BIOSYNTHESIS OF SILVER NANO PARTICLES BY USING THE AGROBACTERIUM AND RHIZOBIUM AND THE EFFECT ON ANTI- BACTERIAL ACTIVITY

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1 BIOSYNTHESIS OF SILVER NANO PARTICLES BY USING THE AGROBACTERIUM AND RHIZOBIUM AND THE EFFECT ON ANTI- BACTERIAL ACTIVITY Rajkumar.G * and Tamizharasi.P Department of Botany, Government Arts College, Thiruvannamalai , Tamilnadu. * corresponding author; Mail: Gv.rajjkumar@yahoo.com ABSTRACT Nanobiotechnology was foreseen to significantly influence science, economy and everyday life in the 21 st century and also one of the driving forces of the next industrial revolution. Nanotechnology entails the tailoring of materials at atomic levels to acquire unique properties for the desired applications. The Nanomaterials of Silver, Copper, Zinc and Gold prepared by using various chemical and biological materials being used industrially for several applications including amendments to textiles, cosmetics, medicines, pharmaceuticals, food, chemical sensing, environmental cleaning, sprays, plastics and paints. The development of reliable processes for the synthesis of silver nanoparticles was an important aspect of nanotechnology today. Among the methods like physical, chemical and biological processes involved in synthesis of nanomaterials, biological methods were currently gaining importance and reliable because they are ecofriendly, cost effective and not involve any toxic materials for synthesis. The biological methods involve with the applications of plant compounds and microbial preparations used in synthesis of silver nanoparticles preparation. Among the microbial organisms (bacteria, fungi, algae and yeast), bacteria are considered as manufactories as they play vital role in remediation of toxic metals through reduction of the metal ions. Therefore, the present work was focused on the synthesis of silver nanoparticles using the extracellular and cell biomass preparations of microorganisms especially bacteria include Rhizobium, Agrobacterium and Bacillus species using silver nitrate solution. KEY WORDS: Silver Nano-particles, Anti bacterial activity, Rhizobium, Agrobacterium. 1.INTRODUCTION Nano-biotechnology has attracted global attention because the nano-particles (NPs) have properties unique from their bulk equivalents. Nanopmiicles of Silver, Copper oxide, Zinc oxide and Gold prepared by using various biological materials like plant compounds and microbial biomass being used industrially for several purposes including amendments to textiles, cosmetics, medicines, pharmaceuticals, food, chemical sensing, environmental cleaning, sprays, plastics and paints. Among the noble metals like Silver, platinum, gold etc., Silver (A g) is the metal of choice of preparation of NPs and has potential applications in the field of Biological systems. Synthesis of NPs from the above metals has reported using chem.- ical, physical and biological methods. Among the methods involved, biological methods are currently gaining importance because they are eco-friendly, cost effect-tive, and don t involve the use of any toxic chemicals for the synthesis of NPs. The biosynthesis of silver and gold NPs has been performed earlier using microorganisms primarily bacteria and eukaryotic organisms such as fungi and higher plants. There 1

2 have been several reports on the synthesis of Silver NPs using different species of bacteria such as Bacillus, Lactobacillus, Proteus, E.coli, Streptomyces,Klebsiella, rynenobacterium, Geobacter, Brevibacterium, Sinorhizobium, Pseudomonas sp. for various applications including pharmaceutical and biological process. A green synthesis of nanosilver particles using a various solvent extracts of many microorganisms including bacteria and fungi were well reported and studied for its various properties including the presence of antimicrobial compounds. One of the common features of these NPs is their antimicrobial activity against wide variety of microorganisms including pathogenic bac-teria and fungi. The antimicrobial activity of NPs largely has been studied with human pathogenic bacteria, mainly Escherichia coli and Staphylococcus aureus and pro-ved effective. Nano-silver is inl;1ibitory to E. coli and S. aureus. Recent studies have shown that specially formulated Silver NPs (Ag-NPs) have good antibacterial activity and the bacteria usually are incapable of developing resistance against Ag-- NPs, because these NPs can at the same time attack a broad range of target molecules in microorganisms such as pro-teins with thiol groups, cell walls and cell membranes. A recent study on Escherichia coli has shown that Ag-NPs react with cell walls and cytoplasmic membranes, resulting in pits in the cell wall of bacteria, and finally killing them. How-ever, there is no significant study found from the literature about study on effect of NPs against microbial plant pathogens mainly surviving in soil which include bacteria and fungi. The bacteria such as Pseudomonas, Xanthomonas, and the fungi such as Alternaria, Fusarium, Cladosporium, Verticillium, Pyricularia, Helminthosporium sp. were reported to be sukrivive in soil and influence the diseases in plants including crop plants. Therefore, in the present project, it is proposed to study the biosynthesis and characterization of silver NPs by using Rhizobium sp. and Agrobacterium sp. by a biosynthesis method which is ecofriendly and cost effective. The synthesized NPs shall be confinned and identified by using chemical reaction method, UV spectroscopy, IR spectrophotometer and XRD. Secondly, the antibacterial and antifungal activity of the synthesized silver NPs by using the above bacteria against the selected species of microbial plant pathogen in the laboratory. The antimicrobial and antifungal testing techniques including agar well diffusion, agar disc diffusion and tube dilution method. The Minimum Inhibitory Concentration (MIC) test for bacterial cells and fungal spore germination also has to be done. 2. MATERIALS AND METHODS: Chemicals, Glassware and Reagents The chemicals and reagents used in the present were belonging to laboratory and analytical grade purchased from licensed scientific companies. The following chemicals and reagents are used in the present study. 1. Silver nitrate 2. Potassium dichromate 3. Sulphuric acid 4. Antibiotic discs 5. Agar agar 6. Yeast extract 7. Beaf extract 8. Bacterial peptone 9. Dextrose The glasswares like conical flask, beakers, test tubes, boiling tubes, measuring jars, pippetes etc. used in the present study were belong to Borosil.The reagents were prepared from the respective chemicals by using double distilled water prepared in the laboratory. Cleaning of glassware The glass wares such as conical flasks, beakers, test tubes, measuring jars, pippetes 2

3 etc. were first soaked in chromic acid solution, (10% potassium dichromate in 25% sulphuric acid) for a few hours to remove tough residues. Then it were washed twice in tap water and rinsed with distilled water. After draining the water completely from the glassware, it was dried in an drying chamber at 80 o C. The glassware was cooled before it being taken for further use in the experiments. Sterilization Sterilization of culture media and glassware were carried out in an autoclave at 121 o C, 15 psi for 20 minutes. Thermo labile substances are sterilized through Millipore filter. All the experiments were conducted under laminar hood with strict aseptic conditions. However, the glasswares are also sterilized by using hot air oven at 120 o C for 3 hrs period. Chemicals and plant samples Silver nitrate solution purchased from Hi Media Laboratories Pvt. Limited, Mumbai, India. The Leaves of Aegle marmelos were collected from the forest region of Thiruvannamalai hills. METHODOLOGY Preparation of aqueous silver nitrate For the preparation of silver nitrate reagent solution, 1 mm AgNO3 solution was prepared by dissolving 3.8 g of Silver nitrate in 1000 ml of double distilled water. The silver nitrate solution was filtered to remove any debris and the clear reagent is stored in amber colour bottle in the laboratory for further use. Preparation of plant extract by conventional method: The Aegle marmelos (Vilvam) and Azadirachta indica (Neem) leaves were washed several times with tap water and finally rinsed by using deionised water. About 100gm of finely cut Aegle marmelos and Azadirachta indica leaves were taken and boiled in 300 ml of double distilled water for 3min. The extracts were collected and filtered through muslin cloth to remove any debris present. Then the extracts were centrifuged at 10,000 rpm for 15mins. And the supernatants were collected and store at 4 C for further experiments. Preparation of plant extracts by homogenization method: The Aegle marmelos (Vilvam) and Azadirachta indica (Neam) leaves were washed several times with tap water and finally rinsed by using deionised water. About 100gm of finely cut Aegle marmelos and Azadirachta indica leaves were taken and homogenized with 300 ml water with the help of mortar and pestle. The extracts were collected and filtered through muslin cloth to obtain clear extract. Then the filtrate was centrifuged for 15 min at 10,000 rpm and supernatant was collected to store at 4 C for further experiments. Optimization and synthesis of silver nanoparticles Synthesis of silver nanoparticles under Sunlight irradiation For synthesis of nanoparticles from plant extract, 1 mm AgNO3 solution was used. First, different concentration of leaf extracts with silver nitrate solution was prepared by taking 1ml, 3ml and 5ml of plant extracts in a conical flask separately and to this 10 ml of 1 mm AgNO3 solution was added with constant stirring. The stirred solution was exposed under sunlight radiation and observed the colour change. The colour change of the solution was checked periodically and the bioreduction of silver ions in the solution was monitored by measuring UV-Vis absorption spectrum by using ELICO UV-VIS Spectrophotometer. Then the conical flasks containing reaction 3

4 mixture was incubated at room temperature for 48 hours. The colour change of the leaf extract from yellow to dark brown indicated the synthesis of silver nanoparticles from the extracts of leaves. Then the contents of reaction mixtures were centrifuged at 10,000 rpm for 15 minutes. The pellet was used for the characterization of the silver nanoparticles. The same was performed for both conventional and homogenized method of extracts. Synthesis of silver nanoparticles under UV irradiation For synthesis of nanoparticles from plant extract, 1 mm AgNO3 solution was used. First, different concentration of leaf extracts with silver nitrate solution was prepared by taking 1ml, 3ml and 5ml of plant extracts in a conical flask separately and to this 10 ml of 1 mm AgNO3 solution was added with constant stirring. The stirred solution was exposed under UV irradiation and observed the colour change. The colour change of the solution was checked periodically and the bioreduction of silver ions in the solution was monitored by measuring UV-Vis absorption spectrum by using ELICO UV-VIS Spectrophotometer. Then the conical flasks containing reaction mixture was incubated at room temperature for 48 hours. The colour change of the leaf extract from yellow to dark brown indicated the synthesis of silver nanoparticles from the extracts of leaves. Then the contents of reaction mixtures were centrifuged at 10,000 rpm for 15 minutes. The pellet was used for the characterization of the silver nanoparticles. The same was performed for both conventional and homogenized method of extracts. Synthesis of silver nanoparticles under Direct Boiling For synthesis of nanoparticles from plant extract, 1 mm AgNO3 solution was used. First, different concentration of leaf extracts with silver nitrate solution was prepared by taking 1ml, 3ml and 5ml of plant extracts in a conical flask separately and to this 10 ml of 1 mm AgNO3 solution was added with constant stirring. The stirred solution was undergone direct boiling and observed the colour change. The colour change of the solution was checked periodically and the bioreduction of silver ions in the solution was monitored by measuring UV-Vis absorption spectrum by using ELICO UV-VIS Spectrophotometer. Then the conical flasks containing reaction mixture was incubated at room temperature for 48 hours. The colour change of the leaf extract from yellow to dark brown indicated the synthesis of silver nanoparticles from the extracts of leaves. Then the contents of reaction mixtures were centrifuged at 10,000 rpm for 15 minutes. The pellet was used for the characterization of the silver nanoparticles. The same was performed for both conventional and homogenized method of extracts. Production and Recovery of silver nanoparticles by centrifugation: Among various concentration and methods used, sunlight irradiation method was very effective and 1ml of homogenized leaf extract was shown more synthesis of nano particles. Further it was chosen for bulk production as 10ml leaf extract in 100 ml of 1mM AgNO3. After bioreduction, the solution consisting of hydrosols of silver nanoparticles was subjected to centrifugation at 10,000 rpm for 15 minutes, and the supernatant was discarded. The pellet formed was dissolved in 0.1 ml of toluene water and air dried. CHARACTERIZATION OF SILVER NANOPARTICLES: UV-visible spectroscopy: The work described in this thesis, UV-visible spectroscopy was used for monitoring the signature of silver nanoparticles. UV- 4

5 visi-ble spectroscopy is a powerful tool for the characterization of colloidal Particles. In particle, noble metal particles are ideal candidates for study with UV-Vis spectroscopy, since they exhibit strong surface plasmon resonance absorption in the visible region and were highly sensitive to the surface modification. Procedure for sampling: The electron gun usually consists of a tungsten wire filament, which was bent into a hairpin ("V") shape and surrounded by a shield with a circular aperture (1-3 mm diameter) centered just below the filament tip. Electrons in the gun were accelerated across a potential difference of the order of 100,000 volts between the cathode (at high Negative potential) and anode (at ground potential). The function of the condenser lens was to focus the electron beam emerging from the electron gun onto the specimen to permit optimal illuminating conditions for visualizing and recording the image. The optical enlarging system of an electron microscope consists of an objective lens followed by one or more projector lenses. The objective lens determines resolution and contrast in the image, and all subsequent lenses bring the final image to a convenient magnification for observation and recording. The objective lens is most critical lens since it determines the resolving power of the instrument and performs the first stage of imaging. The specimen image generated by the objective lens was subsequently magnified in one or two more magnification stages by the intermediate and projector lens and projected onto a fluorescent screen or photographic plate. XRD Measurement The air dried nanoparticles were coated onto XRD grid and analysed for the formation of Ag nanoparticle by Philips X-Ray Diffractometer with Philips PW 1830 X- Ray Generator operated at a voltage of 40kV and a current of 30mA with Cu Kal radiation. The diffracted intensities were recorded from 10' to 80' of 20 angles. FTIR Analysis The bioreduced silver nitrate solution was centrifuged at 10,000 rpm for 15 min and the dried samples were grinded with KBr pellets used for FTIR measurements. The spectrum was recorded in the range of cm-1 using Thermo Nicolet Nexus 670 spectrometer in the diffuse reflectance mode operating at resolution of 4 cm RESULTS Formation of silver nanoparticles and visible observation When the plant extracts of both Aegle marmelos and Azadirachta indica of boiled solutions were involved in the formation of silver nanoparticles formation after few mins sunlight irradiation and kept in room atmospheric condition. The colour of the reaction mixture indicates in brown to ruby red colour after the presence of formation silver nanoparticles. The appearance of the reaction solution with control sample without addition of plant extract was appear in yellow colour. UV-Visible spectroscopy The reaction mixture contained silver nanoparticles formation during reaction appeared in brown to ruby red showed an absorption spectrum at 390 nm to 440 nm which is shown in Figure: 5.The maximum absorption on both Aegle marmelos and Azadirachta indica show from 400 nm to 440 nm which indicates the formation of silver particles in brown to ruby red colour. This was a typical absorption band of spherical Ag nanoparticles due to their surface plasmon. The presence broad absorption spectrum otherwise called resonance indicates the aggregation of the silver nanoparticles in the solution. The maximum absorption value (OD) of 2.64 shows 5

6 at 420 nm in the UV Visible absorption region. Fig. 5. Appearance of silver nanoparticles formation during the reaction The colour of solution before formation of silver nanoparticles appear yellow and after formation of SNPs it is appear in Ruby red colour. The spread of SNPs in petriplates Series2 Series nm wave length Fig. 6. UV-Visible Absorption spectrum of Silver nanoparticles in thereaction mixture Transmission Electron Microscopy (TEM) The dried silver nanoparticles prepared from both Aegle marmelos and Azadirachta indica after centrifugation were taken for the preparation of Transmission Electron Microscopic (TEM) analysis. A TEM image of Ag nanoparticles dispersed on a TEM copper grid (a, scale bar: 30 nm). The TEM picture shows an individual silver particles as well as a group of crystals in clusters of aggregates which was represented by the TEM micrograph (Fig. 6).The morphology of silver 6

7 nanoparticles were slightly variable from spherical to triangular in shape in the photomicrograph. Under these observation, these nanoparticles were found to be in the size range of 20 to 50 nm. Fig. 7 TEM Images of silver nanoparticles prepared from Aegle marmelos 7

8 Fig. 8 TEM Images of silver nanoparticles prepared from Azadirachta indica. X-Ray Diffraction Measurement The dried silver nanoparticles prepared from both Aegle marmelos and Azadirachta indica were coated onto XRD grid and analysed for the formation of Ag nanoparticle by Philips X-Ray Diffractometer with Philips PW 1830 X- Ray Generator operated at a voltage of 40 kv and a current of 30mA with Cu Kal radiation. The diffracted intensities were recorded from 10' to 80' of 20 angles. Fig. 9. XRD pattern from drop-coated films of synthesized silver nanoparticles. Aegle marmelos 8

9 Fig. 10. XRD pattern from drop-coated films of synthesized silver nanoparticles. Azadirachta indica. 9

10 Fig. 11 Absorption spectrum of FTIR analysis of Silver nanoparticles from Rhizobium Antibacterial activity Well diffusion method On growth inhibition test of silver nanoparticles against Escherisia coli, Proterus vulgaris and Vibrio cholearae were tested on well diffusion method. Silver nanoparticles, antibiotics and Silver nanoparticles with antibiotics were prepared and the effect was studied. The silver nanoparticles alone prepared having inhibition effect on E. coli and Vibrio cholerrae and there was no inhibition on Proteus vulgaris. However, the antibiotic mixed with silver nanoparticles showed moderate inhibition on all the above 3 organisms. This indicates that the silver nanoparticles having inhibitory effect on both E.coli and Vibrio cholera at the concentration of 50 µl in the well.theexperimental results shown in the Fig

11 Fig. 12 The antibacterial effect of silver nanoparticles by well diffusion method A-SNPs, B-Antibiotic, C-SNPs+Antibiotic A- Escherichia coli B- Proreus vulgaris C- Vibrio cholerae Disc diffusion method Silver nanoparticles synthesized using Aegle marmelos and Azadirachta indica extract of were tested for its potential antibacterial activity against pathogenic and nonpathogenic bacteria as staphylococcus aureus, klebsella and salmonella typhi and Bacillus licheniformis were used as the test organisms. The results indicates that the silver nanoparticles impregnated discs showing nil to very mild inhibitory effect on all the 4 species of bacteria used in the present study. However, the silver nanoparticles mixed with antibiotics showed no significant difference in the zone of inhibition area which may be due to less concentration. Fig. 13 Antibacterial effect of silver ananoparticles by using disc diffusion assay method a - Control, b - SNPs, c - Antibiotic, d - SNPs+Antibiotic A- Escherichia coli, B- Pseudomonas spc- Vibrio cholera, D- Bacillus licheniformis 11

12 ANTIBACTERIAL ACTIVITY: Antibacterial activity of silver nitrate and silver nanoparticles against human pathogenic bacteria: Silver nanoparticles synthesized using Azadirachta indica extract of were tested for its potential antibacterial activity against few human pathogens staphylo-coccus aureus, klebsella and salmonella typhi were used as the test organisms. Well diffusion assay method: Well diffusion assay method was followed, which involves swabbing the cultures in pre-sterilized nutrient agar plates and four wells cut in the same using sterile cork borer. Each well was loaded with 50~tl of the solutions in the following order: water as negative control, solution of silver nanoparticles, silver nitrate solution and liquid filtrate obtained.then the plates were incubated at 370C for 24hrs and observed for the formation of Zone of Inhibition. DISC DIFFUSION METHOD The paper disc (No.1 Whatmann) was cut downed into small disc (6mm diameter)- and sterilized at 180 o C/30 m in hot air oven impregnated with the test solution and the standard solution. The dried discs were placed on the surface of the medium. The dishes were left standing for 1-4 hrs, at room temperature as a period of preincubation solutions Subsequently diffusion to minimize the effects of variation in time between the application of different incubated for about 18 hrs at about 37 o C and the diameter the circular inhibition zones were measured. DISCUSSION AND CONCLUSION There have been several reports on the synthesis of Silver NPs using different species of bacteria such as Bacillus, Lactobacillus,Proteus,E.coli,streptomyces, Thermomonaspora,Klebsiella, Corynenobacterium, Brevibacterium, Sinorhizobium, Pseudomonas sp. for various applications including pharmaceutical and biological process. A green synthesis of nanosilver particles using a various solvent extracts of many microorganisms including bacteria and fungi were well reported and studied for its various properties including the presence of antimicrobial compounds. It is almost similar to the present study and there is no report of synthesis of silver nanoparticles by using Rhizobium sp. and Agrobacterium sp., hence the present study was designed. An important area of research in nanotechnology is the biosynthesis of nanoparticles such as nanosilver and nanogold particles. Biologically synthesized silver nanoparticles could have many applications such as spectrally selective coatings for solar energy absorption and intercalation material for electrical batteries. The nanotechnology industry is growing at an incredible rate. Yet very little is known about the environmental impact of these tiny particles. Nano particles are particles measured on the nano scale a nano meter is one billionth of a meter elemental properties become very different on the nanoscale. Because of their large surface area compared to their volume. Those different properties are what make nanoparticles so useful. Another reason for nanoparticles are so useful is because they can be placed into fabrics, Plastics and other materials much easier than other larger particles. The nano technology industry is growing faster than the government can regulate it. This is mainly because the different agencies are not sure under whose domain it falls. There is no exact regulation on them and it is just assumed they fall under regulations already in place (Heing Robin Marantz, 2007). Most of the techniques are capital intensive as well as inefficient in materials and energy use. Hence there is an ever 12

13 growing need to develop clean non-toxic and environmentally synthetic procedures. Consequently researchers have used biological synthesis. Since this technique provides particles with good control over the size distribution. The main reason for this may be that the processes devised by nature for the synthesis of inorganic materials on nano and micro scales have contributed to the development of a relatively new and largely un explored area of research based on the use of microbes in the biosynthesis of nano materials (Mandal et al., 2006). The metabolic activity of microorganism can lead to precipitation of nanoparticles in external environment of a cell.the fungi being extremely good candidate for such processes.the extra cellular synthesis of silver and gold nano particles by the fungus colletotricheem sp (Mandal et al., 2006). The extra cellular production of metal nanoparticles by several strains of the fungus Fusarium oxysporum has been described by (Duran et al., 2005). Some bacteria reduce Ferridoxides by producing and secreting small diffusible redox compounds that can serve as electron shuttle between the mirobe and the insoluble iron substrate (Newman, 2000). This extracellular enzyme shows an excellent redox properties and it can act as an electron shuttle in the metal reduc-tion. It was evident that electron shuttles or other reducing agents (e.g. hydroquinone) released by microorganisms are capable of reducing ions to nanoparticles. (Basker, 1998). Among the bacterial species used in the present study for the synthesis of silver nanoparticles such as Rhizobium, Agrobacterium and Bacillus, Rhizobium and Bacillus show good and moderate level of synthesis of silver nanoparticles respectively. The report on synthesis of silver nanoparticles from Bacillus sp. isolated from air sample was demonstrated by Harfenist SA., et al., 2002 and confirmed the particles by using TEM and XRD analysis. Both the extracellular extracts and bacterial cell biomass from both the species were analysed, the extracellular extract supported the synthesis of nanoparticles which confirms that the extracelluar compounds including enzymes and secondary metabolites having the property of bioreduction reactions with inorganic salts. There have many reports on reduction of metals by bacteria otherwise called is biomining properties. The Microorganisms such as bacteria yeast and new fungai play an important role in remediation of toxic metals through reduction of the metal ions. This was considered interesting as nano factories very recently (Jain.D et al.,2009 ) using this dissimilatory properties of fungi may be used to grow nanoparticles of gold and silver (Mukharjee et al., 2001) is extracellularly in Vertieillium fungal cells (Kannan Natarajan et al., 2010). The Kirby-Bauer method was originally standardized for test of antimicrobial studies. This method is well documented and standard zones of inhibition have been determined for susceptible and resistant values (Wilson et al., 2007). The multi drug resistant gram positive and gram negative bacteria strains. The zone of inhibition seems extremely good showing a relatively large zone of inhibition in both gram positive and gram negative bacterial strains. The Uv spectrum of the reaction mixture during the synthesis of silver nanoparticles show the absorption of wavelength between 400 nm to 500 nm in the present study similar to many analysis reports made by many authors (Pugazhenthiran et al., 2009; Kumar & Sastry, 2001; Sadowski et al., 2008). Similarly the SEM images demonstrate the appearance of silver particles in clusters to individuals in the shape of spherical to irregular and larger size may be due to the 13

14 reaction conditions are not quite sufficient to get finer particles. Therefore, the reaction condition may be optimized and standardization of procedure must required for obtaining uniform silver particles for the application suit various purposes including medical and cosmetics. Therefore, it is concluded from the present study as Rhizobium and Bacillus can be used for the synthesis of silver nanoparticles by using the extracellular extracts rather than the cell biomass. It was confirmed that from the UV-Visible absorption spectrum, SEM analysis, XRD and FTIR analysis for the presence of silver nanoparticles formed from the reaction mixture with silver nitrate solution using extracellular bacterial extract. By optimizing the process and standardize the methods, uniformed size smaller nanoparticles can be synthesized to suit various applications. REFERENCES 1. Heing Robin Marantz Our silver coated future. Retrive December from on earth magazine from NRDC < (2007) Mandal D., Balander, ME, Mukhopadhy, P. Sarkar G. Mukharjee P.The use of micro organism for the formation of metal nanoparticles and their application appl. Microbial Biotechnology (2006). 69, Doi : /Os Mandal. D.Bolander M.E. Mukhopadhay D.Sarlear G. Mukdharjee P.Appl. Microbiol Biotechnology (2006), Duran. N., Marcoto D.P. Alverl.O. Pe So 439 I.H.G. exposito E.J. Nan Biotechnology (2005), Newman PK and Kolter R.Nature (2000), 405, Basker R.A., and Tatum JH. J Fermentbioeng (1998), 85, Jain.D, kumar Daimar.H,kachhnaha.s., Kothari.S.L., sept: synthesis of plant - ediated silver nanoparticles using papays fruit extract and evaluation of their miuobial activities (2009) vol.4 no:3 page Mukharjee, P, Ahmad, A., Mandal D, Senapadhy, 8, Sarkar, S.R., Khan, MI Ramani, R, Parishca, R, Ajay Kumar, D.V. Alam, M. Sastry, M. Kumar, R. Bioreduction of Aucl4 ions by the fungur. Verticillium SP and surface tropping of the gold nanoparticles formed. Agnelo. Chem. Int(2001).Ed Kannan Natarajan, subbalaxmi selvaraj, Ramachandra murty.v. Microbial Production silver nanoparticles. Degest Journal of Nanomaterials and biostructures (2010) vol.5 No.1 P: Sadowski.Z, maliszewska.i.h., Grochowalska.B., Polowczyk.I., Kozlecki.T., synthesis of silver nanoparticles using microorganism. Materials science Poland(2008)., Vol. 26. No: 2 P.n :