoparticles (AgNPs) could be synthesized in different sizes and sta- bilities with various biological materials obtained from Desmodesmus sp. LAC-AgNPs had size of 10-30 nm, RAE-AgNPs had size of 4-8 nm and BAE-AgNPs had size of 3-6 nm. Also, the antibacterial activity of silver nanoparticles synthesized as intracel- lular and extracellular showed a strong antibacterial effect against pathogens such as Salmonella sp. and Listeria monocytogenes. Additionally, they have effective antifungal activity against Candida parapsilosis. The broth microdilution method was used for examining antibacterial antifungal effect of synthesis AgNPs. The minimum inhibitory concentration against Salmonella sp., Listeria monocytogenesis and Candida parapsilosis were recorded as 3,125 μl, 1,5625 µl and 0,78125 µl synthesis AgNPs, respectively. As a result, it has thought that different sizes of synthesis AgNPs may have a great potential for biomedical applications. Keywords. Green synthesis, nanoparticle, algae, antimicrobial, Desmodesmus sp. INTRODUCTION In nanotechnology studies, the most attract attention topics is the syn- thesis of nanoparticles (NPs) with strong potency in different sizes and shapes or by using various variables. For example, metal NPs. The area of use for metal NPs are very broad. Even silver NPs alone are used in count- less areas with optics, electronics, catalysis, home furnishings and extensive medical applications, and the annual production is estimated to be hundreds of tons worldwide (Ge et al. 2014). 30 Betül Yılmaz Öztürk Synthesis of nanomaterials containing noble metal requires alternative strategies because of their high cost. Green synthesis or green chemical synthesis leads the list of these strategies and here the target is a biological synthesis of nanomaterial and to obtain products with positive effects in terms of the environment (Sondi et al. 2000). Green synthesis has preferred because of the high costs of materials used in conventional chemical meth- ods and due to the toxic substances released into the environment. Because the toxic effect of nanoparticles is proven in many studies on aquatic organisms in particu- lar. For example, studies on algae (Dağlıoğlu and Öztürk 2016; Dağlıoğlu and Öztürk 2018; Öztürk and Dağlıoğlu 2018) have shown that aquatic invertebrates (Artemia salina) (Dağlıoğlu et al. 2016a), terrestrial invertebrates, honey bee (Apis mellifera) (Özkan et al. 2016), aquat- ic plants (Lemna minor and Myriophyllum spicatum) (Dağlıoğlu and Türkiş 2017a, b). Nevertheless, it more attracts the attention of researchers for the production of metal nanoparticles because of its low cost, environ- mental friendliness, and simple approach. In the green synthesis does not use reducing agents like sodium boro- hydride (NaBH4) used in chemical synthesis (Kozma et al. 2015). These reducing agents are both expensive and may produce oxidized boron species bound to NPs after synthesis. As a result the commercially produced NPs are not appropriate for biological applications. NPs produced by green synthesis are of great importance in terms of environmentally friendly production processes and low costs. Many organisms or a variety of extracts produced by them may be used in the green synthesis process bacteria (Joerger et al. 2000), plant (Khatami et al. 2018a), fungi (Boroumand et al. 2015) and algae (Sin- gh et al. 2013). Algae will have a great platform for products to be used for various purposes over the next few years has a long-term sustainable potential, especially in the pro- duction of food and liquid fuels (Koothari et al. 2017). For this reason algae are commonly chosen for green synthesis because their structures are a rich source of biologically active compounds like chlorophyll, carot- enoids, astaxanthin, phenol, flavonoid, protein, vitamin and minerals (Faulker 2000). Additionally, these phy- tochemical materials are each effective metal reduc- ing agents and their structures contain agents ensuring that hydroxyl, carboxyl, and amino functional groups coat metal NPs (Annamalia and Nallamuthu 2015). It has been supported by various literatures that NPs syn- thesis can be carried out by using various algae. For example, Kannan et al. (2013) performed AgNP synthe- sis with extraction from Chaetomorpha linum species. The researchers succeeded in synthesizing spherical NPs with nearly 30 nm size without using synthetic reactives. Barwal et al. (2011) used a Chlamydomonas reinhardtii model and focused on understanding the role of a vari- ety of cellular proteins in the synthesis and coating of silver NPs. Prasad et al. (2013) studied the extract of the brown algae Cystophora moniliformis species and report- ed that the sizes of NPs may change linked to tempera- ture. Studies mainly use marine macroalgae with insuf- ficient numbers of studies about microalgae. It has been known for many years that silver (Ag) is an antimicrobial agent and it draws much attention due to its application in fields such as colloidal Ag, catalysis and water purification. It is reported that using AgNP may purify drinking water (Pradeep 2009). Further- more, the effect of Ag NPs size variability on antibacte- rial properties has been a matter of interest. As a result, researchers who have successfully achieved green syn- thesis have simultaneously assessed the antibacterial and antifungal effects on a variety of microorganisms (like bacteria and yeast) (Govindaraju et al. 2009; Rajeshku- mar et al. 2014; Salari et al. 2016; Suriya et al. 2012). Our aim is to use the microalgae Desmodesmus sp., which can easily be produced in a laboratory environ- ment, to compare synthesis using both intracellular and extracellular routes. For this, the differences between NPs forming under the same conditions were deter- mined using characterization techniques like UV-Vis, TEM, SEM-EDS, FTIR, DLS and zeta potential. At the same time, antibacterial effects of synthesized NPs on bacterial strains of important food pathogens such as Salmonella sp. and Listeria monocytogenesis were inves- tigated. Additionally, the antifungal effect on the human pathogen of Candida parapsilosis species was investigat- ed. MATERIAL AND METHOD Algae Culture The test organism used in our study, Desmodes- mus sp. (KR261937), was taken from the algae culture collection of Selçuk University Hydrobiology Labora- tory. Algae taken from stock cultures were transferred to the fluid BG-11 medium (NaNO3, 15; K2HPO4, 0.4; MgSO4·7H2O, 0.75; CaCl2·2H2O, 0.36; citric acid, 0.06; iron(III) ammonium citrate, 0.06; Na2-EDTA, 0.01; Na2CO3, 0.2 g/L, 1 mL; trace elements solution, (H3BO3, 61; MnSO4·H2O, 169; ZnSO4 ·7H2O, 287; CuSO4·5H2O, 2.5; (NH4)6Mo7O24·4H2O, 12.5 mg/L) in accordance with the procedure stated in Rippka (1988). The microalgae were transferred to 250 ml erlenmeyer flask and left to proliferate under sterile conditions. The cultures were 31Intracellular and extracellular green synthesis of silver nanoparticles using Desmodesmus sp. left under 3000 lux fluorescent light appropriate for pho- tosynthesis for 12 hours light and 12 hours darkness, at 28±2 °C degrees, and 120 rpm for 15-20 days. When algae passed the log phase stage, cells were centrifuged at 1000 rpm and the biomass was obtained. All chemi- cals used in this study were analytical quality. Intracellular AgNP synthesis using live algae cells Silver nitrate (AgNO3,  ≥99.0%; Sigma-Aldrich) was prepared in 100 mM stock solution. Microalgae passing the logarithmic phase were counted with the automatic cell counting device (LUNA-II™ Automated Cell Coun- ter, South). Using LUNA ™ Cell counting slides, counting and viability of cells were determined with trypan blue. Afterwards, live algae cells (LAC) were harvested by cen- trifugation at 4000 rpm for 20 minutes at 4 °C. The cul- ture filtrate was removed and the pelleted biomass was washed with sterile deionized water to remove foreign absorbed material. The washing procedure was repeated five times and the washed biomass was brought to sus- pension again in distilled water. The suspension of algal biomass had 5mM AgNO3 added the from stock solu- tion. In the control setup, AgNO3 was not added to the algal biomass. Cultures were incubated at 28 °C under similar conditions to those stated above for 72 hours. Intracellular Ag NPs (LAC-AgNPs) synthesis from LAC was completed. After the reaction, the biomass was sepa- rated by the centrifuge and stored at -20 °C until charac- terization. Extracellular synthesis of AgNP Two different algal extracts were obtained using Desmodesmus sp. for the extracellular synthesis of AgNP. These extracts were raw algal extract (RAE) and boiled algal extract (BAE). Preparation of raw and boiled algae extracts For the preparation of R AE extract, 3.0 g (wet weight) algal biomass was suspended in 20 ml of deion- ized water for 5 days. At the end of the 5th day, it was centrifuged at 7000 rpm for 20 minutes. The supernatant (raw algal extract) was separated from cells and prepared for extraction. To begin the reaction, the final concentra- tion of 5 mM AgNO3 was added. After this procedure, continuous mixing began. Later the mixing procedure, a colloidal structure was obtained and after this process repeated centrifugation was completed at 3500 rpm for 5 minutes. These centrifuge cycles were completed to puri- fy AgNP. Extracellular Ag NPs (RAE-AgNPs) synthesis from RAE was completed. After the reaction, the bio- mass was separated by the centrifuge and stored at -20 °C until characterization. To prepare the BAE extract, 3.0 g (wet weight) algal biomass was suspended in 20 ml deionized water and heated to 100 °C for 20 minutes in an erlenmeyer flask. After the boiling procedure, it was cooled and filtered with Grade GF/A glass microfiber filters (Whatman™, pore size: 1,6µm). The obtained filtrate had the final con- centration of 5 mM AgNO3 added at room temperature to begin the reaction. After the procedure continuous mixing was completed. Later the mixing procedure, a colloidal structure was obtained and after this process repeated centrifugation was completed at 3500 rpm for 5 minutes. These centrifuge cycles were completed to puri- fy AgNP. Extracellular Ag NPs synthesis (BAE-AgNPs) from boiled algal extract was completed. After the reac- tion had completed, the biomass was separated by the centrifuge and stored at -20 °C until characterization. Characterization of AgNPs Biological reduction of silver ions with the green synthesis route was observed by taking 3 ml aliquot samples at different time intervals. Absorption measure- ments were made with a UV-Vis (AE-S90-2D UV-VIS Spectrophotometer, China.) spectrophotometer between 190 and 1100 nm. Transmission electron microscope (TEM) images of the intracellular and extracellular synthesized AgNPs were obtained using TEM (JEOL JEM  1220 brand, Japan) working at 100 kV acceleration voltage. Sam- ples used to obtain TEM micrographs were prepared by dropping on a carbon-coated copper grid and dried under a vacuum before investigation. With the aim of investigating the cells at the ultrastructural level, routine TEM monitoring procedure was performed and samples were submerged in epoxy resin. Thin sections were taken at 60 nm thickness with the aid of an ultramicrotome (Leica ultracut UCT, Leica, Germany) (Ilknur et al. 2012; Li et al. 2012; Zhang et al. 2016). Elemental analysis of samples was completed with SEM (a JEOL JSM-5600 LV brand, Japan) device fitted with EDS (E2V Scientific Instruments, United Kingdom). Algae cells were freeze-dried and algae biomass was obtained. Dried biomass was prepared using the KBr pellet technique and ATR technique. The surface chem- istry of reduced Ag samples and the biologically active portions of the live microalgal cells were analyzed to check for comparisons. The fourier transform mid-infra- 32 Betül Yılmaz Öztürk red FTIR spectra (Perkin Elmer Spectrum 100 FTIR- ATR unit, Germany) were collected with conduction mode from 400–4,000 cm-1 with 0,4 cm-1 spatial resolu- tion. Dynamic light scattering (DLS) and zeta potential are necessary parameters to define the size distribution, particle size, homogeneity and stability of LAC-AgNPs, RAE-AgNPs and BAE-AgNPs. Particle size and polydis- persity index (PDI) were determined using the dynamic light scattering technique. DLS studies of LAC-AgNPs, RAE-AgNPs and BAE-AgNPs diluted in deionized water were measured by a Malvern-Zetasizer (Nano-Z590, United Kingdom) device. The zeta potential is a meas- urement of the attraction or repulsion values between particles. Particles with certain load attractions with opposite polarity within the suspension, as a result, a strong bond surface is formed on the surface of the load- ed particle and then a surface extending outward from the loaded particle forms. The behavior of particles with- in polar fluids is determined not by electric load but by zeta values. Zeta potential studies were measured with a Malvern (SN: MAL1064144, United Kingdom) brand Zeta Sizer ZS device. Antimicrobial Susceptibility Testing of synthesis NPs In this study with the aim of determining the anti- bacterial and antifungal susceptibility of LAC-AgNPs, RAE-AgNPs and BAE-AgNPs, Salmonella sp. (gram-) and Listeria monocytogenesis (gram+) bacteria and Can- dida parapsilosis yeast was used. To determine the mini- mal inhibition concentration, the broth microdilution method was taken as a basis. Bacteria cells were left in nutrient broth medium at 37 °C for 1 night, while yeast cells were incubated for 1 night in a shaking incubator at 30 °C in yeast extract-peptone-dextrose (YPD; %1 yeast extract, %2 peptone %2 dextrose) medium and cultures were taken. The density of bacteria and fungal cells were set according to the McFarland 0.5 standard. Tests of minimal inhibitory concentration (MIC) were made in accordance with the CLSI (Clinical Laboratory Stand- ards Institute) criteria M27-A8 for bacteria and M27-A2 for yeast (Zgoda and Porter 2001). The extraction con- taining lowest NPs that inhibited bacterial and fungal development was determined as the volume MIC value. During this process 40 minutes sonication was applied to obtain the LAC-AgNPs. The 96-well plates (Lp Itali- ana Spa) was added 100 µl RPMI 1640 and 100 µl the LAC-AgNPs, RAE-AgNPs and BAE-AgNPs. Later they were diluted with microdilution and left for 24 hours incubation. The plate with resazurin added had results assessed in parallel with the colour change. RESULTS The test organism Desmodesmus sp. (KR261937) is in the Chlorophyceae class and is a water alga gener- ally forming colonies with an oval or shuttle-shaped body. Additionally, it is a photosynthetic microalga with 6-10 horn-like protrusions on the body. In the colonial structure, generally twin cells are found. Additionally, sequences of 4 colonial cells may be seen in low num- bers. The count of the automatic cell counting device is given in Table 1. In the current study when algal bio- mass (LAC) was exposed to Ag ions (Ag+), the colour of the algal biomass changed from natural bright green to brown and compared with the control biomass, the Ag+ ion was biologically transformed (Ag metal accumula- tion) to Ag0. During exposure the colour change began in the first 24 hours; however, the largest colour differ- ence compared to the first day occurred after 72 hours. LAC-AgNPs was researched with UV-Vis for 72 hours (Figure 1A). Simultaneously RAE and BAE were treated with 5 mM AgNO3 and extracellular Ag NPs (RAE-AgNPs and BAE-AgNPs) the formation was researched with UV- Vis spectroscopy. The RAE-AgNPs were colourless and had a high peak at 280-300 nm (Figure 1 B). The RAE exposed to Ag displayed a peak at 420 nm especially after the 48 hours day on UV-Vis spectral analysis and it was considered AgNP formation had begun. It is known that the concentration of the reducing agent in the reac- tion mixture plays an important role in the formation of nucleation points and then controls the size of AgNP; this may have caused less stable AgNP in the RAE (Hiramutsu and Osterloh 2004; Jena et al. 2014). As a result, another experiment was made to increase the concentration of this type of material in the reaction mixture. The biomass was boiled in water to obtain more reducing and stabilizing agents. Initially, the boiled extract had a light yellow colour, which trans- formed to brown when exposed to the Ag nitrate solu- tion procedure. The formation of this colour is due to stimulation of surface plasmon resonance (SPR) effect and the reduction in AgNO3 and may indicate the for- mation of AgNP. With the increase in the reaction dura- tion, the colour of the reaction mixture turned a darker Table 1. Desmodesmus sp. cell counting and cell viability analysis report. Total cell Live cell Dead cell Viability 4,48x108 4,08x108 4,00x107 80,6 % *Stain: Trypan blue. 33Intracellular and extracellular green synthesis of silver nanoparticles using Desmodesmus sp. shade for up to 72 hours. The formation of BAE-AgNPs were proven with the UV-Vis spectrum showing the characteristic surface plasmon resonance (SPR) band for AgNPs. Figure 1 C shows the UV-Vis spectra series recorded for the reaction mixture at a variety of time intervals. The BAE showed a peak at 280-320 nm which may be linked to the presence of peptides. The BAE exposed to Ag displayed a peak at 420 nm especially after the 24 hours day on UV-Vis spectral analysis and it was considered BAE-AgNPs formation had begun. Our UV-Vis results show a steady increase in reduction of Ag ions in LAC up to 420 nm, then it sta- bilized and appeared to pass to a downward trend. This section is defined as the “surface plasmon resonance band” and is due to the stimulation of free electrons in the NPs. The symmetric shape of the band is an indi- cator of the regular distribution of the spherical NPs (Travan et al. 2009). After intracellular synthesis, the exposed biomass had TEM analysis was made to research the morphology Fig. 1. Examination of intracellular and extracellular synthesis of silver nanoparticles by UV-Vis. A. LAC-AgNPs; B. RAE-AgNPs; C. BAE- AgNPs. 34 Betül Yılmaz Öztürk and dimensions of the NPs. TEM images revealed the cells were nearly 4.5-5 µm (length) × 2-3 µm (width) size with an oval shape and with 6-9 colonial protrusions (Figure 2A). Fig. 2. Morphological characterization of the silver nanoparticles. A, B, C Intracellular synthesis, TEM Image of Desmodesmus sp. cell medi- ated synthesized silver nanoparticles. D, E, F Cellular localization of in Intracellular silver nanoparticles, TEM micrograph of thin section (~ 60 nm). NP:nanoparticle, S starch, V vakuol, CM Cytoplasmic membrane. 35Intracellular and extracellular green synthesis of silver nanoparticles using Desmodesmus sp. When the algae cells exposed to AgNO3 were inves- tigated with TEM on a grid with the dropping method, distributed metal NPs were observed, as shown in Fig- ure 2 A, B and C. As seen at different magnifications of the metal nanoparticle shapes, the periphery of the cells is observed more clearly and homogeneously, while the interior sections were not fully identified due to a more compact and electron-dense zone observed (Figure 2 A, B). When micrographs of LAC-AgNPs at high mag- nifications are investigated, the spherical structures of the AgNPs are clearly observed. The intracellular LAC- AgNPs are in the range of 10-30 nm in size and appear to display a homogeneous distribution. In TEM analysis, a section of 60 nm in thickness was taken from the cells in order to be able to see the LAC-AgNPs. Additionally, sections were taken of these cells to identify where the NPs were localized (Figure 2 D, E, F). According to the results of these sections, NPs were localized especially in areas close to the cell membrane, while also in other regions of the cell, e.g., in areas close to starch storage areas (Figure 2 F). The nanoparticle dimensions obtained after RAE- AgNPs were mean 4-8 nm (Figure 3 A, B). NPs synthe- sized in this manner were less stable according to both TEM and zeta potential results. When morphology and size of NPs are examined after extracellular synthesis, the NPs produced by BAE-AgNPs were mean 3-6 nm. These NPs had a homogeneous distribution without aggregation or flocculation (Figure 3 C, D). The rea- son for this is that some stabilizing agents in the algal extract were released by boiling and entered the reac- tion (Mohseniazar et al. 2011; Nithya and Ragunathan, 2009). At the same time, control of dimension and struc- ture may be associated with interactions between bio- components like polysaccharides, proteins, polyphenols and phenolic compounds with metal atoms (Shao et Fig. 3. TEM Image of extracellular synthesized silver nanoparticles A, B. Desmodesmus sp. cell RAE mediated synthesized silver nanoparti- cles (RAE-AgNPs); C, D. Desmodesmus sp. cell BAE mediated synthesized silver nanoparticles (BAE-AgNPs). 36 Betül Yılmaz Öztürk al. 2004). As a result, BAE-AgNPs showed the NPs had equal distribution. Analysis through Energy dispersive SEM-EDS spec- trometers confirmed the presence of an elemental Ag signal of the Ag NPs. Recognition lines for the major emission energies for Ag are displayed and these match with peaks in the spectrum, thus giving confidence that Ag has been correctly identified. In this study, the syn- thesized AgNPs were subjected to elemental analysis with SEM-EDS, the Ag element was observed in all three situations (LAC-AgNPs, BAE-AgNPs, RAE-AgNPs) at the rate (Table 2) (Figure 4 A, B, C). The highest Ag was identified in the BAE-AgNPs groups (Figure 4 B). When these results are considered, they provide information about the purity of the formed NPs. Extracts obtained with different methods (BAE and RAE) and live cell algae (LAC) the AgNPs obtained from these extracts (LAC-AgNPs, R AE-AgNPs and BAE-AgNPs) had FTIR analysis completed on these samples to define the functional groups of chemical components. The analysis results observed many peaks (transmittance a.u) at 3284, 2919, 2851, 2161, 2027, 2034, 1638, 1535, 1380, 1242, 1149, 1023, 812, 717, and 551. When vibrations of biomass exposed to Ag are investigated, the dense broadband at 3400 cm-1 is vibra- tions from alcoholic, phenolic and carboxylic groups (Figure 1 F). Primarily in addition to hydroxyl groups equivalent to O-H strain, vibrations equivalent to pri- mary and secondary amines and amides are shown with N-H strain were observed. The band at nearly 2920 cm-1 reflects the C-H strain of alkanes. The lack of absorp- tion in this region shows hydrogen linked to aliphatic carbon is not present (Rónavári et al. 2017). The band at 2851 cm-1 from the aldehyde group reflects C-H strain. The band at 2161 cm-1 is -S-CΞN thiocyanate, while the bands at 2027 and 2034 cm-1 are -N=C=S isothiocy- anate. This situation shows that cyanate, elemental car- bon and thiocyanate may be found within total organic carbon. The peak at nearly 1,638 cm-1 is equivalent to C = C vibration of aromatic structures, with the peak at 1242 cm-1 equivalent to C-O strain of phenolic groups. The peak at 1380 cm-1 is NO2 asymmetric strain of Table 2. EDS reported from biosynthesized AgNPs. AgNPs Intensity (c/s) Error 2-sig Conc. Units Control 0.060 0.129 0.023 wt.% LAC-AgNPs 130.34 7.217 34.385 wt.% RAE- AgNPs 130.01 7.209 20.975 wt.% BAE- AgNPs 293.68 10.837 54.920 wt.% *kV 20.0, Takeoff Angle 35.0°. Elapsed Livetime 10.0. Conc: Concentration. Fig. 4. SEM-EDS spectrum recorded from biosynthesized AgNPs A. Control group; B. LAC-AgNPs; C. RAE-AgNPs; D. BAE-AgNPs. 37Intracellular and extracellular green synthesis of silver nanoparticles using Desmodesmus sp. alkyl groups, and the peak at 1535 cm-1 is equivalent to C = C strain of biphenol group. Around 1145 cm-1 the C-N strain vibration of aromatic primary and second- ary amines is observed. Aromacity may be mentioned between 900-690 cm-1 (Jena et al. 2014). The extracellular BAE-AgNPs obtained with the boiling method may have bonded to amine groups, while the R AE-AgNPs may have bonded to alkyne groups. Our FTIR results comply well with the literature data, with the surface of AgNPs obtained from intracel- lular Desmodesmus sp. coated with organic components found in the extract, as shown in Figure 3 B. As a result, successful green synthesis is revealed with the soluble organic components or proteins able to bind to Ag ions and reduce Ag ions to form NPs (Jegadeeswaran et al. 2012) (see 800-2919 cm-1 region). Particle size and zeta potential are very important parameters for green synthesized Ag NPs. The particle size of Ag NPs, especially, has a large effect on the anti- microbial properties. Another important value for parti- cle size is PDI. PDI reveals homogeneity (Sharma et al. 2018). Both intracellular and extracellular AgNPs were well distributed in colloidal solution and the mean size distribution of these particles were as follows; the mean particle distribution for LAC-AgNPs were 10-30 nm, with this value 4-8 nm and 3-6 nm from RAE-AgNPs and BAE-AgNPs (Figure 6 A, C, E). According to PDI values for the results, homogeneous particle size distri- bution within the solution was observed to be highest for LAC-AgNPs. Though different sizes were found dur- ing synthesis, higher numbers of small-scale NPs were observed in terms of numbers (Table 3). Zeta potential values are obtained from the high repulsion and attraction forces between each nanopar- ticle and these values define particle stability. Intracel- lular and extracellular AgNPs have high negative zeta potential values. The high negative value affects the push between the particles, thereby increasing the stability of the formulation (Rao et al. 2013). In our study, the zeta potential of AgNPs were measured as -20.2 mV for LAC- AgNPs (Figure 6 B), -19.9 mV for BAE-AgNPs (Figure 6 D) and -14.2 mV for RAE-AgNPs (Figure 6 F). All values for particle sizes, PDI and zeta potentials are shown in Fig. 5. FT-IR spectrum for A. Desmodesmus sp. control group; B. Desmodesmus sp. formed LAC-AgNPs. Table 3. The particle size of silver nanoparticles, polydispersity index and Zeta potential. Particle size (nm) DLS PDI Zeta potential (mV) LAC-AgNPs 20-40 0.445 -20.2 BAE-AgNPs 10-15 0.452 -19.9 RAE-AgNPs 10-20 0.613 -14.2 38 Betül Yılmaz Öztürk Table 3. The observed result contains some differences compared with the TEM studies but is well-correlated. The differences may be due to the tendency of Ag NPs to agglomerate within an aqueous solution; thus the val- ues obtained from DLS will be higher than TEM values (Domingos et al. 2009). Antimicrobial and antifungal effect of synthesis AgNPs LAC-AgNPs, RAE-AgNPs and BAE-AgNPs have been tested on Salmonella sp. (gram +) and Listeria monocytogenesis (gram -), the most frequently encoun- tered bacteria in food poisoning (Cantero et al. 2018; Ma et al. 2018). The MIC values of the synthesized NPs were calculated on these bacteria. It was initiated by adding 100 µl of synthesized NPs so that the MIC val- ues could be found. The LAC-AgNPs, RAE-AgNPs and BAE-AgNPs inhibited 3,125 μl of Salmonella sp. Thus, this value was determined as the MIC value. There was a larger effect on Listeria monocytogenesis compared to Salmonella sp. with 1,5625 µl MIC value determined. Candida parapsilosis is a pathogenic yeast strain with Fig. 6. Size distribution by dynamic light scattering and zeta potential measurement of biyosenthesis silver nanoparticles A, B. LAC-AgNPs; C, D. RAE-AgNPs; E, F. BAE-AgNPs. 39Intracellular and extracellular green synthesis of silver nanoparticles using Desmodesmus sp. high biofilm formation capacity (Soldini et al. 2017). Candida parapsilosis appeared to be more affected com- pared to bacteria and the MIC value was determined as 0,78125 µl (The synthesized AgNPs were initially used in 100 µl). The LAC-AgNPs, BAE-AgNPs and RAE-AgNPs obtained from Desmodesmus sp. with different methods showed significant degree of effect with different MIC values for bacteria and yeast. DISCUSSION AgNP is used in a variety of applications like cataly- sis, biosensing, imaging and antibacterial activity. As result researchers in recent times have performed many studies to synthesize AgNP more economically and eas- ily. Green synthesis is an alternative method developed to produce metal NPs using natural compounds or plant components. The most important advantages of these methods are the lack of toxic material involved in the chemical synthesis and the lack of high costs. In addi- tion to green synthesis and sustainable synthetic meth- ods can decrease environmental pollution to some extent (Khatami et al. 2018c) . Another significant advantage is that NPs may be synthesized in an easy and reliable manner using only live organisms (algae, bacteria, fun- gus and plant), without requiring reducing or stabiliz- ing agents (Tippayawat et al. 2016; Khatami et al. 2018b). Microorganisms such as bacteria and algae have proven to be well suited for nanoparticle synthesis, where par- ticle size and morphology must be stabilized by different methods in green synthesis (Metuku et al. 2014). Algae or plants or their extracts, many prokaryotic organisms and biocompatible macromolecules are very important for nanoparticle synthesis (Poulose et al. 2014). Algae are unique due to the lipids, minerals and some vita- min wealth contained in these organisms (Namvar et al. 2012; Zuercher et al. 2006). Additionally, polysaccha- rides, proteins, and polyphenols are known as functional food as a variety of bioactive material with some medical uses like in cancer, oxidative stress, inflammation, aller- gies, thrombosis and lipidemia (Mahdavi et al. 2013). As a result, hydroxyl, carboxyl and amino functional groups are found among this phytochemical material that is capable of acting in a single step as both effective metals reducing agents and as capping agents. Generally many natural essences in cell contents or released after extraction are biologically active compounds and may be responsible for the reduction of Ag ions and stabilization of the obtained NPs. The carbonyl groups in proteins have strong binding ability against metal NPs and as a result, proteins may form a coating layer on the surface of AgNPs (Dhand et al, 2016; Vivek et al. 2012). This may prevent agglom- eration and increase the stability of NPs synthesized in aqueous media. In our study, UV-Vis data from algal cells exposed to Ag nitrate support the view that of Ag + ions were taken into cells within 24 hours and L-AgNPs formed (Peaked at 420 nm within 24 h.). As metal ions (Ag+) are on the algal surface, they were identified to be held by electrostatic interaction between the functional groups with the negative load on the cell surface, and following this, the metal ions were reduced (Barwal et al. 2011). In this way, high intracellular accumulation and formation of metal particles may be linked to several probable mechanisms in the process. These mechanisms vary according to type and metal accumulation occurs with two processes. The first is adsorption or biosorp- tion at the cell surface independent of metabolism, while the second is metabolism-dependent absorption by orga- nelles or cytoplasmic ligands (Chakraborty et al. 2006). Metal NPs synthesized with green methods may produce colloids with different sizes, shapes, and dis- tributions. If changing the method used provides better results, the green approach may be chosen. As a result of our study, the focus was on the biosynthesis of both intracellular and extracellular AgNP using different extraction techniques with Desmodesmus sp. microal- gae and AgNP production was successfully achieved. In this study during intracellular synthesis, algal cells have pores of 3-5 nm width ensuring passage of low molecu- lar weight material like water, inorganic ions, gases and other small nutritional material required for growth and metabolism (Wang and Chen 2009). Large molecules or macromolecules cannot pass these pores. Similarly, in our study, the intracellular LAC-AgNPs appeared to be larger than the size of the pores in the algal cell walls (10-30 nm) (Figure 2 A, B, C). The BAE-AgNPs and RAE-AgNPs also had spherical shape without agglom- eration. Sections through the cells transversely and lon- gitudinally observed nanoparticle formation distributed through intracellular cytoplasm, with larger size NPs in some regions, e.g., in regions close to vacuoles. In pre- vious research, it was reported that maximum metal accumulation occurred within the cy toplasm, peri- plasm, nucleus and pyrenoids of organisms like Chla- mydomonas (Ag), Chlorella (Au, Pd, Ru, Rh), Klebsor- midium flaccidum (Au) and Shewanella (Au, Pt) (Bar- wal et al. 2011; Dahoumane et al. 2012; Luangpipat et al. 2011). FTIR can be used to identify the type of functional groups and biomolecules that are responsible for cap- ping and efficient stabilization of NPs and qualitative and quantitative identification of the molecular structure 40 Betül Yılmaz Öztürk of organic compounds in the NPs structure (Khatami et al. 2018d). In thi study, the results of FTIR spectro- scopic investigations showed the presence of organic particles especially in the 800-2919 cm-1 region. Linked to this result, the protein building block of amino acids was confirmed as having strong binding ability to metals and formed a layer surrounding metal NPs. They acted as a coating material preventing agglomeration and thus are considered to have ensured high stability of metal NPs. These results confirm the presence of proteins or peptides with possible function as stabilizing agents for AgNPs (Singhal et al. 2011). The extracellular R AE-AgNPs synthesized was found to be less stable and have a tendency to agglom- erate compared to extracellular synthesized BAE-AgNPs and intracellular synthesized L-AgNPs. Additionally, the RAE-AgNPs synthesis process was slower compared to the others. When BAE was used, both high rates of BAE-AgNPs biosynthesis occurred and the stability of these NPs were higher. However, according to the UV results, L-AgNPs were more rapid biosynthesised than extracellular BAE-AgNPs and RAE-AgNPs. This situ- ation may be related to macromolecules like protein or peptide released by boiling directly reducing Ag+ ions. A study by Jena et al. (2014) produced similar results. The researchers explained that in this situation the protein or peptide concentration has a vital role in AgNP for- mation and stabilization. A similar study by Barwal et al. (2011) prepared Chlamydomonas cell extract treated with Ag nitrate with both whole cell esence and protein- removed extraction. When the results are compared, the protein-removed cell extraction was identified to synthe- size larger sizes of AgNP. The researchers explained that the protein concentration is directly correlated with par- ticle formation rate and inversely correlated with particle size (Barwal et al. 2011; Jena et al. 2014). Due to a variety of reasons in the antimicrobial mechanism, Ag ions or salts only have limited use as an antimicrobial agent. However, the use of AgNPs may overcome these limitations. In biological systems (ani- mal cell culture, plants, bacteria) the effect of AgNPs in different concentrations has been investigated (Sobieh et al. 2016, Khatami et al. 2018). For the use of Ag against microorganisms in a variety of areas, it is important to prepare Ag with appropriately priced methods and to know the antimicrobial effect mechanism to increase this effect (Kim et al. 2007). Green synthesized NPs may expand these areas of use significantly. In this study, AgNPs may be a potential antibacterial and antifungal agent and could be prepared cost-effectively. Because, Desmodesmus sp., a green algae, could be a low-cost pro- duction house for intracellular and extracellular AgNPs synthesis because of the minimum growth regimens required for growth, such as water, sunlight and com- mercial fertilizers, and high biomass efficiency. CONCLUSION In this study different sizes of AgNP were success- fully synthesized from Desmodesmus sp. microalgae using both intracellular and extracellular green synthe- sis routes. In particular, intracellular UV results of LAC- AgNPs peaked at 420 nm within the first 24 hours. The purity of Ag NPs was confirmed with SEM-EDS. Nano- particle size and stability were determined according to DLS and Zeta potential results. AgNPs with optimized size were determined to have lethal potential against bacteria and yeast. Thus this study is considered to sup- port sustainable development of green synthesis using the green microalgae of Desmodesmus sp. ACKNOWLEDGEMENTS The author are thank ful to Associate profes- sor İlknur Dağ, Dr. Bükay Yenice Gürsu, Dr. Yeşim Dağlıoğlu and Phd Tay fun Şengel from Eskişehir Osmangazi University for their valuable discussions, advices, and shares on their expertise related to the sub- ject. REFERENCES Annamalai J and Nallamuthu T. 2015. Characterization of biosynthesized gold nanoparticles from aqueous extract of Chlorella vulgaris and their anti-pathogenic properties.  Applied Nanoscience,  5(5), 603-607. DOI 10.1007/s13204-014-0353-y Barwal I, Ranjan P, Kateriya S, Yadav SC. 2011. Cellular oxido-reductive proteins of Chlamydomonas rein- hardtii control the biosynthesis of silver nanopar- ticles.  Journal of nanobiotechnology,  9(1), 56. DOI 10.1186/1477-3155-9-56 Boroumand Moghaddam A, Namvar F, Moniri M, Md Tahir P, Azizi S, Mohamad R. 2015. Nanoparti- cles biosynthesized by fungi and yeast: a review of their preparation, properties, and medical applica- tions.  Molecules,  20(9), 16540-16565. DOI 10.3390/ molecules200916540 Cantero G, Correa-Fiz F, Ronco T, Strube M, Cerdà- Cuéllar M, Pedersen K. 2018. Characterization of Campylobacter jejuni and Campylobacter coli Broiler 41Intracellular and extracellular green synthesis of silver nanoparticles using Desmodesmus sp. Isolates by Whole-Genome Sequencing.  Foodborne pathogens and disease,  15(3), 145-152. DOI 10.1089/ fpd.2017.2325 Chakraborty N, Pal R, Ramaswami A, Nayak D, Lahi- ri S. 2006. Diatom: a potential bio-accumulator of gold.  Journal of radioanalytical and nuclear chemis- try, 270(3), 645-649. DOI 10.1007/s10967-006-0475-0 Dağlıoğlu Y, Çelebi SM, Önalan Ş. 2016. Determination of acute toxic effects of poly (Vinylferrocenium) sup- ported palladium nanoparticle (Pd/PVF+) on Arte- mia salina.   Pakistan Journal of Zoology, 48(1), 187– 193. Dağlıoğlu Y, Kabakcı D, Akdeniz G, Çelebi MS. 2016. Determining the acute toxic effects of poly(vinylferrocenium) supported platinum nanopar- ticle (PT/PVF+ NPs) on Apis mellifera. Dağlıoğlu Y, Öztürk BY. 2016. The assessment of biologi- cal accumulation on exposure in boron particles of Desmodesmus multivariabilis. Biological Diversity and Conservation. 9(3), 204-209. Dağlıoğlu Y, Öztürk BY. 2018. Effect of concentration and exposure time of ZnO-TiO2  nanocomposite on pho- tosynthetic pigment contents, ROS production ability, and bioaccumulation of freshwater algae (Desmodes- mus multivariabilis). Caryologia, 71(1), 13-23, DOI 10.1080/00087114.2017.1400262 Dağlıoğlu Y, Türkiş S. 2017. Effect of nano and micro- particle boron on hydrogen peroxide (H2O2) and lipid peroxidation (MDA) enzyme activity superox- ide dismutase (SOD) of Myriophyllum spicatum. 6(2), 62-70. Dağlıoğlu Y, Türkiş S. 2017. Effect of TiO2 nanoparticles application on photosynthetic pigment contents of duckweed (Lemna minor L.). Acta Biologica Turci- ca, 30(4), 108-115. Dahoumane SA, Djediat C, Yéprémian C, Couté A, Fiévet F, Coradin T, Brayner R. 2012. Recycling and adapta- tion of Klebsormidium flaccidum microalgae for the sustained production of gold nanoparticles.  Biotech- nology and bioengineering,  109(1), 284-288. DOI doi.org/10.1002/bit.23276   Dhand V, Soumya L, Bharadwaj S, Chakra S, Bhatt D, Sreedhar B. 2016. Green synthesis of silver nanopar- ticles using Coffea arabica seed extract and its anti- bacterial activity. Materials Science and Engineering: C, 58, 36-43. DOI 10.1016/j.msec.2015.08.018 Domingos RF, Baalousha MA, Ju-Nam Y, Reid MM, Tufenkji N, Lead JR, Wilkinson, KJ. 2009. Charac- terizing manufactured nanoparticles in the environ- ment: multimethod determination of particle sizes. Environmental science & technology, 43(19), 7277- 7284. DOI 10.1021/es900249m Faulkner DJ. 2000. Marine natural products. Natural Product Reports, 17(1), 7-55. Ge L, Li Q, Wang M, Ouyang J, Li X, Xing MM. 2014. Nanosilver particles in medical applications: synthe- sis, performance, and toxicity. International journal of nanomedicine, 9, 2399. DOI 10.2147/IJN.S55015 Govindaraju K, Kiruthiga V, Kumar VG, Singaravelu G. 2009. Extracellular synthesis of silver nanoparti- cles by a marine alga, Sargassum wightii Grevilli and their antibacterial effects. Journal of Nanoscience and Nanotechnology, 9(9), 5497-5501. DOI 10.1166/ jnn.2009.1199 Hiramatsu H, Osterloh FE. 2004. A simple large-scale synthesis of nearly monodisperse gold and sil- ver nanoparticles with adjustable sizes and with exchangeable surfactants. Chemistry of Materials, 16(13), 2509-2511. DOI 10.1021/cm049532v Ilknur D, Yasemin O, Nuri K. 2012. Effect of disinfectants on biofilm development by five species of Candida. African Journal of Microbiology Research, 6(10), 2380-2386. DOI 10.5897/AJMR11.1427 Jegadeeswaran P, Shivaraj R, Venckatesh R. 2012. Green synthesis of silver nanoparticles from extract of Padi- na tetrastromatica leaf. Digest Journal of Nanomateri- als and Biostructures, 7(3), 991-998. Jena J, Pradhan N, Nayak RR, Dash BP, Sukla LB, Panda PK, Mishra BK. 2014. Microalga Scenedesmus sp.: a potential low-cost green machine for silver nanopar- ticle synthesis. Journal of Microbiology and Biotech- nology, 24(4), 522-533. DOI 10.4014/jmb.1306.06014 Joerger R, Klaus T, Granqvist CG. 2000. Biologically Produced Silver–Carbon Composite Materials for Optically Functional Thin‐Film Coatings. Advanced Materials, 12(6), 407-409. DOI 10.1002/(SICI)1521- 4095(200003)12:6<407::AID-ADMA407>3.0.CO;2-O Kannan RRR, Arumugam R, Ramya D, Manivannan K, Anantharaman P. 2013. Green synthesis of silver nanoparticles using marine macroalga Chaetomor- pha linum. Applied Nanoscience, 3(3), 229-233. DOI 10.1007/s13204-012-0125-5  Khatami M, Alijani HQ, Heli H, Sharifi I. 2018a. Rectan- gular shaped zinc oxide nanoparticles: Green synthe- sis by Stevia and its biomedical efficiency.  Ceramics International. DOI 10.1016/j.ceramint.2018.05.224 Khatami M, Sharifi I, Nobre MA, Zafarnia N, Aflatooni- an MR. 2018b. Waste-grass-mediated green synthesis of silver nanoparticles and evaluation of their anti- cancer, antifungal and antibacterial activity.  Green Chemistry Letters and Reviews,  11(2), 125-134. DOI 10.1080/17518253.2018.1444797 Khatami M, Alijani HQ, Nejad MS, Varma RS. 2018c. Core@ shell nanoparticles: greener synthesis using 42 Betül Yılmaz Öztürk natural plant products.  Applied Sciences,  8(3), 411. DOI 10.3390/app8030411 Khatami M, Alijani H, Sharifi I. 2018d. Biosynthesis of bimetallic and core shell nanoparticles: their biomed- ical applications: A review.  IET Nanobio, 1-19. DOI 10.1049/iet-nbt.2017.0308 Khatami M, Varma RS, Zafarnia N, Yaghoobi H, Sarani M, Kumar VG. 2018. Applications of green synthe- sized Ag, ZnO and Ag/ZnO nanoparticles for making clinical antimicrobial wound-healing bandages.  Sus- tainable Chemistry and Pharmacy,  10, 9-15. DOI 10.1016/j.scp.2018.08.001 Kim JS, Kuk E, Yu KN, Kim JH, Park SJ, Lee HJ, Jeong, DHo, Hwang CY. 2007. Antimicrobial effects of sil- ver nanoparticles. Nanomedicine: Nanotechnology, Biology and Medicine, 3(1), 95-101. DOI 10.1016/j. nano.2006.12.001 Kothari R, Pandey A, Ahmad S, Kumar A, Pathak VV, Tyagi V. 2017. Microalgal cultivation for value-added products: a critical enviro-economical assessment. 3 Biotech, 7(4), 243. DOI10.1007/s13205-017-0812-8 Kozma G, Rónavári A, Kónya Z, Kukovecz A. 2015. Environmentally benign synthesis methods of zero- valent iron nanoparticles. ACS Sustainable Chem- istry & Engineering, 4(1), 291-297. DOI 10.1021/ acssuschemeng.5b01185 Li L, Shi C, Yin Z, Jia R, Peng L, Kang S, Li Z. 2014. Antibacterial activity of α-terpineol may induce mor- phostructural alterations in Escherichia coli. Brazil- ian Journal of Microbiology, 45(4), 1409-1413. DOI 10.1590/S1517-83822014000400035  Luangpipat T, Beattie IR, Chisti Y, Haverkamp RG. 2011. Gold nanoparticles produced in a microalga. Journal of Nanoparticle Research, 13(12), 6439-6445. DOI doi.org/10.1007/s11051-011-0397-9 Ma X, Xu X, Xia Y, Wang Z. 2018. SERS aptasensor for Salmonella typhimurium detection based on spiny gold nanoparticles. Food Control, 84, 232-237. DOI 10.1016/j.foodcont.2017.07.016 Mahdavi M, Namvar F, Ahmad MB, Mohamad R. 2013. Green biosynthesis and characterization of magnetic iron oxide (Fe3O4) nanoparticles using seaweed (Sar- gassum muticum) aqueous extract. Molecules, 18(5), 5954-5964. DOI 10.3390/molecules18055954 Metuku RP, Pabba S, Burra S, Gudikandula K, Charya MS. 2014. Biosynthesis of silver nanoparticles from Schizophyllum radiatum HE 863742.1: their charac- terization and antimicrobial activity. 3 Biotech, 4(3), 227-234. DOI 10.1007/s13205-013-0138-0 Mohseniazar M, Barin M, Zarredar H, Alizadeh S, Shane- hbandi D. 2011. Potential of microalgae and Lacto- bacilli in biosynthesis of silver nanoparticles. BioIm- pacts: BI, 1(3), 149. DOI 10.5681/bi.2011.020 Namvar F, Mohamed S, Fard SG, Behravan J, Mustapha NM, Alitheen NBM, Othman F. 2012. Polyphenol- rich seaweed (Eucheuma cottonii) extract suppresses breast tumour via hormone modulation and apopto- sis induction. Food chemistry, 130(2), 376-382. DOI 10.1016/j.foodchem.2011.07.054 Nithya R, Ragunathan R. 2009. Synthesis of silver nano- particle using Pleurotus sajor caju and its antimicro- bial study. Digest Journal of Nanomaterials and Bio- structures, 4(4), 623-629. Özkan Y, İrende İ, Akdeniz G, Kabakçı D, Sökmen M. 2015. Evaluation of the comparative acute toxic effects of TiO2, Ag-TiO2 and ZnO-TiO2 composite nanoparticles on Apis mellifera (Honey Bee). J. Int. Environmental Application & Science, 10 (1), 26–36. Öztürk BY, Dağlıoğlu Y. 2018. ZnO-TiO2 nanocomposite in Chodatodesmus mucranulatus. Fresenius Environ- mental Bulletin, 27(5), 2951-2962. Poulose S, Panda T, Nair PP, Theodore T. 2014. Biosyn- thesis of silver nanoparticles. Journal of Nanosci- ence and Nanotechnology, 14(2), 2038-2049. DOI 10.1166/jnn.2014.9019 Pradeep T. 2009. Noble metal nanoparticles for water purification: a critical review. Thin solid films, 517(24), 6441-6478. DOI 10.1016/j.tsf.2009.03.195 Prasad TN, Kambala VSR, Naidu R. 2013. Phyconano- technology: synthesis of silver nanoparticles using brown marine algae Cystophora moniliformis and their characterisation. Journal of applied phycology, 25(1), 177-182. DOI 10.1007/s10811-012-9851-z Rajeshkumar S, Malarkodi C, Paulkumar K, Vanaja M, Gnanajobitha G, Annadurai G. 2014. Algae mediated green fabrication of silver nanoparticles and exami- nation of its antifungal activity against clinical path- ogens. International Journal of Metals, 2014. DOI 10.1155/2014/692643 Rao YS, Kotakadi VS, Prasad T, Reddy A, Gopal DS. 2013. Green synthesis and spectral characterization of silver nanoparticles from Lakshmi tulasi (Ocimum sanctum) leaf extract. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 103, 156- 159. DOI 10.1016/j.saa.2012.11.028 Rippka R. 1988. [1] Isolation and purification of cyano- bacteria. In Methods in Enzymology, Academic Press, 167, 3-27. DOI 10.1016/0076-6879(88)67004-2 Rónavári A, Kovács D, Igaz N, Vágvölgyi C, Boros IM, Kónya Z, Pfeiffer I, Kiricsi M. 2017. Biological activ- ity of green-synthesized silver nanoparticles depends on the applied natural extracts: a comprehensive study. International journal of nanomedicine, 12, 871. DOI 10.2147/IJN.S122842 43Intracellular and extracellular green synthesis of silver nanoparticles using Desmodesmus sp. Salari Z, Danafar F, Dabaghi S, Ataei SA. 2016. Sustain- able synthesis of silver nanoparticles using macroal- gae Spirogyra varians and analysis of their antibacte- rial activity. Journal of Saudi Chemical Society, 20(4), 459-464. DOI 10.1016/j.jscs.2014.10.004 Shao Y, Jin Y, Dong S. 2004. Synthesis of gold nanoplates by aspartate reduction of gold chloride. Chemi- cal Communications (9), 1104-1105. DOI 10.1039/ B315732F Sharma P, Pant S, Rai S, Yadav RB, Sharma S, Dave V. 2018. Green synthesis and characterization of sil- ver nanoparticles by Allium cepa L. to produce silver nano‐coated fabric and their antimicrobial evalua- tion. Applied Organometallic Chemistry, 32(3). DOI 10.1002/aoc.4146 Sharma VK, Yngard RA, Lin Y. 2009. Silver nanoparticles: green synthesis and their antimicrobial activities. Advances in colloid and interface science, 145(1), 83-96. DOI 10.1016/j.cis.2008.09.002 Singh CR, Kathiresan K, Anandhan S. 2015. A review on marine based nanoparticles and their potential appli- cations. African Journal of Biotechnology, 14(18), 1525-1532. DOI 10.5897/AJB2015.14527 Singhal G, Bhavesh R, Kasariya K, Sharma AR, Singh RP. 2011. Biosynthesis of silver nanoparticles using Ocimum sanctum (Tulsi) leaf extract and screen- ing its antimicrobial activity. Journal of Nanoparticle Research, 13(7), 2981-2988. DOI 10.1007/s11051- 010-0193-y Sobieh SS, Kheiralla ZMH, Rushdy AA, Yakob NAN. 2016. In vitro and in vivo genotoxicity and molecular response of silver nanoparticles on different biologi- cal model systems. Caryologia, 69(2), 147-161. DOI 10.1080/00087114.2016.1139416 Soldini S, Posteraro B, Vella A, De Carolis E, Borghi E, Falleni M, Losito AR, Maiuro G, Trecarichi EM, Sanguinetti, M, Tumbarello M. (2017). Microbio- logic and clinical characteristics of biofilm-form- ing Candida parapsilosis isolates associated with fungaemia and their impact on mortality. Clini- cal Microbiology and Infection. DOI 10.1016/j. cmi.2017.11.005 Sondi I, Siiman O, Koester S, Matijević E. 2000. Prepara- tion of aminodextran− CdS nanoparticle complexes and biologically active antibody− aminodextran− CdS nanoparticle conjugates. Langmuir, 16(7), 3107- 3118. DOI 10.1021/la991109r Suriya J, Raja SB, Sekar V, Rajasekaran R. 2012. Biosyn- thesis of silver nanoparticles and its antibacterial activity using seaweed Urospora sp. African Journal of Biotechnology, 11(58), 12192-12198. DOI 10.5897/ AJB12.452 Tippayawat P, Phromviyo N, Boueroy P, Chompoosor A. 2016. Green synthesis of silver nanoparticles in Aloe vera plant extract prepared by a hydrothermal meth- od and their synergistic antibacterial activity. PeerJ, 4, e2589. DOI 10.7717/peerj.2589 Travan A, Pelillo C, Donati I, Marsich E, Benincasa M, Scarpa T, Semeraro S, Turco G, Gennaro R, Paoletti, S. 2009. Non-cytotoxic silver nanoparticle-polysac- charide nanocomposites with antimicrobial activity. Biomacromolecules, 10(6), 1429-1435. DOI 10.1021/ bm900039x Vigneshwaran N, Nachane R, Balasubramanya R, Vara- darajan P. 2006. A novel one-pot ‘green’synthesis of stable silver nanoparticles using soluble starch. Carbohydrate research, 341(12), 2012-2018. DOI 10.1016/j.carres.2006.04.042 Vivek R, Thangam R, Muthuchelian K, Gunasekaran P, Kaveri K, Kannan S. 2012. Green biosynthesis of sil- ver nanoparticles from Annona squamosa leaf extract and its in vitro cytotoxic effect on MCF-7 cells. Pro- cess Biochemistry, 47(12), 2405-2410. DOI 10.1016/j. procbio.2012.09.025 Wang J, Chen C. 2009. Biosorbents for heavy metals removal and their future. Biotechnology advances, 27(2), 195-226. DOI 10.1016/j.biotechadv.2008.11.002 Zgoda J, Porter J. 2001. A convenient microdilution method for screening natural products against bac- teria and fungi. Pharmaceutical Biology, 39(3), 221- 225. DOI 10.1076/phbi.39.3.221.5934 Zhang Y, Liu X, Wang Y, Jiang P, Quek S. 2016. Antibac- terial activity and mechanism of cinnamon essential oil against Escherichia coli and Staphylococcus aureus. Food Control, 59, 282-289. DOI 10.1016/j.food- cont.2015.05.032 Zuercher AW, Fritsche R, Corthésy B, Mercenier A. 2006. Food products and allergy development, prevention and treatment. Current opinion in biotechnology, 17(2), 198-203. DOI 10.1016/j.copbio.2006.01.010 Substantia An International Journal of the History of Chemistry Vol. 2, n. 1 - March 2018 Firenze University Press