EXTRACTION OF INTRACELLULARLY SYNTHESISED SILVER NANOPARTICLES USING FUNGI VERTICILLIUM TERRESTRE. By: Author: Mr. Imran Latif Patel, (T. Y. B. Sc. Biotechnology) Co-Author: Prof. Mrs. Ragini Deshpande. (M. Phil, Biophysics) New Arts, Com. And Science college, Ahmednagar. (University of Pune. ) ABSTRACT: Nanobiotechnolgy has become the subject of discussion at global level. Nanoparticles are proving themselves “VITAL” in various fields like biomedical sciences, magnetics, optoelectronics, nanochips production,etc. Nanoparticles are been commonly used in various drug therapies.
Uptil now, pioneers from this field have atttained success of synthesising nanoparticles from micro-organisms like fungi, bacteria, yeast, etc. Nanowires and nanotubes can be prepared by extraction of this nanoparticles from their source. This study is concerned with extraction of intracellularly produced silver nanoparticles from the fungus Verticillium terrestre . Further innovations in this nanoparticles synthesis are also included in this project.. INTRODUCTION: The study of nanomaterials(1nm=10-9m) has increased a lot due to their fundamental properties, organizations to appear as superstructures.
Nanoparticles are having many applications such as optoelectronics2,3,catalysis4,5, reprography6, single electron transistors and light emitters 7,8, non linear optical devices9,10, photoelectrochemical applications11. They are also applied in biomedical sciences, magnetics, mechanics and energy sciences12. Innovations in this nanoparticles will play an important role in various fields for upcoming century12. Various techniques involved in production of nanoparticles resemble intake of high capital as well as inefficient use of energy and material.
Hence many researchers have turned their attention for biological systems for preparation of these nanoparticles. Various types of organisms like bacteria, fungi, yeast, etc. are involved for intra or extra cellular biosynthesis of nanoparticles. E. g unicellular organisms like magnetotic bacteria produces magnetite nanoparticles13, diatoms synthesize siliceous materials 14. The above micro organisms are mainly used in bioleaching process. Multicellular organisms like geranium(plant) are also utilized for the synthesis of nanoparticles.
Even though these micro organisms involved in toxic matter remediation , they are the best employees in nanofactories15. Thus procedures for biological production of nanoparticles have welcomed all over the world. Organisms shows presence of hydrogenasae enzyme which reduces ions from the solution. Various fungi like verticillium 1, F. oxysporum16, actinomycete like thermomonospora1 are been used for the production of gold, silver1, iron16, nanoparticles. Fungus verticillium produces silver and gold nanoparticles when exposed to their respective salt solution.
Verticillium grows at 25 0C1. Nanoparticles which are synthesized intracellularly can be observed using various methods like Scanning Electron Microscope. UV-vis spectroscopy and X-ray diffraction method. Fungus verticillium when exposed to silver nitrate solution produces intracellular nanoparticles1. The appearance of distinct faint brown colour after exposure indicates the intracellular formation of silver nanoparticles12. It gives maximum absorption at 450 nm12. This resonance is clearly missing in the biomass of unexposed vertcillium and the filtrate.
Further evidence of intracellular formation is provided by SEM12. The nanoparticles produced after this procedure range between 25+12 nm. These are highly stable in form . The use of fungi is potentially increasing since they secrete large amounts of enzymes and are simpler to deal within laboratory. Application of nanoparticles needs extraction of these particles from their source. This study is concerned with extraction of silver nanoparticles produced intracellularly within Verticillium. This can be done using various techniques like treatment with detergents, enzymes, etc. followed by centrifugation.
These nanoparticles can be applied in fields like biodetections of pathogens, detection of proteins, probing of DNA, DNA engineering, tumour destruction and separation and purification of biological molecules. In future these nanoparticles will serve a great part in curing various deadly diseases like AIDS by blocking reverse transcriptase . Having known these potential of nanoparticles, this study was planned to confirm the growth conditions of the fungus Verticillium terrestre. Similarly to extract the nanoparticles synthesised and to carry out qualitative tests for the same.
MATERIALS AND METHODS: Fungus verticillium terrestre used by Dr Absar Ahmad for the synthesis of silver nanoparticles. This fungus was grown in MGYP medium in petri plates1. This inoculate was transferred to MGYP broth aseptically. Biomass was separated after 4 day incubation at shaking conditions. Then it was exposed to AgNO3 solution for 3 days. Initial pale yellow colour of the medium was changed to faint brown colour. Scanning electron microscope and UV-visible spectrophotometric readings were taken for the confirmation of the result. These reading corroborates with the reading of Dr.
Absar Ahmad1. Extraction of silver nanoparticles from the fungal cells: Fungal biomass exposed to silver nitrate solution was separated by centrifugation at 5000 rpm for 20 mins. (Remi centrifuge, Remi). The fungal biomass was treated with SDS(Sodium Dodecenyl Sulphate) for 30 mins at 370C. Chitinase and Pectinase treatment was given to the sample for 45 mins at 370C. Cellular biomass was centrifuged in cooling centrifuge(Systronics mech. ) at 15000 r. p. m at 100C for 20 mins. Supernatant was separated and optical density was measured from 400-700 nm. 21 Negative control:
Fungal biomass which was not exposed to silver nitrate was given the same treatments as above. Supernatent was collected and absorption was measured from 400-700 nm. ( spectrophotometer 106, systronics mech. )21 RESULTS : The verticillim biomass was pale yellow coloured before exposure to silver ions. Due to the completion of reaction, transformation of colour took place. This clearly indicates the presence of silver nanoparticles in the fungal cells. This was confirmed by measuring absorption of the supernatent . Maximum absorption was obtained at 410-420 nm range. Since the nanoparticles are extracted, this absorption was obtained. Graph 2) Graph2 shows the absorption spectra of unexposed biomass. So no such maximum absorption can be seen. Since no nanoparticles were produced, no absorption was seen. This indicates the presence of silver nanoparticles in extracted supernatent. CONCLUSION: Even though various nanoparticles have been synthesised using bacteria, fungi, yeast,etc, nanoparticles are synthesised intracellularly. Inthis case, Verticillium terrestre strain which was used had exhibited silver nanoparticles production capacity. This was concluded by Spectrometric readings and Scanning electron micrograph1.
Due to treatment of SDS and enzymes, nanoparticles were released in supernatent. This was observed from the maximum absorption at 410-420 nms. This absorbance was clearly lacking in for the unexposed biomass. This concludes that “SILVER NANOPARTICLES ” were extracted from Verticillium terrestre. Figure: 1 [pic] GRAPH: 1 –Absorption of treated biomass GRAPH :2-Absorption of Supernatent [pic][pic] Discussion: -When the nanoparticles are produced, new proteins are induced. These proteins can be isolated and studied by various techniques like 2D PAGE electrophoresis.
Their molecular weight can be calculated using electrophoresis and the protein can be identified. These proteins can be identified by measuring molecular weight and applying bioinformatics.. Acknowledgements: -Special thanks to the Dr. Absar Ahmad and Atul Bharde working in NCL on nanoparticles on his co-operative guidance in the project. -Special thanks to Dept. of Physics, University of Pune, Pune for providing the Scanning Electron Microscope photographs for the results. -Special thanks to my Father,my guide and my colleages for their helpful co-operation and support. Reference: . “Biosynthesis of metal nanoparticles using fungi” by Murali Sastry, Absar Ahmad, M. Islam Khan and Rajiv Kumar from “current science”, vol 85 no:2 25 july 2003. 2. Brus , L. E. , Journal of Chemistry, Physics, 1984, 80, 4403-4409 3. Wang Y & Herron N. J. Physics. chemistry(1991) , 7, 525-532 4. Schimd. G. Chemistry. Rev. , (1992), 92, 1709-1727 5. Hoffman, A J Mills. G Yee. H . M. R. J. of physics and chemistry,1992, 96, 5546-5552 6. Hamilton, J. F and Baetzold, R. C “Science”, 1979, 205, 1213-1220. 7. Klein, D L roth, R. linn. , A. K. L. , “Alivisators”, A. P and McEuen, P. L. “Nature”, 1997,389, 699-701 8. Weller, H Angew. Chemistry. , Int ED. England. , 1993,32,, 41-53 9. Wang Y. Acc. Chemistry. Res. 1991,24,133-139. 10. Yoffe, A. D. , Advance Physics. , 1993,42,173-226 11. Simkiss, Wilbur KM(1989) Biomineralisation, Academic press, New york 12. “Germanium and silver resistance, accumulation and toxicity in micro-organisms” by Slawsons, Van Dyke(1992) Plasmid. 27, 73-79. 13. “Anaerobic production of magnetite by Fe reducing micro organisms. ” By Lovely Et Al. (1987) Nature 330, 252-254. 14. “Molecular tectonics in biomineralisation ” by Mann S(1993) Nature 365,499-505. 5. “Formation of minerals by micro organisms” by Lowenstam H A(1981) Science 211. 1126-1131. 16. “Development in terrestrial bacterial remediation of metals” by Stephen J R (1981) Nature 306, 311-345. 17. “Mechanistic aspects of biosynthesis of silver nanoparticles from F. oxysporum” by Nelson Duran, Priscyla d Marcato from J Nanobiotechnology 2005, 3:8 18. “From biology to biotechnology” by Fortin D, Beveridge TJ(2000)Biomineralisation, Wiley VCH Weinheim, pp 7-22. 19. “Nanobiotechnology” by Wiley VCH pp 68-92. 20. “Practical Biochemistry” by Wilkinson 35, 125-200. [pic]